Genomics and biotechnological advances in veterinary, poultry, and fisheries 9780128163528, 0128163526

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Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries

Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries Edited by

Yashpal Singh Malik Division of Biological Standardization, ICAR-Indian Veterinary Research Institute, Izatnagar, India

Debmalya Barh Institute of Integrative Omics and Applied Biotechnology (IIOAB), Nonakuri, Purba Medinipur, West Bengal, India

Vasco Azevedo Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), Brazil

S.M. Paul Khurana Amity Institute of Biotechnology, Amity University, Haryana, Gurugram, Manesar, India

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-816352-8 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Charlotte Cockle Acquisition Editor: Patricia Osborn Editorial Project Manager: Lindsay Lawrence Production Project Manager: Mohana Natarajan Cover Designer: Matt Limbert Typeset by MPS Limited, Chennai, India

List of contributors G.K. Aseri Amity Center for Mycobacterial Disease Research, Amity Institute of Microbial Technology, Amity University Rajasthan, Jaipur, India Asha Augustine ICAR-Central Marine Fisheries Research Institute, Kochi, India Aiman Aziz Shifa College of Pharmaceutical Sciences, Shifa Tameer-e-Millat University, Islamabad, Pakistan Mustafeez Mujtaba Babar Shifa College of Pharmaceutical Sciences, Shifa Tameer-e-Millat University, Islamabad, Pakistan Sana Rasul Baloch Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Islamabad, Pakistan Krisztia´n Ba´nyai Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary Megha Kadam Bedekar Aquatic Environment and Health Management Division, ICAR-Central Institute of Fisheries Education, Mumbai, India Jaya Bharati ICAR-Indian Veterinary Research Institute, Bareilly, India; ICAR-National Research Centre on Pig, Guwahati, India Raja Aadil Hussain Bhat ICAR-Directorate of Coldwater Fisheries Research, Bhimtal, Nainital, India Sudipta Bhat Division of Virology, Indian Veterinary Research Institute, Bareilly, India Madan L.B. Bhatt Centre for Advanced Research (CFAR)-Stem Cell/Cell Culture Unit, King George’s Medical University (KGMU), Lucknow, India Emı´lia Oliveira Alves Costa Replicon Research Center, Masters in Genetics, School of Agrarian and Biological Sciences, Pontifical Catholic University of Goia´s, Goiaˆnia, Brazil Alex Silva da Cruz Replicon Research Center, Masters in Genetics, School of Agrarian and Biological Sciences, Pontifical Catholic University of Goia´s, Goiaˆnia, Brazil

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List of contributors

Aparecido D. da Cruz Replicon Research Center, Masters in Genetics, School of Agrarian and Biological Sciences, Pontifical Catholic University of Goia´s, Goiaˆnia, Brazil; Postgraduate Program in Biotechnology and Biodiversity, Federal University of Goia´s, Goiaˆnia, Brazil Rakesh Das Central Institute of Freshwater Aquaculture (ICAR), Kausalyaganga, Bhubaneswar, India Rajib Deb ICAR-Central Institute of Research on Cattle, Meerut, Uttar Pradesh, India Pallavi Deol Division of Virology, ICAR-Indian Veterinary Research Institute, Bareilly, India Kuldeep Dhama Division of Pathology, ICAR-Indian Veterinary Research Institute, Bareilly, India Andor Doszpoly Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary Michael J. D’Occhio The University of Sydney, Sydney, New South Wales, Australia Ablesh Gautam Central Research Institute, Kasauli, India Souvik Ghosh Department of Biomedical Sciences, Ross University School of Veterinary Medicine, St. Kitts and Nevis, West Indies Afsaneh Golkar-Narenji Prestage Department of Poultry Science, North Carolina State University, Raleigh, NC, United States Alvina Gul Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Islamabad, Pakistan Vikas Gupta Department of Veterinary Microbiology, Faculty of Veterinary and Animals Sciences, IAS, RGSC, Banaras Hindu University, Mirzapur, India Mukta Jain Amity Center for Mycobacterial Disease Research, Amity Institute of Microbial Technology, Amity University Rajasthan, Jaipur, India Imelda Joseph ICAR-Central Marine Fisheries Research Institute, Kochi, India Karim Kaleh Department of Biological Sciences, Tennessee State University, Nashville, TN, United States

List of contributors

Eszter Kaszab Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary Azhar Khan Amity Center for Mycobacterial Disease Research, Amity Institute of Microbial Technology, Amity University Rajasthan, Jaipur, India Sajal Kole Aquatic Environment and Health Management Division, ICAR-Central Institute of Fisheries Education, Mumbai, India; Department of Aqualife Medicine, Chonnam National University, Gwangju, Republic of Korea Wilfried A. Kues Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Ho¨ltystr10, Neustadt, Germany Amit Kumar ICAR-Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India Deepak Kumar Genetic Engineering-Virus Laboratory, Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute, Bareilly, India Dharmendra Kumar Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar, Haryana, India Naveen Kumar ICAR-National Institute of High Security Animal Diseases, Bhopal, India Ravi Kumar Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, India Swatantra Kumar Centre for Advanced Research (CFAR)-Stem Cell/Cell Culture Unit, King George’s Medical University (KGMU), Lucknow, India Nayaab Laaldin Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Islamabad, Pakistan Gianvito Lanave Department of Veterinary Medicine, University of Bari, Valenzano, Bari, Italy Supreeti Mahajan Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, India Yashpal Singh Malik Division of Biological Standardization, ICAR-Indian Veterinary Research Institute, Izatnagar, India Szilvia Marton Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary

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List of contributors

Vimal K. Maurya Centre for Advanced Research (CFAR)-Stem Cell/Cell Culture Unit, King George’s Medical University (KGMU), Lucknow, India Lysa Bernardes Minasi Replicon Research Center, Masters in Genetics, School of Agrarian and Biological Sciences, Pontifical Catholic University of Goia´s, Goiaˆnia, Brazil S.S. Mishra Central Institute of Freshwater Aquaculture (ICAR), Kausalyaganga, Bhubaneswar, India Namita Mitra Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States Paul E. Mozdziak Prestage Department of Poultry Science, North Carolina State University, Raleigh, NC, United States Chandra Sekhar Mukhopadhyay College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India Ramadevi Nimmanapalli Department of Veterinary Microbiology, Faculty of Veterinary and Animals Sciences, IAS, RGSC, Banaras Hindu University, Mirzapur, India Aneeqa Noor Department of Neurology, National Reference Center for TSE, Georg-August University, Gottingen, Germany Amit Pande ICAR-Directorate of Coldwater Fisheries Research, Bhimtal, Nainital, India Hitesh N. Pawar Department of Diagnostic and Biomedical Sciences, School of Dentistry, University of Texas Health Science Center at Houston, Houston, TX, United States James N. Petitte Prestage Department of Poultry Science, North Carolina State University, Raleigh, NC, United States Alexis Pigg Department of Agriculture and Environmental Sciences, Tennessee State University, Nashville, TN, United States Irene Plaza Pinto Replicon Research Center, Masters in Genetics, School of Agrarian and Biological Sciences, Pontifical Catholic University of Goia´s, Goiaˆnia, Brazil Rathnagiri Polavarapu Genomix Biotech Inc., Atlanta, GA, United States

List of contributors

Meeti Punetha ICAR-Indian Veterinary Research Institute, Bareilly, India Tausif Ahmed Rajput Shifa College of Pharmaceutical Sciences, Shifa Tameer-e-Millat University, Islamabad, Pakistan S.N. Sahoo Central Institute of Freshwater Aquaculture (ICAR), Kausalyaganga, Bhubaneswar, India B.A.A. Sai Kumar ICAR-Indian Veterinary Research Institute, Bareilly, India Mihir Sarkar ICAR-Indian Veterinary Research Institute, Bareilly, India Ankur Saxena ICAR-Directorate of Coldwater Fisheries Research, Bhimtal, Nainital, India Shailendra K. Saxena Centre for Advanced Research (CFAR)-Stem Cell/Cell Culture Unit, King George’s Medical University (KGMU), Lucknow, India Gyanendra Singh Sengar ICAR-Central Institute for Research on Cattle, Meerut, Uttar Pradesh, India Deepansh Sharma Amity Center for Mycobacterial Disease Research, Amity Institute of Microbial Technology, Amity University Rajasthan, Jaipur, India Laruen E. Shields Department of Agriculture and Environmental Sciences, Tennessee State University, Nashville, TN, United States Danilo Conrado Silva Graduate Program in Animal Sciences, School of Veterinary Medicine and Animal Science, Federal University of Goia´s, Goiaˆnia, Brazil Raj Kumar Singh ICAR-Indian Veterinary Research Institute, Bareilly, India Jagdip Singh Sohal Amity Center for Mycobacterial Disease Research, Amity Institute of Microbial Technology, Amity University Rajasthan, Jaipur, India P. Swain Central Institute of Freshwater Aquaculture (ICAR), Kausalyaganga, Bhubaneswar, India Ashish Tiwari University of Kentucky, Lexington, KY, United States Shailly Tomar Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, India

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List of contributors

Gayatri Tripathi Aquatic Environment and Health Management Division, ICAR-Central Institute of Fisheries Education, Mumbai, India Mudit Tyagi The George Washington University, Washington, DC, United States Divyang Vats Amity Center for Mycobacterial Disease Research, Amity Institute of Microbial Technology, Amity University Rajasthan, Jaipur, India Atul Verma Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, United States; Division of Biological Standardization, ICAR-Indian Veterinary Research Institute, Izatnagar, India Ramneek Verma Department of Microbial and Environmental Biotechnology, College of Animal Biotechnology, Guru Angad Dev Veterinary And Animal Sciences University, Ludhiana, Punjab, India G.M. Vidyalakshmi ICAR-Indian Veterinary Research Institute, Bareilly, India Xiaofei Wang Department of Biological Sciences, Tennessee State University, Nashville, TN, United States Rebecca L. Welch Department of Chemistry, Tennessee State University, Nashville, TN, United States Mohamad Zamani-Ahmadmahmudi Department of Clinical Science, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran

About the editors Dr. Yashpal Singh Malik is an expert in viral diseases’ epidemiology, virus-host interactions, microbial biodiversity, characterization, and diagnosis of pathogens. Since 2014, he has been the principal scientist and national fellow working at Indian Veterinary Research Institute (ICAR), Izatnagar, India. He acquired advanced training in molecular virology at University of Minnesota, Minneapolis, United States (2005), Division of virology, University of Ottawa, Ontario, Canada (2013), and Wuhan Institute of Virology, China (2019). He did his postdoctoral research (2002) at University of Minnesota, Saint Paul, United States. Due to his significant contributions in the field of animal virology, he has been recognized by several prestigious national, state, and academy awards and honors including ICAR-Jawaharlal Nehru Award. He is also a fellow of Indian Virological Society and member of the study group on Picobirnaviridae and Birnaviridae of International Committee on Taxonomy of Viruses (ICTV). He is a member of the managing committee of the World Society of Virology. He has supervised 3 PhD and 17 MVSc students. Has authored 5 books, 25 book chapters, and published 185 scientific research and review articles in reputable journals. He is the editor-in-chief of Journal of Immunology Immunopathology and also edited special issues of Springer’s journal Virus Disease, Bentham’s journal, The Open Virology Journal, and Current Drug Metabolism on emerging themes. Dr. Debmalya Barh is a MSc (applied genetics), MTech (biotechnology), MPhil (biotechnology), PhD (biotechnology), PhD (bioinformatics), postdoctoral (bioinformatics), and PGDM (postgraduate in management). He is honorary principal scientist at the Institute of Integrative Omics and Applied Biotechnology (IIOAB), India. He has blended both academic and industrial research for decades and is an expert in integrative omics-based biomarker discovery, infectious disease genomics and drug discovery, molecular diagnosis, and precision medicine for various complex human diseases and traits. He has worked with over 400 scientists from more than 100 organizations across over 40 countries. He has published over 150 research publications, over 32 book chapters, and has edited over 20 cutting-edge, omics-related reference books published by Taylor & Francis, Elsevier, and Springer. He frequently reviews articles for Nature publications, Elsevier, AACR journals, NAR, BMC journals, PLoS One, and Frontiers, to name a few. He has been recognized by

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About the editors

Who’s Who in the World and Limca Book of Records for his significant contributions in managing advance scientific research. Dr. Vasco Azevedo is a senior professor of genetics and deputy head of the department of genetics, ecology, and evolution of Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. He is a member of the Brazilian Academy of Sciences and is a knight of the National Order of Scientific Merit of the Brazilian Ministry of Science, Technology, and Innovation. He is also a researcher 1A of the National Council for Scientific and Technological Development (CNPq), which is the highest position. He is a molecular geneticist who graduated from veterinary school, Federal University of Bahia in 1986. He obtained his Master (1989) and PhD (1993) degree in molecular genetics, at Institut National Agronomique Paris-Grignon (INAPG) and Institut National de la Recherche Agronomique (INRA), France. He did a postdoctorate in Microbiology Department of Medicine School in 1994 from University of Pennsylvania, Philadelphia, United States. Recently, in 2017, he did another PhD in the field of bioinformatics. His total research publications include over 400 research articles, 3 books, and over 30 book chapters. He is a pioneer of genetics of lactic acid bacteria and Corynebacterium pseudotuberculosis in Brazil. He has specialized and currently researching bacterial genetics, genome, transcriptome, proteome, development of new vaccines, and diagnostics for infectious diseases. Prof.(Dr.) S.M. Paul Khurana PhD (1969); had Post-Doc research in advanced virology at Kyushu University, Japan (1970 72), University of Minnesota, Saint Paul (United States, 1987 88) specializing in immunodiagnostics. Currently he is a professor of biotechnology and the head of University Science Instrument Centre, Amity University Haryana, Gurgaon, India. Earlier he served as the vice chancellor of Rani Durgavati University, Jabalpur and director of Central Potato Research Institute, Shimla. He is also the founding director of Amity Institute of Biotechnology, Gurgaon, Haryana. He has received several awards including SARC Outstanding Achievement Award 2011, ISMPP Lifetime Achievement Award, 2012; CHAI-Hony Fellowship and Lifetime Achievement Award, 2016; International Award for Excellence recognizing eminent contributions to the promotion of significant research in medicinal and aromatic plants, at 7th GOSMAP, Nov, 2018, Thailand, and Fellow National Academy of Agriculture Sciences, National Academy of Biological Sciences among others.

About the editors

He has more than 53 years of experience in research in pathology, virology, and pioneered use of ELISA and ISEM for detection of potato viruses. He was invited/deputed to many international conferences, in India and abroad by the GOI/ICAR/UGC/Confr organizers to the United Kingdom, the United States, the Netherlands, Canada, China, France, Greece, Japan, Italy, Peru, South Africa, Uganda, etc. He has published 230 research papers, 105 reviews/chapters, authored and edited 16 books, and 12 technical bulletins. He was chief editor for journals from Indian Virological Society, Indian Potato Association, Indian Phytopathological Society, and Aphidological Society.

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Foreword The exponential population growth is exerting pressure on the quantity and quality food of animal origin. In spite of attaining recent record-breaking production, a high proportion of the world’s population remains undernourished. Healthy and productive livestock are important to protect and promote people’s livelihoods. Within the veterinary domain, farm animals, poultry, and fisheries are indispensable. Although with the increasing world population, demand for livestock products is also increasing yet several factors affect the growth of animal husbandry sector to meet the growing demand. Innovations in genomics and biotechnology have accomplished some recent advances in the field with many applications to hasten animal growth, enhance reproductive capacity, improve livestock health, and develop new animal products. The role of biotechnology in the management of several animal diseases through vaccines, particularly genetically engineered or DNA vaccines, improved diagnostics and have made a significant effect on human health by ensuring safe food and preventing zoonotic diseases. Books summarizing applications of genomics and biotechnology in veterinary, pets, poultry, and fisheries are limited. This multiauthored book on Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries has wellillustrated chapters and up-to-date reviews and references. This book provides a comprehensive knowledge in the field through a team of more than 70 experts from 9 countries covering cattle genomics, metagenomics revealing new virus species in farm and pet animals, and aquaculture, and genome editing in animals. This book also covers applications for poultry and fishery elaborating the transgenic birds, genome editing in poultry, concepts, and potential applications of gene editing in fish and aquaculture and marine animal biotechnology for food. The concluding section deals with innovations in the field of aquaculture vaccines, diagnosis, and prevention of diseases in farm and pet animals, fishery and aquatic animals, vectored vaccines for animal diseases and bioinformatics, contemporary vaccine approaches and next-generation adjuvants, structure-assisted drug design for animal viral diseases, and vaccines. My compliments to all the editors and authors for having penned down their expertise and long research experience. I hope this book will be useful to teachers and students and also provide critical inputs to various stakeholders including academicians, researchers, planners, policymakers, etc. Joy Krushna Jena Deputy Director General (Animal Sciences), Indian Council of Agricultural Research, Krishi Bhavan, New Delhi, India

Place: New Delhi Dated: June 4, 2019

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Preface Despite that we are in the 21st century and have most of advanced technologies in hand, malnutrition, food scarcity, and death due to starvation are frequently found in various parts of the world. Biotechnology-based food revolution is restricted mainly to cereals, vegetables, and few meat producing animals and poultry birds. Currently, an overwhelming number of research articles and books are available on both plant and animal biotechnology. However, a book summarizing applications of genomics and biotechnology related to veterinary, pets, poultry, and fisheries are not available. Such a resource is essential for the animal biotechnology research community to understand the latest knowledge and trends in this field so that it can be utilized for improving the fodder-animal traits and meet the needs of human beings. To overcome these issues and fill the gaps, we have come up with Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries to provide a literature resource in this area. This is a comprehensive reference book based on up-to-date published literature covering important topics so that it helps guide animal biotechnologists, veterinary clinicians, and fishery scientists to understand the latest advances in these fields in detail; to get a brief overview and applications for cattle, pet animals, poultry, marine, and animal pathogen genomics; to understand the markers to improve farm animals and fishery; to learn recent approaches in cloning and transgenic cattle, poultry, and fish production; to get an idea of genome editing in veterinary animals and poultry birds; to understand molecular diagnosis of cattle, poultry, and fish; and to get an account of next-generation vaccines for cattle, poultry, and fish. In this book, we have included 20 chapters divided into 4 sections. Section 1 (Introduction) comprises three chapters: The first chapter (Chapter 1: Cattle genomics: genome projects, current status, and future applications) by Dr. Mukhopadhyay and colleagues provides an overview of cattle genomics and related projects, their current status, and future applications. In Chapter 2, Metagenomics revealing new virus species in farm and pet animals and aquaculture, metagenomics applications for virus species identification for fishery, farm, and pet animals are discussed by Dr. Banyai’s group. An overview of genome editing in economically important animals is given in Chapter 3, Genome editing in animals: an overview, by Dr. Sarkar and colleagues. Section 2 discusses biotechnological approaches for farm and pet animals in five chapters. Dr. Deb and team in Chapter 4, Genetic markers for improving farm animals, have provided a detailed account on genetic markers to help improve farm animals. In the next two chapters genome editing is discussed by Dr. Kumar’s team on the applications of genome editing in farm animals (Chapter 5: Applications of genome editing in farm animals) while Dr. Sohal’s

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Preface

group discusses this for pet animals (Chapter 6: Applications of the genome editing in pet world). Reverse genetics applications to improve animal health is given in Chapter 7, Modulation of animal health through reverse genetics applications, by Dr. Pawar and colleagues. The last chapter (Chapter 8: Animal models: bridging cross-species variation through animal biotechnology) in this section, by Dr. Babar’s team, deals with animal models used in animal biotechnology. Four chapters, dedicated to biotechnological advances in poultry and fishery are in Section 3. Chapter 9, Transgenic chicken/poultry birds: serving us for survival, by Dr. Mozdziak and colleagues discusses transgenic poultry birds. The next chapter (Chapter 10: Transgenesis and genome editing in chickens) by Dr. Wang’s group elaborates the genome editing technologies applied to poultry birds. Dr. Pande in the next chapter (Chapter 11: Concepts and potential applications of gene editing in aquaculture) has overviewed the potential applications of gene editing in fish. The last chapter in this section is concluded with the description of marine animal biotechnology for food (Chapter 12: Marine biotechnology for food) by Dr. Joseph and Dr. Augustine. In Section 4, we have included eight chapters discussing biotechnology for animal disease diagnosis and prevention. Dr. Malik and colleagues in Chapter 13, Biotechnological innovations in farm and pet animal disease diagnosis, explain various biotechnological innovations for diagnosis of various diseases in farm and pet animals. In the next chapter (Chapter 14: Biotechnological tools in diagnosis and control of emerging fish and shellfish diseases), Dr. Mishra’s group has given a detailed account on biotechnological approaches in developing diagnostics, therapeutics, and vaccines for fishes and aquatic (fodder) animals. Dr. Tiwari’s team deals with advances and applications of vectored vaccines in animal diseases in Chapter 15, Advances and applications of vectored vaccines in animal diseases. Bioinformatics approaches are discussed in Chapter 16, Bioinformatics for animal diseases: focused to major diseases and cancer, by Dr. Zamani-Ahmadmahmudito who explored solutions for animal diseases including cancers. Biotechnological tools for diagnosis and control of emerging fish diseases are discussed in Chapter 17, Biotechnological approaches to fish vaccine, by Dr. Mishra’s group. Dr. Saxena and colleagues deal with the contemporary and next-generation vaccines in managing various viral diseases in Chapter 18, Contemporary vaccine approaches and role of next-generation vaccine adjuvants in managing viral diseases. A detailed account of structure-assisted antiviral drug design for animal viruses is discussed in Chapter 19, Advances in structure-assisted antiviral discovery for animal viral diseases, by Dr. Tomar and colleagues. The concluding chapter (Chapter 20: Vaccines the tugboat for prevention-based animal production) of this book by Dr. Nimmanapalli and Dr. Gupta provides insights on different vaccines for livestock and poultry.

Preface

More than 70 experts from 9 countries have contributed chapters for this book. We hope that this book will be helpful in various ways to scientists, researchers, and students working in the advanced fields of animal biotechnology and veterinary, poultry, and fishery sciences. Yashpal Singh Malik Division of Biological Standardization, ICAR-Indian Veterinary Research Institute, Izatnagar, India

Debmalya Barh Institute of Integrative Omics and Applied Biotechnology (IIOAB), Nonakuri, Purba Medinipur, West Bengal, India

Vasco Azevedo Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), Brazil

S.M. Paul Khurana Amity Institute of Biotechnology, Amity University, Haryana, Gurugram, Manesar, India

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CHAPTER

Cattle genomics: genome projects, current status, and future applications

1

Chandra Sekhar Mukhopadhyay1, Amit Kumar2 and Rajib Deb3 1

College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India 2 ICAR-Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India 3 ICAR-Central Institute of Research on Cattle, Meerut, Uttar Pradesh, India

1.1 Introduction The science of genetics and genomics has traversed a long way through groundbreaking discoveries of cytogenetic maps, synteny analysis, somatic cell genetics, in situ hybridization, radiation-hybrid maps, and constructing high-resolution comparative mapping, molecular markers (hybridization and polymerase chain reaction based), rudimentary genome maps, association studies for quantitative trait loci-marker aided selection (QTL-MAS) [with economically important traits (ETL)], and whole genome shotgun sequencing to whole genomic mapping that inducted considerable positive implications on molecular breeding. The scientific breakthroughs in human genomics have played a pivotal role to harbinger the research works and discoveries in bovine as well as other livestock species. Livestock and chicken were initially considered as model animals for biological studies (pathophysiological, drug discovery etc.) for human research (Bai et al., 2012). Over time, the focus has changed to ameliorate the productive, reproductive, and growth performances and disease resistance in animals to serve mankind. Genomic studies in cattle have mostly been reported prior to that in other livestock species. The discovery of 1/29 centric fusion (Robertsonian translocation) in cattle (Gustavsson and Rockborn, 1964) followed by chromosomemapping through somatic cell hybridization (Heuertz and Hors-Cayla, 1978) flagged the marathon of step-by-step scientific breakthroughs (Larkin, 2011; Womack, 1998). Further research work was focused on linkage analysis and chromosomal organization study (Womack and Moll, 1986), microsatellite-based (Barendse et al., 1997) and single nucleotide polymorphism (SNP)-based (Snelling et al., 2005) linkage maps (Gallagher et al., 1993; Fries et al., 1993; Iannuzzi et al., 1993), gene mapping (Itoh et al., 2003), high-resolution physical map construction (Everts-van der Wind et al., 2005; Snelling et al., 2007), finally genome sequencing and whole genome assembly of Bos taurus (Zimin et al., Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00001-1 © 2020 Elsevier Inc. All rights reserved.

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CHAPTER 1 Cattle genomics

2009), and humped cattle (Canavez et al., 2012) which founded the cornerstone for the much coveted genome-wide association studies (GWAS) and molecular breeding of present days. While in other domestic species the research progress was a bit lagging. The somatic cell hybrid of ovine was reported by Burkin et al. (1993). Horse genomics (Lindgren et al., 1998; Gue´rin et al., 1999, 2003; Swinburne et al., 2000), swine genomics (please vide European PiGMaP initiative) (Archibald et al., 2010), etc. were reported later. On the other hand chicken genome projects have revealed valuable information. Note that the first livestock species to be genome sequenced was the Red Junglefowl. The chicken is considered as a model animal to study genetic linkage owing to high recombination rate of its microchromosomes (Burt, 2005). In this chapter, we will discuss the prime scientific discoveries that have elevated the research platform of cattle genomics and the current scenario of important cattle genomics projects.

1.2 Sequencing cattle genome Cattle genomics has ushered in an era where the voluminous big data obtained through massive parallel sequencing of cattle genome were biocomputationally analyzed to study evolutionary perspectives, domestication, and to explore accurate and reliable methods for the selection of breedable animals with high genetic merits. The large scale, as well as collaborative genome research projects being undertaken in European countries, the United States, and Australia, has explored new information and updated the genome annotation. A number of databases on cattle genome are now available. Cattle variation databases viz. Ensembl variation (https://www.ensembl.org/info/genome/variation/sources_documentation.html? redirect 5 no#bos_taurus), NCBI dbSNP (https://www.ncbi.nlm.nih.gov/snp), genome variation map (http://bigd.big.ac.cn/gvm/) etc. contain valuable information on the genes and their variants, SNPs and QTLs, orthologs of human disease genes, etc. The taurine genome has been sequenced, analyzed, and partially annotated by the Bovine Genome Project which is a collaborative project involving several groups and funding from the United States, Canada, United Kingdom, France, Australia, and New Zealand (Burt, 2009). The first assembly of cattle genome sequence (sevenfold coverage) was published on April 24, 2009 in the Bovine Genome Sequencing and Analysis Consortium (http://bovinegenome.org/? q 5 bovine_genome_consortium; Bovine Genome Sequencing and Analysis Consortium, 2009). The draft sequencing of the taurine genome was conducted at the Baylor College of Medicine Human Genome Sequencing Center, Houston, Texas, United States using the DNA from a Hereford dam L1 Dominette 01449 [Bacterial artificial chromosome (BAC) library was constructed]. The biocomputational analyses of the whole genome sequencing data were carried out by two consortia. The detail of first bovine genome sequencing can be obtained at https://www.hgsc.bcm.edu/other-mammals/bovine-genome-project/. More than

1.2 Sequencing cattle genome

22,000 genes and a core set of 14,345 orthologs (shared among seven mammalian species) were reported in this project (Bovine Genome Sequencing and Analysis Consortium, 2009). Later further improvement with the latest sequencing technologies and refinement of the assembly (that included holandric genes also) has been made over the time with improved versions of the genome assemblies. Table 1.1 is verbatim as reported in the HGSG Bovine Genome Project page (https://www.hgsc.bcm.edu/other-mammals/bovine-genome-project). Two genome assemblies of cattle (B. taurus), namely, BCM4 (of Baylor College of Medicine) and UMD2 (University of Maryland) were sequenced using WGS at the Baylor genome sequencing center (Burt, 2009). The BCM4 reads were assembled using the Atlas assembly program (Havlak et al., 2004), while the assembly of UMD2 (comprising of around 24 million whole genome sequencing reads and 11 million BAC-reads) was constructed using suitable assembly and mapping tools (Zimin et al., 2009). Table 1.1 Releases of bovine genome assemblies and the relevant technical detail. S. no.

Date released

Release name

Coverage

Technique used

1

September 2004

Btau_1.0

3x

2

March 2005

Btau_2.0

6.2x

3 4

August 2006 October 2007

Btau_3.1 Btau_4.0

7.1x 7.1x

5

April 2009

Btau_4.2

7.1x

6

October 2009

Btau_4.5

7.1x

7

July 2012

Btau_4.6.1

7.1x

8

December 2015

Btau_5.0.1

25x

Whole genome shotgun reads from clones of small inserts were assembled Bacterial artificial chromosome (BAC) end sequences were used along with small insert clones as well Same as above Draft assembly same as above. The sequences were mapped to bovine chromosomes Baylor College of Medicine Human Genome Sequencing Center in Houston, Texas released the improved draft assembly Btau_4.2 Additional WGS contigs from Btau_2.0 were incorporated into Btau_4.0 Data of previous Btau_4.5 have been replaced with high quality finished data (as per availability) The most improved version: 19x PacBio data with UMD 3.1 using PB Jelly has been used

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1.3 Bovine single nucleotide polymorphism arrays SNP array is a kind of DNA microarray that allows for detecting polymorphisms at specific SNP loci, within or between population(s). Principally, probes (i.e., DNA fragments of known sequence) impregnated on silicon chip hybridize through hydrogen bonding between the complementary cDNA/cRNA (i.e., target) sequence that is prelabeled with a fluorophore. The strength of the signal (fluorescence) emitted by each spot (or “feature”) is detected with an automated scanner. The intensity of fluorescence is proportionate to the extent of a number of hybridized molecules. SNP arrays are developed after conducting systematic studies on polymorphism existing in the globally divergent breeds of a livestock species and thereafter determining the key SNPs associated with the ETL. The divergent breeds/strains of a species evolve over time due to environmental segregation and other stochastic and deterministic natural forces (mutation, natural, and artificial selection), population size, etc. The natural forces craft the genomes of the segregating breeds by impregnating SNPs in the coding and noncoding regions in the genome (Mukhopadhyay and Kumar, 2013). SNPs that are associated with the causative mutations are used as direct markers or haplotype block for determining association with the traits of interest. While constructing SNP chip, these SNPs are individually probed and the accuracy of the results are checked. The most predictive SNPs (consisting of 30
300 SNPs per trait) that account for most of the trait variation are considered as “key SNPs.” The genotypic values predicted by the “key-SNPs” are summed up to obtain an estimate of the genetic worth of the animal under study. The predicted genetic merit of the animal is termed as the molecular breeding value (MBV) or genomic estimated breeding value (GEBV) since it is estimated from the molecular data. The global players in SNP-chip manufacturing have produced bead chips for bovines that have a varying level of density. Illumina has produced SNP chips like BovineHD Beadchip, BovineLD Beadchip, BovineSNP50 Genotyping Beadchip, etc. in collaboration with United States Department of Agriculture-Agricultural Research Service (USDA-ARS), University of Missouri, French National Institute for Agricultural Research (INRA), National Association of Livestock and Artificial Insemination Cooperatives, and other frontiers in this area. Selection of SNPs is very crucial and is the most cumbersome background work. The SNPs to be selected should be highly informative with a high MAF calculated over divergent breeds. A pertinent problem regarding the SNP density is the lack of flanking information at the end of each chromosome that contributes to lower imputation efficiency in preliminary tests. Construction of bovine SNP chip was challenging. While constructing the BovineLD BeadChip the SNP density was doubled in the first and last segments of each chromosome, in order to mitigate this problem (Mukhopadhyay and Kumar, 2013). Some of the important features of these SNP chips are:

• These Illumina products have got 99% call rates and 99.9% reproducibility (http://www.illumina.com/products/bovinehd_whole-genome_genotyping_kits. ilmn).

1.3 Bovine single nucleotide polymorphism arrays

• The chips also include holandric and mitochondrial SNPs which can be •

exploited for determining subspecies classification and certain paternal and maternal breed lineages (Boichard et al., 2012). The reference minor allele frequency (MAF) has been estimated from divergent breeds (European Holstein, Jersey, Brown Swiss, Swedish Red and White, Charolais, Limousin, Brahman, N’Dama, Santa Gertrudis, etc.) spanned over 10 countries (of North America, Europe, and Oceania).

The low-density BeadChip came into existence after a rigorous work exercised on the Illumina GoldenGate Bovine3K Genotyping Beadchip. The 3000 chip was a low-density bovine SNP chip and was developed and launched by Illumina (http://www.illumina.com/documents/products/datasheets/datasheet_bovine3K.pdf). The major drawback associated with this chip was that the imputation accuracy was compromised due to the dependence of the genotyped individual on the reference population (Wiggans et al., 2012). GoldenGate chemistry, on the contrary to single hybridization event of “Infinium Chemistry,” is based on doublehybridization events to ensure accurate SNP detection. In order to cover this gap, the Illumina Infinium BovineLD Genotyping Beadchip (http://www.illumina.com/ documents/products/datasheets/datasheet_bovineLD.pdf/) was developed, which had high imputation accuracy for higher density SNP genotypes in dairy and beef cattle. The Illumina BovineLD BeadChip included the SNPs available throughout the nuclear genome (including sex chromosomes) and mitochondrial genome, as well. It has been designed to incorporate the SNPs from an array of distant breeds belonging to B. taurus, Bos indicus, and their crosses maintained in different parts of the world. The SNPs incorporated in the chip have high MAF as well as uniform spacing across the genome except at the telomeric regions (reduced spacing between SNPs due to higher density). Thus it supports imputation to higher density genotypes in milk and meat breeds of cattle. The total number of SNPs included in the low-density chip is 6909. The accuracy of imputation to Illumina BovineSNP50 genotypes using the BovineLD chip was over 97% for most dairy and beef populations (Boichard et al., 2012). The BovineSNP50 (54,609 SNPs included) chip was produced from known and validated SNPs. The selection priority of SNP was given to high MAFs in the breeds in the offset; uniform spacing of SNPs (approximately 500 kbp); maintaining the quality of SNP and high reproducibility and inclusion of Y-chromosomal and mitochondrial SNPs for determination of sex, parentage, Y haplotypes vis-a`vis subspecies, and maternal lineages (Boichard et al., 2012). It can run up to 24 samples in parallel. It covers evenly distributed polymorphic SNPs (validated in economically important cattle breeds) with a mean gap of 49.4 kb. The mean MAF is 0.25 over the loci included. The BovineHD BeadChip is improved over its predecessor as it covers SNPs from tropically and temporally adapted taurine and indicine breeds as well as their crosses. The average MAFMAF across all loci is 0.25 for nonhumped and 0.17 for humped cattle. More than 7,49,000 SNPs were validated across all the

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breeds with a MAF more than 0.05. Over 99% of the markers are mapped to the UMD3 bovine genome assembly, which includes coverage of autosomal, mitochondrial, and sex-linked SNPs. Uniform genomic coverage with an average gap of 3.43 kb and a median gap size of 2.68 kb provides excellent SNP density to power robust genome association studies and CNV detection in cattle. It is the most comprehensive tool in Illumina’s bovine portfolio products (http://www. illumina.com/products/bovinehd_whole-genome_genotyping_kits.ilmn). It can be used to identify the genetic variation across worldwide dairy and beef breeds of cattle for genomic selection (GS), identification of QTL, cross-breed mapping, linkage disequilibrium (LD) studies, comparative genetic studies, and breed characterization for evaluating biodiversity (http://www.illumina.com/documents/ products/datasheets/datasheet_bovineHD.pdf). Haploblocks are demonstrated as better explanatory variables, than SNPs, for genomic prediction of MBV (Cuyabano et al., 2014).

1.4 Genome-wide association studies in dairy cattle Whole genome association study evaluates the genome-wide distributed markers (around 1 M apart), obtained from about 1000 or more individuals in each of the treatment vis-a`-vis control groups, in order to identify the positional candidate genes for the traits being investigated. The objective of GWAS is to identify the causative mutations (SNPs) and other variants in DNA which are associated with a trait (Mukhopadhyay and Kumar, 2012, 2015). The limitation of GWAS is that it cannot specify the causal genes on their own. The completion of the Human Genome Project in 2003
04 (International Human Genome Sequencing Consortium, 2004) and the International HapMap Project in 2005 (International HapMap Consortium et al., 2010) has laid the cornerstone for GWAS. The researchers discovered the disease-causing mutations in diseased and risked human population by mining the whole genome data (Table 1.2). GWAS is a process for inspection and screening of detectable common genetic variants (SNPs, indels) in individuals to identify which variant(s) are significantly associated with the trait being studied. The GWAS compares the DNA profiles of individuals having altered traits (viz. disease susceptible, poor production, reproduction, or growth parameters) with the control ones (healthy ones or with normal parameters). The DNA specimen from thousands of individuals (divided into treatment and control groups) is subjected to microarray analysis (or NGS followed by in silico analysis) for detection of specific SNPs that are more prevalent in any one group. The sample size should be in thousands to increase the robustness of the statistical analysis. It has been reported that the associations between SNPs and causal variants generally show low (,1.5) odds ratios. Larger sample size thus improves the power of the test so that it can pass the multiple testing corrections. After the discovery of markers for age-related macular

1.4 Genome-wide association studies in dairy cattle

Table 1.2 Genome size and reported SNPs (and/or refseqs) in various domestic animal.a,b Species

Scientific name

Genome size (Gb)

Number of rs’s in genesc

Nonhumped cattle Humped cattle Goat Sheep Swine Rabbit

Bos taurus

2.67

46,238,047

2.63 2.61 2.8 2.73

5,131,669 60,087 30,029,327 28,699,285 21

2.7 1.21 1.06

1,897,985 14,926,051 6942

Horse Chicken Turkey

Bos indicus Capra hircus Ovis aeris Sus scrofa Oryctolagus cuniculus Equus caballus Gallus gallus Meleagris gallopavo

a

Fan et al. (2010); Dalloul et al. (2010); Yang and Tempelman (2012). Stock and Reents (2013). c Number of SNPs obtained from SNP database of NCBI (http://www.ncbi.nlm.nih.gov/snp/) and https://www.ncbi.nlm.nih.gov/projects/SNP/snp_summary.cgi?view 1 summary 5 view 1 summary& build_id 5 150 with following filters: Variation class: SNP; Annotation: Cited in PubMed, nucleotide, structure; Function Class: 30 splice site, 30 UTR, 50 splice site, 50 UTR, coding synonymous, frameshift, intron, missense, nonsense, stop gained; Global MAF; Validation Status: by-cluster, by-frequency, and no-info. b

degenerations in human in 2005 (Klein et al., 2005), a number of advanced research works on GWAS have identified the causative mutations contributing to a number of human diseases, viz. childhood and adult obesity (Frayling et al., 2007), autoimmunity (Wellcome Trust Case Control Consortium et al., 2007), susceptibility to Crohn’s disease (Parkes et al., 2007), type 1 diabetes (Todd et al., 2007) and type 2 diabetes (Zeggini et al., 2007; Scott et al., 2007), coronary artery disease (Samani et al., 2007), etc. High-density SNPs have been used for GWAS (Hirschhorn and Daly 2005) in humans and other species. The biallelic nature of SNPs (Brookes, 1999) (as assumed in GWAS and GS experiments) is exploited to identify the SNPs that are associated with QTL in animals and disease susceptibility in humans and animals (McCarthy et al., 2008). The generalized methodology GWAS is: two groups (healthy vs diseased) of individuals are compared for the allelic or haplotype differences. All subjects, as mentioned above, in each group are genotyped, using microarray or DNA-seq, for a million SNPs. The healthy and diseased groups are tested for the odds ratio (i.e., the ratio between the proportions of the two groups) of the allele frequencies for each of these SNPs (Clarke et al., 2011). If the two groups are not differing significantly, the odds ratio is one. The P-value, that is, the probability of exclusion of the null hypothesis of odds ratio for the significance of the odds ratio, is calculated using a chi-squared test. It is important that the functional enrichment

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of the genes is found in the modules in order to identify the genes governing the trait of interest. This also requires validation of the generated data with biological samples.

1.5 Marker-assisted selection and genomic selection Marker-assisted selection (MAS) is a process of selecting breeding individuals where a marker (biochemical or genetic) is used for indirect selection of a genetic determinant or determinants of a trait of interest (i.e., productivity, disease resistance, abiotic stress tolerance). MAS is an indirect selection process where a trait of interest is selected, not based on the trait itself, but on a marker linked to it. For instance, if MAS is being used to select individuals with a disease, the level of disease is not quantified but rather a marker allele which is linked with the disease is used to determine disease presence. The assumption is that the linked allele is associated with the gene and/or QTL of interest (Mukhopadhyay and Kumar, 2015). Considerable developments in molecular genetics have led animal breeders to develop more efficient selection systems to replace traditional phenotypic-pedigree-based selection. Smith first suggested the amalgamation of genomic data in the selection process of future breeding animals in the 1960s (Smith, 1967). Gradually, the concept of QTL and major genes acting on a polygenic trait developed. MAS is best suited for the traits which show limited progress through conventional breeding programs, viz.: 1. Sex-limited traits (milk production, fecundity); 2. Traits having low heritability, viz. fitness traits, reproduction parameters, etc.; 3. Traits expressed later in life (survivability, the occurrence of disease in the later stage of life); 4. Traits difficult to measure; 5. Slaughter traits (dressing percentage). GS has been theoretically studied (by simulation studies) and practically implemented in almost all important domestic species (Sellner et al., 2007; Ralph, 2012; Aslam et al., 2012; Petersen et al., 2013; Meuwissen et al., 2014; Dodds et al., 2014; Liu et al., 2014). The advantages of GS could be maximum for dairy cattle breeding programs (Joerg et al., 2014) because the generation interval in traditional progeny testing (PT) schemes is large and selection of young bulls for PT is inaccurate (Schaeffer, 2006). Besides, thousands of bulls that have been progeny tested in the last decades are available as a reference population with very reliable phenotypes, leading to GEBV with high reliabilities (VanRaden et al., 2009). While, for the species with shorter generation interval (viz. poultry, swine), the prediction accuracy is the most important parameter in order to bring about genetic change using GS (Stock and Reents, 2013). The cost of raising male calves in bull mother farms can be reduced to 10% of the previous due to

1.6 Status and attainments of cattle genome projects

the selection of males at an early age based on marker data (Schaeffer, 2006). Pryce and Daetwyler (2012) reported that the market share of non-PT “genomic bulls” (i.e., devoid of milking daughters) internationally ranged between 25% and 50% in 2011. It reflects the gradual increase in the application of GS in the dairy industry. GS may decrease the rate of inbreeding because Mendelian sampling effects can be estimated more accurately, which reduces the coselection of relatives (Daetwyler et al., 2007; Calus et al., 2013). Very strong selection intensity necessarily hikes the rate of inbreeding due to the erosion of genetic variability in the population (De Cara et al., 2011). Thus, GS may double the rate of genetic gain while keeping the rate of inbreeding per generation constant. Young bulls will be superior to proven bulls, and the number of progeny test bulls can be greatly reduced, which then drastically reduces the cost of a PT program (Li et al., 2014).

1.6 Status and attainments of cattle genome projects The next section will discuss the status of cattle genome projects, with major emphasis on GS, worldwide.

1.6.1 Cattle genome projects in Canada Cattle industries in Canada have a significant influence on the livelihood and domestic economy (Shankland, 2011). Canadian cattle production is mainly based on the breeds of highly productive beef cattle and as per the OECD-FAO Outlook projects the value of Canadian beef exports will climb 8.4% by the end of 2018 (https://www.fcc-fac.ca/content/dam/fcc/knowledge/ag-economist/2017). Owing to their excellent adaptability, hardiness as well as foraging capabilities, Hereford and Angus are the traditional breeds of Canadian herd. Further, to expanse, the genetic base of cattle herd as well as to improve the economic traits, certain continental European breeds such as Charolais and Simmental were infused since the 1960s (Athwal, 2002). The crossbreeding program has been initiated by the commercial sectors to capture the advantages of superior economical traits and better genetic gains (Schaeffer et al., 2011). Thus, developing cattle genomic resources has been a strong rationale toward the genetic counseling of animals for excellent milk and beef production, resistance to various infectious diseases, improving feed efficiency as well as better reproductive performance. The Canadian Cattle Genome Project was a large international research project, that involved eminent scientists and data from Canada as well as across the world (Alberta, Ontario, Australia, the United States, Ireland, Scotland, and New Zealand) with the aim to develop an economical genome-wide selection (GWS) tools for efficient beef cattle production (Stothard et al., 2015a). The project was completed on April 1, 2014. A large dataset of animals with feed efficiency and

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carcass/meat quality data were collated by the team members. A total 379 whole genomes of the individual animal from 10 different Canadian beef cattle breed populations were sequenced using the SOLiD 5500xl system and genotyped with a combination of Illumina Bovine BeadChip 50,000 and HD 770,000 platforms. Out of 10, seven (Limousin, Holstein, Angus, Simmental, Gelbvieh, Charolais, and Hereford) and three (Beefbooster, Alberta, and Guelph research populations) were pure and composite breeds, respectively. Animals chosen for sequencing and genotyping were based on an analysis of the pedigree as well as the breed structure for each individual breed. The top 30 key ancestors for each breed were selected for sequencing with the goal of demonstrating 50%
60% of the effective genome, and further animals were chosen for genotyping based on strategies developed to cover over 90% of the genetic base as well as to avoid duplication of close relatives (http://www.canadacow.ca/). Obtained data were subjected for quality control prior to conducting principal component analysis to visualize clustering and bio-banking the sequence repository with the appropriate information of animals like identification number, location, and breed to serve as a reference library for future breeding decisions (Stothard et al., 2015b).

1.6.2 Achievements and status of cattle genome sequencing in European countries Spain, Germany, France, the United Kingdom, and Italy held the largest populations of livestock among the European countries, however, the highest numbers of bovine populations (19.0 million) were recorded in France. In European countries, cattle have been used for the production of milk or meat; draught power, transportation as well as pets/ritual animals, like bulls in Spain (Gkiasta et al., 2003; Cymbron et al., 2005). Half of the European beef cattle production were accounted in France (18.7%), Germany (14.7%), and the United Kingdom (11.7%) (http://ec. europa.eu/eurostat/statistics-explained/index.php/Agricultural_production-animals). As per the FAO database, 464 cattle breeds were classified as local or regional breeds out of total listed 534 breeds in European countries (www.fao.org/dad-is). Genetic diversity existed within European cattle breeds highlights the conservation value of traditional unselected breeds with high effective population size (Medugorac et al., 2009). Thus, characterization of cattle population structures and genetic differentiation play a crucial role in conservation, genealogy, and selection programs. Understanding the genomes enables the detailed assessment of genetic resources as well as origins. Recently, Chung et al. (2017) have analyzed whole genome sequences of 432 unrelated bulls (B. taurus) from 13 different breeds (Angus, Brown Swiss, Charolais, Gelbvieh, Holstein, Jersey, Limousin, Montbeliard, Normandy, Piedmont, European Red Dairy, Holstein Red & White, and Simmental/Fleckvieh), representing 16 different countries, to characterize their population structure as well as genetic diversity. Latent variable probabilistic models for individual-specific allele frequencies were used to analyze these 432

1.7 INTERBULL concept for genetic evaluation of breeding bulls

unrelated bull genomes, as part of the 1000 Bull Genomes Project (Daetwyler et al., 2014), which provided the detailed assessment of population structure among a diverse panel of whole genome sequences (B4.0 million SNPs/bull). Chung et al. (2017) discovered the diverse and pervasive genetic differentiation with respect to the bull’s population structure. They identified that, among all the chromosomes, chromosome 6 contained substantially a large number of SNPs with high R2; which harbors 166 (39.0%) out of 426 SNPs with R2 . 0.6, and all 29 (100%) SNPs with R2 . 0.7. On the contrary, the X chromosome showed the lowest variation with respect to the logistic factors, containing zero SNP with R2 . 0.5. Further, for better understanding the evolutionary as well as biological processes, they conducted the gene set analyses using genomic annotations of SNPs which identified enriched functional categories such as energy-related processes and multiple developmental stages. Thus their population structure analysis of bull genomes can sustenance the genetic management strategies that capture structural complexity as well as promote sustainable genetic breadth.

1.7 INTERBULL concept for genetic evaluation of breeding bulls INTERBULL is an international nongovernmental, nonprofit organization, involved mostly in the international genetic evaluation of breeding bulls with the goal to promote the development as well as the standardization of strategies for genetic evaluation of cattle to make accurate comparisons between animals performing both within and across countries. INTERBULL was a permanent subcommittee developed by joint venture between the International Committee for Animal Recording, the European Association for Animal Production (EAAP), and the International Dairy Federation consisting of 9 members from 47 different countries. INTERBULL uses an advanced scientific method multiple across country evaluation (MACE) to calculate international genetic evaluations. MACE combines information gathered from each country using all known relationships both within and across populations. Further, MACE accounts for the possibility of animals re-ranking between certain countries and it occurs when the performance of animals is better in particular environments than they are in others or even when the genetic evaluation methods vary between countries. Thus, different sets of the result are calculated for every participating country as demonstrated in Fig. 1.1 (http://www.interbull.org/ib/interbullactivities). Important breeds used for genetic evaluation are Ayrshire, Brown Swiss, Guernsey, Holstein-Friesian, Simmental, Jersey, etc. Different trait groups for considering the genetic evaluation of sires are production traits, longevity traits, udder health, calving traits, fertility traits, etc. This can give the benefit of individual countries being able to identify those animals from around the globe that will perform better under their own unique farming systems.

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National evaluations

Country A 1. Sire A1 2. Sire A2 3. Sire A3

MACE

INTERBULL

International evaluations

Country B 1. Sire B1 2. Sire B2 3. Sire B3

Country A 1. Sire A1 2. Sire A2 3. Sire B2 4. Sire B1 5. Sire A3 6. Sire B3

Country B 1. Sire B1 2. Sire A2 3. Sire B2 4. Sire B3 5. Sire A1 6. Sire A3

FIGURE 1.1 International genetic evaluations calculated for different sires from country A and B (and their succeeding rankings), which can be different from one country to the next. Again a different list of international genetic evaluations for all the traits as well as sires evaluated was provided to each member country, expressed in their own units, and relative to their own base of animals. http://www.interbull.org/ib/interbullactivities.

1.8 Achievements and status of cattle genome sequencing in Australia In the year 2001, a Commonwealth Scientific and Industrial Research Organisation workshop recognized the strategic limitations as well as opportunities in Australian livestock industries that could be addressed based on livestock genome sequencing (https://csiropedia.csiro.au/cattle-genome-project/). Hawken et al. (2004) developed an interactive bovine in silico SNP (IBISS) database through the clustering and aligning of bovine expressed sequence tag (EST) as well as mRNA sequences. The IBISS database represents a genomic database that contains consistently annotated predicted gene, mRNA and protein sequences, gene structure, and genomic organization information. To produce 29,965 clusters and 48,565 singletons, approximately 324,000 EST and mRNA sequences were clustered. Further, to determine which SNPs were more likely to be real, an SNP screening regime was positioned on variations detected in the multiple sequence alignment files and predicted SNPs were validated on a diverse set of cattle

1.9 Achievements and status of cattle genome sequencing in Brazil

genomic DNA samples through in vitro amplification as well as sequencing. They identified that 50% of the predicted SNPs were polymorphic in the population. An assembled draft of dairy cattle genome sequence was released in 2007 (Snelling et al., 2007; Bovine Genome Sequencing and Analysis Consortium et al., 2009), which was a comprehensive database containing phenotypic as well as pedigree data, an important resource to capture by Australian dairy industries (Doyle, 2007). In 2009, the complete sequence of the bovine genome was published along with a study of global cattle genetic diversity, which identified 37,470 SNPs in 497 cattle from 19 geographically as well as biologically diverse breeds (Gibbs et al., 2009). Daetwyler et al. (2014) sequenced the whole genomes of 234 bulls including data for 129 individuals from the global Holstein-Friesian population, 43 individuals from the Fleckvieh breed, and 15 individuals from the Jersey breed. For each individual, a total of 28.3 million variants with an average of 1.44 heterozygous sites per kilobase were identified. They further demonstrated the use of this database for identifying a recessive and lethal mutation, underlying embryonic death and chondrodysplasia, respectively. They also executed GWAS for milk production as well as the development of curly coat in cattle, using assigned sequence variants, and further identified variants were associated with these traits.

1.9 Achievements and status of cattle genome sequencing in Brazil Though the majority of the Brazilian cattle herd has B. indicus contribution, only less than 7000 purebred Zebu animals were introduced from India from 1868 to 1962. The Brazilian major B. indicus herd, Nelore (originally Ongole in India), was developed by crossing the imported B. indicus bulls with B. taurus cows (Ferraz and Felı´cio, 2010). In North America, GS is in its preliminary way and being applied to some beef breeds like Angus (Gill et al., 2009; Garrick, 2011); and dual purpose breeds like Simmental and Braunvieh Schweiz (Gredler et al., 2009; Croiseau et al., 2012) to improve EBV’s reliability. However, GS is still not a standard tool for genetic improvement of Nelore cattle herd in Brazil, mostly due to the lack of cost-effective strategies and absence of centralized DNA repositories (Garcia et al., 2013). The pattern of LD in the Nelore breeds is reported being present at lower levels of LD at shorter distances than taurine breeds (Espigolan et al., 2013). Illumina BovineHD (HD) panel (B777K SNPs) based obtained the average LD between adjacent markers in Nelore breeds (B0.29) was similar to the obtained values in Holstein breeds (Hayes et al., 2009a,b). Meuwissen et al. (2001) and Calus et al. (2008); however, stated that these levels of LD are adequate to generate accurate genomic predictions, provided with sufficient phenotypic data. Neves et al. (2014) evaluated the quality of GS for 13 growth, carcass composition and reproduction traits in 685 Nelore

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bulls, from different breeding programs, with high accuracy proofs and genotyped with the HD panel. The genomic predictions accuracies were ranged from 0.17 (navel at weaning) to 0.74 (finishing precocity). They estimated that the accuracy obtained for either weaning traits or both weaning and yearling traits were equivalent to the accuracies of regular proofs (indexes based on EBVs) presented by calves and yearling animals with their own performance. In spite of a small number of genotypes, this study speaks the technical feasibility for GS in Brazilian Nelore cattle breeds. In order to improve the cost-effectiveness of genotyping of Nelore animals, Carvalheiro et al. (2014) carried out genotyping part of the animals with a panel containing around 15,000 useful SNPs and imputing their HD missing genotypes. Since the imputation accuracy from 15,000 to HD was very high (B0.98), thus this strategy is expected to have similar reliabilities of genomic predictions than the more expensive strategy of genotyping using only the HD panel.

1.10 Status of genomic selection across the world in bovine GS has been found to be especially well-suited to bovine breeding and was very rapidly adopted by the breeding industry. For many quantitative traits, such as production and health traits in dairy cattle, a large number of loci are affecting the trait, with a locus capturing only a limited proportion of the total genetic variance (Hayes and Goddard, 2001a,b; VanRaden et al., 2009). Automated methods for SNP genotyping and the use of dense SNP arrays that cover the bovine genome and that explain the majority of genetic variation in important traits have been proposed by GS or whole genome selection (Meuwissen et al., 2001). GS has revolutionized dairy cattle breeding in developed countries like the United States, Canada, Great Britain, Ireland, New Zealand, Australia, France, the Netherlands, Germany, and the Scandinavian countries. Adoption of this technology in the major dairy producing countries has led to significant changes in the worldwide dairy industry (Weller et al., 2017).

1.10.1 Genomic selection in dairy cattle Most of the initial reports on GS were on simulated data (Meuwissen et al., 2001; VanRaden et al., 2008, 2009; Brito et al., 2011). These works revealed that GS can practically be implemented in a domestic animal, especially large animals with longer generation intervals in order to increase the genetic gain and to address the limitations of MAS. Later, GS studies have been done in a number of domestic animals either for selection of breeding animals or for genome-wide association with ETL s (Hayes and Goddard, 2001a,b; Hayes et al., 2006).

1.10 Status of genomic selection across the world in bovine

The reliability of predicted GEBV in dairy cattle has already been evaluated in some countries, like, the United States, New Zealand, Australia, and the Netherlands. These experiments used reference populations between 650 and 4500 progeny-tested Holstein-Friesian bulls which were genotyped for approximately 50,000 genome-wide markers. Reliabilities of GEBV for young bulls without progeny test results in the reference population were between 20% and 67% (Hayes et al., 2009a,b). A GS project has been initiated in Canada, which proposes to genotype 1000 Holstein-Friesian dairy sires for developing a reference population to “train” the SNP chip. Technology platforms are now available to examine the variation among animals for 54,001 SNPs. Cost-benefit analysis in the Canadian dairy population suggests a doubling of genetic gain at 92% of the cost. Gray et al. (2011) studied the possibility of implementing GS by determining the effectiveness of genomic prediction of milk flow traits in Italian Brown Swiss population at the North Carolina State University (United States). The genetic worth for milk flow traits estimated from genomic markers indicated an increase in reliability in most cases compared to traditional pedigree-based evaluations. Across country evaluation of female fertility, parameters have been done in Holstein-Friesian (HF) cows, distributed over Ireland, the United Kingdom, the Netherlands, and Sweden. Bayesian stochastic search variable selection using Gibbs sampling was done for bivariate genome-wide associations of traditional fertility parameters (viz. days to the first service, days to first heat, pregnancy rate to the first service, number of services, and calving interval) and fertility phenotype derived from milk progesterone profiles. It was concluded that sharing of data vis-a`-vis utilizing the physiological measures of the trait under investigation may increase the power of the GWAS by Berry et al. (2012). The composite phenotype of genetic merit for tuberculosis susceptibility among the daughters of HF elite sires was studied at Trinity College Dublin, Ireland (Finlay et al., 2012). They compared around 44,000 SNPs in 307 animals which revealed that 3 SNPs spanned over 65 kb region of chromosome 22 were associated with tuberculosis susceptibility. This genomic region harbors a transporter gene, SLC6A6, or TauT, which is known to function in the immune system. Cuyabano et al. (2014) used haplotype blocks (or haploblocks) as explanatory variables for genomic prediction, which are better than individual SNP as a marker for genomic prediction of MBV. While in an exhaustive study, Brard and Ricard (2015) showed that no specific rules are applicable to predict the reliability of GS using the formulae in vogue. GWAS has also been done for anatomical anomalies, like claw disorder in dairy cattle (van der Spek et al., 2015). The efficiency of feed utilization of multibreed cattle population (measured in term of residual feed intake) has been studied by Khansefid et al. (2014) for detecting the underlying SNPs, using GWAS. In another GWAS conducted by Joerg et al. (2014), to identify the QTLs occurrence of supernumerary teats in the daughters of 1097 Holstein bulls. Van Eenennaam et al. (2014) has discussed the importance and future potential of GS in dairy animals vis-a`-vis other domestic species. Capitan et al. (2015) also indicated the applicability of GS to ameliorate the fertilizing potential of dairy cattle.

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GS has profoundly affected the genetic improvement of dairy and beef cattle. The benefit of genomics is greatest for traits that are difficult to measure, have low heritability, or cannot be quickly, inexpensively, and correctly measured in a large number of animals because the increase in reliability provided by genomics is greatest for those traits (Garcı´a-Ruiz et al., 2016). Genetic trend was predicted to be doubled (Schaeffer 2006) with a decrease or at least no increase in cost. Accuracy for selection of bull dams has increased and the mean generation interval has declined from 4 to 2.5 years (Weller et al., 2017). Besides genetic evaluation, genomics can be used for pedigree discovery, determination of breed composition, mating programs, and tracking of inbreeding (Wiggans et al., 2017; Pryce et al., 2014; VanRaden et al., 2014). Breed composition for crossbred animals can be estimated based on the breed-specific markers, and in 2016 the United States Department of Agriculture (USDA) developed a breed base representation value to report the percentage of DNA contributed to an animal by each of the five breeds currently evaluated genomically (VanRaden and Cooper., 2015). Implementation of genomic evaluation has also led to the inclusion of more traits in the selection indices, and reduction of the emphasis on protein and fat production and conformation traits (Egger-Danner et al., 2015; Chesnais et al., 2016; Weller et al., 2015). Implementing GS in dairy cattle has resulted in an increased genetic gain, which has now been demonstrated by genetic trend analysis in a number of countries. In December 2007, the first commercial SNP genotyping chip was released with a set of 54,001 SNPs for cattle. Over 15,000 animals were genotyped to determine panel SNPs should be used in genomic evaluation of US dairy cattle. Official USDA genomic evaluations were first released in January 2009 for Holsteins and Jerseys, in August 2009 for Brown Swiss, in April 2013 for Ayrshires, and in April 2016 for Guernseys in April 2013 for Ayrshires, and in April 2016 for Guernseys (Wiggans et al., 2017). Smaller countries that are not able to meet this requirement, such as Israel and Ireland, have initiated joint genomic evaluations with larger countries (e.g., Weller et al., 2015; Lund et al., 2016). Hayes et al. (2009c) has compared the results from GS in dairy cattle breeding programs from Australia, New Zealand, Netherland, and the United States and found that the reliabilities of GEBV were substantially greater than breeding values from parental averages. In all countries, the dairy cattle breeding companies are likely to take advantage of the GEBV both to improve rates of genetic gain and to reduce the cost of their breeding programs. In December 2015, the French national database included 400,000 genotyped animals, including 100,000. Now, this large number of genotyped cows is the major resource for population reference replacement and updating. In 2016, 12 French cattle breeds, including several small ones, used GS in their breeding program. This is a crucial evolution because initially, only the largest breeds were able to benefit from this innovation, creating a technological gap with the smaller ones (Boichard et al., 2016). A feature of dairy genomic predictions is collaboration between countries to assemble large reference sets of animal, with three consortiums established

1.10 Status of genomic selection across the world in bovine

(Eurogenomics, including the Netherlands, Germany, France, the Nordic countries, Spain, and Poland; The North American Consortium including the United States, Canada, Italy, and Great Britain; and a “rest of the world” consortium consisting of a number of remaining countries) (Meuwissen et al., 2016). Almost all the countries of the world are pooling resources to conduct multinational GS programs such as the United States and Canada (N.A.) collaboration—GEBVs obtained by USDA in collaboration with Canada for Holstein bulls have been released in public every year since 2008. A project at Guelph with 820 bulls was carried out with increased reliabilities of 8%, 5%, 18%, and 8% for protein yield, fat yield, somatic cell count, and conformation, respectively, for GS (Pryce and Daetwyler, 2011). New Zealand (LIC): LIC had the foresight to store DNA from every sire that was progeny tested since 1980. This enabled LIC to genotype sires that were the best, and the worst too, of their progeny test cohort and thus evaluate markers across the genetic range. The degree of accuracy of GEBVs was measured by their correlation with progeny test breeding values and was found to be ranging from 0.45 to 0.60 for production traits in HF breed (Hayes et al., 2009c). Netherlands (CRV)—CRV launches InSire bulls—designated to GS selected bulls, since 2008, for Holstein and Jersey breeds. Increases in the reliability using GEBVs were also reported by CRV for GS as 17%, 14%, and 11% for protein production, overall conformation, and somatic cell count respectively (Hayes et al., 2009c). Australia (ADHIS & co.): Australian Dairy Herd Improvement Scheme (ADHIS) estimated genomic-based breeding values for bulls in September and December 2010. Some 2381 Holstein bulls were included in the September 2010 analysis (2193 reference bulls and 188 young bulls). In this group of 188 young bulls with almost no daughter performance data, an improvement in reliability across all key traits was evident. Improvement in reliability in Australian breeding values from GS as compared to those from parent average increased from 21% to 53%, 14% to 42%, 8% to 36%, and 12% to 46% for production traits, overall type, fertility, and survival respectively (Pryce and Daetwyler, 2012). Denmark and Sweden (Viking Genetics): Viking Genetics got the first genomic indexes of Holstein, Jersey, and Red Breeds during the latter part of 2007. Today they have genomic indexes every 2 months for purposes of purchasing new bulls for their breeding program. They assign the label of Gen-VikPLUS to their sires with best GEBVs (Pryce and Daetwyler, 2012). Worldwide, approximately 2 million dairy cattle have now been genotyped for the purposes of genomic prediction. In the United States alone, 934,780 Holstein animals, 120,439 Jersey animals, 19,588 Brown Swiss, and 4767 Ayrshire animals have been genotyped. Similar numbers of animals have been genotyped by other countries combined, including 360,000 in France alone (Meuwissen et al., 2016).

1.10.2 Global scenario of genomic selection in beef cattle Van Eenennaam et al. (2014) reviewed accuracies in the range of 0.3
0.7 in beef cattle. In general, however, accuracies of genomic predictions in beef cattle have

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been lower than in dairy cattle. The lower accuracy is because the reference populations are of higher quality in dairy cattle. In beef cattle, the reference population contains fewer animals within a breed, and these animals have not been progeny tested. In addition, the target population and validation animals may be less closely related to the reference population in beef cattle than in dairy cattle. However, in some beef breeds, GS is now applied on a large scale. In the United States, more than 52,000 Angus animals have now been genotyped for GEBV evaluation (Lourenco et al., 2015). GS is advantageous for several important traits in beef cattle which are difficult to measure for, such as feed conversion efficiency and beef quality. Because these traits are expensive to record, it is costly to set up a large training population. For these traits, a multibreed training population and nonlinear analysis based on high-density SNPs or genome sequence data are practiced (Meuwissen et al., 2016).

1.10.3 Genomic selection in multibreed cattle populations GS has been a valuable tool for increasing the rate of genetic improvement in purebred cattle populations. However, there is availability of a large reference population, which is the limitation of GS. To compensate for the small number of reference animals within a breed, it is advantageous to use a multibreed reference population. The principal difference between single- and multiple-breed genomic prediction is in the relationship matrix used to relate SNP effects to phenotypes. Harris and Johnson (2010), used three breed groupings, New Zealand Holsteins, foreign Holsteins, and Jerseys and showed that use of a multibreed genomic evaluation rather than a single-breed produced similar reliabilities for proven bulls, and slightly higher reliabilities for young bulls and Harris et al. (2011) subsequently reported that increasing SNP density in a reference population including purebred and crossbred animals improved prediction accuracy of one pure breed from another, but not crossbred animals. Bolormaa et al. (2014) found that accuracy is increased slightly (0.33
0.38) by using multibreed but not as much as if the same number of animals had been from the same breed. Hoze et al. (2014) recently showed that multibreed genomic evaluation can be very helpful for breeds with small (,500 animals) reference populations, but that the gains decrease as the size of the reference population increases.

1.11 Conclusion Genetic improvement of cattle paved through a long and eventful path. At present, the GS looks set to be the technology that has delivered the largest increase in the rate of genetic gain for the dairy industry in the past 20 years. GS has led to enhanced rates of genetic improvement, shortening of generation intervals, reduction in the number of bulls with daughter records, and fewer genetic ties

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73. Scott, L.J., Mohlke, K.L., Bonnycastle, L.L., Willer, C.J., Li, Y., Duren, W.L., et al., 2007. A genome-wide association study of type 2 diabetes in Finns detects multiple susceptibility variants. Science 316 (5892), 1341
1345. Sellner, E.M., Kim, J.W., McClure, M.C., Taylor, K.H., Schnabel, R.D., Taylor, J.F., 2007. Board-invited review: applications of genomic information in livestock 1,2. J. Anim. Sci. 85 (12), 3148
3158. Shankland, A., 2011. Farming Alone: Canada’s Small Farmers as Key Stakeholders in the Federal Government’s Agricultural Policies (Thesis). Ryerson University, Toronto. URL https://digital.library.ryerson.ca/islandora/object/RULA%3A1102. Smith, C., 1967. Improvement of metric traits through specific genetic loci. Anim. Sci. 9 (3), 349
358. Snelling, W.M., Casas, E., Stone, R.T., Keele, J.W., Harhay, G.P., Bennett, G.L., et al., 2005. Linkage mapping bovine EST-based SNP. BMC Genomics 6 (1), 74. Snelling, W.M., Chiu, R., Schein, J.E., Hobbs, M., Abbey, C.A., Adelson, D.L., et al., 2007. A physical map of the bovine genome. Genome Biol. 8 (8), R165.

References

Stock, K.F., Reents, R., 2013. Genomic selection: status in different species and challenges for breeding. Reprod. Domest. Anim. 48, 2
10. Stothard, P., Liao, X., Arantes, A.S., De Pauw, M., Coros, C., Plastow, G.S., et al., 2015a. A large and diverse collection of bovine genome sequences from the Canadian Cattle Genome Project. GigaScience 4 (1), 49. Stothard, P., Liao, X., Arantes, A.S., Pauw, M.D., Coros, C., Plastow, G.S., et al., 2015b. Bovine whole-genome sequence alignments from the Canadian Cattle Genome Project. GigaScience Database . Available from: https://doi.org/10.5524/100157. Swinburne, J., Gerstenberg, C., Breen, M., Aldridge, V., Lockhart, L., Marti, E., et al., 2000. First comprehensive low-density horse linkage map based on two 3-generation, full-sibling, cross-bred horse reference families. Genomics 66 (2), 123
134. Todd, J.A., Walker, N.M., Cooper, J.D., Smyth, D.J., Downes, K., Plagnol, V., et al., 2007. Robust associations of four new chromosome regions from genome-wide analyses of type 1 diabetes. Nat. Genet. 39 (7), 857. van der Spek, D., van Arendonk, J.A.M., Bovenhuis, H., 2015. Genome-wide association study for claw disorders and trimming status in dairy cattle. J. Dairy Sci. 98 (2), 1286
1295. Van Eenennaam, A.L., Weigel, K.A., Young, A.E., Cleveland, M.A., Dekkers, J.C., 2014. Applied animal genomics: results from the field. Annu. Rev. Anim. Biosci. 2 (1), 105
139. VanRaden, P.M., Cooper, T.A., 2015. Genomic evaluations and breed composition for crossbred US dairy cattle. Interbull annual meeting proceedings. Interbull Bull. 49, 19
23. VanRaden, P.M., Van Tassell, C.P., Wiggans, G.R., Sonstegard, T.S., Schnabel, R.D., Taylor, J.F., et al., 2009. Invited review: reliability of genomic predictions for North American Holstein bulls. J. Dairy Sci. 92 (1), 16
24. VanRaden, P., Sun, C., Cooper, T., Null, D.J., Cole, J.B., 2014. Genotypes are useful for more than genomic evaluation. In: International Committee on Animal Recording (ICAR), May, pp. 19
23. Wellcome Trust Case Control Consortium, 2007. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 447 (7145), 661. Weller, J.I., Stoop, W.M., Eding, H., Schrooten, C., Ezra, E., 2015. Genomic evaluation of a relatively small dairy cattle population by combination with a larger population. J. Dairy Sci. 98 (7), 4945
4955. Weller, J.I., Ezra, E., Ron, M., 2017. Invited review: a perspective on the future of genomic selection in dairy cattle. J. Dairy Sci. 100 (11), 8633
8644. Wiggans, G.R., Cooper, T.A., VanRaden, P.M., Olson, K.M., Tooker, M.E., 2012. Use of the Illumina Bovine3K BeadChip in dairy genomic evaluation1. J. Dairy Sci. 95 (3), 1552
1558. Wiggans, G.R., Cole, J.B., Hubbard, S.M., Sonstegard, T.S., 2017. Genomic selection in dairy cattle: the USDA experience. Annu. Rev. Anim. Biosci. 5, 309
327. Womack, J.E., Moll, Y.D., 1986. Gene map of the cow: conservation of linkage with mouse and man. J. Hered. 77 (1), 2
7. Womack, J.E., 1998. The cattle gene map. ILAR J. 39 (2
3), 153
159. Yang, W., Tempelman, R.J., 2012. A Bayesian ante-dependence model for whole-genome prediction. Genetics 19 (4), 1491
1501.

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Zeggini, E., Weedon, M.N., Lindgren, C.M., Frayling, T.M., Elliott, K.S., Lango, H., et al., 2007. Replication of genome-wide association signals in UK samples reveals risk loci for type 2 diabetes. Science 316 (5829), 1336
1341. Zimin, A.V., Delcher, A.L., Florea, L., Kelley, D.R., Schatz, M.C., Puiu, D., et al., 2009. A whole-genome assembly of the domestic cow, Bos taurus. Genome Biol. 10 (4), R42.

Further reading Boichard, D., Ducrocq, V., Fritz, S., 2015. Sustainable dairy cattle selection in the genomic era. J. Anim. Breed. Genet. 132 (2), 135
143. Elsik, C.G., Tellam, R.L., Worley, K.C., 2009. The genome sequence of taurine cattle: a window to ruminant biology and evolution. Science 324 (5926), 522
528. Pryce, J., Hayes, B., 2012. A review of how dairy farmers can use and profit from genomic technologies. Anim. Prod. Sci. 52 (3), 180
184. Van der Werf, J.H.J., 2009. Potential benefit of genomic selection in sheep. In: Proceedings of the Association for the Advancement of Animal Breeding and Genetics, Vol. 18. pp. 38
41. VanRaden, P.M., Cooper, T.A., Wiggans, G.R., O’Connell, J.R., Bacheller, L.R., 2013. Confirmation and discovery of maternal grandsires and great-grandsires in dairy cattle. J. Dairy Sci. 96 (3), 1874
1879.

CHAPTER

Metagenomics revealing new virus species in farm and pet animals and aquaculture

2

Eszter Kaszab1, Andor Doszpoly1, Gianvito Lanave2, Atul Verma3, Krisztia´n Ba´nyai1, Yashpal Singh Malik3 and Szilvia Marton1 1

Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary 2 Department of Veterinary Medicine, University of Bari, Valenzano, Bari, Italy 3 Division of Biological Standardization, ICAR-Indian Veterinary Research Institute, Izatnagar, India

2.1 Introduction Metagenomics means the genomic analysis of a microbial community from a specific environment. Consequently, viral metagenomics is a culture-independent method that is a useful tool for determining the complexity of viral communities in any type of sample (Bibby, 2013; Handelsman, 2004; Kulski, 2016). Viral nucleic acid suitable for metagenomic analysis could be derived from environmental samples (aquatic or terrestrial environments, surface waters, sediments, agriculture products, human or animal habitations, human and animal vaccines, etc.) or biological specimens (blood, feces, serum, plasma, respiratory, secretion, etc.) (Delwart, 2007; Kulski, 2016; Li et al., 2015a,b). Over the past decade massively parallel sequencing technologies have become the focus of development. Its potential to gain insight into the structure, function, or regulation of genomes and genes has revolutionized many disciplines of life science. The utilization of these techniques in molecular diagnostics has been exploited in microbiology with the potential to detect novel microorganisms, including viruses in diverse specimens and ecosystems. It can also be used for inspection of viral diversity, evolution and spread, for investigation of pathogenesis of viral agents and for characterization of the viral community (Barzon et al., 2011, 2013; Capobianchi et al., 2013). A popular and widely used approach to describe viral communities without prior virus isolation step is shotgun metagenomics (Capobianchi et al., 2013). In this chapter we summarize commonly used laboratory methods that had helped to describe viral diversity and structure of viral communities and some Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00002-3 © 2020 Elsevier Inc. All rights reserved.

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achievements of this approach related to virus discovery with possible implications in veterinary medicine.

2.2 Technical aspects of viral metagenomics In the laboratory space viral metagenomics consists of four major steps: (1) viral enrichment to minimize background of prokaryotic and eukaryotic nucleic acids thus increasing the relative proportion of viral nucleic acids, (2) amplification of viral nucleic acids, (3) sequencing with or without cloning of amplified polymerase chain reaction (PCR) fragments, and (4) bioinformatic analysis of the resulting sequence output (Alavandi and Poornima, 2012; Delwart, 2007).

2.3 Virus enrichment and nucleic acid amplification If necessary, the first step of sample procession is homogenization (physical, e.g., homogenizer, mortar and pestle, freeze-thaw cycles; or enzymatic techniques, e.g., salt solution, detergents, alkaline lysis), centrifugation of the sample and filtration of the supernatant (through 0.22 and 0.45 μm pore membranes) to remove nonviral nucleic acids (i.e., host cellular debris and bacteria). The filtrates can be treated with a mixture of DNases and RNases to further reduce background nucleic acids originating from the host cells and bacteria. The method chosen in this step depends on the physical properties and other characteristics of the sample type (Datta et al., 2015; Mokili et al., 2012; Shah et al., 2014; Vo and Jedlicka, 2014). Following sample homogenization and reduction of the amount of debris and background nucleic acids, viral particles can be concentrated at various efficacies. Commonly used methods include tangential-flow filtration, polyethylene glycol precipitation, and ultracentrifugation. Density gradient ultracentrifugation using cesium chloride gradient provides highly purified virus particles. Concerning the step of viral nucleic acid extraction, the picture is more complex. Viral particles are disrupted by using “lysis buffer,” which may contain chaotropic acids (e.g., guanidine hydrochloride), detergents (sodium dodecyl sulfate, Triton X-100), and/or proteases (e.g., proteinase K). During the subsequent separation phase, the nucleic acids could be isolated from other components. This can be done by liquid
liquid extraction or liquid
solid extraction. During the liquid phase extraction different types of alcohol are used (e.g., phenolchloroform-isoamyl alcohol, isopropanol, etc.). The solid phase extraction may include one of the following procedures: gel filtration, where nucleic acid is separated through gel matrix (e.g., Sephadex), ion exchange chromatography (e.g., anion exchange resin, DEAE-C), and affinity chromatography (silica surface, paramagnetic beads). In general, liquid
solid extraction methods use less hazardous chemicals and provide increased throughput. Various formats have been

2.4 Sequencing technologies

marketed providing flexible, fast, and scalable viral nucleic acid extraction (Datta et al., 2015; Thatcher, 2015; Thurber et al., 2009). The amount of extracted nucleic acids is often too low for sequencing on available next generation sequencing (NGS) platforms; in such a case the amplification of viral nucleic acids is inevitable. Sequence-independent single primer amplification (SISPA) is among the most common amplification methods (Reyes and Kim, 1991). Initially this method was carried out by starting with restriction digestion and followed by ligation of an asymmetric primer or adaptors to both ends of the DNA molecules. By using a primer complementary to the ligated oligonucleotide, the nucleic acid fragments present in the sample could be amplified by PCR. Since then the original method has been modified to achieve more effective enrichment of nucleic acids or identification of viruses, such as DNaseSISPA and Virus discovery based on cDNA-AFLP. Another widely used method is random PCR. It uses a single primer with a known sequence at 50 end and with a random hexamer or heptamer sequence at 30 end in the first reaction. After that, the second PCR reaction is executable with a specific primer complementary to the defined 50 of the first random primer. To amplify circular DNA viral genomes, rolling circle amplification is an alternative option. This method requires short oligonucleotide primers composed of random sequences and polymerase enzyme with strand displacement activity (e.g., bacteriophage φ29 DNA polymerase). Another φ29 DNA polymerase-based method is the multiple displacement amplification. These techniques are often called whole genome amplification methods (Bexfield and Kellam, 2011; Blomstrom, 2011; Delwart, 2007).

2.4 Sequencing technologies Owing to efforts in research and innovation (Fig. 2.1), sequencing capacity and speed have dramatically increased over the past decade, while the cost is continuously decreasing. These achievements together led to the capacity to produce billions of nucleotide bases in a single sequencing run, which was unconceivable some time ago (Escobar-Zepeda et al., 2015; Kulski, 2016).

2.4.1 First-generation sequencing Since 1970s two methods have been developed to determine the precise order of nucleotides (DNA sequencing). In 1975, “plus and minus” method was published by Sanger and Coulson and historically it was the first DNA sequencing method. In 1977, Allan Maxam and Walter Gilbert invented a new protocol (MaxamGilbert sequencing or chemical sequencing), which is based on chemical cleavage of the radiolabelled bases and subsequent separation of the resulting DNA fragments by electrophoresis. This method, however, has not become widely adopted. Finally, in 1977, Sanger et al. accomplished the method of DNA sequencing

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FIGURE 2.1 Metagenomics timeline and milestones. Timeline showing advances in sequencing technologies from Sanger sequencing to Nanopore sequencing.

which ultimately led to the development of the “dideoxy method” or chaintermination sequencing technique. This process—which definitively amended the progress of DNA sequencing technology—depends on the application of dideoxynucleotides (ddNTPs) to cause base-specific termination of primed DNA synthesis. Initially, the radio-labeled fragments were separated by denaturating polyacrylamide-urea gel electrophoresis. These days the technique is coupled with fluorescence and capillary electrophoresis-based sequencers. Sequencing methods have benefited from the development of PCR, recombinant DNA technologies, discovery and molecular cloning of thermostable sequencing polymerases (e.g., Taq polymerase), and reverse transcriptase, as well as the development of fluorescent dye-labeled ddNTPs and process parallelization, which facilitated the birth of genome projects and the field of metagenomics (Heather and Chain, 2016; Hutchison, 2007; Kulski, 2016; Mardis, 2013; Schuster, 2008). Initially, laboratory methods of viral metagenomics relied on molecular cloning of amplified genomic fragments coupled with Sanger sequencing of hundreds to thousands of individual clones (Allander et al., 2005; Finkbeiner et al., 2008). Collectively, the major advantages of this technology are the long read lengths and the excellent resolution of sequence repeats and homopolymer regions. The drawbacks include the limited throughput and the relatively high per base sequencing cost (Hutchison, 2007; Schuster, 2008).

2.4 Sequencing technologies

2.4.2 Second-generation sequencing These methods can handle the parallel sequencing of several samples by molecular barcoding at very high-throughput and at reduced per nucleotide costs due to the miniaturization of the sequencing reactions. For metagenomic applications an added advantage was the lack of need for prior cloning of amplified target genomic DNA/RNA. With the initiation of NGS methods, the efficiency of DNA and RNA-base examinations explosively increased and the relative costs of sequencing have dramatically decreased (Escobar-Zepeda et al., 2015; Kulski, 2016; Metzker, 2010).

2.4.2.1 Pyrosequencing The first commercially available NGS technology was the “pyrosequencing technique” developed by 454 Life Sciences in 2005 and acquired by Roche in 2007, which is a sequencing-by-synthesis method (Barzon et al., 2011; Hutchison, 2007; Kulski, 2016; Schuster, 2008). During the process, pyrophosphate synthesis is measured with the utilization of chemiluminescent method. In brief, when a nucleotide is incorporated into the DNA chain, pyrophosphate is released (Fig. 2.2). The Adenosine triphosphate sulfurylase converts pyrophosphate into ATP, which is used as a substrate for luciferase, hence visible light is detected and measured (chemiluminescence) with charge-coupled device (CCD) camera. The process is reinitiated by adding the next complementary dNTP in the next dispensing cycle (Heather and Chain, 2016; Hutchison, 2007; Kulski, 2016). The method has disadvantages, including the high error rates in homopolymer repeats, the appearance of indels (insertions, deletions), as well as the time consuming sample preparation protocols. In contrast, the number of sequencing reactions and reads dramatically extended, the quality of data improved by reducing cross-talk between reaction wells, and the system was miniaturized (PicoTiterPlates) enabling the reduction of reaction volume. Furthermore, this platform provides long read lengths (400 bp to 1 kb) (Heather and Chain, 2016; Kulski, 2016; Metzker, 2010; Schuster, 2008).

2.4.2.2 Illumina/solexa sequencing Solexa developed the Genome Analyzer in 2006 that was acquired by Illumina in 2007. Currently, it is the most widespread sequencing system with the technology of sequencing-by-synthesis (Fig. 2.2). It differs from the Roche 454 sequencer in that it uses reversible chain-terminating nucleotides, which are fluorescently labeled. The fluorescent tags block the 30 -OH of the new nucleotide and so the next base can only be added when the tag is removed. When a nucleotide is incorporated into DNA template strands, the photon emission from each cluster is recorded by a CCD camera. Illumina provides currently benchtop sequencers (iSeq 100, MiniSeq, MiSeq/MiSeq Dx/MiSeq FGx, and NextSeq 550/NextSeq 550 Dx) and production-scale sequencers (HiSeq 2500/HiSeq 3000/HiSeq 4000, HiSeq X series Five or Ten, and NovaSeq 6000).

33

FIGURE 2.2 Principle of next generation sequencing platforms currently available. Roche 454, specific adaptor containing DNAs are denatured into single strands and captured by amplification beads followed by their emulsion PCR then pyrosequencing where oxyluciferin release is monitored. SOLiD, this is a ligation based method that uses DNA ligase enzyme to identify the presence of nucleotide at a given position in a DNA sequence. Illumina, selected DNA fragments are ligated with adaptors, and primers designed against them are used for the synthesis. Bridge amplification is carried by PCR and polony generations take place. Oxford Nanopore, This is actually based on the tunneling of polymer molecules passing through a protein nanopore, separating two compartments. Movement of DNA through this pore causes disruption in the current set against the voltage across the pore. Change in the current helps to identify the specific molecule and real time advances the quick data analysis.

2.4 Sequencing technologies

With the first sequencer (Genome Analyzer) it was possible to sequence 1 gigabase (Gb) of data in a single run. Nowadays, the output reaches 6000 Gb when using NovaSeq6000. The technology allows advantages and drawbacks too. It provides the lowest per base cost, huge amount of data, better performance along homopolymeric regions, and low error rate. The read length limitation (maximum 2 3 150 bp or 2 3 300 bp) remains an unsolved problem [Ari and Arikan, 2016; Heather and Chain, 2016; Kulski, 2016; Liu et al., 2012; Mardis, 2013; ,https:// www.illumina.com/systems/sequencing-platforms.html. (accessed: 2018.09.22)].

2.4.2.3 Sequencing by oligonucleotide ligation and detection Another commercially available NGS platform, the Sequencing by oligonucleotide ligation and detection (SOLiD) technology, also known as polony sequencing, was developed by George Church. The technology was purchased and improved by Applied Biosystems in 2007. The sequencer adopts the technology of two-base sequencing based on ligation sequencing. These probes are fluorescently labeled and compete for ligation to the sequencing primer by DNA ligase. The fluorescent signal is captured when the probe complement to the template is ligated. The cycle can be repeated by using cleavable probes to remove the fluorescent dye or by removing and hybridizing a new primer to the template. Due to the di-base encoding system and the exact call chemistry (ECC), the accuracy is greater than 99.94% (5500 Series SOLiD System’s accuracy is 99.99%). It provides inherent error correction, because the probes interrogate two base per reaction. In addition, sequencing of homopolymeric regions by the SOLiD technology is not a major issue. Besides more efficient sequencing, the throughput is increased on 5500 series (up to 10
15 Gb on 5500, up to 120 Gb per run on 5500xl W). Nonetheless, the major drawback of the method is the very short read lengths (75 bp (fragment), 75 bp 3 35 bp (paired-end), up to 60 bp 3 60 bp (mate-paired)) and the need for advanced computational infrastructure [Ari and Arikan, 2016; Heather and Chain, 2016; Hutchison, 2007; Kulski, 2016; Liu et al., 2012; Metzker, 2010; ,http://www.appliedbiosystems.com/ absite/us/en/home.html. (accessed: 2018.09.22)].

2.4.2.4 Semiconductor sequencing With Ion Torrent semiconductor sequencing a new paradigm in next generation sequencing was established in 2010. The sequencing technology exploits the fact that addition of a dNTP to a DNA polymer releases an H 1 ion and changes the pH of the solution that is proportional to the numbers of incorporated nucleotides. If there are two identical bases on the DNA strand, the sequencing chip will record two identical bases. If the next nucleotide is not a match, no base will be recorded. This process can be detected by a sequencing chip, which functions like a small solid-state pH meter. Overall, the method provides shorter workflow and simpler, faster, and more affordable sequencing to users. Life Technologies offers several next generation sequencing systems and sequencing chips: Ion Proton

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with Ion PI Chip, Ion S5 System with Ion 520, 530 540 Chip Kit, Ion Personal Genome Machine (PGM) with the Ion 314, 316, 318 Chip. The read lengths (200
400 bp), the accuracy (99%) and the run time (2
7 hours) are similar, but there are differences in the output (reads per run ranges from 0
5 to 60
80 million reads/run; with up to 2
10 Gb per run). The major disadvantages of this technology are biases in detection of insertions, deletions, and homopolymer sequences. However, there are numerous advantages like stable quality of sequencing, better GC depth distribution, higher map rate, relatively longer read lengths, and fastness [Kulski, 2016; Liu et al., 2012; Mardis, 2013; ,https:// www.thermofisher.com/hu/en/home/life-science/sequencing/next-generationsequencing.html. (accessed: 2018.09.22)]. In conclusion, of the four NGS systems described above, the Illumina HiSeq2500
4000 and NovaSeq6000 feature the biggest output and lowest per base reagent cost, the Ion Torrent has the fastest run time, the SOLiD has the highest accuracy, while the Roche 454 system has the longest read length.

2.4.3 Third-generation sequencing To overcome some inconveniences related to second-generation sequencing technologies (e.g., the lengthy procedure of library preparation step, difficult genome assembly due to the relatively short read lengths, biases in identification of indels and homopolymer regions), the third-generation sequencing methods has been developed. At present two approaches are available in the market.

2.4.3.1 Single molecule real-time sequencing Pacific Biosciences developed the single molecule real-time (SMRT) sequencing. SMRT is based on DNA replication via DNA polymerase. When a fluorescent nucleotide is incorporated into the growing DNA chain, the enzyme cleaves off the fluorescent dye. The emitted signals are detectable in real time by a CCD camera placed inside the machine. The other key point of the method is the usage of zero-mode waveguides (ZMW), which are fine wells in a metallic film covering a chip. Each ZMW contains single DNA polymerase attached to their bottom and single DNA target. This allows the observation of a fluorescent labeled single molecule in real time, kept apart from the signal noise background (other fluorescent labeled molecules). With this new approach the read length has dramatically increased with maximum read lengths up to .100 kbp. A major drawback is the relatively high error rate (single pass accuracy B86%, but the consensus accuracy .99.99%), which stems mostly from indel errors. Currently Pacific Biosciences offers two devices, the Sequel System and the PacBio RS II [Ari and Arikan, 2016; Heather and Chain, 2016; Kulski, 2016; Liu et al., 2012; Metzker, 2010; ,http://allseq.com/knowledge-bank/sequencing-platforms/pacific-biosciences/. (accessed: 2018.09.22)].

2.5 Bioinformatics

2.4.3.2 Nanopore sequencing Oxford Nanopore Technologies developed a new, incomparable system. The first nanopore based sequencer was the MinION, in 2014. Recently, the company offers miniature handheld devices (MinION, SmidgION) and high-throughput installations (PromethION) as well. Nanopore devices perform DNA and RNA sequencing directly and in real time. A nanopore is a nano-scale hole, which is created by pore-forming proteins and they act as channels embedded on lipid bilayer or artificial membrane (Fig. 2.2). They are suitable for the detection and quantification of biological and chemical molecules. During the sequencing, a voltage is applied across the membrane continuously, causing ionic current. When the DNA fragment is passed through the nanopore, the conductivity of ion currents in the pore changes, since the nucleotides have different shapes and they have a different effect on the change of the ionic flow. At this time, the smallest instrument commercially available is the MinION. It is a portable, pocket-sized device; when it is attach to a laptop or computer freshly generated data are displayed on the screen in real time. The technology provides long read length (up to 1 Mbp). The method does not require an amplification step and the detection of bases is fluorescent-tag free. However, the technology provides relatively high error rate which can be counterbalanced by increasing the sequencing depth [Ari and Arikan, 2016; Escobar-Zepeda et al., 2015; Heather and Chain, 2016; Kulski, 2016; Liu et al., 2012; ,https://nanoporetech.com. (accessed:2018.09.22)].

2.5 Bioinformatics NGS has dramatically increased the amount of sequenced data. Storage and handling of such data is challenging. Bioinformatics provide software and several workflows (bioinformatics pipelines) to analyze biological data. The main purposes include the conversion of NGS-based signals to sequence data that can be readily processed and the adequate interpretation of the processed sequence data to obtain the desired biological information (Fig. 2.3). Nucleotide sequence analysis has a couple of steps, including base calling, the quality check of the sequencing runs. The read sequences are commonly stored in FASTQ file format or in the native raw data file formats depending on the sequencing instrument and the associated onboard software. This step is followed by quality control of the reads (searching for sequence errors and artifacts, platform-specific error profiles, etc.), then trimming (cleavage of adapters, tags, primers), and the alignment and assembly of contigs (mapping to reference sequences or de novo assembly). In general, there are several assembly tools and software (e.g., Geneious, Velvet, and ABySS); but nowadays viral genome specific genome assemblers, such as VICUNA, Viral Assembly Pipeline (VrAP), VFAT, AV454, RIEMS, and metagenome assemblers like Omega, Genovo, MEGAHIT, MetaSpades, MetaVelvet are also available (Holzer and Marz, 2017;

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CHAPTER 2 Metagenomics revealing new virus species

FIGURE 2.3 Schematic workflow for viral metagenomics. Flowchart represent major steps of metagenomics analysis. For detailed protocol please see the manuscript, Conceic¸a˜o-Neto et al. (2015).

2.6 Practical aspects of viral metagenomics

Rose et al., 2016; Roy et al., 2018). During de novo assembly, there is no available reference sequence for alignment; this technique is key to identify novel virus genome sequences. The next step is genome identification and annotation [open reading frame (ORF), the coding sequence, repeated elements, untranslated regions, etc.], search for single nucleotide polymorphism and insertion/deletion, and comparative sequence analysis. Commercially available analysis tools (e.g., CLCBIO Genomic Workbench, Avadis NGS, and Softgenetics Nextgene) offer user-friendly solutions for this purpose. Virus-specific data bases are useful to collect relevant information on virus families and also sequence data for subsequent taxonomic and phylogenetic classification (Holzer and Marz, 2017; Kulski, 2016; Rose et al., 2016; Roy et al., 2018). Online platforms are also available to make rapid viral taxonomy in a metagenomic data set [,http://kaiju.binf.ku.dk/. (accessed: 2018.09.22), ,https://www.taxonomer.com/. (accessed: 2018.09.22)]. In response to the need for effective detection, assembly, and classification of pathogens, several tools were put on the market in recent times for ease of bioinformatics analysis. The analysis of extremely high-throughput data is very time consuming, therefore development of faster and more user-friendly software will be essential in the near future.

2.6 Practical aspects of viral metagenomics The application of viral metagenomics is being continuously adopted and used in veterinary medicine. In clinical diagnostics, the recognition and the treatment of novel and rare pathogens is a real challenge. With the new technical inventions, this problem could be solved. Another application is the real-time investigation of outbreaks caused by viral pathogens and the prevention of potential epidemics. The environmental monitoring of pathogens by viral metagenomics may also have an important role in efforts of infectious disease control. In this case, the quick reaction, the regulation, and risk assessment based on laboratory confirmed evidence is very important. Moreover, broad-range detection and identification of novel viruses or novel types of known viruses will be able to help us to broaden our range of vision, facilitate protection against viral agents, and understand viral diversity (Table 2.1). The origin of novel viruses or novel variants is diverse, one of the potential sources is the cross-species transfer, hence surveying of potential reservoirs and characterizing the intra-host variability is necessary (Barzon et al., 2013; Bibby, 2013; Capobianchi et al., 2013; Mokili et al., 2012). In several cases, the co-occurrence of the newly identified virus with a disease symptom does not proof the causation, although experimental and epidemiological verification is required. To provide disease causation, Mokili et al. proposed the metagenomics Koch’s postulates. Viral metagenomics has the opportunity to identify the components of multifactorial infectious diseases (Belak et al., 2013; Kapgate et al., 2015).

39

Table 2.1 Selection of novel animal viruses identified with NGS technologies. Host Pig

RNA virus

DNA virus

Cattle

RNA virus

Virus

Taxonomy

Disease

References

Sequencing

Porcine stool-associated RNA virus 1 Porcine stool-associated RNA virus 2 Swine pasivirus 1 (SPaV1) Atypical porcine pestivirus Enterovirus species G (EVG) (EVG 08/NC_USA/ 2015) Ndumu virus

Picornavirales

E

Shan et al. (2011)

Picornavirales

E

Shan et al. (2011)

Picornaviridae

?

Sauvage et al. (2012)

Flaviviridae

O

Hause et al. (2015)

454 pyrosequencing 454 pyrosequencing Illumina Hiseq 2000 Illumina MiSeq

Picornaviridae

E

Shang et al. (2017)

Illumina MiSeq

Togaviridae

O

Masembe et al. (2012)

Porcine astrovirus 4

Astroviridae

E

Pig stool-associated circular ssDNA virus Porcine parvovirus 7 Porcine circovirus 3 Porcine parvovirus 2,4,5,6 Porcine bocavirus 1-H18 Bovine astrovirusNeuroS1 Bovine astrovirus-CH13 Bovine astrovirus-BH89/ 14

Unclassified CRESS

E

Parvoviridae Circoviridae Parvoviridae

E O E

Padmanabhan and Hause (2016) Sachsenröder et al. (2012) Palinski et al. (2016) Phan et al. (2016) Bovo et al. (2017)

454 GS-FLX platform Illumina MiSeq

Parvoviridae Astroviridae

E E&O

Bovo et al. (2017) Li et al. (2013c)

IonTorrent PGM Illumina MiSeq

Astroviridae Astroviridae

O O

Bouzalas et al. (2014) Schlottau et al. (2016)

Illumina HiSeq2500 Illumina MiSeq

454 pyrosequencing Illumina MiSeq Illumina MiSeq IonTorrent PGM

DNA virus

Small ruminants

Chicken

RNA virus

RNA virus

DNA virus

Yak astrovirus S8

Astroviridae

E

Chen et al. (2015)

Bovine enterovirus AN12/Bostaurus/JPN/ 2014 Rotavirus B

Picornaviridae

E

Mitra et al. (2016)

Reoviridae

E

Illumina MiSeq

Retroviridae

O

Hayashi-Miyamoto et al. (2017) Wüthrich et al. (2016)

Picornaviridae Papillomaviridae

O O

Moreira et al. (2017) Bauermann et al. (2017)

Illumina MiSeq Illumina MiSeq

Polyomaviridae

O

Zhang et al. (2014a,b)

Illumina MiSeq

Astroviridae

O

Pfaff et al. (2017)

Ovine astrovirus type 2

Astroviridae

O

Reuter et al. (2012)

Statovirus D1

Unclassified RNA virus (phylogenetically related to Tombusviridae and Flaviviridae) Picornaviridae

O

Janowski et al. (2017)

454 pyrosequencing 454 GS-FLX platform 455 GS-FLX platform

O

Bullman et al. (2014)

Illumina Miseq

Picornavirus QIA01 Chicken phacovirus 1 Sunguru virus Chicken astrovirus PL/ G059/2014

Picornaviridae Picornaviridae Rhabdoviridae Astroviridae

O E O O

Illumina Illumina MiSeq IonTorrent PGM Illumina MiSeq

Gyrovirus GyV7-SF

Anelloviridae

O

Kim et al. (2015a,b) Boros et al. (2016) Ledermann et al. (2014) Sajewicz-Krukowska and Domanska-Blicharz (2016) Zhang et al. (2014a,b)

Bovine beta-retrovirus (BoRV-CH15) Aichivirus B Bovine papillomaviruses BPV22 Bovine polyomavirus BPyV2-SF Ovine astrovirus

Sicinivirus 1

Illumina HiSeq 2000 Illumina MiSeq

Illumina HiSeq2500

Illumina Miseq (Continued)

Table 2.1 Selection of novel animal viruses identified with NGS technologies. Continued Host Turkey

RNA virus

DNA virus

Other birds

RNA virus

DNA virus

Virus

Taxonomy

Disease

References

Sequencing

Turkey heptatitis virus

Picornaviridae

E&O

Honkavuori et al. (2011)

Turkey gallivirus strain turkey/M176/2011/HUN Turkey avisivirus strain turkey/M176-TuASV/ 2011/HUN TuASV-USA-IN1 Picobirnaviruses Turkey stool-associated circular virus Turkey poxvirusHU1124/2011 Mesivirus-1 and 22

Picornaviridae

E

Boros et al. (2012)

Picornaviridae

E

Boros et al. (2013)

454 GS-FLX platform 454 GS-FLX platform 454 GS-FLX platform

Picornaviridae Picobirnaviridae Unclassified single-stranded (ss) circular small DNA viruses Poxviridae

O E E

Ng et al. (2013a,b) Verma et al. (2015) Reuter et al. (2014)

O

Banyai et al. (2015)

Illumina Illumina HiSeq 454 GS-FLX platform IonTorrent PGM

Picornaviridae

O

Phan et al. (2013)

Illumina MiSeq

Bornavirus

Bornaviridae

O

Honkavuori et al. (2008)

Avian coronaviruses Avian gammacoronavirues Avian orthoreoviruses Bunyavirus

Coronaviridae Coronaviridae

R O

Chen et al. (2013) Liais et al. (2014)

454 GS-FLX platform IonTorrent PGM Illumina MiSeq

Reoviridae Bunyavirales

O O

IonTorrent PGM Illumina

Pigeon adenovirus 2

Adenoviridae

O

Farkas et al. (2018) Alkovskhovskii et al. (2013) Teske et al. (2017)

Aviparvovirus Gyrovirus V8

Parvoviridae Anelloviridae

O O

Phan et al. (2013) Li et al. (2015a,b)

Illumina MiSeq Illumina MiSeq

Illumina MiSeq

Dog

RNA virus

DNA virus

Cat

RNA virus

Kobuvirus

Picornaviridae

E

Li et al. (2011)

Norovirus Sapovirus

Caliciviridae Caliciviridae

E E

Martella et al. (2008) Li et al. (2011)

Vesivirus Astrovirus Hepacivirus Rotavirus I

Caliciviridae Astroviridae Flaviviridae Reoviridae

E/R E O E

Rotavirus C Bocaparvovirus 2

Reoviridae Parvoviridae

E R

Martella et al. (2015) Toffan et al. (2009) Kapoor et al. (2011) Mihalov-Kovács et al. (2015) Marton et al. (2015) Kapoor et al. (2012b)

Bocaparvovirus 4

Parvoviridae

R

Li et al. (2013b)

Protoparvovirus 2 Bocaparvovirus, NC

Parvoviridae Parvoviridae

R/E E

Polyomavirus Papillomavirus 1
18 Sakobuvirus A

Polyomaviridae Papillomaviridae Picornaviridae

R O E

Martella et al. (2018) Conceição-Neto et al. (2017) Delwart et al. (2017) Lange et al. (2016) Ng et al. (2014)

Astrovirus Astrovirus Rotavirus I

Astroviridae Astroviridae Reoviridae

E E E

Picobirnavirus Morbillivirus

Picobirnaviridae Paramyxoviridae

E O

Ng et al. (2014) Zhang et al. (2014a,b) Ng et al. (2014), Phan et al. (2017) Ng et al. (2014) Marcacci et al. (2016)

Hepadnavirus

Hepadnaviridae

O

Aghazadeh et al. (2018)

454 GS-FLX platform ? 454 GS-FLX platform ? Primer walking!! Primer walking!! IonTorrent PGM IonTorrent PGM ? 454 GS-FLX platform IonTorrent PGM Illumina Hiseq 2500 Illumina MiSeq ? Illumina MiSeq Illumina MiSeq Illumina MiSeq Illumina MiSeq, Illumina HiSeq Illumina Miseq Illumina NextSeq 500 Illumina Hiseq 2500 (Continued)

Table 2.1 Selection of novel animal viruses identified with NGS technologies. Continued Host DNA virus

Fish

RNA virus

DNA virus

Virus

Taxonomy

Disease

References

Sequencing

Cyclovirus

Circoviridae

E

Zhang et al. (2014a,b)

Illumina MiSeq

Bocaparvovirus 2 Bocaparvovirus 3 Bocaparvovirus Cowpoxvirus

Parvoviridae Parvoviridae Parvoviridae Poxviridae

E E O O

Ng et al. (2014) Zhang et al. (2014a,b) Garigliany et al. (2016) Dabrowski et al. (2013). Mauldin et al. (2017)

Ectromelia-like virus Cyprinid herpesviruses, CyHV-1, -2, -3 Anguillid herpesvirus, AngHV-1 Ictalurid herpesvirus 2 Fisavirus 1

Poxviridae Herpesviridae

O O

Lanave et al. (2018) Davison et al. (2013)

Illumina MiSeq Illumina MiSeq IonTorrent PGM 454 GS-FLX platform, Illumina Hiseq 2000 Illumina MiSeq Illumina

Herpesviridae

O

Alloherpesviridae Picornavirales

O O

van Beurden et al. (2010) Borzak et al. (2018) Reuter et al. (2015)

Piscine reovirus

Reoviridae

O

Palacios et al. (2010)

Tilapia lake virus

Orthomyxoviridae

O

Bacharach et al. (2016)

European sheatfish virus

Iridoviridae

O

Feher et al. (2016)

Totivirus

Totiviridae

O

Lovoll et al. (2010)

Sparus aurata papillomavirus 1 Sparus aurata polyomavirus 1

Papillomaviridae

O

Polyomaviridae

O

Lopez-Bueno et al. (2016) Lopez-Bueno et al. (2016)

Disease: E, enteric; R, respiratory; O, other.

Illumina Illumina HiSeq2000 454 GS-FLX platform 454 GS-FLX platform Illumina HiSeq 2500, Ion Torrent IonTorrent PGM 454 GS-FLX platform 454 GS-FLX platform, Illumina 454 GS-FLX platform, Illumina

2.7 Viral metagenomics and discovery of new viruses in livestock

2.7 Viral metagenomics and discovery of new viruses in livestock 2.7.1 New viruses in pigs Several new astro- and boca-viruses were detected as well as RNA virus’s posavirus 1 and 2 in porcine fecal samples from North Carolina using 454 pyrosequencing (Shan et al., 2011). Similarly, a novel virus alike Chimpanzee stool-associated circular ssDNA virus (ChiSCV) was discovered on 454 pyrosequencing platform and presumptively named pig stool-associated circular ssDNA virus (PigSCV) (Sachsenro¨der et al., 2012). In yet another study, a novel virus was discovered in the fecal samples of healthy piglets, on sequencer Illumina Hiseq 2000, and was named swine pasivirus 1 (SPaV1) (Sauvage et al., 2012). Pestiviruses are prevalent among varied animal species ranging from all farm animals to wild animals, like wild boar and deer species. They were initially discovered through the classical and conventional methods. Now their detection rate has increased with the aid of metagenomics. In the United States, recently discovered pestiviruses have been through the metagenomic studies, under the porcine reproductive and respiratory syndrome virus metagenomic sequencing project (Hause et al., 2015). One of the novel viruses discovered was named atypical porcine pestivirus. Through critical and various cross-validating analysis, identity of a novel virus is set. Later, this virus was found to be widespread in European countries as well (Beer et al., 2017; de Groof et al., 2016; Postel et al., 2016; Schwarz et al., 2017). Metagenomic analysis deciphers for a varied and extended viral flora from single-stranded RNA to circular DNA viruses. A new parvovirus (PPV7) in rectal swabs of porcine was discovered having a significant identity 42.4% and 37.9%, with fruit bat (EhPV2) and turkey parvovirus, respectively, with NS1 gene (Palinski et al., 2016) and deciphered prevalence in US swine. A new enterovirus G (EVG) (EVG 08/NC_USA/2015) in diarrheic pigs has surfaced with the expressive feature of Torovirus Deubiquitinase (Shang et al., 2017). A putative novel rotavirus C VP6 genotype has been reported in Belgian piglets (Theuns et al., 2016). Novel picornavirus has also been reported in swine feces in Japan (Naoi et al., 2016) and another one having similarity with Posavirus (Hause et al., 2016). In pigs, several novel viruses are being constantly detected through metagenomic analysis like circovirus PCV3 (Phan et al., 2016), parvovirus (strain swine/ Zsana3/2013/HUN) (Hargitai et al., 2016), Parvovirus 7 (Palinski et al., 2016), etc. Porcine parvovirus 2 (PPV2), PPV4, PPV5, PPV6, and porcine bocavirus 1H18 isolate (PBoV1-H18) (Bovo et al., 2017), first report for Ndumu virus in Uganda (Masembe et al., 2012), 2009 H1N1, pandemic influenza A virus (Greninger et al., 2010), Torque Teno virus, Porcine boca-like virus (Blomstrom et al., 2010), a new variant of Porcine astrovirus (Karlsson et al., 2015), all have been reported by metagenomics approach. Novel genotype of astrovirus 4 in nasal swab of pigs (Padmanabhan and Hause, 2016) has also been reported.

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2.7.2 New viruses in cattle Novel bovine astroviruses (BoAstV-NeuroS1) in California (Li et al., 2013c), BoAstV-CH13 in Switzerland (Bouzalas et al., 2014), and BoAstV-BH89/14 (Schlottau et al., 2016) have been identified in the brain tissues of bovine with encephalitis, having phylogenetic similarity with that of ovine astrovirus, were sequenced through metagenomics. Metagenomics helped in establishing the relationships between various neurologic diseases among various farm animals like bovine, mink, sheep, etc., and comparison was drawn with the outbreak in Denmark, Finland, and Sweden (Quan et al., 2010; Blomstrom et al., 2010). Initially, BoAstV-NeuroS1 etiology was misdiagnosed. But metagenomics led to the proper diagnosis of an astrovirus-related neurologic disorder. A novel astrovirus has also been reported in diarrheic Yak having genetic closeness with deer in Qinghai-Tibetan plateau (Chen et al., 2015). Schmallenberg virus is among the first novel viruses discovered through metagenomic analysis. It was identified from Germany in cattle suffering from pyrexia, milk drop syndrome, and intermittent diarrhea. The virus belonged to the Simbu serogroup Orthobunyavirus (Hoffmann et al., 2012). Complete genome of bovine enterovirus (BEV) (BEV AN12/Bostaurus/ JPN/2014), an enterovirus (EV) has been sequenced in bovines and the distant relation with EVs has been established on the basis of capsid VP1 proteincoding region (Mitra et al., 2016). A new strain of rotavirus A (RVA) G15P [14] in adult cows has been detected through next generation sequencing in Japan during epizootic diarrhea. This strain was associated with decreased milk production and a conclusion was drawn for its derivation from multiple reassortments among Japanese cattle. Various diverse genotypes have been discovered in Japanese cattle in VP3, NSP3, and NSP4 genes of rotavirus B (RVB). Their identity was found to be between around 52%
68% between human, murine, and porcine RVBs. Analysis and comparative studies were revealed for the independent evolutions of bovine RVBs (Hayashi-Miyamoto et al., 2017). In the Asia-Pacific region, a number of picornaviruses have been discovered in various hosts like bats, canines, felines, etc., in quick succession. Similarly, a number of picornaviruses have been found in bovines in Japan and named bovine Japanese Picornaviruses, due to having close relations with Chinese picornaviruses (Nagai et al., 2015). Bovine papillomavirus type in the genus Dyokappa papillomavirus (Bauermann et al., 2017), bovine beta-retrovirus termed BoRV-CH15 (Wu¨thrich et al., 2016), have been identified in bovines. On Illumina MiSeq, a novel bovine polyomavirus species (BPyV2-SF) was detected along with other known viruses, in store purchased beef from stores in San Francisco (Zhang et al., 2014a,b). In Holstein bull calf, Aichivirus B, was tentatively identified in cerebrospinal fluid by next generation DNA sequencing (Moreira et al., 2017), only after its initial misdiagnosis for infection with RV and Cryptosporidium.

2.7 Viral metagenomics and discovery of new viruses in livestock

2.7.3 New viruses in small ruminants Tunesian sheep pestiviruses strains Aydin/04 and Burdur/05, among ruminants discovered through next generation sequencing in Turkey, have been linked to have a close relation with classical swine fever virus (Becher et al., 2012). New variants of bluetongue virus have also been reported through metagenomics. A novel boca parvovirus 6 has been documented. Similarly, novel astroviruses have been detected in domestic sheep with nonsuppurative encephalitis and ganglionitis (Pfaff et al., 2017), and one was discovered serendipitously in feces of sheep in Hungary (Reuter et al., 2012). Various other viruses have also been identified in farm animals and a new taxa of RNA viruses harbored in the gastrointestinal tracts were found to be related to Tombusviridae and Flaviviridae have been classified. Statoviruses (stool-associated Tombus-like viruses) is one such virus and have diverse range of mammal hosts from humans to bovine (Janowski et al., 2017).

2.7.4 Novel viruses in chickens In 2014, Zhang et al. detected viral sequences in chicken meat purchased from stores in San Francisco by using a combination of MiSeq Illumina platform and Sanger method. In this study chicken meat contained a novel divergent gyrovirus species (GyV7-SF) besides other known gyrovirus sequences (chicken anemia virus; chicken gyrovirus 3,2,4; humangyrovirus 1). Bullman et al. (2014) characterized a novel picornavirus (sicinivirus) from fecal samples of broilers which was proposed to represent a novel genus within Picornaviridae family. It was considered as a putative avian pathogen, because only a few picornavirus have been detected in birds. Zhou et al. (2015) identified the first sicinivirus isolate (strain JSY) from layer chickens by using viral metagenomics technique. The chickens showed serious symptoms associated with high mortality. Later, Kim et al. (2015a,b) identified the QIA01 strain of the novel picornavirus from chicken. It is similar to two chickens’ megriviruses from Hungary. It was suggested that both viruses belong to the same species, Melegrivirus A, but based on the nucleotide identity analysis they may represent different serotypes. The young commercial chickens often suffer from transmissible viral proventriculitis and the novel picornavirus is thought to be responsible for this disease. Boros et al. (2016) reported the complete genome of six known picornaviruses and a novel virus, called chicken phacovirus 1, from a diarrheic chicken. Ledermann et al. (2014) characterized the complete nucleotide sequence a novel Sunguru virus (family Rhabdoviridae), which was first isolated from the blood of domestic chickens from a market in Uganda in 2011. The semiconductor sequencer, Ion Torrent PGM, was used in this study. There is no evidence for this agent to be able to cause disease, moreover the mode of transmission is unknown; thus, further investigations will be needed to determine these missing but important details.

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In 2016, a novel chicken astrovirus has been discovered within the genus Avastrovirus in Poland, which caused “white chicks” condition. This disease may be associated with economic losses, because the infected embryos and chicks have increased mortality (Sajewicz-Krukowska and Domanska-Blicharz, 2016).

2.7.5 Novel viruses in turkeys Day et al. (2010) performed metagenomics analysis on RNA virus community in the turkey gut by using Genome Sequencer. Their study targeted the presence of turkeyorigin picornaviruses, picobirnaviruses, and caliciviruses. The detection of putative novel enteric viruses in turkey is very important for the poultry industry, as these discoveries may allow new insight into the etiology of multifactorial diseases, such as severe enteric disease which is associated with high mortality in turkey flocks. Liver samples were analyzed from eight flocks in California to identify an etiologic agent for turkey viral hepatitis, which is a highly infectious disease among young turkeys. Picornavirus sequences, which are probably responsible for the disease, were revealed by Honkavuori et al. (2011). Boros et al. (2012) identified and characterized a complete genome of novel picornavirus in a fecal sample from commercial turkey meat by using the 454 GS-FLX technology. Also Boros et al. (2013) characterized another novel picornavirus, detected in Hungarian flocks of turkeys, which is distantly related to the members of the genus Avihepatovirus. Newly established genera of picornaviruses, avisivirus, megriviruses, and mesiviruses, were also described from turkey (Boros et al., 2014; Ng et al., 2013a,b). Day and Zsak (2014) analyzed a novel turkey-origin picobirnavirus by pyrosequencing. In that study turkey intestinal tracts served as samples. The picobirnaviruses were identified in Minnesota turkeys (Verma et al., 2015) with “light turkey syndrome” (LTS). They examined fecal samples by metagenomics analysis (Illumina HiSeq) and characterized picobirnavirus strains associated with LTS and non-LTS turkeys. In 2016, Day et al. characterized enteric picornaviruses in turkey and chicken samples also, which may participate in the development of the enteric disease syndromes. Based on metagenomics data, Reuter et al. (2014) characterized a novel single-stranded DNA virus, called turkey stool-associated circular virus in a fecal sample from a 1-year-old domestic turkey with diarrhea in Hungary. In 2015, Ba´nyai et al. described a novel clade of avipoxviruses (turkey poxvirus-HU1124/2011) in turkeys associated with cutaneous and oral cavity lesions. Interestingly, the flock was vaccinated by commercial fowlpox virus vaccines. This study reaffirms that for better prevention, it is important to characterize genetic differences between strains and monitor avipoxviruses in birds.

2.7.6 Novel viruses in other birds Pigeons are reared as pet birds, and pigeon racing is a popular sport. These birds also have close relationship with humans. Phan et al. (2013) collected fecal

2.8 Viral metagenomics and discovery of new viruses in pets

specimens from feral pigeons. They identified and proposed a new genus, namely aviparvovirus. Two related picornaviruses, Mesivirus-1 and -2, were also characterized. Moreover, they described group G RV from pigeons. Teske et al. (2017) identified novel aviadenovirus (pigeon adenovirus 2) in the fecal virome of domestic pigeons, sequenced on Illumina MiSeq platform. This novel virus may have immunosuppressive potential, which has a role in the evolution of a multifactorial disease, the young pigeon disease syndrome. Honkavuori et al. (2008), after pyrosequencing-based analysis, reported two strains of novel bornaviruses from brain samples of parrots with proventriculus dilatation syndrome. Chen et al. (2013) identified a potential novel species of avian coronaviruses in the genus of Gammacoronavirus specific to ducks or some water fowls. The samples were originated from feces from ducks and swab samples from domestic fowls in poultry farms, backyard flocks, slaughtering houses, and live bird markets in 17 provinces in China. More recently, putative new orthoreovirus species were detected in Pekin ducks in Germany (Farkas et al., 2018). In France, novel avian gammacoronavirus has been identified, which was distantly related to turkey coronavirus. Sequencing of the intestinal contents from guinea fowls was performed by using MiSeq platform. The guinea fowls showed the symptoms of fulminating disease (Liais et al., 2014). Alkovskhovskii et al. (2013) reported the Khurdun virus genome (on Illumina platform) and classified it as a novel prototypic bunyavirus. The virus was isolated from coot in 2001, in the Volga River delta. Li et al. (2015a,b) characterized the genome of highly divergent gyrovirus (GyV8) from fulmar (Fulmarus glacialis). For analysis, spleen and uropygial gland tissues were used. It is the first gyrovirus detected in other avian than chicken.

2.8 Viral metagenomics and discovery of new viruses in pets 2.8.1 Novel viruses in dogs The first study describing the use of high-throughput sequencing for exploring the virome of dogs dates back to 2011 (Li et al., 2011). Deep sequencing of nucleic acids from the feces of 18 diarrheic dogs were identified as known canine parvoviruses, coronaviruses, and RVs. Also, the study discovered the genomes of canine kobuvirus (Picornaviridae) and sapovirus (Caliciviridae). Canine sapovirus constituted a novel genogroup within the genus Sapovirus, a group of viruses also associated with human and animal diarrhea. Deep sequencing of pooled stools of wild and domestic canids (wolfs and dogs) in Portugal (Conceic¸a˜o-Neto et al., 2017) has revealed at least 15 different viruses of 12 virus families, including Parvoviridae (densovirus, protoparvovirus, dependoparvovirus, and bocaparvovirus), Picornaviridae (kobuvirus),

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CHAPTER 2 Metagenomics revealing new virus species

Nodaviridae, Mimiviridae, Totiviridae, Paramixoviridae (canine distemper virus), Mycodnaviridae, Picornaviridae, Vigaviridae, and a number of unassigned viruses. Some of the identified viruses (caninovirus, gemycircularvirus, and densovirus), which are commonly found in arthropods, were likely associated with diet, as wild canids may feed on carcasses infested with a wide range of insects. A novel wolf bocavirus was identified, distantly related to other canine and feline bocaparvoviruses. A shotgun metagenomics approach was also used to explore the enteric virome diversity of dogs in a case
control study in Australia (Moreno et al., 2017). Eight eukaryotic viral families were detected, including Astroviridae, Coronaviridae, Reoviridae, Picornaviridae, Caliciviridae, Parvoviridae, Adenoviridae, and Papillomaviridae. Families Astroviridae, Picornaviridae, and Caliciviridae were found only in dogs with acute diarrhea, with Astroviridae being the most common family identified in this group. A beta polyomavirus (dsDNA) was identified in 2017 by metagenomics in respiratory secretions of two dogs with severe pneumonia, which tested negative for all canine respiratory pathogens except Mycoplasma cynos (Delwart et al., 2017). Canine circovirus (ssDNA) was first identified in serum samples from six out of 205 dogs in 2012 (Kapoor et al., 2012a). Subsequently, a similar virus was found in the liver of a dog with severe hemorrhagic gastroenteritis, vasculitis, and granulomatous lymphadenitis (Li et al., 2013a). Screening with specific molecular assays failed to find a firm association with any disease in dogs, although the virus appeared as a common component of canine virome (Anderson et al., 2017; Dowgier et al., 2017; Hsu et al., 2016; Gentil et al., 2017). Porcine-like circoviruses have also been identified in dogs in China (Zhang et al., 2018). At least four different novel canine parvoviruses (ssDNA 1 ) have been identified starting from 2011 in canids. Bocaparvoviruses distantly related to minute virus of canine (canine parvovirus 1) have been identified in dog respiratory samples (Kapoor et al., 2012b) and in the liver (Li et al., 2013b). Another unclassified bocaparvovirus was identified in pooled stools of wolves from Portugal (Conceic¸a˜o-Neto et al., 2017). A novel protoparvovirus, distantly related to canine parvovirus 2, was identified in young dogs with respiratory disease that tested negative to a panel of respiratory pathogens. The virus was also detected in collections of respiratory and enteric swabs, and its presence was significantly associated only in young animals (,6 months) with respiratory disease (Martella et al., 2018). Papillomaviruses (PVs) are nonenveloped, dsDNA viruses with a circular genome of about 8000 base pairs. They are generally host species-specific with some exceptions. Numerous of known PVs are associated with benign and malignant neoplasias of the skin and mucous membranes in humans and animals, but there is evidence that asymptomatic infections are more common. More than 200 human and 140 animal PVs have been characterized, illustrating broad genetic diversity (http://pave.niaid.nih.gov/) (Bernard et al., 2010; de Villiers, 2013;

2.8 Viral metagenomics and discovery of new viruses in pets

Howley et al., 2013). At least 18 PV types have been identified in dogs (Lange et al., 2016). Canine PVs have been found associated with classical exophytic papillomas such as the common canine oral papillomatosis, with endophytic papillomas, with pigmented plaques and in rare cases with squamous cell carcinomas (Lange and Favrot, 2011). RVs, family Reoviridae, are important enteric pathogens causing gastroenteritis in many mammals and birds (Martella et al., 2010; Bernstein, 2009; Dhama et al., 2015). Non-group A rotaviruses have been identified in young dogs with enteritis in Hungary in 2015. The genome of a RV strain was genetically related to bovine and porcine rotavirus C strains (Marton et al., 2015) whilst two pups were infected by a novel rotavirus species (defined as rotavirus I) (MihalovKova´cs et al., 2015). Picornaviruses (ssRNA 1 ) constitute one of the largest virus families and include several human and animal pathogens. However, picornaviruses were not known in the canine host until 2011, when a canine kobuvirus, genetically related to human Aichi virus, was discovered in fecal samples of dogs (Li et al., 2011). Later, the virus has been subsequently discovered in studies in Europe (CarmonaVicente et al., 2013; Di Martino et al., 2013). In 2012, a novel canine picornavirus, strain 209, was identified in Asia; the picodicistrovirus was classified as a novel species, Cadicivirus A, in the new genus Dicipivirus (Woo et al., 2012a). In the same year, another canine picornavirus, strain 325F, still unclassified, was identified in Asia (Woo et al., 2012b). The virus is related to members of the Sapelovirus and Enterovirus genera. Several advances have been gathered using sequence-independent assays and deep sequencing for caliciviruses (ssRNA 1 ) of pets. Caliciviruses include important enteric pathogens of humans (noroviruses and sapoviruses) (Lindsay et al., 2015; Oka et al., 2015) and animal viruses (Harrison et al., 2007; Radford et al., 2009; Ohlinger et al., 1990). In 2008 noroviruses were first discovered in dogs (Martella et al., 2008). The virus was genetically related to Alphatron-like (GIV) human noroviruses. In the subsequent years, norovirus strains genetically heterogeneous have been reported in several countries (Mesquita et al., 2010; Martella et al., 2009; Tse et al., 2012) and exposure of dogs to noroviruses has been confirmed in several epidemiological investigations (Di Martino et al., 2017; Caddy et al., 2013; Mesquita et al., 2014). In 2011 the genome of a canine sapovirus was reconstructed in a metagenomic study in the United States (Li et al., 2011). Similar sapoviruses have been identified later on in Italy (Bodnar et al., 2016) and Japan (Soma et al., 2015). Also, specific antibodies and age-related patterns of prevalence have been identified in dogs, confirming that the virus is common in dogs (Melegari et al., 2018). In 2015, a novel vesivirus was reported in Italy (Martella et al., 2015). The canine vesivirus was highly related to caliciviruses found as contaminants of Chinese hamster ovary cells in the United States and Europe (strain 2117-like) and distantly related to the prototype canine calicivirus strain 48. The virus was detected at a low frequency in household dogs but at a high frequency in kennel dogs. Genome sequencing of old (1960s
70s) canine

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calicivirus isolates in the United States has characterized the isolates as either 2117-like or strain 48-like vesiviruses (Binn et al., 2018). Also, an acute hemorrhagic gastroenteritis outbreak caused by vesiviruses has been described in the United States in 2015 (Renshaw et al., 2018). The outbreak affected 11 dogs following a stay in a pet housing facility and four dogs died. A novel flavivirus (ssRNA 1 ) with considerable genomic similarity to human hepatitis C virus was discovered in 2011 in respiratory samples of domestic dogs and tentatively named as canine hepacivirus (Kapoor et al., 2011). More recently it has also been detected in horse sera (Burbelo et al., 2012), and both viruses are currently referred to as nonprimate hepacivirus (NPHV). Epidemiological investigations in the UK dog population identified NPHV by reverse-transcription polymerase chain reaction (RT-PCR) in tracheal tissues of 48 out of 210 dogs and in the liver, lung, and/or tracheal tissues of 12 out of 20 dogs. The presence of NPHV RNA was confirmed by in situ hybridization. Histopathological examination demonstrated a trend toward higher histopathological scores in NPHVpositive respiratory tissues, although, this was not statistically significant (El-Attar et al., 2015).

2.8.2 Novel viruses in cats Metagenomic studies have also made attempts to explore the composition of feline virome (Table 2.1). Analysis of the stools of a healthy cat from Portugal (Ng et al., 2014) has revealed at least five different viruses of five virus families, including Picornaviridae (Sakobuvirus), Astroviridae (Astrovirus), Parvoviridae (Bocaparvovirus), Reoviridae (Rotavirus), and Picobirnaviridae (Picobirnavirus). The presence of these viruses was confirmed by PCR screening in 10 out of 55 cats and coinfections were common. Almost all (nine out of10) cats positive for enteric viruses appeared healthy. A novel picornavirus, named feline sakobuvirus A, was identified and appeared distantly related to other feline picornaviruses of the genera Sapelovirus (Lau et al., 2012b) and Kobuvirus (Chung et al., 2013). The feline sakobuvirus represents the prototype species for a novel genus, tentatively proposed as Sakobuvirus (Ng et al., 2014). In the same study, the authors identified a novel feline bocaparvovirus (Ng et al., 2014), distantly related to other bocaparvoviruses identified in cats from Hong Kong (Lau et al., 2012a). The pathogenic role of bocaparvovirues is unclear. The nearly complete genome sequence of a feline bocaparvovirus has been subsequently retrieved in the brain tissues of a cat also infected with feline panleukopenia virus. This was the first evidence of nervous system infection by bocaparvovirus (Garigliany et al., 2016). Also, a novel feline non-A rotavirus, strain Viseu, was identified in the Portuguese study. The virus was phylogenetically related to RV species B, G, and H (Ng et al., 2014; Mihalov-Kova´cs et al., 2015). Similar non-A rotaviruses have been identified in a distinct metagenomic study in the United States (Phan et al., 2017).

2.8 Viral metagenomics and discovery of new viruses in pets

The etiology of diarrhea in three cats was investigated. The cats tested negative for a panel of bacteria, viruses, and protozoans. NGS analysis revealed the presence of rotavirus RNA and the virus was found to resemble species I rotaviruses detected in sheltered dogs in Hungary (Mihalov-Kova´cs et al., 2015). The study by Ng et al. (2014) also identified a feline picobirnavirus closely related to a GII human picobirnaviruses. A 2014 metagenomic investigation has provided insights into the enteric virome of 10 different small carnivores of the Mustelidae, Canidae, Viverridae, and Felidae families in wildlife areas of northern Spain (Bodewes et al., 2014). Viruses belonging to the families of Anelloviridae, Astroviridae, Bunyaviridae, Caliciviridae, Circoviridiae, Parvoviridae, Picobirnaviridae, Picornaviridae, Rhabdoviridae, and Retroviridae were detected in 26 out of 42 of the analyzed samples. A comprehensive picture of the viruses shed in the feces of 25 healthy cats from a shelter in China (Zhang et al., 2014a,b) has identified either complete or partial viral genomes of astroviruses, bocaparvoviruses, and cycloviruses. The near complete genome sequences of three astrovirus strains, D1, D2, and D3, were obtained (Zhang et al., 2014a,b), with strain D1 being distantly related to other feline astroviruses (Lau et al., 2013; Ng et al., 2014). In the same study, the nearly complete genomes of two bocaparvovirus strains, FBD1 and FBD2, were retrieved (Zhang et al., 2014a,b), with FBD1 representing a novel parvovirus species (Lau et al., 2012a; Cotmore et al., 2014). Also, a cyclovirus (ssDNA) (family Circoviridae) strain, FD, was identified. Upon sequence and phylogenetic analysis, the virus differed from other cyclovirus species (Zhang et al., 2014a,b). Cycloviruses have been found recently in different biological samples from a number of host species, including mammals and insects (Li et al., 2010a,b; Ge et al., 2011; Rosario et al., 2011; Dayaram et al., 2013; Padilla-Rodriguez et al., 2013; Phan et al., 2014). At least five papillomavirus types have been described so far in domestic cats. Feline papillomavirus type-1 was originally detected in a cutaneous viral plaque (Tachezy et al., 2002). Feline papillomavirus type-2 was detected in a preneoplastic feline Bowenoid in situ carcinoma (BISC) (Munday et al., 2007). The full genome sequence of a third papillomavirus, type-3, has been obtained by NGS from a cat presenting multiple BISCs (Munday et al., 2013), closely related to canine PVs from the genus Taupapillomavirus. Other two feline PVs, types 4 (Dunowska et al., 2014) and 5 (Munday et al., 2017b), were retrieved from a cat presenting multifocal ulcerative gingivitis (Dunowska et al., 2014) and from a cat with preneoplastic facial lesions (Munday et al., 2017a), respectively. Morbilliviruses (genus Morbillivirus, family Paramyxoviridae) cause severe and often fatal infections of humans and animals including measles, canine distemper, and pest of small ruminants (Lamb and Parks, 2013). Morbilliviruses have been very recently identified in cats. Feline morbillivirus was first detected in Hong Kong (Woo et al., 2012c) and associated with tubule-interstitial nephritis. Further studies demonstrated the presence of the virus in domestic cats in Japan

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(Sakaguchi et al., 2014; Furuya et al., 2014), Europe (Sieg et al., 2015; Lorusso et al., 2015; Marcacci et al., 2016), and the United States from healthy cats and from cats with chronic kidney disease (Sharp et al., 2016). Orthopoxviruses (OPXVs), family Poxviridae, are viruses of high interest for scientists because of their potential use as bioterroristic agents and in gene therapy. Reports of OPXV infections in animals and humans have been increasing during the last few decades leading to the perception of an increasing risk for humans (Vorou et al., 2008). In cats, there are a number of reports of poxvirus infections, but the causative agent has been characterized mostly as cow OPXV (Schaudien et al., 2007; Scho¨niger et al., 2007; Schulze et al., 2007; Johnson et al., 2009; Kaysser et al., 2010; Herder et al., 2011). The first full genome sequences of two feline ortopoxviruses were obtained by NGS approach (Dabrowski et al., 2013) from strains collected as part of the routine diagnostic work at the German consultant laboratory for poxviruses. Phylogenetic analysis showed that the two cat strains clustered into cowpox clade. Later, five novel feline OPXV whole genome nucleotide sequences were generated by NGS from a collection of isolates obtained from several localities in the United Kingdom and Norway (Mauldin et al., 2017). Phylogenetic analyses demonstrated the presence of five different clades (A
E) from cowpox. More recently, a cat with multicentric nodular ulcerative dermatitis was found positive for OPXV (Lanave et al., 2018). The virus differed from cowpoxes common in cats and was grouped with an OPXV strain identified from captive macaques in Italy (Cardeti et al., 2017), related to ectromelia viruses (Chen et al., 2003; Mendez-Rios et al., 2012; Mavian et al., 2014). A recent evolution toward the diagnostic application of NGS takes advantage of an enrichment/selection step of cDNA with a mixture of oligonucleotide DNA and RNA probes (targeted genome capture, TGC) (Gnirke et al., 2009). TGC coupled with NGS has been performed to detect and generate sequence data of multiple pathogens from three felid species (domestic cats, bobcats, and mountain lions) (Lee et al., 2017). Thirty-one pathogens in 28 samples representing nine of 11 targeted taxa were retrieved from the NGS experiments. Eleven out of 31 pathogens had not been previously detected despite being subjected to standard diagnostic methods. Seven of 11 pathogens were completely or partially sequenced, including feline immunodeficiency virus (FIV), feline coronavirus, feline foamy virus, felid alphaherpesvirus 1, and feline leukemia virus. Viruses of the genus Orthohepadnavirus, family Hepadnaviridae, are partially ds DNA viruses that infect a variety of mammals including primates, bats, and rodents (Seeger et al., 2013). The type species, human hepatitis B virus (HBV), is a major public health problem, and an estimated 257 million people are currently living with HBV. A new feline hepadnavirus was retrieved in a virus discovery project in Australia (Aghazadeh et al., 2018) from a domestic cat presented with multicentric, large cell, high-grade, B-cell lymphoma, and infected with FIV. A molecular survey for hepadnavirus revealed the presence of the novel hepadnavirus in the blood of 6/60 (10%) FIV-infected cats and two out of 63 (3.2%) uninfected cats.

2.9 Metagenomics revealing new virus species in aquaculture

2.9 Metagenomics revealing new virus species in aquaculture Diseases caused by viruses have a devastating economic effect on aquaculture worldwide, which could be estimated in billions of US dollars annually. NGShas speeded up and revolutionized the viral research of aquatic viruses, as well. The high abundance of aquatic viruses has been known since the 1990s (Bergh et al., 1989; Hara et al., 1991; Proctor and Fuhrman, 1990). Subsequent studies revealed that viruses are the most abundant organisms in the marine environment (Angly et al., 2006; Suttle 2005; Wen et al., 2004). The large number of viral ORFs showing no homologies to any known sequences in databases suggest that the diversity of aquatic viruses may be even greater than imagined (Yin and Fischer, 2008). However, these aquatic viruses infect a much wider range of marine and freshwater species than fishes and shrimps, which are the economically most important species for humans, majority of the detected viruses have hosts among phyto- and zooplankton, and some of them were found in marine mammals, as well. Four major application of NGS in aquaculture could be distinguished and will be discussed below in this order: (1) Metagenomic characterization of the viromes of aquatic environment. (2) Whole genome sequencing of already known/ isolated viruses. (3) Discovering novel viruses in diseased or asymptomatic hosts. (4) Molecular epidemiology (tracking the mutations causing changes in the virulence/pathogenicity of viruses), and studying the evolution of viruses (Nkili-Meyong et al., 2016).

2.9.1 Virome characterization Several papers were published on the microbial ecology of different seas and coral atolls in the last decade (Hwang et al., 2016, 2017; Mahmoud and Jose, 2017; Tseng et al., 2013; Zeigler Allen et al., 2017). In these studies, the overwhelming majority of the viral sequences detected by NGS are unknown, while among the viruses identified, bacteriophages (order Caudovirales) infecting a wide range of microbial hosts (Cyanobacteria, Proteobacteria, and Bacteroidetes) and members of the family Phycodnaviridae (large eukaryotic phytoplankton viruses) showed the highest abundance. For example in a study on the virome of the marine environment in Goseong Bay (Korea), the BLAST searches showed that only 0.74% of the sequences were associated with known viruses, and 87% of these known sequences were originating from bacteriophages and only 13% showed homology to viruses of eukaryotes (Hwang et al., 2016). Nonetheless, already known DNA and RNA viruses of fish were also detected in these studies, these viral sequences were similar to those of members of diverse families, such as Nodoviridae, Retroviridae, Iridoviridae (Zeigler Allen et al., 2017), and Alloherpesviridae (Wood-Charlson et al., 2015; Hwang et al., 2016).

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In the last decade, a huge amount of circular replication-associated protein encoding single-stranded (CRESS) DNA viruses were discovered in different ecosystems including marine and freshwater environments (Angly et al., 2006; Kim et al., 2015a,b; Labonte and Suttle, 2013; Rosario et al., 2009; Rosario and Breitbart, 2011). CRESS DNA viruses have a very broad host-range: vertebrates, mollusc, arthropods, etc. Some of them, classified into the family Circoviridae, were described from diseased fish, however the connection between the circoviruses and the diseases were not yet proved (Doszpoly et al., 2014; Lorincz et al., 2011, 2012). An interesting study, demonstrating the potential significance of a shipmediated virus spread was carried out lately (Kim et al., 2015a,b). In this survey, the composition of viral communities in ballast and harbor waters were determined by metagenomic methods. The results showed that a majority of the viral sequences do not have any homologs in public databases, hence they could not be assigned to any taxa. The minority, the assigned viruses, were prevailed by dsDNA viruses, especially bacteriophages. However, sequences associated with emerging, important viruses of fish and shrimp were also detected, for example, the koi herpesvirus (KHV), infectious spleen, and kidney necrosis virus (ISKNV), white spot syndrome virus Thus, drawing attention to the fact, that ballast water of cargo ships could be an important vector for the transport of nonnative species to new aquatic environments including viruses (Kim et al., 2015a,b).

2.9.2 Complete genome sequencing by next generation sequencing NGS could be used for the full-length sequencing of already known viruses, this method proved to be very useful for the complete genome characterization of large dsDNA viruses (adeno-, herpes-, iridovirus). For example, the complete genomes of all members of the genus Cyprinivirus (Cyprinid herpesviruses, CyHV-1, -2, -3 and Anguillid herpesvirus, AngHV-1) were sequenced by NGS, their genome sizes proved to be the largest among herpesviruses (248
295 kbp). Cyprinid herpesvirus 3 (KHV) is the most ill-famed among the above mentioned viruses, revealing its complete genome was necessary for developing reliable diagnostic methods (PCR, qPCR) and designing DNA vaccine for KHV (ElMatbouli et al., 2007; Zhou et al., 2014). In another study, the deep sequencing of poly(A) RNA of the formerly mentioned AngHV-1 was carried out, providing the first data about the transcriptome of a fish herpesvirus. They found surprisingly lots of RNA splicing, and a low level (1.5/%) of antisense transcription in predicted protein-coding regions unlike in the genome of mammalian herpesviruses (Van Beurden et al., 2012). Another herpesvirus genomes belonging to the genus Ictalurivirus were also sequenced, namely the Ictalurid herpesvirus 2 (Borzak et al., 2018) and the Acipenserid herpesvirus 2 (Doszpoly et al., unpublished). These viruses have a significantly shorter genome (143
165 kbp) than that of the cypriniviruses.

2.9 Metagenomics revealing new virus species in aquaculture

Ranaviruses of fish (family Iridoviridae) were also sequenced by NGS. The European sheatfish virus was isolated from wels catfish (Silurus glanis) and from brown bullhead (Ameiurus nebulosus) in Europe (Ahne et al., 1998; Feher et al., 2016), while the epizootic hematopoietic necrosis virus, another ranavirus was described in Australia earlier (Steiner et al., 1991). These viruses share very high nucleotide identity alongside their entire genome (99%). After sequencing their whole genome by NGS, regions showing insertions/deletions were identified, which allows us to design PCR-based diagnostic that differentiates between the viruses without sequencing them (Mavian et al., 2012). NGS could also be used, if the isolation of a virus fails. Another well-known virus family that encompasses large dsDNA viruses, among them the feared pathogen of smallpox, is Poxviridae. This virus family has a rather wide host-range spanning from invertebrates to human, including fish. The presence of pox-like viruses in fish was discovered in carp (Cyprinus carpio) 40 years ago (Murakami et al., 1976) causing the carp edema disease (Ono et al., 1986). The causative agent could not be isolated yet, in spite of the several attempts, however its complete genome sequencing using metagenomics is in the pipeline (Haenen et al., 2016). Another poxvirus (salmon gill poxvirus) was reported from salmon (Salmo salar), and its full genome was determined by NGS using the organs of diseased fish. Phylogeny inference suggested that the fish poxvirus represented the most basal branch of the subfamily Chordopoxvirinae (Gjessing et al., 2015).

2.9.3 Discovery of novel viruses If all the traditional methods to identify viruses have failed and when the viral pathogen is not culturable in vitro, then NGS is a very powerful tool to discover novel viruses. For example, a novel ssRNA virus was detected from carp (Cyprinus carpio) in Hungary by metagenomics. Its genome proved to be 8712 nucleotide in lengths. The virus was named fisavirus, phylogenetic calculations clustered it under the order Picornavirales, and it shows a distant relation to posaviruses, however a novel family should probably be established for this virus (Reuter et al., 2015). Another example is the heart and skeletal muscle inflammation, it was reported from salmon farms in Norway and the United Kingdom in 1999 causing high mortality, but the causative agent remained undiscovered for more than a decade. In 2010, a virus was identified using NGS, the phylogenetic reconstructions clustered the virus into the family Reoviridae, and it was named piscine reovirus (Palacios et al., 2010). A new virus belonging to the genus Totivirus (family Totiviridae) was also identified in salmon affected by cardiomyopathy syndrome (Haugland et al., 2011; Lovoll et al., 2010). Tilapia lake virus, an orthomyxo-like virus, was also detected by NGS from brain tissues of diseased fish (Bacharach et al., 2016). In an interesting case, three

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viruses from three different virus families were described in a single sea bream (Sparus aurata) using NGS. Fish showed the symptoms of lymphocystis disease, usually caused by an iridovirus (lymphocystis disease virus, LCDV). However, in this case a papilloma- and a polyomavirus were also detected in the same specimen, their complete genome sequences were recovered, providing the first description of a papillomavirus in fish. Moreover, the genome of the sequenced iridovirus (termed as LCDV-Sa) proved to be much longer (the largest known vertebrate iridovirus genome) then the formerly described LCDVs, thus LCDV-Sa should be considered a novel and distinct virus species within the genus Lymphocystivirus (Lopez-Bueno et al., 2016). As for crustacean, NGS also contributed for discovering novel viral pathogens (Van Aerle and Santos, 2017). Recently, an iridescent virus causing severe disease and high mortality in farmed whiteleg shrimp (Litopenaeus vannamei) has been reported and termed as shrimp hemocyte iridescent virus (SHIV). Partial sequences were obtained by viral metagenomics, and phylogenetic calculations based on the sequences of major capsid protein and ATPase showed that SHIV is a new iridescent virus, which could not be clustered into any of the five known genera of Iridoviridae. Based on the sequences a diagnostic PCR was developed for detecting this novel virus threatening shrimp farming industry in China (Qiu et al., 2017). Novel viral sequences were obtained from another shrimp species, the northern pink shrimp (Farfantepenaeus duorarum), using NGS on the hepatopancreas of 12 healthy shrimp captured from the Gulf of Mexico. Among the sequences, a novel nodavirus (F. duorarum nodavirus, FdNV) and a new CRESS DNA virus showing highest similarity to circoviruses were identified. The genome analysis of the FdNV suggested that the virus is closely related to nodaviruses causing white tail disease in giant river prawn (Macrobrachium rosenbergii) and muscle necrosis disease in whiteleg shrimp (Ng et al., 2013a,b). These studies underline the potential of metagenomic approaches in fisheries and other aquaculture industries to identify unknown pathogens causing diseases or even in asymptomatic animal stocks.

2.10 Conclusion Viral metagenomics based on sequence-independent amplification techniques coupled with massive parallel sequencing on new generation sequencers have led to the discovery of numerous novel animal DNA and RNA viruses in intensively reared livestock and poultry, pets, and selected aquaculture species over the past decade. In most cases, the pathogenic role, if any, of the newly discovered viruses has remained unclear. Direct and indirect epidemiological investigations, coupled with animal experiments, will be necessary to obtain clearer indications for an etiologic and clinical connection. Nonetheless, new portable next generation

References

sequencers are on the horizon and MinIon sequencer has proven to serve a promising approach in the field application of disease outbreaks. Currently, the application of this laboratory approach is still in the developmental stage and will likely remain so until per sample sequencing costs becomes acceptable for farmers or pet owners, clinical sensitivity approaches the sensitivity of other multiplexed molecular methods commonly used in diagnostic laboratories and, importantly, bioinformatics pipelines become an easy task not requiring special skills.

Acknowledgements KB was funded by the Lendu¨let Program. SM was supported by the Bolyai Scholarship Program. AD obtained support from the Hungarian Scientific Research Fund (OTKA, K127916). YSM obtained support from Indian Council of Agricultural Research (National Fellow scheme).

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Further reading Abolnik, C., Wandrag, D.B., 2014. Avian gyrovirus 2 and avirulent newcastle disease virus coinfection in a chicken flock with neurologic symptoms and high mortalities. Avian Dis. 58, 90
94.

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Day, J.M., Zsak, L., 2016. Molecular characterization of enteric picornaviruses in archived Turkey and chicken samples from the United States. Avian Dis. 60, 500
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564. Rose, T.M., 2005. CODEHOP-mediated PCR - a powerful technique for the identification and characterization of viral genomes. Virol. J. 2, 20.

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CHAPTER

Genome editing in animals: an overview

3

Jaya Bharati1,2, Meeti Punetha1, B.A.A. Sai Kumar1, G.M. Vidyalakshmi1, Mihir Sarkar1, Michael J. D’Occhio3 and Raj Kumar Singh1 1

ICAR-Indian Veterinary Research Institute, Bareilly, India 2 ICAR-National Research Centre on Pig, Guwahati, India 3 The University of Sydney, Sydney, New South Wales, Australia

3.1 Introduction Genome editing is the process of insertion, deletion, or replacement of DNA at a specific site in the genome of an organism or cell by using nucleases also known as molecular scissors. Broadly speaking, it refers to the process of making targeted modifications to the genome, its contexts (e.g., epigenetic marks), or its outputs (e.g., transcripts). Oligonucleotide templates are used to create efficient single nucleotide changes to the genome and thus permit the transmission of both natural and novel DNA sequence variants into naive livestock breeds and species. Genome editing allows genetic variants to be directly introgressed into livestock genomes, and transient exposure of cells to sequence-targeted editors stimulates homologydirected repair (HDR) to levels that eliminate the need for transgene-dependent selection. Recent advances in genome editing provide the unprecedented ability to introduce almost any targeted modification into genomic DNA and have propelled genome editing from being a technical possibility to a practical reality.

3.2 Existing methods The most commonly used existing methods of genome editing are sequencespecific programmable nucleases, which efficiently catalyze genome editing in a wide variety of organisms (Moscou and Bogdanove, 2009; Bibikova et al., 2002). These are broadly classified into two categories: 1. Protein guided: a. Zinc finger nucleases (ZFNs); b. Transcriptional activator-like effector nucleases (TALENs), and;

Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00003-5 © 2020 Elsevier Inc. All rights reserved.

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2. RNA-guided endonucleases (RGENs): Two-component CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated nuclease) system. They can be programmed to generate targeted double-strand DNA breaks (DSBs) in genomic DNA. Thus, the process of genome editing relies on DNA repair system that occurs when DSBs induced by sequence-specific nucleases in DNA occur. In eukaryotic cells, DSBs are commonly repaired by the endogenous cellular DNA repair pathways of two main types (Lieber, 2010; van den Bosch et al., 2002; Barnes, 2001; Takata et al., 1998), both of which can be exploited to edit the genome as shown in Fig. 3.1. 1. Nonhomologous end joining (NHEJ) and, 2. Homologous recombinational (HR) repair or HDR. NHEJ involves direct ligation of the broken ends and is often accompanied by loss/gain of some nucleotides. Thus, the outcome of NHEJ is variable: nucleotide insertions, deletions, or nucleotide substitutions in the broken region. It is an error-prone process that can create disruptive insertions and deletions (indels) at targeted cleavage sites. If indels cause a frame shift in the reading frame, this can result in the creation of premature stop codons (Swiech et al., 2015); if insertions or deletions comprise three bases (or multiples of three bases) they may affect protein function (Yin et al., 2014) creating nonfunctional protein.

FIGURE 3.1 Different strategies for introducing blunt double-stranded DNA breaks into genomic loci, which become substrates for endogenous cellular DNA repair machinery that catalyze nonhomologous end joining (NHEJ) or homology-directed repair (HDR).

3.2 Existing methods

HDR uses homologous DNA as a template to restore the DSBs and the outcome of this kind of repair is precise and controllable by supplying an exogenous donor repair DNA template that has homology with the sequence flanking the DSBs. Thus, HDR can be exploited to precisely edit genomic sequence or insert exogenous sequence, such as genetically encoded tags in the presence of a donor template, to induce specific insertions, deletions or designer mutations (Chu et al., 2015). It is possible to precisely manipulate any gene and establish knock in and knockout (KO) animal models with HDR-mediated targeting. HDR mechanism can also be used to modify the genome via original nucleotide sequence replacing; and synonymous substitutions can be introduced, which can carry a new restriction site or a mutant allele into the genome, for example, in genetic correction, a wild-type allele replaces a hereditary disease sequence. However, HDR occurs vigorously only in dividing cells and its incidence is low in postmitotic cells and differentiated cells. Also, its efficiency is highly dependent on the cell type, stage of life, as well as the target locus of the genome and template itself (Saleh-Gohari and Helleday, 2004). Since, the desired recombination events are very infrequent (1 in 106
109 cells) (Capecchi, 1989), it presents huge challenges for large-scale applications of genome editing following HDR mechanism.

3.2.1 Zinc finger nucleases The discovery and application of ZFNs made an important contribution to genome editing toolbox. It is based on the principle that different zinc fingers recognize different sets of nucleotide triplets, a hybrid protein containing specific zinc finger DNA binding domains, and the endonuclease FokI is generated to target specific DNA sequences (Kim et al., 1996; Urnov et al., 2010). The coassembly of multiple zinc finger in single ZFNs can alter how the zinc finger and DNA interact, reducing specificity and posing challenges for optimal ZFNs design. Also, off-target cleavage is a significant concern for ZFNs (Carroll, 2014). Although considerable progress has been achieved, the use of ZFNs has not been picked up as widely as anticipated mainly due to: 1. Existence of context effects on the specificities of individual finger in an array; 2. The zinc fingers corresponding to all nucleotide triplets have not been discovered yet; 3. Generation and selection of DNA-specific zinc finger modules/ZFN proteins with high selectivity is costly, laborious, and time consuming (Bibikova et al., 2002, 2003; Urnov et al., 2010; Cradick et al., 2011).

3.2.2 Transcriptional activator-like effector nucleases TALENs consist of a group of special effector proteins working in pairs, which contain N- and C-termini for localization and activation and a central domain for specific DNA binding (Boch and Bonas, 2010; Miller et al., 2010). The genetic

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construct comprising a nuclear localization signal, half-repeat, n-terminal domain, the FokI catalytic domain and an artificial DNA binding domain are generated. TALENs binding sites are located on opposite DNA strands, which are separated by a small fragment, a spacer sequence usually 12
25 bp in length. Once in the nucleus, TALENs bind to target sites and FokI domains located at the c-termini dimerize to cause DSBs in the spacer sequence (Li et al., 2011; Mahfouz et al., 2011). The effective and rapid construction of customized TALE tandem repeats determines the full utilization of TALEs. However, the highly repetitive feature of TALE repeats makes it often inefficient, time consuming, laborious, and expensive to construct long TALE repeats. This has pushed the demand of developing new simpler, more rapid, more efficient, robust, and cost-effective genome editing techniques to meet the needs of new era of biomedical research.

3.2.3 RNA-guided endonucleases It is the most recent addition to the genome editing toolbox. RGENs derived from CRISPR/Cas system enable efficient genome editing compared to its predecessors. The CRISPR/Cas system is an adaptive immune response in bacteria and archaea (Barrangou et al., 2007), which functions by recognizing and cleaving foreign DNA from phages and plasmids via Cas9 protein and guide RNAs, whose sequences are partially derived from the invading genetic elements (Horvath and Barrangou, 2010; Wiedenheft et al., 2011). This system was exploited to develop RGENs that enable targeted genome editing. The rapid pace of improvements, new applications, and adoption for use in diverse organisms have proved the CRISPR/Cas9 system to be an exciting and significant technical leap forward for biomedical applications and research. The CRISPR systems are widespread in prokaryotes: they are found in 87% of archaea and 48% of eubacteria (Grissa et al., 2007). The different species widely in the number of CRISPR cassettes (1
18), repeats (60), size of repeats (23
37 bp), and size of spacers (17
84 bp). The length of spacers and repeats in one cassette is constant and repeat sequences are almost identical (Makarova et al., 2011). Subsequently, with the discovery of the Cas gene, Cas protein, protospacer adjacent motif (PAM), CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and many details regarding the CRISPR systems were quickly unveiled (Bolotin et al., 2005; Brouns et al., 2008; Haurwitz et al., 2010).

3.3 Types of CRISPR/Cas system There are at least 11 different CRISPR/Cas systems, which have been grouped on the basis of combined phylogenetic, structural, comparative genomic analyses, and diverse homology of Cas protein into three categories: type I, type II, and type III. They use distinct molecular mechanisms to achieve nucleic acid recognition and cleavage.

3.3 Types of CRISPR/Cas system

Type I, type II, and type III exhibits Cas3, Cas9, and Cas10 signature genes respectively. The type II system needs only one Cas protein to recognize and cleave target sites, whereas type I and type III CRISPR systems require a set of Cas proteins (Brouns et al., 2008; Makarova et al., 2011; Wiedenheft et al., 2011).

3.3.1 Type II CRISPR/Cas9 system for genome editing The type II system genomic CRISPR locus includes the tracrRNA gene, Cas gene, and CRISPR repeat-spacer array, which are transcribed into tracrRNA, Cas9 proteins, and pre-crRNA, respectively. With the cooperation of tracrRNA and RNaseIII, pre-crRNA can be cut into crRNAs that can interact with tracrRNAs and subsequently lead Cas9 to recognize the specific DNA sites (Chylinski et al., 2013). The Cas9:RNA complex searches DNA sequences randomly and rapidly dissociates from non-PAM sites. PAM sequence is a short motif adjacent to the target sequence (usually NGG motif for SpCas9) which is recognized by Cas9 protein. Once PAM is recognized, the Cas9:RNA complex interrogates the flanking DNA sequences for gRNA complementarity in a Watson-Crick base-pairing interaction (Sternberg et al., 2014). At the target sites that match the tracrRNA:crRNA duplex known as single guide RNA (SgRNA) and are flanked by PAMs, the HNH nuclease domain of Cas9 cleaves the DNA strand complementary to the guide RNA sequence (target strand of DNA), and the RuvC nuclease domain cuts the noncomplementary strand (nontarget strand) resulting in DSBs at the specific sites (Haurwitz et al., 2012) as shown in Fig. 3.2. As a result of its simplicity, the type II CRISPR/Cas system has been developed as a robust programmable tool, which is known as CRISPR/Cas9. Mali et al. (2013a) first utilized CRISPR/Cas9 to induce specific DSBs synthesizing an artificial Cas9 protein and cloned it into a mammalian expression system. In addition, they expressed a chimeric SgRNA to replace the tracrRNA:crRNA duplex. Desired mutations can be introduced in several human cell lines by using a

FIGURE 3.2 The Cas9 enzyme has two cutting domains viz. RuvC and HNH, each for making nicks three base pairs upstream of protospacer adjacent motif (PAM) in each strands, targeted by Single guide RNA (SgRNA), slices DNA to initiate genomic editing.

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custom CRISPR system, with targeting rates ranging from 2% to 38% (Peng et al., 2016). Meanwhile, Cong et al. (2013) designed two different type II CRISPR/Cas systems and demonstrated that Cas9 nucleases can induce precise cleavages at specific sites under the guidance of short RNAs in human and mouse cells. With the gradual perfection of CRISPR/Cas9 systems, Streptococcus pyogenes Cas9 (SpCas9) coexpressed with custom guide RNAs (SgRNAs) have been successfully used in bacteria, fungi, viruses, parasites, plants, animals, and human cell lines (Swiech et al., 2015; Ratz et al., 2015; Zhu, 2015).

3.3.1.1 Cas9 activity Cas9 enzymes from type II CRISPR/Cas systems are emerging as the sequencespecific nucleases of choice for genome engineering for several reasons. As a genome editing tool, the CRISPR/Cas9 system cleaves specific nucleotides based on sequence complementarity with only two significant components: the Cas9 protein and SgRNA. DSBs can be introduced by the CRISPR/Cas9 components which can be injected as DNA, RNA, or protein into most developing organisms and cell types to generate modifications early in the development (Jinek et al., 2014). Moreover, PAM is critical for binding DNA and in its absence, even the target sequences fully complementary to the guide RNA sequence are not recognized by Cas9 (Sternberg et al., 2014) which offers specificity to the Cas9 activity.

3.3.1.2 Multiple gene editing SgRNAs are also successfully applied to multiplex genome engineering which refers to editing several different genome sites in one procedure (Mans et al., 2015). Multiplexing is made easier by the modular nature of the two-component CRISPR/ Cas9 system and the small size of the gRNA. Cas9 nuclease in conjunction with multiple gRNAs has been used to introduce mutations in several genes simultaneously in cultured mammalian cells as well as genetic model organisms such as mice, zebra fish, Arabidopsis, and is efficient in terms of successes per attempt (Chang et al., 2013; Cong et al., 2013; Hwang et al., 2013; Li et al., 2013; Wang et al., 2013). Multiplexing was also successful in introducing mutations in monkeys and silkworms (Niu et al., 2014; Wang et al., 2013). Multiplex all-in-one transient expression vector containing Cas9 and SgRNA was transiently expressed to generate a mutation in five PI3K genes in Dictyostelium cells (Sekine et al., 2018).

3.4 Potential pitfalls A large number of studies have investigated diverse factors affecting the CRISPR/Cas9 system, such as: 1. off-target effects, 2. delivery methods, and 3. incidence of HDR.

3.4 Potential pitfalls

By addressing these potential pitfalls, we can take better advantage of this technique, as well as improve its efficiency and specificity.

3.4.1 Off-target effects A number of studies have demonstrated that the CRISPR/Cas9 system can induce a substantial amount of off-target mutagenesis (Mali et al., 2013b; Yang et al., 2013). The existing strategies for reducing genome-wide off-targets of the broadly used SpCas9 are imperfect, possessing only partial or unproven efficacies. For biological studies and genetic therapies, off-target phenomena generate undesired mutations at random sites, thus impacting precise gene modification (Peng et al., 2016). For reducing the potential off-target effects, points that need consideration are:

3.4.1.1 SgRNAs design Rational design of highly active SgRNAs and selecting proper target sites are a prerequisite to increasing efficiency of genome editing. Target sequences carrying double, triple, or quadruple mismatches were well tolerated with mutation frequencies equal to that of the wild-type sequence (Hsu et al., 2013). Mismatches distal from the PAM sequence are well tolerated (Liu et al., 2012) but mismatches proximal to the PAM sequences can also produce an off-target effect (Fu et al., 2013; Hsu et al., 2013) which should be avoided. It was demonstrated by Fu et al. (2013), that use of truncated SgRNAs with a length of 17
18 nucleotides can decrease undesired off-target effects by more than 5000-fold, as well as retain ontarget efficiency.

3.4.1.2 Cas9 nickase Cas9 enzyme can be converted into Cas9 nickase, a variant protein with singlestrand DNA cleavage capacity, by mutating either the HNH or the RuvC-like domain. In one study, two Cas9 nickases guided by two gRNA targeting the sense and antisense strands were used to generate DSBs (Cradick et al., 2013; Ran et al., 2013), which improved the overall specificity of the system. dCas9: FokI fusions have been reported to significantly reduce the off-target activities (Guilinger et al., 2014; Tsai et al., 2014).

3.4.1.3 “Enhanced Specificity” SpCas9(eSpCas9) The CRISPR/Cas9 gene editing system has been known to be overzealous, unwilling to quit after cleaving the desired target site. Instead, it persists inside a cell, cutting additional sites, introducing unwanted edits, and thereby propagating “off-target” effects. This is the major limitation of the available CRISPR/Cas9 system. A modified CRISPR/Cas9 was proposed by scientists at the Broad Institute, MIT which makes a precise editing tool. These scientists developed a modified Cas9 that binds with the DNA more weakly. The team used structureguided protein engineering to improve the specificity of SpCas9. This approach involved swapping out some of Cas9s 1400 or so amino acids. Cas9 has

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positively charged amino acids that form a positively charged groove that binds negatively charged stretches of DNA. The scientists replaced the positively charged amino acids with neutral amino acids, and the resulting Cas9 was more precise and specific. After experimenting with various possible changes, it was found that mutations in three amino acids dramatically reduced off-target cuts. For the various guide RNAs tested, off-target cutting was so low as to be undetectable and was demonstrated that “enhanced specificity” SpCas9 (eSpCas9) variants reduced off-target effects and maintained robust on-target cleavage (Slaymaker et al., 2016).

3.4.1.4 Cpf1 The limitations of SpCas9 in making specific genetic edits via HDR led to the discovery of other variants of Cas proteins which may increase the efficiency of gene editing. Zetsche et al. (2015) described two RGENs from the Cpf1 family that displayed cleavage activity in mammalian cells. Unlike Cas9 nucleases, Cpf1-mediated DNA cleavage results in sticky-end-like DNA DSBs with a short (4
5 nucleotides) 30 overhang. Cpf1 has a staggered cleavage pattern, which opens up the possibility of directional gene transfer, equivalent to the traditional restriction enzyme cloning. Cpf1 enables the new targeting possibilities of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes in contrast to the NGG PAM sites favored by SpCas9.

3.4.1.5 Cas9-HF1 A Cas9 nuclease that exhibits the effectiveness of the gold standard S. pyogenes CRISPR/Cas9 editing system but shows effectively no detectable off-target activity has been discovered and this protein is named as SpCas9 high-fidelity variant number 1 (SpCas9-HF1). It proved to have comparable on-target efficiency of greater than 90%, compared to wild-type SpCas9 and all of the off-target effects caused by wild-type Cas9 decreased to undetectable levels. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with .85% of SgRNAs tested in human cells. With its exceptional precision, SpCas9-HF1 provides an alternative to wildtype SpCas9 for research and therapeutic applications (Kleinstiver et al., 2016). It is a high-fidelity variant harboring alterations designed to reduce nonspecific DNA contacts, and is a manifestation of how a structural and mechanistic understanding of the Cas9 enzyme can continually improve genome editing tools.

3.4.1.6 HypaCas9 Efforts that increase the cleavage efficiency of CRISPR/Cas9 have encouraged the development of high fidelity (SpCas9-HF1) and enhanced specificity eSpCas9 (1.1) variants. They contain amino acid substitutions that weaken the energetics of target site recognition and cleavage (Slaymaker et al., 2016; Kleinstiver et al., 2016) and exhibit reduced off-target cleavage in human cells (Tsai et al., 2015). Single-molecule Forster resonance energy transfer (smFRET) experiments, demonstrated that both SpCas9-HF1 and eSpCas9(1.1) were trapped in an inactive

3.4 Potential pitfalls

state (Dagdas et al., 2017) when they were bound to mismatched targets. It contains a noncatalytic domain REC3, which recognizes target mismatches and governs the HNH nuclease to regulate overall catalytic fitness. Chen et al. (2017) identified residues within REC3, which is involved in mismatch sensing and a novel hyperaccurate Cas9 variant (HypaCas9) and retains robust on-target activity in human cells (Chen et al., 2017). These findings offer an inclusive model for precision genome editing with minimum off-target effects.

3.4.2 Delivery methods Optimization of delivery methods to achieve efficacy and specificity is of prime importance. There are three ways to deliver the components of CRISPR/Cas9 system. The most commonly used method is of plasmids that simultaneously encode SgRNA and Cas9. With the demand for large fragment expression, multiple plasmids have been used to target different sites (Mans et al., 2015). However, all or part of the plasmids are often randomly integrated into the host genome (Kim et al., 1996) or it can also be inserted into both on-target and off-target sites, which can lead to difficulties in detection. Furthermore, host immune responses can be induced by these inserted bacterial sequences and may interrupt the process of genome editing (Wagner, 2001). Also, these methods of transfection are inefficient in primary cells and may lead to cytotoxicity. Once transfected, plasmid DNA can also persist inside the cells for several days, which may aggravate off-target effects (Gaj et al., 2012). The second approach can be delivery of Cas9 mRNA and in vitro transcribed SgRNA. This method too comes with a major drawback, because translation begins only at the two-cell stage, injection of mRNA encoding Cas9 into singlecell embryos often results in chimerism (Sung et al., 2013). Using Cas9 protein can circumvent this limitation. Moreover, the detection of Cas9 protein is much easier than the detection of Cas9 mRNA before transfection. The introduction of components of CRISPR/Cas9 system into target cells by electroporation, nucleofection, or lipofectamine represents a common and rapid method that can be applied to a wide variety of cell lines. Although these methods of delivery can reduce off-target effects relative to plasmid-mediated delivery, the efficiency is still considered insufficient and needs new optimized delivery mechanism. A nonviral approach, known as CRISPR-Gold that uses gold nanoparticles to encapsulate all the elements needed for CRISPR/Cas9 gene editing and deliver them efficiently directly to cells was developed recently (Lee et al., 2017). It was tested in the mouse model of Duchenne muscular dystrophy (DMD) to repair the faulty DMD gene, leading to improved strength and agility and reduced fibrosis with minimal off-target DNA damage (Long et al., 2014). Thus, CRISPR-Gold, more broadly speaking, CRISPR-nanoparticles open a new way for safer, accurate, and controlled delivery of gene editing tools. Ultimately, these optimized delivery techniques of CRISPR/Cas9 genome editing system could be developed into a new strategy for a number of research and therapeutic purposes.

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3.4.3 Incidence of HDR In general, even in the presence of donor templates, NHEJ is the more frequently repair pathway when using CRISPR/Cas9 systems (Maruyama et al., 2015). Because NHEJ is error-prone but highly efficient, it is suitable for generating indels (insertion deletion). However, it cannot edit the genome precisely or in the same way as HDR. Generally, the efficiency of Cas9-mediated gene editing in mice via NHEJ can reach 20%
60%, whereas the efficiency of HDR is only 0.5%
20% (Tsai et al., 2014). As a result of the low incidence of HDR, use of CRISPR/Cas9 for improving the incidence of precise insertional mutagenesis is a challenge. Cell lines deficient in NHEJ components increase levels of HDR. This suggests that these two repair processes are intensely competitive. Hence, suppressing key NHEJ molecules, such as KU70, KU80, or DNA ligase IV, by gene silencing (Chu et al., 2015), microinjection of ssRNAs (Basu et al., 2015) or the use of small molecule inhibitors (Chu et al., 2015; Robert et al., 2015) has been shown to increase the frequency of repair by the Cas9-mediated HDR pathway. Use of the inhibitor Scr7, which targets DNA ligase IV can increase the efficiency of HDR-mediated genome editing up to 19-fold (Chu et al., 2015; Srivastava et al., 2012). Two small molecules, L755507 and Brefeldin A, can increase the HDR efficiency by two- to threefold (Yu et al., 2015). Robert et al. (2015) also confirmed that two small molecule inhibitors of DNA-PKcs (NU7441 and KU0060648) could reduce the incidence of NHEJ and increase the frequency of HDR. In addition, short hairpin RNA sequences to knockdown KU70/80 or DNA ligase IV can also be used for promoting the efficiency of HDR in both human and mouse cells (Chu et al., 2015). Cells have different abilities with respect to repairing DSBs either using NHEJ or HDR and also the phase of the cell cycle regulates repair pathway selection. NHEJ always occurs during the entire cell cycle, whereas HDR is restricted to the late S and G2 phases (Lin et al., 2014). Therefore, a strategy that combines well-established cell cycle synchronization techniques with direct nucleofection of preassembled Cas9 ribonucleoprotein complexes to achieve controlled nuclease action at the optimal phase of the cell cycle for HDR (Lin et al., 2014) can be utilized for precise genome editing.

3.5 Comparing the CRISPR/Cas9 system versus zinc finger nucleases and transcriptional activator-like effector nucleases As compared to its genome editing forerunners ZFNs and TALENs, CRISPR/Cas9 has several advantages. Despite having efficient gene editing properties, ZFNs and TALENs, due to their low specificity and interference between contiguous modules in larger arrays, they failed to be widely adopted. CRISPR/Cas9-mediated genome

3.6 Applications of CRISPR/Cas9 genome editing technology

editing system adopts the Watson-Crick complementary rule to recognize and cleave target DNA sequence via a short RNA molecule and the endonuclease Cas9, respectively. Its specificity is secured by the method with which it targets nucleic acid base-pairing, and is a remarkable feature similar to the fidelity of DNA replication and transcription. The CRISPR/Cas9 system functions with a universal Cas9 protein framework that dispenses without the need to design a different protein for each DNA target unlike in TALEN or ZFN-mediated genome editing. TALEN-mediated genome editing requires engineering a pair of large repetitive sequences encoding domains for site-specific DNA recognition and cleavage in the genome, whereas in the case of CRISPR/Cas9-mediated genome editing, it requires only a short RNA molecule, which is much easier to manipulate than to assemble TALE repeats. The targeting efficiency varies for different TALEN pairs and in different loci, and it is known that there are engineered TALENs which fail to mediate any genome modifications. A modification in a new target sequence can be done by modifying the 20-nucleotide guide sequence of SgRNA. Also, Cas9 causes a break strictly between the 17th and 18th nucleotides in the target sequence (counting from the 50 -end of the spacer), that is, at a distance of three nucleotides upstream from the PAM. Multiplexing is greatly simplified by introducing a combination of SgRNAs, when compared to the tedious designing of TALENs repeats. Cas9 can be easily modified to become a nickase to facilitate HDR (Cong et al., 2013). The use of nickase and flexibility of the SgRNA construction allow for more accurate target recognition in the genome. Thus, the use of the CRISPR/Cas9 system has a number of advantages over the ZFNs- and TALENs-based methods: it is much easier to execute, more efficient, and is suitable for high-performance and multiplex genome editing in a variety of cell lines and in living organisms. With time, CRISPR/Cas9 system has emerged as a highly versatile, rapid, and economically viable genome editing technology for precise gene modification. The present treatise highlights the present status and future prospects of CRISPR/Cas9 genome editing technology in animal agriculture.

3.6 Applications of CRISPR/Cas9 genome editing technology in animal agriculture 3.6.1 Study of developmental biology The interplay between genome activity and developmental events such as cell proliferation, differentiation, and morphogenesis can be studied by utilizing CRISPR/Cas9 system. ZFNs and TALENs have been used in proof-of-concept studies to achieve locus-specific targeting of epigenetic modifying enzymes. A modification of Cas9 without endonuclease activity, that is, dCas9 platforms, can be programmed and used for investigating genome dynamics in vivo and studying the effects of methylation or certain chromatin states on cellular differentiation

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(Beerli et al., 2000; Konermann et al., 2013, 2015; Maeder et al., 2013; Mendenhall et al., 2013). CRISPR/Cas9 can be targeted to virtually any desired genomic location by a SgRNA enabling the dissection of functional properties of specific genes and their regulatory elements. This will allow mapping of the detailed role of that gene in growth and development; for example, CRISPR/Cas9 genome editing system has been used for the study of steroidogenic function, transcriptional regulation and validation of functional role of regulatory genes viz. Thrombospondins in bubaline corpus luteum (Paul et al., 2019a, 2019b). The causal effects of epigenetic modifications in shaping the regulatory networks of the genome can be probed by Cas9 epigenetic effectors (Hsu et al., 2013). Thus, CRISPR/Cas9 technology has thus provided an efficient tool to study epigenetic control mechanism, which could prove to be a landmark in the study of developmental biology.

3.6.2 Better food production CRISPR/Cas9 provides unprecedented opportunities for identifying specific alleles that affect livestock performance and for locus centric or whole genome
based selection for improvement of animal genetics (Tan et al., 2013). Myostatin (MSTN) a member of TGF-super family, is a negative regulator of skeletal muscle growth (McPherron et al., 1997) determining both muscle fiber number and size, is a potential candidate gene for genetic manipulation for enhancing meat production. Ni et al. (2014) successfully mutated MSTN gene in goat by transfecting goat fibroblast cells with a plasmid expressing both Cas9 and gRNA targeting MSTN. The CRISPR/Cas9 system efficiency to edit MSTN in sheep and generate KO animals with the aim to promote muscle development and body growth has proven to be a valuable system to generate gene-targeted modified animals (Crispo et al., 2015). CRISPR/Cas9 system has been used to develop an efficient gene knock in approach, to generate cisgenic bulls carrying an extra copy of the endogenous bovine SRY gene in the nonpseudoautosomal region of the X chromosome (XSRY). Thus, increased muscling due to SRY gene will lead to more meat production, ultimately leading to increased economic returns. Egg production is a trait that is expressed over a long interval of time and is influenced by both genetic and environmental effects. The male chicks from elite egg laying chicken breeds have no use in the egg industry so they are generally culled on the day of hatching. In order to avoid culling of male chicks, a new technique of knock in of marker gene responsible for green fluorescent protein to chicken sex chromosome has been evolved using CRISPR/Cas9 genome editing system. Eggs with male chicks will glow under the ultraviolet light and thus can be easily eliminated before hatching. The non-meiotic introgression of the Celtic polled allele, referred to as Pc (duplication of 212 bp that replaces 10 bp), into fibroblasts derived from horned dairy bulls was carried out by Tan et al. (2013). For this purpose TALENs were designed such that they could cleave the horned allele but leave the Pc allele unaffected. CRISPR/Cas9 technique has been used to KO

3.6 Applications of CRISPR/Cas9 genome editing technology

the Holstein horned gene and subsequently knock in with a polled gene from Angus cattle. The DNA from these “genome-edited” cells were transferred into bovine eggs to create embryos and then transplanted into recipient cows, so that healthy polled Holstein bull calves are born. These studies suggest that genome editing using CRISPR/Cas9 system can be incorporated into selection programs to accelerate genetic improvement for better food production when selective breeding is either inefficient or impossible.

3.6.3 Disease control The transformative technology of genome editing by CRISPR/Cas9 is being exploited to cure diseases by disrupting endogenous disease-causing genes, correcting disease-causing mutations or inserting new genes with protective functions. More specifically CRISPR/Cas9 and other genome editing technologies have been widely used to control animal diseases by the following ways:

3.6.3.1 Producing disease-resistant animals 3.6.3.1.1 African swine fever African swine fever (ASF) is a contagious hemorrhagic disease of pigs. Domestic pigs are more susceptible to ASF virus than the warthog pig. The difference in susceptibility to virus can be attributed to the difference in amino acid RELA as the potential cause for the difference in response between two species (Lillico et al., 2016). The genome editing techniques using molecular scissor can snip pig genome at RELA which confers resistance against ASF. According to previous research, CD163 is considered as the molecular marker for ASF virus (SanchezTorres et al., 2003). A pig possessing a complete KO of CD163 via CRISPR/ Cas9 and inoculated with Georgia 2007/I demonstrated that CD163 was not necessary for infection with Georgia 2007/I isolate.

3.6.3.1.2 Porcine reproductive and respiratory syndrome Porcine reproductive and respiratory syndrome (PRRS) is a pressing animal husbandry concern as vaccines are not useful in controlling the spread of this disease and it continues to bring heavy economic losses. Two types of strains phenotypically similar but antigenically and genotypically different: European (type 1 exemplified by the prototypical Lelystad virus) and Asian (type 2:VR2332) have been found to infect pigs. The predilection site for virus are pulmonary and the surface receptor CD163, found in macrophages especially of the respiratory system, has been proved to be indispensably required for the invasion of the virus into the alveolar macrophages cells (Reiner, 2016). CD163 is a member of the scavenger receptor cysteine-rich (SRCR) super family and the SRCR domain-5 (SRCR-5) is the region of CD163 which is involved in interaction with the PRRS virus. Acknowledging the crucial role of SRCR-5 in facilitating the invasion of virus into cells (Whitworth et al., 2015), the development of CD163 mutant

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(KO, deletion of SRCR-5, or replacement) cell lines has been seen as a possible research platform to introduce PRRS resistant pigs. CRISPR/Cas9 technology was used by Whitworth et al. (2015) to test the role of CD163 in infection. Forty-five live piglets were born with insertions ranging from 1 bp to 2 kb, deletions from 11 bp to 1.7 kb, as well as a partial domain swap in CD163. It was then demonstrated that these animals were resistant to the PRRSV isolate NVSL 97-7895 and remained healthy for the 35 days after infection. Using CRISPR/Cas9, the exon 5 of CD 163 encoding SRCR-5 region was deleted in pig zygotes following which no adverse effects were demonstrated in the growing pigs (Burkard et al., 2017). Further on challenge of SRCR-5 deleted mutants with PRRS virus, no replication structures were visualized in confocal microscopy revealing that inhibition of infection occurs prior to gene expression and corroborates the presence of checkpoint at the invasion process of the virus (Burkard et al., 2017). The finding that CD163 is required for infection with one PRRSV isolate paves the way for vaccine development and genetic selection programs. Thus, the adoption of genome-edited animals in agriculture promises substantial reduction in PRRSrelated economic losses.

3.6.3.1.3 Tuberculosis Tuberculosis (TB) is one hugely dreaded disease from amongst the list of many chronically debilitating afflictions which is caused by highly resistant bacterium, Mycobacterium tuberculosis in humans and Mycobacterium bovis in cows. To revive the dwindling animal sector from the damaging effect of this disease, the research focus has shifted from novel therapeutics for disease control to the development of TB-resistant cattle. Using CRISPR/Cas9 genome editing method, a TB-resistant gene namely, NRAMP-1, was inserted into bovine fetal cells which were later used as donor cells for the creation of TB- resistant cattle embryos by utilizing the technique of somatic cell nuclear transfer (SCNT) (Gao et al., 2017). A site-specific knock in of TALE nickase-mediated SP110 nuclear body protein gene through homologous recombination to develop TB-resistant cattle was reported by Wu et al. (2015). Thus, TB-resistant cattle developed through genome editing technology could be the hope for future healthy cattle stock for economic livestock farming.

3.6.3.1.4 Pseudorabies Pigs are the natural reservoirs of the pseudorabies virus (PRV), which results in reproductive problems like abortion, still births, mummified fetus, and brings huge economic loss to the pig industry. Xu et al. (2015) manipulated PRV genome using the CRISPR/Cas9 system wherein, PRV UL30 protein responsible for viral DNA replication was targeted and was transfected in PK15 cells, to inhibit viral infection. However, the gene was disrupted by CRISPR/Cas9 endonuclease, the PRV escaped inhibition in a passage-dependent manner to generate variants.

3.6 Applications of CRISPR/Cas9 genome editing technology

3.6.3.2 Cell therapeutics—next generation of cure 3.6.3.2.1 Cancer Genomic manipulations to control tumor and cancer growth in animals through adoptive T-cell transfer, car T cells, synthetic lethal interactions, and antichaperon therapy present an ideal candidate for treatment of animal malignancy. CRISPR/ Cas9 genome editing technology has been employed to turn immune cells against tumors, thus presenting an array of opportunity in cancer therapy. 3.6.3.2.1.1 Adoptive T-cell Transfer. Adoptive cell therapy (ACT) technology involves the collection of tumor-reactive T cells, culturing, and expanding those cells in vitro, and transferring cells back into a patient with cancer (Lorentzen and Straten, 2015). In a study conducted by Rupp et al. (2017), it was found that the expression of programmed death ligand 1 on tumor cells can make human CAR T cells (anti-CD19) hypofunctional, resulting in impaired tumor clearance in a subcutaneous xenograft model. Su et al. (2016) showed that the gene KO of programmed cell death protein I (PD-1) resulted in significant reduction of PD-1 expression but did not affect the viability of primary human T cells. Their results established an approach for efficient checkpoint inhibitor disruption, providing a new strategy for targeting checkpoint inhibitors to improve the efficacy of T-cellbased adoptive therapies based on CRISPR/Cas9 genome editing technology. In order, to overcome this suppressed antitumor response, a protocol for combined Cas9 ribonucleoprotein (Cas9 RNP)-mediated gene editing and lentiviral transduction to generate PD-1 deficient anti-CD19 CAR T cells was developed. PD-1 KO augmented CAR T cell
mediated killing of tumor cells in vitro and enhanced clearance of PDL1 1 tumor xenografts in vivo. Hence, generation of PD-1 KO T cells through CRISPR/Cas9 system can be used as an effective cancer immunotherapy in animals in a similar principle as done in humans. 3.6.3.2.1.2 Harnessing CAR T cells. Chimeric antigen receptors (CARs) are synthetic receptors that help in mediating tumor rejection by redirecting and reprogramming T cells (Jensen and Riddell, 2015). ACT based on the administration of genetically-engineered cytotoxic T cells to express a CAR recognizing CD19, which is expressed by B-cell malignancies has been shown to induce complete lasting responses in chronic lymphocytic leukemia and acute lymphoblastic leukemia (ALL). Eyquem et al. (2017) knocked out the T-cell receptor α constant (TRAC) locus and knocked in the CD19-specific 1928z CAR (Brentjens et al., 2013) under its transcriptional control (TRAC-CAR), CRISPR/Cas9 genome editing technology. Directing a CD19-specific CAR to the TRAC locus results not only in homogenous and consistent expression of TRAC-CAR in human peripheral blood T cells, but it also enhances T-cell potency, via edited cells vastly outperforming conventionally generated CAR T cells in a mouse model of ALL. It was later demonstrated that targeting the CAR to the TRAC locus prevents tonic CAR signaling and establishes effective internalization and re-expression of the CAR following single or repeated exposure to antigen (Eyquem et al., 2017). These findings represent the enormous potential of CRISPR/Cas9 as a genome editing tool to advance Car T-cell therapies.

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3.6.3.2.1.3 Studying synthetic lethal interactions. Simultaneous mutation of two genes can produce a phenotype that is unexpected in light of each mutation’s individual effect (Mani et al., 2008). This kind of genetic interaction have implications for therapeutic development in cancers, in which negative or “syntheticlethal” interactions via simultaneous disruption of both genes cause cell death (Lord et al., 2015). To enable systematic mapping of genetic interaction networks, Shen et al. (2017) developed a CRISPR/Cas9 screening methodology for targeting single genes and pairs of genes in a high-throughput format. Multiplex targeting was combined with array-based oligonucleotide synthesis (Wong et al., 2016; Bassik et al., 2013; Horn et al., 2011; Laufer et al., 2013) to create dual-gRNA libraries covering up to 105 defined gene pairs. In these libraries, each construct bears two gRNAs, each of which is designed to target either a gene or a scrambled nontargeting sequence absent from the genome. All combinations of gene
gene and gene
scramble are exhaustively assayed for effects on cell growth. The experiments were performed on different cell lines and the differences in genetic interaction across cell lines, were identified by systematic CRISPR screens. The results were reproduced as drug
drug interactions in small-scale assays. This combinatorial CRISPR/Cas9 technology for identifying synthetic lethal mutations of oncogenic targets may pave the way for systematic determination of cancer pathways, with manifold applications in the study of gene network influencing tumorigenesis and aiding in the development of novel therapeutics. 3.6.3.2.1.4 Antichaperon therapy. Chaperones, like heat shock protein 70, have been linked to the development of resistance against cancer drug and new therapeutics needed to be developed to resensitize the cancer cells to existing chemotherapy compounds. CRISPR/Cas9 can be used to inactivate a set of genes completely or to regulate their expression, thus it becomes feasible to identify the molecular mechanisms that control a particular pathway, relating to cancer and this forms the basis of antichaperon therapy. CRISPR/Cas9 technology would allow researchers to locate essential genes that cancer cells need to survive, identify drug targets, and rapidly validate their effectiveness. 3.6.3.2.1.5 Dysregulation of Notch signaling. Glycosylation is the covalent linking of a sugar to lipids or proteins which is added post or cotranslationally (Haltom and Jafar-Nejad, 2015). Protein glycosylation is either N-linked or Olinked. O-linked modifications involve sugar N-acetylgalactosamine attached with amino acids serine or threonine, called “mucin-type” glycosylation as they are most commonly found in proteins of mucus membranes; together with N-linked sugars. “Notch” is one such receptor protein, important for cell development and differentiation. Its signaling is deregulated in cancers such as leukemia, breast cancer, and prostate cancer. The enzymes, POFUT1 and POGLUT1 are responsible for linking Notch with glucose and fucose, allowing Notch to be transported to its final destination in the cell membrane. Using CRISPR/Cas9 technology when POFUT1 or POGLUT1 were knocked out, cells displayed much less or no Notch on the cell surface (Takeuchi et al., 2017). Thus, CRISPR/Cas genome

3.6 Applications of CRISPR/Cas9 genome editing technology

editing technology can be used for manipulation of sugars linked to Notch, which could help correct the deregulation and thus help in the fight against cancer.

3.6.3.2.2 Diabetes Obesity and diabetes have become a major health concern issue worldwide (Ahima, 2011; Ashcroft and Rorsman, 2012). The hormone glucagon-like peptide 1 (GLP-1) is a major physiological incretin that controls homeostasis of blood glucose by stimulation of glucose-dependent insulin secretion, inhibition of glucagon secretion, delay of gastric emptying, and protection of islet beta cell mass (Sandoval and D’Alessio, 2015). Native GLP-1 must be delivered through a parental route to achieve its effect and has an extremely short circulating half-life. Yue et al. (2017) developed skin grafts from mouse and human epidermal stem cells, which were engineered by genome editing tool CRISPR/Cas9 to controllably release GLP-1. The expression of inducible GLP-1 from engineered cells grafted onto immunocompetent hosts increased insulin secretion and reversed high-fat-diet-induced weight gain along with insulin resistance. Thus, precise genome editing technology has provided an ideal tool for somatic gene therapy (Wright et al., 2016).

3.6.4 Diagnostics development The diagnostic possibilities inherent in CRISPR/Cas system have a wide horizon. A novel tool, specific high-sensitivity enzymatic reporter unLOCKing (SHERLOCK), which has the potential to simplify the research landscape and revolutionize the global veterinary and public health by detecting cancer, monitoring antibiotic resistance, and responding to viral and bacterial pandemics has been developed. SHERLOCK can provide rapid DNA or RNA detection with attomolar sensitivity, single-base mismatch specificity and faster diagnostics with no requirements of refrigeration. SHERLOCK tests help to distinguish between strains of Zika and Dengue fever and also for determining the difference between mutations in cell-free tumor DNA and could be useful in diagnosing clinical diseases of animals. SHERLOCK greatly differs from CRISPR, and uses a different enzyme called Cas13a (originally dubbed C2c2 by the researchers who discovered it). It combines the collateral effect of Cas13a with isothermal amplification, providing rapid nucleic acid detection which can be easily reconstituted on paper (Gootenberg et al., 2017) and is a cost-effective, rapid, bedside diagnostic test for infectious disease outbreaks in resource-poor areas. Since, it can be lyophilized hence, it is cold-chain independent and long-term storage is feasible at room temperature.

3.6.5 Vector control Among vectors, female mosquitoes (Anopheles gambiae) are the major contributor to the transmission of various diseases worldwide namely flavivirus infection.

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Gene drive/CRISPR-Cas9 constructs is used in altering certain genes (AGAP005958, AGAP011377, and AGAP007280) in A. gambiae which upon disruption confer a recessive female sterility with a progeny transmission rate of 91%
99.6% (Hammond et al., 2016). This acts as an effective deterrent to the increasing mosquito population worldwide. This technique was used for insertion of certain antimalarial genes into the mosquito population (Anopheles stephensi) and was found to be highly efficient in potentially hampering the parasite transmission ability (Gantz et al., 2015). In an attempt to reduce flavivirus infection, various host genes were identified and edited through CRISPR/Cas9 system.

3.6.6 Fighting antimicrobial resistance Bacteria having antibiotic-resistant gene can be selectively killed with a CRISPR/ Cas9 agent that specifically targets the chromosomally integrated resistance gene (Parmley, 2014). A gene cassette encoding a small tracrRNA, Cas9 and a single guide RNA (SgRNA) is delivered into the cell resulting in expression and assembly of the CRISPR/Cas9 agent. The targeted resistance gene is cleaved by the CRISPR/ Cas agent, leading to chromosomal degradation and bacterial cell death. CRISPR/ Cas9 was targeted against carbenicillin resistance gene of Escherichia coli and methicillin resistance gene in Staphylococcus aureus using bacteriophage delivery system (Citorik et al., 2014; Bikard et al., 2014). The results indicated that predominantly resistant organisms have been killed. In a mouse model of Gram-positive bacteria skin infection, a CRISPR/Cas9 bacteriophage targeting a kanamycin resistance gene decreased by fivefold the proportion of resistant bacteria in a mixed population of bacterial colonies on the skin. CRISPR/Cas systems can be effectively introduced into S. pneumoniae and be reprogrammed to prevent capsule switching, and thus CRISPR interference can prevent DNA transformation of pneumococci.

3.6.7 Producing disease models CRISPR/Cas9 genome editing technology can be used to create animal models which mimic diseases or study development by mutating or silencing genes. By changing the DNA at specific targeted level or anywhere in the genome, the model animals can be created at the germline level (Maggio and Goncalves, 2015), which can be utilized to gain an insight into the pathogenesis of disorder and to find treatments for them. Induced pluripotent stem (iPS) cells produced by CRISPR technique used as a “disease-in-a-dish” in vitro model for diseases like Duchenne and Becker muscular dystrophy, Parkinson’s disease, Huntington’s disease, and down syndrome/trisomy 21 (Park et al., 2008; Dimos et al., 2008; Ebert et al., 2009; Soldner et al., 2009) have been developed. CRISPR/Cas9 has been used to obtain the retrovirus-free tissue organs in pigs that could be suitable for xenotransplantation (Yang et al., 2015). Engineered nucleases were used in pigs to KO vWF gene (Hai et al., 2014). A large number of genes in a diversity of

3.7 Ethical issues

human cell types have already been modified using CRISPR/Cas9 system and present a window for modification animal cell types providing the capacity to study mechanistic effects of these mutations in causing animal diseases.

3.7 Ethical issues An important ethical issue in scientific research is that the benefits must be greater than the risks. The application of CRISPR/Cas9 technique involves risks since it may produce off-target mutations, which can be deleterious (Yang et al., 2013). CRISPR/Cas9 may also cleave unintended sequences causing mutations which may cause cell death or transformation (Fu et al., 2013). Efforts have been made to reduce off-target mutations, by using new variants of Cas9 like eSpCas9, Cas9HF-1, hypaCas9 but further improvement is required, especially precise modifications are needed for therapeutic interventions (Cong et al., 2013; Hsu et al., 2013). While the genome editing technology continues to offer important advancements in biomedical research, it has many challenges before it.

3.7.1 Ecosystem disequilibrium It is necessary to probe specificity in experiments using RNA-guided engineered gene drives based on CRISPR/Cas9 technique. The phenomenon of biased inheritance of a genetic trait in a population in a non-Mendelian fashion is called gene drive (Collins, 2018). There exists possibility of mutations off-target which can amplify in each generation. There is a risk of transferring genes and modified sequences to other species. The negative trait may be transmitted to related organisms far and wide and the dispersion of the gene drive trait may be difficult to control (Esvelt et al., 2014). There are risks of accidental release of genetically modified (GM) experimental organisms in the environment (Oye et al., 2014) which may cause extinction of the whole population as a result of targeted gene drive. and hence create drastic consequences to the ecosystem equilibrium.

3.7.2 Regulatory hurdles The efficacy of CRISPR/Cas9 technique to obtain precise genetic modifications makes it more difficult to identify a GM organism once outside the lab. The major concerns include: health effects such as an allergic response to GM products, environmental effects due to uncontrolled release of transgenes (i.e., GM animals), reduced diversity of natural genomes, and sociocultural concerns such as “playing God” (Hackett et al., 2014). The CRISPR zoo is expanding fast, which could be unacceptable to the public. Another issue outstanding is the regulation of patents and economic interests involved. On one hand, patenting gives too much power and profit to biotechnological companies, but on the other hand patenting also helps to regulate the field. The practice of patenting may instigate litigations and frictions among biotechnological companies and researchers.

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3.7.3 Genetic enhancement The efficiency of CRISPR/Cas9 technique increases the possibility for genetically modifying somatic cells. The development of human/animal chimeras for organ transplantation may provide hope in the case of unavailability of a human organ donor. These chimeras may carry human neural and germ cells (Polcz and Lewis, 2016) which have raised many ethical concerns. The International Summit on Human Gene Editing on December 2015, discussed the ethics of germline modification, in which they agreed to proceed with basic and clinical research under appropriate legal and ethical guidelines and generating inheritable changes in humans via altering of gametocytes and embryos was deemed unethical. In February 2016, British scientists were given permission to genetically modify human embryos by using CRISPR/Cas9 and related techniques only for research purpose (Gallagher, 2015).

3.8 Future prospects 3.8.1 Deextinction Deextinction, also known by resurrection biology and species revivalism, aims to recreate or cause the rebirth of the once extinct species. Using CRISPR/Cas9 genome editing system, revival of woolly mammoths (Mammuthus primigenius) and extinct passenger pigeon (Ectopistes migratorius) is a near possibility. The approach involves cloning of mammoths and passenger pigeon gene obtained from preserved samples, followed by SCNT into a genetically similar species (Indian elephants and modern day pigeon) (Reardon, 2016). Another approach involves selective breeding, using CRISPR technology to alter/edit the genome of the genetically similar species by the introduction of various genes that could be helpful in their revival (Lynch et al., 2015).

3.8.2 Customization of pets Improvement of the pets also referred to as customization is being looked upon as an ambitious objective as the editing of the natural genome has become technologically feasible and it has intensified the significance of pet improvement. CRISPR/Cas technology can be programmed to enhance traits in dogs such as behavior and agility, for skill enhancement in working/hunting dogs and to clone deceased pets.

3.8.3 Drug discovery CRISPR/Cas9 has a potential to revolutionize gene and cell replacement therapies. It can be utilized for identification of novel drug targets through functional

3.8 Future prospects

genomics screens and has simplified the production of disease models. The CRISPR-based genetic screens can be used for identifying mutations that confer drug resistance (Shalem et al., 2014). The in vivo treatments in diseases of the liver, eye, lung, and others involve administering CRISPR/Cas9 in cells inside the human body and ex vivo treatments in cases of hemoglobinopathies, such as sickle cell disease, beta thalassemia, immunodeficiencies, and specific approaches to personalized immune therapies for cancer. In intestinal organoids of cystic fibrosis patients, CRISPR/Cas9 technique is used for the functional repair of CFTR gene (Schwank et al., 2013). Another new version of CRISPR, the RNA editing for programmable A
I (G) replacement (REPAIR) has been developed which can target and edit RNA. The REPAIR system utilizes Cas13b, a variant of Cas13. It treats disease by modifying the function of proteins involved in diseaserelated signal transduction and is useful for treating diseases caused by temporary changes in cell state, such as local inflammation Cox et al. (2017). As a possible tool for slowing the degeneration of neurons, RNA editing could someday prove useful in treating diseases as Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis.

3.8.4 Future farming The mutagenic chain reaction, a method based on CRISPR/Cas9 technique generates autocatalytic mutation to produce recessive loss of function mutations, spreading efficiently from their chromosome of origin to homologous chromosome in the vast majority of somatic and germline cells (Gantz and Bier, 2015). At the population level, this chain reaction can drive an allele through a population at super-Mendelian rates of inheritance. Thus, gene drive presents an effective method of genetically engineering an entire population and increasing genetic gain in livestock breeding programs which is brought about by increasing the frequency of favorable alleles (Gould, 2008; Burt and Trivers, 2006). Targeted gene drive mechanisms based on CRISPR/Cas9 editors have been reported to have conversion efficacies of more than 98% (Gantz et al., 2015), demonstrating the potential of this technology in spreading alleles in populations. For quantitative traits, gene drive could be combined with genome editing methods to fix edited alleles more quickly in livestock populations. Each edited allele can have a gene drive based on a CRISPR/Cas9 editor. Furthermore, gene drives increase the efficiency to convert genetic variation into genetic gain. The gene drive can also be coinherited with the edited allele across generations. This ensures complete homozygosity for the favorable allele amongst all descendants of an edited individual, regardless of the genotype of the other parent (Gonen et al., 2017). This means, for a given level of inbreeding, breeders could achieve more genetic gain with gene drives than with genome editing or genome selection alone. Simulation was used to assess the use of genome editing
based gene drives in increasing the genetic gain for quantitative traits in livestock breeding. A diversity of scenarios were tested, each having different editing strategies within the breeding program.

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Among other uses, gene drives can also be used to control damaging invasive species, reverse pesticide, and herbicide resistance in insects or prevent the spread of disease (Esvelt et al., 2014). Using this possibility, researchers were able to generate mosquitoes possessing genes which prevented them from harboring malaria parasites and also rendered female mosquitoes infertile (Gantz et al., 2015). Other interesting areas where CRISPR/Cas9 genome editing technology has potential to change the world of animal agriculture are as follows: 1. For modification of fecal/dung/soil microbes, leading to better efficiency in waste degradation and management; 2. Modifying fungi to efficiently convert straw, stovers, fibrous nonconventional feed, and farm waste into digestible animal feed; 3. Making wildlife animal species resistant to diseases; 4. Modifying microbes to produce fuel which can serve as an alternative to fossil fuel; 5. Single-cell protein production as feed supplement; 6. Engineering microbes for efficient degradation of leather, biowastes, crop residue, and plastics.

3.9 Conclusion The ability to precisely modify animal genomes was limited until the emergence of efficient genome editing technology utilizing engineered nucleases. The technique of genome editing has revolutionized the foundation of functional studies in modern biology and has also led to significant discoveries and has found its way to wide applications in animal agriculture. It can be used to introduce targeted sequences in the germline of any organism and modify somatic genes. Proteinguided ZFNs and TALENs were no doubt cutting-edge technology but during the past three decades RNA-guided CRISPR/Cas9 has evolved from “curious sequences of unknown biological function” into a promising genome editing tool which is employed globally to obtain precision genome editing with greater efficiencies in animal systems. Targeted modifications in animals are done to fulfill varied objectives: to obtain more muscling growth to sustainably feed an increasing global population, to gain food security, development of cell therapeutics, combat antimicrobial resistance, to develop functional genomics screens, vector control, transcriptional modulation, epigenetic control, for live imaging of the cellular genome, and to produce appropriate disease model animals for research and therapeutics. Owing to its facile engineering, reproducibility and affordability, CRISPR/Cas9 has become the system of choice. Genome editing offers a powerful method for accelerating the genetic improvement of livestock, for societal benefits, for exploitation of animal resources in the most economic and logical way possible. CRISPR/Cas9 field requires better, more sensitive off-target detection methods, especially as it moves toward clinical applications of genome

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892. Wagner, H., 2001. Toll meets bacterial CpG-DNA. Immunity 14 (5), 499
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Wiedenheft, B., Lander, G.C., Zhou, K., Jore, M.M., Brouns, S.J., van der Oost, J., et al., 2011. Structures of the RNA-guided surveillance complex from a bacterial immune system. Nature 477 (7365), 486. Wong, A.S., Choi, G.C., Cui, C.H., Pregernig, G., Milani, P., Adam, M., et al., 2016. Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM. Proc. Natl. Acad. Sci. U.S.A. 113 (9), 2544
2549. Wright, A.V., Nun˜ez, J.K., Doudna, J.A., 2016. Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell 164 (1
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1104. Yin, H., Xue, W., Chen, S., Bogorad, R.L., Benedetti, E., Grompe, M., et al., 2014. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32 (6), 551. Yu, C., Liu, Y., Ma, T., Liu, K., Xu, S., Zhang, Y., et al., 2015. Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell 16 (2), 142
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263. Zetsche, B., Gootenberg, J.S., Abudayyeh, O.O., Slaymaker, I.M., Makarova, K.S., Essletzbichler, P., et al., 2015. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163 (3), 759
771. Zhu, L.J., 2015. Overview of guide RNA design tools for CRISPR-Cas9 genome editing technology. Front. Biol. 10 (4), 289
296.

Further reading Zhang, R., Miner, J.J., Gorman, M.J., Rausch, K., Ramage, H., White, J.P., et al., 2016. A CRISPR screen defines a signal peptide processing pathway required by flaviviruses. Nature 535 (7610), 164. Zhang, M.M., Wong, F.T., Wang, Y., Luo, S., Lim, Y.H., Heng, E., et al., 2017. CRISPR
Cas9 strategy for activation of silent Streptomyces biosynthetic gene clusters. Nat. Chem. Biol. 13 (6), 607.

CHAPTER

Genetic markers for improving farm animals

4

Rajib Deb1, Chandra Sekhar Mukhopadhyay2, Gyanendra Singh Sengar1, Alex Silva da Cruz3, Danilo Conrado Silva4, Irene Plaza Pinto3, Lysa Bernardes Minasi3, Emı´lia Oliveira Alves Costa3 and Aparecido D. da Cruz3,5 1

ICAR-Central Institute for Research on Cattle, Meerut, Uttar Pradesh, India College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India 3 Replicon Research Center, Masters in Genetics, School of Agrarian and Biological Sciences, Pontifical Catholic University of Goia´s, Goiaˆnia, Brazil 4 Graduate Program in Animal Sciences, School of Veterinary Medicine and Animal Science, Federal University of Goia´s, Goiaˆnia, Brazil 5 Postgraduate Program in Biotechnology and Biodiversity, Federal University of Goia´s, Goiaˆnia, Brazil

2

4.1 Introduction The basic goal of a genetic evaluation program is to identify genetically superior animals so that germplasms from selected animals could be employed to improve the genetic gain of future generations. Thus, the genetic improving of farm animals will ultimately help to promote production yield and efficiency of individual animals and subsequently from the entire herd, augmenting producer’s profitability. Cumulative genetic perfection through selection as well as grading-up programs will likely lead to a rise in the performance of the farm animals. Molecular tools associated with conventional breeding techniques are of utmost importance to achieve better goals in animal breeding programs (Olsen et al., 1999). Advanced molecular development led to the use of genetic markers for the holistically improvement of production traits, taking into consideration most of the factors that may influence breeding strategies (Bayer and Feldmann, 2003; Mirkena et al., 2010). Although conventional selective breeding in terms of progeny testing as well as assisted reproductive tools like artificial insemination, multiple ovulation, and embryo transfer have been proven to be attractive tools for dramatically improve farm animal productivity (Lindhe and Philipsson,1998; Sartori et al., 2006; Singh et al., 2006), they require a long time period before results are reached and do not efficiently take into account all sources of genetic discrepancy. Again, traditional Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00004-7 © 2020 Elsevier Inc. All rights reserved.

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breeding, although highly efficient, is generally restricted to sex-limited, low heritable or late expressed traits (Meuwissen and Goddard, 1996). Thus, the application of genetic markers may contribute to solve constraints associated with traditional breeding strategies for better selection of genetically superior farm animals. Genetic markers are any visible or assayable phenotype or any DNA sequences located on chromosomes associated with individual or species-specific variation that can be used to identify traits of interest. Thus, genetic markers are useful to assess the genetic basis of observed phenotypic variability (Teneva and Petrovic, 2010). Consequently, genetic markers could be used to identify a partial DNA sequence in unknown pools of genetic material (Chauhan and Rajiv, 2010). In general, the term “genetic marker” refers to variations in a specific DNA sequence among individuals, which have been found to be linked with certain phenotypic traits, including insertions, deletions, translocations, duplications as well as point mutations. A particularly useful marker must have certain biological properties that allow it to be detected and measured in animal tissues or biological fluids (Haley and de Koning, 2006). Different genetic markers have been documented from 1980 onwards, playing an important role in the assessment of genetic diversity in farm animals. The detection of molecular markers in general is based on two approaches, namely hybridization-based and polymerase chain reaction (PCR)-based (Agarwal et al., 2008). A variety of genetic markers including restriction fragment length polymorphism (RFLP), mini- or microsatellites as well as single nucleotide polymorphism (SNP) have been used extensively in animal sciences and husbandry (Kiplagat et al., 2012). In order to develop DNA linkage map, RFLP is known to generate the first tier genetic markers. Different families of hypervariable repetitive DNA sequences, mostly microsatellite and minisatellites, can also be applied in order to disclose different DNA fingerprinting patterns. Due to being userfriendly, applications based on PCR and electrophoresis are the most popular markers for livestock genetic characterization, mostly using microsatellites (Sunnucks, 2001). Again, due to their high mutation rate within and between breed, microsatellite markers are used to measure the genetic diversity as well as genetic admixture among breeds. PCR-based markers are further classified into (1) sequence-targeted PCR assays such as cleaved amplified polymorphic sequence, allele-specific PCR, PCR amplification of specific alleles, simple sequence length polymorphism, and sequence-targeted microsatellite site and (2) arbitrary PCR assays that include random amplified polymorphic DNA (RAPD), RAPD-RFLP, DNA amplification fingerprinting as well as microsatellite-primed PCR. During the last decade, SNP has gained high popularity as they are the most abundant of all marker systems known so far, both in animal and plants (Mitra et al., 1999; Naqvi, 2007). Identifying adequate and efficient genetic markers directly or indirectly associated with different animal phenotypic traits remains a major challenge. This chapter deals with different genetic markers available to identified and differentiate phenotypic traits in major farm animals.

4.2 Genetic markers related to farm animal productivity

4.2 Genetic markers related to farm animal productivity Although phenotypic characterization is one of the most important conventional tools for judging farm animals with high genetic merit, the polymorphic information carried by these markers are meager and restricted to the coding region of the chromosome and are also sex- and/or age-dependent. Currently, genetic markers have been recognized on a massive variety of genes related to traits of economic importance and have been widely accepted. Different candidate genes have been identified in livestock species and some are included in Tables 4.1
4.3.

4.2.1 Genetic markers in large ruminants 4.2.1.1 Markers for dairy production traits Few genetic conditions are measured by a single gene (monogenic or qualitative traits), although many others are controlled by many genes (polygenic or quantitative traits). Healy (1996) estimated that about 87% of qualitative traits in cattle are recessively inherited. It is not astonishing to assume genetic makeup is breed specific, assuming cattle breeds were established in a relative genetic isolation as well as independently of each other (Healy, 1996). Until the beginning of modern molecular biology, the technology was unavailable to identify genes associated with quantitative traits and the variants within a gene that affected differentially its expression with a major positive or negative impact on animal productivity (Casas and Kehrli, 2016). Genome-wide association studies (GWAS) are conceivable due to the accessibility of technology that permits high-through output genotyping of SNPs allowing for decoding the genetics behind the expression of economically important traits (Casas and Kehrli, 2016). Mining animal databases has revealed around 344 quantitative trait loci (QTLs) associated with milk production and 71 with mastitis-related traits in cattle (Ogorevc et al., 2009). Association between DNA polymorphism and milk production has been studied for a number of genes. Farrell et al. (2004) identified milk proteins in cattle having variability at the DNA level, affecting the chemical structure of those proteins and, consequently, their function. However, allelespecific single versus multilocus traits are dependent on the genetic background as well as environmental interactions in experimental models. Some of the milk proteins like beta casein, kappa casein, leptin (LEP), prolactin (PRL), and DGAT1 are proven to be associated with milk production in dairy cattle (Farrell et al., 2004; Ageitos et al., 2006; Navani et al., 2008; Singh et al., 2013). Among different genetic markers, LEP (Singh et al., 2012), PRL (He et al., 2006), ABCG2 (Cohen-Zinder et al., 2005; Ron et al., 2006; Olsen et al., 2007), and DGAT1 (Grisart et al., 2002; Thaller et al., 2003; Grisart et al., 2004; Kaupe et al., 2007; Anton et al., 2008; Banos et al., 2008; Wu et al., 2012) genes are reported to be associated with milk traits among different breeds of cattle. LEP is known to be a powerful biomolecule for augmenting productivity in livestock.

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Table 4.1 Candidate genes influences economic traits in cattle and buffaloes. Genes

Species

Traits

References

Beta casein Kappa casein

Cattle Cattle

Milk Milk

Leptin

Cattle Buffaloes

Milk

Prolactin DGAT1

Cattle Cattle

Milk Milk Meat Reproductive

Growth hormone receptor

Cattle

Milk

ABCG2

Cattle

Milk

Melanocortin-4 receptor Beta-lactoglobulin SCD1 BoLA-DRB3

Buffaloes Cattle Cattle Cattle

Milk Milk Meat Milk

CSN1S1 Protease inhibitor gene Osteopontin PPARGC1A Growth hormone

Cattle Cattle Cattle Cattle Cattle

Milk Milk Milk Milk Milk

STAT5A

Cattle

STAT1

Cattle

Milk Reproductive Milk

OLR1 CYP11B1 Fatty acid synthase

Cattle Cattle Cattle

Milk Reproductive Milk

Ganguly et al. (2012a) Ageitos et al. (2006) Robitaille et al. (2007) Liefers et al. (2005) Mihaiu et al. (2011) Singh et al. (2012) Singh et al. (2013) He et al. (2006) Grisart et al. (2002) Thaller et al.(2003) Grisart et al. (2004) Kaupe et al. (2007) Anton et al. (2008) Banos et al. (2008) Wu et al. (2012) Blott et al. (2003) Hradecka et al. (2008) Cohen-Zinder et al. (2005) Ron et al. (2006) Olsen et al. (2007) Deng et al. (2016a) Ganai et al. (2009) Wu et al. (2012) Sharif et al. (1999) do Nascimento et al. (2006) Kuss et al. (2005) Khatib et al. (2005) Leonard et al. (2005) Weikard et al. (2005) Zhou et al. (2005) Hradecka et al. (2008) Brym et al. (2004) Khatib et al. (2008) Cobanoglu et al. (2006) Khatib et al. (2006) Kaupe et al. (2007) Morris et al. (2007) (Continued)

4.2 Genetic markers related to farm animal productivity

Table 4.1 Candidate genes influences economic traits in cattle and buffaloes. Continued Genes

Species

Traits

References

CARD15 TG POU1F1 Insulin-induced gene 2 Stearoyl CoA desaturase Protamine 1 and 2 CCL2, IL8, CCR2, and IL8RA

Cattle Cattle Cattle Buffaloes Cattle Cattle Cattle

Milk Milk Milk Milk Milk Reproductive Milk

AP2 box region of Hsp70. 1 Mannan-binding lectin 2 OPN

Cattle Cattle Buffaloes

Milk Milk Reproductive

HSP90AB1 FSH receptor gene LHCGR Inhibin alpha Progesterone receptor Growth differentiation factor 9 Y-specific microsatellite loci Calpastatin, micro-calpain

Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle

Milk Reproductive Reproductive Reproductive Reproductive Reproductive Reproductive Meat

CAST Thyroglobulin PRLR Glutathione peroxidase and selenoprotein P

Cattle Cattle Cattle Cattle

Meat Meat Meat Draught power

Pant et al. (2007) Anton et al. (2008) Huang et al. (2008) Deng et al. (2016b) Macciotta et al. (2008) Ganguly et al. (2012b) Leyva-Baca et al. (2007) Deb et al. (2013a) Zhao et al. (2012) Lin et al. (2006) Rolim Filho et al. (2013) Sajjanar et al. (2014) Yang et al. (2010) Yang et al. (2012) Tang et al. (2011) Yang et al. (2011) Tang et al. (2013) Deb et al. (2013b) Schenkel et al. (2006) Casas et al. (2006) Li et al. (2013) Hou et al. (2011) Lu et al. (2011) Meplan et al. (2013)

Due to its role in lactogenesis, galactopoiesis, colostrum secretion, and immunity to mastitis, LEP has become a significant candidate gene for genetic correlation with dairy production (Singh et al., 2012). Various SNPs have been reported on the promoter as well as in exonic regions of the LEP gene and observed to be highly correlated with milk production (Liefers et al., 2005). The PRL gene is mapped to BTA23 at 44.7 cM close to the QTLs for milk yield in bovine. PRL is an important candidate affecting milk production and is one of the genetic markers associated with milk yield in dairy cattle. Based on SNP analyses, it has been hypothesized that CHBP2 and diplotype H2H8 of PRL are useful genetic markers to select heifers for milk production in a dairy program at least in Holstein dairy cattle (Banos et al., 2008); diacylglycerol acyltransferase

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Table 4.2 Candidate genes influencing economic traits in goat and sheep. Genes

Species

Traits

References

Growth hormone Growth hormone receptor Insulin-like growth factor I Leptin Caprine pituitary-specific transcription factor-1 Caprine MSTN Ovine MSTN Bone morphogenetic protein FOXL2

Goat Goat Goat Goat Goat

Meat Meat Meat Meat Meat

Yu et al. (2004) Maj et al. (2007) Lan et al. (2007a) Whitley et al. (2005) Lan et al. (2007b)

Goat Sheep Goat Goat

Meat Meat Meat Reproduction

SRY Amelogenin

Goat Goat Sheep

Reproduction Reproduction

BMP15

Goat Sheep

Reproduction

Casein family genes

Goat

Milk

Melatonin receptor 1A

Sheep

Reproduction Prolificacy

Sheep Sheep Sheep Sheep Sheep Goat Sheep Sheep

Wool Wool Wool Wool Wool Fiber Wool Wool

Li et al. (2006) Clop et al. (2006) Chu et al. (2007a,b) Uhlenhaut and Treier (2006) Shi et al. (2008) Chen et al. (2007) Pfeiffer and Brenig (2005) Malan (2000) Juengel et al. (2003) Chu et al. (2007a,b) Ramunno et al. (2001) Ramunno et al. (2004) Hayes et al. (2006) Othman and Ahmed (2007) Messer et al. (1997) Pelletier et al. (2000) Notter et al. (2003) Chu et al. (2003) Chu et al. (2006) Chu et al. (2007a,b) Chen et al. (2011) Chen et al. (2011) Zhou et al. (2015) Ling et al. (2014) Li et al. (2017) Zhao et al. (2008) Rong et al. (2015) Ma et al. (2017)

Keratin-associated protein Keratin-associated protein Keratin-associated protein Keratin-associated protein Desmoglein 4 Keratin-associated protein Methionine synthase Follistatin MSTN, Myostatin.

8.1 1.3 6.1 22.1

4.2 Genetic markers related to farm animal productivity

Table 4.3 Candidate genes influences economic traits in pig, equine, and poultry. Genes

Species

Traits

References

Porcine CAST

Pig

Meat

Ankyrin repeat domain 1 Fatty acid binding protein 4 HSPs Myogenesis-related genes Cysteine-rich secretory protein-3

Pig Pig Pig Pig Equine

Meat Meat Meat Meat Reproductive

Metalloproteinases 9 Microsatellite loci

Chicken Ostriches

Egg Egg

Malek et al. (2001) Rothschild et al. (2004) Damon et al. (2013) te Pas et al. (2013) Lebret et al. (2013) Cagnazzo et al. (2006) Giesecke et al. (2010) Doty et al. (2011) Novak et al. (2010a,b) Jobim et al. (2011) Zhu and Jiang (2014) Kawka et al. (2012)

(DGAT1) gene—another important candidate gene for milk yield—is involved in triglyceride synthesis. Several, research groups have claimed DGAT1 polymorphisms are associated with milk production traits (Grisart et al., 2002; Thaller et al., 2003; Grisart et al., 2004; Kaupe et al., 2007; Anton et al., 2008; Banos et al., 2008; Wu et al., 2012). Moreover, DGAT1 is now recognized as an important candidate marker particularly for fat content in milk. Moreover, ABCG2 gene was found to have a significant effect on milk fat and protein contents (CohenZinder et al., 2005; Ron et al., 2006; Olsen et al., 2007). Further, certain microsatellite markers have been found to be associated with traits of milk quality in cattle (Olsen et al., 2004). Mihaiu et al. (2011) identified polymorphisms in the LEP hormone are associated with milk and protein yield in buffaloes. In this context, LEP TT genotype was associated with increased protein and milk yield compared to the LEP CC genotype. Deng et al. (2016a,b) reported that genetic variation at the melanocortin-4 receptor (MC4R) was associated with milk production traits and suggested MCR4 was a potential biomarker for water buffalo breeding. A total of 8 SNPs were identified by direct PCR sequencing of samples from 18 buffaloes, and SNPs were genotyped through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method in 332 buffaloes. The authors observed three SNPs, namely H1 (AGT), H2 (GAT), and H3 (GAC), accounted for 93% of the production for all tested animals. Further statistical analysis revealed the SNP g.1104C . T was significantly (P , .05) associated with milk yield and protein and fat percentage in water buffaloes. Deng et al. (2016a,b) further identified polymorphisms at insulin-induced gene 2 were associated with milk production traits, suggesting those polymorphisms could be used as candidate markers to assisted selection in Chinese buffaloes. Four SNPs viz

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g.621272A . G, g.621364A . C, g.632543G . A, and g.632684C . T were detected using DNA pooled sequencing, and it was observed that all the SNPs were significantly (P , .05) associated with 305-day milk yield or protein percentage in Murrah buffaloes. However, the same SNPs had no significant effect on milk production traits in Nili-Ravi buffaloes (P..05). Studies from Nadeem and Maryam (2016) provided an insight for the potential role of genetic variation in the PRL gene in milk-producing abilities of buffalo and also suggested new directions for the investigation of additional candidate genes that may have a promising role to enhance future milk production capabilities of river buffalo breeds in Asian regions through marker-assisted selection.

4.2.1.2 Genetic markers related to reproductive performance Although extremely heritable, traits related to reproductive performance have been effortlessly perceived and used as the base for genetic selection deprived of using genomic methods. However, more complex as well as less heritable traits such as fertility, are not responsive to traditional selection methods (Rothschild and Ruvinsky, 2011). Naturally, there are noteworthy costs and extended time associated with the collection of accurate phenotypical data (Diether and Dyck, 2017). To evaluate sire fertility and dairy fitness, the evaluation is carried on the basis of the recorded data of his daughters’ performance. Thus, the evaluation requires relatively large numbers of animals to be produced by inseminations. It is vital and essential that lots of the stud’s progeny is tested before the potential young male reaches sexual maturity to establish the breeding value of him and to attain a consistent relatively efficient phenotype (Diether and Dyck, 2017). Investigations of sperm fertilizing capability depend on different in vitro trials, due to the high contribution in terms of labor, time consuming, and costs associated with in vivo trials (Chabory et al., 2010). However, selection at the molecular levels through identifying genomic regions, and ultimately individual genes that contribute to heritable differences in sire fertility can help take an early choice in the life of an animal, which will considerably reduce the costs and times for selection program. Consequently, there has been substantial curiosity in genome mapping and identifying genes involved in the regulation of reproductive traits as well as in elucidating the effect of their polymorphic patterns on the animal’s fitness (Syva¨nen, 2005; Hirschhorn and Daly, 2005). SNP information has been used to identify recessive lethal mutations that cause spontaneous abortions and this mutation in cattle. Recessive mutations could be transmitted to the next generation resulting in lower reproductive ability, a situation especially disadvantageous in sires (Sonstegard et al., 2013). In bulls, SNPs associated with low conception rates have also been reported, many of which are situated nearby genes implicated in the acrosomal reaction, chromatin remodeling, and/or meiosis (Pen˜agaricano et al., 2012). Increased concentrations of osteopontin (OPN) are linked with improved acrosome reaction as well as decreased zygote cleavage and blastocyst formation in bovine embryos

4.2 Genetic markers related to farm animal productivity

(Monaco et al., 2009). Some bovine genes such as follicle stimulating hormone receptor (Yang et al., 2010), inhibin alpha (Tang et al., 2011), luteinizing hormone receptor, progesterone receptor (Yang et al., 2011), and growth differentiation factor 9 (Tang et al., 2013) are known to play a crucial role in cattle reproduction, affecting the total number of ova in superovulation as well as the number of transferable embryos in artificial insemination regimens. Henceforth, these genes can be used as potential genetic markers for superovulation response and embryo quality. Again, to obtain superior conception rates, male fertility is equally important. It is reported that male germplasm origin plays a significant role in early pregnancy wastage. Different genetic markers viz cation channel of sperm 1 (Catsper1), protamine 1 & 2, A kinase (PRKA) anchor protein 4 (Akap4), cytochrome oxidase (COX3), sperm-specific NHE (Slc9a10), pyruvate kinase (PKM2), reproductive homeobox 5 (Rhox5), cysteine-rich secretory protein 2 (CRISP2), phosphatidylethanolamine binding protein 1 (PEBP1), tissue inhibitor of metalloproteinase (TIMP2), enolase 1 (ENO1), ATP synthase H 1 transporting mitochondrial F1 complex beta subunit (ATP5B), apoptosisstimulating of p53 protein 2 (ASPP2); alpha-2-HSglycoprotein (AHSG), glutathione peroxidase 4 (GPX4), voltage-dependent anion channel 2 (VDAC2), ropporin-1 and ubiquinolcytochrome-c reductase complex core protein 2 (UQCRC2) have been identified, which are related to semen quality as well as fertility traits in bulls (Reviewed by Kumar et al., 2012). Rawat et al. (2016) identified a certain potential biomarker for pregnancy detection at earlier stages (16
25 days) in Karan Fries heifers using difference gel electrophoresis (DIGE) and label-free quantitation (LFQ). A total of 11 differentially expressed proteins (DEPs) were identified through DIGE, out of which 9 were upregulated having fold change $ 1.5 in all time points. The aforementioned LFQ analysis detected about 200 DEPs. Twenty-eight of these DEPs were upregulated and 40 were downregulated with significant fold change $ 1.5 and # 0.6, respectively. In silico analysis revealed the majority of proteins were involved in immunity and endopeptidase inhibitor, regulation of peptidase and polysaccharide binding activities. Certain DEPs viz insulin-like growth factor (IGF), MBP, and SERPIN were found to be actively involved in various cellular functions related to pregnancy viz embryo implantation and the establishment and maintenance of pregnancy. Hence, these proteins have the potential to act as biomarkers for pregnancy detection. SNPs in the introns of OPN have been identified to be associated with sperm cell motility as well as morphology in water buffalo (Lin et al., 2006; Rolim Filho et al., 2013). Genetic markers connected with fertility phenotype traits may offer information that can be used early in the selection of sires, earlier then the collection of ejaculate. Identification of genetic markers associated with subfertility could permit for the screening of sires for high genetic merit very early in their lives. Overall, improvements to the identification of subfertile sires provide an additional tool for assessing the reproductive quality in a herd.

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4.2.1.3 Genes associated with meat production Ruminant meat products are a key source of protein supply and food security for humans in many areas of the world. The core objective in beef cattle research is concurrently to control muscle development and the qualities of beef cuts. Quality of beef is a complex phenotype that is only measurable at postslaughter and highly variable (Cassar-Malek and Picard, 2016). In the last two decades, most studies about beef quality traits have focused on meat tenderness, and several studies in postmortem muscles have analyzed the evolution of protein profiles in order to identify biomarkers related to meat conversion (Chaze et al., 2013; Sawdy et al., 2004; Jia et al., 2006; Morzel et al., 2008). Jia et al. (2006) identified decreased levels of cofilin, Hsp27, and Hsp20 in the proteomic profiles of different bovine muscles differing in their tenderness at slaughter and 24 hours later. Sawdy et al. (2004) identified fragments of the contractile protein myosin heavy chain considered to be a good indicator of the tenderness of beef after 7 days of aging. Several other biomarkers were recognized through transcriptomic and proteomic approaches (for a comprehensive review refer to Cassar-Malek and Picard, 2016). Biomarkers discovered so far belong to different molecular and biological processes in the muscle cells, like energy metabolism, both glycolytic and oxidative, calcium metabolism, ultrastructure and contraction, oxidative stress, apoptosis, and cell protection with a special focus on heat shock proteins (HSPs). Some other biomarkers associated with beef quality were bovine calpastatin (CAST) (Schenkel et al., 2006; Li et al., 2013), mu-calpain (CAPN1) (Casas et al., 2006; Allais et al., 2011), and thyroglobulin (Hou et al., 2011).

4.2.1.4 Genes related to draught power The identification of molecular markers on draught ability of various cattle breeds is scanty. Effects of glutathione peroxidase 1 (GPX1) gene in Malvi and Nimari cattle (Bos indicus) for draught capacity have been studied which were found to have significant relations on animal adaptability to draught (Meplan et al., 2013).

4.2.2 Genetic markers in small ruminants 4.2.2.1 Meat and milk production A comprehensive study in goats revealed 271 candidate genes and genetic variations of some other genes that are associated with certain economic traits in goats (Supakorn, 2011). Candidate genes might influence certain physiological and metabolic pathways and, thus, affect phenotypic expression. Genes like growth hormone (GH) (Yu et al., 2004), growth hormone receptor (GHR) (Maj et al., 2007), IGF-I (Lan et al., 2007a), LEP (Whitley et al., 2005), caprine pituitary-specific transcription factor-1 (POU1F1) (Lan et al., 2007b), caprine myostatin (MSTN) (Li et al., 2006), and bone morphogenetic protein (BMP) (Chu et al., 2007a,b) are reported to be necessary for bone formation, birth weight, weaning weight, body condition as well as muscular growths. The casein gene and their family are chief

4.2 Genetic markers related to farm animal productivity

candidate genes for the traits of milk yield and composition in goats (Ramunno et al., 2001, 2004; Hayes et al., 2006; Othman and Ahmed, 2007).

4.2.2.2 Reproductive traits In goats, certain candidate genes viz forkhead box L 2 (FOXL2) (Uhlenhaut and Treier, 2006), sex determination region of Y chromosome (SRY) (Shi et al., 2008), and amelogenin (AMEL) (Chen et al., 2007) have been identified in association with sex determination and proliferation. Pfeiffer and Brenig (2005) reported that X- and Y-chromosome-specific variants of the AMEL gene allow sex determination in sheep. Genetic polymorphisms of the BMP15 gene were reported to be associated with both augmented ovulation rates as well as litter size in heterozygous carriers and sterility in homozygous carriers in both sheep and goats (Malan, 2000; Juengel et al., 2003; Chu et al., 2007a,b). Melatonin receptor 1A (MTNR1A) is a well-known and studied gene in sheep associated with reproductive parameters and prolificacy (Messer et al., 1997; Pelletier et al., 2000; Notter et al., 2003; Chu et al., 2003; Chu et al., 2006; Chu et al., 2007a,b).

4.2.2.3 Wool production Wool quality traits are well-known to be polygenic, and several genes have been described to be associated with wool quality traits in sheep. SNPs in keratinassociated protein 8.1 (KAP 8.1) and keratin-associated protein 1.3 (KAP 1.3) genes were reported to be associated with average wool fiber diameter in Chinese Merino sheep (Chen et al., 2011). Deletion of a 57-bp fragment from keratinassociated protein 6.1 (KAP 6.1) gene was associated with wool fineness and fiber diameter in Southdown 3 Merino cross lambs (Zhou et al., 2015). SNPs in keratin-associated protein 22.1 (KAP 22.1) and desmoglein 4 (DSG 4) genes were found to be associated with wool crimp in Southdown 3 Merino cross lambs and Tan sheep, respectively (Ling et al., 2014; Li et al., 2017). KAP is also reported to be associated with the formation of cashmere fiber in goats (Zhao et al., 2008). Rong et al. (2015) reported that, nine SNPs of methionine synthase (MTR) gene were associated with average wool fiber diameter in Chinese Merino sheep. Recently, it was observed that two SNPs viz SNP2 (Chr 16. 25,633,662G . A) and SNP4 (Chr 16. 25,633,569C . T) of Follistatin (FST) gene were associated with wool quality traits in Chinese Merino sheep (Ma et al., 2017). Their observation revealed SNP2 was significantly (P , .05) associated with averages of wool fiber diameter, wool fineness SD, and wool crimp, while SNP4 was significantly associated with wool fineness SD and CV of fiber diameter.

4.2.3 Genetic markers in swine 4.2.3.1 Meat quality traits Though a major part of the world pork market is encompassed of fresh meat, a wide variety of processed products are consumed. A QTL related with pork

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tenderness has been stated on pig chromosome 2, nearby to the CAST locus (Malek et al., 2001) and several polymorphisms have been described in the porcine CAST gene (Rothschild et al., 2004). Both QTL and SNP affected protein structure and had a large effect on pork tenderness. te Pas et al. (2017) reviewed comprehensively measurable biomarkers linked to meat quality from different pig production systems. Transcriptome analysis of the longissimus muscle of pigs was conducted using a custom 15K microarray, equivalent to around 9000 unique genes. As results, the study identified 1233 genes differentially expressed between breeds (Damon et al., 2012). Functional annotation clustering highlighted four main clusters associated with transcriptome breed differences viz metabolic processes, skeletal muscle structure, extracellular matrix, lysosome, and proteolysis. Thus, porcine transcriptome analysis revealed many genes involved in muscle physiology as well as meat quality development in pigs (Damon et al., 2012). Uniting meat quality with transcriptomic data obtained on 50 animals, thousands of correlations were established between microarray gene expression and meat quality traits (Damon et al., 2013). ANKRD1 (Ankyrin repeat domain 1) was found to be a biomarker of ultimate pH (Damon et al., 2013). Pierzchala et al. (2014) validated certain biomarkers associated with loin meat quality among 100 Danish commercial large white pigs. Their results confirmed the microarray data and found in addition associations for other quality traits such as meat color and intramuscular fat (IMF) as well. The modeling presented that the highest clarification of the phenotypic variance of a given quality trait in a best-fit model was for the ultimate pH at 55%, followed by the meat color traits. Proteomics analysis revealed a single protein fatty acid binding protein 4 having larger effects on IMF depot regulation (te Pas et al., 2013).

4.2.3.2 Reproductive traits Incomplete fertilization in swine, where only part of the oocytes in the oviduct are fertilized, leads to lessened litter size and may be connected to the fertile lifespan as well as functionality of sperm cells (Soede et al., 1995). Low heritability in swine may be, in part, resulting from the difficulties to determine the contribution of the sires to the litter size. Nevertheless, the lower heritability makes this trait appropriate for better forecasts with GWAS than a more extremely heritable and easier to select trait. However, the fruitful studies in bovine and equine populations using SNPs correlated with sire conception rate, propose that GWAS on markers related to sire pregnancy rate in swine is also defensible (Pen˜agaricano et al., 2012). Though, in Bos taurus, OPN is found to be positively correlated with nonreturn rates while in the pig, a negative correlation has been observed among OPN concentration and litter size along with farrowing rate (Cancel et al., 1997; Novak et al., 2010a,b).

4.2 Genetic markers related to farm animal productivity

4.2.4 Genetic markers in equine GWAS have been applied in equine to detect various SNPs that are significantly related with conception rate, and the location of these SNPs has assisted to identify candidate genes including some which are associated with lethal phenotypes in other species (Sieme and Dist, 2012). In equine, CRISP3 is considered to be an important candidate in sperm defensive mechanisms by protecting the cells from the mare’s immune response in terms of avoiding neutrophil binding as well as sperm elimination from female reproductive tract (Giesecke et al., 2010; Doty et al., 2011). CRISP3 has also been reported to be positively associated with the ability of sperm to tolerate freezing and first cycle conception rate in equine (Novak et al., 2010a,b; Jobim et al., 2011).

4.2.5 Genetic markers in poultry 4.2.5.1 Meat Meat quality in chickens is an imperative trait that includes pH, meat color, drip loss, tenderness, and intramuscular, abdominal and subcutaneous fat contents. Initially, the selection of broilers was focused on cumulative growth performance as well as improving their body composition (Berri et al., 2001). However, broiler selection led to an indirect and frequently deleterious effects on their meat quality traits, predominantly extreme deposition of abdominal fat, which represented incompetent use of feed ingredients (Abasht et al., 2006; Nadaf et al., 2007). The molecular mechanisms of fundamental meat quality traits in chickens will have both biological as well as economic consequences. Over the past 20 years, many QTLs for many traits in chicken have been well documented by linkage analysis and candidate gene analysis. A total of 272 QTLs for abdominal fat and 52 QTLs for meat quality traits have been identified in a diversity of chicken chromosomal regions (Chicken QTLdb. http://www.animalgenome.org/cgi-bin/QTLdb/GG/ index). Sun et al. (2013) conducted a GWAS of F2 populations derived from a local Chinese breed (Beijing-You chickens) and a commercial fast-growing broiler line (Cobb-Vantress) using Illumina 60K Chicken SNP Beadchips, comprised of a total of 6695 independent SNP markers, distributed on all the chicken autosomes. Among those SNPs, 33 association signals were detected from the compressed mixed linear model for 10 meat quality traits, including dry matter in breast muscle, meat color lightness and yellowness, IMF content in breast muscle, dry matter in thigh muscle, etc. (Sun et al., 2013).

4.2.5.2 Eggs A single cytosine deletion/insertion at the promoter region of chicken matrix metalloproteinases 9 was found to be genetically associated with total egg numbers at 28 weeks and thus the identified polymorphism could be used for markerassisted selection to improve chicken laying performance (Zhu and Jiang, 2014). Further, certain microsatellite loci were found to be associated with laying

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performance in ostriches. Out of a total of 30 microsatellite loci examined in high vis-a`-vis low egg producing experimental groups (n 5 12 each), 23 microsatellite loci (40 alleles of total 243 alleles identified) corresponded to the high-production group (Kawka et al., 2012).

4.3 Conclusion Functional attributes of the genetic markers of different farm animal economically important traits are diverse and some genes have agonist or antagonist effects in nature for expression of their phenotypic traits. Again, some genes could regulate more than one trait and thus the producers should be concerned with these phenotypic effects as the selection of animals with a single trait by using a single gene could influence other traits. Consequently, discovery of candidate genes and their variations, which effects expression of genes and their phenotype of economic traits, will aid breeders to develop some genetic markers for those traits. Markerassisted selection is convenient for traits with low heritability as well as those that can only be measured later in life. Thus, adequate genetic marker identification may add to genomic-enhanced expected progeny differences, especially considering the potential to best estimate an animal’s genetic worth as a parent at their early age in livestock production systems.

Acknowledgment RD got an opportunity for scientific collaboration with the other team members of this book chapter during his post-doctoral research at PUC Goia´s and UFG in Goiaˆnia Brazil under a TWAS Fellowship, Italy.

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Further reading

Zhou, H., Gong, H., Li, S., et al., 2015. A 57-bp deletion in the ovine KAP6-1 gene affects wool fibre diameter. J. Anim. Breed. Genet. 132 (4), 301
307. Zhu, G., Jiang, Y., 2014. Polymorphism, genetic effect and association with egg production traits of chicken matrix metalloproteinases 9 promoter. Asian Australas. J. Anim. Sci. 27 (11), 1526
1531.

Further reading Glowatzki-Mullis, M.L., Gaillard, C., Wigger, G., et al., 1995. Microsatellite-based parentage control in cattle. Anim. Genet. 26 (1), 7
12. Ibeagha-Awemu, E.M., Kgwatalala, P., Ibeagha, A.E., et al., 2008. A critical analysis of disease-associated DNA polymorphisms in the genes of cattle, goat, sheep, and pig. Mamm. Genom. 19 (4), 226
245. Kumar, A., Singh, U., Mehra, M., et al., 2009a. Performance of crossbred cattle under field conditions. Ind. Vet. J. 86 (1), 43
45. Kumar, A., Singh, U., Khanna, A.S., et al., 2009b. Genetic and non-genetic variability in selective value of Hariana cows. Ind. J. Anim. Sci. 79 (4), 388
391. Liu, Y.X., Zhou, X., Li, D.Q., et al., 2010. Association of ATP1A1 gene polymorphism with heat tolerance traits in dairy cattle. Genet. Mol. Res. 9 (2), 891
896. Singh, U., Kumar, A., Beniwal, B.K., et al., 2007. Performance evaluation of Ongole cows by birth weight and sex of their calves. Ind. J. Dairy Sci. 60 (1), 30
32. Singh, U., Kumar, A., Beniwal, B.K., et al., 2008. Evaluation of breeding values of Hariana bulls on organized farms. Ind. J. Anim. Sci. 78 (4), 388
390. Singh, U., Kumar, A., Khanna, A.S., 2010. Non-genetic factors affecting AFC and first lactation traits in Hariana cows. Ind. Vet. J. 87 (1), 91
93. Swierstra, E.E., Dyck, G.W., 1976. Influence of the boar and ejaculation frequency in pregnancy rate and embryonic survival in swine. J. Anim. Sci. 42, 455
460. Theodore, E., Anthony, C., Rinaldi, M., et al., 2012. Genomic and immunologic strategies to improve milk production efficiency and control mastitis. J. Anim. Sci 90 (Suppl. 3), 9. Yang, L., Fu, S., Khan, M.A., et al., 2013. Molecular cloning and development of RAPDSCAR markers for Dimocarpus longan variety authentication. Springerplus 2, 501.

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Applications of genome editing in farm animals

5

Dharmendra Kumar1 and Wilfried A. Kues2 1

Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar, Haryana, India 2 Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Ho¨ltystr10, Neustadt, Germany

5.1 Introduction Genetic modification of an animal involves the artificial altering of its genetic material. Desired genetic modifications of an animal are required for studying the mechanisms of gene functions, creating models for human disease, and improving productivity and disease resistance in animals (Chien, 1996; Majzoub and Muglia, 1996; Murray et al., 1999; Houdebine, 2002; Felmer, 2004). The number of genetically modified animals with an agricultural application has increased significantly in recent years including sheep (Clark et al., 2006), goats (Maga et al., 2006), pigs (Wheeler et al., 2001), and cows (van Berkel et al., 2002; Wall et al., 2005). The purposes include improved quality of milk production and disease resistance, as well as improving the nutritional value of the products. The manipulation of gametes and embryos in methods such as in vitro fertilization, embryo transfer, cloning, and artificial insemination is a prerequisite for the genetic modification processes (Murray et al., 1999; Izquierdo, 2001). Recent advancement in genetic modification through genome engineering (genome editing) technologies dramatically enhanced the ease and speed of performing precise genetic modifications in farm animals (Bosch et al., 2015; Petersen and Niemann, 2015). The genome editing tools mediate targeted genetic alterations by enhancing DNA mutation frequency via creating doublestrand breaks (DSB) at a predetermined genomic site using designer nucleases. Designer nucleases or molecular scissors make one incision on each of the two strands of DNA. The DSBs can then be healed either by blunt-end, nonhomologous end-joining (NHEJ) to introduce random mutations or by homologous DNA repair by adding an engineered strand of DNA with homology on either side of the DSB. Molecular scissors include, (1) zinc finger nucleases (ZFNs), (2) transcription activator-like endonucleases (TALEN), and (3) clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) systems. ZFNs rely on the zinc finger motif, one of the most Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00005-9 © 2020 Elsevier Inc. All rights reserved.

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common DNA binding domains found in eukaryotes (Miller et al., 1985). It is comprised of B30 amino acid modules that interact with nucleotide triplets. ZFNs have been designed to recognize all of the 64 possible permutations of trinucleotide combinations. ZFNs function in dimers, hence pairs of ZNFs are required to target any specific locus: one ZFN recognizes the sequence upstream and the other ZFN recognizes the sequence downstream of the site to be modified. In TALENs each domain recognizes a single nucleotide (Christian et al., 2010; Miller et al., 2011). Thus both ZFNs and TALENs recognize the target sequence by protein DNA-interactions, which require excessive testing of highly functional scissors. In contrast, the CRISPR/Cas9 system relies on base pairing between the guide RNA (gRNA) and the target sequence, which makes this approach straightforward. The CRISPR/Cas9 system evolved as a bacterial defense mechanism designed to recognize and eliminate foreign DNA from invading bacteriophages (Makarova et al., 2011). The CRISPR/Cas9 system consists of a Cas endonuclease that is directed to cleave a target sequence recognized by the specific base pair hybridization of a gRNA (Gaj et al., 2013). The common feature of all molecular scissors is that they mediate targeted genetic alterations by enhancing the DNA mutation rate via the enzymatically catalyzed induction of a DSB at a predetermined genomic site. Among the important three molecular scissors, CRISPR/Cas9 appears as superior over ZFNs and TALENS due to the capability of simultaneously targeting multiple genomic sites. A comparative list of the features of these molecular scissors is enumerated in Table 5.1. Hence, this chapter provides an update on the use of CRISPR/Cas9 to modify the genome of farm animals, summarizes current knowledge on the underlying mechanism, and discusses new opportunities for generating genetically modified farm animals.

5.2 Development of CRISPR/Cas9 system More than 30 years ago, the discovery of the homology-directed repair (HDR) pathway inspired to modify genomic DNA sequences at a precise genomic locus (Smithies et al., 1985; Thomas and Capecchi, 1987). Using the HDR pathway, gene targeting in murine embryonic stem (ES) cells allowed for the first time to specifically modify desired loci in mice (Capecchi, 1989). HDR are widely used in murine ES cells, but their application in somatic cells is limited by the relatively low recombination efficiency. Rudin et al. (1989) suggested that the presence of a DSB could enhance the HDR recombination rate. It has been shown that a DSB may result in random insertions or deletions (InDels) due to the errorprone NHEJ pathway, offering an approach for a targeted gene knockout (Bibikova et al., 2002). However, at this time no method was available to introduce site-specific DSB in a genomic context to initiate the DNA repair process.

5.2 Development of CRISPR/Cas9 system

Table 5.1 Features of different molecular scissors. Factors

ZFN

TALEN

CRISPR

Origin

Eukaryotic transcription factor family Customized protein design

Plant pathogenic bacteria Xanthomonas Customized protein repeat design

Diverse bacteria

Zinc finger motif Protein DNA Zinc finger sequence especially recognizing 3 bp sequence fused to Fok1 nuclease domain

Transcription activatorlike effector Protein DNA Protein sequence specific to binding a nucleotide sequence fused to Fok1 nuclease domain

Moderate

High

Cloning of 20 base oligonucleotide single-guide RNA RNA DNA 20 base crRNA fused to tracrRNA, and Cas9 endonuclease High

18 36

30 40

20

G/C-rich

Start with T and end with A

Protospacer adjacent motif

High

Medium

Low

Limited

Average

Good

High

Low

Low

Complex

Complex

Simple

High

Moderate

Low

Design

Targeting domain Recognition Construct

Average mutation rate Length of recognition domain (bp) Restriction in target site Complexity to design vector Target efficient Off-target efficient In vitro testing Costs

CRISPR, Clustered regularly interspaced short palindromic repeats; TALEN, transcription activator-like endonucleases; ZFN, zinc finger nucleases.

To overcome this challenge, different programmable nuclease-based genome editing technologies have been developed in recent years, enabling targeted DSB creation in a genome. Among the currently available genome editing technologies, the CRISPR/Cas9 represents the most rapidly developing system. The CRISPR/Cas system was discovered in 1987 in Escherichia coli; sequencing data revealed an array of 29 nucleotide repeats (Ishino et al., 1987), which were named clustered regularly interspaced short palindromic repeats. At this time the function of the repeats were unknown, and only in 2007 first experimental evidence

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supported a role of the repeats in adaptive immunity of bacteria against bacteriophages (Barrangou et al., 2007). This finding led to the idea that natural CRISPR/ Cas systems could be harnessed for immunization against phages, a first successful application of CRISPR/Cas for biotechnological purposes (Horvath and Barrangou, 2010). The chronological development of the CRISPR/Cas system is elucidated in Table 5.2.

Table 5.2 Chronological advancement in the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) gene editing technology. Year

Development

Reference(s)

1987 2000

First discovery of CRISPR arrays in bacteria Recognition and characterization of CRISPR families in prokaryotes Definition of the term CRISPR Identification of the foreign origin of spacer sequences and postulation of an adaptive immunity function CRISPR/Cas genes hypothesized as bacterial defense mechanism against viruses First experimental evidence for CRISPR adaptive immunity RNA molecule associated with CRISPR (crRNA) described that act as small guide RNAs CRISPR activity on DNA targets

Ishino et al. (1987) Mojica et al. (2000)

2002 2005

2006 2007 2008

2009 2010 2011

2012

2013 2014

Type III-B Cmr CRISPR complex cleavage of RNAs Cas9 guidance by spacer sequences Association of tracrRNA and crRNA with Cas9 revealed Type II CRISPR systems are modular and can be heterologously expressed in other organisms In vitro characterization of DNA targeting by Cas9 and experimental demonstration as a gene editing tool The first demonstration of Cas9 genome engineering in eukaryotic cells Genome-wide functional screening with Cas9 Crystal structure of apo-Cas9

Jansen et al. (2002) Mojica et al. (2005), Pourcel et al. (2005), Bolotin et al. (2005) Makarova et al. (2006) Barrangou et al. (2007) Brouns et al. (2008)

Marraffini and Sontheimer (2008) Hale et al. (2009) Garneau et al. (2010) Deltcheva et al. (2011) Sapranauskas et al. (2011)

Jinek et al. (2012), Gasiunas et al. (2012) Cong et al. (2013), Mali et al. (2013) Wang et al. (2014), Shalem et al. (2014) Jinek et al. (2014) (Continued)

5.3 The molecular structure of CRISPR/Cas9

Table 5.2 Chronological advancement in the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) gene editing technology. Continued Year

2015

2016

2017 2018

Development Crystal structure of Cas9 in complex with guide RNA and target DNA Genome editing of nonviable human embryos Multiplexed genome editing of B60 endogenous retroviral sequences in pig embryos in the first step to create pig organs suitable for use in humans An improved version of CRISPR/Cas 9 with less risk of off-target DNA restriction First CRISPR clinical trial approved by US panel New base editing technique offering to alter genome without needing to cleave double-stranded DNA or for a donor DNA template CRISPR targeting of RNA molecule Identification of preexisting antibodies targeting Cas9 protein questioning the utility of CRISPR/ Cas9 for gene therapy

Reference(s) Nishimasu et al. (2014) Cyranoski and Reardon (2015) Yang et al. (2015)

Zhang et al. (2015) Reardon (2016) Komor et al. (2016)

Gootenberg et al. (2017) Charlesworth et al. (2018)

5.3 The molecular structure of CRISPR/Cas9 The bacterial CRISPR/Cas system is composed by the CRISPR loci and the Cas genes (Fig. 5.1). The CRISPR locus is an array of short direct repeats and interspersed spacer sequences. The repeats could differ in different bacterial species, and sequence divergence was also established in the spacers and Cas genes (Jansen et al., 2002; Makarova et al., 2006; Godde and Bickerton, 2006). The repeat size of CRISPR locus varies from 21 to 47 bp, whereas interspersed spacer sizes are between 26 and 72 bp (Deveau et al., 2010). The CRISPR locus contains between 1 and 374 repeat-spacer units (Deveau et al., 2010). Apart from these, a leader region is located upstream of the CRISPR locus (Lillestol et al., 2006). The CRISPR array comprises an AT-rich leader sequence followed by short repeats that are separated by unique spacers (Haft et al., 2005). The Cas genes are located in the vicinity of the CRISPR loci (Deveau et al., 2010). A CRISPR/Cas system usually incorporates 4 20 different Cas genes (Haft et al., 2005; Barrangou et al., 2007; Sorek et al., 2008). Collectively, more than 90 Cas genes have been identified; the Cas genes are grouped into 35 families. Out of 35 families, 11 form the

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CHAPTER 5 Applications of genome editing in farm animals

FIGURE 5.1 Structure of the bacterial clustered regularly interspaced short palindromic repeats/ CRISPR-associated (CRISPR/Cas) system.

Cas core which includes the protein families Cas1 through Cas9. A complete CRISPR/Cas locus has minimally one gene belonging to the Cas core (Hille and Charpentier, 2016). CRISPR/Cas systems were categorized into two classes. Class 1 systems employ complex and multiple Cas proteins to degrade foreign nucleic acids, whereas class 2 systems use a single Cas protein. Class 1 was further divided into types I, III, and IV; and class 2 into types II, V, and VI (Wright et al., 2016). Recently, in class 2, 53 new CRISPR/Cas candidates were added and categorized into three groups defined by the context characteristics (Shmakov et al., 2015). The potential target of one subgroup (C2c2) is single-stranded RNA (ssRNA); therefore it can be applied for gene knockdown or knockout applications at the mRNA level (Abudayyeh et al., 2016). The Class 2 type VI-A CRISPR-Cas effector (C2c2) possesses both an activity for CRISPR-RNA maturation and a RNA degradation activity (East-Seletsky et al., 2016). These dual RNase functions are chemically and mechanistically different from each other and from the crRNA-processing behavior of the evolutionary-unrelated CRISPR enzyme Cpf1 (Fonfara et al., 2016). More recently, Strutt et al. (2018) showed that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave ssRNA by an RNA-guided mechanism that is independent of a protospacer adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and able to reduce infection by ssRNA phage in vivo. They also demonstrate that Cas9 direct PAM-independent repression of gene expression in bacteria. Thus some isoforms of Cas holoenzymes have the ability to specifically interact with RNA molecules, and will expand the CRISPR toolbox for transcriptome targeting.

5.4 Delivery and expression system Currently, the most used molecular scissor is the recombinant Cas9 together with a single-guide RNA (sgRNA). Cas9 and a sgRNA could be delivered to

5.5 Mechanism of action

eukaryotic cells by several methods, such as transient transfections, cytoplasmic or nuclear injections, and by viral transduction (Fig. 5.2). Both components could be delivered as plasmid encoded components, or as transcribed RNAs. Alternatively, the recombinant Cas9 protein and synthetic guide RNAs are available from different producers. The choice of the most suitable combination depends on the experimental approach and aims. In microinjection and electroporation methods, physical energy is used for cell entry and this methodology is more suitable for in vitro delivery (Wells, 2004). However, delivery of molecules into cells could also be performed by viral or nonviral vectors. In the case of nonviral vectors the encapsulation of the programmable nucleases or nuclease proteins with lipofectamine is commonly applied (Thomas et al., 2003), such as lipid nanoparticles, liposome, polymers, and conjugates, as well as some novel ones such as cell-derived membrane vesicles (Van Dommelen et al., 2012; Yin et al., 2014). Viral vectors used for systemic delivery in clinical trials include lentiviral, adenoviral, adeno-associated viral, and herpes simplex-1 viral vectors. After delivery into the cells, Cas9 identifies its target by protein-mediated PAM recognition and base pairing between the sgRNA and the DNA target (Cho et al., 2013). Target recognition activates the nuclease sites, resulting in DSB 3 4 nucleotides downstream from the PAM. NHEJ repair results in indels, which often results in a frameshift mutation. HDR relies on a homologous recombination with a supplied donor DNA molecule, which contains desired sequence mutations. Clinical applications of the programmable nuclease complexes are promising for gene therapeutic approaches, but currently are limited by the lack of cell- and organ-specific delivery protocol.

5.5 Mechanism of action The designer nuclease CRISPR/Cas9 performs a specific DSB by target identification via base-hybridization of the sgRNA (Cong et al., 2013; Mali et al., 2013). The sgRNA is composed of a specific 20-bp crRNA and the universal tracrRNA. The sgRNA can be synthesized in the test tube by a T7 RNA polymerase reaction or in cells by a RNA polymerase III transcription from a U6 promoter (Mali et al., 2013; Wang et al., 2013; Jao et al., 2013). To recognize the target site, gRNA sequence should start with a guanine base for U6directed transcription, and two guanine bases for T7-directed transcription (Cong et al., 2013; Mali et al., 2013; Wang et al., 2013; Jao et al., 2013). The target site of 20 bases must be followed by a PAM sequence. The PAM is a trinucleotide sequence with the consensus sequence NGG, which is essential for the binding of the Cas9 nuclease (Mali et al., 2013). The Cas9 scans the nuclear DNA for PAM motifs and starts melting the double strand to allow hybridization of the sgRNA targets; in case of a complementary matching,

137

FIGURE 5.2 Delivery of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) system to eukaryotic cells by different methods and repair mechanism of the targeted DNA.

5.6 Gene editing using CRISPR/Cas9 in farm Animals

FIGURE 5.3 Mechanism of clustered regularly interspaced short palindromic repeats/CRISPRassociated protein (CRISPR/Cas9) system in genome editing.

the Cas9 cleaves the genomic DNA (Sakuma and Yamamoto, 2017). The resulting DSB stimulates the cellular repair mechanisms and can result in sequence mutagenesis (Fig. 5.3). Alternatively, synthetic sgRNA and recombinant Cas9 protein can be assembled in the test tube, and applied as ribonucleoprotein complex. CRISPR/Cas has been quickly applied to generate mutations in different organisms, to establish various disease models, and for the use in gene correction and therapy (Doudna and Charpentier, 2014).

5.6 Gene editing using CRISPR/Cas9 in farm Animals Genetically engineered animal models are playing a crucial role in the study of complex physiological and pathophysiological processes, as well as for the introduction of new traits with commercial value. The standard approaches to generate transgenic mice via pronuclear injection and ES cell-mediated genetic modification are time-consuming and costly, and most importantly are not welltransferable to other mammalian species. Until recently, the only possibility for targeted modifications in farm animals was the HDR in somatic cells and their use in the somatic cell nuclear transfer (SCNT) (Bosch et al., 2015). All of these approaches were rather time-consuming and limited to few “model” organisms. Currently, the CRISPR/Cas9 approach is the most successful and versatile strategy for genome editing in bacteria, fungi, plants and animals, and has been successfully translated to livestock (Selokar and Kues, 2018). By using the CRISPR/ Cas technology, the genomes of several farm animals, such as pigs, sheep, goats, and cattle have been modified. An incomplete list of genome-edited farm animals

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CHAPTER 5 Applications of genome editing in farm animals

using CRISPR/Cas is provided in Table 5.3. CRISPR provides an easier approach to establish transgenic animal models compared with previous possibilities. More recently, CRISPR/Cas9 technology has successfully been used to insert a tuberculosis resistance gene, natural resistance-associated macrophage protein 1, into the cow genome and produced engineered cows which are more resistance to tuberculosis (Gao et al., 2017). This study is a proof of concept indicating that the CRISPR/Cas system could be used to develop disease resistance animals. The genomes of several farm animals have also been successfully edited and produced

Table 5.3 Clustered regularly interspaced short palindromic repeats/ CRISPR-associated (CRISPR/Cas9)-mediated genome editing in farm animals. Species

Gene

Method

Application

References

Pig

DAZL and APC

SCNT

Tan et al. (2013)

SLA-1,2,3

SCNT

vWF

CPI

GGTA1

CPI

α1,3GT, CMAH, B4GalNT2 CD163

SCNT

Model for studying colon cancer and for development of therapeutics Piglets devoid of all cell surface class I proteins for immunological research Knockout improved bleeding efficiency Knockout of GGTA1 for xenotransplantation research Multiple gene knockouts for xenotransplantation research

CPI

Whitworth et al. (2016)

NANOS2

CPI

CD163 SCRC5 Myostatin (MSTN)

CPI

Resistance against porcine reproductive and respiratory syndrome virus (PRRSV) Lack of germ line which could provide a suitable environment to host germ cells from a genetically superior male PRRSV

SCNT

Generation of isozygous, MSTN knockout cloned pigs free of selectable marker gene increased longissimus muscle size and decreased backfat thickness

MSTN

CPI

Improved muscle differentiation and growth

Sheep

Reyes et al. (2014) Hai et al. (2014) Petersen et al. (2016) Butler et al. (2016)

Park et al. (2017)

Burkard et al. (2017) Bi et al. (2016)

Han et al. (2014), Crispo et al. (2015) (Continued)

5.6 Gene editing using CRISPR/Cas9 in farm Animals

Table 5.3 Clustered regularly interspaced short palindromic repeats/ CRISPR-associated (CRISPR/Cas9)-mediated genome editing in farm animals. Continued Species

Goat

Cattle

Japanese Black Cattle

Gene

Method

Application

References

ASIP

CPI

BMPR-IB

CPI

FGF5

CPI

Alteration of sheep coat color pattern Increased ovulation rate and consequently larger litter size Improved wool quality and yield.

GDF8

SCNT

FGF, GDF8

CPI

Zhang et al. (2017) Zhang et al. (2017) Li et al. (2017) Ni et al. (2014) Wang et al. (2015)

MSTN and FGF5 GDF9

CPI

BLG

SCNT

POLLED

SCNT

NRAMP1

SCNT

IARS

SCNT

CPI

Live-born goats harboring biallelic mutations Production of gene-modified goats with either one or both genes disrupted Enhancement of fiber length Introduction of defined point mutations Generation of beta-lactoglobulin knockout goats Production of hornless dairy cattle Transgenic cattle with increased resistance to tuberculosis Repair of the IARS mutation without any additional DNA footprint

Wang et al. (2016) Niu et al. (2017) Zhou et al. (2017) Carlson et al. (2016) Gao et al. (2017) Ikeda et al. (2017)

CPI, Cytoplasmic microinjection; IARS, isoleucyl-tRNA synthetase; SCNT, somatic cell nuclear transfer.

live animals for xenotransplantation research, disease modeling, and agricultural purposes (reviewed by Tan et al., 2016; Petersen, 2017; Lamas-Toranzo et al., 2017; Ruan et al., 2017; Huang et al., 2018). Apart from this, efforts have also been made to improve the nutritional value of milk, either by knockout of the beta-lactoglobulin milk allergen or by expressing of the lactoferrin-glycoprotein, which absorbs iron from the intestine and suppresses the growth of bacteria. Attempts have been made to express human lysozyme into mammary gland of cows (Liu et al., 2014), which protects the cows from mastitis, a major economic disease of dairy farming. It is likely that in the near future several edited animals will be commercialized; however, editing concepts and proofs have to be ethically accepted and approved.

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CHAPTER 5 Applications of genome editing in farm animals

5.7 Technical challenges of the CRISPR/Cas9 genome editing The CRISPR/Cas9 technology is a highly specific method for genome editing, which accelerates the pace to generate farm animals with desired genotypes. Although the genome editing has been successfully applied in diverse eukaryotes, safety aspects and the absence of mutations in undesired places of the genome are still challenges. A high degree of specificity is the main challenge and would be essential for commercialization of gene-edited livestock. Another challenge is the assessment of off-target mutations, and suitable delivery methods (Zhang et al., 2014). Therefore it is important to improve the specificity of Cas9 and reduce off-target events. Optimized recombinant Cas9 variants with increased specificity have been identified (Slaymaker et al., 2016). In addition, improvements in the efficient capacity of the HDR-mediated repair have been achieved by modulating the activity of the cellular DNA ligase IV to inhibit NHEJ process (Maruyama et al., 2015) or by applying adenoviral proteins to promote HDR process (Chu et al., 2015). Further improvements of the Cas9 resulted in enzyme variants, such as HF-Cas or eCas, which show reduced off-target activities (Kleinstiver et al., 2016; Slaymaker et al., 2016). Other nucleases of the CRISPR/Cas family, such as Cfp1, make the target site selection more flexible since they require different PAM sequences than Cas9 (Zetsche et al., 2015). The recent development of base editors to change a single base within the genome without the need to introduce a double-stranded DNA break further extended the CRISPR toolbox (Komor et al., 2016; Kim et al., 2017; Gaudelli et al., 2017). The chemical modification of synthetic guide RNAs, to increase stability and halve-life, also showed promising results (Yin et al., 2017).

5.8 Premises and promises of genome editing by CRISPR/Cas9 The scientific premise for genome editing by CRISPR/Cas9 and other molecular scissors is the knowledge of the genome organization and of the genome sequences. The ongoing whole genome sequencing approaches and more efficient and rapid high throughput sequencing methods provide the basis for the application of genome editing. The more detailed annotation of protein-coding genes, RNA coding genes, small RNA genes, but also of regulatory regions and noncoding genomic regions will extend the possibilities for more fine-tuned genome editing. Apart from directly modifying the genome, noncutting CRISPR/Cas9 fusion proteins will be used for more subtle epigenetic programming. Currently, genome editing is an emerging area in the laboratory. To exploit the already created genome-edited farm animals and promote the ongoing research, the rules for approval and commercialization must be defined.

References

Acknowledgment Authors acknowledge DST-DAAD for their support.

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Park, K.E., Kaucher, A.V., Powell, A., Waqas, M.S., Sandmaier, S.E., et al., 2017. Generation of germline ablated male pigs by CRISPR/Cas9 editing of the NANOS2 gene. Sci. Rep. 7, 40176. Petersen, B., 2017. Basics of genome editing technology and its application in livestock species. Reprod. Domest. Anim. 52 (Suppl. 3), 4 13. Petersen, B., Niemann, H., 2015. Molecular scissors and their application in genetically modified farm animals. Transgenic Res. 24 (3), 381 396. Petersen, B., Frenzel, A., Lucas-Hahn, A., Herrmann, D., Hassel, P., et al., 2016. Efficient production of biallelic GGTA1 knockout pigs by cytoplasmic microinjection of CRISPR/Cas9 into zygotes. Xenotransplantation 23, 338 346. Pourcel, C., Salvignol, G., Vergnaud, G., 2005. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151, 653 663. Reardon, S., 2016. First CRISPR clinical trial gets green light from US panel. Nature . Available from: https://doi.org/10.1038/nature.2016.20137. Reyes, L.M., Estrada, J.L., Wang, Z.Y., Blosser, R.J., Smith, R.F., et al., 2014. Creating class I MHC-null pigs using guide RNA and the Cas9 endonuclease. J. Immunol. 193, 5751 5757. Rudin, N., Sugarman, E., Haber, J.E., 1989. Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae. Genetics 122, 519 534. Ruan, J., Xu, J., Chen-Tsai, R.Y., Li, K., 2017. Genome editing in livestock: are we ready for a revolution in animal breeding industry? Transgenic Res. 26 (6), 715 726. Sakuma, T., Yamamoto, T., 2017. Magic wands of CRISPR-lots of choices for gene knock-in. Cell. Biol. Toxicol. 33 (6), 501 505. Sapranauskas, R., Gasiunas, G., Fremaux, C., Barrangou, R., Horvath, P., et al., 2011. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 39, 9275 9282. Selokar, N.L., Kues, W.A., 2018. How farm animals are improving human health and welfare. Rev. Sci. Tech. 37, 83 96. Shalem, O., Sanjana, N.E., Hartenian, E., Shi, X., Scott, D.A., et al., 2014. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343 (6166), 84 87. Shmakov, S., Abudayyeh, O.O., Makarova, K.S., Wolf, Y.I., Gootenberg, J.S., et al., 2015. Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Mol. Cell 60, 385 397. Slaymaker, I.M., Gao, L., Zetsche, B., et al., 2016. Rationally engineered Cas9 nucleases with improved specificity. Science 351 (6268), 84 88. Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A., Kucherlapati, R.S., 1985. Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature 317 (6034), 230 234. Sorek, R., Kunin, V., Hugenholtz, P., 2008. CRISPR a widespread system that providesacquired resistance against phages in bacteria and archaea. Nat. Rev. Microbiol. 6 (3), 181 186. Strutt, S.C., Torrez, R.M., Kaya, E., Negrete, O.A., Doudna, J.A., 2018. RNA-dependent RNA targeting by CRISPR-Cas9. Elife 7, e32724. Tan, W., Carlson, D.F., Lancto, C.A., Garbe, J.R., Webster, D.A., et al., 2013. Efficient nonmeiotic allele introgression in livestock using custom endonucleases. Proc. Natl. Acad. Sci. U.S.A. 110, 16526 16531.

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Tan, W., Proudfoot, C., Lillico, S.G., Whitelaw, C.B., 2016. Gene targeting, genome editing: from dolly toeditors, Transgenic Res., 25. pp. 273 287. Thomas, K.R., Capecchi, M.R., 1987. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51 (3), 503 512. Thomas, C.E., Ehrhardt, A., Kay, M.A., 2003. Progress and problems with the use of viral vectors for gene therapy. Nat. Rev. Genet. 4, 346 358. van Berkel, P.H., Welling, M.M., Geerts, M., van Veen, H.A., Ravensbergen, B., et al., 2002. Large-scale production of recombinant human lactoferrin in the milk of transgenic cows. Nat. Biotechnol. 20, 484 487. Van Dommelen, S.M., Vader, P., Lakhal, S., Kooijmans, S.A., van Solinge, W.W., et al., 2012. Microvesicles and exosomes: opportunities for cell-derived membrane vesicles in drug delivery. J. Control. Release 161, 635 644. Wall, R.J., Powell, A.M., Paape, M.J., Kerr, D.E., Bannerman, D.D., et al., 2005. Genetically enhanced cows resist intramammary Staphylococcus aureus infection. Nat. Biotechnol. 23, 445 451. Wang, X., et al., 2015. Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system. Sci. Rep. 5, 13878. Wang, H., Yang, H., Shivalila, C.S., Dawlaty, M.M., Cheng, A.W., et al., 2013. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910 918. Wang, T., Wei, J.J., Sabatini, D.M., Lander, E.S., 2014. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343 (6166), 80 84. Wang, X., Cai, B., Zhou, J., Zhu, H., Niu, Y., Ma, B., et al., 2016. Disruption of FGF5 in cashmere goats using CRISPR/Cas9 results in more secondary hair follicles and longer fibers. PLoS One 11 (10), e0164640. Wells, D., 2004. Gene therapy progress and prospects: electroporation and other physical methods. Nat. Rev. 11, 1363 1369. Wheeler, M.B., Bleck, G.T., Donovan, S.M., 2001. Transgenic alteration of sow milk to improve piglet growth and health. Reproduction 58, 313 324. Whitworth, K.M., Rowland, R.R., Ewen, C.L., Trible, B.R., Kerrigan, M.A., et al., 2016. Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus. Nat. Biotechnol. 34, 20 22. Wright, A.V., Nun˜ez, J.K., Doudna, J.A., 2016. Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell 164 (1 2), 29 44. Yang, L., Guell, M., Niu, D., George, H., Lesha, E., Grishin, D., et al., 2015. Genomewide inactivation of porcine endogenous retroviruses (PERVs). Science 350, 1101 1104. Yin, H., Kanasty, R.L., Eltoukhy, A.A., Vegas, A.J., Dorkin, J.R., Anderson, D.G., 2014. Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 15, 541 555. Yin, H., et al., 2017. Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing. Nat. Biotechnol. 35, 1179 1187. Zetsche, B., Gootenberg, J.S., Abudayyeh, O.O., Slaymaker, I.M., Makarova, K.S., et al., 2015. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759 771. Zhang, F., Wen, Y., Guo, X., 2014. CRISPR/Cas9 for genome editing: progress, implications and challenges. Hum. Mol. Genet. 23 (R1), R40 R46.

References

Zhang, X.H., Tee, L.Y., Wang, X.G., Huang, Q.S., Yang, S.H., 2015. Off-target effects in CRISPR/Cas9-mediated genome engineering. Mol. Ther. Nucleic Acids 4, e264. Zhang, X., Li, W., Liu, C., Peng, X., Lin, J., et al., 2017. Alteration of sheep coat color pattern by disruption of ASIP gene via CRISPR Cas9. Sci. Rep. 7 (1), 8149. Zhou, W., Wan, Y., Guo, R., Deng, M., Deng, K., et al., 2017. Generation of betalactoglobulin knock-out goats using CRISPR/Cas9. PLoS One 12, e0186056.

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6

Jagdip Singh Sohal1, Azhar Khan1, Divyang Vats1, Mukta Jain1, Rathnagiri Polavarapu2, G.K. Aseri1 and Deepansh Sharma1 1

Amity Center for Mycobacterial Disease Research, Amity Institute of Microbial Technology, Amity University Rajasthan, Jaipur, India 2 Genomix Biotech Inc., Atlanta, GA, United States

6.1 Introduction Since the discovery of the DNA double helix in 1953, many basic biological concepts pertaining to the genome, such as transcription, translation, genetic code, epigenetic modification and many more have been established by multiple experimental techniques. Tools for in vitro DNA manipulations (such as polymerases, restriction endonucleases, ligases etc) and techniques such as recombinant DNA technology, in vitro DNA synthesis, the site-specific mutagenesis, whole-genome sequencing etc have also been established. Nonetheless, site-specific modification within genomes has remained a major challenge to the scientific world. To meet this challenge, genome editing technologies in the past two decades have been developed for precise site specific alternation of the genomes. As the name suggests, genome editing (more popular as gene editing) is a set of technologies with the ability to modify or edit the genome of an organism/ individual at the desired site. Using genome editing, DNA can be inserted, deleted, modified or replaced in living beings. Genome editing utilizes engineered endonucleases (popular as molecular scissors) that create a site specific double-strand break for subsequent alterations in the DNA. These genome editing tools employ specially engineered nucleases including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly-interspaced short palindromic repeat (CRISPR) systems. These nucleases, in particular the CRISPR systems, immensely facilitate the wide application of genome editing in various biological fields. Pavletich and Pabo, in 1991, developed first engineered nuclease by coupling nonspecific endonuclease with DNA binding ZFN motif (Pavletich and Pabo, 1991). Subsequently, these engineered ZFNs were employed in genetic

Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00006-0 © 2020 Elsevier Inc. All rights reserved.

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Table 6.1 Comparison of various gene editing techniques.

Parameter

Zinc finger nuclease (ZFN)

Transcription activator-like effector nuclease (TALEN)

Cas9

Length of the target site Off-target effects Accessibility Multiple targeting In vivo delivery Targeting system

18 36 bp Variable Difficult Difficult Easy Protein

30 40 bp Low Moderate Difficult Difficult Protein

22 bp Variable Easy Easy Moderate Protein/RNA

engineering of drosophila and mammalian cells (Maeder et al., 2008; Bibikova et al., 2002, 2003). ZFN was the predominant genome targeting technology for over10 years, but over time better tools emerged (Carroll, 2011; Guo et al., 2010; Cathomen and Joung, 2008). With the identification of genome targeting abilities of transcription activator-like (TAL) effectors in 2009, a new window of genome editing emerged and TAL tagged with nonspecific endonuclease (TALEN) was developed in 2011 (Cermak et al., 2011; Miller et al., 2011). TALEN allowed a larger window of potential target sites in gene editing. Therefore, TALEN was named Method of the Year 2011 by Nature Publishing Group (Baker, 2011). In 2012, CRISPR-CISPR associated (Cas) system was demonstrated as a new genome editing tool (Zhang et al., 2014). CRISPR-Cas is a natural bacterial defence mechanism to deal with foreign plasmids and phages (Mojica and Montoliu, 2016). The CRISPR-Cas-based gene editing method was first used to edit the genome of zebrafish in 2014 (Gonzales and Yeh, 2014). Gene editing tools have been summarized in Table 6.1.

6.2 Overview of gene editing tools 6.2.1 Zinc finger nucleases ZFNs were the first to be used in genome editing. ZFNs are composed of a ZFN mediated DNA binding domain for DNA recognition and a nuclease activity domain of FokI for DNA cleavage (Kim et al., 1996). ZFNs can cause doublestrand breaks for subsequent modifications. They typically comprise of 30 amino acid modules that interact with nucleotide triplets (Szczepek et al., 2007). ZFNs have been designed to recognize many different combinations of tri-nucleotides. However, certain nucleotide triplets cannot be recognized by ZFNs. This reduces the specificity of ZFNs and is the major disadvantage with ZFNs (Gaj et al., 2013).

6.3 Scope of genome editing

6.2.2 Transcription activator-like effector nucleases TALENs are similar to ZFNs in that they use DNA binding motifs to direct the nonspecific nuclease (FokI) to cleave the genome at a specific site, but instead of recognizing DNA triplets, each domain recognizes a single nucleotide (Nelson et al., 2016). The interactions between the TALEN derived DNA binding domains and their target nucleotides are less complex than those between ZFNs and designing TALENs is easier than ZFNs (Nelson et al., 2016). However, TALENs may cause certain off-target cleavage leading to undesirable mutations and therefore affect the specificity of the method (Nelson et al., 2016).

6.2.3 Clustered regularly-interspaced short palindromic repeat/Cas9 system The CRISPR-Cas9 system is an RNA-based bacterial defense mechanism designed to recognize and eliminate foreign DNA from invading bacteriophage and plasmids (Nelson et al., 2016; Barrangou, 2015). This consists of a Cas endonuclease directed to cleave a target sequence by a guide RNA (gRNA). CRISPR/Cas system is complex of ribonucleoprotein, wherein guide RNA is complexed with Cas endonuclease (Nelson et al., 2016). The guide RNA is complementary to invading DNA. Binding of guide RNA to target DNA triggers Cas endonuclease to cut the target DNA at a specific site (Nelson et al., 2016; Gasiunas et al., 2012). Both the Cas endonuclease and the gRNA are encoded by a CRISPR array in the bacterial genome and the system can be coopted to cleave any target sequence of choice by modifying the sequence of the gRNA (Pride et al., 2012; Grissa et al., 2007). Similar to the ZFN and TALEN systems, the CRISPR/Cas system can be used to introduce desired modification at the site of DNA cleavage. For in vitro modification in DNA, CRISPR-Cas method includes a plasmid construction including genes for Cas9 protein and gRNA (complementary to target DNA). As soon as this vector is inserted into a cell, expression of Cas9 and gRNA genes will be executed and the ribonucleoprotein complex of gRNA and Cas9 will be formed. gRNA will bind the target DNA to form RNA-DNA duplex. This duplex is identified by the Cas9 protein which will cleave the target DNA. The Cas9 protein identifies the protospacer adjacent motif sequence and cleaves the target DNA at desired site. CRISPR/Cas9 system can also be delivered in living cells as ribonucleoprotein complex of gRNA and cas9 protein (Dewitt et al., 2017). The specific advantage of CRISPR/Cas9 is that it can be used directly on embryonic cells (Hsu et al., 2014). Moreover, it is more simple and efficient.

6.3 Scope of genome editing Gene editing offers tremendous scope in engineering the genome for the variety of applications. The uses of genome editing have so far been in scientific research

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to investigate models for human diseases, characterize gene function, and improve traits in plants or animals. Genome editing of crop plants is a rapidly advancing technology where in targeted mutations can be introduced into a plant genome in a highly specific manner with great precision. The most widely used instrument in genome editing is the CRISPR/Cas9 system. Gene editing is widely used in plants for characterizing gene function and improving traits. With the rising global temperature rice spikelet sterility is becoming a serious problem globally. Researchers have used CRISPR/Cas9 system to develop a new variety of rice resistant to high temperature induced spikelet sterility. Performance of this new variety has been successfully tested under hot conditions (Hakata et al., 2017). Similarly, using a gene editing tool, a heat resistant variety of magnolia plant has also been developed (Shen et al., 2017). TALEN has been used to develop disease-resistant variety of rice. This variety is resistant to pathogen Xanthomonas oryzae causing a blight that leads to significant annual losses in rice production (Sahu et al., 2018). Gene editing technology has been increasingly applied to study or develop treatment for human diseases including genetic disorders and infectious diseases. CRISPR/Cas9 gene editing studies are focusing on developing programmed cell death-based system for HIV infected cells and cancerous cells (Wang et al., 2016). CRISPR/Cas9 based technique has also been developed to inhibit HIV DNA in infected cells (Yin et al., 2018; Zhu et al., 2015). CRISPR tool is under investigation to deplete telomerase activity in the cancer cell as a potential treatment method (Kim et al., 1996). Further, for tackling vector-born diseases like malaria, mosquito lines naturally resistant to malaria parasites have also been developed using CRISPR method (Ghorbal et al., 2014). Genome editing techniques are also being applied in biotechnology. CRISPR/Cas9-induced genome editing is being used in microalgae to improve biofuel production (Beisson and Peltier, 2013). Use of CRISPR/Cas9 system to identify target molecules is becoming popular and will impact the drug discovery (Scott, 2018). By identifying the target molecules drugs can be developed to inhibit targets.

6.4 Companion animals and gene editing: scope and prospects Throughout history, animals have played a key role in human life. People have dependent on animals for food, clothing, transportation, etc. Although animals still maintain many of those traditional uses around the world, the role of animals in modern society has changed tremendously. There has been a massive increase in the number of animals kept purely for companionship and pleasure (pet animals). The modern day definition of a pet or companion animal is an animal kept primarily for a person’s company, protection, or entertainment. Examples of companion animals are dogs, cats, horses, ferrets, rabbits, fish, gerbils, parrots, etc.

6.4 Companion animals and gene editing: scope and prospects

Table 6.2 Few examples of gene editing in livestock species. S. no.

Species

Technique

Purpose

References

1 2 3

CRISPR/Cas9 CRISPR/Cas9 CRISPR/Cas9

Viral resistance Hypoallergenic eggs High meat production

Hübner et al. (2018) Oishi et al. (2016) Kang et al. (2017)

4

Porcine Avian Bovine, caprine, and ovine Bovine

High milk production

Liu et al. (2013)

5

Ovine

Zinc finger nuclease (ZFN) CRISPR/Cas9

Li et al. (2017)

6

Bovine

CRISPR/Cas9

Higher wool production Reduction in lactose content of milk

Sun et al. (2018)

Gene editing is expected to have a huge impact in the veterinary world (Table 6.2). It is being widely applied to increase production (milk, meat, and wool) and improve nutritional quality. With the use of CRISPR/Cas9 system, pigs fully resistant to porcine reproductive and respiratory syndrome (PRRS) and African swine fever infection have been generated (Hu¨bner et al., 2018; Burkard et al., 2017). Further, TALEN has been used to generate pigs knockout for myostatin gene, thereby doubling the muscle mass (Kang et al., 2017). Using CRISPR/Cas method, Merino sheep knockout for fgf5 gene has been created to increase wool production (Li et al., 2017). Unlike livestock species, gene editing has entered the pet world recently. Scientists are focusing on the creation of super muscular dogs and horses with greater racing stamina and high muscle contractures. Work has also been started on improving pet owner relationships and deextinction of species using gene editing. Although, gene editing in the pet world is a new area of research and very less work has been undertaken in this field, more work is needed to be done. In this chapter, we will discuss the applications of gene editing in pets and its future prospects.

6.4.1 Super muscular dogs In present day, dogs are being used in police and military operations to investigate crime scenes, draw pieces of evidence, and inferences about the crime. To improve their stamina, scientists in China used gene editing to produce customized dogs (Fig. 6.1). They created a super muscular beagle dog with double muscular mass by deleting myostatin gene using CRISPR/Cas9 system (Zou et al., 2015). Myostatin is responsible for the negative regulation of skeletal muscle mass. Dogs with a knockout for myostatin gene roughly doubles muscle mass. Zygotes from pregnant females were collected and modified using

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FIGURE 6.1 (A) Muscular dog formed by gene editing. (B) Normal dog.

CRISPR/Cas9 to knockout myostatin gene. Edited zygotes were transferred back to females to give birth to super muscular dogs. The successful use of gene editing tools will help in creation of new varieties of dogs with favorable traits.

6.4.2 Micro pigs Pigs are intelligent and social animals that can be kept as pets. However, their bigger size and high food demand neglects them as pet. To achieve this, Beijing Genomics Institute (BGI) in China developed Bama micro pigs that can be used as a model for human diseases as well as pet animals (Fig. 6.2). These micro pigs weigh only 15 kilograms. TALENs were used to disable one of two copies of the growth hormone receptor gene in the fetal cells of Bama pigs (Cryanoski, 2015). With one receptor disabled, cells do not receive the full growth signal during development, resulting in stunted pigs. Males grown from the gene-edited fetal cells were used to breed normal Bama pig females. Half of the resulting offspring were micro pigs. Then, these micro pigs were used in breeding to develop the future generation of micro pigs. No adverse health effects have been observed in these second generation micro pigs.

6.4 Companion animals and gene editing: scope and prospects

FIGURE 6.2 (A) Micro pig produced by gene editing. (B) Normal pig.

6.4.3 Pet animals as disease model There are many diseases in companion animals that share genotypic and phenotypic similarities with human diseases. These diseases offer platforms to investigate the course of disease as well as to develop novel therapeutics. Ferrets have been used as successful model for human diseases. Ferrets share many similarities to humans in terms of physiological features of brain function, reproduction as well as pathological characteristics of diseases like cancer, influenza infection, and cystic fibrosis (Maher and DeStefano, 2004). Recently, Kou et al. (2015) developed a gene-edited ferret (knockout for dcx, disc1, and aspm genes) to study the brain development. On the same line, ferret models can also be developed to study diseases like influenza and cystic fibrosis. Similarly, geneedited dogs and cats can be generated as models for human cancers and inflammatory bowel diseases (Alvarez, 2014; Cerquetella et al., 2010) Moreover, dogs and horses can be used to develop models for musculoskeletal diseases (Kol et al., 2016). Further, gene-edited dogs and cats can serve as models for human genetic disorders (hemophilia, narcolepsy, severe combined immunodeficiency X-linked, cleft palate, Duchenne muscular dystrophy, lysosomal storage disease, etc.) and cardiovascular diseases (dilated cardiomyopathy, hypertrophic cardiomyopathy, arythmogenic right ventricular cardiomyopathy, etc.) (Kol et al., 2016). Recently, China-based biotech company, Sinogene, combined gene editing and cloning techniques to develop dogs with blood clotting disorder as a model for human atherosclerotic cardiovascular disease (Humphrey, 2018).

6.4.4 Other prospects of gene editing in pets • Gene editing has the ability to improve the emotional relationship between pet and owner. Since dogs have been part of human civilization for longer

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•

•

•

•

•

•

than any other species, gene editing is being exploited to improve dog and owner attachment. It has been shown that certain sequence variations (2213AG, 294TC, 274CG) in the gene of oxytocin hormone receptor in dogs are associated with high attachment with the owner. Recently, cats have become one of the world’s most popular pets. However, cats have a natural hunting behavior and people may get offended if their domestic cat brings flash of other animals into their house. With the cat genome published in 2014, scientists are trying to explore gene editing to control the hunting behavior of cats. Historically, horses have played a great role for human civilization and have been used in many different ways for travel, work, pleasure, sports, war, etc. Like the gene-edited dogs, work is in progress to develop horses with knockout myostatin gene. This will increase the muscular mass of the horses and these horses will have potential to better perform in sports due to high endurance and speed. Gene editing can serve in conserving the breeds/varieties of the pet species. There are many breeds of dogs (alpine mastiff, Russian tracker, southern hound, etc.), cats (Oregon rex, Mexican hairless cat, sumxu, etc.), and horses (bidet, angevin, jennet, etc.) that are near extinction. Scientists have successfully used gene editing to transfer genes of extinct woolly mammoths in Asian elephants to conserve some of their traits (Saplakoglu, 2018; Lynch et al., 2015). Hence, such tools can be used to conserve the endangered pet breeds. A deextinction project for passenger pigeon is in progress, and scientists are comparing genomes of the passenger pigeon with band-tailed pigeon. After comparison, the genome of the band-tailed pigeon will be edited to develop a new generation of passenger pigeons. We expect that gene editing will improve the health of pets. Similar to the findings of the genome editing for human and livestock diseases, control and therapeutic techniques can also be developed for pet diseases. Cancer is common in dogs and accounts for almost 50% of all mortality (Alvarez, 2014). Therefore strategies like programmed cell death as described for human cancers can also be developed for pet animals. The gene editing tools used to counter infectious diseases like PRRS and African swine flu in pigs can also be extended to pet animals. The first and the most common expectation of owners is a longer lifespan for their pets. Genome and epigenome editing are being used to uncover novel molecular pathways involved in aging and aging-related diseases. This will help in the developments of new therapeutic interventions for extending health span and lifespan. Work is in progress to develop dogs that are not prone of hip problems. Further, gene editing holds the potential to develop designer companion animals. BGI is trying to alter the size, color, and patterns of koi carp fish using CRISPR to make fish with desired patterns more suitable for home aquariums (KOI BITO Forum).

References

6.5 Conclusion The potential of genome editing in improving pet/companion animals has recently been recognized. Tools have been optimized to modify the genome of pet species for the targeted alternations. It is expected that in the future, gene editing tools will help make the lives of our pets more comfortable, healthier, and longer. Furthermore, pets with desired properties like color, low aggression, more emotional attachment, disease resistance, higher stamina, etc. can be generated based on the personalized requirement of an owner. However, it is important to discuss the ethical issues of gene editing because the benefits of any technology should be much higher than the risks. Gene editing may involve risks of off-target mutations that can be deleterious. Larger genomes may contain DNA sequences with identity or homology to the intended target sequence, so gene editing tools can target these sequences leading to cell death. Another important problem is the efficient, safe delivery of CRISPR/Cas9 into cell types or tissues that are hard to transfect and/or infect. The critical issue is the patenting of the organisms. Transgenic organisms have been patented when they have an industrial use. However, patenting the gene-edited germ lines of animals will be accepted or it will not be. Ethical considerations should also be on ground of equality. There is a concern that genome editing will only be accessible to the wealthy community. Hence, there is a need to develop a global consensus on the use of genome editing tools with a defined line of controls and boundaries.

Conflict of interest There is no conflict of interest.

Acknowledgements All the authors of the manuscript thank and acknowledge their respective universities and institutes.

References Alvarez, C.E., 2014. Naturally occurring cancers in dogs: insights for translational genetics and medicine. ILAR J. 55, 16 45. Baker, M., 2011. Gene Therapy Net (website). Available from: ,http://www.genetherapynet.com/gene-editing-tools/talen.html.. Barrangou, R., 2015. The roles of CRISPR-Cas systems in adaptive immunity and beyond. Curr. Opin. Immunol. 32, 36 41.

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Beissona, Y.L., Peltier, G., 2013. Third-generation biofuels: current and future researchon microalgal lipid biotechnology. OCL 20, D606. Bibikova, M., Golic, M., Golic, K.G., Carroll, D., 2002. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161, 1169 1175. Bibikova, M., Beumer, K., Trautman, J.K., Carroll, D., 2003. Enhancing gene targeting with designed zinc finger nucleases. Science. 300, 764. Burkard, C., Lillico, S.G., Reid, E., Jackson, B., Mileham, A.J., Ait-Ali, T., et al., 2017. Precision engineering for PRRSV resistance in pigs: macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function. PLOS Pathog. 13, e1006206. Available from: https:// doi.org/10.1371/journal.ppat.1006206. Carroll, D., 2011. Genome engineering with zinc-finger nucleases. Genetics 188, 773 782. Cathomen, T., Joung, J.K., 2008. Zinc-finger nucleases: the next generation emerges. Mol. Ther. 16, 1200 1207. Cermak, T., Doyle, E.L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., et al., 2011. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82. Cerquetella, M., Spaterna, A., Laus, F., Tesei, B., Rossi, G., Antonelli, E., et al., 2010. Inflammatory bowel disease in the dog: differences and similarities with humans. World J. Gastroenterol. 16, 1050 1056. Cryanoski, D., 2015. Gene-edited ‘micropigs’ to be sold as pets at Chinese institute. Nature. 526, 18. Available from: https://doi.org/10.1038/nature.2015.18448. Dewitt, M.A., Corn, J.E., Carroll, D., 2017. Genome editing via delivery of Cas9 ribonucleoprotein. Methods 121-122, 9 15. Gaj, T., Gersbach, C.A., Barbas, C.F., 2013. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31, 397 405. Gasiunas, G., Barrangou, R., Horvath, P., Siksnys, V., 2012. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci.U.S.A. 109, 2579 2586. Ghorbal, M., Gorman, M., Macpherson, C.R., 2014. Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system. Nat. Biotechnol. 32, 819 821. Gonzales, A.P., Yeh, J.R., 2014. Cas9-based genome editing in Zebrafish. Methods Enzymol. 546, 377 413. Grissa, I., Vergnaud, G., Pourcel, C., 2007. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 35, 52 57. Guo, J., Gaj, T., Barbas, C.F., 2010. Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases. J. Mol. Biol. 400, 96 107. Hakata, M., Wada, H., Masumoto-Kubo, C., Tanaka, R., Sato, H., Morita, S., 2017. Development of a new heat tolerance assay system for rice spikelet sterility. Plant Methods 13, 34. Available from: https://doi.org/10.1186/s13007-017-0185-3. Hsu, P.D., Lander, E.S., Zhang, F., 2014. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 157, 1262 1278. Hu¨bner, A., Petersen, B., Keil, G.M., Niemann, H., Mettenleiter, T.C., Fuchs, W., 2018. Efficient inhibition of African swine fever virus replication by CRISPR/Cas9 targeting of the viral p30 gene (CP204L). Sci. Rep. 8, 1449. Available from: https://doi.org/ 10.1038/s41598-018-19626-1.

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CHAPTER

Modulation of animal health through reverse genetics applications

7

Hitesh N. Pawar1, Namita Mitra2 and Ramneek Verma3 1

Department of Diagnostic and Biomedical Sciences, School of Dentistry, University of Texas Health Science Center at Houston, Houston, TX, United States 2 Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States 3 Department of Microbial and Environmental Biotechnology, College of Animal Biotechnology, Guru Angad Dev Veterinary And Animal Sciences University, Ludhiana, Punjab, India

7.1 Introduction The science of genetics came into existence with the works of Augustinian monk Gregor J. Mendel on pea plants in 1866. His thoughtful experimentation provided the first ever groundwork for modern genetics that concluded with opine that traits are carried by “discrete units” which are particulate in nature. Around the same time, Ernst Haeckel proposed that factors responsible for transmission of hereditary traits are contained in the nucleus. In 1869, Friedrich Miescher showed this hereditary material in the nucleus and termed it “nuclein” which was later replaced by the term “nucleic acid” coined by his pupil Richard Altmann. It took almost 73 years after that to show that DNA, and not proteins or RNA, is the genetic material; made possible by the works of Oswald Avery, Colin MacLeod, and Maclyn McCarty, thus clarifying the chemical nature of genes. After another decade, in 1953, James Watson and Francis Crick made a historic discovery about the threedimensional structure of DNA and described it as a double helical structure held together by hydrogen bonds. Their findings and predictions were largely based on the data from X-ray crystallography work on DNA by Maurice Wilkins and Rosalind Franklin (Maddox, 2002). Just a few years later in 1957, Francis Crick elucidated the concept of “Central dogma of molecular biology.” By this time, DNA had become an enigmatic molecule and the researchers around the world yearned to play with it to find out the long-kept secrets at the molecular level of cell. In the following years, many important molecular concepts like DNA replication, operon systems for regulation of protein synthesis, genetic code, restriction nucleases, and many more were explained that would provide the crucial tools for molecular biology and recombinant DNA technology. The year 1977 marked the development of two rapid DNA sequencing methods namely chemical cleavage method (Maxam and Gilbert) and chain termination method (Sanger), respectively. Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00007-2 © 2020 Elsevier Inc. All rights reserved.

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Hence, genomes from different species could now be compared. Sanger’s method was comparatively less onerous and is therefore widely used even today. A revolutionary breakthrough in the field of molecular biology was the invention of polymerase chain reaction (PCR) by Karry Mullis in 1983 which allows to quickly make billions of DNA copies within a few hours, one of the most commonly used technique in molecular biology. Later, the researchers around the world were increasingly obsessed with finding “genetics”, that is, underpinning the genes responsible for specific phenotypes; however, in present times technological advances and increased affordability in high throughput sequencing have greatly accelerated the accumulation of genetic data leading to the circumstances where the situation has nearly “reversed,” more precisely, scientists are now ever more concerned with discovering the function of an already known gene sequence. “Reverse genetics” in simple words, refers to the method employed for discovering the function or phenotypic effect of already known DNA or protein sequence. This is in contrast to “classical or forward genetics” where the phenotype is known and methods are employed to determine the gene responsible. In other words, forward genetics is going from phenotype to genotype and reverse genetics is going from genotype to phenotype. In this era of automated sequencing, genomic data are heaping at a rapid rate with very little known about their functional characteristics, and reverse genetics is being explored to address this problem. In this chapter, we will discuss the major techniques used in reverse genetics with intended emphasis on advanced methods and their applications in veterinary health.

7.2 In vitro mutagenesis A variety of genetic, molecular, and biochemical methods are often required to create and investigate alteration in a specific region of DNA, mainly protein coding region. Although gene cloning and protein expression can give a rough idea about the protein and its structure but the relationship between protein structure and its mode of action is the major obstacle in functional genetic studies. To tackle this problem, the best way is to create a mutation in the functional gene, and a subsequent effect of change in coding amino acid can be determined by evaluating properties of translated proteins. Mutation in a gene can also occur in natural process but screening of whole genome to identify mutation is cumbersome. The technique by which mutations are purposely created in gene sequence to study functional protein is termed as in vitro mutagenesis. It can be achieved in three different ways. Firstly, deletion: removing one or few nucleotides from DNA sequence; secondly, insertion: addition of nucleotides in given coding region of DNA; and thirdly, substitution: exchange of nucleotides. Many different methods have been used for in vitro mutagenesis, most common of which are chemical mutagenesis, PCR-mediated mutagenesis and viral vector-mediated mutagenesis. Chemical method is one of the earliest techniques developed for creating mutations and is achieved via injecting a mutagenic reagent into the animal

7.3 RNA interference

such that it passes directly into the gonads and into differentiating germ cells to create random mutations. However, this method has not found reasonable success in animal genome editing, except for few lab models (Drosophila and Zebrafish), owing to the nonspecific nature of mutations it results in. Later a more flexible approach, site-directed mutagenesis, was devised to create specific and intentional changes to the DNA sequence of a gene. Also known as PCR-mediated mutagenesis or oligonucleotide-directed mutagenesis, it can be used for shorter DNA segments and one mutation per experiment can be achieved by this method. This method requires two-step amplification of DNA sequences—in each step of PCR amplification, one of two PCR primers is normal while the other one contains the desired mutation (forward primer in one step and reverse primer in other step), with either single base mismatch or multiple base mismatches (1
3 base mismatch). After PCR reactions, both amplified products are pooled in one tube and final PCR is carried out with two complimentary mutated PCR primers from previous reactions to get full length DNA molecule. To scan for the desired mutation, all amplified DNA needs to be cloned and sequenced each time. This procedure is relatively quick and easy to carryout but has limitation of use of DNA polymerase which causes high error rate (0.8
1.1 3 1024 base substitutions/bp of product) as this enzyme lacks 30
50 exonuclease activity, as a result a number of clones have to be scanned in order to find the desired mutant (Kunkel and Bebenek, 2000). Another simple approach is Kunkel’s method, based on phage vector M13, which reduces the need to select for the mutants. Here, uracil-inserted, circular, single-stranded DNA is utilized as a template to synthesize double-stranded DNA (dsDNA) in vitro with an oligonucleotide primer that introduces a desired mutation. After introducing these dsDNA into Escherichia coli, recombinant clones predominate due to cleavage of the uracilated strand in vivo. Kunkel mutagenesis is particularly useful in phagedisplay experiments that are based on M13 bacteriophage, as this viral particle contains a circular, single-stranded genome. While there are numerous methods for achieving site-directed mutagenesis, decreasing costs of oligonucleotide synthesis has paved the way for artificial gene synthesis which is now frequently used as a substitute to site-directed mutagenesis. In this approach, a series of partial overlapping oligonucleotides are synthesized commercially for the desired sequence intended at the target loci. The partial gaps are then filled through amplification with DNA polymerase, to create the complete double-stranded gene. In this DNA synthesis, restriction enzymes are included to create sticky ends and then ligate into a cloning vector.

7.3 RNA interference RNA interference (RNAi) was initially discovered as a natural phenomenon in eukaryotes to regulate endogenous genes and to protect integrity of genome from viruses, transposons, and other intruders. Research on demonstration of antisense

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RNA to inhibit gene expression started with the works of Light and Molin (1982, 1983) in prokaryotes, and eventually the phenomenon was elucidated by Fire and coworkers in 1998 through experimental introduction of dsRNA in nematode Caenorhabditis elegans. Andrew Fire and Craig C. Mello were awarded the 2006 Nobel Prize in Physiology/Medicine for the first identification of etiology of the RNAi phenomenon. RNAi refers to the use of sequence-specific dsRNA to neutralize targeted mRNA molecules in order to suppress gene expression. The phenomenon is also called by names quelling (in fungi); co-suppression/ virus-induced gene silencing/posttranscriptional gene silencing in plants; and gene-knockdown in eukaryotes. Three classes of small RNAs can regulate genes by targeting transcripts in the cytoplasm: (1) micro-RNA (miRNA) that are hairpin-structures with imperfect complementarity to target transcripts and cause translational repression; (2) small interfering RNA (siRNA) with perfect complementarity to targets and cause transcript cleavage; and (3) piwi RNA (piRNA) which target transposon transcripts in animal germlines. Imperfect compatibility of miRNA with the mRNA allows it to silence hundreds of target molecules but in contrast siRNA can silence only specific mRNA molecules on account of its perfect compatibility. miRNAs are chiefly endogenous in origin, that is, they are conscious products of the organism’s own genes. On the other hand, siRNAs are mostly exogenous in origin, derived from foreign or invasive nucleic acids such as viruses, transposons, and transgenes; however, few endogenous siRNA transcripts have also been identified. miRNAs and siRNAs are characterized by the double-stranded nature of their precursors. In contrast, piRNAs derive from precursors that are poorly understood but appear to be single stranded (Malone and Hannon, 2009). Prokaryotic gene expression is regulated by a similar RNA-based system, but which is not identical to RNAi because here the dicer enzyme is not involved. Despite differences, miRNA and siRNA [both B21 2 23 nucleotides (nts)] work in a similar manner with the help of Dicers and Argonaute proteins (AGOs) (Fig. 7.1). RNA polymerase II, inside the nucleus, transcribes primary transcript of miRNA (pri-miRNA). In animals, processing of primary miRNA (pri-miRNA) to mature miRNA takes place in two stages, by the actions Drosha in the nucleus and Dicer in the cytoplasm (Kim et al., 2009). Pri-miRNA is processed to form 65
75 nts long pre-miRNA by a microprocessor complex composed of enzyme Drosha (a class 2 RNase III enzyme) and cofactor DiGeorge syndrome critical region 8 in humans; or cofactor Pasha in C. elegans and Drosophila melanogaster (Denli et al., 2004). Both pri- and pre-miRNAs are hairpin-looped structures. Pre-miRNAs are transported to cytoplasm by Exportin 5 in the presence of the Ran-GTP cofactor that hydrolyzes to Ran-GDP for unidirectional release of premiRNAs. In cytoplasm, another double strand-specific RNase III family enzyme, Dicer, cleaves pre-miRNA into one or more functional miRNA duplexes about 22 nts long. Dicer also cleaves dsRNA into functional siRNA duplexes in cytoplasm. One miRNA/siRNA duplex is incorporated into AGO protein to form an effector complex from which one strand known as passenger strand is degraded while the

FIGURE 7.1 Schematic representation of basic mechanism or pathway for RNA interference (RNAi) in the cell.

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other strand (guide strand) remains bound to AGO as mature miRNA/siRNA. This mature small noncoding RNA is then loaded on to RNA-induced silencing complex to guide the complex to its target mRNA, which is silenced through either translation repression by miRNA, or mRNA degradation by siRNA. However, miRNA can also be responsible for significant reduction in mRNA abundance through mRNA degradation (Bagga et al., 2005). Experimentation has proved RNAi to be a very powerful and prospective gene silencing technology and consequently several knockdown clinical trials are on the way to come into a therapeutic and diagnostic reality. siRNA and short hairpin RNA (shRNA) are the most commonly used synthetic RNA molecules for silencing. Target specificity, which siRNAs and shRNAs provide, has made them promising knockdown molecules in medical applications. shRNA is an artificial, single RNA molecule that is self-complementary resulting in a tight hairpin structure similar to miRNA. Expression of shRNA in cells guided by appropriate promoters is typically accomplished by delivery of plasmids or through viral or bacterial vectors. shRNA or miRNA can be delivered to create genetically modified animals with stably inherited RNAi transgene that can subsequently pass on to generations of animals derived from the original founder animal. On the other hand, use of siRNA would only result in a transient alteration of gene activity. Other synthetic antisense molecules that are being explored and have shown potential are saiRNA (Ribozyme-enhanced single-stranded Ago2-processed interfering RNA) (Shang et al., 2015), shRNAmir (Fellmann et al., 2013; Premsrirut et al., 2011; Stegmeier et al., 2005; Zhu et al., 2007), Morpholino antisense oligonucleotide, miRNA inhibitors (antimiRs), and miRNA mimics (Van Rooij and Kauppinen, 2014). RNAi has an advantage over classical vaccines and small drug molecules in that it is highly selective for the pathogen and can be made in bulk very quickly. By comparison, it can take months to screen the vast number of candidate small drug molecules against a new virus or to develop and test vaccines. Moreover, effective vaccines and drugs are lacking against certain viruses particularly RNA viruses that alter their antigen makeup quite frequently over time through antigenic drifts or mutations. In this context, miRNAs may be explored as suitable therapeutic candidates attributable to their capacity of binding multiple targets with partial complementarity and, thus, likely to tackle the high rate of mutation. Besides animal therapeutics, these knockdown molecules also represent potential prognostic tools as disease biomarkers, diagnostic tools for disease detection, and, to create genetically disease resistant animals. Genome-wide libraries of double-stranded RNAs in C. elegans (Kamath and Ahringer, 2003) and Drosophila (Kuttenkeuler and Boutros, 2004) genomes have been created which target every gene in respective organism for a systematic analysis of gene function by generating loss-of-function phenotypes. Genome wide siRNA screening experiment against vesicular stomatitis virus (infects cattle, pigs, and horses) in HeLa cells identified 23 genes that reduced virus replication when silenced, without affecting cell viability (Lee et al., 2014). Knockdown of one or more of these genes can lead to an important therapeutic treatment.

7.3 RNA interference

With more advances in this field, transgenic animals expressing shRNA targeting disease agents have been produced and have shown reasonable inhibition of disease. These include a goat expressing shRNA targeting the prion protein of transmissible spongiform encephalopathy (Golding et al., 2006); a calf expressing an antiprion shRNA, (Wongsrikeao et al., 2011); pigs expressing antiPRRS (porcine reproductive and respiratory syndrome) virus shRNA (Li et al., 2015a,b) and porcine endogenous retrovirus (PERV) shRNA (Ramsoondar et al., 2009); mice expressing two antiFMDV shRNA (Chang et al., 2014), pigs expressing antiFMDV shRNA (Hu et al., 2015) and goats expressing antiFMDV shRNA (Li et al., 2015a,b). Bovine viral diarrhea virus, African swine fever virus, classical swine fever virus, highly pathogenic avian influenza, chicken anemia virus, and infectious bursal disease virus are other economically important veterinary pathogens that have been shown to be inhibited by RNAi either in vitro or in vivo (Bradford et al., 2016). RNAi approaches have also been explored explicitly to target disease vectors and target genes responsible for colonization of insect vectors. One such approach is the use of RNAi-based sterile insect technique (SIT) to biologically control pest insects that heavily damage agriculture/forestry and cause significant losses to the livestock industry through either disease transmission like trypanosomiasis, or maggot infestation in animal wounds. Conventional SIT is based on sterilizing the insect males through radiation and releasing overwhelming numbers of these sterilized males. Sterile males compete with the wild males for female insects, thus reducing the next generation’s population. Whyard et al. (2015) demonstrated that feeding sex-specific dsRNA to larvae of the dengue vector Aedes aegypti halted sperm production in adult males, producing substantially sterile males. Therefore, sterilizing males by administering RNAi through food sources or by transgenics could provide sustainable and an environmentally-friendly approach to controlling pests. Other studies employing combined cancer immunotherapies with immunomodulatory RNAi targeting immunosuppressive factors/genes have resulted in amplified immune responses with improved clinical effects (Sioud, 2014). Chronologically, laboratory-based experiments of using RNAi to block the infectious agents started in 2003 against influenza A virus (Ge et al., 2003). However, even today, RNAi approaches in animal infections are in the early stages of development but nevertheless, there have been positive experimental demonstrations that assure promising RNAi therapeutics in the near future. It is now widely acknowledged that RNAi can be an efficient way to reduce gene activity. However, as with most emerging technologies, some concerns remain to be overcome. The use of RNAi technologies can arouse the internal immune response within an animal which in extreme situations can lead to cell death. Another correlated concern is the possibility of off-target effects through weak interactions of the RNA molecules to messenger RNAs transcribed from nontarget genes. Apparently if this occurred in vivo, the effect could be quite severe. Random insertion of the shRNA construct into the genome can lead to

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problems including overloading of the RNAi machinery (Grimm et al., 2006) or complete exclusion of a required gene product. At the same time, in vivo delivery of siRNAs into cells remains a significant obstacle for the use of RNAi in throughput studies. It is also laborious, can give dubious results, and can be unsuitable for isolating mutants that have lethal phenotypes (Gilchrist and Haughn, 2005). Newer precision genome engineering tools such as zinc finger nucleases (ZFNs), clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based, RNA-guided endonucleases, and transcription activator-like effector nucleases (TALENs) can be used to avoid some of these problems.

7.4 Targeted genome modification by homologous recombination Homologous recombination (HR) is a natural phenomenon in animals that produces exchange of genes during meiosis in order to create gamete cells. This recombination event generates genetic variation amongst the next generation. This phenomenon is also used by the cells to precisely repair deleterious doublestrand breaks in DNA. In reverse genetics, this technique has been widely exploited to produce mutations in target genes. Gene targeting based on HR is a genetic technique that enables the disruption or mutation of an endogenous gene/ allele through site-specific integration of exogenous DNA (Fig. 7.2). The loss-offunction or altered phenotype created as a result of disruption/mutation of the gene in question will help to identify its unknown function. It has been firstly reported in Saccharomyces cerevisiae, in which specific deletion of a gene was replaced by antibiotic resistant (selection marker) gene. HR has been widely used to inactivate thousands of genes in most of the species, majorly in flies and mice. It also has a critical role in implementation of nuclease-based genome editing techniques. This deletion of target gene and insertion of selection marker is often known as gene “knock-out.” Similarly, HR can also be used to introduce in vitro modified sequence in endogenous allele of target species or cells, this is referred as gene “knock-in.” This knock-in genetic engineering technique is used to (1) insert specific tag coding sequences and the open reading frame (ORF) to isolate knock-in organisms or cells, (2) replace wild type gene by mutated gene to study consequences of gene mutations (Hardy et al., 2010). Similarly, vector-mediated gene targeting is also achieved by circular and linear DNA molecules in which the ends point outwards when hybridized with target sequence was referred as “ends-out” and when hybridized within the region named as “ends-in.” This targeted gene insertion in the genome provided a powerful approach to evaluate the function of several genes in model organisms. Just like all other techniques it has some reasonable limitations, first, spontaneous integration of an exogenous DNA sequence is very low (1 in 105
109 cells); second, modified

FIGURE 7.2 The basic working principle of major nuclease-based genome-editing technologies. Zinc finger nuclease recognizes triplet sequence code while each TALE recognizes an individual base. Similarly, simple RNA to DNA base pairing and the PAM sequence determine CRISPR targeting specificity. All these techniques result in double-strand breaks in DNA sequence, which are subject to be repaired either by nonhomology end joining (NHEJ) or homologous recombination (HR)-directed repair. While NHEJ results in random insertion and gene disruption at the target DNA, HR can be attached to insert a donor DNA template at the target site for precise gene editing.

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gene integration rate depends upon cell type and cellular state; and third, random insertion rate of exogenous sequences in undesired locus is similar or a higher rate than the target site. To overcome these limitations several strategies had been developed, the first one is the use of endonucleases often referred to as meganucleases which recognize 14
40 bp target sequences to make double-stranded breaks. Although each nuclease has unique recognition sites, the probability of finding right nucleases for target sequence is very low, and the majority of double-stranded breaks are through error-prone nonhomologous end joining (NHEJ). In recent times, new nucleases are introduced in genetic engineering field, ZFNs, TALEN, and CRISPR/Cas9 nucleases that can induce DNA doublestrand breaks with specificity for targeted sites, and these breaks can be efficiently repaired by HR.

7.5 Nuclease-based reverse genetics tools 7.5.1 Zinc finger nuclease Zinc finger (ZF) motif was discovered in 1985 as an integral part of transcription factor (TF) IIIa in xenopus oocytes that had specific binding affinity to DNA. The classical ZF is a compactly folded globular structure consisting of two cysteine and two histidine residues (Cys2His2) in each repeated unit of 28
30 amino acids bound to a single zinc atom. This structure has a polypeptide backbone fold consisting of two conserved antiparallel β-sheets forming a hairpin opposing conserved α-helix structure. Each finger uses this α-helix residue to bind to the major groove of the DNA by three specific base pairs (Pavletich and Pabo, 1991). By combining multiple ZF motifs a larger DNA recognition domain can be generated with enhanced target DNA binding specificity and this forms the basic idea behind the ZFN technology. In 1996, the first ZFNs were created (Kim et al., 1996). These are synthetic chimeric proteins consisting of site-specific ZF DNAbinding domain at the amino terminus fused to the nonspecific cleavage domain of the FokI endonuclease at the carboxyl terminus. ZFN has a modular structure, because each ZF domain recognizes one nucleotide triplet. ZFNs can recognize DNA sequences 9
18 bps in length and are used for gene targeting by introducing insertions or deletions at cut sites in the genomes of living cells. These can be designed for any given DNA sequence. FokI endonuclease must dimerize for efficient double-strand cleavage and hence, it requires two ZFNs to target any specific locus in the genome (Fig. 7.2). A pair of two different ZFNs binding to nonidentical sites also increase target specificity. The two ZFN molecules bind to the targeted DNA in a tail-to-tail orientation separated by 5
7 bp, with dsDNA breaks occurring in the spacer region (Shimizu et al., 2009). After breakage at specific locus, the linear dsDNA is repaired by one of two highly conserved processes: NHEJ, which often results in small insertions or deletions and can be exploited for gene disruption, and HR,

7.5 Nuclease-based reverse genetics tools

which can be used for gene insertion or replacement (Fig. 7.2). Previously genomic integration of ZFN sequences and donor DNA were achieved by p-elementmediated transformation until 2008, when high efficiency homologous and non-HR was achieved by injecting ZFN mRNAs and donor DNA into the embryos (Beumer et al., 2008). This method became the basis for editing cultured cells, including pluripotent stem cells, plants, and animal models. Despite a theoretical possibility to target any specific sequence, ZFN approach has in fact a few major disadvantages including the complexity and high cost of protein domains construction for each genome locus and the probability of inaccurate cleavage of target DNA due to single nucleotide substitutions or inappropriate interaction between domains. Therefore, an active search for new methods for genome editing was continued.

7.5.2 Transcription activator-like effector endonucleases The TALEN system, another precise genome editing method, was developed subsequently and was named as method of the year in 2011 by Nature Methods, attributable to relative constructional simplicity and high efficiency (Becker, 2012). History of transcription activator-like effector (TALE) system was natively identified in Xanthomonas genus, a phytopathogenic gram-negative bacteria. These bacteria can infect a wide variety of plant species and cause significant economic losses in agricultural industry. In plants, these bacteria secrete some effector proteins commonly known as, transcription activator-like effectors, TALEs, via their type III secretion system into the cytoplasm of cells that affect certain cellular processes in the plants and make them more susceptible to these pathogens. After thorough investigation of their effector proteins it was revealed that these proteins are capable of binding to host DNA and later acting as TFs to trigger the expression of their target genes that result in increased susceptibility to bacterial infection. To encounter these TALEs, plants have developed a defense mechanism that comprises of certain genes activated by these effectors themselves. Some of these genes appear to have evolved to contain TAL effector binding sites similar to the sites in intended target genes (Voytas and Joung, 2009). This defense mechanism accounts for flexibility to manipulate the TAL effector DNA-binding domain. TALE consists of a central repeat domain responsible for modular DNA binding, a nuclear localization signal which directs a TALE to the nucleus, and a domain composed of TALE proteins that activates the target gene transcription. The DNA-binding domain consists of monomers and each monomer binds to one nucleotide in the target nucleotide sequence. Monomers are tandem repeats of highly conserved 33
34 amino acid residues, of which two amino acids at positions 12 and 13 are highly variable (repeat variable diresidue, RVD), and are responsible for the recognition of a specific nucleotide (Moscou and Bogdanove, 2009). In some cases, RVDs can bind to several nucleotides with variable efficiencies. Thus, a specific DNA sequence can be targeted by selecting a combination of

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repeat segments that contain the appropriate RVDs. As a result, TALEs have attracted great interest as a genome editing tool. Before the 50 -end of a sequence bound by a TALE monomer, the target DNA molecule always contains a thymidine molecule that has a strong correlation with the binding efficiency. To generate a chimeric TALEN protein, TALE is fused to the catalytic domain of FokI endonuclease to create double-stranded breaks in target DNA sequence for genome editing. Commonly used RVDs to target specific DNA sequence include NI (asparagine
isoleucine), NN (asparagine
asparagine) for adenine; NG (asparagine
glycine) for thymine; HD (histidine-aspartic acid) for cytosine and NK (asparagine
lysine), NN, NH (asparagine-histidine) for guanine. As discussed in the ZFN section, FokI cleaves as a dimer, therefore these TALENs function in pairs, binding opposing targets across a spacer region over which the FokI nuclease domains come together to create the break (Fig. 7.2). Double-strand breaks are repaired in nearly all cells by either homologous or non-HR. ZFNs and TALENs differ in three main aspects: (1) TALE repeats are three to four times larger than ZFNs, per base pair of the DNA; (2) the gap between two binding sites (spacer length) is variable, which complicates TALEN design and could lead to a greater off-target activity relative to an identical nuclease with a fixed spacer length; (3) ZFN assembly requires well-characterized modules to achieve specific gene targeting because crosstalk between the individual fingers can lead to imperfect DNA recognition (Defrancesco, 2011). In ZFNs contextdependent effects between the repeat units are reported that have not been reported for TALENs. Various assembly methods have indicated that TALE repeats can be combined to recognize potentially any target sequence, the only limitation is that TALE binding sites must start with thymidine. Nevertheless, TALEN has an advantage over ZFN in terms of its simplicity, costs, and straightforwardness in design and assembly strategies (Peterson, 2017).

7.5.3 Clustered regularly interspaced short palindromic repeats and its associated gene 9 CRISPR and its associated gene 9 (Cas9), discovered just 2 years after the discovery of TALEN, has revolutionized the genome editing arena in a short span of time. The CRISPR/Cas9 system has greater advantage over the ZFNs, TALENs, and other endonucleases in that it uses simple guide RNA to confer target specificity which is very simple to design and construct, unlike ZFNs and TALENs which require modules of repeat domains. Earlier in 1987, researchers identified unknown short and unique repetitive DNA sequences (B20
40 bp) in E. coli called as repeats, separated by spacer regions (nonrepetitive sequences) (Ishino et al., 1987). During that time the function and purpose of these repeats were unresolved. Nearly two decades later, sequencing studies of many other bacterial genomes identified similar repetitive nucleotide sequences with a unique structure in which these short palindromic repeat sequences were separated by

7.5 Nuclease-based reverse genetics tools

nonrepeating DNA sequences (spacers) and also that these interspaced repeats were in close proximity to Cas gene which has helicase and nuclease activity (Jansen et al., 2002; Haft et al., 2005). Far along it was identified that these spacer DNA sequences were homologous to the DNA of many plasmids, transposons, and bacteriophages. The first direct evidence about the function of CRISPER/Cas9 system was provided by bacteriophage challenge experiments in Streptococcus thermophilus where it was demonstrated that bacteria acquire new spacer sequences in their genome, originally derived from the phage genome, complementary to the bacteriophage genomic DNA fragments to direct target specificity of Cas enzymes which provide protection from viral infection (Barrangou et al., 2007). This study showed that CRISPR/Cas9 system is a unique adaptive immune system of bacteria to protect them from foreign DNA infiltration and limit the modification of bacterial genome by bacteriophages. The CRISPR-mediated interference is implemented in three main steps: (1) spacer acquisition, (2) crRNA (CRISPR RNA) transcription and its maturation, and (3) target identification and cleavage. Foreign nucleic acid sequence is identified during the spacer acquisition and processed into short, spaced sequences that are inserted into CRISPR cassette, to be flanked by a pair of repeat sequences. Then CRISPR array is transcribed and processed into small mature RNAs, crRNAs. Each crRNA contains repeated sequences and a single spacer that identifies the complimentary target nucleic acid sequence. The crRNA is complexed with Cas protein (sometimes more than one protein) and additional RNAs to cleave the target sequence. The classification efforts are still to complete for the CRISPR system as continuous experimental findings are heaping to refine the current classification. As of now, the CRISPR system is broadly categorized into two classes: class 1 system with multisubunit Cas protein complex, and the class 2 system with single Cas protein (Cas9 or Cpf1) in the crRNA-effector complex. These two classes include six system types classified based on the content of Cas genes, CRISPR repeats length and sequence, crRNA biogenesis, and effector protein activity (Makarova et al., 2015; Koonin et al., 2017). Class 1 includes type I, type III CRISPR systems, and type IV CRISPR system that includes rudimentary CRISPR-Cas loci which lack the spacer adaptation whereas, class 2 containstype II, V, and VI CRISPR systems. The Cas1 and Cas 2 genes are universally present in all types of CRISPR systems, however, some signature Cas genes distinguish the types from each other (Cas3 for type I; Cas9 for type II; Cas10 for type III; Cpf1 for type V). Class 1 system shares similarities in all stages of CRISPR immunity (acquisition, expression, and cleavage). Type I system use multiprotein complex and type III system uses Cmr/Csm complex to recognize and cleave foreign DNA as well as RNA when guided by crRNA. Both systems have highly palindromic CRISPR repeats which form hairpin loops and allow Cas6, ribonuclease to break the pre-crRNA into individual targeted crRNAs. Class 2 system uses a single signature protein for binding and cleavage of foreign nucleic acids by type II and type V systems (Koonin et al., 2017). Type V

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system uses crRNA with partial repeat and complete spacer sequences and signature protein Crf1 to identity, bind, and cleave foreign nucleic acid sequences (either DNA or RNA). On the other hand, type II system uses signature Cas9 protein to recognize, bind, and cleave foreign genetic material with an additional RNase III activity generated RNA molecule required to make complex with crRNA. The second RNA molecule that complexes with crRNA is transactivating RNA (tracrRNA) which is required for biogenesis of crRNA and Cas9 guided interference against foreign nucleic acid sequences. In experimental systems used for CRISPRmediated genome editing, these two small RNAs (crRNA and tracrRNA) are condensed into one RNA sequence known as the guide RNA (gRNA) or single-guide RNA (sgRNA). Type II CRISPR/Cas9 system from Streptococcus pyogenes (spCas9) is the most widely used system by researchers, because of its simple PAM (protospacer adjacent motif) sequence requirements, that is, any nucleobase followed by two guanine nucleobases. Cas9 and recently identified Cpf1 are two of the most commonly used effector proteins in CRISPR/Cas system, though exploration for other CRISPR-effector proteins is still ongoing. Cpf1 has the advantage that it generates 50 overhangs rather than the blunt ends produced by Cas9 protein. Type IV and type V are the rarest types of the CRISPR/Cas system, which constitute less than 2% of bacterial and archaeal genome. As discussed above, multiple systems have been identified in prokaryotes with type I system being the predominant one in bacterial and archaeal genome (B60%), but Type II CRISPR/Cas9 system obtained more popularity and is the vastly used system so far because of its simple functional architecture. As mentioned before, three major steps are involved in this acquired immune system (CRISPR/Cas9) mechanism of bacteria and archaea. Step1: adaptation or spacer acquisition; a small fragment of foreign DNA that entered a bacterial cell is inserted into the CRISPR locus of the host genome, forming a new spacer. In the viral genome, this fragment is present as a protospacer that is complementary to the spacer and flanked by a short (2
5 bp), conserved sequence called PAM. The new spacer is always inserted on the AT-rich side of the leader sequence located before a CRISPR cassette that also contains promoter elements and landing sites for regulatory proteins. Apparently, this is the way the targets of most of the CRISPR/Cas systems are formed. Step 2: transcription, the entire CRISPR locus is transcribed into a long pre-crRNA (poly-spacer precursor crRNA). The processing of an immature transcript into mature crRNA in most of the CRISPR/Cas systems is implemented by Cas6 endonuclease. Short crRNAs (39
45 nucleotides) contain one spacer sequence, and their ends contain repeats involved in the formation of the stem loop structure with 50 ends forming the stem and 30 ends forming the loop. Step 3: target identification and cleavage, the resistance to foreign DNA or RNA, is provided by the interaction between crRNA and a complex of Cas proteins; crRNA recognizes complementarily the protospacer sequence, and Cas protein cleaves it. This cleaved DNA site is repaired by NHEJ recombination in the cells (Cong et al., 2013; Jinek et al., 2013) that has variable efficacies. Although it repairs cleaved DNA sequence to the original position, it also

7.6 Applications of nuclease-based gene editing

results in loss or gain of nucleotides. The majority of mutations created by Cas9 are generally single nucleotide deletions or insertions (Cradick et al., 2013). The NHEJ method of cleavage repair is simple and fast method to create null mutations in the functional gene. Other than NHEJ, HR is the alternative repair mechanism that occurs in cells, wherein, when a donor sequence comes into contact with the surroundings of target DNA cleavage site, it exchanges or inserts the donor DNA sequence (Cong et al., 2013). This method allows Cas9 system to insert desired DNA sequence at the target site (Fig. 7.2).

7.6 Applications of nuclease-based gene editing tools in modulating animal health Since the successful experimentations of ZFN, TALEN, and CRISPR/Cas9 for targeting specific locus were reported, the number of studies in this direction have dramatically increased. Several applications of these nuclease-based gene editing tools have been proposed in model animals. Although livestock studies remain insufficient, there is a consistent rise in the use of these newer techniques for transgenic livestock production. The major target of current studies is directed toward improved animal health by incorporating disease resistance genes, improvement in animal performance, animal’s use in biomedicine, and most importantly boost animal production or productivity. Production of animals that are resistant to particular diseases causing major outbreaks and mortality is a major challenge. Targeted nuclease-based mutagenesis can be used to incorporate mutations or deletions into the ORF of a chosen gene—either to disrupt its expression or to produce a mutant protein that is associated with a particular disease phenotype. In the earlier studies, PRRS virus resistant pigs were created by the gene knock-out technique which was used to delete CD163 receptor gene. PRRS virus infects monocytes/macrophages and uses CD163 receptor to fuse with host membrane and release viral genetic information for successful infection. CD163 knock-out pigs were completely resistant to PRRS virus and associated syndromes (Whitworth et al., 2016). However, CD163 have other major biological functions like inflammatory response to a variety of pathogens. Deletion of whole CD163 receptor results in loss of other functions of this receptor. Another research group (Burkard et al., 2017), in an attempt to overcome this limitation found that the scavenger receptor cysteine-rich domain 5 (SRCR5) of CD163 was the essential interaction site for successful infection by PRRS virus. They used CRISPR/Cas9 system to cleave SRCR5 domain of CD163 to produce genetically modified pigs, macrophages from which were fully resistant to PRRSV while maintaining biological function all at once. Some studies have demonstrated disease resistance in cattle against Mycobacterium bovis through reverse genetics techniques (Gao et al., 2017; Wu et al., 2015). M. bovis, an intracellular pathogen, causes tuberculosis with potential economic losses in the cattle industry and it also has zoonotic importance. Recently, a Cas9 nickase system was adopted to introduce natural resistance-associated

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macrophage protein 1 (NRAMP1) gene in cattle genome (Gao et al., 2017). NRAMP1 gene is associated with innate resistance to intracellular pathogens by inducing the production of nitric oxide (NO) in addition to other proinflammatory responses. Cas9 nickase system-induced breaks stimulated the insertion of the NRAMP1 gene in cattle genome. The NRAMP1 transgenic cattle obtained through somatic cell transfer exhibited increased resistance to tuberculosis. Another study (Wu et al., 2015) reported that expressing the mouse SP110 gene in bovine macrophages through TALENs increased resistance to M. bovis. Recently an effort was made to cure genetic disease isoleucyl-tRNA synthetase (IARS) syndrome in Japanese black cattle, a major breed for high quality beef production in Japan. IARS syndrome is a recessive disease caused by a single nucleotide substitution. To overcome this mutation CRISPR/Cas9 system was used to create a double-strand break near the mutation site so as to introduce donor DNA that contained a synonymous codon for the correct amino acid. Somatic cell nuclear transfer embryos were generated from the repaired cells and were transferred to donor cows. Fetal genomic DNA analysis showed corrected repair of the IARS mutation without any additional DNA footprint (Ikeda et al., 2017). These studies introduced a new era of gene deletion in animals which further could be bred to significantly reduce the economic losses associated with bacterial and viral infections. Similarly, improved animal performance is another major target of using genome editing tools. The most well-known example includes the successful knocking out of myostatin (MSTN) gene, a negative regulator of growth hormone and restrictive factor for muscle mass growth. MSTN knock-out phenotypes were successfully introduced in cattle, sheep, goats, and pigs by using various nuclease-based genome editing techniques (Bi et al., 2016; Guo et al., 2016; Ni et al., 2014; Proudfoot et al., 2015; Yu et al., 2016). Another application of these gene editing methods includes altering the nutritional value of milk and making it allergen (β-lactoglobulin/blg) free so as to make it available to blg-allergic humans who are otherwise not able to consume milk. It was successfully achieved by using ZFN-derived mutations in blg gene of cattle (Yu et al., 2011) and CRISPR/Cas9-induced knock-out of blg gene in goats (Zhou et al., 2017). In a similar study in chickens, CRISPR/Cas system was used to mutate genes encoding ovalbumin and ovomucoid which are major allergens from chicken eggs and major components of egg white. Although techniques to remove horns are well established in veterinary practice but are painful and stressful for the animals. Use of TALEN technique has also significantly contributed in animal welfare by creation of horn-free cattle (Oishi et al., 2016). Several studies have also been conducted in the field of biomedicine with the use of DNA nucleases.

7.7 Conclusion Infectious diseases have a major economic burden on animal health. Animals suffering with pathogenic infections undergo high treatment costs, deliver reduced

References

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CHAPTER

Animal models: bridging cross-species variation through animal biotechnology

8

Nayaab Laaldin1,
, Sana Rasul Baloch1,
, Aneeqa Noor2,
, Aiman Aziz3, Alvina Gul1, Tausif Ahmed Rajput3 and Mustafeez Mujtaba Babar3 1

Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Islamabad, Pakistan 2 Department of Neurology, National Reference Center for TSE, Georg-August University, Gottingen, Germany 3 Shifa College of Pharmaceutical Sciences, Shifa Tameer-e-Millat University, Islamabad, Pakistan

8.1 Introduction The use of animal models in scientific experimental research has proven to be highly useful to establish an understanding of various biological processes. Historically, animals have been used in education and research since the ancient Greek times. The first reports come from the studies of Aristotle who used them to increase his understanding of living beings (Oparin, 1957). The most important advances in scientific knowledge can be owed to animal studies. Some examples include the role of the pancreas in digestion, development of vaccines especially the oral polio vaccine by Albert Sabin, and the study of deadly viral infections such as the Zika virus. With the advent of biotechnology, scientists have found better ways to modify animal models for mimicking human diseases (Andersen and Winter, 2017). Investigation of human diseases is now being done using knock-out and specific pathogen-free and gnotobiotic animals. Further, majority of transgenesis study use mice as model. The use of animal models for understanding and characterizing disease pathophysiology, identifying drug targets, and testing the efficacy of pharmaceutical drugs is highly useful in modern biotechnology. As many human diseases are heterogeneous and multifactorial, it is difficult to use natural animal models for the




Equal contribution

Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00008-4 © 2020 Elsevier Inc. All rights reserved.

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study. Animals can be modified via gene editing and transgenic technology to mimic the characteristics of the disease. These genetic engineering techniques include knock-in or conditional knock-out of the gene(s). This creates highly specific alterations in the genome, helping to control gene expression levels. These specific disease models are used to corroborate drug targets that have already been identified. Despite the advantages, some believe that the results from animal models cannot be extrapolated to humans because the genetic difference between the species often results in an alteration in disease pathophysiology. However, this can be countered by using different types of animal models for different diseases. One of the most important considerations during scientific research involving animal models is the ethical aspect. Multiple regulatory bodies, institutional, domestic, and international, must review experiments involving animal testing prior to their execution to make sure that that the use of animals is inevitable and is done in an ethical manner. This chapter aims to review biotechnological advancements that have provided better animal models of human diseases along with the limitations surrounding these techniques.

8.2 Animal models of diseases The invention of animal models refers to the use of animals to create a simplified, yet comparable, representation of complex human systems and diseases. Animal models are key players behind the understanding of physiology of various human systems and pathology of human diseases since the 6th century (Ericsson et al., 2013). Several animals have been selected to serve this purpose, based on their physiological and genetic similarity with human beings (Table 8.1). Depending on the nature of study, the selected animal candidate can be manipulated in multiple ways to establish maximum relevance to human systems. These models are classified into five major subtypes, that is, induced, mutant, negative, genetically-modified, and orphan models, which are discussed in the following section.

8.2.1 Induced models Induced model refers to the experimental induction of a disease phenotype in an animal. Induced models can be created using one of the following techniques:

8.2.1.1 Pharmacological or chemical-induced models This procedure involves the treatment of an animal with a drug or toxin that can selectively impact the target organ and disrupt its physiological functions to mimic the pathology of a disease. Scopolamine-induced amnesia is a druginduced model for Alzheimer’s disease whereby the cholinergic system is targeted

8.2 Animal models of diseases

Table 8.1 Relative usage and advantages of species employed in biomedical studies. Relative usage in research (%)

Models

Species

Rodent models

Mus musculus (mouse) Rattus norvegicus (rat) Cavia porcellus (guinea pigs) and Sus (pigs) Mesocricetus (hamsters) Oryctolagus cuniculus (rabbits) Felis (cat), Canis (dog), Capra (goats), Bos (cattle) Equidae (horses, donkeys, and crossbreds)

61 14 1.5

• Easy multiplication,

0.2 3.12

• Shorter lifespan

Nonhuman primate models

Apes (Hominoidea) and monkeys (Ceboidea)

0.052

•

Other nonmammalian models

Insect (Drosophila, Tribolium, etc.), fish, Amphibians, reptiles, birds, and other mammals

18.6

•

Other mammalian models

Advantages housing and use

• Ease of genetic manipulation

• Physiological and

1.5

• •

•

genetic similarities with homo sapiens Exhibit clinical signs of human diseases Comparable size and nutritional requirements Highly developed systems—ideal for neurological and psychiatric disorders Small and easy to manipulate Observation of various growth stages possible with low-power microscopes

Rodents are the most common animals employed in biomedical research in states of European Union because of ease in genetic manipulation. Although larger animals may have more physiological relevance to humans, the economic and biotechnological limitations of their use decrease their contribution towards biomedical science (Stewart et al., 2014; Gonzalez et al., 2015; Grow et al., 2016).

to mimic cognitive aspects of this disorder (Van Dam and De Deyn, 2011). Streptozotocin-induced destruction of pancreatic beta-cells is another example of a chemical-induced model ford mellitus (Graham et al., 2011). Although the chemical-induced models may provide a cheaper and easier way to mimic disease pathology, they are not always ideal for biomedical studies because the effect of the introduced toxin might impact multiple organs.

8.2.1.2 Lesion-induced models Lesion-induced models provide a more specific mode of disease-induction in animals. Lesion-induced models for Alzheimer’s disease and diabetes mellitus

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involve surgical lesions in the cholinergic centers of basal forebrain and pancreas respectively (Van Dam and De Deyn, 2011; Sharma et al., 2013). Lesions can be both mechanical and electrolytic in nature.

8.2.1.3 Stress-induced models Stress-induced models have made significant contributions toward the understanding of behavioral sciences. Application of chronic mild stress through various environmental stimuli is being used to generate models for pain disorders and depression (Greenwood-Vanmeerveld and Johnson, 2017; Moreines et al., 2017) (Fig. 8.1).

8.2.1.4 Induction of disease through biological molecules Application of biological molecules/entities, which trigger the induction of disease, is another way of generating animal models. Application of scrapieassociated prion protein is being extensively used to produce animal models of prion diseases (Jacquemot et al., 2005). Similarly, injection of amyloid-β in animals produces Alzheimer’s disease-like phenotype (Van Dam and De Deyn, 2011).

FIGURE 8.1 Types of animal models of diseases.

8.3 Mimicking clinical conditions in animals

8.2.2 Spontaneous models Spontaneous models, also referred to as mutant models, are tailored to match human disease by nature, therefore, they can be used in biomedical research without any alterations. Such models may be generated due to spontaneous mutations in animals or the natural ageing process of animals. Spontaneous animal models of human diseases bypass the complications that occur due to experimental induction of a disease and might be more advantageous for the study of animal diseases (Kol et al., 2015). Spontaneous models of neurodegenerative diseases, cancer, and diabetes mellitus are popular amongst the biomedical community for understanding the pathophysiological mechanisms and toxicity analysis (Eaton and Wishart, 2017; Pospischil et al., 2016; Wang et al., 2013).

8.2.3 Negative models All the species do not possess the physiological machinery to respond to diseasecausing agents, thereby becoming resistant to a disease. Such animals are referred to as negative models of a disease and contribute toward biomedical research by acting as negative controls for studies (Hau, 2008).

8.2.4 Genetically-modified models Advances in biotechnology have allowed the creation of animal models that would have been impossible with experimental induction. Transgenic technology allows targeted manipulation of a single gene and its products, resulting in a very specific and pathologically-relevant animal model. Various techniques employed for the generation of genetically-modified animals have been discussed in the following sections of this chapter.

8.2.5 Orphan models There are several animal diseases that have no pathological equivalent in humans. Animal models of such diseases are referred to as orphan models (Hau, 2008).

8.3 Mimicking clinical conditions in animals Human diseases can be mimicked in animals through the generation of two types of models, namely phenotypic and genotypic animal models. Phenotypic models (mostly experimentally induced) mimic the clinical symptoms of the disorder observed in patients whereas the genotypic models (primarily mutant or genetically modified) carry the genetic aberrations/mutations observed for that disorder (Oleas et al., 2013). Although the two conditions coexist in human patients, it is difficult to mimic both in one animal model due to the complicated nature of

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most human diseases and the cross-species differences. For most complex multifactorial diseases like diabetes, there are several mouse models since all the characteristics of the disease cannot be mimicked in a single animal model (Cefalu, 2006). Biomedical studies targeted at the generation animal models generally initiate with the selection of ideal animal species. Animals are selected for the study of human diseases based on their genetic, anatomical, and physiological similarity to humans. For crucial preclinical trials involving animal models, it is essential that the animal should mirror the characteristics of the clinical condition under study. Usually, small animals such as mice or rats are used since they have a distinct genotype, are easier to maintain and reproduce quickly. However, for pharmacokinetic studies, the use of larger animals (nonhuman primates, dogs, and rabbits) is advantageous in most cases as their organs are more anatomically like humans and the results can be extrapolated easily. For a successful biomedical study, it is critical that an experiment involving animal models is properly designed and conducted. If there are flaws in the experimental design, the potency of an animal model is compromised. Some of the main factors that determine the success of biomedical studies are: 1. Selection of animal—It is essential to carefully select a specific strain or species of animals since every animal has a different characterization of various clinical conditions. Also, the gender and age of the animal should be close to the condition under study. For example, in the study of diseases such as Alzheimer’s and osteoarthritis (which are diseases of the elderly), it is necessary to use older animals to get accurate results during drug testing (Bouchlaka et al., 2013). Moreover, the cross-reactivity between the drug under investigation and the animal model needs to be ensured. Some drugs are cross-reactive to primate animal models which only makes them specific for undergoing testing. Further the inbred and outbred strains are used as per requirement during experimentation and biological production. The inbred strains of animals possess higher percentage of homozygosity and consequently reflects the minimum within group variation in response to a particular drug/medicine/treatment. But outbred strains of animals, because of higher percentage of heterozygous loci, often create a variation in response to the same stimulus and those results may not be reproducible. 2. The course of treatment—In humans, therapeutic treatment of a condition is normally started when the disease fully manifests itself with distinct symptoms. However, in most animal studies treatment is started before or soon after the initiation of disease pathology. This may cause the overestimation of the effect of the prospective treatment in animal studies. 3. Group size—The number of animals used in an experiment should ensure that there is enough data to generate an effective statistical analysis. Here it is important to note that use of inbred strain reduces number of animals required in a particular group.

8.4 Engineering of animal models

4. Control group—To determine the difference in characteristics of disease animal models, we need to establish a control group with healthy animals. Apart from all these factors, animal models need to be validated to ensure that they can mimic the disease properly. The following factors ensure the validity of animal models: 5. Face validity—This includes the similarity of pathological processes and symptoms between the animal model and the disease under study. 6. Predictive validity—This is done to show that effective drugs exhibit a similar effect on the animal model as on humans. It is usually difficult to ascertain this since not many approved human drugs are active in specific animal systems and often there is an incomplete association between the disease mechanisms in animals and humans. 7. Target validity—There should be a similarity between the target under investigation in an animal model and that in a clinical case. Some receptors have a different function in the animal model as compared to that in humans. For example, in rodents, the beta-3 adrenergic receptor is involved in energy metabolism but not in humans (Weyer et al., 1999). Since the use of animal models is diverse (studying biological processes, identifying drug targets, testing potential drugs), it is necessary that the validation should fit a specific purpose. For example, when studying pathological mechanisms, face validity holds more importance (Denayer et al., 2014).

8.4 Engineering of animal models Drug discovery has been a slow process despite the availability of multiple species, genetically and physiologically comparable to human beings, and various tools for induction of diseases. The primary cause of this delay is the fact that until the 1970s, scientists were limited in their ability to create deliberate, targeted alterations in animals. There are many human diseases that do not occur naturally in animals. The simultaneous evolution of genomics, that provided insights into the differences in the blueprints of various living organisms, and advances in biotechnology, which allowed the translation of this knowledge into animal models, equipped the scientific community to engineer customized preclinical models of various diseases (Ericsson et al., 2013). There are several methods to tailor an animal model that fulfills the requirements of a disorder. The basic mechanism involves transfection of sperms, eggs, or embryos with foreign DNA that can suppress or overexpress certain genes. Since the genes contain blueprints of all cellular functions, they are ideal targets for manipulation of cellular pathways. The successful transfection of animals is traced with selective markers, if necessary, followed by in vitro fertilization (if necessary) and transferred to surrogate females for development. The transfected animals can be cloned to replicate the genetic mutation and increase the number

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of transgenic animal models (National research council, 2004). Although the earlier technologies were not very useful for site-specific mutagenesis and gene delivery, the advent of technologies like clustered regularly interspaced short palindromic repeats (CRISPR) has made the engineering techniques extremely precise and practical. Full genome sequences are now available for various animals, increasing the utility of genetic engineering techniques and making more animals suitable for biomedical research. Various tools used for genetic manipulation and their applications are discussed in the next section. Selection of specific models, engineered to fulfilling the need of study, is extremely important for cost and time-efficient experiments that comply with animal welfare regulations. The biggest advantage of engineered animal models, in comparison to induced and spontaneous models, is the control that they have provided to scientists. Theoretically, it is now possible to manipulate the expression of any single biological molecule at genomic, transcriptomic, and translational stages. The practicality of this technique has surpassed basic research, and personalized medicines are now being generated through humanized (chimeric) models (Walsh et al., 2017). The overall costs are relatively low in comparison to the advantages of these techniques, and genetically-modified organisms are readily available for research. Transgenic and gene-edited mammalian models follow the disease pathology more closely than induced and spontaneous models. The technique is being employed to generate better models of some of the most common, yet complex, debilitating human diseases, including cardiovascular disorders, cancer, diabetes, stroke, dementia, and HIV-AIDS. Any hypothesis concerning human diseases can now be tested in a more personalized, but reliable, manner. In the present era, a well-planned experiment, with a relevant model, has a greater chance to contribute toward translational research than the studies from the 20th century.

8.5 Specific pathogen-free animals Specific pathogen-free animals are animals that are free from certain pathogen specified, but these animals are not necessarily free of the ones which are not specified. It does not necessarily indicate that all pathogens or microbes that can affect the species are excluded. It is important to define the specific pathogens excluded. The synonyms for specific pathogen-free animals are—disease-free animals, healthy animals, pathogen-free animals, clean animals, caesarian-derived animals. According to the International Committee on Laboratory Animals, “Specific pathogen-free (SPF) animals, which are free of specified microorganisms and parasites but are not necessarily free of the ones that are not specified.”

8.5.1 Production methodology The principle behind the production of SPF animals is that to obtain animals from a stage in their life cycle when there is either minimum amount of contamination

8.5 Specific pathogen-free animals

or not at all. Caesarian section technique is used to obtain pathogen-free animals. The placenta acts as a very efficient barrier and it prevents the fetus from becoming infected with most bacteria, virus, and parasites. The most practical and successful system, accepted for obtaining SPF animals, is to first raise them by germ-free methods, and establish them under pathogen-free conditions. Many problems are encountered in maintaining them germ-free, especially in nutrition and fertility. Once established germ-free, considerable care must be exercised to make the switch to controlled contamination. Small laboratory animals are raised in plastic isolators under germ-free conditions, and then transferred to a minimal disease breeding area, from which they will be supplied for experiments. Weaning animals, other than those for breeding replacements, are not kept in the rodent breeding unit, but are transferred to the stock room in the experimental unit. A litter is transferred by placing them in a sterile cardboard box, which is then sealed into a polythene bag and passed out through the “dunk” tank. The litter then enters the experimental unit through another “dunk” tank, where the polythene bag is removed, and the litter is caged. The transport boxes are stored flat and made up as required. Biosecurity begins with personal cleanliness. Showering or washing facilities and supplies should be provided, and personnel should change their clothing as often as necessary to maintain personal hygiene. Personnel should not be permitted to eat, drink, apply cosmetics, or use tobacco in animal facilities. Visitors should be limited as appropriate, and institutions should implement appropriate precautions to protect the safety and well-being of the visitors and the animals. It is essential that the agricultural animal care staff maintain a high standard of biosecurity to protect the animals from pathogenic organisms that can be transferred by humans. Disposable gear such as gloves, masks, coats, coveralls, and shoe covers may be required under some circumstances. Personnel should not leave the work place in protective clothing that has been worn while working with animals. Each SPF room will have serology conducted every 3 months. The serum test will be done annually by a basic panel. The infected animals will be immediately treated or discarded. Swabs from the surface of the walls and floors of the breeding unit and samples of the filtered water supply and of the “dunk” tank fluid are taken four times at weekly intervals. These are examined for the number and type of organisms present: Salmonella, Mycobacterium, Bordetella, Pasteurella, Mycoplasma, and Corynebacterium.

8.5.2 Importance of specific pathogen-free animals in research • SPF animals are free of many zoonotic pathogens which could otherwise be

•

transmitted to users and staff. This method of animal experimentation minimizes disease outbreaks in the animal colony which may otherwise result in the total eradication and loss of the animal colony. In addition to the immunocompromised animals, most of the other laboratory animal lines have much higher breeding, fecundity, and lifespan in SPF standards than in nonSPF standards.

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• SPF rodents for modern biomedical research need defined levels of pathogens

•

and other infectious agents that may change the research results. The quality assurance of a laboratory includes the animal’s microbial quality, genetic quality, and environment. Microbial quality determines the repeatability and reproducibility for animal experiments. These two factors are required for regulatory agencies like Organisation for Economic Co-operation and Development, The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, United States Food and Drug Administration, etc. SPF animals play a vital role in generation of novel genetically-modified mouse models.

8.6 Gnotobiotic animals A gnotobiotic animal is a germ-free animal in which only certain known strains of bacteria and other microorganisms are present. Gnotobiotic animals are also “gnotobiotes” or “gnotobionts” Like SPF animals they are born in aseptic conditions, which may include removal from the mother by caesarean section and immediate transfer of the newborn to an isolator where all incoming air, food, and water are sterilized. The production of gnotobiotic animals or birds depends upon if embryos developing inside an egg or the mammalian uterus are microbiologically sterile, if they come from healthy parent stock. The SPF and gnotobiotic birds are very easy to produce as eggs can be introduced into the sterile environment late in the incubation period and left to hatch, where after the young birds are selfsupporting. Mammals must be aseptically derived by hysterectomy or hysterotomy as late as possible before term. Inside the isolator the young are hand-fed on a sterilized liquid diet similar in composition to the mother’s milk. If the animals are successfully brought to sexual maturity natural breeding can continue in the germ-free environment, and other strains or even other species may be fostered on to lactating females. The gnotobiotic is used in study of interbacterial reaction. Further it is used in the study of nutrition and metabolism, immunology, vaccine production, and diagnostic.

8.7 Biotechnological approaches for generating animal models 8.7.1 Nuclease editors The past two decades have been a revolutionizing era for the field of genomeediting technology. It has given scientists an opportunity to manipulate and explore

8.7 Biotechnological approaches for generating animal models

genetic makeup of a vast range of cell types with different kinds of nucleases that target specific domains. The advancement of genome-editing approach has led to cost-effective ways to generate animal models of human diseases.

8.7.1.1 Clustered regularly interspaced short palindromic repeats/Cas9 CRISPR/Cas9 has opened doors for new possibilities to generate animal models of human disease more easily and accessibly, thus reducing the expenditure and time spent on generating animal models. With recent advancement in genomeediting techniques several approaches, including point mutation, copy number variants, coding and nongene mutation are easier to attain regardless of genetic background and species (Birling et al., 2017). CRISPR/Cas9 has revolutionized the industry of genome editing as it can be applicable to a wide range of animals with its efficiency and diverse use. CRISPR stands for clustered regularly interspaced short palindromic sequence. The Cas system associated with CRISPR cleaves a specific sequence of DNA by using a combination of proteins and short RNAs. Protospacers are collected from foreign DNA and incorporated into the bacterial genome to express short RNAs which are later used to cleave DNA sequence (Cong et al., 2013). In 2014, Feng Li was successful in developing transgenic mice in which Fah gene was targeted at a higher rate in NSG mutant. This animal model has a capacity to play a critical role toward engraftment of human tissues and cells (Li et al., 2014). Generating single-nucleotide polymorphism animal models with the help of CRISPR/Cas9 technology is novel work for scientists these days. SNP models provide a possibility for developing new therapies and gives better insight into human genetics. With the use of CRISPR/Cas9, a mouse model was developed that has a human SNP rs1039084 in the STXBP5 locus which is associated with a decline in thrombosis (Zhu et al., 2016). When compared to zinc finger nucleases (ZFNs), CRISPR/Cas9 does not require large segments of DNA to recode proteins for each target. By replacing the 20 bp protospacer through subcloning of the sequence into guide RNA plasmid, Cas system can alter to target any genomic sequence. Genome-wide libraries and a large set of vectors can be easily generated with CRISPR/Cas which is a significant advantage over zinc finger nuclease and transcription activator-like effectors nuclease (Zhou et al., 2014). CRISPR technology has become indispensable for the development of the animal model of human diseases. Scientists are considering CRISPR technology for therapeutic applications, like viral diseases, metabolism disorders, cancer immune therapy, etc. Keeping CRISPR/Cas9 phenomenal progress in view, it can be anticipated that this technology will take biomedical research to another level by helping us understand the basis of complex diseases and engineer better model organism.

8.7.1.2 Zinc finger nucleases The use of ZFNs to genetically engineer Drosophila and human cells started in 2002 (Bibikova et al., 2003). Other alternative approaches, including reverse

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splicing and oligonucleotide, rely on DNA bp recognition. However, ZFNs work through recognizing DNA/protein binding. ZFNs are comprised of two domains; DNA-binding domain which is zinc-finger mediated and a FokI for cleaving DNA (Kim et al., 1996). ZFNs are designed in a way that it recognizes two flanking site sequences on the forward and other on the reverse strand. Upon its binding to the sites, a FokI domain cleaves the DNA sequence which results in the generation double-strand breaks (Urnov et al., 2010). Once the double-stranded DNA is cleaved by ZFNs, an insertion/deletion or point mutation is induced by nonhomologous end joining and homologous-directed repair, respectively. Before the revolutionization of genome editing through CRISPR/Cas9, ZFNs were widely used for heritable gene disruption in mouse models and zebrafish (Ma and Liu, 2015). ZFNs genome editing does not only come with advantages. It is presented with difficulties when binding with longer bp of nucleotides with high affinity, making it hard for an amateur to engineer ZFNs. To overcome this issue, protocols for screening ZFNs and zinc finger components open library has been developed. In spite of that, it takes a lot of time for an amateur to optimize ZFNs editing (Gupta and Musunuru, 2014).

8.7.2 Somatic cell nuclear transfer Somatic cell nuclear transfer (SCNT) involves a donor and a recipient: somatic cell nucleus and an enucleated oocyte. The transfer has to happen before an oocyte develops into an embryo and activates. Conventionally, an oocyte was enucleated by the use of micropipettes, in which one holds the oocyte while other removes the nucleus. Another method to enucleate an oocyte involves a sharp blade, in which a chunk of an oocyte containing DNA is cut off. Upon enucleating, the oocyte is fused with donor nucleus with the help of electrical pulse. Following fusion, the embryo is activated to grow and develop further (Niemann and Lucas-Hahn, 2012). The activation of the embryo can be achieved with salts, such as strontium chloride, or an electrical pulse. Embryos are then kept in vitro until developed enough to be transferred into surrogate mother (Dicks et al., 2015). An example of the use of SCNT to generate an animal model of disease is cystic fibrosis transmembrane conductance regulator (CFTR) knock-out pig model of human cystic fibrosis. Long after the identification of CFTR as a responsible factor for cystic fibrosis, the disease still remains incurable. Disrupted CFTR gene mice models facilitated the studies of cystic fibrosis but had its limitations. To manifest the disease characteristics properly, Roger and his team generated CFTR-disrupted pigs with the help of SCNT and homologous recombination. The mice models did not manifest the disease characteristic (Rogers et al., 2008) (Fig. 8.2).

8.7.3 Pronuclear microinjection Almost half a century ago, Gordon and Ruddle came up with the idea of pronuclear microinjection. It is a technique in which a pronucleus of a developing

8.7 Biotechnological approaches for generating animal models

FIGURE 8.2 Biotechnological approaches for the development of animal models.

zygote is injected with a foreign DNA with the microinjection (Gordon and Ruddle, 1981). Using this technology, various transgenic animals have been generated till day, including rabbits, mice, and pigs. While the procedure is quite easy to perform, it does not guarantee efficiency. The professional cannot be sure as to which zygote will integrate the injected transgene and where in the genome will the gene be integrated. With its low productivity, only 5%10% of the offspring are born with the transgene. Sometimes, the integrated gene might not even express appropriately, if it has been placed in the gene-silencing areas (Wall, 2001). Microinjection has proved to be of more importance with recent refinements in the biotechnology industry. Recently, several piglets were generated by microinjection of CRISPR/Cas and all were found to be edited (Whitelaw et al., 2016). Also, a diabetes mellitus type 2 animal model has been developed by microinjection lentivirus containing dominant-negative glucose-dependent insulinotropic polypeptide receptor into zygotes (Renner et al., 2010).

8.7.4 RNA interference RNA interference is based on defense mechanism of a host designed in a way that it brings forth the response against any foreign RNA, particularly viral RNA, resulting in reduced inflammatory response and translation of viral mRNA. Upon

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introducing dsRNA into the cell, it is spliced into small interfering RNA (siRNA) by an RNase enzyme. siRNA are then incorporated into RNA inducing silencing complex. Consequently, RISC binds to mRNA which is complementary to siRNA, thus blocking the production of protein. Using these techniques, many have designed siRNA complementary to a specific gene to weaken its expression to produce a knock-down model (Hannon and Rossi, 2004). For its efficiency in transgenic models, a few modifications had to be done to ensure continuous production of siRNA throughout cell’s lifetime. The approach has been useful to generate animal models with attenuated gene expressions, such as atherosclerosis model with decreased activity of Apolipoprotein E (Bordignon et al., 2013). Tangier disease model was also generated by silencing ATP-binding cassette transporter subfamily A number 1 (ABCA-1) in mice, thus creating ABCA-1 deficient mouse line (Chang et al., 2004).

8.8 Translational significance of animal models The course of drug discovery is a hectic process that initiates with preliminary research to understand the fundamentals of a disease, passes through validation in cell culture/animal models, and finally goes through clinical trials to reach bedside. The complexity of human systems is hard to mimic in other organisms however, rodents and nonhuman primates have provided an excellent alternative for comprehensive molecular analysis of human diseases and drug discovery. Although animal models have been involved in drug discovery since the 18th century, their role in translational research became more prominent after 1962 when the Food and Drug Administration passed laws to involve at least two animal models in toxicity analysis before a drug could be approved for clinical trials (Junod, 2014). Each year, approximately 100, 11.5, 3.8, and 3.5 million animals are used for research in the United States, European Union (EU), United Kingdom, and Canada respectively, indicating their significance in biomedical research (U.S. Department of Agriculture, 2016; Canadian Council on Animal Care, 2015). They are being extensively used to predict clinical outcomes of various therapies. While mouse, zebrafish, Drosophila, and Caenorhabditis. elegans have been the primary targets of genetic engineering and serve as valid models for many complex diseases, the larger animals provide the advantage of larger, more developed, and comparable organs for study. Insulin, penicillin, streptomycin, and vaccines for the various infectious disease (diphtheria, tetanus) are amongst the first breakthroughs in medicine made using animal models (Badyal and Desai, 2014). These drugs are now being routinely prescribed for prevention and treatment of various disorders. With the advent of time, animals have no longer remained the test subjects for cosmetic and pharmaceutical industry only. As biomedical scientists got access to various tools of biotechnology, they did not only develop better animal models, they also

8.8 Translational significance of animal models

developed engineered natural compounds for use in medicine. Various molecules, including enzymes, ribozymes, and monoclonal antibodies, are under research to address various physiological phenomena (Williams et al., 1993). Using humanized models, it is now possible to generate molecules and antibodies that can bypass immune system and provide better therapeutic intervention for human diseases (Janne et al., 1992). The research for therapies of multifactorial human diseases is now shifting toward the use of engineered biological entities. A combination of engineered models and drugs has drastically affected the development of therapies. The manipulation of severe combined immunodeficiency mice revolutionized the research for immunodeficient disorders and cancer, allowing the development of immunotherapy and gene-based therapy (Carrillo et al., 2018). These therapies are already under investigation in clinics for various types of cancers (Janiczek et al., 2017). Gene-based immunotherapy for HIV-AIDS is also under clinical trials (Butcher, 2007). Transgenic models of Alzheimer’s disease have made significant contributions to developments of amyloid-β antibodies, a therapy already undergoing clinical trials (Wisniewski and Goni, 2014). Stem-cell therapies for cardiovascular disorders are also undergoing preclinical and clinical studies, thanks to biotechnology (Tsilimigras et al., 2017). The biomedical community has crossed major milestones in its quest to address human diseases and animal models have been their major associates. Nevertheless, it is important to consider that there are still inherent physiological and pharmacological differences among humans and other animals and these differences decrease the translational efficiency of many animal studies. Growing evidence suggests the lack of applicability of animal research in translational studies, making their use debatable (Perel et al., 2007). Animal models have proved effective in the backward validation of drugs, but their utility in forward validation has remained limited (Mogil, 2009). Only one-third of the highly cited animal studies make it to clinical trials (Hackam and Redelmeier, 2006). The cost of drug discovery has soared over the past few decades as many promising therapies prove to be ineffective in clinical trials. However, the decreased translational value of studies in the recent past cannot be blamed solely on animal testing. In this era of scientific revolution, researchers have not only come closer to better animal models, they also have a lot more information about probable drug targets and candidates at our disposal. A critical screening process and effective experimental protocols are therefore needed to increase the transfer of therapies from bench to bedside. Animal testing must be conducted responsibly before the therapy can be tested on humans. History presents several examples of the effects of incomplete and improper animal trials on the human population. One of the most notorious, one being the example Thalidomide was adrug prescribed to pregnant women for morning sickness without testing it in pregnant animals, resulting in thousands of children being born with birth deficits (Rajkumar, 2004). Therefore despite the dividing views about animal models, their contribution to medicine cannot be nullified and must be continued.

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8.9 Pathological and pharmacological considerations The use of transgenic technology has allowed scientists to create pathologicallyrelevant animal models for various neurological, psychological, and metabolic diseases. These models have eliminated the ethical and economic limitations that hinder the study of various diseased phenotypes thereby allowing experiments that would have been otherwise impossible. However, the extent of the use of therapies developed through animal models is limited due to various physiological and pharmacological considerations. Even for most pathologically-relevant animal models, the experimental design and results must be analyzed critically to understand the translational relevance.

8.9.1 Physiological considerations There is an inherent difference in the physiology of humans and other living beings, even our closest evolutionary relatives. Although biotechnology has allowed the elimination of certain differences and slightly bridged the gap between human and animal models, these physiological differences must be considered when designing a study that involves animal testing. The physiological differences in anatomical structures, for example, the blood-brain barrier, or cellular components, like different receptors, might impact the way the studied drug is metabolized and decrease the translational value of a study (Tordjman et al., 2003; Potashkin et al., 2011). Moreover, the differences in regulation of gene expression between human and animal models, with respect to alternative splicing and use of miRNAs, impacts the use of transgenic models to mimic human diseases (Xie et al., 2005; Kim et al., 2006). Even if the basic biology behind certain pathologies is similar, the pace at which various disease-associated events occurs in human beings is different than that seen in animals and can impact the outcome of a study. However, the time-specific induction of mutations is now becoming possible (Housden et al., 2017). Many human diseases are products of multiple genetic, epigenetic, transcriptional, translational, and posttranslational events; therefore, it is not possible to mimic the exact pathology of any disorder in animals by altering or silencing a single gene. Failure of animal models to mimic such diseases completely requires the use of multiple models and several species (Levi et al., 2001; Tordjman et al., 2007). The selected species must have maximum similarity with the human organ under study and different models can be used to study the effect of a disease on different organs (Pearce et al., 2007). However, it must be considered that the outcome of a disease can vary among different species. The models for Parkinson’s disease, for example, rely on changes in studying motor deficits in rodent models. Although the neuroanatomical basis is similar across species, motor deficits may be manifested differently (Potashkin et al., 2011). Furthermore, long-term complications of a disease or drug and the individual variability cannot be brought into consideration through animal testing, therefore, such studies might just provide partial insights (Mogil, 2009; Mix et al., 2010).

8.10 Ethical and regulatory issues

8.9.2 Pharmacological considerations Analysis of toxicity of any candidate drug usually involves multiple models of a single disease. The lack of uniformity of the animal models employed for various studies of a single disorder does not only cause discrepancies in results but may also hinder the advancement of various promising drugs into the translational phase. The researched therapies for prion diseases, for example, comprise of hundreds of agents that target the disease pathology by preventing the accumulation, aiding the clearance or preventing the toxic effects of the accumulated prion protein. Out of the massive chemical libraries and biological molecules studied, quinacrine is among the few agents proven effective in animal models and the efficacy of this drug shows huge discrepancies. These discrepancies and its limited efficacy in clinical trials have been attributed to the differences in the biophasic distribution of the drug in various animal models and patients (Gayrard et al., 2005). The cross-species pharmacokinetic differences must, therefore, be carefully predicted. Antimicrobial drugs serve as another similar example. They have a 69-fold higher half-life in humans in comparison to mice models used for the study, predicting a drastic difference in their effectiveness among the two (Andes and Craig, 2002). Even among similar species, the metabolism of a drug can vary across genders, highlighting the need for studies to include both genders in experimentation (Czerniak, 2001). The translation of results from animal studies to humans requires the tuning of dose-response relationships. Some tested drugs, even when used at a very low and safe dosage might be harmful to humans. A recent clinical trial with anti-CD28 monoclonal antibody TGN1412, a drug against leukemia, involved testing healthy humans with 1/500th of safe monkey dose but all subjects developed multiorgan failure within hours of infusion (Suntharalingam et al., 2006). Moreover, animal models might not be very useful for predicting long-term effects of drug candidates. Several therapies, when translated to clinical trials, lead to unanticipated side effects in human subjects (Mix et al., 2008). Taking the physiological and pharmacological differences among animal models and human patients into account is a key to successful animal testing. The experimental design must include carefully selected animal models for the drug target, be randomized, involve both genders and target both short and long-term effects of a candidate drug. The tests selected for predicting the impact of a drug must be very sensitive to metabolic and behavioral changes in animals. Finally, the husbandry conditions must not be a source of excessive stress on animals as it might impact the outcome of the study.

8.10 Ethical and regulatory issues The use of animal experimentation in biomedical research has been of crucial importance for the study of human disease. It’s a step to scientific progress as it

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directs researchers toward new methods to cure and prevent human diseases. However, the animals used to conduct these experiments are kept in unfavorable conditions and are subjected to painful interventions. Neither do these animals benefit from the suffering nor have they voluntarily consented to it. Keeping the facts in view, the scientific community is often presented with questions on ethical issues that animal research raises. Ethical issues of animal research refer to any issues which employment of animal model and its treatment from an ethical perspective. To minimize the animal suffering during experiments and to control the unethical use of animal, many laws, and acts have been passed. Over the period, many organizations are formed to look over the matter, such as Committee for Purpose of Control and Supervision on Experiments on Animals (CPCSEA) and National Institute of Health. Committees like these have provided the scientific community with some guidelines on animal feeding, housekeeping, transportation, and their use in experimentation (Doke and Dhawale, 2015). The CPCSEA is a statutory body formed by the Act of the Indian Parliament under Prevention of Cruelty Act of in 1960 in India. The CPCSEA was established to ensure that lab animals are well maintained, to oversee that experiments are conducted according to ethical norms, to promote humane care of animals used in biomedical and behavioral research, to enhance animal well being and quality, and also to improve laboratory animal facility. Proper use of animals in experiments and avoidance or minimization (when avoidance is not possible) of pain and suffering inflicted on experimental animals should be an issue of priority for research personnel, and unless the contrary is scientifically established, investigators should proceed on the basis that procedures that cause pain or suffering in human beings will also cause similar pain or suffering in animals. All scientific procedures adopted with animals that may cause more than momentary or slight pain and/or suffering should be performed with appropriate sedation, analgesia, or anesthesia. All establishments engaged in research and education involving animals, are required to comply with the various guidelines, norms, and stipulations set out by CPCSEA. The main functions of CPCSEA are: 1. registration of establishments conducting animal experimentation or breeding of animals for this purpose; 2. selection and appointment of nominees in the Institutional Animal Ethics Committees of registered establishments; 3. approval of Animal House Facilities on the basis of reports of inspections conducted by CPCSEA; 4. permission for conducting experiments involving use of animals; 5. recommendation for import of animals for use in experiments; 6. action against establishments in case of violation of any legal norm/ stipulation. Further, Association for Assessment and Accreditation of Laboratory Animal Care International program started in 1965, is a private, nonprofit organization that promotes the humane treatment of animals in science through voluntary

8.10 Ethical and regulatory issues

accreditation and assessment programs. To date more than 1000 institutions have been accredited across 47 countries. Most animals are killed during or at the end of the experiment. Therefore the use of animal models is only permitted if the killing of a certain animal is executed through humane method of euthanasia. The suffering of animals not only includes physical torture but also psychological. Animals do experience psychological stress when kept in isolation resulting in unpleasant consequences. Suffering is considered intrinsically bad regardless of who the sufferer is. Just because the sufferer is a rat, doesn’t make suffering acceptable at any cost. But according to the consequentialist approach, the benefit of the society is weighed moreover the suffering; regardless of who suffers (Brown and Jones, 2008; Levy, 2012). There isn’t one answer to the permissibility of animal model use. The permissibility varies with experiments, not solely depending on how many benefits from it and how many are harmed. It is possible that larger number of animals may suffer for fewer humans. If this is the case, the permissibility depends on the nature of distress employed on models. The benefit of the experiment should be measured on the current knowledge of its outcome. One can never be certain of the actual outcomes, thus estimating, the experiment’s worth must be based on its known benefits whether it be for medical purposes or pure knowledge (Bentham, 1972). Since the inception of “consequentialism” by Jeremy Bentham, the theory has been a favorite for animal emancipation. Not long ago, Peter Singer ignited the debate again in his book written from a consequentialist point of view. The theory of consequentialism does not encourage the ban on animal models but it asks the animal experimentation to meet stringent requirements (Singer, 1995) For the purpose of animal protection and welfare, European directive 2010/6/ EU was set to provide a regulatory and ethical framework for all animal experimentation. The directive is based on three R’s strategy; which stands for reduction, refinement, and replacement. It suggests that the number of animal models should be restricted, if and when necessary. The reduction of animal use is possible by utilization of video-based and interactive technologies in educational institutions. Other experimental methodologies and approaches should replace the use of animals whenever applicable. Lastly, the experimentation on animals should be carefully refined and adjusted in a way that it inflicts the least amount of pain and distress and minimized on animals (Ranganatha and Kuppast, 2012). The animal models are being replaced in production of biological where option for cell lines/culture are emerging. All experimentations involving the use of animal models are evaluated on the basis of these three principles and conducted in accordance with regulatory provisions. Ethical committees are appointed to watch over these provisions which cover the inspection of animal holding premises, all procedures of animal experimentation, and training. The evaluations are not singularly based on the three R’s strategy, but also on the cost-benefit analysis. If failed to comply with these regulations, the research project can be disapproved by government official/authority or Institutional Animal Care Committee. In some extreme

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cases, the project can be terminated or fined heavily (Conn, 2017). In compliance to the regulation, it is mandatory that committees include members from animal protection and are not directly related to animal research (Barre´-Sinoussi and Montagutelli, 2015; Hau and Schapiro, 2002). After experimentation if animals are not required to be sacrificed then they must be rehabilitated as per 4th “R” principle of CPCSEA. Also rehabilitated animals may be reused (5th “R”) if the previously executed experiment is not going to affect the outcome of current experiments. One of the greatest biomedical challenges of recent times are complicated and multifactorial diseases such as Parkinson’s, Alzheimer’s, Huntington, and cancer. Animal models provide a structure that is controlled, well characterized, and designed for experimentation. The two objectives are, that animals need constant improvement for better and reliable information and animal protection needs perpetual deliberation, must go hand-in-hand for high-quality science.

8.11 Conclusion Animal models have been a key part of the biomedical research for the past two centuries. Initially limited to natural and experimentally induced models, biomedical researchers have skillfully adopted the use of biotechnology to decrease crossspecies variation and engineer animal models that fulfill specific requirements of a disease. Engineered animal models tend to follow pathophysiology of human diseases more closely and produce humanized therapeutic molecules thereby contributing toward both drug testing and drug designing. The experimental and ethical considerations play an important role in successful animal studies and their subsequent progress to translational phase. The debates surrounding the use of animals in biomedical research, primarily due to ethical concerns and physiological differences, are pushing the need for alternative models. The development of organ-on-chip and in silico systems is underway and might replace animal testing in a few decades (Badyal and Desai, 2014; Zheng et al., 2016; Usha et al., 2018). However, until better techniques are developed to understand human diseases and discover therapies, geneticallyengineered models are the best hope for millions suffering from debilitating disorders around the world.

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Further reading

Zhou, Y., Zhu, S., Cai, C., Yuan, P., Li, C., Huang, Y., et al., 2014. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509 (7501), 487. Zhu, Q.M., Ko, K.A., Ture, S., Mastrangelo, M.A., Chen, M.H., Johnson, A.D., et al., 2016. Novel thrombotic function of a human SNP in STXBP5 revealed by CRISPR/ Cas9 gene editing in mice. Arterioscler. Thromb. Vasc. Biol. 37 (2), 264270.

Further reading European Commission, 2014. Seventh Statistical report (viewed 06.05.18.). U.K. Government, 2016. Annual Statistics of Scientific Procedures on Living Animals Great Britain 2016 (viewed 06.05.18.).

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Transgenic chicken/poultry birds: serving us for survival

9

Afsaneh Golkar-Narenji, James N. Petitte and Paul E. Mozdziak Prestage Department of Poultry Science, North Carolina State University, Raleigh, NC, United States

9.1 Introduction The human population is increasing rapidly; therefore, new cost-effective technologies are necessary for nutrition and treatments for human disease. Domestic animals are a very important source of food (Kralik et al., 2012) with a valuable source of high quality and easily digestible proteins in addition to essential vitamins including A and B12 and minerals such as zinc, iron, and calcium (WHO/FAO/UNU Expert Consultation, 2007). In the agriculture industry, there are many efforts to increase the efficiency of animal productions such as milk, eggs, and meat. In classical animal breeding, existing genetic variability is used and the introduction of new alleles is performed by crossbreeding of different populations or selection of animals in an existing population. Also, detection of animals with superior production is done using phenotypic and statistical methods to separate genetic and environmental effects (Wilmut et al., 1992). Classical animal breeding has been one of the most important techniques for changing the animal production system over selective breeding which focuses on specific loci or multiple loci using new genomic tools (Meuwissen et al., 2001). Modern selection techniques have been employed to increase valuable nutritional properties of some animal products; overconsumption of those products causes obesity and other related diseases (WHO/FAO/UNU Expert Consultation, 2007). Agriculture system, which includes animal husbandry, is supposed to change in the future and faces challenges. Animal husbandry is affected by the climate, resources, and demands for animal products (International Assessment of Agricultural Knowledge Science and Technology for Development (IAASTD). Transgenic animal production can probably be one of the solutions for those challenges that animal husbandry and agriculture may confront in the future. In human nutrition, poultry, eggs, and meat are major sources of high-quality protein which is critically needed by millions of people living in poverty (Farrel, 2008). Transgenesis or gene modification is a technique that we use to introduce new

Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00009-6 © 2020 Elsevier Inc. All rights reserved.

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genes into the genome of an organism. So far gene constructs have been introduced to different kinds of farm animals including cattle, goats, pigs, rabbits, sheep, and poultry (Jahav and Siddiqui, 2007). Transgenic technology in poultry science has been mostly around the production of therapeutic proteins in egg whites or blood. However, it has been mentioned that in the poultry industry transgenesis helps to improve economic traits such as meat quality, feed conversion efficiency, residency to diseases, and protection of humans from zoonotic diseases (Okon et al., 2016; McKay, 2008). Furthermore, transgenesis in poultry science is a valuable tool in the poultry industry because it allows manipulation of the genetic pathways that govern important economical traits (Okon et al., 2016). To date, there have been no reports for the introduction of new genes that can modify meat quality in poultry species. In this review, we discuss transgenesis applications for the poultry industry, human nutrition, and medicine.

9.2 Transgenesis usage for the poultry industry and environment protection Poultry production is based on raising fast-growing chickens (Yang and Jiang, 2005). Mass production of meat or eggs with high efficiency and low cost are important in the poultry industry. For maximum productivity, the poultry industry segregates chicken breeds into broiler and egg-laying chickens. Broiler chickens are bred for rapid growth to reach maximum meat production but egg-laying chickens are bred for high production of the egg with the highest efficiency (Arthur and Albers, 2003). Some other factors such as disease and food intake efficiency are effective on the efficiency of poultry production. Improvement in feed efficiency is one of the main factors to reduce the costs of poultry production. Feed efficiency is possible to be improved by genetic selection on growth, feed intake (feed conversion ratio), and architecture of gastro-intestinal tract (de Verdal et al., 2011). Transgenesis technology is helpful to improve those factors for instance growth hormone (GH) transgenesis, a rapid approach to accelerate performance of agriculture species such as rapid growth, food intake, and metabolic rates which has been observed in fish (Devlin et al., 2009; Dalmolin et al., 2015). Among livestock, GH transgenic technique for sheep and pigs exist (Reed and Mann, 1985). Furthermore, GH transgenesis has been utilized to increase goat milk (Zhang et al., 2014). GH transgenesis in the poultry industry may help to increase egg production performance. The poultry industry can affect human health with the excessive production of phosphorus and nitrogen which causes water contamination and spreading of pathogens (Kent, 2005). Poultry species are monogastric and they are unable to digest phytate phosphorus due to the lack of phytase enzyme in the digestive system, an addition of phytase to their diet is necessary (Yu et al., 2004). The benefit of this enzyme in poultry nutrition has been discussed before and the inclusion of this enzyme to poultry diet is one of

9.3 Poultry transgenesis and human nutrition

the most adopted practices. Phytase releases phosphorus from phytate molecule and makes it available. However production of more soluble phosphorus causes phosphorous excretion to surface water (Kleinman et al., 2002). It has been suggested that transfer of bacterial phytase gene to pig helps to digest a large part of phytate in food (Golovan et al., 2001). The introduction of bacterial enzyme gene to poultry is probably possible and helps to prevent the needs for additives like phytase into the diet. Another factor that adversely influences the poultry industry is an avian disease. It has reported that the total economic costs of the disease that are mostly related to vaccines and condemnations, were about 20%of the gross value of production which is about three times the cost of losses from mortality (Trevor, 2013). Bacterial disease causes significant food safety problems for human consumption of contaminated meat or eggs (Klasing et al., 1997). Furthermore, there are concerns about antibiotic residues that may adversely affect human health (Barton and Barton, 2000). Although traditional methods such as vaccines have been effective to control disease, there is still various diseases that threaten\ the poultry industry. Recently the development of molecular techniques and genetic selection helps to produce disease-resistant poultry breeds (Jie and Liu, 2011). There are some important diseases in avian species including influenza and Marek’s diseases (MD). Transgenic technology has been employed to produce resistant chickens for influenza virus (Lyall et al., 2011). There have been efforts to produce transgenic chickens resistant to MD, however it has not been successful (Crittenden and Salter, 1992). Although still there is no flock of transgenic disease-resistant chicken, it is a promising technique to prevent disease scattering in the poultry industry without using expensive vaccines or antibiotics.

9.3 Poultry transgenesis and human nutrition There are two main poultry productions including meat and eggs which are very important in human nutrition and health. Poultry meat consumption tremendously increased and poultry meat marketing has been reported to be in progress and a growing industry (OECD-FAO, 2012). This industry is successful because of poultry being the healthiest meat compared to red meat, which is due to poultry being a high-quality protein with low cholesterol and the presence of some functional components. Furthermore, poultry meat is suitable for food processing by meat producers to offer more attractive, more convenient. and easy-to-use products. The lower price of poultry meat is another reason for this successful industry. Another advantage of poultry meat products is that there are no limitations for consumption in different cultures (Cavani et al., 2009) Chicken meat is known as one of the healthiest and cheapest meats compared to other livestock meats. Facilities for producing broiler chicken can be easily established (Farrel, 2008). Therefore it is the best source of animal protein for low-income

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populations. Quantity and quality of meat are highly effective on human survival and health. The quality of meat is correlated to its nutritional value which is related to the content of high-value protein, unsaturated fatty acids, vitamins, macro- and micronutrients, cholesterol, and other biologically active compounds (Mozdziak et al., 2003; Magdelaine et al., 2008). Meat quality is affected by genetic factors such as appropriate choice of breed, line or hybrid (Dal Bosco et al., 2012; Bianchi et al., 2006; Suganthi, 2014), sex of bird (Lo´pez et al., 2011), rearing system (Tong et al., 2015), and slaughter age (Lo´pez et al., 2011; Franco et al., 2012). Improvement of muscle and meat parameters is related to breeding strategies including selection according to dominant traits or preferred traits by crossbreeding. Also, meat parameters are possible to be modified using endogenous and exogenous hormones. Recently the identification and sequencing of chromosomal regions related to quantitative trait loci affect the carcass traits, growth, and meat quality factors (Niwa, 2007). For instance, myostatin and follistatin are two proteins in which their expression pattern affects muscle mass (Lee, 2007). It has been assumed that gene transfer helps to introduce new genes that can promote increased muscle growth (Toldra´, 2008). In the 1980s Palmiter et al. explained that with the integration and expression of rat or human hormones, mouse weight could be doubled (Palmiter et al., 1982, 1983). After that utilization of this technology was considered in the livestock industry for mass production of meat (Westhusin, 1997). Transgenesis has been used to improve production of healthy meat. For instance, transgenic pigs have been produced that express spinach desaturase to synthesis essential polyunsaturated fatty acids including linoleic and linoleic acid (Saeki et al., 2004). Therefore previous studies on mammalian species indicate that increasing meat production and changing meat parameters are possible in poultry strangeness as well. Chicken eggs are known as the carrier of critical nutrients which are important for nutritional status of human particularly in low-income people who live in developing countries (Farrel, 2008). Eggs offer a moderate amount of calories, an excellent quality of protein, and are low cost. Furthermore, eggs are rich in fat-soluble compounds and suitable for all ages (Natoli et al., 2007). However, due to saturating fat contents (about 3 g/100 g) and cholesterol content (200 300 mg/100 g) (Li et al., 2013) there has been public warning against frequent consumption of such (Eilat-Adar et al., 2013). Egg yolk is largely used in the food industry because of its special properties. Therefore modification of egg components such as fatty acids and cholesterol, which may adversely affect human health, has gained more consideration. Therefore strategies for obtaining eggs with lower amounts of cholesterol were employed, which have been mostly focused on genetic selection or alteration of chicken’s diet with nutrient or nonnutrient factors and pharmacological agents (Elkin, 2006). But those strategies to alter the amount of egg yolk cholesterol have low efficiency (Elkin, 2007). Recently n-3 polyunsaturated fatty acids (PUFA)-enriched eggs have been considered as a healthful food and there is a report about customer willingness to purchase even more expensive eggs (Marshall et al., 1994). The level of PUFA can be modified by changing the

9.4 Poultry transgenesis and medicine

laying hen diet (Lemahieu et al., 2013). Transgenesis technology employment is probably possible for the production of eggs with healthier contents. The high consumption of eggs in the world makes mass production of the egg necessary and the question is how to increase egg production to meet global demands (Zaheer, 2015). So far manipulation of egg size is possible with changing diet (Selman and Houston, 1996), thermoregulation (Nager and Van Noordwijk, 1992), and clutch size (Nol et al., 1997). However, there are still many unknown factors that affect egg quality and its contents (Williams, 1996). In order to use transgenesis technology for this purpose, more research is necessary to find the most effective factors on egg quality and egg development process. There have been some efforts for this purpose, for instance transgenic quail has been utilized to find out the mechanism of lipolysis regulation for yolk development and egg production in poultry species (Chen et al., 2016; Shin et al., 2014). Furthermore, some studies on hormone effects have been performed, such as evaluation of exogenous GH which has been suggested to be effective for increasing egg production in egg-laying hens (Mohammadi and Ansari-Pirsaraei, 2016).

9.4 Poultry transgenesis and medicine Transgenesis has been a useful technique to produce suitable animal models for research on human disease. Transgenic chicken is considered as a bioreactor to produce human recombinant therapeutic proteins and vaccines which can be extracted from their egg. Recently chicken has been introduced as a very efficient bioreactor for purposes of producing human recombinant proteins because of their short reproductive cycle and because they are easier to keep and rise (Li and Lu, 2010). Recently monoclonal antibodies have been produced for treatment of infectious disease, cancer, autoimmune diseases (Marston et al., 2018). Therefore because of increasing demand for these antibodies, mass production is necessary. The hybridoma is one of the technologies developed for antibody production, however it is time expensive, consuming, and needs expert people in cell culture. A large quantity of protein can be deposited in the chicken egg by tubular gland oviduct (Zhu et al., 2005) with more similar glycosylation pattern to humans. Therefore it is possible to produce high quality and the mass amount of monoclonal antibodies that help to offer cheap and easily available drugs to save human life. Recently an anticancer monoclonal antibody against CD20 protein has been produced in transgenic egg white (Kim et al., 2018). Anti-CD20 (Rituximab) is a monoclonal antibody against a protein on B cells which is used for the treatment of non-Hodgkin’s lymphoma, chronic lymphocytic leukemia, and rheumatoid arthritis (Hainsworth et al., 2000; Lopez-Olivo et al., 2015). Also, a recent report showcased the production of human interferon-β in transgenic chicken egg white as the first successful knock-in of a gene in chicken to produce encoded protein (Oishi et al., 2018). Many other proteins have been produced by transgenic

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chickens using different transgenesis techniques including: interferonα-2 (Rapp et al., 2003), erythropoietin (Sun et al., 2007), epidermal growth factor (Park et al., 2015), lysozyme (Dalmolin et al., 2015), and defensing (Liu et al., 2015). In biology and developmental studies, transgenic chicken has been suggested the chicken is a promising model for biological research particularly embryology studies (Mozdziak and Petitte, 2004; Mozdziak et al., 2003, 2018). There are some stable germline transgenic chicken that can be used for biological research such as flocks that expressing enhanced green fluorescent protein mCherry (McGrew et al., 2004). Chicken has been introduced as a suitable model for studies on human craniofacial ciliopathies disorder, and the transgenic chicken line has been developed is inducible. In addition manipulation of the genome using transcription activator-like effector nucleases or clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPER/cas9) technology helps to edit ciliary genes in chicken during development (Schock et al., 2016).

9.5 Conclusion Poultry species particularly chicken (Gallus gallus) are very important in agriculture for human nutrition. Transgenesis in the poultry industry is a useful technique to improve human nutrition in both aspects of quality and quantity to offer enough and high-quality food supply which is necessary for human health and survival. In addition, transgenesis can be used to increase disease resistance in commercial birds, potentially decrease phosphorus excretion, and release presently indigestible plant material. Therefore transgenesis will provide the opportunity for the poultry industry to more efficiently produce meat and eggs with less adverse effects on the environment. Secondly, it may be possible to mass produce high-quality therapeutic proteins using transgenic chicken that may have an enormous effect on human survival from the disease. Therefore all of these benefits of poultry transgenesis in human life may allow for the development of transgenic farms.

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Palmiter, R.D., Norstedt, G., Gelinas, R.E., Hammer, R.E., Brinster, R.L., 1983. Metallothionein-human GH fusion genes stimulate growth of mice. Science 222 (4625), 809 814. Available from: https://doi.org/10.1126/science.6356363. Park, T.S., Lee, H.G., Moon, J.K., Lee, H.J., Yoon, J.W., Yun, B.N.R., et al., 2015. Deposition of bioactive human epidermal growth factor in the egg white of transgenic hens using an oviduct-specific minisynthetic promoter. FASEB J. 29 (6), 2386 2396. Available from: https://doi.org/10.1096/fj.14-264739. Rapp, J.C., Harvey, A.J., Speksnijder, G.L., Hu, W., Ivarie, R., 2003. Biologically active human interferon α-2b produced in the egg white of transgenic hens. Transgenic Res. 12 (5), 569 575. Available from: https://doi.org/10.1023/A:1025854217349. Reed, K.C., Mann, D.A., 1985. Rapid transfer of DNA from agarose gels to nylon membranes. Nucl. Acids Res. 13 (20), 7207 7221. Available from: https://doi.org/10.1093/ nar/13.20.7207. Saeki, K., Matsumoto, K., Kinoshita, M., Suzuki, I., Tasaka, Y., Kano, K., et al., 2004. From the cover: functional expression of a 12 fatty acid desaturase gene from spinach in transgenic pigs. Proc. Natl. Acad. Sci. U.S.A. 101 (17), 6361 6366. Available from: https://doi.org/10.1007/BF00968651. Schock, E.N., Chang, C.F., Youngworth, I.A., Davey, M.G., Delany, M.E., Brugmann, S.A., 2016. Utilizing the chicken as an animal model for human craniofacial ciliopathies. Dev. Biol. 415 (2), 326 337. Available from: https://doi.org/10.1016/j.ydbio.2015.10.024. Selman, R.G., Houston, D.C., 1996. The effect of prebreeding diet on reproductive output in zebra finches. Proc. R. Soc. B Biol. Sci. 263, 1585 1588. Available from: https:// doi.org/10.1098/rspb.1996.0232. Shin, S., Choi, Y.M., Han, J.Y., Lee, K., 2014. Inhibition of lipolysis in the novel transgenic quail model overexpressing G0/G1switch gene 2 in the adipose tissue during feed restriction. PLoS One 9 (6), 1 10. Available from: https://doi.org/10.1371/journal. pone.0100905. Suganthi, R.U., 2014. The uniqueness of immunocompetence and meat quality of native chickens: a specialized review. World J. Pharm. Pharm. Sci. (WJPPS) 3 (2), 2576 2588. Retrieved from: http://www.wjpps.com/current_issue.php. Sun, Y., Chen, X., Xiao, D., 2007. Tetracycline-inducible expression systems: new strategies and practices in the transgenic mouse modeling. Acta Biochim. Biophys. Sin. 39 (4), 235 246. Available from: https://doi.org/10.1111/j.1745-7270.2007.00258.x. Toldra´, F. (2008). Meat Biotechnology. Springer. https://doi.org/10.1007/978-0-387-79382-5 Tong, H.B., Cai, J., Lu, J., Wang, Q., Shao, D., Zou, J.M., 2015. Effects of outdoor access days on growth performance, carcass yield, meat quality, and lymphoid organ index of a local chicken breed. Poult. Sci. 94 (6), 1115 1121. Available from: https://doi.org/ 10.3382/ps/pev032. Trevor, B.J., 2013. Poultry health and disease control in developing countries 1 5. Available from: https://doi.org/10.1158/1078-0432.CCR-14-3111. Westhusin, M., 1997. From mighty mice to mighty cows. Nat. Genet. 17 (1), 4 5. Available from: https://doi.org/10.1038/ng0997-4. WHO/FAO/UNU Expert Consultation, 2007. Protein and amino acid requirements in human nutrition. World Health Organization Technical Report Series, (935), pp. 1 265. ISBN 92 4 120935 6. Williams, T.D., 1996. Intra- and inter-individual variation in reproductive effort in captive breeding zebra finches (Taeniopygia guttata). Can. J. Zool. Rev. Can. Zool. Available from: https://doi.org/10.1139/z96-011.

Further reading

Wilmut, I., Haley, C.S., Woolliams, J.A., 1992. Impact of biotechnology on animal breeding. Anim. Reprod. Sci. 28 (1 4), 149 162. Available from: https://doi.org/10.1016/ 0378-4320(92)90101-I. Yang, N., Jiang, R.-S., 2005. Recent advances in breeding for quality chickens. Proc. Nutr. Soc. 61 (3), 373 381. Available from: https://doi.org/10.1079/WPS200563. Yu, B., Jan, Y.C., Chung, T.K., Lee, T.T., Chiou, P.W.S., 2004. Exogenous phytase activity in the gastrointestinal tract of broiler chickens. Anim. Feed Sci. Technol. Available from: https://doi.org/10.1016/j.anifeedsci.2004.08.011. Zaheer, K., 2015. An Updated review on chicken eggs: production, consumption, Management aspects and nutritional benefits to human health. Food Nutr. Sci. 6, 1208 1220. Available from: https://doi.org/10.4236/fns.2015.613127. Zhang, Q., Chen, J.Q., Lin, J., Yu, Q.H., Yu, H.Q., Xu, X.J., et al., 2014. Production GH transgenic goat improving mammogenesis by somatic cell nuclear transfer. Mol. Biol. Rep. 41 (7), 4759 4768. Available from: https://doi.org/10.1007/s11033-014-3347-7. Zhu, L., Van De Lavoir, M.C., Albanese, J., Beenhouwer, D.O., Cardarelli, P.M., Cuison, S., et al., 2005. Production of human monoclonal antibody in eggs of chimeric chickens. Nat. Biotechnol. 23 (9), 1159 1169. Available from: https://doi.org/10.1038/ nbt1132.

Further reading Cao, D., Wu, H., Li, Q., Sun, Y., Liu, T., Fei, J., et al., 2015. Expression of recombinant human lysozyme in egg whites of transgenic hens. PLoS One 10 (2), e0118626. Available from: https://doi.org/10.1371/journal.pone.0118626.

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10

Xiaofei Wang1, Laruen E. Shields 2, Rebecca L. Welch3, Alexis Pigg2 and Karim Kaleh1 1

Department of Biological Sciences, Tennessee State University, Nashville, TN, United States 2 Department of Agriculture and Environmental Sciences, Tennessee State University, Nashville, TN, United States 3 Department of Chemistry, Tennessee State University, Nashville, TN, United States

10.1 Introduction For thousands of years, humans have been manipulating the genomes of animals and crops to obtain progeny that carry desirable traits, despite that there was little understanding of genetics. Nevertheless, genome manipulation, combined with environmental manipulation, has brought about tremendous improvement in the production of animal and plant products: food, medicine, industrial raw materials. Artificial selective breeding is probably the earliest method for genome manipulation, where natural superior variants were selected to produce succeeding generations of crops and animals, with the intent of passing the superiority to future generations. Artificial selective breeding has been documented in accounts going back to ancient Rome, when a Muslim traveler recorded his observations on Roman cropping techniques (Kingsbury, 2009). It is also possible that natural hybrids were also utilized in animal and plant breeding in early history. Although artificial selective breeding practice began in ancient times, it was not until recently, when the law of heredity was discovered by Mendel, has genome manipulation become guided by genetics. It becomes clear that the genome dictates the performance of crops, farm animals, or other organisms giving the same environment to different individuals of the same species. Modern farming improvement relies heavily on genetic and environmental manipulation for the ultimate subject: the organism being farmed. Guided by genetics, modern genome manipulation has employed a wide array of approaches. Although artificial selection is still an essential technique, many new tools have been added to the toolbox of researchers and breeders. Early artificial selection relies on natural variants and spontaneous mutations in the genome. However, there are limitations in existing variants and spontaneous mutations that are suitable for the breeding goals. Since mutations can be induced artificially, mutation induction has been employed to increase the available gene Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00010-2 © 2020 Elsevier Inc. All rights reserved.

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pool for selection. Researchers have employed radiation to induce mutations in the genome of interest (Abplanalp et al., 1964; Lundqvist, 2014). To date a good number of crop lines have been commercially produced by radiation induction. Radiation breeding is, however, often accompanied by high chromosomal aberrations and detrimental effects on the irradiated organism. Researchers have also employed chemical mutagens for mutation induction. Popularly used chemical mutagens include alkylating agents such as ethyl methanesulfonate, which cause base substitution in DNA. Mutagenesis at predefined sites in a large genome is of great interest, because it could help resolve the function of genes of interest. For this purpose, sitedirected mutagenesis was invented (Flavell et al., 1975; Rawlins and Muzyczka, 1980). In its early stage, site-directed or site-specific mutagenesis was only applicable to in vitro studies (Shortle et al., 1982), or to viral genomes (Pugatsch and Stacey, 1982; Bryant and Parsons, 1982). Combined with gene replacement method, site-directed mutagenesis was soon applied in prokaryotes (Ohman et al., 1985). Later, manipulation of large genomes using site-directed mutagenesis and followed by homologous recombination, called gene targeting in literature, provided the opportunity to manipulate large genomes such as the mouse genome (Capecchi, 1989). This technology is most often used in generating “knock-out” animals, in which the function of a targeted gene is attenuated, modified, or otherwise disrupted. Transgenesis, the generation of transgenic animals, is closely related to mutagenesis, with subtle difference that transgenesis involves exogenous DNA. Transgenesis began with the injection of foreign DNA constructs into animal pronuclei of fertilized eggs (Gordon et al., 1980; Brinster et al., 1981). While foreign genes can be introduced into the animal genome by this method, the exogenous DNA integrates into the host genome in an uncontrolled way, often at random locations. Nevertheless, foreign genes are introduced to animal genome. The expression of the foreign genes can result in dramatic changes of phenotype (Palmiter et al., 1982). Although exogenous genes can be easily introduced into animal cells today, they may not be replicated and transmitted during cell division, because the artificially constructed genes often lack required DNA elements for replication and proper segregation during cell division. After introduction of exogenous genes to the host cells, selection is required to obtain cells with transgene integration into the chromosome. Otherwise, the exogenous DNA must carry necessary components for stable transmission through mitosis and meiosis. Unfortunately, no convenient vector systems contain the necessary elements for self-replication and transmission in higher animal cells to date. Several tools have been engineered to modify a large genome at a predetermined site with fewer unintended modifications. These technologies include zinc finger nucleases (ZFNs), transcription activator-like effectors (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9 (CRISPR/Cas9), collectively termed genome editing technologies, which allowed for a more precise modification at a targeted site. These technologies can generate

10.2 History of chicken genome manipulation

deletions, insertions, and base substitutions. This review will focus on these technologies in more detail.

10.2 History of chicken genome manipulation The chicken has been domesticated in possibly multiple geographic locations for about 2500 years (Xiang et al., 2014; Miao et al., 2013). The red jungle fowl, Gallus gallus, is the primary ancestor of domestic chickens. DNA evidence indicates that several other species in genus Gallus also contributed to the genetic makeup of the domesticated chickens. Since domestication, the chicken has been selected for various traits. Artificial selection apparently played a major role in forming the breed-specific features of the chicken genome. A large number of breeds with distinct, stable characteristics have been created and raised all over the globe, which is an indication of the success of traditional artificial selection. It is the subject of debate as to whether there were artificial crosses between the domestic chicken and wild fowl. If such crosses did occur, then hybridization approach for chicken genome manipulation can be traced far back in history, since the contribution from other wild species is widespread in many chicken breeds. Intensive farming of poultry began in 1930s in the United States, where producers began to provide chicken meat for the everyday dinner table (Godley and Williams, 2009). During and immediately after World War II, the demand for chickens surged. Research in poultry nutrition and breeding improved poultry production enormously. Today, broiler chickens can reach 5
6 pounds in just 6 weeks after hatch, compared with the 5
6 months required by traditional backyard chickens. This rapid growth resulted from genetic, nutritional, and management improvements. Studies show that traditional backyard chickens could not acquire the growth rate of modern broilers even if they were fed the broiler rations. Inversely, modern broiler breeds could not grow as rapidly if they were fed the traditional nonformulated diet. In addition to serving in ritual functions and as a human dietary protein source, the chicken is a convenient model for biological studies (Delany, 2004). Some studies in chickens have resulted in groundbreaking discoveries in biology, including the discoveries of oncogene and virus-caused cancer development. Studies in the manipulation of the chicken genome will certainly continue to contribute to the field of biology. Recent breakthroughs in genome technologies have been soon applied to the study of the chicken genome. The chicken is the first nonmammalian vertebrate whose genome sequence was made available. While the genome of chickens is smaller in size, close to one third of the mammalian counterpart, the estimated number of coding genes in the chicken genome is almost the same as in a mammalian genome. The smaller avian genome is, attributable to smaller intergenic

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regions, smaller introns, and lower content of highly repetitive sequences, advantageous for the study and understanding of the regulatory regions in all vertebrates. Thus, in addition to the commercial value of the species, the value of chickens as a model system for biological studies continues to be important. After invention, recombinant DNA technologies were soon applied to the manipulation of the chicken genome after they were invented. The introduction of artificial foreign genes manipulated in vitro to an organism can be traced back to the discovery of Griffith’s transforming principle in the 1920s. The first true transgenesis was reported in 1974, when Chang and Stanley expressed Staphylococcus aureus genes in Escherichia coli (Chang and Cohen, 1974). The successful transfer of foreign genes to vertebrate embryos was first reported in 1980, when DNA was directly injected to mouse embryos. The earliest transgenic chicken was reported by Salter et al. (1987), who injected retroviral vectors to chicken embryos, and successfully established germline transmission of the transgene. Since then, there have been a large number of articles describing studies on various aspects of generating transgenic chickens (Bosselman et al., 1989; Briskin et al., 1991; Fraser et al., 1993; Naito et al., 1998; Harvey et al., 2002). Early transgenesis approaches allowed lateral transfer of recombinant genes to an organism, but the recombinant gene often integrates at genomic sites that are not intentionally controllable. The exploration for methods that can integrate transgenes at defined sites followed. Several methods for integration of transgenes at predefined site have become available, for example, ZFNs, TALENs, and CRISPR/Cas9. These methods were soon applied to studies in chickens (Fan et al., 2011; Park et al., 2014; Taylor et al., 2017; Veron et al., 2015; Oishi et al., 2016), and fascinating progress was made in the past few years.

10.3 Embryo culture Since the development of transgenic birds and/or genome-edited birds involves the manipulation of embryos, it is necessary to mention the development of techniques in avian embryo culture. The most effective way to modify the genome of a vertebrate individual is to operate on fertilized eggs, because a fertilized egg is capable of developing into an individual animal. A stem cell cultured in vitro can develop in to any cell type, but the current technology is unable to produce a vertebrate animal from a stem cell cultured in vitro without the use of fertilized eggs. An efficient way to obtain a fertilized egg at the single cell stage is by in vitro fertilization. Technologies for in vitro fertilization has been well developed for mammals. While it is possible to conduct in vitro fertilization in chickens (Naito et al., 1998; Tanaka et al., 1994), it is more costly due to the fact that hens rarely release more than one ovum at a time, which is in sharp contrast to mice which release many eggs in each ovulation cycle. However, fertilized,

10.3 Embryo culture

naturally laid chicken eggs are readily available. After fertilization, the chicken embryo immediately begins to develop while in the reproductive tract of the hen. By the time of oviposition, the chicken embryo has developed to stage X (EyalGiladi and Kochav, 1976; Kochav et al., 1980), wherein it may already have 50,000 cells. Clearly, strategies for generating transgenic and genome-edited mammals must be modified for birds (Fig. 10.1). Chicken embryos lacking eggshells or those with compromised eggshells cannot develop normally unless the necessary conditions are met. To access the embryo enclosed in the eggshell, it is necessary to either remove the eggshell, or to make a window through the eggshell. In either case, the integrity of the eggshell is compromised. An embryo in a compromised shell often dies due to infection, water loss, and/or other issues during incubation. Attempts to culture chicken embryos have been reported in the 1970s. Auerbach et al. (1974) showed successful culture of shell-less chicken embryo explants from 3 to around 18, even 21 days of incubation in petri dishes. A similar method was reported by Dunn and Boone (1976). However, none of the embryos hatched under the conditions provided. After the successful establishment of transgenic mammals, it became clear that cultivation of chicken embryos in compromised eggshells to hatch is a necessary step in order to generate heritable transgenic chickens. In order to overcome this difficulty, Rowlett and Simkiss (1986) developed a surrogate eggshell method, with which 3-day old embryos were transferred to surrogate eggshells either from turkeys or from chickens, and then sealed with cling film. The surrogate method allowed near-normal hatching of chicks. Perry (1988) reported a complete culture system for chicken embryos from fertilization to hatch, based on the surrogate eggshell method. The system was designed to accommodate the needs of embryonic development in three stages. In stage (I), the ovum, collected from the magnum of the oviduct, was placed in a glass jar containing 8
16 mL culture media, and cultured for 1 day, which corresponds to the developmental stage of the ovum in the oviduct. In stage (II), the ovum was transferred to a surrogate eggshell which was then filled with culture media, and sealed with saran wrap, leaving no free-space. The embryo was subsequently cultured for 3 days, which corresponds to the embryogenesis stage (incubation days 1
4). In stage (III), the embryo was transferred to another surrogate eggshell, then the surrogate eggshell was supplemented with egg white, leaving some unfilled space. After the opening was covered with saran film, the eggshell was then set in the upright position, and the embryo was cultured to hatch. Using this system, Perry reported up to 22% hatchability. Improvement was made later by Naito et al. (1990), who replaced the thick albumen with thin albumen from laid ovum prior to incubation. Naito and colleagues were able to bring up to 50% of the embryos in surrogate eggshells to hatch. Further development of the surrogate eggshell system was reported by Borwompinyo et al. (2005), who described improvement for transfer of embryos, sealing of the surrogate eggshells. They reported up to 75% hatchability. Similar methods were also used for quail embryos. While the surrogate eggshell method was successful in bringing the

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Transduction Transgene vector Transfection Genome editing tools

Primordial germ cells

v Inject into stage x windowed egg

Inject into stage 14 embryo

v Seal and Incubate 3 d

Seal and incubate

Transfer to surrogate shell

Chimeric chick

Selection and breeding for desired progeny

FIGURE 10.1 Strategies for production of transgenic or genome-edited birds. Appropriate vectors are designed and produced, then introduced by transduction or transfection into primordial germ cells (PGCs) in vitro. Specific PGCs are then selected and those stably carrying the transgene or the genome-edited allele are injected into stage x (before incubation) embryos. After injection, the eggs are sealed and incubated for 3 days, and then, the embryos are transferred to a surrogate shell, where they develop to hatch. Alternatively, the PGCs are injected into HH stage 14 (about 3 days into incubation) embryos, which are incubated to hatch. Transgene vectors or genome-editing vectors can also be package in viral particles, which are injected into stage X embryos. Following viral particle injection, the eggs are sealed and incubated for 3 days, and then the embryos are transferred to surrogate shells, where they are incubated to hatch. Potential chimeric chicks are assessed for the presence and expression of the transgene or the genome-edited allele, then selective breeding is conducted to obtain desired progeny.

10.4 Delivery of transgenes

operated embryos to hatch, direct windowing of the eggshell was also reported to be successful (Speksnijder and Ivarie, 2000). The size of the window and the way by which the window is sealed appear to be critical to the development of embryos. Either way, the air bubble introduced into the egg is believed to be toxic to the embryo during the early stage of development and thus to be avoided (Andacht et al., 2004). The surrogate eggshells for embryo culture must be larger than the donor eggs. However, fresh surrogates may be unavailable, and it is inconvenient to prepare surrogate eggshells. Culture of embryos using artificial vessel has also been explored. Tahara and Obara (2014) reported a successful trial in which chicken shell-less embryos after 3-day of incubation were placed in artificial vessels made up of polymethylpentene film and cultured to hatch. With the supplementation of calcium lactate and distilled water, 90% of the embryos survived to day 17 of incubation. In addition, pure oxygen was provided to the culture vessels from day 17 for the surviving embryos, which allowed a hatchability of 57% from the surviving embryos on day 17. This artificial vessel method achieved an accumulative hatchability of 32%. To date, the majority of transgenic chickens were generated via the surrogate eggshell method. While artificial culture methods have the potential to yield more consistent results, it awaits for further improvement to obtain high hatchability and convenience of preparation.

10.4 Delivery of transgenes Despite that traditional approaches have been effective in improving production traits in crops and livestock, methods that can introduce new genes laterally into a targeted species are also needed. Natural lateral gene transfer rarely occurs among higher organisms. Transgenesis based on recombinant DNA technologies has provided new potential for crop and livestock improvements, and has generated new products, such as new pharmaceuticals. Two prominent goals in transgenesis study are to (1) understand the biological system and (2) produce organisms with desirable traits that were not attainable by traditional approaches. Stable transmission and expression of transgenes are necessary for maintenance of transgenic animals. Transgenes may exist as episomes that do not integrate into the genome of the host cells. In eukaryotes, episomes are not stable, and may diminish rapidly as cells divide without the replication of the episomes. Stable transmission requires the integration of the transgene into the host chromosome in eukaryotic cells. Unless the transgenic DNA molecule contains all elements required for replication and segregation during mitosis and meiosis in eukaryotic cells, a transgene is doomed to disappear from the host progeny. Attempts to create vertebrate artificial chromosomal vectors have been so far proven to be very difficult and inconvenient, despite the many desirable advantages (reviewed in Satoh et al., 2018; Katona, 2015).

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Recombinant DNA can be introduced into animal cells in several ways. A widely used approach is the liposome-based method. Liposome transfection has been very efficient in delivery of exogenous DNA into animal cells in culture. For this reason, attempts have been made to apply liposome-based methods (Wang et al., 2019) or polyethylenimine encapsulation methods (Zuo et al., 2016) for introducing recombinant DNA to chicken embryos. Recombinant DNA can also be introduced into chicken embryos by electroporation (Williams et al., 2018; Sato et al., 2007). However, these methods are suitable for transient expression and short-term analysis of gene expression, but are less efficient for direct introduction of recombinant DNA to chicken embryos for generating animals with heritable transgenes. Several reports have described the use of sperm to mediate the generation of transgenic chickens. In a study using sperm-mediated gene transfer by Collares et al. (2011), chicken semen was collected and washed several times to remove seminal plasma which has DNase activity. The spermatozoa were transfected with an EGFP vector using dimethyl sulfoxide or dimethylacetamine as the transfectant. The transfected spermatozoa were then used for artificial insemination of laying hens. The study showed that the DNA of the transgene was present in 38% of the resulting chicks, and the mRNA from the transgene was present in 21%. Protein level expression of the transgene was also observed, albeit at a very low rate. Cooper et al. (2017) also demonstrated the feasibility of sperm-mediated transgene delivery, called sperm-assisted genome editing, reporting knockout of the transgene for green fluorescence protein with genome editing tools. There are, however, reports indicating that using DNA-sperm mixture could not produce transgenic chickens when DNase inhibitor was used to prevent the transgene from being degraded. Nonetheless, sperm-mediated DNA delivery approach appears to be feasible and convenient for avian genome manipulation. To date, viral vectors have proven to be very effective for transgene delivery. Viral vectors are engineered from viral genomes, which contain basic elements that are necessary for delivery of genetic material into cells. Viral vector engineering harnesses the innate ability of viruses to enter cells. During engineering, elements that are not essential for integration are replaced by transgenes of interest. In addition, some DNA elements are added for the convenience of engineering and the control of transgene expression (Kay et al., 2001). The life cycle of a virus includes infection and replication. Infection is the introduction of the viral genome into the host cell. This involves the interaction of viral surface proteins with cell membrane and membrane proteins. Once the viral genome is in the host cell, early regulatory viral genes will be expressed, followed by the latent phase when viral structural genes are expressed. In typical engineered viral vectors (Fig. 10.2), the structural genes are often replaced by transgenes. Because the removal of some viral genes renders the virus replication deficient, engineered viral vectors are often safer to use. The viral structural genes

10.4 Delivery of transgenes

Transgene promoter cPPT

Kozak

ORF

RRE ψ

WPRE

Δ5’ LTR Packaging promoter Marker promoter Marker pUCori

ΔU3/3’ LTR SV40 early pA

Antibiotic resistance

FIGURE 10.2 Illustration of a typical lentiviral vector (adopted from Vectorbuilder https://en. vectorbuilder.com/design/pLV_Ex.html). Transgene promoter: the promoter driving the transgene expression in the recipient cell. Kozak: Kozak consensus sequence believed to facilitate translation initiation in eukaryotes. ORF: The open reading frame of the transgene. WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element, necessary for viral RNA stability in packaging cells. Marker promoter: promoter for the ubiquitous expression of the marker gene. Marker: a visually detectable gene or a dualreporter gene for selection or visualization of cells transduced with the vector. ΔU3/30 LTR: truncated HIV-1 30 long terminal repeat. It contains polyadenylation signal for termination of all upstream transcripts. SV40 early pA: Simian virus 40 early polyadenylation signal for improving viral titer. Antibiotic resistance: antibiotic resistance gene allowing the maintenance of the plasmid by antibiotic selection in E. coli. pUC ori: pUC origin of replication for high copy number replication of the plasmid in E. coli. Packaging promoter: promoter driving the transcription of downstream DNA sequence in packaging cells. Δ50 LTR: truncated HIV-1 50 long terminal repeat (LTR). In wildtype lentivirus, LTRs are located unidirectionally on two ends of the viral genome. Ψ: HIV-1 packaging signal for the packaging of viral RNA into virus. RRE: HIV-1 Rev response element allowing the nuclear export of viral RNA by the viral Rev protein during viral packaging. cPPT: HIV-1 Central polypurine tract. It creates a “DNA flap” that increases nuclear importation of the viral genome during target cell infection. This improves vector integration into the host genome, resulting in higher transduction efficiency.

are put in separate plasmid vectors so that structural proteins can be produced for production and packaging of viral particles after transfection of appropriate host cells with the plasmid vectors. Today, many engineered viral vectors are commercially available. Major classes of viral vectors are lentiviral vectors, adenoviral vectors, and adeno-associated virus (AAV) vectors. Adenoviral and AAV vectors remain episomal after transduction, thus are not good for permanent transmission

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of transgenes. Lentiviral vectors are derived from the human immunodeficiency virus (HIV), a member of the retroviral family. They retain their ability to integrate into the host genome. The site of integration is almost random. As a result, the integration may interrupt an essential gene and render the allele containing the transgenic insertion defective. Since the delivery of viral vectors into cells and the expression of viral genes depend on the interactions between viral and host proteins, it is necessary to consider tropism during transgene engineering. The commonly used lentiviral vectors have wide tropism and have been successfully used in transducing chicken embryonic cells (Kwon et al., 2018; Cao et al., 2015; Balic et al., 2014; Byun et al., 2013), which clearly indicates the practicality of using this type of vectors in chickens. Transposon-based vectors are another type of gene delivery tools widely used in transgenic research. One type of transposon-based vectors, piggyBac vectors, originates from a short inverted terminal repeat transposable element from the cabbage looper Trichoplusia ni (Handler et al., 1998). This class of transposons is found in many animal taxa, suggesting their versatility in these animals for gene delivery. This type of transposons has two inverted terminal repeats flanking an open reading frame encoding a transposase. Engineered piggyBac vectors are constructed as two plasmids, one retaining the flanking inverted terminal repeats between which the targeted transgene (cargo) is placed, the other encoding the transposase (see review Kim and Pyykko, 2011). When both the cargo plasmid and the transposase plasmid are cotransfected into the host cell, the transposase will recognize the inverted terminal repeats and integrate the cargo transgene into the host genome. The transposase is only transiently expressed. When the host cell divides, the transposase is gradually lost from the dividing cells, leaving the transgene permanently integrated in the genome. The advantage of the piggyBac system includes the easiness of preparation and the large cargo space. Because piggyBac vectors are introduced into cells by transfection, one drawback is the feasibility of transfection in some cell types that are hard to transfect. It has been demonstrated that piggyBac vectors can be successfully integrated into both the genome of chicken primordial germ cells (PGCs; Park and Han, 2012; Glover et al., 2013; Park et al., 2015) and embryos (Jordan et al., 2014). Tol2 vectors are another widely used transposon-based tool for transgene delivery. The Tol2 vector system is very similar to the piggyBac vector system in many ways. However, Tol2 vectors insert in the genome without any significant bias, unlike the piggyBac which typically inserts at sites containing the sequence AATT. Reports also clearly indicate that the use of the Tol2 vector system is quite feasible in chickens. Lambeth et al. (2016) used Tol2 vectors to generate transgenic chickens expressing aromatase. Freeman et al. (2012) demonstrated the successful application of Tol2 vectors in embryonic inner ear with electroporation. Stoller and Fekete (2016) demonstrated the use of Tol2 vectors to introduce miRNA to chickens in order to study ear development.

10.5 Primordial germ culture

10.5 Primordial germ culture Accessibility of avian zygotes immediately following fertilization is problematic, in contrast to their mammalian counterparts whose ovum can be fertilized in vitro and can be easily utilized for genome manipulation. Recent development in chicken PGC culture has significantly advanced the field of genome manipulation studies in chickens. PGCs are destined to become adult germ cells that give rise to sperm and ovum. Chicken PGCs appear in the embryo before oviposition, and migrate through blood to the germinal ridge where future gonads develop around stage 17 during incubation (see review Lee et al., 2015). This special property allows the culture of PGCs in suspension (van de Lavoir et al., 2006). PGCs whose genome is modified with the recombinant DNA technology can be injected into recipient embryos to generate chimeric chickens, in which a portion of the germ cells comes from the injected, genome-manipulated PGCs. These donor PGCs will give rise to genome-manipulated gametes. Mating of the chimeric chickens may produces genome-manipulated progeny, which can be selected for further breeding and analysis. Chicken PGCs can be isolated from blood of embryos incubated for about 54 hours. Isolated PGCs are useful for genome modification by transgenesis and genome editing (Oishi et al., 2016; Dimitrov et al., 2016). Other cell sources that are competent to form germ cells are also valuable resources for genome manipulation. It has been suggested that embryonic cells at stage X maintained their undifferentiated status. However, studies have indicated that transplanted stage X blastodermal cells have limited ability to form germ cells (Carsience et al., 1993; Pain et al., 1996). Research has also shown that transplanted donor testicular cells have the ability to form testicular cells in recipients (Lee et al., 2006; Trefil et al., 2010). Further improvement of the method could make it more effective. Disruption of the endogenous germ cells in the host for the transplant of PGCs or any germline competent cells could increase the efficiency. Nakamura et al. reported the use of busulfan to reduce the number of endogenous PGCs. They first injected a dose of busulfan to chicken embryos before incubation. The busulfan injection reduced the number endogenous PGC. They then injected donor PGCs to the busulfan-treated embryos and generated chimeric chickens which produced donor PGC-derived progeny (Nakamura et al., 2010). Later, this group used X-irradiation to remove endogenous PGCs, and transplanted PGCs from a donor to the recipient Nakamura et al. (2012). They also obtained chimeric chickens derived from donor PGCs, which are transmissible to future generations. In general, much progress has been made in avian PGC culture and manipulation in vitro. In addition, technology for chicken embryonic stem cell (ESC) culture has also advanced significantly (for review, see Farzaneh et al., 2017; Kagami, 2016).

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10.6 Precise genome editing Programmable genome editing systems are the most notable tool to become available in research in the last decade. Precise localization and efficient cleavage of double-strand DNA using programmable genome editing tools have ushered in new advances in biotechnology and transgenic research. Programmable genome editing not only creates loss-of-function of a gene of interest, but also enhances homologous recombination by causing double-strand DNA break. Currently, researchers have several tools to overcome the difficulties of precise genome editing, including ZFNs, TALENs, and CRISPR/Cas9 (CRISPR associated protein) systems. These editing systems enabled both efficient and precise modification of the genome in various ways and to various degrees. These technologies have brought about new breeding techniques that promote biological traits, disease resistance, and the production of biofunctional proteins. The relevant genes and DNA elements are introduced via genome editing, and animals are then bred until stable lines are established. An examination of the various genome editing tools used in avian species will aid in providing a complete picture of current genome manipulation studies in birds today.

10.6.1 Zinc finger nucleases ZFNs were famed as the first programmable tool. They are engineered customizable DNA binding proteins and nucleases. In 1996, ZFNs were invented by fusion of zinc finger domains from various zinc finger proteins and the cleavage domain of Fok I nuclease. Supposedly, ZFNs are sequence-specific, cutting DNA at strictly defined sites (Kim et al., 1996). ZFNs have a modular structure, in which each zinc finger domain is able to recognize a nucleotide triplet. A ZFN can be engineered to bind to and cleave a specific DNA sequence, making a double- stranded break near the recognition site. Zinc finger libraries have been created that are specific for each of all 64 possible trinucleotides combinations. Therefore, engineered ZFNs can target virtually any sequence of interest. ZFNs can be used for editing cultured cells, including pluripotent stem cells, which are of special importance when developing methods that will work for avian gene editing (Lombardo et al., 2011). The first successful germline transmission of a ZFN-induced mutation was reported in 2002 in fruitflies (Bibikova et al., 2002). There have been other published results using ZFNs to target genes of interest, but so far there is no avian ZFN gene editing research published. While ZFN technology opened the door for more precise gene editing in research, it was accompanied by many disadvantages as well. The complexity and high cost of protein domain construction for each particular genome locus and the probability of inaccurate cleavage of target DNA due to single nucleotide substitutions or inappropriate interaction between domains are the most commonly

10.6 Precise genome editing

associated issues. Because of the major barriers inherent in this technology, the active search for new methods in genome editing is unrelenting.

10.6.2 Transcription activator-like effectors Although TALENs appeared recently, this system has proven to be effective and reliable for genome engineering. They are considered the second generation of programmable genome editing tools used in biotechnology. These customizable DNA scissors can be used to disable a functional gene, correct a damaged or mutated gene, replace or insert a DNA sequence at a specifically chosen location in a genome (Nemudryi et al., 2014). TALENs are made up of a catalytic domain from a nuclease fused to a TAL effector (TALE) protein containing a highly conserved, repeated array of 33
34 amino acid residues, varying only at repeat variable diresidues (RVDs) at positions 12 and 13. These RVDs are specific for the particular DNA sequence to which the TALE binds, and once bound, the nuclease cuts at the target locus (Bogdanove and Voytas, 2011). The customizable specificity of the DNA recognition sequence allows precisely targeted editing at specific genomic loci with fewer off-target effects than ZFNs. TALEN editing has been employed in model organisms from mice to zebrafish, but editing of avian species presents additional challenges (Cooper et al., 2018) as researchers lack ESCs that are germ-line competent, and it is difficult to access and manipulate single-cellstage embryos. A report in 2014 described the first successful generation of knock-out chickens through germ-line transmission of TALEN-edited PGCs (Park et al., 2014). This study specifically examined the efficacy of two TALEN constructs designed to induce insertions/deletions (indels) in specific targeted genes in the chicken genome, with the ultimate end of generating chickens with knock-out of ovalbumin (OV) gene function. To verify TALEN functionality in the chicken genome, constructs were created to induce deletions in three genes [DsRed, β1,4-gal-actosyltransferase 1 (B4GALT1), and β-actin]. TALEN1 and TALEN2 plasmid sets were transfected into regular or DsRed-expressing DF1 chicken cells and results were analyzed via T7 endonuclease I (T7E1) assay and DNA sequencing. Both TALENs induced indels that caused frameshifts in DsRed. Next, the ability of TALEN to silence specific chicken genes was confirmed using constructs specifically targeting chicken B4GALT1 and β-actin together with CMV-GFP reporter plasmids. GFP-positive cells were selected by fluorescence-activated cell sorting (FACS) 1 day posttransfection, cultured in vitro for 10
14 days, then analyzed via T7E1 assay. Mutations in B4GALT1 (36.4%
60.0% efficiency) and β-actin (8.3%
9.1% efficiency) included 1
33 nucleotide deletions and 1 nucleotide insertion, indicating that TALEN mutation of endogenous chicken genes was successful in vitro. The researchers addressed whether TALEN-mediated germline transmission would generate gene-targeted chickens. Two OV TALENs were constructed to induce indels in the translational initiation codon of exon 2 in the chicken OV gene. After transfection into chicken DF1 cells and analysis by T7E1

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and gene sequencing, it was found that OV TALEN2 induced mutation (36.7% mutation efficiency), and OV TALEN1 did not. Therefore, PGCs were cotransfected with OV TALEN2 and the GFP vector and FACS separated 1-day posttransfection. Where the DF1 cells sorted at 30%
40%, less than 5% of PGCs expressed GFP. In the SNUhp26 male PGC line, mutations at the targeted locus (33.4% efficiency) included in-frame as well as frameshift mutations due to 6
29 nucleotide deletions. Finally, OV-knockout PGCs from White Leghorn (WL) chickens were transplanted into recipient Korean Oge (KO) chicken embryos to generate germ-line chimeric founders. Sperm cells from these chimera, analyzed by T7E1 assay and DNA sequencing, showed TALENs induced deletions of the OV gene (18.8%). Test-cross analysis of offspring showed 8% mutation rate and hatched progeny were heterozygous for OV knockout, but lacked any phenotypic deformity, demonstrating that TALENs successfully generated mutant chickens by inducing frameshifts which knocked out the function of the OV gene (Park et al., 2014). Another study examined TALEN-mediated targeting of a gene important for the development of germ cell lineage by generating knockout transgenic chickens (Taylor et al., 2017). Vasa is a DEAD box RNA helicase vital to the formation of germ cells in many species. DDX4 is the chicken homolog of Vasa, though its function has not been as well characterized. This study proposed to induce a TALEN-stimulated homology-directed repair (HDR) wherein homologous recombination occurs when cuts in the DNA are repaired by endogenous cellular machinery. This recombination incorporates exogenous DNA sequences into the target site in the genome. In this case, TALEN-stimulated HDR was used to insert a GFP-2a puromycin reporter transgene into exon 2 immediately downstream of the DDX4 ATG start codon. The endogenous regulatory machinery would control reporter expression and a poly-A tail in the transgene would terminate transcription before exon 4, thereby silencing the function of DDX4 and generating a knock-out transgenic chicken that could allow researchers to examine DDX4 effect on germ cell lineage and oogenesis. Additionally, using homologous arms designed to terminate at exon 10 and exon 19 demonstrated the efficacy of a single nuclease pair in affecting large (10.3 and 30.2 kb) genetic deletions. To determine if the TALENs efficiently targeted the desired locus in PGCs, multiple transfections of female and male PGCs were performed, with puromycin selection at 48 hours posttransfection eliminating untransformed cells. Analysis by flow cytometry after 2 weeks in culture showed that cotransfection of the TALEN pair with the reporter yielded PGCs that stably expressed the GFP transgene (8.1%). Southern blot and RT-PCR analysis confirmed that females were hemizygous knockouts while a single allele was targeted in males. Surrogate embryos were injected with transgenic male PGCs, generating chimera which were then testcrossed, producing transgenic offspring with 6% efficiency. Southern blot analysis of G1 transgenes’ genomic DNA indicated females were hemizygous knockouts while a single allele was targeted in males. The G1 female chickens proved sterile after hatching, with female PGCs forming initially but lost during meiosis. These results highlight the efficacy of TALENs for targeted

10.6 Precise genome editing

genomic editing while further illuminating the function of DDX4 in poultry (Taylor et al., 2017).

10.6.3 Clustered regularly interspaced short palindromic repeats Only a few years after the development and utilization of the TALEN gene editing system, the third programmable genome editing system was developed: CRISPR. The CRISPR genome editing system was established and employed in 2012. This editing tool provides vast potential for application in therapeutic, biomedical, and agricultural research. Over the next few years, CRISPR technologies transformed the entire gene editing field (Sorek et al., 2008; Boettcher and McManus, 2015; Cong et al., 2013). In contrast to TALEN proteins, recognition by the CRISPR/Cas system is carried out via the complementary interaction between a noncoding RNA and the target site of DNA. CRISPR functions based on the CRISPR/Cas9 system are present in prokaryotes for nucleic acid editing. In nature, CRISPR was identified in both archaea and bacteria and is a natural part of adaptive immune defense system. Bacteria have naturally occurring clustered repeats called CRISPRs which are composed of regularly spaced repeats and unique spacers. The latter are derived from invading pathogens, such as viruses. The spacer sequences form the immune memory in the infected cells and descendants. When cells with the immune memory are invaded by viruses, the spacer transcripts will serve as a guide to direct the CRISPR defense system to attack the invading viruses using the Cas9 protein (Barrangou et al., 2007). The protection is sequence-specific (Jinek et al., 2012; Mali et al., 2013). CRISPRCas loci are present in 48% of bacteria and 84% of archaea (Barrangou et al., 2007). Although the presence of clustered repeats of genetic elements were initially observed in the genome of E. coli in 1987, the term CRISPR and the utilization of the system did not appear until a decade later (Sorek et al., 2008). Subsequently, the CRISPR system was recognized to target invading DNA elements (Grissa et al., 2007; Jansen et al., 2002; Brouns et al., 2008; Han and Krauss, 2009). As with ZFN and TALEN systems, the CRISPR/Cas9 system also causes double-stranded breaks in DNA. Repair of double-stranded DNA breaks by nonhomologous end joining often results in the deletion of a small segment of DNA at the breaking point. If the breaks are repaired by the homologous recombination mechanism, exchange between DNA molecules may occur, which may result in incorporation of foreign DNA at the site of the breakage. Since the site-specificity of the CRIPR/Cas9 system is dictated by the spacer RNA sequence (called guide RNA) in the Cas9 enzyme, it is very convenient to design a guide RNA to target any site in the genome for generation of doublestranded DNA breaks. It is for this reason that this programmable genome editing tool is so widely used in so many living organisms. The CRISPR/Cas9 system has demonstrated success in several avian species. The system has been used to modify genes in somatic cells of birds, chicken

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ESCs, and spermatogonial stem cells (Veron et al., 2015; Ahn et al., 2017; Zhang et al., 2017; Zuo et al., 2017). Introduction of CRISPR vectors into PGCs resulted in production of chickens with a modified genome. Examples of genome-edited chickens include those with indel mutations in the ovomucoid gene and those with gene cassettes in the immunoglobulin gene replaced (Oishi et al., 2016; Dimitrov et al., 2016). These studies indicate that CRISPR/Cas9 is more effective than the previous two genome editing tools mentioned (ZFNs and TALENs) in avian species as it has an extremely effective rate in germline transmission.

10.6.4 Cre/LoxP The Cre/Lox-P system is a site-specific gene editing tool discovered and refined in the late 1980s and early 1990s. Cre (abbreviation for “causes recombination”, or “cyclization recombinase”) is a 38 kD protein stemming from a P1 bacteriophage (see review Ray et al., 2000). It catalyzes recombination between two loxP loci, thus is also called Cre recombinase. LoxP (locus of crossing P1) is a 34-bp long DNA element on bacteriophage P1, made up of two lateral segments of 13bp inverted repeating sequence, and a center 8-bp asymmetric, variable sequence, which gives the orientation of a LoxP site. When Cre is expressed, the protein binds to each of the LoxP sites, creating a dimer. The two LoxP sites then align in a parallel orientation, forming a tetramer. The activity of Cre results in doublestranded DNA breaks and crossover events (Fig. 10.3). For genome manipulation, LoxP sites are inserted to the flanking regions of targeted genes, in other words the gene is floxed, and the Cre recombinase is also introduced to the cell. The activity of Cre recombinase results in the removal of targeted DNA sequences, Cre-dependent gene expression, or recombination (Fig. 10.3). Cre/LoxP system can be used in gene editing protocols because it is highly unlikely that the exact loxP sequence occurs in organisms other than the bacteria. The outcome of Cre recombination relies on the location and orientation of the loxP sites. There are typically three different outcomes of a Cre-mediated recombination event: inversion, deletion/knockout of the floxed DNA segment, or reciprocal translocation of DNA segments. Inversion occurs if the loxP loci are on the same DNA strand and have opposite orientation. Deletion happens when the two sites face the same direction on one DNA strand. Translocation stems from loxP sites located on two separate DNA molecules. The Cre/LoxP system is widely used in tissue-specific gene expression studies, wherein Cre recombinase is expressed in a tissue-specific manner by engineering it under the control of tissue-specific promoters and target genes are accordingly floxed (Hennet et al., 1995; Sieburth et al., 1998; Miniou et al., 1999) (for review, see Kos, 2004). Despite the potential benefit in studies of gene functions, there are limited studies in chickens using this system. Leighton and colleagues generated transgenic chicken lines expressing Cre recombinase (Leighton et al., 2016), further use of the transgenic lines for functional studies or another application was not reported. Park and colleagues demonstrated an efficient transgene

10.7 Conclusion

Inversion

loxP

Gene X

loxP

loxP

Cre

Gene X

loxP

loxP

Gene X

Cre

loxP

Deletion Cre

loxP

Gene X

loxP

loxP

Cre

Gene X

loxP

Gene X

loxP loxP

Cre

Translocation

loxP

Gene Y

loxP

Gene X

loxP

Gene X

loxP

Gene Y

FIGURE 10.3 Illustration of Cre/LoxP mediated DNA editing. Depending on the orientation of loxP sites, Cre recombinase activity causes the inversion or removal of floxed (flanked by loxP sites) DNA element in the genome, or causes reciprocal translocation between two DNA molecules.

expression system using a cumate-inducible promoter and Cre-loxP recombination in chicken DF1 and QM7 cells (Park et al., 2017).

10.7 Conclusion In the last two decades, DNA sequencing technologies have laid a solid foundation for high throughput studies of the chicken genome. After Homo sapiens, the chicken was the second vertebrate species whose genome is “completely” sequenced. Almost simultaneously, efforts to characterize the chicken transcriptome were reported (Cogburn et al., 2003; Boardman et al., 2002; Carre et al.,

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2006). The availability of a large collection of chicken transcript sequence data heralded the era of functional genomics studies in chickens using microarray technology (Burnside et al., 2005; Bourneuf et al., 2006; Ellestad et al., 2006). These tools and resources allowed the fruitful interrogation of the genetic network that governs the development, immunity, and physiology of chickens (Wang et al., 2007; Resnyk et al., 2013, 2017; Cogburn et al., 2018). The arrival of the massive parallel sequence technology made the sequencing of both genome and transcriptome inexpensive and readily accessible. Numerous studies have been published in the last decade using RNA sequencing and genome resequencing technologies. Studies on chicken genome variations and their phenotypic effects were also progressed tremendously. Significant progresses were also seen in the application of SNP arrays for genotyping (Groenen et al., 2011; Jia et al., 2013; Kranis et al., 2013), genome tiling arrays for copy number variation discovery (Wang et al., 2010; Crooijmans et al., 2013). Recombinant DNA-based genome manipulation biotechnology has transformed the landscape of genome studies in vertebrates. However, the application of these technologies to poultry has lagged. Genome editing in chickens using CRISPR tools has only begun to flourish in the last few years. It is reasonable to expect that, in the foreseeable future, combinations of long-term PGC culture, genome editing technology, and transgenic technology will lead to more convenient and cost-effective modifications of the chicken genome. The creation of readily available genome-engineered resource lines will be especially helpful for studies of the genetic circuitry. Precise genome manipulation can reveal functional elements that are especially important in regulating production and reproductive traits. Genome engineering has the potential to facilitate breeding programs aimed at improving production efficiency and creating new animal farming products using the chicken as a bioreactor. As functional elements and their organization in the genome result from millions of years of natural selection and probably thousands of years of artificial selection, precision modification of the genome warrants meticulous investigation if it is ever to achieve the level of superiority demanded by various applications. In its dedication to the pursuit of that superiority, the research community has a responsibility to vigilantly uphold those rigorous ethical standards that are laid out for the appropriate study of animals.

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90. Lee, Y.M., Jung, J.G., Kim, J.N., Park, T.S., Kim, T.M., Shin, S.S., et al., 2006. A testismediated germline chimera production based on transfer of chicken testicular cells directly into heterologous testes. Biol. Reprod. 75, 380
386. Lee, H.J., Lee, H.C., Han, J.Y., 2015. Germline modification and engineering in avian species. Mol Cells 38, 743
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Concepts and potential applications of gene editing in aquaculture

11

Amit Pande1, Raja Aadil Hussain Bhat1, Ankur Saxena1 and Mudit Tyagi2 1

ICAR-Directorate of Coldwater Fisheries Research, Bhimtal, Nainital, India 2 The George Washington University, Washington, DC, United States

11.1 Introduction Scientists have employed gene engineering techniques for several years to answer complex questions in biology. The deciphering of DNA structure, its replication, transcription, and translation have been landmarks in the history of genomics. The discovery of restriction endonucleases enabled the cleavage of the genetic material (DNA) at precise locations within the gene which facilitated its characterization, cloning, sequencing, and finally expression to obtain protein in large quantity. Genetic engineering tools further enabled the generation of useful recombinant proteins, vaccines, high-yielding transgenic plants and animals, besides identifying the molecular signatures of various pathogenic and genetic diseases. Moreover, genetic manipulation proved highly useful in the development of numerous molecular diagnostic techniques, such as assays involving polymerase chain reaction (PCR), quantitative PCR (q-PCR), high throughput sequencing, and microarray. Notably, genetic engineering techniques, namely RNA interference, has revolutionized the field of genetic manipulation and exerted a great impact on modern medicine, especially gene therapy. Unfortunately, RNA interference has its own limitations, involving nonspecific and incomplete depletion of target protein. These limitations prompted the hunt for “novel” approaches that are able to selectively and completely eliminate expression of a given gene, which led to the discovery of genome editing. In this chapter, the concept of genome editing, principles governing them, and their application in life sciences with a focus on aquaculture are discussed.

11.2 Genome editing Basically, genome editing refers to the technique of introducing sequence-specific modifications by means of engineered nucleases in a variety of organisms and cells (reviewed by Ma and Liu, 2015; Gaj et al., 2016; Hu et al., 2016; Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00011-4 © 2020 Elsevier Inc. All rights reserved.

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Barman et al., 2017). In the last few decades, several genome editing techniques had been developed for manipulating the genome of an organism. These approaches allow the molecular biologists to precisely edit, delete, or alter the genomic sequences of an organism using a specific set of engineered nucleases. Phenotypic characteristics and genotypic composition of an organism can be altered by making precise, specific, and accurate changes in the genetic material of an organism. Gene editing involves a specific double-strand break (DSB) by endonucleases, followed by DNA repair either with error-prone nonhomologous end joining (NHEJ) or by less-frequent but precise homologous recombination (HR) (Fig. 11.1). Both the precise changes to the nucleotide sequences of a gene or, disruption of a gene due to imperfect repair of a genomic segment are expected to have a tremendous impact on numerous branches of scientific development and applications, which include development of novel gene-based therapies, besides acceleration of discoveries in both basic and translational scientific fields. The technology of gene editing can be very well applied in the treatment of genetic diseases, cancer, elimination of pests as well as diseases.

FIGURE 11.1 Targeted nucleases [zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly-interspaced short palindromic repeats (CRISPR)] cleave targeted DNA at the desired site. Site-specific double-strand breaks (DSBs) are repaired by either nonhomologous end joining (NHEJ) or via high fidelity repair system homology-directed repair (HDR) pathway by providing exogenous ssDNA or dsDNA template. Imprecise NHEJ pathway produces insertion and/or deletion mutations of variable length, whereas HDR incorporates precise point mutations or modifications.

11.3 Zinc finger nucleases

This novel concept initiated with the discovery of engineered nucleases like the zinc finger nucleases (ZFNs) and transcriptional activator-like effector nucleases (TALENs). These nucleases induce DSB at a precise DNA locus of interest, which triggers the events of DNA repair, primarily through error-prone NHEJ. The repair of DNA breaks via NHEJ results in several mutations to the targeted gene. Subsequently, the induced stop codons due to mutations restrict the protein expression through mutated mRNA. The technology has been used for numerous applications, such as to disrupt or replace genes for activating, repressing, visualizing, and modifying the loci of interest. However, the discovery of clustered regularly-interspaced short palindromic repeats (CRISPR) and CRISPRassociated protein 9 (Cas9) has transformed science, besides inviting public debate. CRISPR-Cas9 technique was judged as the “breakthrough of the year” by the Science Magazine in 2015. This technique allows precise editing of the genetic code to enable novel gene drive that could be spread to other organisms. CRISPR-Cas9 has a cutting edge over other techniques because of its ability to deliver a gene to a precise location in addition to its affordability and relative ease of use in the lab. The ease with which CRISPR-Cas9 and TALENs can be configured to recognize new genomic sequences has revolutionized genome editing to enable significant discoveries in the fields of synthetic biology, human and animal health, disease modeling, drug discovery as well as agriculture. Their ability to induce double-stranded breaks followed by activation of cellular DNA repair enables site-specific genome modifications. The technique can be employed to carry out knockouts by introducing insertions or deletions using NHEJ. Gene integration or base correction via homology-directed repair (HDR) can be carried out in the presence of donor template with homology to the targeted chromosomal site. The flexibility of the genome modifying enzymes serves as nitty-gritty for artificial transcription factors that are capable of modulating gene expression within a genome. Moreover, CRISPR-Cas9 system can also be employed to edit mRNA sequences, which provides the advantage of transient disruption of any gene expression (Abudayyeh et al., 2017; Cox et al., 2017).

11.3 Zinc finger nucleases ZFNs are artificial restriction enzymes developed by covalently linking zinc finger DNA-binding domain (DBD) with a DNA-cleavage domain of FokI endonuclease (Fig. 11.2). ZFNs domain architecture consists of approximately three to four zinc-finger domains. Each domain is composed of 30 amino acid residues that are structurally arranged in ββα motif (Pavletich and Pabo, 1991). The amino acid residues that assist in recognition of target DNA are located within the α-helical domain which typically recognizes 3
4 bp of the target sequence (Wolfe et al., 2000). These fused nucleases function only as dimers,

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FIGURE 11.2 Graphical representation of zinc finger nucleases (ZFNs). The depicted structure shows dimeric ZFNs containing four zinc finger DNA-binding domains, each recognizing three DNA bases of target sequence fused with a FokI endonuclease. Spacer sequence shown in red is generally 4
7 bp.

therefore, pairs of ZFNs are designed to cleave a specific locus within the genome. One pair recognizes the forward sequence while the other is designed to recognize the reverse strand. Dimerization of FokI endonuclease domain results due to binding of ZFNs on the two flanks of the target sequence which results in cleavage of the DNA within a 4 or 7 bp spacer sequence. This separates the two inverted “half-sites” and leads to the formation of DSB with 50 overhangs (Urnov et al., 2010). DSB triggers the cells’ intrinsic DNA-repair process, namely, HR and NHEJ. Generally, error-prone NHEJ occurs in all phases of cell cycle while precise HR takes place during the sister chromatid available stages of cell cycle. Reports have shown that if repair template is cotransfected with the ZFNs, the repair templates integrate into 1%
20% of the cells at target locus through HR (Miller et al., 2007; Do et al., 2012). ZFNs offer various advantages like the development of knock-in and knock-out animal models by manipulation of germline-competent cells, knockout of genes not responsive to RNAi and heritable mutations. Off-target mutations are an alarming concern associated with ZFNs genome editing (Gabriel et al., 2011; Pattanayak et al., 2011). Scientists have applied several approaches to increase the target specificity of DNA- binding domains of ZFNs. The most successful among all is designing of obligate heterodimeric ZFN architectures governed by charge
charge repulsion to avoid unwanted homodimerization of the FokI cleavage domain (Miller et al., 2007; Doyon et al., 2011). Another approach for refining the ZFN specificity is to introduce them into cells as purified proteins. Inherent cell penetrating property of purified ZFN proteins introduce DSBs in the target sequence with less offtarget effects as compared with when translated into the cells. (Gaj et al., 2012, 2014). Another approach to minimize the off-target effects is by designing ZFNickases. These consist of one inactivated ZFN monomer in combination with

11.4 Transcriptional activator-like effector nucleases

another enzymatically active ZFN monomer which can facilitate gene editing by cleaving one of the DNA strands without the formation of DSBs (Kim et al., 2012; Ramirez et al., 2012; Wang et al., 2012).

11.4 Transcriptional activator-like effector nucleases TALEN system was developed by researchers working on an economically important bacterial pathogen of the genus Xanthomonas which causes severe damage to agricultural crops. As this pathogen causes a significant impact on agricultural production, extensive study has been conducted on its interaction with the host. An indepth study has shown that this bacterial genus secretes effector proteins known as transcription activator-like effectors (TALEs) into the cytoplasm of plant cells as important virulence factors (Boch et al., 2009). The mechanism of these secreted effector proteins revealed their DNA- binding ability and caused the activation of target genes by mimicking the eukaryotic transcription factors. TALENs are flexible artificial restriction enzymes developed by fusing TALE DBD at aminoterminus site with nonspecific DNA-cleavage domain and nuclear localization signal (Fig. 11.3) (Schornack et al., 2006; Christian et al., 2010; Miller et al., 2011). Binding of TALENs with DNA was first reported in 2007 by Ro¨mer and his coworkers, and a year later two groups of researchers decoded the binding sites of TALE proteins for the target DNA (Boch et al., 2009; Moscou and Bogdanove, 2009). DNA-binding domain consists of monomers with 10 to 30 repeats, each binds with one nucleotide of the target sequence. Monomers are tandem repeats of highly conserved 33 amino acid residues with the highly divergent repeat variable diresidue (RVD) amino acids located at positions 12 and 13. These two highly variable amino acids recognize one of the four DNA base pairs (Lamb et al., 2013; Moscou and Bogdanove, 2009). Natural TALE transcription factors secreted from bacterial secretion III system target avirulent genes of plants that have 50 thymidine. (Bogdanove and Voytas, 2011; Boch et al., 2009; Cermak et al., 2011). There is a similar requirement in synthetic TALE transcription factors, thus the presence of thymidine as No residue is responsible for the increased activity of TALENs (Lamb et al., 2013). Generally all the designed TALE repeat arrays contain four domains with the RVDs containing asparagine and soleucine (NI), asparagine and glycine (NG), two asparagine (NN), and histidine and aspartic acid (HD) for binding the nucleotides A, T, G, and C, respectively (Boch et al., 2009; Moscou and Bogdanove, 2009; Cong et al., 2012; Streubel et al., 2012). Several reports have found hypervariable residues with asparagine and lysine (NK) or asparagine and histidine (NH) offer more specificity toward G with slightly lower activity than NN residue which binds with A as well (Cong et al., 2012; Christian et al., 2010).

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FIGURE 11.3 The principle of DNA targeting by clustered regularly-interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) genome editing tool, an example showing the CRISPR-binding site of N-terminal CARD domain of melanoma differentiation associated gene 5 (MDA5) of green chromide, “Etroplus suratensis” (Bhat et al., 2015). The target site (50 -N20-TGG) is shown in bold, where green-boxed TGG trinucleotide is PAM sequence. Complementary and noncomplementary strands to the gRNA are cleaved (depicted as the scissors) by the RuvC and HNH nucleases of Cas9 to produce double-strand breaks (DSBs)’ three base pairs proximal to the PAM. CARD, Caspase activation and recruitment domains; PAM, protospacer-adjacent motif; SS, seed sequence (12 nucleotides upstream to PAM).

There are certain recommendations for designing of TALEs. TALEs should consist of at least three to four properly spaced strong RVDs for its activity, such as HD or NN. Stretches of weak RVDs (NI, NG or NK) should be avoided at the ends besides including strong RVDs as well. For high guanine specificity, NH should be used to promote TALE activity or NK should be incorporated if strong RVDs are present and finally, NN for guanine should be incorporated only if few strong RVDs are present. It has been reported that the first amino acid residue in the RVD (H and N) are indirectly involved in nucleotide binding through stabilizing the spatial conformation. The second amino acid residue of strong RVDs binds to DNA bases through hydrogen bonds while the second amino acid residue of weak RVDs makes van der Waals contacts with targeted nucleotides.

11.5 Clustered regularly-interspaced short palindromic

11.5 Clustered regularly-interspaced short palindromic repeats/CRISPR-associated protein 9 CRISPR-Cas9 is the latest gene editing tool among all target endonucleases which is composed of CRISPR and Cas9. This targeted endonuclease (CRISPR-Cas9) has created a lot of eagerness among the scientific community because of its quick, inexpensive, efficient, and precise gene editing other than existing genome editing tools. Since the discovery of these mysterious repeats in the late 1980s in some bacterial genes, their functional evidence as an adaptive immune system of bacteria was provided by Horvath and colleagues (Barrangou et al., 2007). They have reported that Streptococcus thermophilus cells consisting of complementary CRISPR locus provide immunity against bacteriophages. CRISPR-Cas9 gene editing tool was developed from naturally occurring gene editing system of bacteria. The genetic material of invading bacteriophages which are incorporated within the CRISPR locus are known as CRISPR arrays. These arrays remind the bacteria about exogenous DNA of the viruses or plasmids during reinfection and transformation respectively. During reinfection with same virus or a related virus, CRISPR arrays are transcribed into small guide CRISPR RNA (crRNA) that guides the effector endonucleases (Cas9 proteins) to degrade the DNA of invading virus at complementary sites (Jinek et al., 2012). Further, it has been reported that target recognition by the Cas9 protein requires only seed sequence (12 bp of guide RNA, gRNA) within crRNA and conserved protospacer-adjacent motif (PAM) having NGG nucleotide sequence present downstream of the crRNA binding site (Jinek et al., 2012). gRNA directs the Cas9 to bind with PAM of the target sequence. The size of gRNA is about 100 bp which consists of scaffold sequence required for binding the Cas9 and 20 nucleotides toward 50 end which are complementary to the target locus. Conformational changes in Cas9 result in the binding to nonprotospacer part of gRNA results in which leads to hybridization with 20 bp-protospacer of the target. The binding of guide strand with target strand leads to cleavage of DNA by Cas9 enzymes at a position 3 bp upstream of PAM. Once the double stranded DNA (dsDNA) break is formed, researchers use DNA repair machinery of the cell to add or delete sequences of genetic material or to make changes to the DNA by switching an existing segment with a modified DNA sequence. Thus with CRISPR, scientists have generated short RNA templates that match a target DNA sequence in the genome. The crystal structure of Streptococcus pyogenes CRISPR-Cas9 with its target ˚ resolution has been revealed (Nishimasu et al., 2014). It has sequence at 2.5 A been observed that the bilobed architecture of Cas9-target sequence complex is composed of target recognition and nuclease lobes, “sgRNA:DNA heteroduplex” housed in a positively-charged groove between RuvC and HNH nuclease domains (Nishimasu et al., 2014). Formation of heteroduplex leads to conformational changes that bring the noncomplementary strand into the RuvC active site, which

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places the HNH domain near the target site (Jiang et al., 2016). PAM recognition is mediated by the arginine-rich motif of the carboxyl-terminal domain of the nuclease lobe (Anders et al., 2014). A user-friendly approach to genome editing has been developed by cutting down the requirement of CRISPR components resulting in a feasible, flexible, and robust system. Presently, the CRISPR-Cas 9 requires only Cas9 nuclease and a single, synthetic gRNA containing essential crRNA and transactivating CRISPR RNA (tracrRNA) elements (Cho et al., 2013; Cong et al., 2013; Jinek et al., 2013). Till now, three different variants of Cas9 nuclease have been designed for carrying out genome modifications. The first is known as wild-type, which recognizes the specific locus of dsDNA and cleaves it, resulting in stimulation of DSB repair machinery. Repair of DSBs by NEHJ results in site-specific mutagenesis (Overballe-Petersen et al., 2013). The DSBs are repaired by HDR pathway allowing the incorporation of accurate and precise insertions, whereas Cas9 is delivered with complementary sequences of targeted locus. However, a mutant Cas9 system known as Cas9D10A with only nickase activity breaks only single-strand DNA thus leading to inhibition of NHEJ repair pathway. When provided with the exogenous template, single-strand breaks are repaired only by highly precise HDR pathway, thus resulting in a reduction of insertion mutations (Cong et al., 2013; Jinek et al., 2012). Furthermore, the offtarget effects can be reduced by designing paired Cas9 nickases which produce adjacent DNA nicks (Ran et al., 2013). A nuclease-deficient Cas9 model (dCas9) has been developed by inactivating cleavage activity of endonucleases after mutating their certain active sites. Thus, this variant is used to target any region of the gene without inducing the DSBs/cleavage (Jinek et al., 2012; Deltcheva et al., 2011). The dCas9 system can be used to upregulate or downregulate genes by fusing them with various effector domains (Qi et al., 2013; Maeder et al., 2013). Moreover, dCas9 fused with enhanced green fluorescent protein and gRNA can be used to visualize the repetitive DNA sequences of the genome (Chen et al., 2013a,b). The CRISPR-Cas9 system is classified into three distinct classes with cas1 and cas2 present in all classes that are necessary for the integration of spacer during the adaption stage. The type I CRISPR-Cas9 system requires multiple Cas proteins for degradation of foreign DNA with cas3 protein forming the major portion of the cleaving cascade complex (Makarova et al., 2015). Type II class contains single and very large Cas9 protein, consisting of two nuclease domains, a RuvC-like nuclease domain near the N-terminus and the HNH (or McrA-like) nuclease domain in the middle of the protein (Makarova et al., 2015). Type III CRISPR-Cas systems contain polymerase and processers of spacer repeat transcripts. Type III systems can be further divided into two subtypes III-A and III-B which are involved in degradation of plasmid and RNA targeting. It has been demonstrated that subtype III-A limits the horizontal gene transfer in Staphylococcus. epidermidis by cleaving the transfected plasmid DNA via HD

11.6 Comparison of three genome editing platforms

FIGURE 11.4 Dimerization of paired transcriptional activator-like effector nucleases (TALENs). Fused transcription activator-like effector DNA (TALE) repeats bind with the target DNA and the FokI nuclease domain cleaves the target DNA at a specific site. Each TALE repeat is designed to recognize one nucleotide of target DNA, the specificity to recognize each target nucleotide is determined by repeat variable diresidue (RVD) within each TALEN repeat.

domain of the polymerase-like protein (Marraffini and Sontheimer, 2008). Whereas, subtype III-B from Pyrococcus furiosus has been reported to cleave RNA and act as a RNA-guided RNA cleavage complex (Hale et al., 2009). Out of all three CRISPR-Cas systems, type II permits its application in all organisms due to its simplicity in design. CRISPR-Cas type II systems consist of fused Cas9 endonucleases with short “guide” sequence of RNA that guides the endonucleases to act on a specific target sequence in a genome (Fig. 11.4). The designing of synthetic RNA sequences is much easier than engineering proteins as required for ZFNs and TALENs. In 2012, Jinek et al., published the first report of engineered type II CRISPR-Cas system of S. pyogenes and confirmed that SpCas9 could be guided by a programmable chimeric dual-RNA to target and cleave various DNA sites in vitro.

11.6 Comparison of three genome editing platforms All genome editing tools can be used for incorporating/inducing point or frameshift mutations to disrupt the function of a gene, integration of desired DNA bases and precise editing, with their usage depending upon a given context. The comparison of different genome editing tools on the basis of their attributes are briefly described below and tabulated in Table 11.1.

11.6.1 Efficiency The gene editing efficiency of different targeted nucleases depends upon the transfection efficiency of given cell type, method of transfection, and target

257

Table 11.1 Comparison of different genome editing tools.

Transcriptional activator-like effector nucleases (TALENs)

Clustered regularlyinterspaced short palindromic repeats (CRISPR)

S. no.

Feature

Zinc finger nucleases (ZFNs)

1.

Structure

Fusion of zinc finger DNA-binding domain (DBD) with DNA-cleavage domain of FokI endonuclease

2. 3. 4. 5.

Size of recognition site Ease of designing Multiplexing Off-target

9
18 bases in DNA Difficult than TALENs and CRISPR No Same as that of TALENs

6.

Difficult, require recoding of large DNA segments (500
1000 bp)

Easy, requires only change in 20-bp protospacer of gRNA

7.

Ease of redesigning/ adaptability to target new site Viral delivery

Difficult than other two because it requires polyadenylation signal and promoter

8.

Efficacy

Using Lenti and adenoviruses. Needs cotransduction with two lentiviral vectors each encoding a monomer to form functional heterodimer 11

9.

Application

Indels, obligate ligation-gated recombination (ObLiGaRe). Can insert a 15-kb inducible gene expression cassette at a defined locus in human cell lines, Tagligation

Indels

10.

Cost

Higher than CRISPR

Less than other two

Fusion of transcription activator-like effector DNA (TALE) repeats with DNA-cleavage domain of FokI endonuclease 30
36 bases in DNA Easier than ZFNs No Same as that of ZFNs

Using adenovirus

11

Higher than CRISPR

Cas9 endonuclease and guide RNA (gRNA)

23 DNA bases in DNA Easy than other two Yes More than other two

11 1

11.7 Delivery system

nucleotide sequence. It has been reported that TALENs are more mutagenic than ZFNs using context-dependent assembly. The study suggests that TALENs’ specificity does not depend only on the target sequence, but also on the neighboring sequence in the genome (the sequence context). The context sequence should be taken into consideration before designing TALENs. Overall the targeting efficiencies of TALENs and ZFNs show a slight variation in introducing mutation of a particular gene (Kim et al., 2013). Another study has reported that CRISPRCas9 are more mutagenic than TALENs in producing the mutant clones of human embryonic stem cells (Ding et al., 2013).

11.6.2 Specificity The ZFNs and TALENs function as dimers and the DBD provide them target specificity which is typically designed to recognize 9
18 and 30
36 bp of DNA per cleavage site respectively. A recent report has found less specificity in larger TALEN arrays (Guilinger et al., 2014). One study has opined that TALENs and ZFNs target the same site in C-C chemokine receptor type 5 (CCR5) gene, the off-target mutation induced by TALENs in a highly homologous CCR2 gene is 1%, while as the off-target activity of ZFNs was 11% at CCR2. This is comparable to mutation produced at target CCR5 locus (Mussolino et al., 2011). CRISPR-Cas9 being RNA-guided recognizes the target sequence by the PAM sequence and the 20 nucleotides upstream to PAM. A seed sequence of 12 DNA bases in targeted locus present upstream to PAM is important in its recognition (Cong et al., 2013; Jiang et al., 2013). Similar to ZFNs, CCR5 specific- CRISPR-Cas9 induced cleavage of the nontarget CCR2 gene with mutation rates of 5%
20% (Cradick et al., 2013). Reports have shown that off-target effects can be minimized by calculating the precise Cas9 mRNA concentration (Fujii et al., 2013; Pattanayak et al., 2013). Moreover, by using the paired Cas9 nickase the off-target mutations can be reduced up to 1500-fold (Mali et al., 2013a,b; Ran et al., 2013). However, there is limited knowledge.

11.7 Delivery system cDNA encoding for ZFNs can be delivered with lentivirus and adenovirus (Lombardo et al., 2007; Ha¨ndel et al., 2011), while a TALEN monomer being approximately 3 kb and less stable is delivered by adenovirus because of their larger cargo delivery capacity (Holkers et al., 2012). In addition, highly unstable repetitive sequences in TALENs impair their activity by delivering them through some viral vectors (Holkers et al., 2012), although it can be reduced by minimizing the repetitive sequence in the coding region (Yang et al., 2013). Being significantly larger than ZFNs’ genes (approximately 1 kb), TALENs are harder to deliver and difficult to get translated into a TALEN pair within a cell

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when delivered as mRNA. Thus, reducing the size of TALENs by eliminating the repeats has allowed the delivery of TALEN genes by using lentivirus (Yang et al., 2013). The size of cDNA encoding Cas9 protein is generally larger than 3 kb making them difficult to deliver through some viral vectors due to their cargo size and need of providing the polyadenylation signal as well as the promoter. Several emerging small- sized CRISPR-Cas systems from other species could be delivered by almost all viral vectors, for example, the size of Cas9 in Neisseria meningitides is 3.2 kb that permits its delivery through adeno-associated virus which is not possible with Cas9 of S. pyogenes. This is because some vectors have limited packaging capacity which limits the delivery of TALENs. Therefore designing of TALENs should be carried out with utmost care in order to make it small in size while retaining its efficiency to permit the delivery of TALENs with almost all viral vectors. However, the efficiency of gene editing methods, their specificity, and off-target effects are yet to be explored in the context of aquaculture and fisheries.

11.8 Ease of designing TALENs are easier to design than ZFNs because RVD code can easily be customized to develop many TALEN repeats that bind with each nucleotide of the target with high activity (Moscou and Bogdanove, 2009). Designing ZFNs that can specifically recognize the DNA triplets, particularly of 50 -CNN-30 and 50 -TNN-30 variety has hindered their widespread adoption, thus lacking the targeted flexibility and versatility present in other genome-editing technologies. The ease with which CRISPR-Cas9 can be designed helps in engineering a large number of vectors for targeting various loci or even screening genomic libraries (Mali et al., 2013a; Wang et al., 2014; Shalem et al., 2014; Koike-Yusa et al., 2014; Zhou et al., 2014). The main requirement for designing the gRNA is the presence of PAM (downstream NGG trinucleotide) adjacent to target nucleotide sequence, which on an average comes once every 8 bp in the mammalian genome (Cong et al., 2013). CRISPR-Cas9 can be easily adapted to target new sites by changing the 20 bp protospacer of the gRNA with no change in Cas9 protein component.

11.9 Multiplexing Multiplexing is the process of targeting several loci simultaneously in one cell is another advantage of CRISPR-Cas9 over ZFNs and TALENs (Cong et al., 2013; Mali et al., 2013b). In comparison to TALENs and ZFNs, CRISPR-Cas9 has been a method of choice for multiplexing studies in mice. It can be employed to target multiple genes simultaneously due to its higher activity and lower requirement of gRNA thus facilitating the introduction of mutations in multiple genes

11.10 Applications of genome editing

(Wang et al., 2013). Practically multiplexing with ZFNs and TALENs becomes difficult because the amount of RNA load needed to incorporate mutations at multiple sites can be cytotoxic before each individual nuclease reaches its working concentration.

11.10 Applications of genome editing 11.10.1 Research and development Genome editing can be used to alter the DNA in cells, tissues, or model organisms to understand their biology and how they behave in an environment with the altered genome. Genome editing helps in xeno-transplantation, by attempting the transfer of cells, tissues, and organs from animals to treat loss or dysfunction in patients (Perota et al., 2016). Several authors have found CRISPR-Cas9 as an important tool in identifying and characterizing important bacterial virulent genes and elucidating the immune evading strategies adopted by bacteria. Genome editing has been used in agriculture to obtain genetically-modified crops for achieving desirable traits such as high yield, resistance to disease and drought. Thus, genome editing could reduce input cost by minimizing the use of water and fertilizers, besides improving the quality and yield of agricultural produce (Bortesi et al., 2016). Similarly, genetically-modified animals could provide a high yield of milk and meat. This technology could also be used for rapid growth to enhance meat production as well as the development of disease-resistant animals.

11.10.2 Treatment of diseases Genome editing could be helpful to induce changes in human blood cells to treat conditions including leukemia and AIDS. It has a lot of potential to treat various pathogenic infections and simple genetic conditions like hemophilia. For instance, T-cell genome engineering can be a promising tool for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases (Kim et al., 2017; Delhove and Qasim, 2017). To achieve this feat, improved tools are required to either knock-out or knock-in targets in order to introduce genome modifications to modulate T-cell function and correct disease-associated mutations. Genome editing can assist correction or inactivation of deleterious mutations, introduction of protective mutations, therapeutic transgenes, and even in disruption of viral DNA (Cox et al., 2015).

11.10.3 Functional genomics CRISPR-Cas9 has allowed the screening of noncoding regions within a gene like enhancers and promoters with minimum off-target effects and high efficiency

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(Shalem et al., 2015). Furthermore, the CRISPR-Cas9 system has provided answers to genetic regulations that are beyond the control of the coding region of the genome, as in epigenetics. The targeted nucleases have tremendous potential in finding out the function of the particular gene by inducing mutations. CRISPR-Cas9 technology is a novel way to perform genome-wide genetic screens like single guide RNA (sgRNA) screening, CRISPR-Cas9 knock-out screening, CRISPR mutagenesis for genetic screens to discover gene function and to understand the mechanism of gene dysfunction that results in disease (Cox et al., 2015).

11.10.4 Fishery science Several researchers have used zebrafish as a model organism to study genetic modifications. Gene editing tools have been extensively used in zebrafish research, essentially making it an important model to answer the significant problems in the field of fish genetics, reproductive biology, toxicology, drug-receptor and hostapathogen interaction. CRISPR-Cas9 system has shown to be the most promising and efficient genome editing tool in the development of genetically-modified medaka, Atlantic salmon, zebrafish, and tilapia as model organisms for studying various biological mechanisms (Auer et al., 2014; Edvardsen et al., 2014; Qiu et al., 2014; Wang et al., 2015).

11.10.5 Production of the mono-sex population Sexual dimorphism is a well-established fact in fish, and a large number of fish species exhibit sexual dimorphism in body growth. For example, male tilapia grow faster than females, whereas female rainbow trout and Indian major carps grow faster than their male counterparts. The difference in growth rates can be harnessed to produce a mono-sex population of a particular fish, which could increase the production rates per unit area. Furthermore, unwanted reproduction of prolific fishes can be tackled by producing mono-sex population thereby minimizing the threat of their establishment in wild. In aquaculture, sex reversal is generally done by administration of androgen or gynogen hormones, which has posed serious issues like bioaccumulation, biomagnification, and other concerns on water quality and biodiversity. Targeted nucleases have a tremendous potential to produce mono-sex and sex-reversed fishes directly by disrupting the sex-determining genes without causing any serious impact on biodiversity. For example, knockout of female sex-determining genes in tilapia (with XX sex-determining chromosome) like foxl2, sf-1, or cyp19a1a disrupted by targeted nucleases resulted in testicular development (Li and Wang, 2017). Thus genome editing tools offer a versatile and environmentally-friendly approach to producing mono-sex population.

11.10 Applications of genome editing

11.10.6 Production of fast-growing fishes Some endemic coldwater fish species like snow trout, have a slow growth rate due to their genetic makeup, physiology, and the environmental constraints of their habitat. These coldwater fish species have the potential to become a candidate aquaculture species due to their thriving ability in stagnant water (ponds) as compared to rainbow trout which require continuous clean and well-aerated water. The genes inhibiting the skeletal muscle growth could be knocked out or the expression of growth-promoting genes could be increased with the help of targeted nucleases. Such an attempt was performed in common carp, a gene coding for myostatin (suppressor of muscle growth) was disrupted by CRISPR-Cas9, which resulted in the production of larger phenotypes in F0 generation (Zhong et al., 2016). Similar techniques could be adapted to slow-growing coldwater fishes like snow trout for increasing coldwater fish production. The detailed investigation can be carried out on other growth-related factors like growth hormone and insulin-like growth factor-1, which could provide potential target sites for increasing the fish growth by targeted nucleases.

11.10.7 Sterility Production of sterile fish is often desired in aquaculture for controlling the unwanted reproduction in weed and predatory fishes, and the establishment of exotic and transgenic fishes in the wild in case of their accidental escape from confined environments like ponds and raceways. This problem could easily be tackled with targeted nucleases by disrupting the sex-determining genes. ZFN was used to obtain sterile channel catfish by disrupting the β subunit gene of pituitary luteinizinghormone (Qin et al., 2016).

11.10.8 Development of pollution markers Several genes play major roles in minimizing/neutralizing the effects triggered by various pollutants. Expression of some specific genes induced by a particular pollutant could be used as a genetic marker to check the level aquatic pollution. The targeted nucleases could be used to produce homozygous mutants for a specific gene for monitoring the pollution level in the aquatic environment. This was achieved in Atlantic killifish by producing the homozygous mutants of AHR2 gene (aryl hydrocarbon receptor 2) by CRISPR-Cas9 for monitoring the levels of aryl hydrocarbon pollutants in marine ecosystem (Aluru et al., 2015).

11.10.9 Production of ornamental fishes Genes involved in pigmentation could be targeted by genome editing tools for producing ornamental fishes with desired color and pigmentation. These genomeediting platforms prevent the wastage of time as compared to classical genetic

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techniques. In zebrafish, genome editing tools (ZFN, TALEN, and CRISPR-Cas9) have resulted in mutation of golden gene resulting in production of light-colored eyes that are heritable up to F1 generation (Jao et al., 2013; Dahlem et al., 2012; Doyon et al., 2008).

11.10.10 Functional characterization of genes The function of a gene in an immunological or physiological pathway could be elucidated by incorporating mutations in the selected gene with genome editing tools. It has been demonstrated that spermatogenesis, folliculogenesis, and reproductive capability, are not impaired in both sexes when kiss-1 and kiss-2 genes including their receptors are knocked down in both sexes of zebrafish with designed TALENs (Tang et al., 2015). This report was contradictory to the current view on the role being played by kiss genes in fish reproduction by acting as a key regulator of gonadotropin-releasing hormone (Mechaly et al., 2013). Hence, extensive study is required to confirm the roles played by kisspeptins in some aquaculture species before designating the purified kisspeptin proteins as an efficient alternative for induced breeding hormones. Another disruption attempt was done in Labeo rohita toll-like receptor-22 through homologous-directed repair using CRISPR-Cas9 technique (Chakrapani et al., 2016). Development of model fish with altered genes would provide deep insights about the distinctive roles being played by a particular gene in an immunological or physiological pathway. These studies would help in the development of vaccines, molecular adjuvants, and alternatives molecules with high specificity and potency. For example, efficient, induced breeding hormones like ovaprim and ovatide could be replaced with efficient synthetic-induced breeding hormones in coming years.

11.11 Conclusion The potential of genome editing tools has not been tapped yet in commercial scale as far as aquaculture is concerned. They are still confined to the laboratory and the in near future may bring radical changes in aquaculture production by modifying the genome of fish as per needed. In comparison to traditional transgenesis, targeted nucleases generally do not introduce foreign DNA into the genome of target species. Thus, removing the public health concerns and increasing the human acceptability as compared to genetically-modified organisms. Furthermore, it is expected that genome editing tools could be used for improving traits important to aquacultures such as disease resistance, feed conversion efficiency, growth, and reproductive performance and tolerance to biotic and abiotic stressors. Genome editing may be potentially useful in monitoring the level of pollutants in the aquatic environment; disease modeling and drug screening could be their other potential uses.

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1845. Mechaly, A.S., Vin˜as, J., Piferrer, F., 2013. The kisspeptin system genes in teleost fish, their structure and regulation, with particular attention to the situation in Pleuronectiformes. Gen. Comp. Endocrinol. 188, 258
268. Miller, J.C., Holmes, M.C., Wang, J., Guschin, D.Y., Lee, Y.L., Rupniewski, I., et al., 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat. Biotechnol. 25 (7), 778
785. Miller, J.C., Tan, S., Qiao, G., Barlow, K.A., Wang, J., Xia, D.F., et al., 2011. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29 (2), 143
148. Moscou, M.J., Bogdanove, A.J., 2009. A simple cipher governs DNA recognition by TAL effectors. Science 326 (5959), 1501. Mussolino, C., Morbitzer, R., Lu¨tge, F., Dannemann, N., Lahaye, T., Cathomen, T., 2011. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res. 39 (21), 9283
9293. Nishimasu, H., Ran, F.A., Hsu, P.D., Konermann, S., Shehata, S.I., Dohmae, N., et al., 2014. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156 (5), 935
949. Overballe-Petersen, S., Harms, K., Orlando, L.A., Mayar, J.V.M., Rasmussen, S., Dahl, T. W., et al., 2013. Bacterial natural transformation by highly fragmented and damaged DNA. Proc. Natl. Acad. Sci. U. S. A. 110 (49), 19860
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770. Pattanayak, V., Lin, S., Guilinger, J.P., Ma, E., Doudna, J.A., Liu, D.R., 2013. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31 (9), 839
843. Pavletich, N.P., Pabo, C.O., 1991. Zinc finger-DNA recognition: crystal structure of a Zif268
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References

Perota, A., Lagutina, I., Quadalti, C., Lazzari, G., Cozzi, E., Galli, C., 2016. The applications of genome editing in xenotransplantation. J. Genet. Genomics 43 (5), 233
237. Qi, L.S., Larson, M.H., Gilbert, L.A., Doudna, J.A., Weissman, J.S., Arkin, A.P., et al., 2013. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152 (5), 1173
1183. Qin, Z., Li, Y., Su, B., Cheng, Q., Ye, Z., Perera, D.A., et al., 2016. Editing of the luteinizing hormone gene to sterilize channel catfish, ictalurus punctatus, using a modified zinc finger nuclease technology with electroporation. Mar. Biotechnol. 18 (2), 255
263. Qiu, C., Cheng, B., Zhang, Y., Huang, R., Liao, L., Li, Y., et al., 2014. Efficient knockout of transplanted green fluorescent protein gene in medaka using TALENs. Mar. Biotechnol. 16 (6), 674
683. Ramirez, C.L., Certo, M.T., Mussolino, C., Goodwin, M.J., Cradick, T.J., McCaffrey, A.P., et al., 2012. Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects. Nucleic Acids Res. 40 (12), 5560
5568. Ran, F.A., Hsu, P.D., Lin, C.Y., Gootenberg, J.S., Konermann, S., Trevino, A.E., et al., 2013. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154 (6), 1380
1389. Ro¨mer, P., Hahn, S., Jordan, T., Strauß, T., Bonas, U., Lahaye, T., 2007. Plant pathogen recognition mediated by promoter activation of the pepper Bs3 resistance gene. Science 318 (5850), 645
648. Schornack, S., Meyer, A., Ro¨mer, P., Jordan, T., Lahaye, T., 2006. Gene-for-genemediated recognition of nuclear-targeted AvrBs3-like bacterial effector proteins. J. Plant Physiol. 163 (3), 256
272. Shalem, O., Sanjana, N.E., Hartenian, E., Shi, X., Scott, D.A., Mikkelsen, T.S., et al., 2014. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343 (6166), 84
87. Shalem, O., Sanjana, N.E., Zhang, F., 2015. High-throughput functional genomics using CRISPR-Cas9. Nat. Rev. Genet. 16 (5), 299
311. Streubel, J., Blu¨cher, C., Landgraf, A., Boch, J., 2012. TAL effector RVD specificities and efficiencies. Nat. Biotechnol. 30 (7), 593
595. Tang, T.H., Liu, Yun, Luo, Daji, Ogawa, Satoshi, Yin, Yike, Li, Shuisheng, et al., 2015. The kiss/kissr systems are dispensable for zebrafish reproduction: evidence from gene knockout studies. Endocrinology 156 (2), 589
599. Urnov, F.D., Rebar, E.J., Holmes, M.C., Zhang, H.S., Gregory, P.D., 2010. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11 (9), 636. Wang, J., Friedman, G., Doyon, Y., Wang, N.S., Li, C.J., Miller, J.C., et al., 2012. Targeted gene addition to a predetermined site in the human genome using a ZFNbased nicking enzyme. Genome Res. 22 (7), 1316
1326. Wang, H., Yang, H., Shivalila, C.S., Dawlaty, M.M., Cheng, A.W., Zhang, F., et al., 2013. One-step generation of mice carrying mutations in multiple genes by CRISPR/Casmediated genome engineering. Cell 153 (4), 910
918. Wang, T., Wei, J.J., Sabatini, D.M., Lander, E.S., 2014. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343 (6166), 80
84. Wang, F., Shi, Z., Cui, Y., Guo, X., Shi, Y.B., Chen, Y., 2015. Targeted gene disruption in Xenopus laevis using CRISPR-Cas9. Cell Biosci. 5 (1), 15.

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Wolfe, S.A., Nekludova, L., Pabo, C.O., 2000. DNA recognition by Cys2His2 zinc finger proteins. Annu. Rev. Biophys. Biomol. Struct. 29 (1), 183
212. Yang, L., Guell, M., Byrne, S., Yang, J.L., De Los Angeles, A., Mali, P., et al., 2013. Optimization of scarless human stem cell genome editing. Nucleic Acids Res. 41 (19), 9049
9061. Zhong, Z., Niu, P., Wang, M., Huang, G., Xu, S., Sun, Y., et al., 2016. Targeted disruption of sp7 and myostatin with CRISPR-Cas9 results in severe bone defects and more muscular cells in common carp. Sci. Rep. 6, 22953. Zhou, Y., Zhu, S., Cai, C., Yuan, P., Li, C., Huang, Y., et al., 2014. High-throughput screening of a CRISPR-Cas9 library for functional genomics in human cells. Nature 509 (7501), 487
491.

Further reading Fellmann, C., Gowen, B.G., Lin, P.C., Doudna, J.A., Corn, J.E., 2017. Cornerstones of CRISPR-Cas in drug discovery and therapy. Nat. Rev. Drug Discov. 16 (2), 89
100. Handel, E.M., Cathomen, T., 2011. Zinc-finger nuclease-based genome surgery: it’s all about specificity. Curr. Gene Ther. 11 (1), 28
37.

CHAPTER

Marine biotechnology for food

12

Imelda Joseph and Asha Augustine ICAR-Central Marine Fisheries Research Institute, Kochi, India

12.1 Introduction Developed as well as developing countries are facing challenges in sustainable supply of nutritious food and energy, climate change and environmental degradation, health and aging populations. Marine biotechnology can make an increasingly important contribution toward meeting these societal challenges. It can substantially contribute to the growing demand for high quality and healthy food and other products from fisheries and mariculture in a sustainable way. The growing demand for seafood will have to be delivered through improved and innovative culture systems and practices. Efficient and environmentally responsible mariculture and a greater diversity of marine food products are available now due to the biological and biotechnological progress in the past few decades. Biotechnology has paved the way for increasing production efficiency and product quality, introduction of new species for farming; and the development of sustainable practices through a better understanding of the molecular and physiological basis of health, reproduction, development and growth, and a better control of these processes in mariculture. Challenges in understanding and controlling reproduction, early lifestage development, growth, nutrition, daisease and animal health management, environmental interactions, and sustainability are the challenges faced by mariculture. It needs a focused approach to tackle the issues in the coming years. The marine ecosystem represents a largely untapped reservoir of bioactive ingredients that can be applied to numerous aspects of food processing, storage, and fortification. Due to the wide range of environments they survive in, marine organisms have developed unique properties and bioactive compounds that, in some cases, are unparalleled by their terrestrial counterparts. Enzymes extracted from marine organisms provide numerous advantages over traditional enzymes used in food processing due to their ability to function at extremes of pH and temperature. Polysaccharides derived from algae like algins, carrageenans, and agar are widely used as thickeners and stabilizers in foods as well as for gels (Rasmussen and Morrissey, 2007). Fish proteins like collagens and gelatin derivatives could operate at low temperatures and can be used in heat-sensitive processes such as gelling and clarifying (Kim, 2015). Polysaccharides from algae Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00012-6 © 2020 Elsevier Inc. All rights reserved.

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like algin, carrageenan, and agar are widely used in a variety of food products. Besides applications in food processing, marine-derived compounds such as omega-3 polyunsaturated fatty acids and pigments, find applications in the nutraceutical industry. These bioactive ingredients provide health benefits, including reduction of heart diseases, diabetes, anticarcinogenic and antiinflammatory activities. Despite the vast possibilities for the use of marine organisms in the food industry, tools of biotechnology are required for successful cultivation and isolation of these bioactive compounds. Marine biotechnology could exploit the enormous potential of the majority of unexplored functional diversity of marine life. It includes new genes, new molecules, and unusual microorganisms and their biochemical processes for the benefit of different sectors like food, industry, medicine, energy, etc. The contribution of the marine environment to the world’s food supply is very significant and according to the Food and Agricultural Organization (FAO), fisheries and aquaculture provide almost 50% of the animal protein supply. Today, consumers often demand minimally processed food, without the addition of chemical preservatives. The application of marine biotechnology could contribute to address the global challenges of food, energy, security, and health as well as contribute to green growth, sustainable industries, etc., leading to a considerable contribution to the economic growth of the country. Biotechnology along with conventional biomass technologies play a major role in converting the huge aquatic biomass into value-added chemicals applying biorefinery concepts. Unlike terrestrial environments, the seas offer a broader variety of useful constituents to be used in foods. There has been a growing interest in functional food ingredients, nutraceuticals, probiotics, prebiotics, enzymes, and various dietary supplements resulting from the processing of marine organisms. In this chapter, developments and upcoming areas of research that utilize advances in biotechnology in the production of food ingredients from marine sources are included (Fig. 12.1).

12.2 Food from marine sources 12.2.1 Marine fish Fish occupy the highest position in marine animal consumption and fish provide approximately 16% of the world’s protein requirements with herring, salmon, cod, flounder, tuna, mullet, and anchovy being the most common species of fish used for food. One of the largest commercially canned fishery products in the world is tuna (e.g., Thunnus obesus). According to the FAO of the United Nations (2018), the total catch of the commercial tuna species increased from 162,980 metric tons in 1950 to more than 5 million metric tons in 2016. The nutritional benefits of fish consumption are due to the presence of proteins, unsaturated

12.2 Food from marine sources

Marine resources Products Culture

• Food • Food supplements • Nutraceuticals • Carotenoids • Vitamins • Minerals • Enzymes

Biotechnology tools/protocols • Mariculture: Breeding–seed production– farming • Genetics-Molecular biology–selection– mariculture • Health management-Vaccines: Probiotics • Environment management—Ecosystem based farming for sustainability • Bioprospecting: food–nutraceuticals

FIGURE 12.1 Marine biotechnology for food.

essential fatty acids, minerals (e.g., calcium, iron, selenium, and zinc), and vitamins (vitamin A, B3, B6, B12, E, and D).

12.2.2 Molluscs, echinoderms, and crustaceans Molluscs, together with echinoderms, have been widely consumed as marine foods and are considered natural functional foods. Many Asian populations consume cuttlefish, squid, octopus, and nautiloids due to their therapeutic effects, for example, rickets are cured with the bones of cuttlefish, as well as gastrointestinal disorders and ear inflammation. Crustaceans are the biggest and most economically important class of marine arthropods in the global fisheries markets; they also have significant roles in nutraceutical industries. Crabs, prawns, and shrimp have gained great attention due to their effective utilization and health benefits. Nutrient composition of marine crustaceans like shrimp and krill was analyzed and found to decrease the total blood lipids in humans, and improve vitamin A levels, specific proteins, and eicosapentaenoic acid, an omega-3 fatty acids. These are suggested to be used in the development of value-added health food products and for human consumption due to high nutritional value (Suleria et al., 2015).

12.2.3 Marine algae Algae include microscopic and macroscopic plants in the marine environment. Seaweeds or marine macrophytes are classified based on various properties such

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as pigmentation, chemical nature of photosynthetic storage product, the organization of photosynthetic membranes, and other morphological features. Seaweeds belong to four different groups, distinguished on the basis of color: blue-green algae (phylum: Cyanophyta, up to 1500 species), red algae (phylum: Rhodophyta, about 6000 species), brown algae (phylum: Ochrophyta, classes: Phaeophyceae, about 1750 species), and green algae (phylum: Chlorophyta, classes: Bryopsidophyceae, Chlorophyceae, Dasycladophyceae, Prasinophyceae, and Ulvophyceae, about 1200 species). Each of these groups also has microscopic representatives. Seaweeds as well as microalgae are crucial primary producers in oceanic aquatic food webs. Seaweeds are rich in minerals and essential trace elements, and raw materials for the pharmaceutical and cosmetics industry (Chapman, 1970). Seaweed is a very versatile product widely used for food in direct human consumption.

12.3 Mariculture technologies for food Mariculture involves farming of marine plants or animals for food, medicine, or any industrial applications. Mariculture is the fastest growing food sector in the world. Global aquaculture production (including aquatic plants) in 2016 was 110.2 million tons, with the first-sale value estimated at USD 243.5 billion. Farmed food fish production included 54.1 million tons of finfish (USD 138.5 billion), 17.1 million tons of molluscs (USD 29.2 billion), 7.9 million tons of crustaceans (USD 57.1 billion), and 938,500 tons of other aquatic animals (USD 6.8 billion) such as turtles, sea cucumbers, sea urchins, frogs, and edible jellyfish. Farmed aquatic plants included mostly seaweeds and a much smaller production volume of microalgae. The nonfood products included only ornamental shells and pearls. Mariculture started by catching wild juveniles and feeding them in a controlled environment. As more knowledge was gained, the degree of control with the production process increased and the farmers increased their influence on growth and reproduction. The degree of control is often categorized by the intensity of the farming operation. Traditional, extensive, semiintensive, and intensive are the existing farming practices (Quentin et al., 2010). Mussel farming is an example of an extensive method of mariculture used around the globe, whereby the farmer provides a rope or a stake for the juveniles to attach to and undertakes some culling so that the density does not get too high, but otherwise leaves the mussels to grow without further interference.

• Marine ponds: Mainly grow prawns and some finfishes either by tide-fed systems or by pumping in seawater at periodic intervals.

• Tanks (broodstock tanks; larval rearing, intensive culture tanks): Some species grow well in well-aerated tanks with regular exchange of water to keep the dissolved oxygen levels high and remove wastes.

12.4 Biotechnology in mariculture

• Sea cage farming (salmon, breams, snapper, seabass, grouper): High density, • • • •

•

low volume system with maximum production in unit area than in any other culture systems. Long line farming of bivalves is a nonfed culture system. Raceway farming: Usually large concrete tanks having higher flow rates than ponds are raceways which are used for farming of many fish species. Hatcheries: Hatcheries are land-based seed production units set up in a protected environment. Integrated multitrophic aquaculture (IMTA) and polyculture: Polyculture and integrated aquaculture are methods of raising diverse organisms within the same farming system, where each species utilizes a distinct niche and distinct resources within the farming complex. Recirculating aquaculture systems (RAS): RAS are closed and low discharge systems which have concerns for water conservation and reduced waste discharges.

12.4 Biotechnology in mariculture 12.4.1 Genetic manipulation The scope for genetic manipulation is greater in fish and bivalves than in domesticated livestock, which are considerably improved by a long history of artificial selection. Because of the higher market value, genetic advances in aquaculture are much ahead for fish than for any other farmed species. Genetic markers play an important role in the construction of high-resolution genetic linkage maps for aquaculture species, in identifying genes involved in quantitative trait loci for marker-assisted selection and in the assessment of implementation of genetic manipulations such as polyploidy and gynogenesis.

12.4.1.1 Selective breeding Since marine organisms are largely wild and relatively little is known about their genetic constitution, genetic improvement studies have wider implications in mariculture than in allied sectors including agriculture. The application of genetics to the breeding and management of cultivable marine organisms result in considerable improvement as in the case of poultry and livestock. In fish breeding studies, both the traditional selective breeding strategies of established animals breeders, and the more novel schemes of gynogenesis, self-fertilization, sex manipulation, and induced polyploidy may be feasible. The appropriate genetic studies can be considered as encompassing those prior to farming, on-farming, and postfarming activities. Genetic studies are important in fisheries for the conservation of genetic resources. The application of genetics to the breeding and management of cultivable marine organisms can very well result in considerable improvement in their farming qualities (Wilkins, 1981).

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12.4.1.2 Polyploidy In addition to transgenic research, advances in chromosome manipulation (polyploidy) also show potential for improving production in the aquaculture industry, particularly in the case of shellfish. Use of polyploidy in aquaculture can result in sterility, along with enhanced growth and survival rates and increased quality of final products. Polyploidy has been experimented in edible oysters in many countries including India.

12.4.1.3 Transgenics Transgenics are organisms into which transgene has been artificially introduced and the transgene stably integrated into their genomes. Transfer of transgene into the nucleus of a target cell where integration into the host genome takes place. The development of transgenic fish can serve as excellent experimental models for basic scientific investigations, environmental toxicology, and in biotechnological applications. The first transgenic fish were produced in and as of 2003, more than 35 species had been genetically engineered in research laboratories worldwide (Wakchaure et al., 2015). Transgenic fish show better gross food conversion regarding the increase in fish weight per unit of food fed rather than their unmodified relatives. The creation of transgenic fish and shellfish is a topic of great interest in aquaculture research due to the potential improvements in production that this technology can offer (Zbikowska, 2003; Dunham, 2010). Major areas of transgenic research in fish include use of growth hormones (GHs) to increase growth and feed conversion efficiency; use of antifreeze proteins for enhanced cold tolerance and freeze resistance; use of antimicrobial peptides for increased disease resistance; use of metabolic genes to promote low-cost, land-based diets; and genetic methods for inducing sterility. Research with transgenic GHs has made the most progress, with the patented production of a line of Atlantic salmon capable of increased growth and feed conversion efficiency. This product has been licensed to a major biotechnology company and is currently awaiting regulatory approval for commercial use in the United States and in Canada. Despite the potential for GMOs in aquaculture, a number of environmental and human health concerns remain. Major concerns include escapement of transgenic fish into the wild, where they could disrupt natural gene pools through breeding with wild species, and the possible detrimental effects of introducing transgenics into the human and aquatic food chains (Dona and Arvanitoyannis, 2009). One way to alleviate fears of escapement and breeding with natural populations has been the creation of sterile organisms. Recent work using the gene excision method has shown a potential way to produce sterile transgenic organisms by crossing two specific lines of transgenic parents. A more traditional method for inducing sterility has been the creation of triploid organisms through chromosome manipulation. Although fish have not responded very well to this technology, it has been very advantageous in the shellfish industry. Because of their sterility, triploid shellfish also have increased growth and flesh

12.4 Biotechnology in mariculture

palatability. The success of these products will ultimately be determined by consumers and their perspectives on the advantages (such as price and availability) of aquatic GMOs versus environmental and human health concerns.

12.4.2 Health management In the past, fish health research has been limited primarily to understanding diseases in aquaculture facilities, where abnormal conditions could be relatively easily observed, treated, and prevented. Disease impacts to wild marine populations are difficult to observe because sick fish are often consumed by predators before they die directly from disease. Alternatively, fish that die directly from disease are often difficult to locate if their bodies sink to the bottom or if mortalities occur at offshore areas. The problem is further increased by the unavailability of marine test fishes with a known disease history that can be used as experimental animals. By rearing colonies of specific pathogen-free test animals, this uncertainty was overcome. These fishes are also useful in developing predictive tools that forecast and prevent diseases in wild populations. The research approach involves a combination of field-based disease observations, followed by hypothesis derivation and testing using SPF fish. Fish health management is a critical component to disease control and is invaluable to improved harvests and sustainable production. Efficient health management tools, such as disease surveillance, farm biosecurity protocols, vaccination regimes, use of immunostimulants, and other tools are helpful in mitigating most losses due to diseases. Vaccination has been proven to be a very effective way of protecting fish from viral and bacterial diseases. Viral vaccines with improved techniques for delivery at affordable prices have been developed in finfish. The majority of the commercial vaccines are targeted at the high-value salmonid industry with vaccines against diseases such as IPN, ISA, IHN, and pancreatic disease (Dhar et al., 2014). The first vaccines were based on inactivated (by heat or chemical) viruses. Inactivated virus vaccines are still a major portion of the overall vaccine supply. The Alpha Jects Micro 1 ISA (Novartis) and Alpha Jects 1000 vaccines are examples of this type of vaccine, targeting ISA and IPN, respectively. However, the use of inactivated viruses as a vaccine is hampered since some fish viruses are not easily culturable, such as SalHV3, a herpesvirus of Atlantic cod and lymphocystis virus, making the production of vaccines for these viruses based on whole inactivated virus difficult. With these and developments in improved and powerful scientific tools, new variations in the types of vaccines available are playing an increasingly important role in fish health management. Ideally, vaccines will allow the differentiation of a vaccinated fish from an infected (or previously infected) fish to aid in epidemiology and disease surveillance/control. These attributes of the ideal vaccine are most likely to be met either by a recombinant subunit vaccine or by an inactivated viral vaccine, as a live attenuated vaccine could potentially lead to carrier formation. Attenuated virus vaccines based on live

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viruses have been selected for cross reactivity (a less virulent virus that elicits an immune response to the target virus), genetically modified to attenuate the virus, and/or cultivated under conditions that disable viral virulence. Subunit vaccines are another class of vaccines that have emerged with the advent of molecular biology. Highly successful examples of subunit vaccines are the IPNV VP2-based vaccine from Microtek International and the ISAV recombinant hemagglutinin esterase gene from Centrovet. Recombinant DNA vaccines involve injecting an organism with histone-free (“naked”) DNA representing a gene of the pathogen itself. A DNA vaccine against IHNV was the first effective DNA vaccine tested in fish. Subsequently, DNA vaccines were tested against a number of fish viruses, including IPNV, VNNV, and SVCV. So far only one DNA vaccine, a vaccine against IHN, has been approved for use in Canada and this vaccine is not approved in Europe and the United States for commercial application due to safety concerns (Dhar et al., 2014).

12.4.3 Environment management This involves ecosystem-based farming for sustainability of aquaculture production system. This is done by rearing several aquatic organisms together. Each species utilizes a distinct niche and distinct resources within the same farming system and optimum utilization of resources with minimum wastage is attained. Several aquatic organisms, which are compatible, are grown together in IMTA and polyculture. Environmental health and sustainability is ensured by such farming complexes. RAS generally consists of land-based tanks with constantly flowing water. RAS conserve water and allow control of all the environmental factors that might affect the stocked organism. RAS has less impact upon environment because, wastes and uneaten feed are not simply released into the ambient environment in the manner that they are with other culture systems and exotic species, and diseases are not introduced into the environment. Another environment management technique is the use of probiotic application for inadequate control of water quality in aquaculture systems.

12.5 Bioprospecting for food 12.5.1 Functional foods and nutraceuticals from marine organisms Marine resources are a source of high-value compounds with nutraceutical value to be used as functional ingredients: omega-3 fatty acids, chitin, chitosan, protein hydrolysates, algal constituents, carotenoids, collagen, taurine, and other bioactive compounds. Nutraceutical refers to raw foods, fortified foods, or dietary supplements containing bioactive molecules that provide health benefits beyond basic nutrition. These bioactive compounds include certain polysaccharides, peptides,

12.5 Bioprospecting for food

phytochemicals, vitamins, and fatty acids that are naturally present in foods, and can be added to foods producing fortified or functional foods or can be formulated into dietary supplements. Macroalgae, also called seaweed, are the most popular type of algae in the nutraceutical industry as it provides a great variety of food and food ingredients especially in Asian countries like Korea, Japan, and China. Agarose is one of the main products from macroalgae. Other metabolites and natural products with unique nutritional and therapeutic properties isolated from seaweeds include proteins, furanone, polyunsaturated fatty acids, L-α kainic acid, phenotics, pigments, phlorotannins, phycocolloids (carrageenan and agar), and minerals. Red and brown seaweeds are alternative sources of vitamins, minerals, and proteins, and are good sources of essential fatty acids. They have been used to prepare bioactive peptides and to improve protein digestibility. Recently, antihypertensive bioactive peptides have been isolated which may act as angiotensin-converting enzyme inhibitors. Macroalgae are also rich sources of insoluble and soluble dietary fiber as they are not digested by enzymes in the gut and are mainly composed of indigestible sulfated polysaccharides. Examples of structural and storage polysaccharides found in red and brown seaweeds include fucan, agar, laminaran, carrageenan, and alginate. The alginates from brown seaweeds are utilized as hydrocolloids due to their biological activity. Food and cosmetic industries use fucans from brown seaweeds. However, all bioactive molecules from algae have not yet been identified and molecules from marine algae may provide different health benefits and biological activities.

12.5.2 Marine sources of bioactive molecules Marine ecosystems have a high diversity of living organisms compared to terrestrial ecosystems providing numerous resources for human nutrition and health. Marine invertebrates are a diverse group with habitats in all ocean ecosystems, ranging from the intertidal zone to the deep sea environment. Marine invertebrates are classified into several phyla, viz., Porifera (sponges), Cnidaria (corals, sea anemones, hydrozoans, jellyfish), Annelida (Polychaetes, marine worms), Bryozoa (moss animals or sea mats), Mollusca (oysters, abalone, clams, mussels, squid, cuttlefish, octopuses), Arthropoda (lobsters, crabs, shrimps, prawns, crayfish), and Echinodermata (sea stars, sea cucumbers, sea urchins). This diverse group also includes seaweeds, microalgae, bacteria, cyanobacteria, certain fish species, and crustaceans that produce secondary metabolites as an adaptation to their hostile marine environment. The global nutraceutical market comprised of functional foods and beverages and dietary supplements, was valued at around USD 250 billion in 2014. Consumer demand for nutraceuticals is rapidly increasing with the market expected to reach around USD 385 billion by 2030 (Suleria et al., 2015). Marine organisms such as sponges, tunicates, bryozoans, molluscs, bacteria, microalgae, macroalgae, and cyanobacteria have recently been utilized for biotechnology. Compounds produced from these organisms are effective as

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therapeutics for infectious and noninfectious disease, with a high specificity for target molecules, usually an enzyme. In the nutraceutical industry, marine algae are used as sources of food and food ingredients. Microalgae, the most simple and primary organized members of marine plant life, are rich sources of food ingredients, such as β-carotene, vitamins C, A, E, H, B1, B2, B6, and B12, astaxanthin, polysaccharides, and polyunsaturated fatty acids. As such, bioactive molecules from microalgae are commercially produced, used as food additives, and also incorporated into infant milk formulations and dietary supplements. Marine fish contains proteins, unsaturated essential fatty acids, minerals, and vitamins and is highly nutritional. Crustaceans with eicosapentanoic acid also have significant roles in nutraceutical industries. Marine organisms are one of the most important sources of bioactive compounds for the food and pharmaceutical industries. Bioactive compounds can be isolated from various sources including marine plants, animals, and microorganisms. Marine bioactive compounds that have been most extensively studied include carbohydrates, pigments, polyphenols, peptides, proteins, and essential fatty acids. These compounds have rheological properties, deeming them useful in the food industry, as well as various biological functions like antioxidant, antithrombotic, anticoagulant, antiinflammatory, antiproliferative, antihypertensive, antidiabetic, and cardio-protection activities making them attractive nutraceuticals and pharmaceutical compounds. Agar, alginate, and carrageenans are high-value seaweed hydrocolloids, which are used as gelation and thickening agents in different food, pharmaceutical, and biotechnological applications. The annual global production of these hydrocolloids has reached 100,000 tons with a gross market value just above USD 1.1 billion. Seaweed polysaccharides are widely utilized in the food industry and are of particular technological importance due to their broad spectrum of functionality. The physical properties (e.g., gelling, viscosity enhancement, etc.) are tunable by controlling molecular properties of the chains and the environmental conditions (e.g., pH, ionic strength, etc.). This results in hydrocolloid systems with a remarkably wide spectrum of physical properties that find applications across food industry. The most industrially relevant types of carrageenan are kappa-, lambda-, and iotasulfated anionic galactans. Agar is also a linear galactan with backbone of two alternating disaccharides, agarobiose, and seaweed polysaccharides (agar, alginate, carrageenan), and neoagarobiose consisting of two major polysaccharide fractions, namely agarose and agaropectin. Use of seaweed polysaccharides in foods is well established and its potential in or drug industries are also being established.

12.5.3 Bioactive compounds of importance in farming 12.5.3.1 Carotenoids Carotenoid pigments, obtained by animals from their diets, give most of the bright red, yellow, and orange colors well appreciated in aquaculture (Toyomizu

12.5 Bioprospecting for food

et al., 2001). Only plants, bacteria, fungi, and algae can synthesize carotenoids; animals cannot biosynthesize them thus, they must be obtained from the diet (Schiedt, 1998). They play a critical role in the photosynthetic process and they carry out a protective function against damage by light and oxygen. Carotenoids also play other important functions as pro-vitamin A, antioxidants, immunoregulators, and they are mobilized from muscle to ovaries which suggest a function in reproduction (Shahidi et al., 1998; Nakano et al., 1999). It has also observed that fishes with a high level of carotenoids are more resistant to bacterial and fungal diseases (Shahidi et al., 1998). Carotenoids have been included in diets of salmonids, crustaceans, and other farmed fish, mainly as pigments to provide a desirable coloration to the cultured organisms. Carotenoids not only contribute in improving quality by enhancing color, but could also help to give a better image in the minds of consumers of aquaculture products, in view of increasing information available on carotenoids’ positive effect on human health. Aside from their quality enhancing properties, carotenoids seem to improve certain production parameters of farmed species. In crustaceans, such as shrimp, a bright and appropriate color is also associated with freshness and quality and the desired coloration preserved through storage, processing, and cooking (Boonyaratpalin et al., 2001). In the sea urchin industry, based on the production of marketable gonads, the highest commercially valuable sea urchin gonads are bright yellow-orange (Shpigel et al., 2004). Astaxanthin is a high-value keto-carotenoid pigment renowned for its commercial application in various industries comprising aquaculture, food, cosmetic, nutraceutical, and pharmaceutical. Among the verified bioresources of astaxanthin are red yeast Phaffia rhodozyma and green alga Haematococcus pluvialis. The supreme antioxidant property of astaxanthin reveals its tremendous potential to offer manifold health benefits among aquatic animals which is a key driving factor triggering the upsurge in global demand for the pigment. Carotenoids play a significant role in shrimp aquaculture also. Crustaceans cannot synthesize carotenoids, thus it must be supplied in their diet. Astaxanthin is the optimal carotenoid for the proper pigmentation of Penaeiid shrimps. A nutritional deficiency of astaxanthin in the diet causes blue color syndrome. Additional benefits of this essential carotenoid include roles as an antioxidant and precursor of vitamin A, as well as enhancing immune response, reproduction, growth, maturation, photoprotection, and defense against hypoxic conditions in culture ponds. Astaxanthin dramatically improves the nauplii quality and zoea survival of shrimp broodstock. One first strategy would be to supplement shrimp diets with 75 150 ppm of commercial astaxanthin 2 months prior to harvest to achieve a total body carotenoid content in excess of the critical threshold of 30 40 mg/kg. Broodstock supplemented with 150 ppm of astaxanthin has been found to significantly improve nauplii quality and zoea survival (Lorenz, 1998).

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12.6 Conclusion As more food of high nutritional quality are in need for the ever-increasing human population, biotechnology is looked at as the solution for enhanced production as well as improved varieties. Marine biotechnology is untapped compared to plant and other terrestrial animal biotechnology. The future development in mariculture, which would be the major food supplying system in the world, is to develop innovative methods based on molecular biology for selective breeding of mariculture species; develop biotechnological applications and methods to increase sustainability of ecosystem-based aquaculture production, including alternative preventive and therapeutic measures to enhance environmental welfare; sustainable production technologies for feed supply; intensive farming through recirculating farming systems with zero wastes; and integration of new, low environmental impact feed ingredients including alternatives for fishmeal and fish oil.

References Boonyaratpalin, M., Thongrod, S., Supamattaya, K., Britton, G.E., Schlipalius, L., 2001. Effects of β-carotene source, Dunaliella salina, and astaxanthin on pigmentation, growth, survival and health of Penaeus monodon. Aquacult. Res. 32, 182 190. Chapman, V.J., 1970. Seaweeds and Their Uses. Methuen & Co Ltd, London, 304 pp. Dhar, A.K., Manna, S.K., Allnutt, F.C.T., 2014. Viral vaccines for farmed finfish. Virus Disease 25 (1), 1 17. Dona, A.I., Arvanitoyannis, I.S., 2009. Health risks of genetically modified foods. Crit. Rev. Food Sci. Nutr. 49 (2), 164 175. Dunham, R.A., 2010. Aquaculture and Fisheries Biotechnology, Genetic Approaches, second ed. Stylus Cabi Publishing, 495 pp. FAO, 2018. The State of World Fisheries and Aquaculture. FAO, Rome, 227 pp. Kim, S.K., 2015. Handbook of Marine Biotechnology. Springer, Berlin, Heidelberg. Lorenz, R.T., 1998. A review of the carotenoid, astaxanthin, as a pigment source and vitamin for cultured Penaeus prawn. NatuRosea Tech. Bullet. 51, 1 7. Nakano, T., Miura, Y., Wazawa, M., Sato, M., Takeuchi, M., 1999. Red yeast Phaffia rhodozyma reduces susceptibility of liver homogenate to lipid peroxidation in rainbow trout. Fish. Sci. 65, 961 962. Quentin, R.G., Ray, H., Dale, S., Maree, T., Meryl, W., 2010. Handbook of Marine Fisheries Conservation and Management. Oxford University Press, Oxford, New York, 770 pp. Rasmussen, R.S., Morrissey, M.T., 2007. Marine biotechnology for production of food ingredients. In: Taylor, S.L. (Ed.), Advances in Food and Nutrition Research. Elsevier, New York, pp. 237 292. Schiedt, K., 1998. Absorption and metabolism of carotenoids in birds, fish and crustaceans. In: Britton, G., Liaaen-Jensen, S., Pfander, H. (Eds.), Carotenoids Biosynthesis and Metabolism. Birkha¨user, Basel, pp. 285 358.

References

Shahidi, F., Metusalach, J., Brown, J.A., 1998. Carotenoid pigments in seafoods and aquaculture. Crit. Rev. Food Sci. 38, 1 67. Shpigel, M., McBride, S.C., Marciano, S., Lipatch, I., 2004. The effect of photoperiod and temperature on the production of European sea urchin, Paracentrotus lividus. Aquaculture 245, 101 109. Suleria, H.A., Osborne, S., Masci, P., Gobe, G., 2015. Marine-based nutraceuticals: an innovative trend in the food and supplement industries. Mar. Drugs 13, 6336 6351. Toyomizu, M., Sato, K., Taroda, H., Kato, T., Akiba, Y., 2001. Effects of dietary Spirulina on meat color inmuscle of broiler chickens. Br. Poultry Sci. 42, 197 202. Wakchaure, R., Ganguly, S., Qadri, K., Praveen, P.K., Mahajan, T., 2015. Importance of transgenic fish to global aquaculture: a review. Fish. Aquac. J. 6, 124. Wilkins, N.P., 1981. The rationale and relevance of genetics in aquaculture: an overview. Aquaculture 22, 209 228. Zbikowska, H.M., 2003. Fish can be first—advances in fish transgenesis for commercial applications. Transgenic Res. 12 (4), 379 389.

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Biotechnological innovations in farm and pet animal disease diagnosis

13

Yashpal Singh Malik1, Atul Verma2, Naveen Kumar3, Pallavi Deol4, Deepak Kumar5, Souvik Ghosh6 and Kuldeep Dhama7 1

Division of Biological Standardization, ICAR-Indian Veterinary Research Institute, Izatnagar, India 2 Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, United States 3 ICAR-National Institute of High Security Animal Diseases, Bhopal, India 4 Division of Virology, ICAR-Indian Veterinary Research Institute, Bareilly, India 5 Genetic Engineering-Virus Laboratory, Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute, Bareilly, India 6 Department of Biomedical Sciences, Ross University School of Veterinary Medicine, St. Kitts and Nevis, West Indies 7 Division of Pathology, ICAR-Indian Veterinary Research Institute, Bareilly, India

13.1 Introduction Livestock, poultry, and aquaculture are among the fastest growing and expanding agriculture sectors to fulfill the need of the growing population of humans. However, the growth in this sector is under the continuous increasing threats of infectious diseases worldwide. This menace is further aggravated by globalization in animal trade for various purposes. The sudden entry of an infectious disease in a new country or geographical location could lead to delayed diagnosis and rapid spread into the susceptible animal population. In response to climate change, vector-borne diseases are also increasing worldwide. To prevent the spread of infectious diseases, one of the basic and critical requirements as prescribed by the World Organization of Animal Health (OIE) is the application of rapid, accurate, and highly sensitive identification of infectious agents. Though the term “biotechnology” was coined in the year 1919 by Karl Ereky, the tangible biotechnological advancements in improving the human and animal health were started in the late 20th century. Since then, biotechnological applications have been making significant contributions in the development of novel powerful diagnostic assays for the efficient diagnosis and control of animal infectious diseases. Importantly, Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00013-8 © 2020 Elsevier Inc. All rights reserved.

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biotechnology has made the availability of pen-side tests for use at the field level to detect the causative infectious agent during a disease outbreak. In this chapter, we primarily describe and discuss the innovative biotechnological advancements made in the animal disease diagnosis in a step-wise manner.

13.2 Infectious diseases’ impact The impact of infectious diseases is immense and is felt all across the world. Infectious diseases have affected the whole society, economy, and political system. Vital sectors are under continuous economic loss and unrelenting development. The infectious diseases have taken a huge physical toll on animals and humans. This has pressed on humanity and has caused substantial economic, social, and mental losses. Thus, it is a matter of animal health and economic interest to invest in strategies to give a blow to infectious diseases and put them under control. Elimination of the pathogens and/or their vectors from their natural reservoirs would always be a first thought, but the removal is not easy, as they are constantly emerging and it is always very difficult to predict the emergence of infectious agents. Evolution of pathogens is putting extra challenges, pressing on humanity to look at the new strategies and forcing the researchers to look for innovative ones. The newly evolved pathogens are always more advanced and deadly from the previous ones and put up a strong resistance. “From the evolutionary perspective, they [viruses and bacteria] are ‘the fittest’ and the chances are slim that human ingenuity will ever get the better of them.” With the increase in the knowledge of infectious diseases and science, the degree of pace in pathogen discovery has increased. To keep pace and for better diagnosis, new tools and techniques need to keep on evolving. This is not only to quickly detect the pathogens, but also to make predictions with probable and possible outbreaks. To understand the scenario and to reach a definite conclusion, knowledge of epidemiology and pathogenic etiology also needs to be studied. This will facilitate in understanding the ancestry of the pathogen, and provide an insight and a mechanism for the epidemic, endemic, and even pandemic outbreaks. This system will also assist in understanding the interface transmission between and the directional flow of the zoonotic infectious diseases. Thus, phylogenetic analysis and epidemiology would aim toward strategizing the challenges during pathogen surveillance and discovery. SARS, a coronavirus was pandemic in 2003. But epidemiology and microbiology mediated to stem its disastrous results and also the causative agent of SARS was identified. Bacteria, viruses, and parasites, present in feces, contaminate foodstuffs and cause disease in humans and animals, affecting the social set up and consumer demands. To increase the productivity and for the maintenance of good health of animals, antibiotics are frequently administered resulting in the growth and emergence of antibiotic-resistant bacteria. This further aggravates the condition, and makes the situation more appalling. Overseas

13.3 Diagnosis of pathogens

imported pets were also found to transmit and carry over the diseases to humans (Smith et al., 2012). Even aquaculture is at risk of contaminants with the virus directly affecting the marine lives, as in the case of the new virus discovered in salmon (Finstad et al., 2012). Even honeybees and other pollinators are transmitting pathogens like fungi, bacteria, and viruses through the contaminated food items (Cox-Foster et al., 2007).

13.3 Diagnosis of pathogens An array of classical and conventional techniques have been developed and used for the laboratory diagnosis of infectious agents or pathogens. The techniques include serological, cell culture, and electron microscopy based methods, which are either time-consuming or labor-intensive or both. However, with the advancement in the biotechnology field, new and robust diagnostic techniques are continuously evolving and taking over the conventional methods (Caliendo et al., 2013). Presently, molecular detection-based methods such as polymerase chain reaction (PCR) or its variants, and serological methods such as enzyme-linked immunosorbent assay (ELISA), are being used worldwide for the accurate diagnosis of many animal diseases. However, point-of-care (POC) and high-throughput novel assays have been developed recently. Furthermore, we discuss the pros and cons of frequently used diagnostics assays for animal diseases in accordance with the following sections: 1. Serological diagnostic assays 2. Nucleic acid-based diagnostic assays a. Hybridization methods b. Amplification methods 3. Novel and high-throughput assays a. Microarray b. Peptide nucleic acid and aptamers c. Biosensors d. Next-generation sequencing based methods e. POC diagnostics f. Patented diagnostic technologies

13.3.1 Serological diagnostic assays Serological methods were introduced in the early 1930s for the diagnosis of pathogens. Various serological diagnostics have been developed such as complement fixation, counter-immunoelectrophoresis, immunofluorescence in cell culture, ELISA, radio immunoassay, immune adherence haemagglutination assay, reverse passive hemagglutination assay, latex agglutination (LA), chemiluminescent immunoassay, and immunochromatography test (ICT). Among these, ICT

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and ELISA especially sandwich ELISA and competitive ELISA are used frequently in the commercial diagnostic kits for animal diseases worldwide. ICT assays mostly utilized mammalian IgG in commercial diagnostic kits, however, avian IgY antibodies, with added advantages over the mammalian IgG, have been employed for the detection of norovirus, rotavirus, and astrovirus in the fecal samples with good sensitivity and specificity ranging between 90% and 95% (Khamrin et al., 2009). Modifications in ELISA format or combinations with other diagnostic methods have proved novel ways to detect pathogens more efficiently and accurately. For example, recently a novel ELISA for the detection of group A rotavirus antigen in the fecal samples of multiple host species has been developed (Kumar et al., 2016). This assay utilizes the potential use of synthetic peptides and is based on the detection of conserved VP6 protein using anti-recombinant VP6 antibodies as capture antibodies and anti-multiple antigenic peptide (identified and constructed from highly immune-dominant epitopes within VP6 protein) antibodies as detector antibodies. Another assay, which is simple to perform without the requirement of laboratory facilities, is dot-ELISA. A highly sensitive and specific dot-blot assays for rapid detection of staphylococcal enterotoxin-A in food has been reported (Singh et al., 2017). Dot-ELISA has been employed in diagnosing various important poultry diseases (Alam et al., 2012; Dhama et al., 2011; He et al., 2010; Majumder et al., 2018; Manoharan et al., 2004). Immuno-PCR is another powerful assay that has been used for the immunodetection of viral nucleic acids. By combining ELISA with PCR, sensitivity of detection can be increased up to 200 times and is especially useful in detecting low quantity viruses in the stool samples (Bonot et al., 2014). The major advantage of immune-PCR is that several viral nucleic acids can be detected simultaneously. Recently, a combination of nanoparticles with the immuno-PCR, also known as nanoparticle amplified immune PCR (NPA-IPCR), has been reported which increases the sensitivity 1000-folds compared to ELISA and several folds to RT-PCR. Antigen detection using an antibody bound to gold nanoparticle cofunctionalized with thiolated DNA complementary to a hybridized DNA has been developed (Perez et al., 2011). Here, the presence of antigen/virus particles activates the formation of a “sandwich” complex of gold nanoparticle construct, virus, and an antibody functionalized nanoparticles used for extraction. Now, this complex is heated to 95 C, thus releasing DNA tags followed by the detection through real-time PCR. NPA-IPCR offers a viable platform for the development of an early-stage diagnostics requiring an exceptionally low limit of detection.

13.3.2 Nucleic acid-based diagnostic assays Nucleic acid-based detections are used through the amplification methods, hybridization methods, which could be in situ, in vitro, and in vivo.

13.3 Diagnosis of pathogens

13.3.2.1 Hybridization-based methods The most common and widely used hybridization-based method is in situ hybridization, which could utilize fluorescent (FISH) or chromogenic (CISH) molecules. The CISH-based assays for the rapid characterization of microorganisms, such as Mycobacterium species and the dimorphic fungi in positive culture samples have been described (Louro et al., 2001; Scarparo et al., 2001). Recently, a FISH-based assay has been developed for the identification and differentiation of Mycobacterium tuberculosis complex from nontuberculous mycobacteria (Baliga et al., 2018).

13.3.2.2 Amplification-based methods Nucleic acid amplification methods are amongst the best in detecting pathogens with high sensitivity and specificity in the clinical samples. Various modifications in nucleic acid amplification methods have provided collectively robust methods to yield better and accurate results. These modifications could be categorized into two amplification methods viz PCR and its variants, and isothermal amplification methods.

13.3.2.2.1 Polymerase chain reaction and its variants These are the most common tools used for the pathogen detection worldwide. The three basic variants include (1) real-time PCR, which is a modified version of conventional PCR, where quantification of DNA sequence is possible without any further step of running the amplified product on agarose gel; (2) multiplex PCR, where multiple sequences can be detected in a single reaction mixture, and (3) reverse transcriptase PCR (RT-PCR) where RNA is transcribed to cDNA and this cDNA is used in the amplification as template. The real-time PCR can utilize different fluorescence chemistries such as SYBR green, TaqMan, or molecular beacon probes. Recently, a TaqMan real-time RT-PCR assay has been developed for rapid detection and quantification of Japanese encephalitis virus in swine blood and mosquito vectors (Pantawane et al., 2018).

13.3.2.2.2 Isothermal amplification methods In isothermal amplification, a number of target DNA copies increase at a constant temperature in just one cycle without the need of a thermocycler. Various techniques have been developed using isothermal amplification methods viz nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), signal-mediated amplification of RNA technology (SMART), strand displacement amplification (SDA), rolling circle amplification (RCA), loopmediated isothermal amplification (LAMP), isothermal multiple displacement amplification (IMDA), helicase-dependent amplification (HDA), circular helicasedependent amplification (cHDA), single primer isothermal amplification (SPIA), and strand invasion-based amplification (SIBA). In all these methods, isothermal temperature amplified products can be visualized on gel through the various

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structures visible on gel or by the incorporation of dyes in the special structures formed on amplification serving in real-time detection. Among all these techniques, LAMP is the most widely used isothermal amplification method which is in fact an autocycling strand displacement DNA synthesis, which deploys four primers forming a stem-loop DNA by self-primed DNA synthesis and a DNA polymerase with strand displacement activity (Malik et al., 2013; Parida et al., 2008). Recently, an improved strategy using a double-labeled probe to overcome the problem of false positivity of LAMP together with target gene real-time quantification is devised for detection of avian orthoreovirus (Kumar et al., 2017) and Salmonella spp. (Mashooq et al., 2016). Other potential isothermal amplification techniques are recombinase polymerase amplification (RPA) and NASBA. The former has brought a breakthrough in the detection of nucleic acids as it does not require denaturation of the template. RT-RPA is an extension of the above method, in which bacterial RT is used in the amplification of RNA. RT-RPA was developed to study the outbreak of footand-mouth disease (FMD) disease in Egypt (Abd El Wahed et al., 2013). High degree of fidelity, portability, cost efficiency, simplicity, sensitivity, and tolerance to inhibitors, put this method into the category of resounding techniques, and implementation is quite easy at quarantine stations (Moore and Jaykus, 2017); while the latter one requires initial denaturation of the template followed by temperature labile polymerase dependent isothermal amplification and was designed specially to detect RNA (Compton, 1991). A multiplex real-time nucleic acid sequence-based amplification (qNASBA) system for the simultaneous detection of rotavirus A, norovirus genogroup II/astrovirus has recently been developed (Mo et al., 2015a). RT-NASBA proved as more efficient than the conventional RT-PCR and TaqMan RT-PCR assays (Mo et al., 2015b).

13.3.3 Novel and high throughput assays 13.3.3.1 Microarray A microarray is a multiplex lab-on-a-chip test. It is an arrangement of the large amount of biological materials for high-throughput screening on a solid support generally a glass slide, through the detection-based assays. Microarray has done wonders in the high-throughput screenings and for the breakthrough causes of the outbreaks. Simultaneous detections of coinfections and other more phenomenal changes during the outbreaks are the crucial developments to study the infectious diseases in endemic regions. Their easiness has brought the working systems onto the platform on global diagnostics. Multiple diagnostics with hybridizing ability put it at more ease to strategize the control management programs. But, this technique comes at high expenditures. Data management skills and their interpretations need off to the most important tasks to be worked on. With the advent, new kits for point-of-care detections, bioelectric arrays, and liquid microarrays are in the development process. This would be an easy and an improved hybridization

13.3 Diagnosis of pathogens

method for individual probe and target combinations with accurate detections. This will reduce the effort from clinical diagnosis to the personal level. These all will help in understanding the proper and common pathogens with scaling down the time.

13.3.3.2 Peptide nucleic acids and aptamers Peptide nucleic acids (PNAs) are highly versatile synthetic oligonucleotides, in which the native sugar-phosphate backbone of DNA is replaced with amino acids. PNAs bind to complementary DNA strands with higher specificity and strength. Furthermore, they are resistant to nucleases and proteases, making them a highly stable diagnostic reagent. The PNA-based assay has greater sensitivity than direct sequencing and is significantly more affordable and rapid (Ray and Norde´n, 2000). The potential diverse uses of PNA have been exhaustively described in a recent review (Gambari, 2014). A rapid label-free visual PNA-based assay for detection and pathotyping of Newcastle disease virus has also been reported (Joshi et al., 2013). Similarly, PNA-based beacons have also been used in HIV genotyping with high specificity (Zhang and Apella, 2010). Aptamers are artificial nucleic acid ligands that are isolated from combinatorial libraries of synthetic nucleic acid by an iterative process of adsorption, recovery, and reamplification. DNA aptamers in particular have many advantages over antibodies (Brody and Larry, 2000). Aptamers, first reported in 1990, are attracting interest in the areas of diagnostics and offer themselves as ideal candidates for use as biocomponents in biosensors (aptasensors), possessing many advantages over state of the art affinity sensors (O’Sullivan, 2002). The aptamers have proved to be potential diagnostic assays, especially in the detection of toxins such as brevetoxin-2, potent marine neurotoxins (Shimaa et al., 2015), marine biotoxinpalytoxin (Shunxiang et al., 2017), β-bungarotoxin (β-BuTx), and a neurotoxin from the venom of Bungarus multicinctus (Ye et al., 2014a). Furthermore, aptamers have been used for the serological detection of Mycobacterium bovis (Fu et al., 2014), Cryptosporidium parvum (Iqbal et al., 2015), and prion disease (Saijin et al., 2012).

13.3.3.3 Biosensors Biosensors are portable, easy to handle, ultrasensitive, quick, and may be quite specific with less probability of a false positive. Biosensors work on various principles viz detecting the changes in the pH, the ion concentrations, mass by specific hybridization, enzymatic reaction, loss of functionality, change in the electrical potential, change in color, and temperature. Based on these principles, many biosensors have been devised for the detection of animal pathogens; for example, an extended-gate field-effect transistor for the direct potentiometric serological diagnosis of the BHV-1 (Tarasov et al., 2016), nanowire-based immunosensor for bovine viral diarrhea virus (BVDV) (Montrose et al., 2015), luminescence resonance energy transfer based biosensors for the ultrasensitive detection of the H7 strain (Ye et al., 2014b), quartz crystal microbalance (QCM) based

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immunosensors to detect H5N1 (Li et al., 2011), and SpectroSensTM optical microchip sensors for foot-and-mouth disease virus (FMDV) (Bhatta et al., 2012).

13.3.3.4 Next-generation sequencing The limited methods for detection of microbial signatures and the advent of new technology for quick and parallel gene expression capacities have eased in the detection of microbial disease. Next-generation sequencing (NGS) is now being increasingly applied in understanding the molecular epidemiology, transmission, and characterization of animal pathogens. Instead of gene-by-gene analysis, large deposits of genes available in the clinical sample can be detected in a single test. Applications of NGS are considered as more resourceful. Thus, it is widely accepted as a diagnostic tool and speedily is being replaced with most other molecular diagnostic technologies and has brought revolution in the diagnosis of pathogens. Various modifications and improvements have brought a huge change in the sequencing and identification of genomes. It all started with pyrosequencing on Roche 454, with small read lengths and less efficiency. Roche 454 was followed by ion-torrent Illumina platform. The NGS has made it possible to sequence the complete viral genomes of many viruses cost-effectively such as including an avian influenza virus (Croville et al., 2012), classical swine fever virus (Leifer et al., 2013), and Bluetongue viruses (Rao et al., 2013). Recently, nanopore technology, with the promising improvement has brought a wonderful efficiency with emerging science and technology (Goodwin et al., 2016). Nanopore systems can sequence both DNA and RNA viral genome in real time. This technology is based on the principle that when a strand of DNA/RNA is allowed to pass through a nanopore, the current is changed as the based A, T, C; and C passes through the pore in different combinations. Using these systems, sequencing can be performed on the portable MinION device, the benchtop GridION and the high-throughput, high-sample number PromethION. Recently, nanopore sequencing has proved a revolutionary diagnostic tool in detecting the Ebola virus (Hoenen et al., 2016), influenza viruses (Keller et al., 2018; Wang et al., 2015) and porcine viral enteric disease complexes (Theuns et al., 2018). Overall, the biotechnological innovations have equipped us now to have high-resolution sequencing tools that are revolutionizing the ability of veterinary diagnostic laboratories to detect emerging animal pathogens.

13.3.3.5 Point-of-care diagnostics With the advent of many biotechnological advances in veterinary diagnostics, point-of-care diagnostics (POCD) are now available for economically important animal diseases. The POCD is basically a simple, rapid, and portable diagnostic device that can be applied at the field level in effective monitoring the disease status. Most of the commercially available POCDs utilize either antigen/antibody or nucleic acid detection technologies. The former is usually available in the format of lateral flow assays or immunochromatographic strip tests. These assays are

13.3 Diagnosis of pathogens

simple to use, rapid, inexpensive, disposable, and thus make them the ideal assay for POCD for animal pathogens. The commercially available immunochromatographic strip tests for economically important animal diseases are summarized in Table 13.1. These assays are equally sensitive as compared to ELISA (Ferris et al., 2010). Furthermore, combining these assays with smartphones has made the increased sensitivity and quick reporting of results possible (Yeo et al., 2016). Therefore, these assays offer a novel herd level surveillance tool, and provide immediate results to the farmers. However, these assays have less analytical sensitivity as compared to nucleic acid-based POCD. The real-time PCR (qPCR) is a well-established tool with high sensitivity of pathogens detection and recently, qPCR has been transitioned into POCD platform. These platforms are fully automated combining nucleic acid extraction, thermal cycling, and reporting of results on-site. For example, MiniLab (Enigma Diagnostics) is a platform (10 35 kg) which can be easily carried to field level and it combines silica paramagnetic-bead-based nucleic acid extraction with lyophilized qPCR reagents in a single cartridge. This platform has been validated for AIV, ASFV, CSFV, and FMDV (Goldenberg and Edgeworth, 2015). However, this platform is still not available commercially. There are other platforms that do not include nucleic acid extraction step (need to be done separately), such as genesig (Primerdesign Ltd, United Kingdom), Genedrive (Epistem Ltd, Manchester, United Kingdom), Cepheid SmartCycler (Cepheid), T-COR 8 (Tetracore), and R.A.P.I.D. (Idaho Technologies) (Takekawa et al., 2010,2011). The genesig is now supplying lyophilized qPCR assay kits for 62 bovine, 42 equine, 47 porcine, 60 avian, 40 canine, and 26 feline different pathogens. However, these kits are not yet licensed for diagnosis of animal pathogens and are for research purposes only.

13.3.3.6 Patented diagnostic technologies As per the agreement on Trade Related Aspects of Intellectual Property Rights (TRIPS) under Paragraph 3 of Article 27, many countries have excluded diagnostic, therapeutic, and surgical methods of humans or animals from the scope of patentable systems. However, the important patented technologies that are being used in the various formats of diagnostic assays are provided in Table 13.2. A high-speed reagent system for qPCR, full velocity technology has been developed by the Stratagene which saves time in addition to highly reproducible results. This technology has been used for infectious diseases, cancer, and drug sensitivities testing and already granted five US patents, US6548250, US6893819, US6350580, US6589743, and US6528254. Besides, a POCD product, Dual Path Platform (DPP) has been developed by the Chembio Diagnostic Systems, on which tests to detect HIV and syphilis have already been developed. This company in collaboration with National Institutes of Health and the Infectious Disease Research Institute, United States is working constantly to use this platform for the detection of infectious diseases of humans and animals.

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Table 13.1 Commercial kits for point-of-care diagnosis of important livestock and companion animal pathogens. S. no.

Commercial kits

Disease conditions

Species

Performancea

Manufacturers/ suppliers

Livestock 1.

BioSign FMDV (Patent No.: 5,559,041)

Foot-and-mouth disease

Ruminants and pigs

Sn 5 98.6%, Sp 5 98.6%

2.

BOVIGAM TB Kit (OIE registered)

Tuberculosis

N/A

3.

SNAP BVDV Test

Bovine viral diarrhea

Cattle, sheep, and goats Cattle

4.

CSFV Ab Test

Classical swine fever

Pigs

Sn 5 95.9% 100%, Sp 5 99.5% 100% depending on the sample types N/A

5.

FASTest CRYPTO strip BioSign PRRSV

Cryptosporidium parvum

Cattle

Sn 5 96.7%, Sp 5 99.9%

Porcine reproductive and respiratory syndrome

Pigs

Sn 5 98.7%, Sp 5 98.5%

Avian influenza (type A virus)

Chicken

Sn 5 99.9%, Sp 5 99.9%

Bovine spongiform encephalopathy

Cattle

N/A

Rabies

Cattle and horse Cattle and buffalo

Sn 5 98%, Sp 5 100%

6.

7. 8. 9. 10.

Anigen Rapid AIV Ag Test Kit Prionics-Check PrioSTRIP BSE Kit Anigen Rapid Rabies Ag Test Anigen Rapid B. Brucella Ab Test Kit

Brucellosis (Brucella abortus)

Sn 5 94.4%, Sp 5 100%

Princeton BioMeditech Corporation, United States Thermo Fisher Scientific, United States IDEXX Laboratories, Inc., United States IDEXX Laboratories, Inc., United States MEGACOR GmbH, Germany Princeton BioMeditech Corporation, United States Bionote, Inc., Republic of Korea Prionics AG, Switzerland Bionote, Inc., Republic of Korea Bionote, Inc., Republic of Korea

Companion Animals 1.

SNAP4DxPlus Test

Lyme disease, ehrlichia, anaplasma, and heartworm disease

Cats and dogs

2.

SNAPFeline TripleTest

Cat

3.

SNAP Parvo Test (USDA-approved) Anigen Rapid Rabies Ag Test FASTest CRYPTO strip

Feline immunodeficiency virus, feline leukemia virua, and Feline heartworm infection Parvovirus

4. 5. a

Rabies virus Cryptosporidium parvum

Dog Dog and cat Dog and cat

Sn 5 90.3% 99.0%, Sp 5 94.3% 99.3% depending on the etiological agent Sn 5 89.3% 100%, Sp 5 98.6% 99.5% depending on the etiological agent Sn 5 100%, Sp 5 98 100% Sn 5 98%, Sp 5 100% Sn 5 96.7%, Sp 5 99.9%

Sn and Sp represent relative diagnostic sensitivity and specificity, respectively in comparison to gold standard assays.

IDEXX Laboratories, Inc., United States IDEXX Laboratories, Inc., United States IDEXX Laboratories, Inc., United States Bionote, Inc., Republic of Korea MEGACOR GmbH, Germany

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Table 13.2 Important patented technologies frequently used in the distinct diagnostic assays. S. no.

Diagnostic methods

1.

PCR

Company Roche molecular systems Hoffmann-La Roche Inc.

2.

RT-PCR

Invitrogen

3.

qPCR

University of Utah (licensed first to Idaho technology then to roche diagnostics)

Applera (Applied Biosystem) Roche diagnostic 4.

5.

Sequencing

Nucleic acid extraction

California Institute of Technology licensed to Applied Biosystems Pyrosequencing AB Amersham (GE Healthcare) Dynal

6.

7.

Recombinant Stanford and UCSF proteins production Stanford and UCSF

Point-of-care Chembio Diagnostic diagnostics Systems Stratagene

Technology/ product name

IP rights

PCR machine

US5656493

Thermostable reverse transcriptases Reverse transcriptase lacking RNase H activity Monitoring nucleic acids with probes or dyes during or after amplification, that is, basic for SYBR Green and FRET technology Real-time cycler

US5322770

Real-time PCR reagents Automated sequencer

Methods for sequencing DNA Nucleic acid separation using magnetic beads Oligonucleotide-linked particles for specific nucleic acid separation Proteins produced using recombinant prokaryote DNA Proteins from recombinant eukaryote DNA Dual Path Platform Full Velocity nucleic acid amplification technology (qPCR)

US6063608

US6174670

US6814934 US6171785 US5171534

US6210891 US5523231

US5512439

US4237224

US4468464

US7189522 US6350580

13.4 Applications of biotechnology

13.4 Applications of biotechnology in farm and companion animal’s disease diagnosis 13.4.1 Biotechnological tools in farm animal’s disease diagnosis Farm animals reared all over the world for major agricultural and production purposes majorly include cattle, buffalo, sheep, and goats. Since the last few decades, a number of infectious diseases have been found associated with farm animals, causing colossal loss to the livestock rearing community and few of them being zoonotic in nature, becoming a problem for the public health. Highly contagious livestock diseases such as FMD, hemorrhagic septicemia, peste-des-petits ruminants, and surra cause irreparable economic losses. Several other infectious diseases of dairy cows such as BVD, Johne’s disease, tuberculosis, infectious bovine rhinotracheitis, and liver fluke infestations are generally regarded as being widespread and endemic. The best known and arguably most important discovery of farm animal diseases in the last few decades is bovine spongiform encephalopathy (BSE), and others include digital dermatitis, neosporosis and bovine abortion, bovine neonatal pancytopenia, Arcanobacterium pluranimalium, and Schmallenberg virus. Among all, the world organization for animal health classifies FMD and BSE as diseases of major interest in cattle. These diseases are known to have a significant effect on dairy production either directly due to death or indirectly due to effects on fertility or milk production, and subsequently, culling. The disease conditions are usually identified based on history and clinical profile of the affected population, but for affirmative diagnosis of the pathogens responsible, the identification of the causal agent is done on the samples or clinical specimens for submission to diagnostic labs. The clinical profiles of several diseases overlap, making diagnosis a little tricky and cumbersome, so initially, the isolation of the infectious agent in pure form using cell culture systems or growth on specific and selective medium became a chosen method for the diagnosis of many pathogenic diseases in farm animals. Albeit their usefulness as most sensitive method of detection, they are not used routinely due to time-lapse in confirming illness (Bursle and Robson, 2016). These methods may take hours to several weeks to obtain a confirmatory result. Therefore, other approaches based on morphology/biochemical properties of pathogens took the lead and were favored for pathogen detection and identification. Several infectious viral disease agents viz astrovirus, adenovirus, rotavirus, etc. were identified through electron microscopy (Ong and Chandran, 2005). But, these also have some drawbacks like more time consuming, less sensitivity, and costly instrumentation. Apart from these techniques, approaches like detection of pathogen-specific antibodies or detection of antigenic proteins of pathogens were adopted and categorized under serological assays. Serological assays measure antigen antibody interactions for diagnostic purposes. These assays are continuously being improved with technologies like rapid strip detection, thus becoming the most preferred tools and are broadly referred to as immunoassays.

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Enzyme immunoassays (ELISA) have always been the field applicable diagnostic methods in the detection of various farm animal diseases caused by FMDV, Clostridium perfringens, M. bovis, and Escherichia coli. Hitherto reports have shown the problem of false negative results and cross-reactivity in some of the serological methodologies. Innovations including the use of synthetic biology by making highly reactive peptides help to increase the sensitivity and avoid the cross-reactivity issues to some extent. With the more recent advances in diagnostics with the availability of sequences, nucleic acid-based methods have complemented the established techniques as more specific and sensitive methods in detection of pathogens with lesser false positive results in comparison to serological-based methods (Bursle and Robson, 2016). PCR and real-time PCR methods are regularly used in the detection of Campylobacter, Shigella, bovine respiratory syncytial virus, Eimeria, Salmonella species, and many other pathogens. Likewise, seminested and nested PCR have been developed for the detection of Babesia bovis and Babesia bigemina. These nucleic acid-based techniques are amongst the standard detection methods and are routinely used for testing in diagnostic laboratories. To improve the efficacy and promote the simplicity, modifications in the form of isothermal amplifications, like LAMP and polymerase spiral reaction (PSR) have been adopted and are better in the application process, as these are easy to perform, portable, specific, sensitive, and most importantly, quick and cost-effective. LAMP is established to be an apex, leading diagnostics for the detection of many farm animal-related diseases like FMD, brucellosis, bovine popular stomatitis, sheep pox, and goat pox (Dukes et al., 2006; Song et al., 2012; Zhao et al., 2014). Likewise, PSR has been developed for detection of Brucella spp. (Das et al., 2018), bovine herpesvirus-1 (Malla et al., 2018), and canine parvovirus 2 (Gupta et al., 2017). To further increase the sensitivity and specificity, the combination of ELISA and PCR-like immune-PCR, proximity ligation assay, PCR-ELISA, have been successfully discovered making the detection 1000-fold more sensitive. The pathogen detected with these combinations includes low pathogenic strains of Campylobacter (Ding et al., 2013). NASBA, restriction fragment length polymorphism, amplified fragment length polymorphism, and random amplification of polymorphic DNA, are various biotechnological tools that have been further advancing the diagnosis of various infectious diseases. In addition, NGS has brought a revolution in the diagnosis of many pathogens. It appears helpful in the identification of many pathogens, especially viruses in the fecal matter and those that could not be isolated in cell culture system. Mining of sequences in samples gives varied genome sequences providing the clues of not only pathogens, but also new strains, genotypes, new viruses, and even the zoonotic efficiencies of the viruses. Anis and coworkers demonstrated that targeted bovine NGS is a specific and cost-effective tool for diagnosis of major bovine pathogens in clinical samples (Anis et al., 2018). Even pathogens with low pathogenicity such as bovine enteroviruses (BEV), adenoviruses in wild

13.4 Applications of biotechnology

captive animals, and hepatitis E viruses have been revealed in the mining of sequences in the sample. One of the approaches to disease diagnosis is the development of biosensors. Assays based on biosensors uses the transducers to convert the biological interaction of pathogen with its specific antibodies to measurable signals. Biosensors have been quite useful in the diagnosis in POC detection. Biosensors with specific biochemical recognition helped in the identification of E. coli in cattle (Dharmasiri et al., 2010). Colibacillosis is also seen in a variety of farm animals like cattle, pigs, and goats. In C. perfringens detection, epsilon-toxin-specific monoclonal antibody was immobilized onto single-walled carbon nanotubes and adjusted to detect relevant concentrations of toxin in nanomolars and were comparable to ELISA-based results. Many other methods like mass spectrometry, microarrays, and MALDI-TOF are also under employment for the detection of many farm animals-associated pathogens, like Francisella tularensis, Staphylococcus aureus, Enterococcus faecalis, E. coli (Demirev and Fenselau, 2008; Lundquist et al., 2005; van Baar, 2000).

13.4.2 Biotechnological tools in companion animals’ disease diagnosis Companion animals are the domesticated animals kept for company of human beings or for utilitarian purposes, that is, guarding, herding, military/police activity. They have grown along with the human civilization and evolution and have developed a good bond with humans. Although there is a variety of species which are suitable as companion animals (dogs, cats, rabbit, ferrets, caged birds, fishes, and guinea pigs), dogs and cats are the most common companion species. Their physical, behavioral, social, and emotional needs can be easily met at home. On the other hand, dogs and cats play a fundamental role in the life of human beings with many physiological and psychological benefits (Wood et al., 2005). Also, living with companion animals makes human surroundings happier and prosperous. As these animals enrich our lives, it becomes our responsibility to take care of the companion animals and to protect them from any kind of harm. There is a spectrum of infectious diseases that occur in companion animals. Lyme disease, psittacosis, hookworms, and Salmonella are amongst the most common diseases in pet animals. Some other examples include Rabies, E. coli, Rickettsia spp., infectious canine hepatitis, canine distemper virus, Ehrlichia spp., Helicobacer spp., Brucella canis, Bordetella bronchoseptica, and influenza A virus. Canine parvovirus (CPV) and feline leukemia virus are two of the important viral diseases of dogs and cats, respectively. Many of these infections are zoonotic in nature. The two most important human lifethreatening infections include rabies and zoonotic visceral leishmaniasis, which are exerting their negative impact globally (Lembo et al., 2010; Palatnik-deSousa et al., 2009).

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Since these animals share a close environment and are in direct contact with humans, hence they have a potential to spread these infections to human beings. Chances of introduction of new diseases also arise on the import of animals from foreign lands. Thus, maintenance of strict trade rules and regulations and hygienic conditions becomes a necessity. Once the disease occurs, it is important to identify the causative agent to improve the effectiveness of treatment and to control the disease. Since the clinical observations are not sufficient and can overlap with other diseases leading to misdiagnosis, several validated laboratory assays are used for confirmatory diagnosis. Until 20 years ago, these laboratory tests exploited cell culture (for isolation of specific pathogen) and serological assays (for detection of antibodies generated against a specific pathogen or antigenic proteins). Few examples composed of Vero cells and recombinant Vero-SLAM cells used for culturing Rickettsia rickettsia, and Toxoplasma gondii, Madin-Darby canine kidney cells for canine adenovirus and canine herpesvirus. Similarly, serological methods include a long list. Immunodiffusion testing is most often used to detect antibodies to fungal pathogens in dogs, such as Aspergillus fumigatus, Coccidioides immitis, and Blastomyces dermatitidis. Agglutination tests include the microscopic agglutination test for serologic diagnosis of leptospirosis (agglutination of live leptospires) and the cryptococcal antigen LA test (agglutination of antibody-coated latex beads). Hemagglutination inhibition is used to determine antibody titers to CPV and canine influenza virus, and it evaluates the ability of serum to inhibit erythrocyte agglutination by these viruses. ELISA is commonly used for the detection of feline retroviral, heartworm, Giardia, Leishmania, and tick-borne infections. Indirect IFA for serologic testing in dogs and cats include quantitative serology for some tick-borne infectious diseases (e.g., Ehrlichia canis, Anaplasma spp.). Direct immunofluorescence assay in veterinary medicine include diagnosis of Giardia oocysts, FeLV within monocytes in peripheral blood or bone marrow, or canine distemper virus within epithelial cells from a conjunctival scraping. Many of these assays involve the use of polyclonal antibodies, or, more commonly, monoclonal antibodies-dependent diagnosis. With the recent boom in the database of sequences for pathogens, new diagnostic tools like PCR, real-time PCR, and multiplex PCR have almost replaced the established techniques and are adopted as routine diagnostics for testing clinical samples. Canine respiratory coronavirus, canine adenovirus-2, canine herpesvirus, feline herpesvirus-1, canine distemper virus, West Nile virus, and Encephalitis viruses are some of the examples, which are routinely diagnosed using these techniques. Other biotechnological tools cover hybridization assays, PNA, nanoparticles-based assays, etc. Hybridization-based methods have also been found to be compatible with the diagnosis of many diseases and composed of Taqman-based probes, molecular beacons, and FRET-based probes. Although not yet widely used for veterinary applications, PNA probes are now increasingly available to detect target DNA. Fluorescent PNA probes, followed by signal amplification were used to differentiate between M. tuberculosis complex and

13.5 Conclusion

nontuberculous Mycobacterium spp. (Zerbi et al., 2001). In another example, NGS has also been used for the comparison of the oral microbiome of canines with their owners as they are in direct contact with their pets and many diseases might get transmitted to them (Oh et al., 2015). Gold nanoparticle-based immunochromatographic strip test using a combination of mAb and pAb was developed as an alternative for on-site and cost-effective diagnosis of CPV infection (Sharma et al., 2018). Another use of biotechnology has been observed for rapid and early detection of CPV using a QCM biosensor (Kim et al., 2015). Also, for genotyping of CPV-2, conventional methods are time consuming, therefore, a probe-based duplex fluorescence melting curve analysis (FMCA) for genotyping six different CPV-2 variants (original CPV-2, CPV-2a, CPV-2b, CPV-2c, and vaccine strains of CPVpf and CPVint) using only two Taqman probes has been developed (Liu et al., 2019). Despite the fact that a wide range of diagnostic tools are available, there is a considerable chance for better advancement in diagnostics, in terms of speed and accuracy, to control and eradicate economically important diseases. In the near future, use of new biotechnological tools like biosensors and nanotechnology will pave the way. Further, NGS platforms like MinION (a portable, real-time NGS sequencer) coupled with NanoPipe analysis are promising tools to perform bacterial and viral disease investigation in low throughput laboratories and specifically in the field (Beato et al., 2018; Shabardina et al., 2019). Although, yet not been adopted for animal disease diagnosis, but novel platforms such as smartphonebased diagnosis (which expands nucleic acid-based detection assays toward POCD) like RT-LAMP and fluorescent lateral flow immunoassay (already developed for Zika virus and Dengue virus) provide exciting opportunities for veterinary diagnostics in the near future (Rong et al., 2019).

13.5 Conclusion Biotechnological innovations have brought new generation diagnostic methods for rapid and sensitive diagnosis of various diseases of livestock and pet animals. Infectious diseases entail remarkable economic loss, weak food production system, food insecurity, and high maintenance cost of the agriculture sectors including farm animals, poultry, and aquaculture. Besides, these diseases carry a huge risk of transmission to humans as sporadic and endemic zoonoses. Classical and conventional diagnostic methods are labor intensive, time consuming, less sensitive, and difficult to meet the needs of the emerging pathogen diagnostics. Thus, new innovations have to be worked on and need to be practiced. Over the long term, innovations will be helping in the diagnosis of pathogens with accurate, sensitive and specific detections. NGS, biosensors, and advanced amplification techniques will persist for longer periods in their constant modified forms. Innovations will always be bringing the new applications in the diagnostics for

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the improved versions of techniques. New technique applications come with the cost and unbroken funding will be putting new prospective techniques into the trials. These techniques should be simplified in the innovations for their easy practices at the field itself, without looking for any skilled personnel/highly equipped laboratories.

Conflict of interest There is no conflict of interest.

Acknowledgments All the authors of the chapter thank and acknowledge their respective universities and institutes.

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Perez, J.W., Vargis, E.A., Russ, P.K., Haselton, F.R., Wright, D.W., 2011. Detection of respiratory syncytial virus using nanoparticle amplified immuno-polymerase chain reaction. Anal. Biochem. 410 (1), 141 148. Rao, P.P., Reddy, Y.N., Ganesh, K., Nair, S.G., Niranjan, V., Hegde, N.R., 2013. Deep sequenc-ing as a method of typing bluetongue virus isolates. J. Virol. Methods 193, 314 319. Ray, A., Norde´n, B., 2000. Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future. FASEB J. 14 (9), 1041 1060. Rong, Z., Wang, Q., Sun, N., Jia, X., Wang, K., Xiao, R., et al., 2019. Smartphone-based fluorescent lateral flow immunoassay platform for highly sensitive point-of-care detection of Zika virus nonstructural protein 1. Anal. Chim. Acta 1055, 140 147. Saijin, X., Yongzhong, O.Y., Xinglei, Z., Xue, L., Shuiping, Y., Huanwen, C., 2012. Aptamer-based assay for prion diseases diagnostic. Proc. Environ. Sci. 12, 134 1353. Scarparo, C., Piccoli, P., Rigon, A., Ruggiero, G., Nista, D., Piersimoni, C., 2001. Direct identification of mycobacteria from MB/BacT alert 3D bottles: comparative evaluation of two commercial probe assays. J. Clin. Microbiol. 39, 3222 3227. Shabardina, V., Kischka, T., Manske, F., Grundmann, N., Frith, M.C., Suzuki, Y., et al., 2019. NanoPipe-a web server for nanopore MinION sequencing data analysis. GigaScience 8 (2), 169. Sharma, C., Singh, M., Upmanyu, V., Chander, V., Verma, S., Chakrovarty, S., et al., 2018. Development and evaluation of a gold nanoparticle-based immunochromatographic strip test for the detection of canine parvovirus. Arch. Virol. 163 (9), 2359 2368. Shimaa, E., Mohamed, S., Mohammed, Z., 2015. Aptamer-based competitive electrochemical biosensor for brevetoxin-2. Biosens. Bioelectron. 69, 148 154. Shunxiang, G., Xin, Z., Bo, H., Mingjuan, S., Jihong, W., Binghua, J., et al., 2017. Enzyme-linked, aptamer-based, competitive biolayer interferometry biosensor for palytoxin. Biosens. Bioelectron. 89, 952 958. Singh, M., Agrawal, R.K., Singh, B.R., Mendiratta, S.K., Agarwal, R.K., Singh, M.K., et al., 2017. Development and evaluation of simple dot blot assays for rapid detection of staphylococcal enterotoxin-a in food. Indian J. Microbiol. 57 (4), 507 511. Smith, K.M., Anthony, S.J., Switzer, W.M., Epstein, J.H., Seimon, T., Jia, H., et al., 2012. Zoonotic viruses associated with illegally imported wildlife products. PLoS One 7 (1), e29505. Song, L., Li, J., Hou, S., Li, X., Chen, S., 2012. Establishment of loop-mediated isothermal amplification (LAMP) for rapid detection of Brucella spp. and application to milk and blood samples. J. Microbiol. Methods 90 (3), 292 297. Takekawa, J.Y., Iverson, S.A., Schultz, A.K., Hill, N.J., Cardona, C.J., Boyce, W.M., et al., 2010. Field detection of avian influenza virus in wild birds: evaluation of a portable rRT PCR system and freeze-dried reagents. J. Virol. Meth. 166 (1 2), 92 97. Takekawa, J.Y., Hill, N.J., Schultz, A.K., Iverson, S.A., Cardona, C.J., Boyce, W.M., et al., 2011. Rapid diagnosis of avian influenza virus in wild birds: use of a portable rRT PCR and freeze-dried reagents in the field. J. Vis. Exp. 54, 2829. Tarasov, A., Gray, D.W., Tsai, M.Y., Shields, N., Montrose, A., Creedon, N., et al., 2016. A potentiometric biosensor for rapid on-site disease diagnostics. Biosens. Bioelectron. 79, 669 678.

References

Theuns, S., Vanmechelen, B., Bernaert, Q., Deboutte, W., Vandenhole, M., Beller, L., et al., 2018. Nanopore sequencing as a revolutionary diagnostic tool for porcine viral enteric disease complexes identifies porcine kobuvirus as an important enteric virus. Sci. Rep. 8 (1), 9830. van Baar, B.L., 2000. Characterisation of bacteria by matrix-assisted laser desorption/ionisation and electrospray mass spectrometry. FEMS Microbiol. Rev. 24 (2), 193 219. Wang, J., Moore, N.E., Deng, Y.M., Eccles, D.A., Hall, R.J., 2015. MinION nanopore sequencing of an influenza genome. Front. Microbiol. 6, 766. Wood, L., Giles-Corti, B., Bulsara, M., 2005. The pet connection: pets as a conduit for social capital? Soc. Sci. Med. 61 (6), 1159 1173. Ye, F., Zheng, Y., Wang, X., Tan, X., Zhang, T., Xin, W., et al., 2014a. Recognition of Bungarus multicinctus venom by a DNA aptamer against β-bungarotoxin. PLoS One 9 (8), e105404. Ye, W.W., Tsang, M.-K., Liu, X., Yang, M., Hao, J., 2014b. Upconversion luminescence resonance energy transfer (LRET)-based biosensor for rapid and ultrasensitive detection of avian influenza virus H7 subtype. Small 10 (12), 2390 2397. Yeo, S.J., Choi, K., Cuc, B.T., Hong, N.N., Bao, D.T., Ngoc, N.M., et al., 2016. Smartphone-based fluorescent diagnostic system for highly pathogenic H5N1 viruses. Theranostics 6 (2), 231 242. Zerbi, P., Schønau, A., Bonetto, S., Gori, A., Costanzi, G., Duca, P., et al., 2001. Amplified in situ hybridization with peptide nucleic acid probes for differentiation of Mycobacterium tuberculosis complex and non-tuberculous Mycobacterium species on formalin-fixed, paraffin embedded archival biopsy and autopsy samples. Am. J. Clin. Pathol. 116, 770 775. Zhang, N., Apella, D.H., 2010. Advantages of peptide nucleic acids as diagnostic platforms for detection of nucleic acids in resource-limited settings. J. Infect. Dis. 201 (Supplement 1), S42 S45. Zhao, Z., Fan, B., Wu, G., Yan, X., Li, Y., Zhou, X., et al., 2014. Development of loopmediated isothermal amplification assay for specific and rapid detection of differential goat Pox virus and Sheep Pox virus. BMC Microbiol. 14 (1), 10.

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Biotechnological tools in diagnosis and control of emerging fish and shellfish diseases

14

S.S. Mishra, Rakesh Das, S.N. Sahoo and P. Swain Central Institute of Freshwater Aquaculture (ICAR), Kausalyaganga, Bhubaneswar, India

14.1 Introduction Growing demand of food fish in recent years along with scope of economic gain, has favored both horizontal and vertical expansion of aquaculture all over the world. FAO data indicated that global fish production stands at 167 million ton (MT), of which 44% (73.8 MT) is contributed by the aquaculture sector (FAO, 2016). The fisheries’ sector supplies 17% of animal protein and supports the livelihood of 12% of the world’s population. Globally, India stands second in culture fisheries production, only next to China. Aquaculture in India, has been steadily growing with an annual growth rate of over 7% and more than 14.5 million people are dependent on it for their livelihood security, especially in coastal regions. It has been reported that frequent occurrence of infectious disease of bacterial and viral origin have caused severe damage to the aquaculture sector in India and impeded in sustainable development. There are reports of occurrence of emerging and reemerging diseases in aquaculture (Bondad-Reantaso et al., 2005). In aquatic environment, fishes are subjected to a wide range of factors including pathogens, that result in health status of fish. Disease becomes evident only when a stressful condition dominates, thereby suppressing the immune status of fish (Chapela et al., 2018; Mishra et al., 2018). Although local pathogens combined with other factors, such as poor husbandry and inadequate water quality, are the most common causes of disease outbreaks in fish farming, the introduction of “exotic” pathogens through international trade in live aquatic animals and their products have been the major reason associated with new epizootics (Subasinghe et al., 2001). Aquaculture needs innovative biotechnological interventions to overcome the challenges in terms of development of suitable technologies for water quality management in culture systems, development of rapid disease diagnostics and methods for combating disease outbreaks, supply of disease-free or high health broodstock and seed (Subasinghe et al., 2003). Molecular techniques can be used to solve that type of problems and increase sensitivity and specificity of pathogen Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00014-X © 2020 Elsevier Inc. All rights reserved.

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detection (Altinok and Kurt, 2003). Most importantly, methods that utilize the PCR can lead to identification of an isolate within hours as opposed to days in traditional microbiological methods. Again it can be used on small quantities of cells, including those that are not viable or are otherwise unculturable. This chapter reviews development and application of modern biotechnological tools in disease diagnosis and sustainable development of aquaculture.

14.2 Disease problems in fish culture Cultured aquatic animals including fish and shrimp are susceptible to pathogenic microbes as these are normal inhabitants of the aquatic environment (Ahmed and Kumar, 2005; Idowu et al., 2017). The occurrence of disease in aquatic environment is a complex interaction between the host species, disease agents and the environment. Outbreaks of disease are greatly influenced by the susceptibility of the hosts, the virulence of the pathogens, and adverse environmental conditions. Intensive and semiintensive farming practices favor disease occurrence, due to high stocking densities, increased stress to cultured stocks and inadequate water exchange (Idowu et al., 2017; Kaoud, 2015). Thus fish become more susceptible to various infectious organisms such as protozoa. helminths, crustaceans, bacteria, fungus, and virus (Faruk and Anka, 2017; Mishra et al., 2017a,b). On the other hand, disease outbreaks are less common in open water environments, even though the pathogens and host species may be present (Kaoud, 2015). Fish parasites infest the gills, skin, gut, or as grub-like worms in fish muscle tissue causing irritation, impaired function, weight loss, and eventually kill the fish. It has been observed that high organics favor sessile ciliates, including Heteropolaria/ Epistylis, Apiosoma, and Ambiphyra, which attach to fish but feed on bacteria and other nutrients in the water column. Other protist parasites that thrive in rich, organic environments include the trichodinids (e.g., Trichodina, Tripartiella), and Tetrahymena, Uronema (Yanong and Francis-Floyd, 2019). Among all parasites infestation with Argulous is maximum followed by Dacylogyrous sp. affecting gills. Occurrence of Myxobolous, Trichodina, and Ergasilus sp. are also reported in cultured fish (Mishra et al., 2018). A wide variety of bacterial pathogens also cause major losses to fish culture. It has been estimated that around 34% of total diseases are caused due to bacterial pathogens. These microorganisms are essentially opportunistic pathogens which invade the tissues of a fish host rendered susceptible to infection by stress factors (Ahmed and Kumar, 2005; Idowu et al., 2017). Common bacterial pathogens, Aliivibrio salmonicida, Vibrio spp., Edwardsiella ictaluri, E tarda, Streptococcus spp., and other related Gram-positive cocci, cause disease in aquaculture species (Yanong and Francis-Floyd, 2019). Common Gram negative bacteria responsible for causing disease in cultured fish include: Vibriosis (V. anguillarum, V. harveyi clade, V. parahaemolyticus, Ali. salmonicida (V. salmonicida), V. vulnificus, Photobacterium damselae), Aeromonasis (Motile Aeromonas spp.: Aeromonas

14.2 Disease problems in fish culture

caviae, A. hydropila, A. sobria, A. veronii, A. jandaei, and Aer. salmonicida) Edwardsiellosis (Edwardsiella anguillarum, E. ictaluri, E. piscicida, E. tarda, and Yersinia ruckeri); Pseudomonasis (Pseudomonas anguilliseptica, P. fluorescens) Flavobacteriosis (Flavobacterium branchiophilum, F. columnare, F. psychrophilum, Tenacibaculum maritinum) (Haenen, 2017). Again common Gram-positive bacteria involved in disease conditions are Mycobacteriosis (Mycobacterium fortuitum, M. marinum, Nocardiaasteroides, N. crassostreae (ostreae), N. seriolae); Streptococcosis (Streptococcus agalactiae, S. iniae, Lactococcus garvieae, and Aerococcus viridans); Renibacteriosis (Renibacterium salmoninarum). There are some infections caused by anaerobic bacteria (Clostridium botulinum) and intracellular bacteria (Piscirickettsia salmonis, Hepatobacter penaei, Francisella noatunensis, and Chlamydia spp. (Faruk and Anka, 2017; Haenen, 2017; Idowu et al., 2017; Mishra et al., 2017a,b). Bacterial kidney disease (BKD), caused by R. salmoninarum (Rs) is economically important in cultured salmonids. E. ictaluri causes enteric septicemia of catfish, the most important infectious disease in the channel catfish industry. F. columnare, the most prominent member of this group responsible for columnaris disease, is the most common in warm-water species of fish (Yanong and Francis-Floyd, 2019). Bacterial gill disease, caused by F. branchiophilum, is the most frequently reported in young cultured salmonids or fish cultured under conditions of high organic loading. Deterioration of water quality due to high stocking density, high organic load mostly due to accumulation of unused feed, less water exchange causes stress to animals making them prone to diseases (Idowu et al., 2017). Some of the fish pathogens like Streptococcus agalactiae, Streptococcus iniae, Edwardsiella tarda, Vibrio vulnificus, Photobacterium damselaedamselae, Mycobacterium marinum, and Mycobacterium fortuitum are of zoonotic importance (Haenen, 2017). Occurrence of viral diseases in aquaculture has been a major area of concern, although their occurrence in freshwater fish culture has not been so drastic. Prevalence of more than 125 different viruses have been reported in fish culture around the globe (Ahmed and Kumar, 2005; Hamera and Bondad-Reantaso, 2001; Sahoo and Goodwin, 2012). The identification and characterization of previously undescribed viral diseases in aquaculture is increasing because of recent technologic advances and increasing expertise in disease diagnosis and characterization (Yanong and Francis-Floyd, 2019). Occurrence of epizootic haematopoietic necrosis, infectious haematopoietic necrosis (IHN), Oncorhynchus masou virus, infectious pancreatic necrosis, viral encephalopathy and retinopathy (VER), spring viraemia of carp (SVC),viral haemorrhagic septicaemia (VHS) and lymphocystis are of great concern to fish culture worldwide (Ahmed and Kumar, 2005; Hamera and Bondad-Reantaso, 2001; OIE, 2000, 2018). The following diseases of fish are listed by the OIE (2018).

14.2.1 Fish diseases
Infection with Aphanomyces invadans (epizootic ulcerative syndrome);
Infection with epizootic haematopoietic necrosis virus;
Infection with Gyrodactylus salaris;

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Infection Infection Infection Infection Infection Infection Infection

with with with with with with with

HPR-deleted or HPR0 infectious salmon anemia virus; infectious haematopoietic necrosis virus; koi herpesvirus; red sea bream iridovirus; salmonid alphavirus; spring viraemia of carp virus; viral haemorrhagic septicemia virus.

14.2.2 Crustacean diseases The following diseases of crustaceans are listed by the OIE:










Acute hepatopancreatic necrosis disease; Infection with Aphanomyces astaci (crayfish plague); Infection with Hepatobacter penaei (necrotizing hepatopancreatitis); Infection with infectious hypodermal and haematopoietic necrosis virus; Infection with infectious myonecrosis virus; Infection with Macrobrachium rosenbergii nodavirus (white tail disease); Infection with Taura syndrome virus; Infection with white spot syndrome virus; Infection with yellow head virus genotype 1.

Besides the above, there are many other bacterial and viral diseases prevalent in many aquaculturing countries, which are of regional importance and some are not notifiable to OIE. One of the oldest recognized fish viral disease is, “carp pox” which is caused by cyprinid herpesvirus-1. Pox lesions may be seen on other species of fish and are sometimes referred to as fish pox. Lesions typically are smooth and raised and may have a milky appearance (Petty and Francis-Floyd, 2019). In India, the impact of fish viral diseases has been minimal due to typical culture systems adopted and type of fish species being cultured. Cases of viral nervous necrosis virus (VNNV) orVER disease in Asian sea bass have been reported in India (Azad et al., 2005; Jithendran et al., 2011). Azad et al. (2005) reported VNN in larvae of the Asian sea bass Lates calcarifer (Bloch) which suffered heavy mortalities (60%
90%) during the hatchery-rearing phase. Another emerging viral disease of fish is Tilapia lake virus (TiLV), affecting farmed tilapia (Oreochromis niloticus) and other Tilapia (Oreochromis spp.) in the AsiaPacific region (FAO, 2017). TiLV is an orthomyxo-like RNA virus and originally observed and reported in Israel, Ecuador, Colombia, and Egypt (NACA, 2017). This disease has serious socioeconomic consequences and is a direct threat on food security for millions of people in Africa, Asia, and South America (Nicholson et al., 2018). There are reports of mass scale mortality in tilapia culture in Thailand, causing mortality up to 90% of stocks (NACA, 2017). World Organization for Animal Health (OIE) reported TiLV, had been officially detected on three continents covering eight countries and subsequently in Tanzania, Uganda, Indonesia, Taiwan, and Peru. In India, there were some reports TiLV

14.3 Diseases in shrimp (shellfish)

disease outbreaks in farmed tilapia in West Bengal and Kerala states with .85% mortality (Behera et al., 2018), although disease occurrence with mass scale mortality have not been reported in other parts. There are some reports of isolation of viral pathogens from ornamental fish in India (Rathore et al., 2012; Swaminathan et al., 2016). Viral pathogens like cyprinid herpesvius-2, koi ranavirus (KIRV), carp edema virus (CEV), megalocytiviris and goldfish haematopoietic necrosis herpes virus, have been reported in ornamental fish culture in India (Sahoo et al., 2017). Some reports indicated KIRV causing huge mortality of koi Cyprinus carpio in a farm in south India (Sahoo et al., 2017). In addition to these, koi sleepy disease caused by CEV has been reported in C. carpio (Swaminathan et al., 2016). Spring viraemia of carp virushas emerged in several regions of the world where it has been associated with heavy losses in common carp and koi carp (Walker and Winton, 2010). The details of emerging viral diseases in fin fish has been presented in Table 14.1.

14.3 Diseases in shrimp (shellfish) Unlike in fish culture, viral diseases are a major cause of concern in shrimp aquaculture industry worldwide and several viral outbreaks often cause catastrophic losses in shrimp farming around the globe. Many viral diseases like yellow-head disease (YHD), infectious hepatopancreas and haematopoietic necrosis (IHHN), white spot disease (WSD), bacterial white spot syndrome, baculoviral midgut gland necrosis, gill-associated virus, spawner mortality syndrome (“midcrop mortality syndrome”), Taura syndrome, nuclear polyhedrosis baculovirosis have caused havoc in shrimp culture industry worldwide (Ahmed and Kumar, 2005; Hamera and Bondad-Reantaso, 2001). Out of these occurrence of monodon baculo virus (MBV), yellow head virus (YHV), white spot disease (WSD), and Taura syndrome Virus (TSV) disease were reported in India, out of which WSD has become endemic in India, responsible for significant mortality and loss to shrimp farmers (Mishra et al., 2017a,b). Infectious myonecrosis virus (IMNV) has been responsible for causing severe damage in L. vannamei culture. Similarly, occurrence of white tail disease or white muscle disease (WMD) has caused severe loss to prawn industry in many southeast Asian (SE) countries including India. The causative organisms were identified as M. rosenbergii nodavirus (MrNV) and its associated extra small virus (Sahul Hameed et al., 2004). Among bacterial diseases, iluminescent bacterial disease, vibriosis, bacterial septicemia and larval mycosis are common, mostly occur due to poor hygiene and water quality management. Adoption of better management practices along with use of antiseptics can significantly reduce such disease occurrence. However, acute hepatopancreatic necrosis disease (AHPND) also commonly called early mortality syndrome (EMS) is currently the most important nonviral disease threat for cultured shrimp P. vavvamei. It is usually characterized by mass

315

Table 14.1 Common emerging diseases of fish and shellfish. S. no.

Disease

Etiological agent

Species affected

Salmonids, rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), grayling (Thymallus thymallus), white fish (Coregonus sp.), and turbot (Scophthalmus maximus). Rainbow or steelhead trout (Oncorhynchus mykiss), Pacific salmon including chinook (O. tshawytscha), sockeye (O. nerka), chum (O. keta), and Atlantic salmon (Salmo salar). Redfin perch (Perca fluviatilis), rainbow trout (Oncorhynchus mykiss), sheatfish (Silurus glanis) and catfish (Ictalurus melas) Several carp species including common carp and koi carp (Cyprinus carpio), grass carp, silver carp, bighead carp (Aristichthys nobilis), Goldfish (Carassius auratus), Common carp (Cyprinus carpio) and Koi carp

Known geographic distribution

A. Fish diseases 1.

viral hemorrhagic septicemia (Egtved disease)

Novirhabdovirus family: Rhabdoviridae

2.

Infectious hematopoietic necrosis

Novirhabdovirus family: Rhabdoviridae

3.

Epizootic haematopoietic necrosis

Ranavirus family: Iridoviridae

4.

Spring viraemia of carp (SVC)

Vesiculovirus family: Rhabdoviridae

5.

Koi herpes virus disease (KHV) Viral encephalopathy and retinopathy (VER)/viral nervous necrosis virus

Cyprinid herpesvirus-3 Family: Alloherpesviridae viral nervous necrosis virus (VNNV) (1) ssRNA, Nodaviridae, and Betanodavirus

Tilapia lake virus disease (TiLV) disease Red sea bream iridoviral disease Infectious salmon anemia

TiLV (an Orthomyxo-like RNA virus). Megalocytivirus Family: Iridoviridae

6.

7. 8. 9.

Isavirus family: Orthomyxoviridae

Larval or juvenile barramundi (sea bass, Lates calcarifer), European sea bass Turbot, halibut, Japanese parrotfish, red-spotted grouper and striped jack Tilapia species (Oreochromis niloticus), other (Oreochromis spp. Red sea bream, many other estuarine, marine species Atlantic salmon brown trout, sea trout, and rainbow trout

United States, Europe, and North America

North America, Europe, and Asia

Australia, Europe, Asia, North America, and Africa Europe, Asia, North, and South America

Asia, Europe, North America, Israel, and Africa Asia, the Mediterranean, and the Pacific.

Asian region Japan, Taiwan United States, Scotland, and Norway

10.

11.

Salmonid alphavirus disease (pancreas disease or sleeping disease) Bacterial kidney disease (BKD)

Alphavirus Family: Togaviridae

Atlantic salmon, rainbow trout, and brown trout

Ireland, United Kingdom, France, Germany, Italy, Spain, Switzerland, Poland, and Norway North America, Japan, Western Europe, and Chile

Caused by Renibacterium salmoninarum (Rs) belonging to the genus Renibacterium

Salmonidae family are clinically susceptible, in particular the Oncorhynchus species (Pacific salmon and rainbow trout).

Macrobrachium rosenbergii nodavirus (MrNV), non-enveloped virus 26
27 nm, ssRNA (RNA1 and RNA2) MBV, dsDNA, size, 75 3 300 nm, Baculovirus, and occluded

Giant freshwater prawn Macrobrachium rosenbergii

YHV. (1) ssRNA virus, genus: Okavirus, family: Roniviridae, order: Nidovirales Baculovirus, dsDNA, enveloped, family: Nimaviridae, genus: Whispovirus Picorna virus, family: Dicistroviridae, order: icornavirales

P. monodon, P. vennamei, P. indicus, and P. stylorostris

China, Taiwan Thailand and other southeast Asian (SE) countries, and Australia Thailand, Indonesia, Taiwan and Philippine, and other SE countries Asian countries, North America, and in Mexico

Most commercially cultivated penaeid shrimp species.

China, Taiwan, Japan. Then in Latin America

Pacific white shrimp Penaeus vannamei, P. stylirostris, P. setiferus, and P. schmitti

Single stranded, DNA Parvovirus family Parvoviridae

All penaeid species—P. monodon, P. merguiensis, P. vennamei, P. semisulcatus, P. indicus, P. japonicus

Ecuador, China, Taiwan, Thailand, Malaysia, Indonesia and other southeastern countries Worldwide distribution among cultured penaeid shrimp SE countries

HPV, ssDNA virus, Parvovirus (22B24 nanomicrons), family: Parvoviridae

P. monodon, P. vennamei, P. semisulcatus, P. indicus, and P. chinensis

Asia, Africa, Australia and North and South America

B. Diseases in shellfish 1.

White tail disease/white muscle disease (WMD) of freshwater prawn

2.

Monodon baculo virus (MBV) disease

3.

Yellow head disease (YHD)

4.

White spot disease (WSD)

5.

Taura syndrome Virus (TSV) disease

6.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) disease Hepatopancreatic Parvovirus (HPV) disease

7.

P. monodon, P. merguiensis, P. vennamei, P. semisulcatus, P. indicus, and P. japonicas

(Continued)

Table 14.1 Common emerging diseases of fish and shellfish. Continued S. no.

Disease

Etiological agent

Species affected

Known geographic distribution

8.

Infectious Myonecrosis virus (IMNV) disease

Penaeus vannamei. Experimental infections in Penaeus stylirostis and P. monodon

Brazil, spread to Indonesia China, and SE Asia

9.

Spawner-isolated mortality virus disease/ midcrop mortality syndrome (MCMS) Acute hepatopancreatic necrosis disease/early mortality syndrome (AHPND/EMS), Hepatopancreatic microsporidiosis (HPM)

IMNV, Size: B40 nm, unenveloped, dsRNA, family: Totiviridae SMVD is caused by a singlestranded icosahedral DNA virus measuring 20
25 nm, related to family: Parvoviridae Bacteria: Vibrio parahaemolyticus

Penaeus monodon. Experimentally in P. esculentus, P. japonicus, P. merguiensis, and Metapenaeus ensis

Queensland, as well as the Philippines, and Sri Lanka

P. monodon, P. vennamei, P. indicus, and P. japonicus

Southern China, Vietnam, Thailand, and Malaysia

P. monodon, P. vennamei, P. indicus, and P. japonicus

Thailand, then in China, Vietnam, Thailand, Indonesia, and Malaysia

10.

11.

Enterocytozoon hepatopenaei (EHP), microsporidian parasite

Adapted from Mishra, S.S., Das, R., Choudhary, P., Debbarma, J., Sahoo, S.N., Giri, B.S., et al., 2017b. Present status of fisheries and impact of emerging diseases of fish and shellfish in Indian aquaculture. J. Aquat. Res. Mar. Sci. 2017, 5
26; Pantoja, C.R., Lightner, D.V., Poulos, B.T., Nunan, L., Tang, K.F.J., Redman, R.M., et al., 2008. Paper Presented on Overview of Diseases and Health Management Issues Related to Farmed Shrimp, on 17.4.2008. OIE Reference Laboratory for Shrimp Diseases Department of Veterinary Science & Microbiology, University of Arizona, Tucson; Petty, B.D., Francis-Floyd, R., 2019. Viral diseases of fish. In: MSD Manual Veterinary Manual. Merck Sharp and Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ. Available from: ,https://www.msdvetmanual.com/exotic-andlaboratory-animals/aquarium-fishes/viral-diseases-of-fish.; OIE, 2000. Diagnostic Manual for Aquatic Animal Diseases, third ed. Office International des Epizootics, Paris.

14.3 Diseases in shrimp (shellfish)

mortality during the first 35 days of culture. The details of emerging diseases of shrimp relevant to SE countries have been presented in Table 14.1.

14.3.1 Diagnostic methods For a successful disease control program in aquaculture, detailed information in terms of disease status of a farm, zone, or country, need to be collected. It is an effective and fundamental foundation for any method of disease prevention and control (Mohan et al., 2008). Several advanced technologies are now being adopted for better health management practices in aquaculture, with prevention and control of fish diseases. The modern diagnostic assays are based on interactions of certain important biomolecules of the pathogens such as antigen, nucleic acids, etc. following their isolation, purification, and characterization for preparing suitable diagnostic and/or prophylactic tools. As elaborated by Subasinghe et al. (2001) and Bondad-Reantaso et al. (2005), the essential principles of disease treatment and control are to (i) establish an accurate diagnosis; (ii) select an appropriate and environmentally-responsible treatment, and (iii) evaluate management practices within the farm and determine if future outbreaks could be prevented by changes in procedure or design. OIE (2000) and FAO/NACA (Hamera and Bondad-Reantaso, 2001) have recommended the use of three levels of diagnostics for emerging and reportable fish and shellfish diseases. All three levels of diagnostic specialization are necessary for confirmation of newly or rarely encountered disease. The different levels of disease diagnosis which can be undertaken when investigating a disease situation are as follows. a. Level I: Farm/production site observations, record keeping, and health management. This is important, as this forms the basis for triggering the other diagnostic levels (II and III). b. Level II: It includes the specializations of parasitology, histopathology, bacteriology, and mycology, which require moderate capital and training investment. c. Level III: It comprises the types of advanced diagnostic specialization, which requires significant capital and training investment. Isolation of the causative bacteria, fungus, and virus, biochemical characterization of pathogens, immunoassays (enzyme-linked immunosorbent assays or fluorescent, antibody technique), transmission electron microscopy, molecular diagnostics like polymerase chain reaction and nucleic acid assays using specific probes, etc. come in this category.

14.3.1.1 Immunoassays Immunoassays for the detection of the antigens of microorganisms remain important tools for the diagnosis and management of infectious diseases. Several antibody-based technologies are now being used in disease diagnosis program in aquaculture. Serology methods, used for screening for the presence of specific

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antibodies in the serum of animals, have been a useful indicator of previous exposure to a pathogen and are being routinely used in clinical and veterinary medicine (Campbell and Landry, 2006). The advantage of serology is that it is able to indicate infection, before it is possible to detect the pathogen by culture or other methods (Kaoud, 2015). Antigen detection methods are also very valuable for the rapid and specific identification of infectious agents (Adams and Thompson, 2011). An ideal immunoassay to detect antibodies against infectious agents will have high sensitivity so to detect low concentrations of antibodies, as well as high specificity to avoid cross-recognition of antigenically-related antigens and reduce the possibility of no false-positive results. Enzymes, radioisotopes, and fluorescent dyes are used in serological techniques like enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay, and fluorescent antibody test (FAT). The immunoassays can be grouped into several categories according to the type of detection systems used (1) colorimetric, (2) radiometric, (3) chemiluminescent, or (4) fluorescent (Tang and Stratton, 2006). ELISA is an indirect or colorimetric enzyme immunoassay. Immunoblotting which includes Western blot, is another technique for antibody detection. Capture antigens such as proteins are electrotransferred to a nitrocellulose membrane. If target antibodies are present in the specimen, they will bind to the antigens present on the nitrocellulose strips. Visualization of the antibodies bound to antigen is accomplished using a series of reactions with goat antihuman IgG conjugated with biotin, avidin conjugated with horseradish peroxidase enzyme (HRP), and the HRP substrate. The bands corresponding to the antigens will be seen on the nitrocellulose strip (Tang and Stratton, 2006). These immunodiagnostic tests are currently used to screen and/or confirm diagnosis of many fish diseases. These are of significant use for detection of exotic pathogens of cultured finfish, for example, channel catfish virus, infectious hematopoeitic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), viral haemorrhagic septicaemia virus (VHSV), viral nervous necrosis virus, and BKD, as well as shrimp diseases [e.g., white spot syndrome virus (WSSV), YHV, infectious hypodermic and haematopoeitic necrosis virus (IHHNV), and TSV world (Lightner, 1996; Lightner and Redman, 1998; Tang et al., 1997)]. Lateral flow diffusion method has been a recent development of serological test, designed more for the antigen-specific immunoassay than for antibody detection. It uses colloidal gold, carbon, paramagnetic or color latex beads to create a visible line in the capture zone where there is a nitrocellulose or nylon membrane. Lateral flow assays have been available on the commercial market. The assays are typically designed on nitrocellulose or nylon membranes contained within a plastic or cardboard housing. In the antibody detection format, a capture antigen is bound to the membrane, and a second labeled antibody is placed on a sample application pad. As the sample migrates down the membrane by capillary action, antibody present in the sample binds to the labeled antigen and is captured as the complex passes. Colloidal gold, carbon, paramagnetic, or colored latex beads are commonly used particles that create a visible line in the capture zone of the assay membrane for a positive result. These assays are simple to use, require minimal

14.3 Diseases in shrimp (shellfish)

training, and require no special storage conditions. In most cases, the manufacturer provides simple instructions that include pictures of positive and negative results. This technique has the potential for future use once validated, particularly for detecting preexposure of fish to viral pathogens (Adams and Thompson, 2011).

14.3.1.2 Molecular diagnostics for fish diseases The basics of a successful health management program includes prompt, efficient, and accurate diagnosis of causative agent responsible for disease outbreaks and mortality. Molecular methods have significantly revolutionized diagnostic methods in human and animal medicine including in fish disease diagnosis and control programs. These techniques are more convenient than conventional methods as they are generally faster, more specific, precise, and preceding cultivation of microorganisms is not necessary. Again, the identification and characterization of unculturable and slow-growing pathogens is easier to detect using such molecular methods (Subasinghe et al., 2001; Tang et al., 1997). Various molecular biology techniques continue in becoming more important in fish and shrimp farming, particularly in detection and prevention of various diseases. The common molecular tools used in fish disease diagnosis and characterization of pathogens have been elaborated in Table 14.2. Table 14.2 Common molecular tools used in fish and shellfish disease diagnosis and characterization of pathogens. Molecular Diagnostics for Detection of Pathogens

Genotyping techniques in characterization of pathogens

1. 2.

Polymerase chain reaction (PCR) Multiplex PCR (mPCR)

3.

Nested PCR

4.

Reverse transcriptase PCR (RT-PCR)

5. 6.

8.

Real-time PCR (qPCR) DNA probe-based hybridization techniques: Dot blot, in situ hybridization (ISH), fluorescence in situ hybridization, or FISH, colony hybridization Loop-mediated isothermal amplification (LAMP) Microarrays

Pulse field gel electrophoresis Arbitrarily primed PCR (AP-PCR) or random amplified polymorphic DNA (RAPD amplified fragment length polymorphism (AFLP) assays ERIC-PCR, Rep-PCR, BOX-PCR, ISPCR, and VNTR-PCR Ribotyping techniques (16SrDNA) Amplified ribosomal DNA restriction analysis (ARDRA)

9. 10.

MALDI-TOF mass spectrometry Nanotechnology and nanosensors

7.

DNA sequence analysis Multilocus sequence typing (MLST) analysis

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14.3.1.2.1 Polymerase chain reaction One of the most prominent techniques is the PCR. No technique has had a greater impact on disease diagnosis than PCR. PCR is a technique for amplifying a specific region of DNA, defined by a set of two “primers” at which DNA synthesis is initiated by a thermostable DNA polymerase. It is a rapid and simple means of producing relatively large number of copies of DNA molecules from minute quantities of source DNA. Usually, at least a million-fold increase of a specific section of a DNA molecule can be realized for detection and analysis (Kaoud, 2015). Initially, American biochemist Kary B. Mullis conceived the idea of PCR in 1983 and was later developed into a technique at the Cetus Corporation in Emeryville, California. This technology has proven to be a revolutionary method. The regions amplified are usually in the range of 150
3000 base pairs (bp) in length. Primer design is important to obtain higher sensitivity and specificity. The technique is continuously being improved by different researchers with advent of newer PCR-based techniques. PCR has a wide scope of applications, including analysis of ancient DNA from fossils, DNA fingerprinting, mapping the human genome, and detection of microorganisms present in low numbers in water, food, soil, or other systems (de la Pen˜a, 2001, 2002). There are many forms of PCR-derived amplification techniques being undertaken in different laboratories: (i) hot-start PCR, (ii) touchdown PCR, and (iii) degenerate PCR. Hot-start PCR technique focuses on the inhibition of DNA polymerase activity during reaction setup. This is accomplished by physically separating or chemically inactivating one or more of the reaction components until high temperature triggers mixing or reactivation to give a complete reaction mixture. This procedure limits nonspecific annealing of the primers and generally improves yield of the desired amplicon. As elaborated by Tang and Stratton (2006), touchdown PCR, was originally used to simplify the process of determining optimal PCR annealing temperatures. In this case, a range of annealing temperatures are introduced. The earliest cycles of touchdown PCR have high annealing temperatures. In subsequent cycles, the annealing temperature is decreased by small increments (usually 1 C) every several cycles to a final “touchdown” annealing temperature, which is then used for the remaining 10 or so cycles. This gradual decrease in annealing temperature selects for the most complementary primer-target binding in early cycles. This is most likely the sequence of interest. As the annealing temperature decreases, primers will anneal to nonspecific sequences, however, amplification of these products will lag behind that of the specific product. This favors synthesis of intended product over any nonspecific products (Tang and Stratton, 2006). PCR is very fast, sensitive, and highly specific and has been widely used in the detection of fish and shrimp viruses like WSSV, MBV,IHHNV, hepatopancreatic parvo virus (HPV), TSV, YHV, etc. (Lightner 1996; Lightner and Redman 1998). The assay has also been used in the detection of Vibrio penaeicida in shrimp and Aer. salmonicida subspecies salmonicida (de la Pen˜a, 2002). The basic requirement of PCR is to design specific primer sequence for target gene of importance that can be able to

14.3 Diseases in shrimp (shellfish)

characterize and differentiate specific strain/species of organisms or animals. Hence work on recognizing specific target genes must be carried out for different fish and shellfish pathogens to fully exploit the potential application of PCR technology in diagnosis. Common diagnostic tests used for different fish and shrimp diseases have been presented in Table 14.3. Table 14.3 Fish vaccines commercially available against major infectious bacterial and viral diseases of fish. SL. no. 1. 2. 3. 4. 5. 6. 7. 8.

Name of vaccine Flavobacterium columnare vaccine Free-cell Aeromonas hydrophila vaccine Edwardsiella ictalurii vaccine Streptococcus agalactiae vaccine Streptococcus iniae vaccine Enteric red mouth (ERM) vaccine Vibrio anguillarum-Ordalii

9.

Aeromonas salmonicida bacterin Aeromonas hydrophila vaccine

10.

Edwardsiella ictaluri bacterin

Used in fish species vaccinated

Against diseases

Channel Catfish, Salmonids, and Caprs Indian Major Carps

Dropsy

Catfish Tilapia

Edwardsiellosis Streptococciosis

Tilapia Salmonids

Streptococciosis Enteric red mouth disease Vibriosis

Salmonids, Rainbow trout Salmonids Salmonids Channel Catfish, Japanese flounder

Columnaris disease

Furunculosis Motile Aeromonas Septicemia Enteric septicemia

Vaccines against viral diseases 1.

Spring viremia of carp vaccine

Common carp

2.

Koi herpes virus (KHV) Vaccine

Koi carp

3.

Nodavirus vaccine

Seabass

4.

Infectious hematopoietic necrosis virus vaccine

Salmonids

5.

Infectious pancreatic necrosis virus (IPNV) vaccine Iridoviral disease vaccine Infectious salmon anemia vaccine

Salmonids

6. 7.

Red sea bream Salmonids

Spring viremia of carp Koi herpes virus disease Viral nervous necrosis Infectious hematopoietic necrosis Infectious pancreatic necrosis Iridoviral disease Infectious salmon anemia

Adapted from Shefat, S.H.T., 2018b. Use of probiotics in shrimp aquaculture in Bangladesh. Acta Sci. Microbiol. 1 (11), 20
27.

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14.3.1.2.1.1 Reverse transcriptase polymerase chain reaction. Taq polymerase does not work on RNA samples, so PCR cannot be used to directly amplify RNA molecules. Because DNA polymerase requires a double-stranded DNA template, RNA must be transcribed into complementary cDNA prior to PCR by the enzyme reverse transcriptase (RT) because Taq has limited RT activity. This method of RNA amplification is called reverse transcriptase-polymerase chain reaction (RTPCR). Two RT enzymes commonly used in RT-PCR are moloney murine leukemia virus RT and avian myeloblastosis virus RT. Both enzymes have the same fundamental activities but differ in some characteristics, including temperature and pH optima. These enzymes are available in preoptimized RT-PCR kits. Onetube RT-PCR incorporates both the RT enzyme and a thermostable DNA polymerase in a single tube for synthesis and amplification of the target RNA sequence. This is the preferred procedure for routine analysis. Commercial RTPCR kits are available and alternatively, reagent mixes can be prepared also from separate component parts (Tang and Stratton, 2006). There are several different kinds of primers that can be used to make cDNAs, like oligo-dT will prime cDNA synthesis on all polyadenylated RNAs, random-primed cDNA synthesis gives a broad range of cDNAs, and is not limited to polyadenylated RNAs and lastly, oligo-nucleotide primers complementary to the RNA(s) of interest may be used to synthesize highly-specific cDNAs (de la Pen˜a, 2001). RT-PCR is commonly used for detection of RNA shrimp virus, which have caused havoc in aquaculture. Different researchers have used RT-PCR for detection of TiLV in fish (Behera et al., 2018; Nicholson et al., 2018) and the test was found to be very sensitive and specific. 14.3.1.2.1.2 Nested polymerase chain reaction. Nested and heminested PCR are designed to increase the sensitivity of PCR by directly reamplifying the product from a primary PCR with a second PCR. Nested PCR primers are ones that are internal to the first primer pair. The larger fragment produced by the first round of PCR is used as the template for the second PCR (de la Pen˜a, 2002). Nested PCR can also be performed with one of the first primer pair and a singlenested primer. In heminested PCR, the second round of PCR uses one of the firstround primers and one new, internal primer. The amplicon from the second round of PCR is shorter than that of the first (Tang and Stratton, 2006). The advantage of nested PCR is that the sensitivity and specificity of both DNA and RNA amplification can be dramatically increased by using this method. The specificity is particularly enhanced because this technique almost always eliminates any spurious nonspecific amplification products. Disadvantages of nested PCR include extra time and cost associated with two rounds of PCR and the increased risk of contamination incurred during transfer of first-round amplification products to a second tube. The physical separation of amplification mixtures with wax or oil and designing the second primer set with a higher annealing temperature are two variations used to reduce the potential for contamination (de la Pen˜a, 2001, 2002; Tang and Stratton, 2006). PCR technique has been applied by different researchers for detection of WSSV, MBV in shrimp, and this would be more useful in

14.3 Diseases in shrimp (shellfish)

screening of samples under field condition. Besides detection of shrimp viruses, PCR has been used for detection of bacterial pathogens of public health importance in fish and fish products indicating its scope of application in food quality control. 14.3.1.2.1.3 Multiplex polymerase chain reaction. The detection of multiple pathogens in a single test and its implementation in routine disease monitoring procedures constitute a step further in the development of techniques for rapid, specific, and effective diagnosis (Chapela et al., 2018). In multiplex PCR, also called mPCR, two or more unique DNA sequences in the same specimen are amplified simultaneously. Primers are designed so that each amplification product is a unique size, has a unique melting temperature, or unique probe binding sequence. This allows the detection and identification of different microorganisms in the same specimen. Primers used in multiplex reactions must be designed carefully to have similar annealing temperatures and to lack complementarity to avoid dimerization (Altinok et al., 2008; Tang and Stratton, 2006). Multiplex PCR has the potential to produce considerable savings of time and effort within the laboratory without compromising test utility. Since its introduction, multiplex PCR has been successfully applied in many areas of nucleic acid diagnostics, including gene deletion analysis. In the field of infectious diseases, the technique has been shown to be a valuable method for identification of viruses, bacteria, fungi, and parasites (Kaoud, 2015). Altinok et al. (2008) used multiplex PCR for the simultaneous detection of the five major fish pathogens, Aeromonas hydrophila, Aer. salmonicida subsp. salmonicida, F. columnare, Rs, and Y. ruckeri. Each of the five pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. Similarly, Del Cerro et al. (2002) developed a multiplex PCR assay based on the 16S rRNA genes for the simultaneous detection of three major fish pathogens: Aer. salmonicida, Flavobacterium psychrophilum, and Y. ruckeri. Altinok and Kurt (2003) also used a mPCR for the simultaneous detection of four major fish pathogens: Flavobacterium psychrophilum, L. garvieae, Pseudomonas aeruginosa, and P. putida. Each of the four pairs of oligonucleotide primers exclusively amplified the 16S rDNA gene of their targeted microorganism. The average detection limits for each organism amplified by mPCR were two colony-forming units (CFU) of F. psychrophilum, three CFU of L. garvieae, three CFU of P. aeruginosa, and five CFU of P. putida in mixed cultures. Multiplex PCR did not produce any nonspecific amplification products when tested against 28 related species of bacteria.

14.3.1.2.2 Real-time polymerase chain reaction Another development in PCR technology is “real-time PCR” also popularly called as qPCR, where monitoring DNA amplification is done in real-time mode through monitoring of fluorescence at different times. This technique is the most practical, because it does not require time-consuming post-PCR

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manipulation and gel electrophoresis for detection of amplified product. Realtime PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon accumulated by every PCR cycle. In initial cycles the fluorescence is too low to be distinguishable from the background. However, the point at which the fluorescence intensity increases above the detectable level corresponds proportionally to the initial number of template DNA molecules is called the quantification cycle (Cq). This allows determination of the absolute quantity of target DNA in the sample according to a calibration curve constructed of serially diluted standard samples with known concentrations or copy numbers (Kralik and Ricchi, 2017). Fluorescence detection is considered to be more sensitive than immunological or radioactive isotope detection (Tang and Stratton, 2006). qPCR offers additional speed, sensitivity, and specificity over traditional PCR methods and its application in multiplex format increases the number of pathogens that can be analyzed in the same reaction, thus reducing costs (Chapela et al., 2018). Four different principles are commonly used for real-time PCR detection. All four technologies are based on the measurement of fluorescence during the PCR. The amount of emitted fluorescence is proportional to the amount of PCR product and enables the monitoring of the PCR reaction. The resulting PCR curve is used to define the exponential phase of the reaction, which is a prerequisite for accurate calculation of the initial copy number at the beginning of the reaction (Klein, 2002). The simplest and cheapest principle is based on intercalation of doublestranded DNA-binding dyes that emit fluorescent light when intercalated into double-stranded DNA (dsDNA). The light unit of the fluorescence signal is proportional to the amount of all ds-DNA present in the reaction (Klein, 2002). SYBR green is a fluorogenic minor groove binding dye that exhibits little fluorescence when in solution but emits a strong fluorescent signal upon binding to double-stranded DNA (Tang and Stratton, 2006). This technology can be easily applied to already established PCR assays. SYBR green I is also lower in cost compared with other intercalating agents on the market and is easy to use. However, since these dyes do not make a distinction between the various dsDNA molecules in a PCR reaction, the production of nonspecific amplicons must be prevented. The other three principles are based on the introduction of an additional fluorescence-labeled oligonucleotide. Sufficient amounts of fluorescence are only released either after cleavage of the probe (hydrolysis probes like TaqMan Probe) or during hybridization of one (molecular beacon) or two (hybridization probes) oligonucleotides to the amplicon. Introduction of the additional probe increases the specificity of the quantified PCR product and allows the development of multiplex reactions (Klein, 2002). TaqMan is a homogenous PCR test that uses a fluorescence resonance energy transfer probe typically consisting of a green fluorescent “reporter” dye at the 50 -end and an orange “quencher” dye at the 30 -end. When the probe anneals to a complementary strand of an amplicon during PCR, Taq polymerase cleaves the probe during extension of one of the primers, and the

14.3 Diseases in shrimp (shellfish)

dye molecules are displaced and separated. The electronically excited reporter dye is no longer suppressed by the quencher dye, and the significant increase in green emission can be monitored by a fluorescence detector. The intensity of the green fluorescence directly correlates with the concentration of PCR product in the reaction. The TaqMan probe is monitored with a blue light-emitting diode as an excitation source and two photodiode detectors with band-pass filters centered at 530 and 590 nm wavelength. Molecular beacons are stem-loop (hairpin) shaped hybridization probes with a fluorescent dye and a quencher dye on the opposite extremities brought to their proximity by the complementary stem. The commonly used fluorescent dyes are FAM, TAMRA, TET, and ROX paired with a quenching dye, typically DABCYL. Molecular beacons are suitable for mutation analysis and single nucleotide polymorphism detection when specific mutations are known (Tang and Stratton, 2006). qPCR has become preferred method for validating results obtained from array analyses and other techniques that evaluate gene expression changes. The sensitivity and specificity achieved in a well-designed qPCR make it an ideal tool for use in the surveillance and monitoring of covert infections (Altinok and Kurt, 2003). qPCR assays are also used to determine the presence of specific genes and alleles, for example, typing of strains and isolates, antimicrobial resistance profiling, toxin production, etc., (Klein, 2002). However, the mere presence of genes responsible for resistance to antimicrobial compounds or fungal toxin production does not automatically mean their expression or production. Therefore although qPCR-based typing tests are faster, their results should be correlated with phenotypic and biochemical tests (Kralik and Ricchi, 2017). Sebastiao et al. (2018) used qPCR for the diagnosis of Aeromonas hydrophila infections in fish, targeting an adhesin gene (ahaI) based on SYBR green I. The assay had 100% specificity and the sensitivity was 1.9 log gene copies of detection for total DNA from tissue. ´ lvarez et al. (2016) used SYBR green I real-time PCR assay for speFerna´ndez-A cific identification of the fish pathogen Aer. salmonicida subsp. salmonicida. The specificity test proved that 100% (40/40) of the Aer. salmonicida subsp. salmonicida strains tested showed a positive amplification. Nicholson et al. (2018) detected tilapia lake virus using RT-PCR and SYBRgGreen RT-qPCR. The use of RT-qPCR for the detection of viruses is advantageous because of its quantitative nature, high sensitivity and specificity. Jansson et al. (2008) developed qPCR for detection of Rs, the causative agent of BKD, which is a serious threat to salmon in aquaculture. The probe was labeled with the fluorescent reporter dye, 6-carboxyfluorescein at the 50 end and with the black hole quencher (BHQ-1) at the 30 end. The PCR was based on detection of unique parts of the 16S rRNA gene and DNA equivalent to 1
10 Rs genomes could be detected per reaction. No cross-reactivity with other fish pathogenic or related bacteria could be demonstrated. Keeling et al. (2013) used molecular beacon based real-time PCR assay for detection of the vapA (surface array protein) gene in the fish pathogen, Aer. salmonicida. The assay showed 100% analytical specificity and analytical sensitivities of 5 6 0 fg (DNA).

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14.3.1.2.3 Hybridization techniques Hybridization method is a promising tool for rapid and reliable detection and identification of target agent. The term “hybridization” refers to the chemical reaction between the probe and the DNA or RNA to be detected. The assay is based on a direct hybridization of labeled (e.g., isotopes, fluorophores) oligonucleotide probes with specific DNA/RNA in sample. The oligonucleotide probes used to identify bacteria are usually short DNA molecules, between 15 and 25 nucleotides long. The shorter the probe, the lower the probe can tolerate mismatches. A variety of labeling and detection systems are available for nucleic acid probes. Radioisotopes (35S, 32P, or 125I) were earlier had great demand and use in labeling the DNA probes but health safety has been a great issue. Hence, other labeling methods have become increasingly popular. These include labeling with a variety of haptens such as biotin or digoxygenin and detection by antibody binding coupled with fluorescent, chemiluminescent, or colorimetric detection methods. The use of fluorescent labeled probe is ideal as it usually produces strong signal and less background. Probes can be designed on different phylogenetic levels, specific for domain, phylum, family, genus, or species (Tang and Stratton, 2006). If hybridization is performed on actual tissue sections, cells, or isolated chromosomes in order to detect the site where the DNA or RNA is located, it is said to be done “in situ.” By contrast, “in vitro” hybridization takes place in a test tube or other apparatus, and is used to determine sequence similarity of two nucleotide segments (Kaoud, 2015). ISH combines the specificity and sensitivity of nucleic acid hybridization with the ability to obtain histological and/ or cytological information. In ISH, digoxigenin-labeled probes are detected enzymatically with antidigoxigenin antibodies conjugated with alkaline phosphatase or horseradish peroxidase. These enzymes convert soluble substrates into insoluble precipitates that appear as dark, localized cellular or subcellular staining. Biotin is another popular nonisotopic label that can be detected with enzyme conjugates of avidin, streptavidin, or antibiotin antibodies (Tang and Stratton, 2006). Fluorescence in situ hybridization, or FISH, is a method used to label cells or chromosomes according to the sequences of nucleic acids contained within them. In microbiology, the nucleic acid that is labeled as RNA or DNA of the ribosomes and the target is usually whole cells. The process works by taking fluorescently labeled pieces of DNA or RNA called probes that are around 20 nucleotides in length. The probes are incubated in the presence of cells under appropriate conditions to permit specific hybridization of probe to target nucleic acid. Cells types that contain ribosomes with complementary RNA sequences become labeled by the binding of the fluorescent probe in situ. These labeled cells can then be visualized by flow cytometric or fluorescence microscopy (Altinok and Kurt, 2003; Arun et al., 2018). Colony hybridization is another hybridization-based method using labeled polynucleotide probes complementary to a unique sequence of DNA of the suspected pathogen. The method is used in human medicine on pathogens isolated

14.3 Diseases in shrimp (shellfish)

from clinical samples by culture. The resulting bacterial colonies are lysed, and the DNA is denatured and fixed onto an inert support, which is then subsequently hybridized with the DNA probe. The method allows specific-pathogen DNA to be detected in mixed cultures of bacteria and there is no need to isolate the pathogen DNA before performing the assay. However, the method is time consuming and the method can only be performed on samples where it has been possible to culture the microorganism (Adams and Thompson, 2011). A very important factor that influences the sensitivity and specificity of probe hybridization is hybridization stringency. This is the only condition that can be adjusted during the reaction, which defines the number of mismatches that can be tolerated in a hybrid molecule. Optimizing stringency is the key for successful hybridization assay. At high stringency, mismatches are rare. An overly stringent reaction may decrease the sensitivity of the assay due to well-matched hybrids may be disrupted. A less stringent reaction may detect unwanted, nonspecific reaction. Stringency can be easily adjusted by varying the washing conditions. Stringency is increased by increasing temperature and formamide concentration or lowering salt concentration. Most commercial hybridization assays have been optimized, however, each laboratory may still need to work out its own hybridization/washing conditions (Tang and Stratton, 2006). Commercially-available molecular probes have been developed for detection of shrimp viruses like white spot, SEMBV, MBV, TSV, HPV, YHV,IHHNV, and type-A baculovirus (FAO and NACA, 2001). These probes can be designed to be highly specific, thereby allowing more accurate identification of pathogens than was possible with many nonmolecular techniques. This clearly helps with differentiation between significant and closely related infectious agents.

14.3.1.2.4 Loop-mediated isothermal amplification Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions. This technique has been used by a number of authors to detect bacterial, parasitic, and viral fish pathogens. When combined with reverse transcription, this method can also amplify RNA sequences with high efficiency (Kaoud, 2015). The method relies on the autocycling strand displacement DNA synthesis, using Bst DNA polymerase and a set of at least four specially designed primers (two inner and two outer primers) to recognize a total of six distinct sequences on the template DNA (Notomi et al., 2000). The reaction is carried out at isothermal condition, as the denaturation of strands takes place by strand displacement. In the initial stages of LAMP reaction all four primers are involved, however, in the latter cycling reaction only the inner primers are used for strand displacement DNA synthesis. The LAMP reaction is initiated by an inner primer containing sequences of sense and antisense strands of the target DNA. This is followed by the release of a single-stranded DNA through the priming by an outer primer. This single-stranded DNA will serve as a template for DNA synthesis primed by the second inner and outer primers that can hybridize

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at the other end of the target. This process will result in the formation of a stemloop DNA structure. In the subsequent step of LAMP cycling, one inner primer will hybridize to the loop on the product and initiate strand displacement DNA synthesis which will result in the original stem-loop DNA and a new stem-loop. Cycling continues for a period of approximately 1 hour and results in the accumulation of 109 copies the target. The final products of the reaction are stem-loop DNA with several inverted repeats of the target and cauliflower-like structures with multiple loops. (Arun et al., 2018). The reaction time can be reduced using two further primers. Products of LAMP amplification can be visualized by eye with the addition of SYBR green I to the mixture, changing from orange to green in color if the reaction is positive, or can be detected by photometry for turbidity caused by increasing the quantity of magnesium pyrophosphate in solution. Some commercial LAMP kits use an enzyme substrate system to visualize the reaction on a membrane (Adams and Thompson, 2011). The amplification of the target gene is completed within 1 hour at temperatures ranging from 60 C to 65 C (Notomi et al., 2000; Mori et al., 2001). A large amount of product is formed, due to the strand displacement activity of Bst polymerase enzyme. Because of this property, identification of a positive reaction does not require any special processing or electrophoresis (Mori et al., 2001; Rathore, 2016). Several methods can be used to detect positive LAMP reactions. The results can be interpreted with naked eye due to turbidity. Because of this feature it can be easily applied in the field as a diagnostic technique and a semiskilled person can interpret the results. Amplified products can be visualized in the presence of fluorescent intercalating dye viz; SYBR Green I, calcein, ethidium bromide, picogreen, propidium iodide, hydroxyl naphthol blue, etc. by illuminating with a UV lamp or daylight. LAMP products electrophoresed on the agarose gel can be stained with ethidium bromide and visualized on UV light that appears as ladder like pattern. Turbidity can be recorded or monitored in real-time’ in the form of O.D. units at 400 nm every 6 seconds by using a Loopamp Realtime turbidmeter, that is comparatively inexpensive than real-time PCR machine (Rathore, 2016). It is faster and more sensitive than conventional PCR, and capable of detecting as few as six copies of DNA in the reaction mixture (Notomi et al., 2000). The complete LAMP procedure can be performed in 90 minutes and as it is carried out under isothermal conditions, it can be performed without the use of a thermocycler, making it suitable as a field test if a small oven is available. A number of LAMP kits are now available on the market for aquaculture. Soliman and El-Matbouli (2006) used one step reverse transcription LAMP (RT-LAMP) assay for detection of VHS. A set of six primers were designed, based on the G-protein sequence of the VHS virus serotypes. The assay was optimized to amplify VHS RNA by incubation at 63 C for only 1 hour, and required only a simple water bath or heating block to provide a constant temperature of 63 C. RT-LAMP amplification products were detected by visual inspection using SYBR green I stain and had a ladder-like appearance when electrophoresed on an agarose gel. The detection limit of the RT-LAMP assay was found to be similar

14.3 Diseases in shrimp (shellfish)

to the commonly used RT-PCR method and both methods detected VHS RNA at a dilution of 106. LAMP has high specificity and sensitivity. Its specificity is due to the four primers which bind to six different regions of the target DNA. It provides high amplification efficiency, with DNA being amplified 109
1010 times in 60 minutes. The high amplification efficiency of LAMP is attributed to no time loss of thermal change because of its isothermal reaction. However there are some disadvantages associated with LAMP assay like chances of carry over contamination is higher in this method. Complicated primer design and restricted availability of reagents are some of the problems to undertake LAMP test under field conditions (Rathore, 2016).

14.3.1.2.5 Microarrays Microarrays are an extension of traditional blotting technique, where a labeled probe molecule is hybridized to target DNA or RNA attached to a membrane. However, this process is reversed in microarray analysis with the probe attached to the support substrate, usually a nonporous solid surface, and the labeled DNA or RNA then hybridized to the probe. The major advantage of this approach is that the sample can be screened with thousands of probes simultaneously. This technology allows, in one assay, for simultaneous assessment of the expression rate of thousands of genes in a particular sample. Gene expression profiling using DNA microarrays holds great promise for the future of molecular diagnostics. In diagnostics, microarrays has been shown to be a valuable method for identification and diagnosis of fish diseases caused by viruses, bacteria, fungi and protozoa in one step (Kaoud, 2015). Arrays with potential application to diagnostic clinical research can be divided into at least four major categories based on what genes are represented on the array: (1) phylogenetic oligonucleotide arrays, which are designed based on a conserved marker such as the 16S rRNA gene and are used to detect specific organisms and compare the relatedness of microbial communities; (2) functional gene arrays, which are designed for key functional genes involved in various physiological processes, such as antibiotic resistance, and provide information on the genes and microbial populations involved with these processes; (3) community genome arrays , which contain the whole genomic DNA of cultured microorganisms and can describe an isolate or community based on its relationship to these cultivated organisms; and (4) metagenomic arrays, which contain probes produced directly from environmental DNA itself and can be a potentially powerful technique because, unlike the other arrays, they can applied with no prior sequence knowledge of the community (Tang and Stratton, 2006). There are a number of ways of using DNA microarrays for the detection of unique DNA (or RNA) sequences. One method is to fluorescently label all the DNA sequences in the test sample. The sample DNA that hybridizes to a specific location on the microarray can be detected by fluorescent array detection and the data analyzed by computer programs. Often more practical is to use competitive hybridization in which the test sample competes for hybridization to the tethered oligonucleotide, on the chip, with a fluorescent labeled competitor

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oligonucleotide. When the test DNA is perfectly complimentary to the tethered oligonucleotide, it will hybridize to the chip. When the test DNA is not perfectly complementary to the tethered oligonucleotide, the fluorescent labeled competitor oligonucleotide will bind to the tethered oligonucleotide on the chip and displace the test DNA. A fluorescent microarray detector and computer program can then analyze the fluorescent array for the presence or absence of the species/strain specific DNA sequence. Compared with traditional nucleic acid hybridization with membranes, microarrays offer the additional advantages of high density, high sensitivity, rapid detection, lower cost, automation, and low background levels. Microarrays may provide a better option for largescale diagnostic testing and can survey a sample for a multitude of sequences simultaneously. Since most of the pathogens genetic sequences are available in the GenBank, oligonucleotide probes complementary to all pathogens can be made and inserted into microarray so that variety of microbes could be detected by a single microarray chip. Warsen et al. (2004) developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides. Microarray probes (22to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silanederivatized glass slides using a robotic arrayer. PCR products (199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55 C) to the microarray. Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546. This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens. Similarly, Cao et al. (2011) developed a microarray for the simultaneous detection and identification of diverse putative pathogens often associated with fishery products by targeting specific genes of Listeria monocytogenes, Salmonella, Shigella, Staphylococcus aureus, Streptococcus pyogenes, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, and Yersinia enterocolitica and the 16S-23S rRNA gene internal transcribed spacer region of Proteus mirabilis and Proteus vulgaris. The microarray contained 26 specific probes and was tested against a total of 123 target bacterial strains that included 55 representative strains, 68 clinical isolates, and 45 strains of other bacterial species that belonged to 8 genera and 34 species, and it was shown to be specific and reproducible. A detection sensitivity of 10 ng DNA or 10 CFU/mL for pure cultures of each target organism demonstrated that the assay was highly sensitive and reproducible. Mock and real fishery product samples were tested by the microarray, and the accuracy was 100%. The DNA microarray method was found to be is specific, sensitive, and reliable and has several advantages over traditional methods of bacterial culture and antiserum agglutination assays

14.3.1.3 Matrix-assisted laser desorption/ionization-time of flight mass spectrometry Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is a technology increasingly used in diagnostic identification

14.3 Diseases in shrimp (shellfish)

of microorganisms. MALDI-TOF mass spectrometry generates a characteristic spectrum, called a peptide mass fingerprint, formed as a result of the presence of up to 2,000 proteins found in a unique pattern in each organism. This approach identifies species in a microbial community and even differentiates strains based on molecular signatures (e.g., rRNA). A characteristic spectrum is recorded representing a specific fingerprint for each species/strain It has been described as “a revolution in clinical microbial identification” (Pomerleau-Normandin et al., 2018). MALDI-TOF-MS can be applied to biological samples with minimal preparation. The prepared sample is mixed with a low molecular weight compound that strongly absorbs laser light but is stable in the presence of biological samples. The dried matrix-sample is then exposed to multiple pulses of an ultraviolet laser, which causes the matrix to sublime and the sample to ionize. The charged ions are then accelerated under an electrical field towards a detector, and the time of flight under acceleration is used to calculate the mass to charge ratio (m/z) of each individual peak. The resulting spectrum can then be algorithmically matched against a database of reference spectra. Microbial identifications may be performed directly from a single colony smeared directly onto the target plate or disposable slide, an aliquot of microbial suspension, or following cell lysis and protein extraction of an aliquot of culture broth or a suspended colony or colonies (CADTH, 2015). In general,  ES ionization produces multiply charged ions of an analyte, and this more complex spectral pattern requires deconvolution into the simpler singly charged pattern prior to interpretation. An advantage of ES ionization, however, is that the production of multiply charged ions for large biomolecules results in a lowering of the mass range of the spectrum to typically 2000 Da, because for a doubly charged ion, where z 5 2, the mass of the observed spectral peak (m/z) is halved. The mass range detected is then compatible with most types of detectors. Preparation of the sample, however, requires solubilization in a suitably volatile solvent together with the removal of salts/debris that might block the nebulizer (Dare, 2006). The diagnostic accuracy with MALDI-TOF mass spectrometry against established methods of identifying microbes is high and well established, with the exception of a few organisms (depending on the system and database interrogated) that still require additional testing, and those rare organisms not well represented in the various databases in use. MALDI-TOF reduces the time from positive culture or isolate to identification by at least 24 hours in most cases, though that depends upon the organism, the system and database used, and on individual laboratory workflow modifications. There is limited direct evidence on the effect of this reduction on clinical outcomes, with no randomized controlled trial data and sparse observational evidence on improvement of outcomes such as 30-day mortality, length of hospital stay, length of  ICU stay, and recurrence and readmission. In addition, to translate the reduced time to identification to quicker and more specific therapy there must be effective communication between laboratory,  ID specialists, and treating physicians. Studies of costs are limited to cost calculations and budget impacts, without considering cost-effectiveness or system

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updates (CADTH, 2015). This technique has fast, easy and high-throughput characteristics, does not need bacterial cultivation and generates simple and easily interpretable spectra. However, clinical samples are usually rich in host proteins and normal flora, which might result in overlapping mass spectra. Moreover, MALDI-TOF MS is limited in sensitivity since a sufficient number of cells is necessary to prevent their lost in background noise. Furthermore, the initial investments and maintenance costs are very high, whereas the overall operating costs are low (Dare, 2006).

14.3.1.4 Nanotechnology and nanosensors The term nano, derived from the Greek word for dwarf, is usually combined with a noun to form words such as nanometer, nanorobot, and nanotechnology. Nanotechnology is broadly defined as systems or devices related to the features of nanometer scale (one billionth of a meter). In the last two decades, nanoscience and nanotechnology have seen a plethora of new developments in almost every field of science and technology, especially in biology and medicine. The small dimensions of this technology have led to the use of nanoarrays and nanochips as test platforms. The recent advancements in nanotechnology have led to the development of nanoparticle-based facile assays for specific detection of the bioanalytes of clinical interest (Kaoud, 2015). Nanotechnology presents a great opportunity to develop fast, accurate and cost effective diagnostics for the detection of pathogenic infectious agents. The properties observed in nanomaterials are different from those observed in the bulk (micron-size) material due to their small size (1
100 nm) and large surface area, resulting in enhanced surface reactivity, quantum confinement effects, enhanced electrical conductivity and enhanced magnetic properties, among others. Most importantly, modifications of the nanostructures’ surface can alter dramatically some of their properties. Hence, a single binding event can be potentially recorded. Because of these phenomena, multiple nanostructures have been engineered to detect particular molecular targets in biodiagnostic applications, including pathogen detection (Kaittanis et al., 2010). The surface functionalization of nanomaterials by biomolecules has led to the development of new interdisciplinary research areas like biomedical nanotechnology, nanomedicine, diagnostic devices, theranostics, contrast agents, nanobiosensors, and targeted drug delivery vehicles. Functionalities can be added to nanomaterials by interfacing them with biological molecules or structures (Shanker et al., 2014). One advantage of this technology is the potential to analyze a sample for an array of infectious agents on a single chip. Applications include the identification of specific strains or serotypes of disease agents or the differentiation of diseases caused by different viruses but with similar clinical signs (Kaoud, 2015). Another facet of nanotechnology is the use of nanoparticles to label antibodies. The labeled antibodies can then be used in various assays to identify specific pathogens, molecules or structures. Example of nanoparticle technology includes the use of gold nanoparticles, nanobarcodes, quantum dots and nanoparticle probes. Gold nanoparticles (GNPs) with unique optical properties and high surface area

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are being extensively used for facile detection of bioanalytes of interest in the samples. The colloidal solution of GNPs exhibits intense red and blue/purple colors depending on the size, shape, and degree of aggregation of nanoparticles (Kaoud, 2015; Shanker et al., 2014). The need for rapid detection and characterization of pathogenic microorganisms has led to the evolution of the biosensor. Nanotechnology has tremendous potential to enhance the performance of biosensors. A biosensor consists of two components: a molecular recognition element and a transducer. The recognition element or bioreceptor is a molecule that recognizes and specifically interacts with a target analyte. The transducer converts the interaction into a measurable signal for quantification. In a biosensor, the bioreceptor is designed to interact with the specific analyte of interest to produce an effect measurable by the transducer. High selectivity for the analyte among a matrix of other chemical or biological components is a key requirement of the bioreceptor. While the type of biomolecule used can vary widely, biosensors can be classified according to common types bioreceptor interactions involving: antibody/antigen, enzymes, nucleic acids/DNA, cellular structures/cells, or biomimetic materials (Kaoud, 2015). The use of molecular self-assembly and gold nano-particles plus EIS detection rendered a detection limit of 30 virus particles/mL for adenovirus 5 and 100 cells/mL for E. coli 0157: H7 (Lee et al., 2015). The gold nano-particle sensor surface could be selfassembled and regenerated on the electrode at least 30 times without losing analytical performance. The combination of nanotechnology and EIS is an attractive and powerful concept for future chemical and biological sensors research and integration in to lab-on-a-chip devices for field deployable sensors (Lee et al., 2015). For detection of Salmonella in water samples, a hetero-structured silicon/gold nano-rod array is fabricated by the glancing angle deposition (or GLAD) thin film method and functionalized it with anti-Salmonella antibodies and organic dye molecules. Due to the high aspect ratio nature of the silicon nanorods, dye molecules attached to the silicon nanorods produce an enhanced fluorescence upon capture and detection of Salmonella. This novel nanobiosensor could have broad appeal to the food industry, food safety inspection agencies, government agencies overseeing food safety, and researchers focused on safety and biosecurity research. In addition to above mentioned nanobiosensors, QCM based nanoimmunosensors can also be developed and modified with gold/silver nanoparticles for the detection of Salmonella using piezoelectric quartz crystal microbalance (Srivastava et al., 2014). Biosensors are frequently coupled to sophisticated instrumentation to produce highly-specific analytical tools, most of which are still in use only for research and development due to the high cost of the instrumentation, the high cost of individual samples analysis, and the need for highly trained personnel to oversee the testing.

14.3.1.5 Genotyping techniques in characterization of pathogens The advent of molecular biology has caused a significant shift in the types of approaches used to characterize and identify microbial pathogens and to devise

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disease management strategies The shortcomings of phenotypically based typing methods have led to the development of typing methods based on the microbial genotype or DNA sequence, which minimize problems with typeability and reproducibility and, in some cases, enable the establishment of large databases of characterized organisms (Olive and Bean, 1999). Genotyping methods are less subject to natural variations, although they can be affected by insertions or deletions of DNA into the chromosome or random mutations that can delete or create RE sites or the gain or loss of extrachromosomal DNA (Tenover et al.,1997). DNA-based bacterial characterization methods can be divided roughly into PCR amplification-dependent and independent genomic analysis approaches. A reproducible method is one that yields the same result upon repeat testing of a bacterial strain. This is very important in the context of epidemiological study, this means the same strain recovered from epidemiologically linked patients will give the identical or similar typing results. The discriminatory power of a technique refers to its ability to differentiate among epidemiologically unrelated isolates. Traditional phenotyping methods such as antibiogram typing, serotyping and biotyping usually show lower discriminatory power than molecular method (Tenover et al.,1997). Along with considerations related to a particular method’s ease of interpretation, its ease of use is also important. The technical difficulty, cost, and time to obtain a result must also be evaluated in assessing the utility of a particular typing method (Olive, and Bean, 1999).

14.3.1.5.1 Pulse field gel electrophoresis The predominant technique used for strain characterization has been pulsed-field gel electrophoresis (PFGE). PFGE was first developed in the early 1980s, to genotype microorganisms by electrophoretic separation of chromosomal DNA by molecular weight. Over the years, this technology has proved to be a powerful tool used alone or in conjunction with restriction endonuclease digestion of the DNA in order to understand the evolution of antimicrobial resistance generated within a single clone and to determine genetic relatedness among microbial strains in industrial and agricultural settings, as well as health care associated epidemiologic investigations (Tang and Stratton, 2006). Principle of Pulsed-field gel electrophoresis (PFGE) was first described in 1984 as a tool for examining the chromosomal DNA of eukaryotic organisms (Schwartz and Cantor, 1984). Subsequently PFGE has proven to be a highly effective molecular technique for many different bacterial species (Tenover et al., 1997). In this method, the bacterial genome which typically is 2000
5000 kb in sizes digested with a restriction enzyme that has relatively few recognition sites and thus generated approximately 10
30 restriction fragments ranging from 10 to 800 kb. These fragments can be resolved as a part of distinct bands by PFGE, using specially designed chamber that positions the agarose gel between three sets of electrodes that form a hexagon around the gel. PFGE is often considered the “gold standard” of molecular typing methods and has been applied successfully to a wide range bacterial species. PFGE has proven to be superior to most other methods for biochemical and

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molecular typing. It is highly discriminatory and superior to most methods for analysis. PFGE was also more discriminatory than repetitive element sequencebased PCR (Rep-PCR) for differentiating strains of many bacteria (Olive, and Bean, 1999). With the aid of the computerized gel scanning and analysis software, it is possible to create data banks of PFGE patterns for all organisms, enabling the creation of reference databases to which any new strain could be compared for identifying its phylogenetic relationship to other similar strains (Olive, and Bean, 1999). PFGE has been widely used in genetic and epidemiological analyses of different pathogens, including Gram-positive and Gram-negative bacteria and fungi.

14.3.1.5.2 Polymerase chain reaction -based strain typing techniques PCR-based strain typing techniques are designed to generate multiple bands that provide a unique fingerprint for a particular species or strain of microorganism (Olive and Bean, 1999). Unlike diagnostic tests that determine presence or absence of a microorganism (or its nucleic acid) in a specimen, these procedures are used to differentiate epidemiologically unrelated organisms at the species or subspecies level. They must generally produce multiple DNA bands to provide sufficient discrimination power, and these banding patterns must be reproducible run-to-run and among isolates of the same predefined group while clearly distinguishing isolates that epidemiologically or phenotypically fall outside of that group (Tang and Stratton, 2006). 14.3.1.5.2.1 Arbitrarily primed - polymerase chain reaction and random amplified polymorphic DNA. Arbitrarily primed PCR (AP-PCR) or Random amplified polymorphic DNA (RAPD) are methods of creating genomic fingerprints from species, even if little is known about the target sequence to be amplified. RAPD is one of the many modifications of PCR principle, which can be used for molecular characterization of microbes, pants and animals (Oakey et al., 1998). RAPD was described simultaneously by Welsh and McClelland (1990) and Williams et al. (1990) and this technique is best suited for molecular genetic characterization of the microbes. The band pattern represents a “genetic fingerprint” characterizing a particular bacterial strain (Welsh and McClelland, 1990). RAPD fingerprinting allows detection of DNA polymorphism by randomly amplifying multiple regions of the genome through PCR using arbitrary primers designed independently of the target DNA sequence (Lawrence et al., 1993). Strainspecific arrays of amplicons (fingerprints) are generated by PCR amplification using arbitrary, or random sequence oligonucleotides that are often less than 10 nucleotides in length, and low-temperature annealing. A single primer is often used, because it will anneal in both orientations. Detectable PCR product is generated when the primers anneal at the proper orientation and within a reasonable distance of one another (Tang and Stratton, 2006). These fingerprints have been used for typing and identification of bacteria and increasingly for the study of genetic relationship between strains and species of microorganisms, plants and animals (Oakey et al., 1998).

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The technically demanding method of RAPD has been applied to the study of crayfish plague fungus, Astacus astaci. RAPD uses a single primer in lowstringency polymerase chain reactions. Random binding of primers results in different sizes of fragments from samples with non-identical DNA. Application of the RAPD technique grouped different isolates of the fungus and provides the means to carry out epidemiological investigations. The method has also been used to examine another Aphanomyces species that has resulted in serious losses in both farmed and wild fish in Asia. Other fish pathogens have been studied using RAPD, but problems with reproducibility and risks of contamination render the method unsuitable as a stand-alone method of diagnosis. However, RAPD can be a useful as a first step in the development of specific primers or probes and has been used in such a way in the study of bacteria (Kaoud, 2015). Vogel et al. (2000) observed that RAPD analysis had the highest discriminatory capacity for typing E. coli isolates. RAPD analysis, ribotyping and serotyping could all be used for assessment of strain relationships. Among the 32 epidemiologically unrelated E. coli isolates, 29 types were distinguished by RAPD analysis, 25 by ribotyping and 27 by serotyping. In RAPD a single primer has mostly not proven sufficient to yield band patterns complex enough to permit the separation of different E. coli strains. A solution has been to run a series of RAPD analyses with different primers and combine the patterns but this reduces the speed and cost effectiveness of the method. Similarly, Hopkins and Hilton (2001), used RAPD analysis using two or more primers has been reported to provide additional discriminatory ability over one primer used individually. This may be of particular application in epidemiological typing of clonal organisms, such as Shiga toxinproducing E. coli O157, where strain differentiation can be difficult. Using four arbitrary primers individually, and in all possible permutations, E. coli O157 isolates and other arbitrarily chosen E. coli strains were typed using RAPD analysis. 14.3.1.5.2.2 Amplified fragment length polymorphism assays. Amplified Fragment Length Polymorphism (AFLP) technology is a technique for fingerprinting genomic DNA. AFLP technology is based on classical, hybridization based fingerprinting and PCR based fingerprinting. It is based on the selective amplification of a subset of genomic restriction fragments using PCR. It is based on the selective amplification of a subset of genomic restriction fragments using PCR. The AFLP technology was developed by KeyGene in the early 1990s and has become one of the most popular genetic fingerprinting technologies worldwide. AFLP can be used for typing prokaryotes and eukaryotes. Selected markers are amplified in a PCR, which makes AFLP an easy and fast tool for strain identification in agriculture, botany, microbiology, and animal breeding. AFLP involves the restriction of genomic DNA, followed by ligation of adapters or linkers containing the restriction sites to the ends of the DNA fragments. The linkers and the adjacent restriction site serve as primer binding sites for subsequent amplification of the restriction fragments by PCR. Selective nucleotides extending into the restriction fragments are added to the 30 ends of the PCR primers such that only a subset of the restriction fragments are recognized. Only restriction fragments in

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which the nucleotides flanking the restriction site match the selective nucleotides will be amplified. The amplified fragments are visualized by means of autoradiography, phosphoimaging, or other methods. Like AP-PCR and RAPD, AFLP can be applied to organisms without previous knowledge of genomic sequence (Tang and Stratton, 2006). Originally applied to the characterization of plant genomes, AFLP has been applied to bacterial typing (Janssen et al., 1997). A major advantage of the technology is the high marker density that can be obtained. A typical AFLP fingerprint contains between 50 and 100 amplified fragments. The frequency with which AFLP markers are detected depends on the level of sequence polymorphism between the tested DNA samples. The application of the AFLP technology requires no prior sequence information. Studies to date have demonstrated that AFLP is reproducible and has good ability to differentiate clonally derived strains (Janssen et al., 1997; Olive, and Bean, 1999). The differentiation power of AFLP appears to be greater than that of PCR-based ribotyping (Olive, and Bean, 1999). The reproducibility of the banding patterns for a given strain facilitates storing patterns in databases for use in identifying new bacterial strains. This combined with strong discriminatory power may make AFLP attractive to those laboratories performing frequent epidemiological studies such that a DNA sequencer becomes cost-effective. 14.3.1.5.2.3 Enterobacterial repetitive intergenic consensus - polymerase chain reaction, repetitive element - polymerase chain reaction, BOX- polymerase chain reaction, insertion sequence - polymerase chain reaction, and variable number tandem repeat - polymerase chain reaction. Enterobacterial repetitive intergenic consensus (ERIC)-PCR, repetitive element (Rep)-PCR, insertion sequence (IS)-PCR, and variable number tandem repeat (VNTR)-PCR are examples of PCR-based typing methods that target repetitive, conserved sequences found in bacteria and, in some cases, fungi. Versalovic et al. (1991) described the presence of repetitive sequences in a wide range of bacterial species and demonstrated their use to directly fingerprint bacterial genomes. Specific repetitive sequences include the 124
127 base-pair ERIC sequence, the 154 base pair BOX sequence, and the 35
40 base-pair repetitive extragenic palindromic sequence. These sequences are located intergenically throughout the chromosome. Some repetitive sequences translocate to new locations in the genome and are called transposons or insertion sequences. Some ISs are species-specific, whereas others have no species restriction. VNTR’s are repeated sequences of non-coding DNA. Whether ERIC, IS, VNTR, or other repetitive element or sequence, the basis of the strain typing is the same. The ability of repetitive element
based PCR methods to distinguish unrelated strains or species is based on the random distribution of elements within the genome and the time required for these to become established. Three primer sets are commonly used for rep-PCR genomic fingerprinting analysis, corresponding to REP, ERIC, and BOX sequences. The protocols are referred to as REP-PCR, ERIC-PCR, and BOX-PCR, respectively, and rep-PCR, in general. The primers are designed to amplify intervening DNA between two

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adjacent repetitive elements (Louws et al., 1999). ERIC sequences are 126-bp elements which contain a highly conserved central inverted repeat and are located in extragenic regions of the bacterial genome. They have been defined primarily based on sequence data obtained from E. coli and Salmonella typhimurium. This PCR uses primers to match short consensus repetitive sequences. A complex array of 10
30 or more PCR fragments is generated per genome, ranging in size from less than 200 bp to more than 6 kb. Rep-PCR has been extensively used to identify pathogens, to differentiate strains, and to assess the genetic diversity of plant pathogens. Recent reviews have provided detailed protocols and applications of rep-PCR including applications to medical and environmental microbiology. Each primer set (REP, ERIC and BOX) is useful to fingerprint diverse bacteria, including Gram-negative and Gram-positive bacteria, as well as plant-associated actinomycetes (Louws et al., 1999). 14.3.1.5.2.4 Ribotyping. Rationales for the application of ribotype-based differentiation of independent isolates within a species have included taxonomic classification, epidemiological tracking, geographical distribution and population biology and phylogeny (Bouchet et al., 2008). Because the 16S rRNA gene is the most conserved of the three rRNA genes, 16S rRNA gene sequencing has been established as the “gold standard” for identification and taxonomic classification of bacterial species. As in the eukaryotes, the prokaryotic rRNA genes contain highly conserved sequences. The potential utility of conserved regions to identify or amplify the rRNA genes, followed by exploitation of more variable regions of the genes or spacers to detect or identify bacteria that may be difficult or even impossible to culture has long been recognized. The rRNA genes have been used in PCR assays for Renibacterium salmoninaru, Aer. salmonicida, Y. ruckeri, Vibrio anguillarum, Flavobacterium, and many other fish pathogens (Altinok and Kurt, 2003). Knowledge of intraspecies conservation of the 16S rRNA gene sequence and basic 16S-23S-5S ribosomal operon structure led Grimont and Grimont (1986) to the first insights into its usefulness in developing ribotyping for bacterial classification. Different variants of ribotyping are used. In RFLP-Ribotyping, genes coding for rRNA uses a labeled probe containing 16S or 23S or both 16 and 23 ribosomal cDNA. Before hybridization the DNA is digested with BamHI, EcoRV, PvuII or NarI and transferred on a membrane by Southern blot. The hybridization patterns or ribotypes produced by hybridization of probe to different fragments of DNA digested allow to do the differentiation between the bacterial species. For the hybridization with a specific probe, the nucleic acid is fixed on a solid support, nitrocellulose or nylon membrane, by dot blot or colony hybridization. The probe can be a single oligonucleotide or cloned and characterized DNA fragment, labeled with biotin or digoxigenin to produce a colorimetric reaction or radiolabeled. With the radiolabeled probes the amount of hybrid formed is determined by autoradiography. Fluorescence in-situ hybridization (FISH) on a microscopic slide was also used to detect and to determine the population of Bifidobacterium spp. in different samples (Ward and Roy, 2005).

14.4 DNA sequence analysis

In PCR-RFLP-Ribotyping, the specific locus to be examined is amplified with gene-specific primers and subjected to RFLP analysis. The DNA fragments are separated on an agarose or small polyacrylamide gel, and the digestion patterns are visualized following ethidium bromide staining. The 16S, 23S, and 16S
23S spacer regions have also been used as targets for locus-specific RFLP (Vila et al., 1996). In this variation of ribotyping, the ribosomal DNA is amplified and subjected to digestion with a restriction enzyme, and the DNA fragments are visualized following separation by gel electrophoresis, alleviating the need for Southern blotting. In addition to length variation, many species demonstrate high degrees of sequence variability among multiple copies of the ISR. Such variability is due, in part, to the fact that intergenic regions often encode one or two tRNAs. Ribotyping can be successfully applied for differentiation of bacterial strains that display a high degree of heterogeneity within the rRNA operons. However, the discriminatory power of locus-specific PCR is generally not as good as that of other methods, due primarily to the limited region of the genome which can be examined. It has been noted PCR-ribotyping to have poor discriminatory power in comparison to PFGE and biochemical typing methods. 14.3.1.5.2.5 Amplified ribosomal DNA restriction analysis. An alternate variation of PCR ribotyping, amplified rRNA gene restriction analysis (ARDRA) is based on PCR amplification of the 16S rRNA gene followed by restriction digestion. Jayarao et al. (1991) developed this technique to determine subspecies of Streptococcus uberis, as a means to avoid methods involving DNA hybridization or sequencing. Bacterial culture and DNA isolation are needed with the amplified ribosomal DNA restriction analysis (ARDRA) technique. The DNA is used to the PCR amplification of the totality or only a region of the 16S rRNA gene. This amplification is followed by a restriction digestion of the PCR products. The selection of restriction enzymes is important to have a clear distinction in ARDRA pattern to differentiate the larger amount of species. The digestion products are visualized under UV-light after agarose gel electrophoresis and ethidium bromide staining. Analysis and comparison of more than one restriction profile can be necessary to have a differentiation between some close species (Ward and Roy, 2005). As 16S rRNA gene sequence variation for interspecies differentiation of isolates is well established, it is not surprising that ARDRA, has proven useful for species differentiation rather than intraspecies epidemiological discrimination or phylogenic organization of independent isolates.

14.4 DNA sequence analysis All molecular genetic methods for distinguishing organism subtypes are based on differences in their DNA sequence. Thus, DNA sequencing would appear to be the best approach to differentiating subtypes. DNA sequencing was originally performed by using radioactive labels for detection of the reaction products. Current

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DNA sequencing protocols employ fluorescent nucleotides to label the DNA. The sequence is then read with an automated instrument (Olive, and Bean, 1999). There are several considerations that must be evaluated before undertaking the use of DNA sequencing for subtyping. First, for practical purposes, DNA sequencing must be directed at only a very small region of the chromosome of an organism. It is impractical to sequence multiple or large regions of the chromosome of an organism. Thus, in contrast to techniques like PFGE, Rep-PCR, or RAPD analysis, which examine the entire chromosome, DNA sequencing examines only a very small portion of the sites which can potentially vary between bacterial or fungal strains. Furthermore, the short region of DNA used must meet several criteria before it can be used for strain differentiation. The structure of the region of DNA selected must consist of a variable sequence flanked by highly conserved regions. This enables PCR amplification and typing of all members of a species. The variability within the selected sequence must be sufficient to differentiate different strains of a particular species. Unfortunately, for bacteria and fungi, few sequences meet these criteria. While the 16S rRNA genes have been used to identify new species of organisms, they show limited variability between strains of a bacterial species. The intergenic region between the 16S and 23S rRNA genes has also been used with variable results. In contrast, DNA sequencing is considered the gold standard for viral typing. The regions used for viral genotyping and detection of drug resistance mutations are in short, well-defined sequences that fulfill the criteria needed for the application of nucleic acid sequencing methodologies (Olive, and Bean, 1999).

14.5 Multilocus sequence typing analysis In long term epidemiological investigation, the relationship between the strains recovered over greater periods of time and often from a broader geographic range are studied. Several different kinds of intra-species variation are used for molecular typing using a variety of molecular tools like RAPD, RFLP, ERIC-PCR, BOX, Rep-PCR etc. and the results vary between the techniques employed and between the laboratories. Again, since highly variable genes that are targeted in these approaches are highly variable for a season and environmental pressure and thus their high rates of evolution will obscure the true relationship between isolates. Genes that are not subject to any unusual selective forces and which diversify slowly by the random accumulation of neutral variation, should provide more reliable information about the relationship between the isolates. This is the very concept of MLST for characterization and typing of microbial isolates using housekeeping genes as the target genes. MLST is actually the modified extension method of an old but reliable method called multilocus enzyme electrophoresis (MLEE) that has been used for genetic characterization of species. In MLST the alleles at multiple house keeping loci are assigned or characterized directly by nucleic acid sequencing than indirectly from the electrophoretic mobilities of their

14.6 Preventive and control measures

gene products. MLST makes use of rapid sequencing technique to uncover allelic variants in conserved genes or house keeping genes of microbes for the purpose of characterizing, sub-typing and classifying them. Internal approximately 450 bp fragments of house-keeping genes are used in MLST. For each gene fragment, every unique sequence is assigned as a different allele. Bacterial strains are therefore defined unambiguously by a string of integers or the allelic profiles or sequence types. It has been particularly useful in studying the population genetics of a variety of microbial pathogens including and their epidemiological prevalence study. MLST has been evaluated to characterize Klebsiella isolates. Amplification of seven housekeeping genes, for example, rpoB (beta-subunit of RNA polymerase), gapA (glyceraldehyde 3-phosphate dehydrogenase), pgi (phosphoglucose isomerase), tonB (periplasmic energy transducer), mdh (malate dehydrogenase), phoE (phosphorine E) and infB (translation initiation factor 2) was carried out using PCR (Mishra and Goyal, 2010) and following the specified annealing temperatures for primers given at the K. pneumoniae MLST data base (http://pubmlst.org/kpneumoniae). Each distinct sequence within a locus was assigned a unique allele number. Each nucleotide sequence was analyzed by BLASTn algorithm against the K. pneumoniae MLST database and a matching allele number in the database was searched. Nucleotide sequences having no matching allele in the MLST database were considered new and got their respective allele number and sequence type(ST) assigned by the curator of the K. pneumoniae MLST database web site. After getting the allele numbers and sequence types, cluster analysis was performed by Bionumerics software version 4.0 (Applied Maths, Martens-Latem, Belgium) using categorical coefficient. MLST analysis has indicated that in many species, recombinational replacements contribute more to clonal diversification than due to point mutation. A major advantage of this new technique is that the sequence data are unambiguous and electronically portable, allowing molecular typing of bacterial pathogens via internet (Spatt, 1999). MLST was initially evaluated using Neisseria meningitides because it provides a good example of species in which the rate of recombination is high but in which distinct clones can clearly be distinguished by MLEE. MLST has also been used for characterizing many other bacterial and fungal isolates (Mishra and Goyal, 2010). The biggest advantage of this technique over other molecular typing methods is that sequencing data are repeatable, reproducible and portable between laboratories. This has led to creation of global database for many microbes on the line of Genebank, that allows the exchange and verification of molecular typing data via internet or web among various laboratories around the world.

14.6 Preventive and control measures 14.6.1 Vaccines for fish diseases A holistic approach should be adopted for treatment that considers the interaction between the host, the environment and pathogens. Clearly, the most appropriate treatment for the disease will depend on the diagnosis determined by a fish health

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specialist and also on the specific management practices and resource of the farm. The main objectives of a good management practice is to prevent introduction of disease to the farm system, prevent propagation of existing disease agents, enhance the natural resistance of fish and produce healthy, high quality fish (Idowu et al., 2017). In most cases, healthy fish have ability to withstand considerable environmental changes and thus can resist diseases. However prophylactic care need to be taken. Vaccination is an established, proven and cost-effective method for controlling certain infectious diseases in cultured animals. Vaccines reduce the severity of disease losses, reduce the need for antibiotic use, leave no residues in the product and do not induce pathogen resistance (Subasinghe et al., 2001). An optimal vaccine must be able to induce innate mechanisms, a sufficient antibody response, induce T-cell response(s) and generate specific immune memory in the host fish species. Besides, the adverse effects of infectious diseases has also demanded the strategic development of vaccine design because indiscriminate use of antibiotics in aquaculture can make rise in problems of developing bacterial resistance, food safety hazards and environmental problems. Vaccination has become the most important, easy and effective approaches to prevent and control infectious diseases in fish. Several significant progress have been made for developing effective fish vaccines. But until now, only a few vaccines are commercially available against infectious viral and bacterial diseases. There are currently many commercially available vaccines for finfish diseases, and a few more are under development (Shefat, 2018a). In Japan, the use of injection vaccines is now widespread among maricultured animals, proved effective against bacterial (e.g., L. garvieae infection of yellowtail) and viral infections (e.g., Iridoviral infection of red sea bream) and has changed the patterns of disease occurrences (Bondad-Reantaso et al., 2005). Some of the important bacterial diseases against with fish vaccines are commercially available include Aer. salmonicida, Vibrio salmonicida, V. anguillarum, Y. ruckeri, R. salmoninarum, Flavobacterium psychrophilum, F. columnare, P. salmonis, L. garvieae, Streptococcus iniae, Photobacterium damselae subsp. Piscicida, and E. ictaluri (Haenen, 2017). Some of the vaccines commercially available against fish bacterial and viral diseases, have been presented in Table 14.3. Similarly, more than 20 different virus DNA vaccines have been developed experimentally for prophylactic use in fish targeting viruses such as rhabdoviridae, orthomyxoviridae, togaviridae, and nodaviridae. The rhabdoviridae DNA vaccines (e.g., VHSV and IHNV) have shown high levels of efficacies, whereas others have in most instances possessed moderate to low efficacies (Dalmo, 2017). Apex-IHN (Aqua Health Ltd., an affiliate of Novartis Animal Health Inc.) is the recombinant vaccine recently developed and commercialized for the protection of salmonids against IHNV (Salonius et al., 2007). Apex-IHN DNA vaccine has proved to be very successful, while other DNA vaccines against other piscine viruses are in the advanced pipeline. In 2016, the European Medicines Agency (EMA) recommended granting a marketing authorization in the EU for “Clynav,” a DNA vaccine against salmon pancreas disease (salmonid alphavirus-3)

14.8 Probiotics

(Dalmo, 2017). It is very important to identify safe host species and production of protective antigens is probably the most feasible strategy towards for low cost commercial vaccines development. Advances in genome sequencing of pathogens can accelerate the opening of opportunities to investigate new generation vaccines. To meet the challenge to develop efficacious vaccines, systems vaccinology approach using both transcriptomics, epigenetic, proteomics and metabolomics platforms together with bioinformatics may be necessary (Dalmo, 2017).

14.7 Immunostimulants The alternative technique to prevent the diseases has been proposed that the strengthening of fish immune systems through the application of immunostimulants is one of the most promising methods. Immunostimulation is one method that is gaining popularity and is considered a promising development in aquaculture. An immunostimulant is defined as a chemical, drug, stressor or action that enhances the innate or non-specific immune response by interacting directly with cells of the system activating them. Immunostimulants can be grouped under chemical agents, bacterial preparations, polysaccharides, animal or plant extracts, nutritional factors and cytokines (Barman et al., 2013). Immunostimulants were found to be effective in enhancing parameters of non-specific immunity and resistance to diseases of fish and crustaceans. Such methods, however, are still very limited, especially for shrimp, however the large number of commercial immunostimulants available on the market clearly reflects the interest in this area as an alternative method to enhance survival from disease challenges (Apines-Amar and Amar, 2015). The most proven effect of immunostimulants is to facilitate the function of phagocytic cells and increase their bactericidal and fungicidal activities. Immunostimulants can promote recovery from immunosuppressive states caused by any form of stress (Barman et al., 2013). Some commercially available immunostimulants or aquaculture use has been presented in Table 14.4. In conjunction with good health management and good husbandry practices, there is great potential for the use of vaccine technology for specific use in Asian aquaculture (Bondad-Reantaso et al., 2005).

14.8 Probiotics Probiotics are live-beneficial microorganisms which confer health benefits to the host if administered in sufficient quantity. Use of probiotics have gained interest as an alternative to the antibiotics in shrimp disease management in aquaculture (Shefat, 2018b). Fuller (1989) defined probiotics as a live microbial feed supplement which beneficially affects the host animal by improving its microbial balance. In aquaculture systems, the interaction between the microbiota and the host

345

Table 14.4 Some immunostimulant preparations being marketed for use in fish and shellfish culture. Name

Company

Contains

Method of application

Penstim

AURUM Aquaculture Ltd, United States Immundyne, United States

Beta-glucan B (1,6) branchedB (1,3) glucan from yeast

Immersion for larvae/PLs and feed ingredient for grow-out shrimp Immersion for larvae/PLs and feed ingredient for grow-out shrimp As feed ingredient

Immustim Calcium spirulan SP 604

Kelly Moorhead, Hawaii

Sulfated polysaccharide from spirulina

Alltech Inc, United States

Vitastim Laminarn

Taito and Company, Japan Pronova, Norway

Levamisole DS 1999

Janssen Pharmaceutica, Belgium International Aquaculture Biotechnologies Ltd Biotec-Mackzymal, Norway

Premix of mannan (Biomos), Cr and Se yeast, probiotics B (1,6) branchedB (1,3) glucan from fungi B (1,6) branchedB (1,3) glucan from brown algal laminariae Tetrahydro-6-phenylimidazolthiazole hydrochloride Bacterin

Macroguard Lactoferrin Selenium yeast Lysozyme Hydochloride nutri-care

As feed ingredient As feed ingredient As feed ingredient

DMV international, the Netherlands Alko, Finland

Lactoferrin from bovine milk

As feed ingredient in bath Add directly in culture medium(larval culture)
With vaccines in injection
in feed ,6
8 weeks cont. As feed ingredient

Selenium yeast

As feed ingredient

Belovo, Belgium

Lysozyme from hen’s eggs

As feed ingredient

Nutricorp Animal Bio Solutions, Nellore, Andhra Pradesh, India

Extract of herbs, vitamins, enzymes, probiotics, amino acids, and organic minerals

In Feed: Below 30 ppt salinity: 10 g/kg of feed for 5 days, Above 30 ppt salinity: 15 g/kg of feed for 5 days.

B (1,6) branchedB (1,3) glucan from yeast

CHARGERGEL

Growwel Formulation Pvt. Ltd., Hyderabad 500092, India

i-Booster

Hayagreeva Bio Organics Pvt Ltd, Kondapalli, Krishna District, Andhra Pradesh, India Advance Aqua Biotechnologies India Pvt. Ltd., Vijayawada, Andhra Pradesh, India Padmaja Laboratories Pvt. Ltd., Chinnoutapalli, Andhra Pradesh, India BIOSTADT INDIA LIMITED, Worli, Mumbai 400018, India

BIOSTIM

PHYTO PROTECT CITROMAX

1
3 D-Glucan, polysaccharides, betaine, and betaglucans, improves the functional capacity of haepatopancreas Sea herb Fucoidan, herbal extract, Agent-V (ISC1227), vitamins and minerals. Boosts immune system Feed probiotics, enzymes, amino acids, mineral mixture, and anti-oxidants, immunity by production of haemocytes, and phagocytes Herbal immune stimulant, herbal gut health improves, digestive system stimulants

As feed ingredient: 8 g/kg of feed pellets in the evening

It is a herbal immune stimulant. Best molecule for EMS protection, controls vibrio bacteria effectively

In feed, 0.5
1 gm/kg of feed

As feed ingredient 3
5 g/1 kg feed 5 days per week As feed ingredient 4
5 g/kg feed

In feed, 300 g/tonne of feed

Adapted from Barman, D., Nen, P., Mandal, S.C., Kumar, V., 2013. Immunostimulants for aquaculture health management. J. Mar. Sci. Res. Dev. 3, 134. doi:10.4172/ 2155-9910.1000134. and Mishra, S.S., Das, R., Das, B.K., Choudhary, P., Rathod, R., Giri, B.S., et al., 2017c. Status of aqua-medicines, drugs and chemicals use in India: a Survey Report. HSOA J. Aquac. Fish 1, 004.

348

CHAPTER 14 Biotechnological tools

is not only limited to the intestinal tract. Merrifield et al. (2010) defined probiotic for aquaculture as “Live, dead, or component of a microbial cell that, when administered via the feed or to the rearing water, benefits the host by improving either disease resistance, health status, growth performance, feed utilization, stress response or general vigor, which is achieved at least in part via improving the hosts or the environmental microbial balance”. The use of probiotics has opened a new era in industrialized fish farming and its use are gaining increasing scientific and commercial interest and are now quite commonplace in healthpromoting foods to therapeutic, prophylactic, and growth supplements (Verschuere et al., 2000). Probiotics are generally administered as live microbial feed supplements which affect the host animal by improving the intestinal microbial balance to optimize the presence of non-toxic species. A stable gut microflora helps the host resist pathogenic invasions, particularly via the gastrointestinal tract. Probiotics are widely used in animal husbandry but their use in aquaculture is increasing considering positive results in fish and shrimp farming in Indian aquaculture (Mishra et al., 2017a,c). Several probiotic products extracted from different species of bacteria and a yeast are commercially available for use in aquaculture in Bangladesh. Probiotics have gained popularity in shrimp aquaculture in Bangladesh as an alternative to the antibiotic in prevention of infectious diseases of fish by (Shefat, 2018b). In many cases, probiotics have been reported to significantly reduce antibiotic use in shrimp hatcheries. Suppression of proliferation of certain pathogenic bacteria (e.g., Vibrio spp.) in shrimp hatcheries has been achieved by introducing non-pathogenic strains or species of bacteria, that compete for microbial metabolite resources. Several beneficial effects have been reported to provide by probiotics in shrimp aquaculture such as increased survival rate of shrimp, improve the population density of beneficial bacterial flora, reduce concentrations of nitrogen and phosphorus, and increase yields of shrimp, and improve water quality (Shefat, 2018b; Wang et al., 2005). Different probiotic products being used in Indian aquaculture have been presented in Table 14.5. Nandi et al. (2017) evaluated the effects of Bacillus amyloliquefaciens CCF7 probiotic supplemented feed in L. rohita challenged with pathogenic strain of A. hydrophila and observed effectiveness in terms of reduction of stress and enhancement of serum protein, lysozyme, and IgM level in treated group. Vaseeharan and Ramasamy (2003) noted that probiotic treatment with ell-free extracts of Bacillus subtilis BT23 significantly reduced the average mortality rate and controlled the growth of disease causing pathogen Vibrio harveyi. A study reported that the use of probiotics significantly increased DO concentration, reduced dissolved phosphorus and total inorganic nitrogen, and chemical oxygen demand. This indicates that the addition of the commercial probiotics had a noticeable influence on water quality of shrimp ponds and shrimp production (Wang et al., 2005). Probiotics increased the specific amylase activity, increased survival rate and wet weight gain. The feed conversion ratio, specific growth rate, and final production were significantly higher in shrimp receiving the probiotic

Table 14.5 Some commercially available probiotics for use in Aquaculture in India. Sl. no.

Name

Composition

Indications

Dose

1.

Pro Marine

Probiotics fortified with vitamin C and calcium

2.

MICRO FEED SUPERGUT

Bacillus subtilis, Lactobacillus acidophilus, Bifidobactrium bifidus, and Streptococcus faecium. Contains the selective multistrain of probiotics along with sea weed extract and molasses. Probiotics, yeast, enzymes, vitamins, sea weed extract, trace minerals. Lactobacillus, Bacillus subtilis, Cerevisiae, Fungal diastase, vitamin B12, papain, pepsin Active/inactive species of Lactobacillus and growth enhancers. Active/inactive forms of Candida spp., Saccharomyces spp. and growth enhancers Probiotic strains

Removes unwanted microorganism. Accelerates encrustation Arrests the growth of pathogenic bacteria in the fish gut Limit the active of harmful pathogenic bacteria, maintain a healthy and balance gut microflora Prevents and controls the bacterial pathogens in the gut Improve FCR and resistances to disease due to favorable gut flora Enhance the proliferation of beneficial

Growth: 5 g/kg of feed twice daily. White gut: 5
10 g/kg of feed twice daily In feed: 100 g/tonne of feed -regularly 7 days in every month 10
20 mL/kg shrimp feed

3. 4. 5. 6. 7. 8. 9.

P-LACT PLUS PROBAC-G GENLAC PRO ALGUT PRO AQUAGEN PRO VIBRION

13. 14.

GEN
PRO Environ GENTECH P.S. Thiomax Ec-Plus

15.

Spark-PS

10. 11. 12.

Probiotic strains, alkaline protease and lipase, etc. Total 36 Probiotic strains probiotic mix Aqua Probiotics Probiotics and micronutrients. microorganisms incorporated on calcareous ground substance. Probiotics

Single cell protein and growth enhancer the proliferation of beneficial bacteria Enhances the ammonia-oxidizing activity

10
20 g/kg of feed for 10 days 500 g/1 tonne feed For shrimp: 3 kg/tonne feed. For Fish: 4
5 kg/tonne feed For shrimp: 2 kg/tonne feed. For fish: 3 kg/tonne feed 2
3 L/acre in 3
5 ft depth.

Efficiently reduces the harmful microbial load like Vibrio spp. Produces variety of antibacterial anti-fungal substances For eater and soil pollutant management. It helps to degradation of organic load.

250
500 g/ acre. Every 7
10.

Works in the bottom sludge layers Works in the entire water column, as well as in the bottom sludge layers Secretes various hydrolytic enzymes and digest complex organic substances

2
3 kg/acre Prawn/shrimp: 15 kg/acre,

250
500 g/acre. Every 7 days 250
500 g/acre 5 L/ha

2
3 L/ha

Adapted from Mishra, S.S., Das, R., Das, B.K., Choudhary, P., Rathod, R., Giri, B.S., et al., 2017c. Status of aqua-medicines, drugs and chemicals use in India: a Survey Report. HSOA J. Aquac. Fish 1, 004.

350

CHAPTER 14 Biotechnological tools

(Ziaei-Nejad et al., 2006). Feeding of potential probiotics showed the best feed conversion ratio, effectively reduced the occurrence of disease in shrimp by Vibrio parahaemolyticus (Balca´zar et al., 2007). Swain et al. (2009) fed the Penaeus monodon with four probiotics and concluded that the probiotic strains effectively inhibited the pathogens, increased survival rate to Vibrio harveyi, and disease resistance against Vibrio parahaemolyticus. He proved that the Streptococcus phocae and Enterococcus faecium isolated from brackish water shrimp is highly potential to control pathogenic vibriosis in shrimp culture. A LAB strain Lactobacillus plantarum MRO3.12 showed the highest efficiency in reducing Vibrio harveyi pathogen. This shows promise of probiotics application to be effective and economical, however, further refinement of administration and concentration loads required for effective pathogen suppression is required. Effective and economically-viable probiotics also require greater research into optimal strains of probiotic microorganisms and stringent evaluation under field conditions of their economic feasibility.

14.9 Therapeutics in fish diseases Although improvement of water quality, nutrition, and other general husbandry factors may be enough to improve the health of a population, chemotherapeutics are often required to ameliorate disease outbreaks. Management of fish diseases is challenging because of basic logistics, including the aquatic environment, numbers of fish, and routes of administration, and also because of the pharmacologic and regulatory complexities of chemotherapeutic usage in fish (Yanong and Francis-Floyd, 2019). While chemotherapy will perhaps remain one of the main strategies for controlling transmissible diseases in the foreseeable future, especially in finfish, there is increasing recognition of its limitations in terms of aquaculture situations, host species, and effectiveness against certain pathogen groups. In some cases, rather than providing a solution, chemotherapy may complicate health management by triggering toxicity, resistance, residues, and occasionally, public health and environmental consequences (OIE, 2000). Control methods currently rely on avoidance of pathogens through thorough disinfection of fertilized eggs and stocking materials. FDA’s Center for Veterinary Medicine has identified a number of “low regulatory priority aquaculture drugs”. These compounds have undergone review by the Food and Drug Administration and have been determined to be new animal drugs of low regulatory priority (USFDA, 2017). European Union, USFDA and Japan have notified zero level drug residues of selected antibiotic viz. chloramphenicol, furazolidine, nalidixic acid, neomycine, oxolinic acid (quinoline compound), oxy tetracycline, tetracycline, and sulphamethaxazole/trinethoprim (sulfonamide) in the imported shrimp (Aly and Albutti, 2014). The details of drugs, chemicals, and antibiotics used in Indian aquaculture have been previously

14.9 Therapeutics in fish diseases

reviewed (Mishra et al., 2017c). FDA-approved therapeutic options for fish are limited but increasing. The FDA website (www.fda.gov/AnimalVeterinary/ ResourcesforYou/AnimalHealthLiteracy/ucm213944.htm) is the best resource for basic information on the status of drugs and chemicals, particularly those intended for aquaculture use. In addition, the FDA has listed several compounds as being of “low regulatory concern.” These compounds, although not fully approved, are considered innocuous enough for use in food fish. USFDA approved drugs and chemicals permitted for use in aquaculture along with other drugs and chemicals being used in Indian aquaculture have been presented in Table 14.6.

Table 14.6 USFDA approved drugs and chemicals permitted for use in Aquaculture. Sl. no.

Active ingredient

Application method

Withdrawal time

Medicated feed Medicated feed Medicated feed

15 days

Antimicrobial (gills and skin only) Antimicrobial (gills and skin only) Herbicide antibiotic (gills and skin only)

Immersion

None

Immersion

None

Immersion Immersion

Parasiticide (gills and skin only) Parasiticide (gills and skin only) Parasiticide (gills and skin only)

Immersion

5 days in channel catfish, northern pike; 30 days in all others None

Immersion

7 days

Immersion

7 days

Parasiticide (external crustaceans)

Immersion

Not for use in food fish

Generic use

Antibiotics 1.

Florfenicol

Antibiotic

2.

Oxytetracycline dihydrate Ormetoprim sulfadimethoxine

Antibiotic

3.

Antibiotic

21 days 42 days in salmonids; 3 days in catfish

Antimicrobials—chemicals 5. 6.

Hydrogen peroxide (35%) Chloramine-T

7.

Diquat

8.

Formalin

9.

Copper sulfate (CuSO4) Potassium permanganate (KMnO4) Diflubenzuron

10

11.

(Continued)

351

352

CHAPTER 14 Biotechnological tools

Table 14.6 USFDA approved drugs and chemicals permitted for use in Aquaculture. Continued Sl. no.

Active ingredient

Generic use

Application method

Withdrawal time

Other antiseptics and sanitizers used in aquaculture in India but not in the list by USFDA 13.

14.

14.

15. 16.

17.

18

Di-decyl dimethyl ammonium chloride, di-octyl dimethyl ammonium chloride, and octyl decyl dimethyl ammonium chloride Benzalkonium chloride (BKC)

(Viranex S) Pot. monopersulphate 50% w/w containing triple salt Iodine 20% w/v (Biolin Plus) Formaldehyde: 7.5%, Glutaraldehyde: 7.5%, BKC: 5.0% (SOKRENA-WS) Didecyl dimethyl ammonium chloride 4th generation QAC (Paraclean) benzyl konium chlorides: 5.0%, Formal dehyde: 37%
41%, calcium gluconate: 5.0%

Bactericidal, fungicidal, virucidal

Immersion

None

Microbicidal and detergency properties, Disinfection and antimicrobial activity. Broad spectrum virucidal

Immersion

None

Immersion

None

Pond water sanitizers Special multi-action disinfectant for bacteria, viruses and fungi Sanitization of pond water to prevent water borne diseases of bacterial, viral, and fungal origin Reduces the microbial load in the pond. Prevent all external infections of fish

Immersion Immersion

None None

Immersion

None

Immersion

None

Adapted from Mishra S.S., Das R., Das B.K., Choudhary P., Rathod R., Giri B.S., et al., 2017c. Status of aqua-medicines, drugs and chemicals use in India: a Survey Report. HSOA J. Aquac. Fish 1, 004; USFDA, 2017. Approved Aquaculture Drugs. U.S. Food and Drug Administration. Available from: ,https://www.fda.gov/animalveterinary/developmentapprovalprocess/aquaculture/ucm132954.htm. (updated 24.05.17). and Yanong, R.P.E., Francis-Floyd, R., 2019. Therapeutic considerations in aquaculture. In: MSD Manual Veterinary Manual, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ. Available from: ,https://www.msdvetmanual.com/exotic-and-laboratoryanimals/aquaculture/therapeutic-considerations-in-aquaculture..

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14.10 Conclusion Improved rapid diagnostics are crucial for the sustainable future for aquaculture. Thus rapid and reliable diagnosis of disease and pathogens constitutes an important aspect of the disease management program, so that suitable preventive and control measures could be undertaken to control the mortality and minimize the loss. Traditional diagnostic methods tend to be costly, labor intensive, slow, and might not lead to a definitive diagnosis of disease and too much time is wasted in arriving on a conclusion so that decisive action on management could be taken. Biotechnology encompasses a wide range of approaches that can improve subsistence and commercial aquaculture production and management. Developments in biotechnology can provide many opportunities for developing new and refining existing diagnostic methods, to improve the accuracy, sensitivity, and specificity of rapid tests. There is a need to include such methods in the OIE manual as soon as the new technologies have been validated. Rapid serological tests like ELISA, latex agglutination test, FAT, andlateral flow assay have been found to be very useful for field application. Besides DNA-based diagnostic test like PCR, RTPCR, qPCR, mPCR, microarray, DNA probe- based ISH, etc. proved their worth in rapid and accurate detection of pathogens. Apart from the sensitivity and rapidity of diagnosis, principal advantage of molecular and technological diagnostic methods is in the detection of nonculturable agents. Genotyping techniques like RFLP, RAPD, ribotyping, ERIC-REP PCR, gene sequencing, and MST techniques have been quite useful in differentiation and characterization of new pathogens. These tests will be invaluable for rapid, reliable diagnosis applied to regular screening programs for aquaculture, such as sensitive broodstock and egg screening and regular monitoring of the disease status. Various vaccines and immunostimulants are now commercially available for enhancing protection in fish and shellfish against a variety of diseases, which could be of immune’s use in controlling disease occurrence in aquaculture. Recent developments in nanotechnology and nanosensors have made their way in aquaculture application in pathogen detection and control. These biotechnological tools based on cutting edge technologies in immunology, microbiology, molecular biology, and nanotechnology have potential application in addressing the fish health management, besides their use in quarantine protocols, diagnostics, immunoprophylaxsis, probiotics, bioremediation, and chemotherapeutics.

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Hopkins, K.L., Hilton, A.C., 2001. Use of multiple primers in RAPD analysis of clonal organisms provides limited improvement in discrimination. Biotechniques 30 (6), 12624. 12667. Idowu, T.A., Adedeji, A., Sogbesan, O.A., 2017. Fish disease and health management in aquaculture production. Int. J. Environ. Agric. Sci. 1 (11), 002. Janssen, P., Maquelin, K., Coopman, R., Tjerberg, I., Bouvet, P., Kerstens, K., et al., 1997. Discrimination of Acinetobacter genomic species by AFLP fingerprinting. Int. J. Syst. Bacteriol. 47, 1179
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220. Louws, F.J., Rademaker, J.L.W., de Bruijn, F.J., 1999. The three Ds of PCR-based genomic analysis of Phytobacteria: diversity, detection and disease diagnosis. Annu. Rev. Phytopathol. 37, 81
125. Merrifield, D.L., Dimitroglou, A., Foey, A., Davies, S.J., Baker, R.R., Bøgwald, J., et al., 2010. The current status and future focus of probiotic and prebiotic applications for salmonids. Aquaculture 320, 1
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Diseases Department of Veterinary Science & Microbiology, University of Arizona, Tucson. Petty, B.D., Francis-Floyd, R., 2019. Viral diseases of fish. In: MSD Manual Veterinary Manual. Merck Sharp and Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ. Available from: ,https://www.msdvetmanual.com/exotic-and-laboratory-animals/aquarium-fishes/viral-diseases-of-fish.. Pomerleau-Normandin, D., Heisz, M., Su, M., 2018. Misidentification of risk group 3/security sensitive biological agents by MALDI-TOF MS in Canada: November 2015
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09 August 2016. ICAR-CIFA, Training Manual No. 30. ICAR-Central Institute of freshwater Aquaculture, Bhubaneswar, Odisha, pp. 161
164. Rathore, G., Kumar, G., Raja Swaminathan, T., Swain, P., 2012. Koi herpes virus: a review and risk assessment of indian aquaculture. Indian J. Virol. 23 (2), 124
133. Sahoo, P.K., Goodwin, A.E., 2012. Viruses of freshwater finfish in the Asian
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29. Sahul Hameed, A.S., Yoganandhan, K., Sri Widada, J., Bonami, J.R., 2004. Studies on the occurrence of Macrobrachium rosenbergii nodavirus and extra small virus-like particles associated with white tail disease of M. rosenbergii in India by RT-PCR detection. Aquaculture 238, 127
133. Salonius, K., Simard, N., Harland, R., Ulmer, J.B., 2007. The road to licensure of a DNA vaccine. Curr. Opin. Invest. Drugs 8, 635
641. Schwartz, D.C., Cantor, C.R., 1984. Separation of yeast chromosomesized DNAs by pulsed field gradient gel electrophoresis. Cell 37, 67
75. Sebastiao, F., de Macedo Lemos, E.G., Pilarski, F., 2018. Development of an absolute quantitative real-time PCR (qPCR) for the diagnosis of Aeromonas hydrophila infections in fish. Acta Sci. Microbiol. 1.4, 23
29. Shanker, R., Singh, G., Jyoti, A., Dwivedi, P.D., Singh, S.P., 2014. Nanotechnology and detection of microbial pathogens. In: Verma, A.S., Singh, A. (Eds.), Animal Biotechnology. Models in Discovery and Translation. Academic Press, Elsevier, p. 668. Available from: http://dx.doi.org/10.1016/B978-0-12-416002-6.00028-6. Shefat, S.H.T., 2018a. Vaccines for use in finfish aquaculture. Acta Sci. Microbiol. 2 (11), 15
19. Shefat, S.H.T., 2018b. Use of probiotics in shrimp aquaculture in Bangladesh. Acta Sci. Microbiol. 1 (11), 20
27. Soliman, H., El-Matbouli, M., 2006. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS). Vet. Microbiol. 114, 205
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Spatt, B.G., 1999. Multilocus sequence typing: molecular typing of bacterial pathogens in an era of rapid DNA sequencing and the Internet. Curr. Opin. Microbiol. 2, 312
316. Srivastava, P., Pandey, S., Singh, P., Singh, K.P., 2014. Nanotechnology and its role in pathogen detection: a short review. Int. J. Curr. Sci. 13, E9
E15. Subasinghe, R.P., Bondad-Reantaso, M.G., McGladdery, S.E., 2001. Aquaculture development, health and wealth. In: Subasinghe, R.P., Bueno, P., Phillips, M.J., Hough, C., McGladdery, S.E., Arthur, J.R. (Eds.), Aquaculture in the Third Millennium. Technical Proceedings of the Conference on Aquaculture in the Third Millennium, Bangkok, Thailand, 20
25 February 2000. NACA/FAO, Bangkok/Rome, pp. 167
191. Subasinghe, R., Curry, D., McGladdery, S.E. and Bartley, D. (2003). Recent Technological Innovations in Aquaculture. Review of the State of WorldAquaculture, FAO Fisheries No. 886, pp. 59
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703. Swaminathan, T.R., Kumar, R., Dharmaratnam, A., Basheer, V.S., Sood, N., Pradhan, P.K., Sanil, N.K., et al., 2016. Emergence of carp edema virus in cultured ornamental koi carp, Cyprinus carpio koi, in India. J. Gen. Virol. 97 (12), 3392
3399. Tang, Y.-W., Stratton, C.W., 2006. Advanced Techniques in Diagnostic Microbiology. Springer Science 1 Business Media, LLC, New York, NY, p. 539. Tang, Y.W., Procop, G.W., Persing, D.H., 1997. Molecular diagnostics of infectious diseases. Clin. Chem. 43, 2021
2038. Tenover, F.C., Arbeit, R.D., Goering, R.V., 1997. How to select and interpret molecular strain typing methods for epidemiological studies of bacterial infections: a review for health care epidemiologists. Infect. Control Hosp. Epidemiol. 18 (6), 426
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6831. Verschuere, L., Rombaut, G., Sorgeloos, P., Verstraete, W., 2000. Probiotic bacteria as biological control agents in aquaculture. Microbiol. Mol. Biol. Rev. 64, 655
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87. Walker, P.J., Winton, J.R., 2010. Emerging viral diseases of fish and shrimp. Vet. Res. 41 (6), 51. Available from: https://doi.org/10.1051/vetres/2010022. Wang, Y.-B., Xu, Z.-R., Xia, M.-S., 2005. The effectiveness of commercial probiotics in northern white shrimp Penaeus vannamei ponds. Fish. Sci. 7 (5), 1036
1041.

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Ward, P., Roy, D., 2005. Review of molecular methods for identification, characterization and detection of bifidobacteria. Lait 85, 23
32. Warsen, A.E., Krug, M.J., LaFrentz, S., Stanek, D.R., Loge, F.J., Call, D.R., 2004. Simultaneous discrimination between 15 Fish pathogens by using 16S ribosomal DNA PCR and DNA microarrays. Appl. Environ. Microbiol. 70 (7), 4216
4221. Welsh, J., McClelland, M., 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18 (24), 7213
7218. Williams, J.G., Kubelik, A.R., Livak, K.J., Rafalski, J.A., Tingey, S.V., 1990. DNA polymerase amplified by arbitrary primers are useful as genetic maker. Nucleic Acids Res. 18, 6531
6535. Yanong, R.P.E., Francis-Floyd, R., 2019. Therapeutic considerations in aquaculture. In: MSD Manual Veterinary Manual, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ. Available from: ,https://www.msdvetmanual.com/exoticand-laboratory-animals/aquaculture/therapeutic-considerations-in-aquaculture.. Ziaei-Nejad, S., Rezaei, M.H., Takami, G.A., Lovett, D.L., Mirvaghefi, A.-R., Shakouri, M., 2006. The effect of Bacillus spp. bacteria used as probiotics on digestive enzyme activity, survival and growth in the Indian white shrimp Fenneropenaeus indicus. Aquaculture 252 (2-4), 516
524.

Further reading Altink, I., 2011. Multiplex PCR assay for detection of four major bacterial pathogens causing rainbow trout disease. Dis. Aquat. Organ. 93 (3), 199
206. Available from: https:// doi.org/10.3354/dao02300. Mohan, C.V., Bhatta, R., 2002. Social and economic impacts of aquatic animal health problems on aquaculture in India. In: Arthur, J.R., Phillips, M.J., Subasinghe, R.P., Reantaso, M.B., MacRae, I.H. (Eds.), Primary Aquatic Animal Health Care in Rural, Small-Scale, Aquaculture Development. FAO Fisheries Technical Paper No. 406, Rome, pp. 63
75. Petr, K., Ricchi, M., 2017. A basic guide to real time PCR in microbial diagnostics: definitions, parameters, and everything. Front. Microbiol. 8, 108. Available from: ,https:// doi.org/10.3389/fmicb.2017.00108..

CHAPTER

Advances and applications of vectored vaccines in animal diseases

15

Ashish Tiwari1, Ablesh Gautam2, Sudipta Bhat3 and Yashpal Singh Malik4 1

University of Kentucky, Lexington, KY, United States 2 Central Research Institute, Kasauli, India 3 Division of Virology, Indian Veterinary Research Institute, Bareilly, India 4 Division of Biological Standardization, ICAR-Indian Veterinary Research Institute, Izatnagar, India

15.1 Introduction An ounce of prevention is worth a pound of cure Benjamin Franklin.

Although Benjamin Franklin didn’t imply it for the vaccination, his axiom is perfectly appropriate for the field of infectious diseases. Since the time, Edward Jenner’s first vaccine experiment has been a key intervention in the control of the infectious diseases. It was a successful vaccination regimen that helped to eradicate smallpox in humans and rinderpest in animals (Morens et al., 2011). Viral vectors have been used as potential candidates for therapeutics as well as preventive regimens, that is, gene therapy and vaccines, respectively. Being viruses, these tools infect almost all living cells, and thus have proven to be a successful approach in delivery. Highly efficient gene transduction ability, precise delivery of genes to the target cells, elicitation of optimum immune response, together with accelerated cellular immunity underwrite the success of viral vectors (Nayak and Herzog, 2010). The elicitation of cellular immunity differentiates them from the subunit vaccines, where the latter elicits a humoral response (Ura et al., 2014). This further explains why viral vectors serve as potential tools for therapeutics. They invoke a strong cytotoxic T-lymphocyte (CTL) response by mediating intracellular antigen expression, thereby causing elimination of virusinfected cells (Li et al., 2009). In the early 1970s, viral vector was first introduced with development of a recombinant DNA from the SV40 virus using the genetic engineering techniques (Jackson et al., 1972). Later, in the early 1980s, recombinant vaccinia virus was targeted by incorporating a foreign DNA into its genome (Mackett et al., 1982; Panicali and Paoletti, 1982). Various virus vectors have been developed since Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00015-1 © 2020 Elsevier Inc. All rights reserved.

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FIGURE 15.1 The process of recombinant vector vaccine production. DNA encoding gene of the pathogen is inserted into the vector plasmid by means of molecular cloning. The resultant recombinant plasmid is transfected into the viruses while propagating in tissue culture cells. The process of transfection aids in the phenomenon of homologous recombination, whereby, viruses take up the plasmid DNA. This whole process finally leads to formation of recombinant vector vaccines.

then, and have been used in animals and humans across the globe. However, vaccinia virus and adenovirus prove to be the most widely used vectors to date (Ura et al., 2014). Both these vectors are capable of inducing a robust cellmediated immune response against the expressed foreign antigens. General vectored vaccine design requires inserting a foreign gene along with regulatory elements such as promoter and terminator sequences in to appropriate sites, usually dispensable gene in virus vector genome. The foreign gene cassette is usually transferred to the vector by plasmid transfection. Fig. 15.1 provides a general overview of steps in single-antigen vector generation. Some vectors such as poxvirus vectors can take up large foreign nucleic acid sequences. These have been used to deliver multiple antigens. The antigens could either be from single

15.1 Introduction

FIGURE 15.2 Multivalent vector vaccines. These vaccines have more than one pathogen-derived antigen. The genes encoding the pathogen antigens may be inserted into one insertion site or two different insertion sites.

pathogen or multiple pathogens-offering wider protection. Depending on the dispensability of vector genes, foreign gene could either be inserted at a single site or at separate sites in the vector genome (Fig. 15.2). In general, vectored viral vaccines are able to induce stout immune response even without an adjuvant. There is production of interferons and inflammatory cytokines as a result of elicitation of innate immune response by viral components (Akira et al., 2006). There are several factors upon which efficacy and safety of the viral vectored vaccines are determined, such as immunogenicity, ability for immune evasion, genetic stability, replication deficiency, genotoxicity, and costeffectiveness. Further, the guidelines provided by European Medicines Agency for the quality of clinical and nonclinical aspects, are recommended for administration of live, recombinant viral vector-based vaccines against infectious diseases (Ura et al., 2014).

15.1.1 Vectors used for vaccine delivery 15.1.1.1 Poxvirus vectors Poxviruses belong to the Poxviridae family and contain a double-stranded DNA genome ranging in size between 130 and 300 kb pairs (Panicali and Paoletti, 1982). One of the main characteristics of these viruses is that large foreign DNA sequences can be inserted in their genome, allowing their use as vectors for the expression of heterologous immunogenic proteins from other pathogens, including other viruses (Bhanuprakash et al., 2012). It was in the early 1980s, when scientists developed these vectors by inserting foreign DNA into the vaccinia virus

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genome, thereby recognizing the possibility of recombinant vaccinia viruses to be used as vaccine vectors (Mackett et al., 1982). Currently, poxviruses have become most common and best characterized recombinant vectors. Attenuated strains from different genera of poxviruses have been successfully used as vectors for recombinant vaccines. Many new-generation veterinary vaccines have been developed and licensed using vaccinia virus, fowlpox virus, capripoxvirus, parapoxvirus, and canarypox viruses (Bhanuprakash et al., 2012). Large genome of poxviruses allows packing flexibility. Moreover, due to their cytoplasmic replication there is no risk of persistence and integration into the host genome. Poxvirus vaccines induce long lasting humoral as well as cytotoxic T-cell responses against the heterologous immunogens, and are remarkably thermostable during freeze-drying, economic, and easy to manufacture and administer (Bhanuprakash et al., 2012). Since these vectored vaccines have limited replication ability in the host cells (Robert-Guroff, 2007), therefore, the vaccines for which they used have safety comparable to killed vaccines whereas immunogenicity is comparable to a live virus vaccine.

15.1.1.2 Adenovirus vectors Adenoviruses are members of the family Adenoviridae. These are icosahedral, nonenveloped DNA viruses that replicate in the nucleus. The linear doublestranded DNA genome of adenoviruses codes for early (E) and late (L) genes, expressed before and after the replication of viral genome, respectively. Adenovirus code for at least four early genes. E1 gene products are involved in the control of viral gene transcription, shut-off of host proteins, and cell transformation; E2 gene product E2A codes for a DNA binding protein involved in DNA elongation whereas E2B codes for viral DNA polymerase. Adenovirus E3-coded proteins interfere with host immune response and these are dispensable for the virus (Benedict et al., 2001). Finally, E4 gene controls transition from early to late gene expression, shut-off of host cell protein expression, viral replication, and virion assembly (Imler, 1995). Adenovirus, originally evaluated as a vector for gene therapy, is currently one of the very popular vectored vaccine platforms both in humans as well as in veterinary medicine. Adenoviruses offer several advantages as vaccine vectors. They induce strong humoral as well as antigen specific cellular immune response in the host (Yang et al., 2003) upon delivery of a single dose given either by oral (Xiang et al., 2003) or parenteral route (Fitzgerald et al., 2003; Shiver et al., 2002). Furthermore, adenovirus vectors (Ad vector) can accommodate relatively large foreign genes, are easy to produce in large titers with well-established cell lines and methods of purification and scaleup. Adenoviruses do not integrate in to the host genome and can be maintained for several months as episome providing long- lasting immunity (Ehrhardt et al., 2003). Human adenovirus serotype 5 has been one of the most commonly used Ad vectors, however, in recent years other human and nonhuman (Tatsis et al., 2006) adenoviruses have been evaluated as vaccine vectors (Esparza, 2005).

15.1 Introduction

Recombinant adenoviral vectors can be used as replication competent or replication defective vectors. In replication competent Ad vectors, E3 genes are replaced by foreign gene expression cassette whereas in replication defective Ad vectors E1 genes are replaced by foreign genes (Imler, 1995). Although Ad virus incompetent in replication offers the advantage of safety, studies support use of replication competent vectors in veterinary use. Mucosal immune responses and protection after respiratory challenge are limited following immunization with replication incompetent viruses (Fischer et al., 2002; Papp et al., 1997). Replication competent Ad virus induces higher antibody titer as compared to the replication defective viruses (Reddy et al., 2000). Also, replication competent ad vectors have potential to circumvent maternal-derived immunity (Fischer et al., 2002). Ad viral vectors have already been used against several human and animal infectious agents/diseases like Plasmodium falciparum, Mycobacterium tuberculosis, HIV-1, hepatitis C virus, Avian influenza (AI) virus, M. tuberculosis, and foot-and-mouth disease virus (Draper and Heeney, 2010).

15.1.1.3 Retrovirus vectors Retroviruses are enveloped, single-stranded RNA viruses that contain reverse transcriptase. The retroviruses vectors are usually defective in replication mechanism. Most of them are derived from either murine or avian disease causing viruses (Kurian et al., 2000). The retrovirus vectors have 7 11 kb genome size, and they can easily accommodate 7 8 kb foreign DNA inserts. This enables the retroviruses to provide long-term gene expression. Retroviruses have been found to provide suboptimum immunogenicity (Young et al., 2012). Therefore the retroviral vector-based vaccines have also been studied for their potential use as therapeutics. In one clinical study, X-linked severe combined immunodeficiency (SCID-X1) and malignant glioma patients were provided retroviral vector-based gene therapy (Cavazzana-Calvo et al., 2000; Ram et al., 1997). Although, SCIDX1 patients demonstrated high efficacy for the treatment, nevertheless, four out of the 10 patients were found to develop lymphoma (Gaspar et al., 2004). Similar results have also been found during preclinical trials which directly impact the safety of these vaccines (Li et al., 2002; Modlich et al., 2005). The development of lymphoma is contributed to the integration of the viral long terminal repeats (LTRs) into protooncogenes. The retroviral vectors function by integrating near promoters of the cellular genes that regulate cell replication. Vectors that are either nonintegrating or self-inactivating (SIN) can lower the risk of tumorigenesis (Maruggi et al., 2009; Montini et al., 2009). Since, SIN vectors possess partially deleted LTRs, they are inactive during vector production, therefore, they have been strongly recommended for clinical trials in SCID-X1 patients as a therapeutic tool (Thornhill et al., 2008).

15.1.1.4 Lentivirus vectors Lentiviruses are a subclass of retroviruses. Lentiviruses have a broader host tropism. Unlike retroviruses they can infect both dividing and nondividing cells,

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whereas retroviruses can invade only the dividing cells (Durand and Cimarelli, 2011). Lentiviral vectors are beneficial in similar ways to those of retroviral vectors. However, integration sites of lentiviruses are farther than the cellular promoter sites, therefore, the probability of inducing tumorigenesis by lentiviruses is lower than the retroviral vectors. HIV is one of the popular lentivirus that has been developed into a transgene delivery vector. There are several other types of HIV-based vectors generated by omitting the regulatory genes that are not required during vector production and the HIV viral envelope (Dull et al., 1998). To further customize the vectors according to various cells and tissues, the wild type HIV viral envelopes have been replaced by chimeric or modified envelopes. For instance, since the vesicular stomatitis virus (VSV) has a broad range of tropism, it has been used to generate VSV/HIV-1-based vectors containing the glycoprotein envelope derived from VSV (Schroers et al., 2000). Similarly, therapeutic vectors have been designed by using envelopes from filovirus (Kobinger et al., 2001), Moloney murine leukemia virus (Watson et al., 2002), and measles (Frecha et al., 2008). The HIV-1-based vectors have demonstrated serious concerns of virulence. In general, the virulence of lentiviruses is highly species-specific. Henceforth, feline immunodeficiency virus (FIV)-based vectors have been developed to combat the drawbacks laid by HIV-1 and the herpes simplex virus-based vectors (Sinn et al., 2003). The FIV vector-based vaccines have proven to be significantly efficient in the field (Chiuppesi et al., 2012; Pistello et al., 2010).

15.1.1.5 Cytomegalovirus vectors Cytomegalovirus (CMV) is a member of the herpesviruses. Although, there are a multiple number of species-specific CMVs, human CMV (HCMV) has been greatly studied. CMV, also known as human herpesvirus type 5, often goes asymptomatic as it is mildly pathogenic in healthy individuals (Grinde, 2013). Higher pathogenicity is observed only in pregnant women and immunocompromised people. HCMV has a large double-stranded linear DNA genome of about 235 kb surrounded by a capsid. The envelope of the virus comprises of the glycoproteins gB and gH that bind to the cellular receptors. Protective immunity was observed against simian immunodeficiency virus (SIV) infection by a rhesus CMV (RhCMV) vector-based vaccine in SIV patients (Hansen et al., 2011). Subsequently, the infection was also seen to be cleared. It has been observed that RhCMV-based vaccine works by inducing a characteristic major histocompatibility complex class II specific CTL response that results in recognizing several antigen epitopes (Hansen et al., 2013). The HCMV vectorbased vaccines have been demonstrated as highly promising against HIV infection (Humphreys and Sebastian, 2018).

15.1.1.6 Sendai virus vectors Sendai virus (SeV) belonging to the family Paramyxoviridae, is an enveloped, single-stranded RNA virus. SeV is pathogenic in mice and causes

15.2 Vectors for poultry vaccines

bronchopneumonia. Although, its zoonosis has not been reported so far, however it shares great homology to the human parainfluenza type1 (hPIV-1) virus. Therefore, immunity against hPIV-1 also works against SeV. This has been shown by Hara et al., where the majority of adults have demonstrated SeVspecific neutralizing antibodies (Nakanishi and Otsu, 2012). There are two envelope glycoproteins, HN and F proteins, encoded in SeV genome. These proteins are responsible for mediating host-cell invasion and determining tropism. SeV viruses that do not possess F protein are defective in replication, thereby contributing to make the vector safe. To propagate the SeV vector, the packaging cell lines are transfected with transgenic SeV that have F-gene deleted and foreign DNA inserted into their genome. The packaging cells express the F-protein, which suffice to the SeV viruses lacking this protein. Soon after transfection, viral genome localizes to the cytoplasm that further enables rapid gene expression. The SeV vectors are highly efficient transducing both dividing and nondividing cells. The human respiratory epitheliaare also efficiently transduced by SeV vectors, thus, making the mucosal route, a popular route of administration. There is seemingly less influence of preexisting immunity to SeV when administered by intranasal route as compared to intramuscular route (Moriya et al., 2011). The limitation of SeV vector includes its low transgene capacity (3.4 kb) (Ura et al., 2014). The SeV vectors have been exploited for gene therapy and have served as candidates for vaccine in human trials (Hara et al., 2011; Slobod et al., 2004).

15.2 Vectors for poultry vaccines The poultry industry is one of the rapidly evolving sectors. The industry, however, faces the challenges of various infectious diseases, especially viral diseases (Hess and McDougald, 2013). Thus, administration of multiple vaccines to combat avian diseases has become a regular routine in poultry production. Most of these vaccines were developed by conventional methods such as inactivation or attenuation. But, with advancements in the technologies, recombinant vaccines have also been developed. However, these vaccines due to their complexities and timeline involved in development, are restricted to a limited set of diseases. More recently, genetic modifications have been made to multiple attenuated poultry vaccine viruses and used as vaccine vectors for various poultry diseases. The examples include herpes virus of turkey (HVT) (Darteil et al., 1995; Li et al., 2011), Newcastle disease virus (NDV) (Huang et al., 2004; Nakaya et al., 2001; Park et al., 2006), fowlpox virus (FPV) (Boyle and Coupar, 1988; Bublot et al., 2006; Swayne et al., 2000), adenovirus (Francois et al., 2004), infectious laryngotracheitis virus (Lu¨schow et al., 2001; Veits et al., 2003), and Marek’s disease virus (MDV) (Li et al., 2016; Sakaguchi et al., 1998; Tsukamoto et al., 1999). Although these vectored vaccines have been effective, their performance seems to be greatly influenced by the presence of maternally-derived antibodies (MDA) in

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the neonatal chicks (Bublot et al., 2006; De Vriese et al., 2010; Faulkner et al., 2013; Maas et al., 2011; Sarfati-Mizrahi et al., 2010).

15.2.1 Herpesvirus of turkey The herpesvirus of turkey is unaffected by the presence of MDAs which cause persistent infections in chickens. Therefore lifetime immunity is induced by a single dose of HVT-vectored vaccines when administered to 18-day old embryo or 1-day old chicks (Morgan et al., 1992). Further, long fragments of foreign DNA can be precisely incorporated in the genomes of herpesvirus without affecting their replication efficiency (Afonso et al., 2001). Henceforth, HVT serves to be a safe and effective poultry vaccine for MDV for the past five decades (Hess and McDougald, 2013; Okazaki et al., 1970). Due to the characteristic features of HVT as a vector, recombinant HVT (rHVT) vaccines expressing F protein of NDV, VP2 protein of infectious bursal disease virus (IBDV), and HA antigen of H5 AIV for immunization against Newcastle disease (ND), infectious bursal disease (IBD), and avian influenza (AI), have been produced and undergone registration and commercialization [http://www.merial.ca/en/poultry/productslivevaccines/ Pages/vaxxitek.aspx., https://www.vectormune.com/, https://www.innovax-vaccines. com/]. With the advancement in reverse genetics techniques, incorporation of HVT genome into cosmid or bacterial artificial chromosome (BAC) clones, has resulted in more precise and targeted insertion of genes of interest of various viruses into the HVT genome (Baigent et al., 2006). Vaccine developed using HVT-BAC clone, a rHVT vaccine expressing haemagglutinin (HA) antigen from avian influenza virus (AIV), has shown to elicit delayed protective immunity in vaccinated chickens (Li et al., 2011). However, in ovo vaccination with rHVT-AIV vaccines, showed continuous increase in the level of specific antibodies from day 35 until day 91 (Iqbal, 2012). These studies imply that upon in ovo vaccination, rHVT-AIV vaccines have the potential to provide strong and long-term immunity (Kapczynski et al., 2016; Kilany et al., 2016; Palya et al., 2015; Rauw et al., 2012). The suboptimal performance of rHVT-AIV vaccines in chickens may be probably due to heterogeneity in the antigens in vaccines versus contemporary field virus variants. In order to combat the emergent antigenic variants of viruses, computationally-designed antigenic epitopes of synthetic HA antigens have been developed that provide a broad level of protective immunity (Carter et al., 2016; Crevar et al., 2015; Wong et al., 2017). The HVT-BAC clone recombinant vaccines have also been found to be slower in replication in cell culture as well as vaccinated chicks than the wild-type HVT virus strain (Liu et al., 2015). This suboptimal replication may account for reduced expression of antigens, which in return may have led to a delayed onset of optimum amount of specific antibody production. More recently, CRISPR/Cas9 gene editing technology has been utilized for rapid incorporation of foreign antigens into the wild-type HVT strains (Tang et al., 2018). This will greatly help in the rapid development of rHVT vaccines against various infectious agents of livestock and poultry.

15.3 Vectored veterinary vaccines

15.3 Vectored veterinary vaccines The most common vectored veterinary vaccines are developed by using poxvirus and adenovirus (Baron et al., 2018). Among poxviruses, canarypox virus has been used for different target pathogens including equine influenza virus, West Nile virus (WNV), equine herpes virus (EHV1), rabies virus, feline leukemia virus, and canine distemper virus (Bronson et al., 2007; Paillot and El-Hage, 2016; Poulet et al., 2003). Other poxviruses like vaccinia, modified vaccinia (MVA) virus, FPV, and capripoxvirus have also been used as vectors. A commercialized vaccinia virus vectored rabies vaccine is now available for use in wildlife (Maki et al., 2017). There are also commercial poultry vaccines for Newcastle and AI vaccine vectored in FPV (Draper and Heeney, 2010). Less virulent MVA viruses also proved to be better vectors and used for several veterinary pathogens like Mycobacterium bovis, EHV1 (Draper and Heeney, 2010; Huemer et al., 2000). Also, capripoxvirus has been used as vectors for giving protection against peste-petits-ruminants virus (PPRV) (Caufour et al., 2014). Human adenovirus 5 has been used for several veterinary pathogen which includes AI virus, M. bovis, foot-and-mouth disease virus (FMDV), Rift Valley fever virus, PPRV (Herbert et al., 2014; Toro et al., 2007). Recently, a canine adenovirus 2 vectored FMDV have been shown to provide effective protection (De Vleeschauwer et al., 2018) There are also other less commonly targeted viruses for giving protection against veterinary pathogens. Recently, porcine reproductive and respiratory syndrome virus (PRRSV)-vectored multi-component vaccine against PRRSV, porcine circovirus (PCV) type 2, and swine influenza virus (Tian et al., 2017) have been explored. PPRSV-vectored vaccine, expressing E2 of classical swine fever virus (CSFV) showed protection in pigs from a lethal challenge of highly-pathogenic PRRSV and CSFV (Gao et al., 2018). Recently, C-strain of CSFV was developed to express the Cap gene of PCV type 2 (Zhang et al., 2017). Nonpathogenic PCV1-vectored vaccine induces protective immunity against PCV2 infection in pigs (Fenaux et al., 2004). Recently, inactivated rabies viruses displaying the canine distemper virus glycoproteins showed protective immunity against both pathogens (da Fontoura Budaszewski et al., 2017). Equine herpesvirus type 1 (EHV-1)-vectored vaccines were also used for H3N8 equine influenza virus in equine species giving good protection for both the viruses (Van de Walle et al., 2010). Lately, EHV-1 vector expressing Rift Valley fever virus Gn and Gc was shown to induce neutralizing antibodies in sheep (Said et al., 2017). Flaviviruses have also been used as vectors for rabies virus vaccine (Giel-Moloney et al., 2017). Gallid herpes virus 3, SB-1 strain, has been used as a vector for several poultry vaccines like Marek’s disease (MD), IBD, ND, or AI (Sadigh et al., 2018). Several veterinary vaccines developed employing the vector approaches are detailed in the Table 15.1.

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Table 15.1 A list of veterinary vectored vaccines indicating its current status. Virus vector family Adenoviridae

Poxviridae

Target veterinary pathogen Human adenovirus 5

Attenuated canarypox virus

Fowlpox virus (FPV)

Paramyxoviridae Herpesviridae

Modified vaccinia virus Ankara (MVA) NYVAC or rMVA Vaccinia virus NDV (LaSota strain) Turkey herpesvirus

Flaviviridae

YFV-17D

Avian influenza virus Mycobacterium tuberculosis Foot-and-mouth disease virus

Target species

Target antigen Mycobacterial mycolyltransferase FMDV capsid and protease antigens

Equine influenza virus

Bovine Bovine and swine Horses

West Nile virus (WNV) Equine herpes virus (EHV1)

Horses Horses

Rabies virus Feline leukemia virus Canine distemper virus

Cats Cats Dogs Ferrets Poultry Poultry

PreM Env gB, gC, and gD glycoproteins of the Kentucky strain Glycoprotein G Env, Gag Pol HA and F HA and F H5 HA HN and F

Avian influenza virus and FPV Newcastle disease virus (NDV) and FPV Mycobacterium bovis EHV1 EHV1 Rabies virus Avian influenza virus and NDV Infectious bursal disease virus and Marek’s disease virus (MDV) WNV

Bovine

Wildlife Poultry Poultry Horses

HA (Kentucky and Newmarket strains)

Mycobacterial mycolyltransferase EHV1 gC Immediate early (IE) gene Glycoprotein G H5 HA VP2 of infectious bursal disease virus (IBDV) in herpes virus of turkey (HVT) backbone PreM-Env

Status (commercial or under preparation)

ProteqFlu-Te (Europe) Recombitek (United States) Recombitek equine WNV

Purevax Feline Rabies Purevax FeLV RECOMBITEK Distemper Purevax Ferret Distemper Trovac AI H5 Vectormune FP-N

Raboral NewH5 PreveNile Vaxxitek HVT 1 infectious bursal disease (IBD)

15.5 Conclusion

15.4 Challenges in vectored veterinary vaccine Research in vectored vaccines has progressed rapidly and animal vaccines have been licensed for use in large animals, companion animals, wildlife, and birds (Ura et al., 2014). Despite of several advantages, which viral vectoring can offer, preexisting immunity is a major obstacle for many viral-vectored vaccines, such as against Ad serotype 5 (AdHu5) or herpes simplex virus type 1 (HSV-1), where the rate of seroprevalence to these viruses is very high [40% 45% and 70% (or more) of the US population, respectively] (Hocknell et al., 2002; Pichla-Gollon et al., 2009). Vector-specific antibodies may impede the induction of immune responses to the vaccine-encoded antigens, as they may reduce the dose and time of exposure of the target cells to the vaccinated antigens (Pichla-Gollon et al., 2009). In a large-scale clinical trial (STEP) of anAdHu5-based HIV-1 vaccine, the vaccines showed a lack of efficacy and tended to increase the risk of HIV-1 infection in vaccine recipients who had preexisting neutralizing antibodies to AdHu5 (Buchbinder et al., 2008; Pine et al., 2011). For an HSV-1-based vector vaccine, it has been demonstrated that preexisting anti-HSV-1 immunity reduced, but did not abolish, humoral and cellular immune responses against the vaccineencoded antigen (Hocknell et al., 2002). However, Brockman and Knipe found that the induction of durable antibody responses and cellular proliferative responses to HSV-encoded antigen were not affected by prior HSV immunity (Brockman and Knipe, 2002; Lauterbach et al., 2005). Similarly, preexisting immunity to poliovirus had little effect on vaccine efficacy in a poliovirusvectored vaccine (Mandl et al., 2001). There are several approaches to avoid preexisting vector immunity, such as the use of vectors derived from nonhuman sources, using human viruses of rare serotypes (Kahl et al., 2010; Lasaro and Ertl, 2009), heterologous prime boost approaches (Liu et al., 2008), homologous reimmunization (Steffensen et al., 2012), and removing key neutralizing epitopes on the surface of viral capsid proteins (Gabitzsch and Jones, 2011). The inhibitory effect of preexisting immunity can also be avoided by masking the Ad vector inside dendritic cells (Roberts et al., 2006; Steffensen et al., 2012). In addition, mucosal vaccination or administration of higher vaccine doses can overcome preexisting immunity problems (Alexander et al., 2012; Belyakov et al., 1999; Priddy et al., 2008; Xiang et al., 2003).

15.5 Conclusion Overall, the viral vector-based vaccine approach is an emerging and promising choice for gene therapy as well as for vaccine production. Several viral vectorbased vaccines have reached the commercial windows and similarly, viral vectorbased gene therapies have also been in practice. Here we have elaborated upon viral vectors that are currently in use, as well as the potential candidates that are

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under clinical trials. Vectors such as MVA and Ad are the most widely used against HIV-1 as therapeutics (Cosma et al., 2003; Zhang and Zhou, 2016). The other vectors also exhibit unique features, such as, CMV vector is able to induce a unique immune response, SeV vector enables mucosal immunity, etc. (Moriya et al., 2011). Large-scale manufacturing of viral vector-based vaccines and testing of their safety can be done alongside traditional vaccines. The vectored virus vaccines that are derived from MVA virus, the measles virus, and the poliovirus, have been studied extensively for their potential use. Apart from these, there are several other viral vectors that are being investigated. Thoughtful use of the advantages of these vectors can benefit for enhanced efficacy and safety. These vaccines have great potential in inducing a strong immune response and targeted delivery. On the other hand, genetic alterations in the genome of the vector-based vaccine candidates will enable to surpass the obstacles arising in the development of efficacious and safe vaccines.

Conflict of interest There is no conflict of interest.

Acknowledgments All the authors of the manuscript thank and acknowledge their respective universities and institutes.

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Kapczynski, D.R., Dorsey, K., Chrzastek, K., Moraes, M., Jackwood, M., Hilt, D., et al., 2016. Vaccine protection of turkeys against H5N1 highly pathogenic avian influenza virus with a recombinant turkey herpesvirus expressing the hemagglutinin gene of avian influenza. Avian Dis. 60, 413 417. Kilany, W.H., Safwat, M., Mohammed, S.M., Salim, A., Fasina, F.O., Fasanmi, O.G., et al., 2016. Protective efficacy of recombinant turkey herpes virus (rHVT-H5) and inactivated H5N1 vaccines in commercial mulard ducks against the highly pathogenic avian influenza (HPAI) H5N1 clade 2.2. 1 virus. PLoS One 11, e0156747. Kobinger, G.P., Weiner, D.J., Yu, Q.-C., Wilson, J.M., 2001. Filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo. Nat. Biotechnol. 19, 225. Kurian, K., Watson, C., Wyllie, A., 2000. Retroviral vectors. Mol. Pathol. 53, 173. Lasaro, M.O., Ertl, H.C., 2009. New insights on adenovirus as vaccine vectors. Mol. Ther. 17, 1333 1339. Lauterbach, H., Ried, C., Epstein, A.L., Marconi, P., Brocker, T., 2005. Reduced immune responses after vaccination with a recombinant herpes simplex virus type 1 vector in the presence of antiviral immunity. J. Gen. Virol. 86, 2401 2410. Li, Z., Du¨llmann, J., Schiedlmeier, B., Schmidt, M., von Kalle, C., Meyer, J., et al., 2002. Murine leukemia induced by retroviral gene marking. Science 296, 497. Li, C., Hirsch, M., DiPrimio, N., Asokan, A., Goudy, K., Tisch, R., et al., 2009. CytotoxicT-lymphocyte-mediated elimination of target cells transduced with engineered adenoassociated virus type 2 vector in vivo. J. Virol. 83, 6817 6824. Li, Y., Reddy, K., Reid, S.M., Cox, W.J., Brown, I.H., Britton, P., et al., 2011. Recombinant herpesvirus of turkeys as a vector-based vaccine against highly pathogenic H7N1 avian influenza and Marek’s disease. Vaccine 29, 8257 8266. Li, K., Liu, Y., Liu, C., Gao, L., Zhang, Y., Cui, H., et al., 2016. Recombinant Marek’s disease virus type 1 provides full protection against very virulent Marek’s and infectious bursal disease viruses in chickens. Sci. Rep. 6, 39263. Liu, J., Ewald, B.A., Lynch, D.M., Denholtz, M., Abbink, P., Lemckert, A.A., et al., 2008. Magnitude and phenotype of cellular immune responses elicited by recombinant adenovirus vectors and heterologous prime-boost regimens in rhesus monkeys. J. Virol. 82, 4844 4852. Liu, S., Sun, W., Chu, J., Huang, X., Wu, Z., Yan, M., et al., 2015. Construction of recombinant HVT expressing PmpD, and immunological evaluation against Chlamydia psittaci and Marek’s disease virus. PLoS One 10, e0124992. Lu¨schow, D., Werner, O., Mettenleiter, T.C., Fuchs, W., 2001. Protection of chickens from lethal avian influenza A virus infection by live-virus vaccination with infectious laryngotracheitis virus recombinants expressing the hemagglutinin (H5) gene. Vaccine 19, 4249 4259. Maas, R., Rosema, S., Van Zoelen, D., Venema, S., 2011. Maternal immunity against avian influenza H5N1 in chickens: limited protection and interference with vaccine efficacy. Avian Pathol. 40, 87 92. Mackett, M., Smith, G.L., Moss, B., 1982. Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proc. Natl. Acad. Sci. U.S.A. 79, 7415 7419. Maki, J., Guiot, A.-L., Aubert, M., Brochier, B., Cliquet, F., Hanlon, C.A., et al., 2017. Oral vaccination of wildlife using a vaccinia rabies glycoprotein recombinant virus vaccine (RABORAL V-RG): a global review. Vet. Res. 48, 57.

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Mandl, S., Hix, L., Andino, R., 2001. Preexisting immunity to poliovirus does not impair the efficacy of recombinant poliovirus vaccine vectors. J. Virol. 75, 622 627. Maruggi, G., Porcellini, S., Facchini, G., Perna, S.K., Cattoglio, C., Sartori, D., et al., 2009. Transcriptional enhancers induce insertional gene deregulation independently from the vector type and design. Mol. Ther. 17, 851 856. Modlich, U., Kustikova, O.S., Schmidt, M., Rudolph, C., Meyer, J., Li, Z., et al., 2005. Leukemias following retroviral transfer of multidrug resistance 1 (MDR1) are driven by combinatorial insertional mutagenesis. Blood 105, 4235 4246. Montini, E., Cesana, D., Schmidt, M., Sanvito, F., Bartholomae, C.C., Ranzani, M., et al., 2009. The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy. J. Clin. Invest. 119, 964 975. Morens, D.M., Holmes, E.C., Davis, A.S., Taubenberger, J.K., 2011. Global rinderpest eradication: lessons learned and why humans should celebrate too. J. Infect. Dis. 204, 502 505. Morgan, R.W., Gelb Jr, J., Schreurs, C.S., Lu¨tticken, D., Rosenberger, J.K., Sondermeijer, P.J., 1992. Protection of chickens from Newcastle and Marek’s diseases with a recombinant herpesvirus of turkeys vaccine expressing the Newcastle disease virus fusion protein. Avian Dis. 36 (4), 858 870. Moriya, C., Horiba, S., Kurihara, K., Kamada, T., Takahara, Y., Inoue, M., et al., 2011. Intranasal Sendai viral vector vaccination is more immunogenic than intramuscular under pre-existing anti-vector antibodies. Vaccine 29, 8557 8563. Nakanishi, M., Otsu, M., 2012. Development of Sendai virus vectors and their potential applications in gene therapy and regenerative medicine. Curr. Gene Ther. 12, 410 416. Nakaya, T., Cros, J., Park, M.-S., Nakaya, Y., Zheng, H., Sagrera, A., et al., 2001. Recombinant Newcastle disease virus as a vaccine vector. J. Virol. 75, 11868 11873. Nayak, S., Herzog, R.W., 2010. Progress and prospects: immune responses to viral vectors. Gene Ther. 17, 295. Okazaki, W., Purchase, H., Burmester, B., 1970. Protection against Marek’s disease by vaccination with a herpesvirus of turkeys. Avian Dis. 413 429. Paillot, R., El-Hage, C., 2016. The use of a recombinant canarypox-based equine influenza vaccine during the 2007 Australian outbreak: a systematic review and summary. Pathogens 5, 42. Palya, V., Kova´cs, E.W., Tata´r-Kis, T., Felfo¨ldi, B., Homonnay, Z.G., Mato´, T., et al., 2015. Recombinant turkey herpesvirus-AI vaccine virus replication in different species of waterfowl. Avian Dis. 60, 210 217. Panicali, D., Paoletti, E., 1982. Construction of poxviruses as cloning vectors: insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccinia virus. Proc. Natl. Acad. Sci. U.S.A. 79, 4927 4931. Papp, Z., Middleton, D.M., Mittal, S.K., Babiuk, L.A., Baca-Estrada, M.E., 1997. Mucosal immunization with recombinant adenoviruses: induction of immunity and protection of cotton rats against respiratory bovine herpesvirus type 1 infection. J. Gen. Virol. 78, 2933 2943. Park, M.-S., Steel, J., Garcı´a-Sastre, A., Swayne, D., Palese, P., 2006. Engineered viral vaccine constructs with dual specificity: avian influenza and Newcastle disease. Proc. Natl. Acad. Sci. U.S.A. 103, 8203 8208.

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Pichla-Gollon, S.L., Lin, S.-W., Hensley, S.E., Lasaro, M.O., Herkenhoff-Haut, L., Drinker, M., et al., 2009. Effect of preexisting immunity on an adenovirus vaccine vector: in vitro neutralization assays fail to predict inhibition by antiviral antibody in vivo. J. Virol. 83, 5567 5573. Pine, S.O., Kublin, J.G., Hammer, S.M., Borgerding, J., Huang, Y., Casimiro, D.R., et al., 2011. Pre-existing adenovirus immunity modifies a complex mixed Th1 and Th2 cytokine response to an Ad5/HIV-1 vaccine candidate in humans. PLoS One 6, e18526. Pistello, M., Bonci, F., Zabogli, E., Conti, F., Freer, G., Maggi, F., et al., 2010. Envexpressing autologous T lymphocytes induce neutralizing antibody and afford marked protection against feline immunodeficiency virus. J. Virol. 84, 3845 3856. Poulet, H., Brunet, S., Boularand, C., Guiot, A., Leroy, V., Tartaglia, J., et al., 2003. Efficacy of a canarypox virus-vectored vaccine against feline leukaemia. Vet. Rec. 153, 141 145. Priddy, F.H., Brown, D., Kublin, J., Monahan, K., Wright, D.P., Lalezari, J., et al., 2008. Safety and immunogenicity of a replication-incompetent adenovirus type 5 HIV-1 clade B gag/pol/nef vaccine in healthy adults. Clin. Infect. Dis. 46, 1769 1781. Ram, Z., Culver, K.W., Oshiro, E.M., Viola, J.J., DeVroom, H.L., Otto, E., et al., 1997. Therapy of malignant brain tumors by intratumoral implantation of retroviral vectorproducing cells. Nat. Med. 3, 1354. Rauw, F., Palya, V., Gardin, Y., Tatar-Kis, T., Dorsey, K.M., Lambrecht, B., et al., 2012. Efficacy of rHVT-AI vector vaccine in broilers with passive immunity against challenge with two antigenically divergent Egyptian clade 2.2. 1 HPAI H5N1 strains. Avian Dis. 56, 913 922. Reddy, P.S., Idamakanti, N., Pyne, C., Zakhartchouk, A.N., Godson, D.L., Papp, Z., et al., 2000. The immunogenicity and efficacy of replication-defective and replicationcompetent bovine adenovirus-3 expressing bovine herpesvirus-1 glycoprotein gD in cattle. Vet. Immunol. Immunopathol. 76, 257 268. Robert-Guroff, M., 2007. Replicating and non-replicating viral vectors for vaccine development. Curr. Opin. Biotechnol. 18, 546 556. Roberts, D.M., Nanda, A., Havenga, M.J., Abbink, P., Lynch, D.M., Ewald, B.A., et al., 2006. Hexon-chimaeric adenovirus serotype 5 vectors circumvent pre-existing anti-vector immunity. Nature 441, 239. Sadigh, Y., Powers, C., Spiro, S., Pedrera, M., Broadbent, A., Nair, V., 2018. Gallid herpesvirus 3 SB-1 strain as a recombinant viral vector for poultry vaccination. NPJ Vaccines 3, 21. Said, A., Elmanzalawy, M., Ma, G., Damiani, A.M., Osterrieder, N., 2017. An equine herpesvirus type 1 (EHV-1) vector expressing Rift Valley fever virus (RVFV) Gn and Gc induces neutralizing antibodies in sheep. Virology Journal 14, 154 161. Sakaguchi, M., Nakamura, H., Sonoda, K., Okamura, H., Yokogawa, K., Matsuo, K., et al., 1998. Protection of chickens with or without maternal antibodies against both Marek’s and Newcastle diseases by one-time vaccination with recombinant vaccine of Marek’s disease virus type 1. Vaccine 16, 472 479. Sarfati-Mizrahi, D., Lozano-Dubernard, B., Soto-Priante, E., Castro-Peralta, F., FloresCastro, R., Loza-Rubio, E., et al., 2010. Protective dose of a recombinant Newcastle disease LaSota avian influenza virus H5 vaccine against H5N2 highly pathogenic avian influenza virus and velogenic viscerotropic Newcastle disease virus in broilers with high maternal antibody levels. Avian Dis. 54, 239 241.

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Schroers, R., Sinha, I., Segall, H., Schmidt-Wolf, I.G., Rooney, C.M., Brenner, M.K., et al., 2000. Transduction of human PBMC-derived dendritic cells and macrophages by an HIV-1-based lentiviral vector system. Mol. Ther. 1, 171 179. Shiver, J.W., Fu, T.-M., Chen, L., Casimiro, D.R., Davies, M.-E., Evans, R.K., et al., 2002. Replication-incompetent adenoviral vaccine vector elicits effective antiimmunodeficiency-virus immunity. Nature 415, 331. Sinn, P.L., Hickey, M.A., Staber, P.D., Dylla, D.E., Jeffers, S.A., Davidson, B.L., et al., 2003. Lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha. J. Virol. 77, 5902 5910. Slobod, K.S., Shenep, J.L., Luja´n-Zilbermann, J., Allison, K., Brown, B., Scroggs, R.A., et al., 2004. Safety and immunogenicity of intranasal murine parainfluenza virus type 1 (Sendai virus) in healthy human adults. Vaccine 22, 3182 3186. Steffensen, M.A., Jensen, B.A.H., Holst, P.J., Bassi, M.R., Christensen, J.P., Thomsen, A. R., 2012. Pre-existing vector immunity does not prevent replication deficient adenovirus from inducing efficient CD8 T-cell memory and recall responses. PLoS One 7, e34884. Swayne, D.E., Garcia, M., Beck, J.R., Kinney, N., Suarez, D.L., 2000. Protection against diverse highly pathogenic H5 avian influenza viruses in chickens immunized with a recombinant fowlpox vaccine containing an H5 avian influenza hemagglutinin gene insert. Vaccine 18, 1088 1095. Tang, N., Zhang, Y., Pedrera, M., Chang, P., Baigent, S., Moffat, K., et al., 2018. A simple and rapid approach to develop recombinant avian herpesvirus vectored vaccines using CRISPR/Cas9 system. Vaccine 36, 716 722. Tatsis, N., Tesema, L., Robinson, E., Giles-Davis, W., McCoy, K., Gao, G., et al., 2006. Chimpanzee-origin adenovirus vectors as vaccine carriers. Gene Ther. 13, 421. Thornhill, S.I., Schambach, A., Howe, S.J., Ulaganathan, M., Grassman, E., Williams, D., et al., 2008. Self-inactivating gammaretroviral vectors for gene therapy of X-linked severe combined immunodeficiency. Mol. Ther. 16, 590 598. Tian, D., Sooryanarain, H., Matzinger, S.R., Gauger, P.C., Karuppannan, A.K., Elankumaran, S., et al., 2017. Protective efficacy of a virus-vectored multi-component vaccine against porcine reproductive and respiratory syndrome virus, porcine circovirus type 2 and swine influenza virus. J. Gen. Virol. 98, 3026 3036. Toro, H., De-chu, C.T., Suarez, D.L., Sylte, M.J., Pfeiffer, J., Van Kampen, K.R., 2007. Protective avian influenza in ovo vaccination with non-replicating human adenovirus vector. Vaccine 25, 2886 2891. Tsukamoto, K., Kojima, C., Komori, Y., Tanimura, N., Mase, M., Yamaguchi, S., 1999. Protection of chickens against very virulent infectious bursal disease virus (IBDV) and Marek’s disease virus (MDV) with a recombinant MDV expressing IBDV VP2. Virology 257, 352 362. Ura, T., Okuda, K., Shimada, M., 2014. Developments in viral vector-based vaccines. Vaccines 2, 624 641. Van de Walle, G.R., May, M.A., Peters, S.T., Metzger, S.M., Rosas, C.T., Osterrieder, N., 2010. A vectored equine herpesvirus type 1 (EHV-1) vaccine elicits protective immune responses against EHV-1 and H3N8 equine influenza virus. Vaccine 28, 1048 1055. Veits, J., Lu¨schow, D., Kindermann, K., Werner, O., Teifke, J.P., Mettenleiter, T.C., et al., 2003. Deletion of the non-essential UL0 gene of infectious laryngotracheitis (ILT) virus

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leads to attenuation in chickens, and UL0 mutants expressing influenza virus haemagglutinin (H7) protect against ILT and fowl plague. J. Gen. Virol 84, 3343 3352. Watson, D.J., Kobinger, G.P., Passini, M.A., Wilson, J.M., Wolfe, J.H., 2002. Targeted transduction patterns in the mouse brain by lentivirus vectors pseudotyped with VSV, Ebola, Mokola, LCMV, or MuLV envelope proteins. Mol. Ther 5, 528 537. Wong, T.M., Allen, J.D., Bebin-Blackwell, A.-G., Carter, D.M., Alefantis, T., DiNapoli, J., et al., 2017. Computationally optimized broadly reactive hemagglutinin elicits hemagglutination inhibition antibodies against a panel of H3N2 influenza virus cocirculating variants. J. Virol. 91, e01581 01517. Xiang, Z., Gao, G., Reyes-Sandoval, A., Li, Y., Wilson, J., Ertl, H., 2003. Oral vaccination of mice with adenoviral vectors is not impaired by preexisting immunity to the vaccine carrier. J. Virol. 77, 10780 10789. Yang, T.C., Dayball, K., Wan, Y.H., Bramson, J., 2003. Detailed analysis of the CD8 1 Tcell response following adenovirus vaccination. J. Virol. 77, 13407 13411. Young, G.R., Ploquin, M.J.-Y., Eksmond, U., Wadwa, M., Stoye, J.P., Kassiotis, G., 2012. Negative selection by an endogenous retrovirus promotes a higher-avidity CD4 1 T cell response to retroviral infection. PLoS Pathog. 8, e1002709. Zhang, C., Zhou, D., 2016. Adenoviral vector-based strategies against infectious disease and cancer. Hum. Vaccin. Immunother. 12, 2064 2074. Zhang, L., Li, Y., Xie, L., Wang, X., Gao, X., Sun, Y., et al., 2017. Secreted expression of the cap gene of porcine circovirus type 2 in classical swine fever virus C-strain: potential of C-strain used as a vaccine vector. Viruses 9, 298.

CHAPTER

Bioinformatics for animal diseases: focused to major diseases and cancer

16

Mohamad Zamani-Ahmadmahmudi Department of Clinical Science, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran

16.1 Introduction Bioinformatics is defined in the Oxford English Dictionary as follows: bioinformatics is conceptualizing biology in terms of molecules (in the sense of physical chemistry) and applying “informatics techniques” (derived from disciplines such as applied moths, computer science, and statistics) to understand and organize the information associated with these molecules, on a large scale. In short, bioinformatics is a management information system for molecular biology and has many practical applications. In the last two decades, a huge mass of various large-scale molecular data has been generated using miscellaneous high-throughput technologies such as DNA/RNA microarray, whole-genome sequencing (WGS), RNA sequencing (RNA-seq), 2-dimensional electrophoresis (2-DE), etc. (C. International HapMap, 2003; Vizcaı´no et al., 2016; Apweiler et al., 2004; Auton et al., 2015). The large volumes of the molecular data have been deposited in biological databases. Some of the most important and popular databases include NCBI databases (https:// www.ncbi.nlm.nih.gov/), Ensemble (https://ensembl.org/index.html), GeneCards (https://www.genecards.org/), ArrayExpress (https://www.ebi.ac.uk/arrayexpress/), UniProtKB/Swiss-Prot (https://web.expasy.org/docs/swiss-prot_guideline.html), KEGG (http://www.genome.jp/kegg/), BioCarta (https://www.hsls.pitt.edu/obrc/ index.php?page 5 URL1151008585), Reactome (https://reactome.org/), etc. The bioinformatics databanks continuously and rapidly are being enriched (updated) (Reichhardt, 1999; Benson et al., 2000; Bairoch and Apweiler, 2000). For example, the GenBank repository of nucleic acid sequences increased from 11,545,572 entries on April 2001 to 209,775,348 entries on July 2018. Furthermore, SwissProt repository of protein sequences increased from 95,320 on April 2001 to 557,713 entries on June 2018. The number of samples deposited in gene expression omnibus (GEO), as a public functional genomics data repository, has reached to 2,546,985 (4348 datasets) on July 2018 (http://www.ncbi.nlm.nih.gov/geo/).

Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00016-3 © 2020 Elsevier Inc. All rights reserved.

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Traditional experimental/computational methods fail to handle and analyze these data. Hence, advanced computer sciences, mathematics, and statistics are used to derive meaningful inference from the large quantities of the complex biological data (Luscombe et al., 2001; Can, 2014; Kim, 2002). In other words, the bioinformatics emerged as a multidisciplinary approach employing molecular biology/genetics, computer science, mathematics, and statistics (Can, 2014; Altman, 1998). Traditional statistical approaches were vastly updated and adjusted to accurately interpret and disclose the complex nature of high-throughput data. For example, long-established Student’s t-test is frequently used in the biological experiments, but when the same is employed to compare expression level of thousands of genes measured using microarray technique in two groups of the patients (e.g., cancer patients vs healthy control ones), a large number of false positives result. This is termed as multiple testing problem in bioinformatics, and employing Bonferroni correction and Benjamini Hochberg procedure would fix it (McDonald, 2014). The bioinformatics functions divided into three entities. First, bioinformaticians create and develop databases, where the high-throughput data are stored and managed using precise scientific criteria. These databases are usually accessible and free to the investigators, where they can explore and download the desired data based on options provided in the databases. In addition, new findings resulting from the scientific investigations can be submitted to these databases and will be deposited, if defined criteria are met. The second goal of the bioinformatics is to produce and develop tools, methods, algorithms, and softwares used for analyzing and interpreting raw bioinformatics data. Obviously, in this way, tool/program developers should have enough knowledge of computational and programming sciences. Having a good understanding of cellular biology can have a positive effect on developing more useful and practical programs/algorithms. Finally, the third aim of the bioinformatics is to use such programs and tools for mining large-scale data deposited in the databases (Luscombe et al., 2001; Zaki et al., 2007). In comparison with the traditional experimental studies, which investigated a small part of cellular components, the bioinformatics studies can simultaneously explore and analyze many cellular components at a global level. In other words, the bioinformatics studies integrate the sparse molecular findings and provide a general and accurate view of complex physiological and pathological cellular events (Xu et al., 2014). The scope of the issues studied in the bioinformatics discipline is highlighted below: 1. Exploring and alignment analysis of DNA, RNA, and protein sequences. In such type of the bioinformatics analysis, DNA, RNA, or protein sequences are aligned to find possible structural, functional, or evolutionary associations (Mount, 2004). In general, the alignment analysis is performed in two major categories (i.e., global alignment and local alignment) (Polyanovsky et al., 2011). ClustalW2 (https://www.ebi.ac.uk/Tools/msa/clustalw2), T-coffee

16.1 Introduction

(https://web.archive.org/web/20080918022531/http://tcoffee.vital-it.ch/cgibin/ Tcoffee/tcoffee_cgi/index.cgi), BLAST (https://blast.ncbi.nlm.nih.gov/Blast. cgi), and FASTA3x (https://fasta.bioch.virginia.edu/fasta_www2/fasta_list2. shtml) are examples of well-known tools for alignment analysis. 2. Analysis of macromolecular structure (DNA, RNA, and protein) which can be named as “structural bioinformatics.” In this branch, three-dimensional structure of the macromolecules is predicted using various algorithms. Prediction of three-dimensional structure can be useful for studying binding interactions to selecting fitting targets, evolutionary analysis, X-ray crystallography trials, and assessment of associations between structural information and molecular functions (Gu and Bourne, 2009; Schwede and Peitsch, 2008). The protein data bank (PDB) (http://www.rcsb.org/) is an important database for the three-dimensional structural analysis of the macromolecules (Berman, 2008). 3. Evaluation of interactions of the biological components such as transcripts, proteins, and metabolites, which can be named as “systems biology.” In systems biology, the scientists aim to investigate structure and dynamics of the complex biological system at the molecular level using the computational and mathematical approaches. As simple examples, using the miscellaneous network reconstruction algorithms, gene regulatory networks, signaling networks or protein protein interaction networks can be constructed (Bansal et al., 2007; Blais and Dynlacht, 2005; Hasan and Kahveci, 2015; Sharafi et al., 2017). STRING (http://string-db.org/newstring_cgi/show_network_ section.pl), MiMI (Tarcea et al., 2009), and GeneMANIA (http://genemania. org/) are examples of well-known interaction databases. In systems biology, first quantitative assay of the biological components is performed and mathematical models are reconstructed based on these quantitative data. In addition, using these data, scientists can model how cells behave under various conditions or how different species respond and adapt to the miscellaneous conditions (Kirschner, 2005; Westerhoff et al., 2009). One of the most important and perhaps most important input data for the systems biology researches is omics data. In omics technology, pools of a biological component (e.g., genes, transcripts, proteins, metabolites, carbohydrates, or lipids) are quantified and processed. In other words, in omics technology, the molecules are measured and studied with a holistic view. Based on the investigated molecule, different terminology are applied: genomics (genes), transcriptomics (transcripts), proteomics (proteins), metabolomics (metabolites), glycomics (carbohydrates), lipidomics (lipids), interferomics (RNA interference), etc. (Horgan and Kenny, 2011; Manzoni et al., 2018). Given the importance of some types of omics data (i.e., genomics, transcriptomics, and proteomics) as major inputs for bioinformatics analyses, they will be dealt with in detail. 4. Some other branches of the bioinformatics include issues dealing with storing, sharing, and presenting of the large-scale molecular data, which are generated

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experimentally or computationally. Here, is a brief and concise description of the most popular omics technologies viz, genomics, transcriptomics, and proteomics.

16.1.1 Genomics Genomics is the study of the whole genome of an organism. In this analysis, after DNA sequencing, the function and structure of genome are analyzed using the advanced bioinformatics approaches. In humans, there are approximately 3 billion base-pairs coding B20,000 genes. Coding and noncoding regions include 1% 2% and 98% 99% of the genome, respectively. The noncoding regions function as structural and regulatory constituents (Manzoni et al., 2018; Venter et al., 2015; C. International Human Genome Sequencing, 2004). Information of DNA sequences is obtained using techniques such as shotgun sequencing and other high-throughput sequencing technologies. Using these technologies, first digital copy of human genome was published independently by two research groups supervised by Lander et al. (2001) and Venter et al. (2001) in 2001. Further, many protein-coding genes and genomic aberrations such as single nucleotide polymorphisms (SNPs) or segmental chromosomal instabilities were identified. In 2005, Lindblad-Toh et al. (2005) published the first report on complete canine genome of dog, which is considered as an ideal animal model to study human diseases (especially cancer). Chromosomal instabilities at the genome level fall into two major groups including simple nucleotide variations (SNVs) and structural variations (SVs). The SNPs are the main group of SNVs, while SVs compose of larger chromosomal aberations including chromosomal translocation, inversion, and insertion/ deletions (indels) (Gonzaga-Jauregui et al., 2012). Common methods for genomics studies consist of DNA-microarray (Bumgarner, 2013), Sanger sequencing (Sanger et al., 1977), and next-generation sequencing (NGS). WGS and whole exome sequencing are examples of NGS, which assess chromosomal instability in coding/noncoding regions and coding regions, respectively (van Dijk et al., 2014). Another technology used in the genomics studies is array-comparative genomic hybridization (aCGH), which is used to identify genomic copy number variants (CNVs). Originally, CGH technology has been developed to overcome the constraints of karyotyping or fluorescence in situ hybridization. With conjugation of DNA-microarrays and CGH technique, a new and more specific format of CGH (i.e., aCGH) was developed. In this technology, probes with variable sizes are designed to hybrid with the corresponding regions on the genome and the copy number alterations (CNAs) of the genomic regions were calculated at different resolutions (Kallioniemi et al., 1992; Weiss et al., 1999; Pinkel and Albertson, 2005). Some typical databases containing genomics datasets were summarized in Table 16.1. Furthermore, the researcher can browse the genome of various organisms using genome browser tools such as UCSC Genome Browser (https://

16.1 Introduction

Table 16.1 List of some important databases containing DNA sequence datasets. Name

Website address

A brief description

GenBank

http://www.ncbi.nlm.nih.gov/ genbank/

RefSeq

http://www.ncbi.nlm.nih.gov/ refseq/

EMBLEBI

http://www.ebi.ac.uk/ena

HGP

1000 Genomes 100,000 Genomes

http://www.genome.gov/ 11006929 http://hapmap.ncbi.nlm.nih. gov/ http://www.1000genomes. org/ http://www. genomicsengland.co.uk/

Annotated collection of all publicly available DNA sequences (NCBI-based). It is part of the of the international nucleotide sequence database collaboration. A comprehensive, integrated, nonredundant, well-annotated set of reference sequences including genomes, transcripts, and proteins. It is part of EMBL. When the EMBL-EBI moved to Hinxton it hosted two databases, one for nucleotide sequences (the EMBL data library, which was renamed EMBL-bank and eventually became part of the European nucleotide archive) and one for protein sequences (Swiss-Prot TrEMBL, now known as UniProt). Human genome project.

DDBJ

http://www.ddbj.nig.ac.jp/

HapMap

Haplotype map project. 1000 Genomes project. It is a sequence of 100,000 genomes from around 70,000 people including patients with rare diseases, their families and patients with cancer. DNA data bank of Japan.

EMBL, European molecular biology laboratory.

genome.ucsc.edu/), Ensembl (https://ensembl.org/index.html), and NCBI-Genome (http://www.ncbi.nlm.nih.gov/genome/).

16.1.2 Transcriptomics Perhaps, the transcriptomics is most practical omics technology. In this technology, using array chips, expression level of thousands of genes is quantified to obtain a general and holistic pattern of cellular transcription activities. The most practical method in transcriptomics is gene expression profiling (GEP) using RNA-microarray technology (Duggan et al., 1999). Affymterix, Illumina, and Agilent companies produce these expression GeneChips. For example, Affymetrix GeneChips for human and dog could assess expression levels of .54,000 and .42,000 transcripts, respectively. Another important technology in the transcriptomics is RNA-seq using NGS, which is more percise but costly in comparing

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with RNA-microarray. Since RNA-microarray chips are designed based on current genome information, expression level of some transcripts not included in the platform will be missed, while RNA-seq captures and sequences all possible RNAs in the cellular mixture. In both technologies, total RNA content of cell lysate is extracted and converted to complementary DNA (cDNA). cDNAs are hybridized with probes on microarray chips or are fragmented and sequenced in RNA-seq approach (Shendure, 2008; Cho and Chou, 2016; Nagalakshmi et al., 2010). The raw data generated from RNA-seq/microarray are analyzed using various bioinformatics tools/programs. In these analyses, expression pattern of transcripts (downregulation or upregulation), possible role of the genes as tumor suppressors, oncogenes, or regulators, role of the genes in cell cycle, and prognostic efficacy of genes are evaluated (Ben-Porath et al., 2008; Kim and Orkin, 2011). Through comparing expression pattern of the transcripts between tumor and healthy control samples, possible genes involved in oncogenesis can be detected (ZamaniAhmadmahmudi et al., 2015, 2016a). Molecular phenotyping and development of prognostic gene signatures (modules) in patients with cancer using transcriptome data have been greatly extended in recent investigations. Scientists are trying to develop new methods to subtype various cancers more precisely and more efficiently than traditional classification methods. Furthermore, they attempt to determine novel prognostic genes, which may efficiently predict the clinical outcome in patients with cancer compared to the routine prognostic factors (Zamani-Ahmadmahmudi et al., 2017a; Lossos et al., 2004; Alizadeh et al., 2000; Shaughnessy et al., 2007). There are some databases that save and maintain transcriptome data generated experimentally or computationally. GEO (http://www.ncbi.nlm.nih.gov/geo/), ArrayExpress (https://www.ebi.ac.uk/arrayexpress/), The Cancer Genome Atlas (TCGA) (https://cancergenome.nih.gov/), and Genotype-Tissue Expression (GTEx) (http://www.gtexportal.org/home) are three web resources containing sequence data generated from microarray and RNA-seq (Table 16.2). Furthermore, there are many programs/tools specifically designed for mining transcriptome raw data. Bioconductor is an open source of the bioinformatics sotfwares that contains many packages, which majorly run in R programming environment (Gentleman et al., 2004). Some of the most useful packages/web-tools include geWorkbench (Floratos et al., 2010), BRB-ArrayTools (developed by Dr. Richard Simon and the BRBArrayTools Development Team), and GenePattern (Reich et al., 2006). One of the common interesting features of these tools is that they do not require programming experience. Moreover, alongside the exploration of transcriptomics datasets, GenePattern and BRB-ArrayTools have many features to mine the genomics data.

16.1.3 Proteomics The term proteomics first appeared in 1997. Proteomics is the study of the entire set of proteins (proteome) of a cell line, tissue, or organism under a specific condition(s) (Kellner, 2000). There is little positive correlation between mRNA expression level and protein expression level. For example, a weak correlation

16.1 Introduction

Table 16.2 List of some important databases containing transcriptomics datasets. Name

Website address

A brief description

GEO

http://www.ncbi. nlm.nih.gov/geo/

ArrayExpress

https://www.ebi.ac. uk/arrayexpress/

TCGA

https:// cancergenome.nih. gov/

GTEx

http://www. gtexportal.org/ home/

GEO is an international public repository that archives and freely distributes microarray, NGS, and other forms of high-throughput functional genomics data submitted by the research community. ArrayExpress is one of the repositories to archive functional genomics data from microarray and sequencing platforms in compliance with MIAME and MINSEQE guidelines. TCGA, a collaboration between NCI and NHGRI, has generated comprehensive, multidimensional maps of the key genomic changes in 33 types of cancer. GTEx project aims to investigate the relationship between genetic variation and gene expression in 53 human tissues.

GEO, Gene expression omnibus; NGS, Next-generation sequencing; MIAME, minimum information about a microarray experiment; MINSEQE, minimum information about sequencing experiment; TCGA, the cancer genome atlas; NCI, national cancer institute; NHGRI, national human genome research institute; GTEx, genotype-tissue expression.

coefficient (0.48) was reported between mRNA and protein abundances in the human liver (Anderson and Seilhamer, 1997). This weak association can be attributed to the posttranscription editions on mRNA (such as RNA splicing) and posttranslational modifications (PTMs) (such as glycosylation, acetylation, methylation, biotinylation, phosphorylation, glycylation, etc.) on proteins’ structure (Alfonzo, 2014; Machnicka et al., 2013; Duan and Walther, 2015). Furthermore, unlike cellular genome, cellular proteome has an active and dynamic nature. However, we can conclude that the genomics and proteomics fields are different but complementary (Kellner, 2000). There are a number of techniques, namely mass spectrometry (MS), highperformance liquid chromatography, and 2-DE, which are used in the proteomics experiments. Unlike genomics and transcriptomics studies, proteomics counterparts are difficult to perform because of incomplete and inaccurate annotation of the proteins sequences and problems with technologies used for proteome analysis. For example, MS analysis counters several troubleshooting procedures (Pible and Armengaud, 2015; Bell et al., 2009). A typical proteomics process includes the steps below (O’Farrell, 1975; Klose, 1975): 1. Preparation of cell/tissue lystae using a lysis buffer; 2. Separation of proteins mixture in polyacrylamide gel according to isoelectric point (pI) using isoelectric focusing (IEF);

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3. Separation of proteins in second dimension according to the molecular weight using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method; 4. Staining of the resolved gel using coomassie brilliant blue or silver nitrate; 5. Excision of the interested spots on the gel and submission to identify suspected proteins using MS technology; 6. Bioinformatics analysis on the raw data resulted using MS. Proteomics studies are categorized into three parts including expression proteomics, structural proteomics, and functional (interaction) proteomics (Graves and Haystead, 2002). In expression proteomics, proteins expression is quantified and is compared between different groups (e.g., tumor vs healthy samples) or different conditions (e.g., different stages of a cancer). Techniques such as 2-DE and MS are employed in such investigations (Graves and Haystead, 2002; Chandramouli and Qian, 2009). One typical example of the expression proteomics is a molecular technique named serological proteome analysis (SERPA), which is used for detection of autoantibody in serum of patients with various diseases (especially patients with cancer). In this method, a combination of 2-DE, western blotting, and MS are employed (Klade et al., 2001; Nakanishi et al., 2006). Using this approach, some serum biomarkers are detected in patients with renal carcinoma (Klade et al., 2001; Kellner et al., 2002), human breast cancer (Hamrita et al., 2008), and canine breast cancer (Zamani-Ahmadmahmudi et al., 2014). In structural proteomics, the large-scale three-dimensional proteins structures are determined to clarify structure-function associations of uncharacterized gene products. X-ray crystallography and nuclear magnetic resonance spectroscopy are the usual technologies used in the structural proteomics (Chandramouli and Qian, 2009; Shin et al., 2008). Functional proteomics is a wide-ranging term for some specific proteomics approaches for analyzing biological functions of unknown proteins and unfolding pattern of protein complexes using protein protein interactions (Monti et al., 2007). Again, there are databases containing protein sequences, proteomics datasets, and three-dimensional protein structures (Table 16.3). Among them, Swiss-Prot/ Uniprot and RefSeq are the most important repositories for protein sequences and PRoteomics IDEntifications database (PRIDE) and ProteomeXchange (PX) are the main repositories for proteomics data. In addition, some packages have been developed to analyze 2-DE gels. They include Delta2D (http://www.decodon. com/), Melanie (http://www.expasy.ch/melanie/), PDQuest (http://www.bioradcom/), Progenesis (http://www.nonlinear.com/products/progenesis/samespots/overview.asp), and REDFIN (http://www.ludesi.com/redfin).

16.2 The investigation of the canine cancers using the omics data and bioinformatics methods: comparative aspects to human Nowadays, using the advanced molecular technologies, a big step toward a better diagnosis, prediction of outcome, and treatment of various cancers has been taken.

16.2 The investigation of the canine cancers

Table 16.3 List of some important databases containing protein sequences and proteomics datasets. Name

Website address

A brief description

RefSeq

http://www.ncbi.nlm.nih. gov/refseq/about/

UniProtKB/ Swiss-Prot

https://www.uniprot.org/ uniprot/

UniProtKB/ TrEMBL

https://www.uniprot.org/ uniprot/

PDB

http://www.rcsb.org/ pdb/home/home.do

PRIDE

https://www.ebi.ac.uk/ pride/archive/

PX

http://www. proteomexchange.org/

A comprehensive, integrated, nonredundant, well-annotated set of reference sequences including genomes, transcripts, and proteins. It is the manually annotated and reviewed section of the UniProtKB. It contains high quality annotated and nonredundant protein sequences. It contains high quality, computationallyanalyzed records that are enriched with automatic annotation and classification. Protein data bank (PDB) contains the threedimensional structural data of large biological molecules, such as proteins and nucleic acids. PRIDE is a public data repository for proteomics data, including protein and PTMs, and supporting spectral evidence. PX is a repository of submitted MS proteomics data. PRIDE is one of the founding members of ProteomeXchange.

UniProtKB, UniProt knowledgebase; PDB, Protein data bank; PRIDE, PRoteomics IDEntifications database; PTMs, posttranslational modifications; PX, ProteomeXchange; MS, mass spectrometry.

Although traditional pathology evaluations and some limited molecular techniques are used to diagnose cancers and determine patient prognosis, these methods have many disadvantages, which limit pathologists, oncologists, and clinicians’s efficacy in the clinical processes (Zamani-Ahmadmahmudi et al., 2017a; Lossos et al., 2004; Alizadeh et al., 2000; Shaughnessy et al., 2007; Decaux et al., 2008). The final goal of pathologic classification (subtyping) of the cancers is to help formulate better treatment protocol and to determine patient’s survival time, more precisely. For these reasons, because of limitations of pathologic assessments in many situations, the clinicians cannot accurately and efficiently follow the therapeutic procedure (Lossos et al., 2004; Decaux et al., 2008; Rosenwald et al., 2002). Considering the above problems, pathologic subclassification of the different cancers in human and small animals (especially dog) is being scientifically revised. For examples, subclassification of the lymphoma, according to morphology of the neoplastic lymphocytes, is performed based on three systems [i.e., The National Cancer Institute working formulation, the updated Kiel classification, and the revised European-American classification of lymphoid neoplasms (REAL)]. Both The National Cancer Institute Working Formulation and updated Kiel classification considers the architectural pattern of the lymph node and cellular morphology in their classifications. In addition, the updated Kiel classification also considers B or T lymphocyte origin as another classification criterion (Bienzle, 2011).

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Use of molecular indexes in the cast of tumor biomarkers is also a part of routine pathologic assessments of the cancers. For example, evaluation of the expression patterns of some useful biomarkers using immunohistochemistry in tumor tissues can serve as a helpful diagnostic and prognostic factor. Some cell proliferation biomarkers such as Ki67, agyrophilic nucleolar organiser region, and proliferating cell nuclear antigen are used as suitable prognostic parameters in canine malignant lymphomas, and human non-Hodgkin’s lymphomas (NHL). However, it seems that using a single biomarker rather than combination of multiple biomarkers has low sensitivity and specificity in diagnosing cancer at early stages (Kiupel et al., 1999; Janmohamed et al., 1990). For instance, when some biomarkers including CEA, CA15-3, and CA27-29 are used as single diagnostic biomarkers, they have low efficacy to diagnose patients with breast cancer (Tan et al., 2009). Given all difficulties with the custom clinical diagnostic/prognostic parameters described above, new high-throughput technologies such as DNA/RNA-microarray, WGS, RNA-seq, and findings of these methods (i.e., omics data) show promising results to diagnose the cancers at early stages, subtype them more precisely, and determine the survival time more accurately in patients with cancers. Alongside related studies conducted for human cancers, some investigations exploring animal cancers (especially the canine cancers) have been performed in smaller scale. A brief description about various cancers in dogs with a focus on omics studies investigating canine cancer biology at genome, transcriptome, and proteome levels are given below:

16.2.1 Various types of the canine cancers Similar to humans, diverse types of tumors also occur in dogs. The dog is considered as an ideal model of human cancer, because similar tumors in both species have comparable tumor biology, clinical manifestations, diagnosis protocol, and response to treatment. For example, canine B-cell lymphoma has been proposed as a suitable model of human NHL, where many similar clinical and molecular properties were reported in both tumors (Zamani-Ahmadmahmudi et al., 2015, 2016, 2017b; Zamani-Ahmadmahmudi, 2016a; Richards et al., 2013). Laboratory animals such as mice have some disadvantages, which reduced their use as ideal models of the diseases. Genetic homogeneity, impaired immunity, small size, and metabolic differences are examples of these disadvantages (Richards et al., 2013). Cutaneous tumor, mammary gland tumor, and NHL were reported as the most frequent cancers in dogs. Canine cutaneous histiocytoma, mastocytoma, and hemangiopericytoma are the most prevalent skin tumors (Dobson et al., 2002; Baioni et al., 2017; Merlo et al., 2008). In one study, frequency of tumors in female and male dogs was estimated to 271.1 and 99.3 cases per 100,000 dogs, respectively. In this study, the mammary tumors and the NHL had the highest incidence in bitches, and skin cancer and the NHL had the highest incidence in

16.2 The investigation of the canine cancers

male dogs (Merlo et al., 2008). Other tumors including melanoma, osteosarcoma, brain tumors, liver tumors, and kidney tumors rarely occurred in dogs.

16.2.2 Genomics studies in the canine cancers In 2005, Lindblad-Toh et al. (2005) introduced the first high-quality draft genome sequence of the domestic dog (Canis familiaris). In this draft, the map of SNPs in different canine breeds was also provided. One of the techniques, which is frequently used to detect DNA CNAs in canine genomics studies, is aCGH. Differentiation of acute myeloid leukemia from acute lymphocytic leukemia (ALL) or chronic myeloid leukemia from chronic lymphocytic leukemia (CLL) has been performed using CNAs model with 83.3% and 95.8% precision, respectively. Some common CNAs (i.e., gain of HSA 12 and loss of HSA 13q14 in CLL and gain of HSA 21q in ALL) between human and canine leukemia were reported (Roode et al., 2015). Gain of dog chromosomes 13 and 31 and loss of chromosome 14 were the most common aberrations observed in canine multicentric lymphomas (Thomas et al., 2003). In canine hemangiosarcoma, the rate of DNA aberrations is low in comparison with other canine sarcomas. The loss of chromosome 16 and the gain of dog chromosomes 13, 24, and 31 are the main chromosomal instabilities in the canine hemangiosarcoma (Thomas et al., 2014). In canine transitional cell carcinoma (TCC), gain of chromosomes 13 and 36 and loss of chromosome 19 were detected in up to 84% of the studied cases. It has been suggested that these three chromosomal aberrations can be used as a diagnostic factor for canine TCC (Shapiro et al., 2015). Again, a significant number of shared CNAs were detected in the canine TCC and human counterpart (Shapiro et al., 2015). The most prevalent CNAs in canine melanoma include loss of chromosome 22 and gain of dog chromosomes 13 and 17 (Poorman et al., 2015). The presence of four CNAs in canine mast-cell tumors could find the aggressive phenotypes with sensitivity of 78% 94% (Mochizuki et al., 2017). Wide chromosomal aberrations on the entire genome of the dogs with canine transmissible venereal tumor (CTVT) were obtained, where these abnormalities majorly occurred in centromeric and telomeric regions (Thomas et al., 2009b). Comparative chromosomal aberrations analysis of the human NHL and the canine NHL revealed recurrent DNA copy number aberrations on human chromosomes 8 and 21 and dog chromosomes 13 and 31 which shared similar properties (Thomas et al., 2011). As presented above, two important findings can be concluded from aCGH analyses performed on the different canine cancers. First, the dog chromosome 13 aberrations are frequently reported in different canine tumors such as blood malignancy (Roode et al., 2015), hemagniosarcoma (Thomas et al., 2014), TCC (Shapiro et al., 2015), melanoma (Poorman et al., 2015), and NHL (Thomas et al., 2011). Second, CDKN2A gene is frequently aberrated in various types of the canine cancers viz. hemagniosarcoma (Thomas et al., 2014), TCC (Shapiro et al., 2015), melanoma (Poorman et al., 2015), and histiocytic sarcoma (Hedan et al., 2011).

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CNAs in chromosomal locus of some well-known oncogenes or tumor suppressors were studied. CNAs involving CDKN2A [hemagniosarcoma (Thomas et al., 2014), TCC (Shapiro et al., 2015), melanoma (Poorman et al., 2015), and histiocytic sarcoma (Hedan et al., 2011)], VEGFA [hemagniosarcoma (Thomas et al., 2014)], c-MYC [melanoma (Poorman et al., 2015)], RB1 [histiocytic sarcoma (Hedan et al., 2011)], PTEN [osteosarcoma (Angstadt et al., 2011), histiocytic sarcoma (Hedan et al., 2011)], KIT [mast-cell tumor (Mochizuki et al., 2017)], RUNX2 [osteosarcoma (Angstadt et al., 2011)], and TUSC3 [osteosarcoma (Angstadt et al., 2011)] were revealed. Other genomics technologies were employed to investigate the canine cancer in rare situations. For examples, NGS of DNA was employed to investigate hemangiosacroma, osteosarcoma, lymphoma in dogs. These three types of tumors were considered as sarcoma tumors to investigate the efficacy of new immunotherapy agents such as eBAT [an angiotoxin consisting pseudomonas exotoxin fused to enhanced green fluorescent (EGF)]. The results revealed that canine sarcoma tumor could be an ideal immunotherapy model for the human counterpart (Borgatti et al., 2017). WGS and detection of genomic abnormality using SNP analysis has been performed on canine CTVT (Decker et al., 2015) and dogs with the mammary gland tumors (Liu et al., 2014). DNA sequence of canine breast cancer indicated that simple carcinoma subtype (corresponding to human breast carcinomas) contains more genomic aberrations compared to the complex carcinoma (rare subtype in human). These findings suggested that the canine complex carcinoma is majorly controlled by epigenetic events, but not genetic regulatory elements. Bioinformatics analysis of four canine cancers viz. mammary gland tumor, colorectal cancer, leukemia, and osteosarcoma revealed that canine miRNAs are majorly located in the aberated regions of canine cancers. In addition, incidence of miRNAs was more frequent in CpG islands than all the other regions (incidence rate ratio 5 12.88) (Zamani-Ahmadmahmudi, 2016a).

16.2.3 Transcriptomics studies in the canine cancers Transcriptome studies were performed in the canine cancers majorly aiming to compare GEP and molecular pathways in dogs and humans, to establish molecular subtyping of canine tumors or to detect prognostic gene(s). Though RNA microarray is the most commonly used in canine transcriptome analyzes, NGS of RNA was also performed on some canine sarcoma tumors [i.e., lymphoma, osteosarcoma, and hemangiosarcoma) (Borgatti et al., 2017), canine mammary cancer (Liu et al., 2014), and canine meningioma (Grenier et al., 2017)]. Analysis of comparative gene expression pattern was performed in dogs with the mammary gland tumor (Uva et al., 2009; Klopfleisch et al., 2010a, 2011), osteosarcoma (Selvarajah et al., 2009; Paoloni et al., 2009; Fowles et al., 2016; Zamani-Ahmadmahmudi et al., 2018), invasive urothelial carcinoma (iUC) (Dhawan et al., 2015), and lymphoma (Zamani-Ahmadmahmudi

16.2 The investigation of the canine cancers

et al., 2015, 2016, 2017b; Richards et al., 2013; Mudaliar et al., 2013). In some studies, a great degree of similarities between cancer-related pathways in tumors of both species was suggested. Pathways including phosphatase and tensin homolog (PTEN), PI3K/AKT, WNT-beta catenin, MAPK cascade, and KRAS in canine and human breast cancers (Uva et al., 2009), NF-κB pathway in the canine and human NHL (Richards et al., 2013; Mudaliar et al., 2013), integrin signaling, chemokine/cytokine signaling, and Wnt signaling in canine and human osteosarcoma (Selvarajah et al., 2009) showed similar expression patterns. In addition, the bioinformatics analysis by means of gene-set enrichment analysis show that embryonic stem cell (ES) gene expression signatures viz. nitric oxide synthase (NOS) targets, Myc targets, ES-expressed, and Polycomb targets have similar expression patterns in the canine and human mammary gland tumor (Zamani-Ahmadmahmudi, 2016b) and osteosarcoma (Zamani-Ahmadmahmudi et al., 2018). Differentially-expressed genes in the different canine cancers are mostly associated with cell cycle pathways (Klopfleisch et al., 2010a, 2011; Selvarajah et al., 2009; Giantin et al., 2014; Tamburini et al., 2010), matrix modulation (Klopfleisch et al., 2010a, 2011; Tamburini et al., 2010), DNA replication (Giantin et al., 2014), p53 signaling pathway (Dhawan et al., 2015; Giantin et al., 2014), inflammation (Tamburini et al., 2010), angiogenesis (Klopfleisch et al., 2011; Tamburini et al., 2010), adhesion (Klopfleisch et al., 2011; Tamburini et al., 2010; O’Donoghue et al., 2010), and invasion (Klopfleisch et al., 2011; Tamburini et al., 2010). Many studies focused on prognostic efficacy of GEP. Prognostic genes — IGF2, ADHFE1, CCDC3, SCN1B, AGTR1, NDRG2, FBP1, and IMP1 in canine osteosarcoma (O’Donoghue et al., 2010), FOXM1, GSN, FEN1, and KPNA2 in canine mast tumor (Giantin et al., 2014) and IL-8 and SLC1A3 in canine osteosarcoma (Paoloni) were reported. Using bioinformatics and molecular phenotyping approaches, canine B-cell lymphoma was classified into two molecular subtypes [i.e., activated B-cell (ABC) and germinal center B-cell] with different survival times (Richards et al., 2013). In the same way, Frantz et al. (2013) categorized canine lymphoma into the three molecular subtypes —namely high-grade T-cell lymphoma, low-grade T-cell lymphoma, and B-cell lymphoma, where the clinical outcome in three molecular subgroups was prognostically significant (Frantz et al., 2013). Using the bioinformatics analysis and experimental verifications, prognostic efficacy of 36 human B-cell lymphoma prognostic genes were tested in canine B-cell lymphoma microarray datasets. The results revealed that two genes—namely CCND1 and BIRCS5 could strongly predict the clinical outcome in the studied cases (Zamani-Ahmadmahmudi et al., 2017b). Similarly, on reconstruction of gene regulatory network in canine B-cell lymphoma, it has been found that CCND1 and FOS genes were significantly associated with overall survival and progression-free survival (Zamani-Ahmadmahmudi et al., 2016). Some gene signatures (gene module) can be used as prognostic signatures in canine osteosarcoma and mammary gland tumor. The investigation showed that high

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expression of the ES gene signatures including ES expressed, NOS targets, and Myc targets are associated with short survival, while high expression of Polycomb targets are associated with long survival in dogs with a mammary gland tumor (Zamani-Ahmadmahmudi, 2016b). Also, NOS targets were suggested as a prognostic gene signature that efficiently predicted the clinical outcome in humans and dogs with osteosarcoma (Zamani-Ahmadmahmudi et al., 2018). Furthermore, molecular expression pattern can be used to distinctly identify luminal and basal subtypes of canine iUC (Dhawan et al., 2015).

16.2.4 Proteomics studies in the canine cancers Some of the canine cancers were investigated using bioinformatics approaches and proteomics technologies to identify the proteins involved in cancer biology and prognosis. Zamani-Ahmadmahmudi et al. (2013, 2014) tried to find cancer antigens that elicit immunological responses and result in production of autoantibody in dogs with breast cancer using SERPA method. They have found four serum biomarkers (autoantigens), namely manganese-superoxide dismutase, triose phosphate isomerase, alpha-enolase (ENO1), and phosphoglycerate mutase1; which trigger autoantibody response. These serum biomarkers were previously suggested as important biomarkers in human breast cancer. Using 2-DE and matrix-assisted laser desorption/ionization time-of-flight tandem (MALDI-TOF) MS, proteins expression pattern was compared between the dogs with and without breast cancer metastasis. Overexpression of thioredoxin-containing domain C5, coronin 1A, proliferating cell nuclear antigen, adenosin, and bompain and downregulation of periredoxin 6, isocitrate dehyrogenase, maspin, calretinin, and Vinculin occurred in cases with the metastasis (Klopfleisch et al., 2010b). Through comparing proteins expression pattern in the dogs with B-cell lymphoma and healthy control using 2-DE and MALDI-TOF, upregulation of macrophage capping protein and downregulation of triosephosphate isomerase, glutathione S-transferase, and prolidase (proline dipeptidase) in tumor samples were detected (McCaw et al., 2007). Surface-enhanced laser desorption ionization time-of-flight MS were employed to detect serum biomarkers in canine lymphoma samples (Ratcliffe et al., 2009; Gaines et al., 2007). Tow serum biomarkers with mass spectral peaks of m/z value 7041 and 74 726 Da were proposed as factors, which can differentiate lymphoma and nonlymphoma samples with sensitivity of 75%, specificity of 91%, and positive predictive value (PPV) of 80% (Ratcliffe et al., 2009). Elevated expression of α2 globulins such as haptoglobin, α-antichymotrypsin, α2-macroglobulin, and inter-α-trypsin were shown in sera of the dogs with multicentric lymphoma (Atherton et al., 2013). Expression of endoplasmin, keratin 7, and GRP78 was significantly higher in carcinoma samples compared with normal prostate or bladder tissues (LeRoy et al., 2007). Furthermore, increase expression of TCP1A, TCP1E, PDIA3, HSPA9, ANXA6, ACTR3, and WDR1 in high-grade canine cutaneous mast-cell tumors (MCTs) compared with low-grade ones was identified (Schlieben et al.,

16.4 Genomics, transcriptomics, proteomics

2012). Overexpression of JMJD1C in oral benign tumors, BTBD16 in nontonsillar oral squamous cell carcinoma, ARHGEF28 and INVS in oral melanoma, and BRCA2, WBP2, PTPN1, PSME4, and P2RY1 in all types of oral cancers were previously reported (Pisamai et al., 2018).

16.3 Bioinformatics and omics data in the cancers of other domestic animals Analysis of the various cancers in other domestic animals such as cats, horse, and cattle using advanced omics technologies and subsequent bioinformatics examination has been carried out on a lesser scale compared to canine counterparts. Using CGH method, a wide range of recurrent genomic instabilities were detected in genome of cats with injection-site-associated sarcoma (ISAS). Two deletion aberrations were notably correlated with spontaneous sarcomas (nonISAS) phenotypes (Thomas et al., 2009a). Furthermore, recurrent amplifications of the KIT proto-oncogene and homozygous deletions of PTEN in feline fibrosarcoma were reported (Thomas, 2015). In contrast, trivial genomic aberrations in feline gastrointestinal lymphoma detected using microarray-based CGH analysis were nonrecurrent, and included deletion of PTEN and amplification of the MYC proto-oncogene (Thomas, 2015). A moderate number of CNAs in a cohorts of cats with mammary tumor were obtained. Gain of ERBB2 and PTEN deletion were showed in high-grade feline mammary tumors (Ressel et al., 2009). Via genome-wide association study and EquineSNP70 BeadChip, a missense mutation in damage-specific DNA binding protein 2 (DDB2) was detected in horses with squamous cell carcinoma (Bellone et al., 2017).

16.4 Genomics, transcriptomics, proteomics, and bioinformatics approaches to investigating the other animal diseases: a brief description Genomics research performed in livestock (farm animals) has a major focus on animal breeding for improving production/reproduction and detecting genetic locus of resistance to infectious diseases (Dekkers, 2012; Kadarmideen, 2014; Bishop and Woolliams, 2014; Adam, 2018) with a little attention to studying the pathogenesis of a specific disease. Our core goal in this chapter is to present the application of omics technologies and bioinformatics approaches in the animal cancers (especially the canine cancers). Given vast ranges of animal species and enormous areas of physiologic and pathologic conditions studied using omics and bioinformatics technologies, the readers are referred to (Dekkers, 2012; Kadarmideen, 2014; Bishop and Woolliams, 2014; Adam, 2018) the below links describing application of various genomics and transcriptomics technologies in

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normal and pathologic conditions in the different animals (especially domestic animals). https://www.ncbi.nlm.nih.gov/gds/?term 5 cattle (Filters: Manage Filters: Bos taurus); https://www.ncbi.nlm.nih.gov/gds/?term 5 horse (Filters: Manage Filters: Equus caballus); https://www.ncbi.nlm.nih.gov/gds/?term 5 dog (Filters: Manage Filters: Canis lupus); https://www.ncbi.nlm.nih.gov/gds/?term 5 feline (Filters: Manage Filters: Felis catus); https://www.ncbi.nlm.nih.gov/gds/?term 5 Ovis%20aries (Filters: Manage Filters: Ovis aries); https://www.ncbi.nlm.nih.gov/gds/?term 5 goat (Filters: Manage Filters: Capra hircus); https://www.ncbi.nlm.nih.gov/gds/?term 5 gallus (Filters: Manage Filters: Gallus gallus). Various proteomic approaches are used to explore pathogenesis of animal diseases. Some of the important diseases studied using proteomics methods in different animal species include: leishmaniosis, progressive glomerular disease, European adder bite, meningoencephalitis, weight loss program, and idiopathic pulmonary fibrosis in dog; urinary tract diseases and azotemia in cat; subclinical and clinical mastitis, mastitis caused by Staphylococcus aureus/Escherichia coli/ lipopolysaccharide, subclinical mycobacterial infection, bovine respiratory disease, stress, and paratubercolosis in cattle and small ruminants; osteoarthritis and osteochondritis, chronic equine laminitis, and spontaneous equine recurrent uveitis in horse; and food and mouth disease, stress, classical swine fever, porcine reproductive and respiratory syndrome, and peritonitis-induced sepsis in pig. Further discussion about this matter can be found in Ceciliani et al. (2014).

16.5 The future role of the bioinformatics and omics data in studying animal diseases (especially the cancers) With the advent of flood of omics data, bioinformatics methods/programs are being developed to handle the omics data more robustly and more efficiently. By mining various databases containing diverse biological datasets, one can browse, download, upload, and analyze a huge number of various human cancer types. In addition to omics data, many clinical metadata are also available for some cancer types. As an example, TCGA (https://cancergenome.nih.gov/) contains 2.5 petabytes of data from more than 11,000 patients with 33 human cancer types. For each cancer types, raw sequencing data, transcriptome-profiling data, SNVs, CNVs, DNA-methylation profiling, and clinical metadata have been provided. In

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comparison, many of these databases contain rare and incomplete omics datasets containing the animal cancer samples. Among various animal species, datasets including omics data in canine cancers reach an acceptable rate, but this volume of data is still subtle compared with the human datasets. In other animals (especially domestic animals), oncomics data is very rare and spare in corresponding resources such GEO, ArrayExpress, and GTEx. The hope is that with extending and developing of bioinformatics/omics databases containing larger animal samples and datasets, more robust studies will be conducted, resulting in more realistic and accurate findings. Another issue is problems occurring during working with many bioinformatics tools, programs, and packages in animal studies. Indeed, most of the bioinformatics programs are originally developed based on default set for use with human samples. Hence, in many situations, researchers cannot use programs and should try another one. As an example, none of packages developed to analyze aCGH raw data cannot upload data of canine cancers for subsequent CNAs analysis. Hence, some modifications should be performed on these programs prior to the analysis, if possible (Zamani-Ahmadmahmudi, 2016a). Likewise, entire genome of animals cannot be viewed and explored using current softwares [e.g., the integrative genomics viewer (Robinson et al., 2011)]. Poor annotation of chromosomal regions and gene locus in online/offline genome browsers tools such as UCSC Genome Browser (https://genome.ucsc.edu/) and NCBI-Genome (http:// www.ncbi.nlm.nih.gov/genome/) is a great dilemma in comparative genomics studies (Zamani-Ahmadmahmudi, 2016a). Clearly, development of animal compatible tools, programs, and packages will facilitate and accelerate the bioinformatics investigations in animal cancers and other diseases.

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Biotechnological approaches to fish vaccine

17

Megha Kadam Bedekar1, Sajal Kole1,2 and Gayatri Tripathi1 1

Aquatic Environment and Health Management Division, ICAR-Central Institute of Fisheries Education, Mumbai, India 2 Department of Aqualife Medicine, Chonnam National University, Gwangju, Republic of Korea

17.1 Introduction Vaccination is one of the most efficient and economical remedial measures that has contributed to the significant reduction of disease outbreaks and antibiotics usage in aquaculture. However, production and licensing of effective fish vaccines are limited due to the scarcity of knowledge elucidating immunological mechanisms of vaccine protection. Improving vaccines implies the detailed understanding of the fish immune system, rapid identification of the specific antigen, proper formulation of antigen, use of adjuvants, and employing methods of administration that are more effective and less harmful to the fish. The historical approach for developing fish vaccines was based on the principle of Louis Pasteur’s “isolate, inactivate, and inject” (Zhao et al., 2014). Although, this method was effective for developing a vaccine against some fish pathogenic bacteria, its utility faced major obstacles for developing a vaccine against most other fish pathogens especially viruses. In addition to this, the difficulty in administrating vaccine to a large number of fish of variant sizes for providing long-term protection is the other major impediment against the success of fish vaccinology. To this cause, biotechnological advancements and its applicability in the field of basic and applied research comes as a boon for fish vaccinologists as it paved newer avenues for designing novel and effective vaccines as well as developing easy, nonstressful, and cost-efficient delivery methods. In view of the above background, this chapter briefly reports the usage of biotechnology in the areas of fish vaccinology—a paradigm shift from traditional vaccine to new generation vaccine with the help of biotechnology.

17.2 Biotechnology in developing new generation vaccines As mentioned earlier the traditional approach for developing fish vaccines was based on inactivation of whole cell bacteria followed by administration by Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00017-5 © 2020 Elsevier Inc. All rights reserved.

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immersion or injection route. But apart from a few successful bacterial vaccines, the traditional vaccine formulation proved to be weak and could not induce the desired protective immune response. Also, due to the emergence of a wide range of fish pathogens showing wide antigenic variations, different host invasion, and immune evasion mechanisms, the traditional approach seems to be inappropriate. Furthermore, the in vitro propagation of some microbes is not possible, and development of vaccines against these pathogens is a slow and time-consuming process, which sometimes poses difficulty in timely countering of emerging and reemerging pathogens. Purposively, novel methods were needed for discovering newer types of effective vaccines that can be developed from the advances made in genetics, immunology, chemistry, biotechnology, and molecular biology (Delany et al., 2014; Finco and Rappuoli, 2014; Singh et al., 2015). In this context, gene sequences of bacterial, viral, and metazoan genomes, combined with the knowledge on gene functions and derived proteins have evolved as the novel methods for fish vaccination which are described in the following section and also summarized in Table 17.1 and Fig. 17.1.

17.2.1 Recombinant vaccines The emergence of biotechnology has led to the development of recombinant vaccines where only the immunogenic target regions of a pathogen are used as vaccine antigens and expressed in a heterologous host from which the protective antigen is purified and used in vaccine formulation (Adams et al., 2008). Biotechnological advances have helped in recognition of the gene sequence of pathogen’s protective antigen for designing a recombinant protein. After recognition, the antigens can be inserted in prokaryotic (Noonan et al., 1995) and eukaryotic (Lecocq-Xhonneux et al., 1994) production hosts and can be cultured on a large scale under strictly controlled laboratory conditions by fermentation methodology. The production hosts range from bacteria (Noonan et al., 1995), cell culture (Acosta et al., 2006), yeast (Vakharia, 2008), insect cells (Lecocq-Xhonneux et al., 1994), microalgae as well as transgenic plants (Muktar et al., 2016). Although this technology was found to be very useful in providing protection against various kinds of human and animal pathogens (Diane Williamson et al., 1995; Wilhelm et al., 2006), in the case of fish vaccine, the administration of the recombinant antigens produced through fermentation has found to be inefficient in inducing protective immunity, which might be due to poor immunogenicity of the antigens (Leong et al., 1997; Lorenzen and Olesen, 1997). Thus for developing a protein antigen-based vaccine, immunoproteomics provides a viable alternative (Connolly et al., 2006). Characterization of multiplex protein compounds by 2D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis helps in gaining valuable data regarding the expressed proteins of bacterial pathogens (Chen et al., 2004). In addition, western blot analysis of sera collected from infected fish or from survivor fish helps in revealing the most immunogenic antigen which are easily recognized by host
immune system

Table 17.1 Next generation vaccines in aquaculture. Type of vaccine

Antigens

Name

Fish host

Delivery methods

Remarks

References

Recombinant protein

Infectious pancreatic necrosis virus (IPNV)/ VP2 Spring viraemia of carp virus (SVCV) Salmon rickettsiae

Microtek

Salmon

IP injection

International Inc. Pharos, S.A., Bayovac 3.1 In vitro

carp

IP injection

Safe and low cost method but faces obstacles in glycolysation of the proteins and restoration of the tertiary structure

de Kinkelin (1994), Dhar and Allnutt (2011), Adams and Thompson (2006)

Salmonid

IP injection

Salmon

IP injection

In vitro

Salmon

IP injection

Biosafety production, induction of apoptosis in infected cells but lack of data regarding field performance

Wolf et al. (2012), Adams and Thompson (2006)

In vitro Brivax II

Rainbow trout Rainbow trout

IP injection IP injection

In vitro

Ictalurus punctatus

IP injection

Liu et al. (2015), Lawrence and Banes (2005)

Aqua Health Ltd Novartis In vitro

Salmon

IM injection

Rainbow trout Rainbow trout

IM injection IM injection

Induction of cell mediated, humoral and mucosal immunity but carry the possibility of back-mutation to virulent strains Induces both cellular and humoral immunity but limiting their potential use as genetically-modified organism (GMO)

In vitro

Salmonids

IM injection

In vitro

European sea bass

Oral

Vector technology

Genetically attenuated pathogen

Infectious salmon anemia virus (ISAV) Infectious hematopoeitic necrosis virus (IHNV) IPNV Aeromonas salmonicida Edwardsiella ictaluri

DNA vaccine

IHNV IPNV Pancreatic Disease (PD) Viral hemorrhagic septicemia virus (VHSV) Nervous necrosis virus (NNV)

LaPatra et al. (2001), Meeusen et al. (2007), Kurath (2008), Ballesteros et al. (2014), Valero et al. (2016)

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CHAPTER 17 Biotechnological approaches to fish vaccine

FIGURE 17.1 Next-generation vaccines in aquaculture.

(Chen et al., 2004). Both these approaches can be applied in the reorganization of potential vaccine candidates against fish pathogens. Another important aspect for the development of a vaccine is the ability of large-scale production of antigenic proteins required for commercialization of the vaccine. The recombinant DNA technology is widely used for this purpose due to its economical viability to cultivate adequate quantities of the immunoprotective antigens (Sun et al., 2009; Wilhelm et al., 2006). These vaccines not only yield an alternative pathway to conventional formalin-killed vaccines but also are safe and economical in comparison to live attenuated bacteria-based vaccines, which may revert to pathogenic agents (Clark and Cassidy-Hanley, 2005). The first subunit vaccine in aquaculture was developed for rainbow trout (Oncorhynchus mykiss) wherein, infectious hematopoeitic necrosis virus (IHNV) glycoprotein (G) was expressed in Escherichia coli and purified as vaccine (Gilmore et al., 1988). During the same time, a Belgium-based company, Pharos, S.A., developed a vaccine against carp virus by including recombinant G protein of carp virus (spring viraemia) expressed in baculovirus expression system. Several other studies also reported the effectiveness of recombinant protein vaccine against viral hemorrhagic septicemia virus (VHSV) (Lorenzen et al., 1998), Ichthyophthirius multifiliis (Dickerson and Findly, 2014; He et al., 1997), Aeromonas hydrophila (Poobalane et al., 2010), grouper nervous necrosis virus (GNNV) (Liu et al., 2006; Tanaka et al., 2001), Piscirickettsia salmonis (Wilhelm et al., 2006), and grass carp reovirus (He et al., 2011) in inducing protective immune responses against respective pathogens. The success story of the recombinant vaccine technology in fish

17.2 Biotechnology in developing new generation vaccines

vaccinology lies in the successful development and commercialization of different vaccines against important fish diseases such as., recombinant vaccine comprising of infectious pancreatic necrosis virus (IPNV),’VP2-based vaccine from Microtek International (Canada); infectious salmon anemia virus (ISAV) hemagglutininesterase gene-based vaccine from Centrovet (Chile) (Frost and Ness, 1997); IPNV-VP2 and IPNV-VP3-based oral (marketed as AquaVac and IPN oral manufactured from Merck Animal Health, New Jersey, United States); and intraperitoneal injection (marketed as Norvax and Minova-6 from Intervet-International BV, Netherland) vaccines (Dhar et al., 2014).

17.2.2 Vector technology Similar to the recombinant technology, vector technology implies the same principle for vaccine production but utilizes mainly viral production hosts which express the protein of another pathogen as a vaccine antigen (Adams et al., 2008). The self-assembling ability of viral structural proteins into vectors with the resemblance of a native virus has resulted in the development of this class of subunit vaccine based on virus-like particles (VLPs) (Dhar and Allnutt, 2011). The baculovirus expression system has proven to be an improved approach for fast expression of plentiful recombinant proteins (VLPs) and is suggested to be an inexpensive and efficient method for producing heterologous proteins (Adams and Thompson, 2006; Hu et al., 2008; Shivappa et al., 2004). Baculovirus (family Baculoviridae) are large double-stranded enveloped viruses consisting of circular DNA genomes (80
180 Kbps) and are considered good protein expression systems using insect cell lines. Several researches have demonstrated partial to complete protection against IHN (Laurent et al., 1994), VHS (de Kinkelin, 1994), IPNV (Shivappa et al., 2004), and GNNV (Lu et al., 2011) by using baculovirusexpressed antigens. The capsid protein VP2 of IPNV-based VLPs vaccines is already being marketed by three manufacturers namely Norvax (IntervetInternational BV, The Netherlands), IPNV (licensed in Chile, Centrovet, Chile), and Salmonid Rickettsial Syndrome (SRS)/IPNV/Vibrio (licensed in Canada and Chile, Microtek International Inc., BC, Canada) (Dhar et al., 2014). Apart from baculovirus, alphavirus expression system is a novel and beneficial tool in vaccine development as alphavirus-based replicon has the advantage of the fact that it does not spread/recombinant to other cells after initial replication (Olsen et al., 2013; Wolf et al., 2012). Furthermore, another fascinating property of the particle of alphavirus replicon is its potent ability to improve mucosal immunity (Chen et al., 2002). In the alphavirus-replicon system, the virus structural genes of the 30 -open reading frame are replaced by the gene of interest (GOI) which become capable of expressing the GOI when introduced into cells (Olsen et al., 2013) and may produce 200,000 RNA copies of GOI RNA molecules (Strauss and Strauss, 1994). For developing a fish vaccine, salmonid alphavirus (SAV) replicon vectors are used commonly as these vectors are functional in cells from a wide range of animal classes and express GOI in the temperature

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range of 4 C
37 C (Biacchesi, 2011; Olsen et al., 2013). Vaccine against ISA for Atlantic salmon (Salmo salar) has been reported by Wolf et al. (2012) wherein SAV-based replicon expressing the ISAV hemagglutinin-esterase was used as a candidate vaccine.

17.2.3 Genetically attenuated pathogens The advent of genetic engineering has helped in the production of live-attenuated vaccines which are typically done by deletion, disruption, or insertion of the metabolic pathway or virulence gene that causes attenuation in pathogen (Meeusen et al., 2007; Shoemaker et al., 2009). The resulting mutant pathogens then work as an avirulent pathogen inducing a protective immune response in a host without causing any disease (Adams et al., 2008; Liu et al., 2015; Ma et al., 2010). Although this approach can be effective, its related safety concerns to the vaccinated animals as well as to the environment have been the legitimate reason for unacceptance of these vaccines in aquaculture (Marsden et al., 1998). Nevertheless, several researchers have employed technologies like genetic alteration in LPS, transposon mutagenesis, marker-less deletion system, etc. in developing attenuated live vaccines against Edwardsiella ictaluri (Lawrence and Banes, 2005; Lawrence et al., 1997), Flavobacterium columnare (Klesius and Shoemaker, 1999; Shoemaker et al., 2009), Edwardsiella tarda (Igarashi and Iida, 2002), A. hydrophila (Leong et al., 1997), Streptococcus iniae (Buchanan et al., 2006), and Vibrio anguillarum (Liu et al., 2015).

17.2.4 Vaccines based on naked DNA (DNA vaccines) Use of immunogenic genes via DNA or RNA constitutes the next-generation immunization approaches of scientific improvement following the prophylactic or therapeutic administration of recombinant proteins (Dhama et al., 2008; Gillund et al., 2008; Heppell and Davis, 2000). DNA vaccination or nucleic acid immunization entails the delivery of plasmid DNA (raised in microorganisms such as bacteria) encoding a vaccine antigen to the host which in turn expresses inside the recipient under the control of eukaryotic promoters. The expressed antigenic proteins are recognized by the host
immune system, inducing strong and longlasting humoral and cell-mediated immune responses without the risk of inadvertent infection. DNA-based immunization has attained wide attention in their utility to promote protective immunity against numerous diseases in fish (Donnelly et al., 1996; Ogas Castells et al., 2015; Robertsen et al., 2016). To its advantage, DNA vaccines are stable in dried powder or in a solution and do not need a cold chain unlike traditional vaccines (Ballesteros et al., 2014; Meeusen et al., 2007). Another benefit comprises of the organization of a vector encoding numerous antigens that could be given in a single administration, and thus creating the possibility of a multivalent vaccine for multiple diseases (LaPatra et al., 2015; Lorenzen et al., 2002). Also, DNA vaccines are relatively cheap and are easy to

17.3 Conclusion

produce via identical production processes (Hølvold et al., 2014). Among various DNA vaccines developed against fish pathogens, the intramuscular injection of rhabdoviral (IHNV and VHSV) genes encoding surface glycoproteins are found to evoke a high level of protection due to the inherent capability of rhabdovirus G proteins to promote antiviral responses (Anderson et al., 1996; Hølvold et al., 2014; Lorenzen et al., 1998; Purcell et al., 2006). However, DNA vaccines against other important viruses such as the IPNV with the usage of VP2 gene have been reported to induce different levels of protection (Cuesta et al., 2010; de las Heras et al., 2009, 2010). Oral delivery of chitosan-encapsulated DNA vaccine against nodavirus [nervous necrosis virus (NNV)] has also been successfully developed for protecting European sea bass juveniles against NNV and improving their survival post infection (Valero et al., 2016). Several authors have also developed successful DNA vaccine against fish bacterial pathogens such as V. anguillarum, E. tarda using the genes encoding extracellular virulence factors of the respective pathogens (Denkin and Nelson, 2004; Milton et al., 1992; Norqvist et al., 1990; Shao, 2001; Kumar et al., 2008; Kole et al., 2018). Although, DNAbased vaccine has proven to be a promising vaccination strategy in conferring the substantial protection against various fish pathogens, its usage in food fishes is legally restricted in most countries as they fall under genetically-modified organism (GMO). Few other limitations of DNA vaccines comprise of immune tolerance against the expressed antigen, chromosomal integration, risk of autoimmunity, inflammation in the injection site and tissue damage (Hølvold et al., 2014). Nevertheless, to date, one DNA vaccine against IHNV is being legalized for use in Canada.

17.2.5 Reverse vaccinology Advancement in biotechnology has paved the way for newer technologies in recent years, bringing into focus the latest vaccinology termed as reverse vaccinology. This newer technology utilizes a bioinformatics approach to predict the immunogenic sequences of infectious pathogens in designing vaccines which are difficult to design and require sufficient quantum of time. Reverse vaccinology has gained importance in developing fish vaccines against a number of significant bacterial pathogens viz. Photobacterium damselae subsp. piscicida (Andreoni et al., 2016), Streptococcus agalactiae (Li et al., 2016), E. tarda, and F. columnare (Mahendran et al., 2016).

17.3 Conclusion Evolution of biotechnology and advances in the development of new vaccines against pathogens have made an important contribution in reducing the risk of diseases outbreak and subsequent losses in aquaculture. Aquaculture vaccination

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is becoming a significant area of the aquatic animal health management while it has investigated a cost-effective technology for monitoring significant threatening pathogen. Most of the development and efforts in aquatic animal vaccines are still in their infancy, and challenges toward multicomponent and cost-effective vaccination programs are yet to be addressed. Technical difficulties as well as scientific and biological limitations control the generation or commercialization of vaccines for all economically significant fish disease. Besides the improvement of recombinant approaches in the generation of new vaccines, we are entailed with the task to improve new expression system which can ensure glycosylation of the expressed antigenic proteins and restoration of their tertiary structure. Integrated research works are essential in order to address the safety concerns over DNA vaccine in food fishes and its implication in human health and environment to make it a safer alternative in fish vaccinology. Parasitic infections are capable of evading host
immune response thereby resulted in huge economic losses to the aquaculture industry. These pathogens are mainly controlled by hazardouschemicals which cause a serious threat to human health so vaccines against these parasites are very much essential. In addition, commercial vaccines may or may not be protective in different circumstances, so autogenous vaccines are required for better protection at a specific facility. Furthermore, more suitable and economical delivery methods need to be developed to vaccinate small fish. It is better to consider a vaccination program as a part of comprehensive fish health management, and not the only way for a disease problem. The basic information on immunization of fish could be applied for large-scale vaccination in fish, and for more progress in this field it is necessary to have a cooperation between basic and applied science.

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337.

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18

Contemporary vaccine approaches and role of next-generation vaccine adjuvants in managing viral diseases

Shailendra K. Saxena, Vimal K. Maurya, Swatantra Kumar and Madan L.B. Bhatt Centre for Advanced Research (CFAR)-Stem Cell/Cell Culture Unit, King George’s Medical University (KGMU), Lucknow, India

18.1 Introduction Vaccine is the most efficient intervention of medical sciences to reduce both mortality and morbidity caused by infectious diseases. In modern medicine, vaccinology is the most important cornerstone that provides an improved quality of life by controlling the transmission of diseases across communities (Hajj Hussein et al., 2015). Vaccines are the most economical means for public health interference to focus the global health economic load related mainly with infectious diseases. Over the past century through global vaccination campaign, polio, tetanus, diphtheria, and measles are significantly restricted whereas smallpox has been successfully eradicated form the world. Similarly, other microbial infections that mainly affect the younger populations have been significantly decreased in developed countries (Doherty et al., 2016). Generally, pathogens for which new vaccines are required have extensive variability, complex pathogenesis, and immune evasion properties (Servı´n-Blanco et al., 2016). A conventional method for the vaccine development has several limitations such as they are slow, time consuming, and fail to meet the requirements of a new vaccine during pandemics (Khurana, 2018). Recent advancement in structural biology, systems biology, computational biology, molecular and cellular immunology, molecular genetics, nanotechnology, bioinformatics, and formulation technologies provides novel approaches in immunogenic design with appropriate adjuvant discovery for new diseases (Loomis and Johnson, 2015). Structural vaccinology, recombinant DNA, reverse vaccinology, polysaccharide chemistry, and synthetic RNA vaccines are novel technologies that have been employed to design next-generation vaccines in the last 30 years (Rappuoli et al., 2014) (Fig. 18.1). Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00018-7 © 2020 Elsevier Inc. All rights reserved.

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FIGURE 18.1 Evolution in vaccine development technologies: Meningococcal B (Men B), Group B Streptococcus (GBS), Haemophilus influenzae type b (Hib), Group A Streptococcus (GAS), and Bacillus Calmette-Guerin (BCG).

18.2 Structural vaccinology Structural vaccinology is the knowledge of structural biology, bioinformatics, and human immunology for rational engineering of immunogens (Liljeroos et al., 2015). Structural vaccinology based on the principle of single or multiple-selected epitopes detection, which may be enough to stimulate a protective immunity and efficient host immune response, does not require identification of the full antigenic protein. Structural vaccinology together with human immunology are fast rising approaches for the rational of designing newer vaccines containing various antigenic epitopes for the development of highly effective immunity (Delany et al., 2014). Recently, researchers have developed an immunogen specific to respiratory syncytial virus (RSV) by using a structure-based approach that produces defensive responses against the fusion glycoprotein of RSV (McLellan et al., 2013). Another major advantage of structural vaccinology is the development of a better antigen that prevents HIV infection by inhibiting HIV replication. The envelope protein of HIV is the main target of HIV neutralizing antibodies (Benjelloun et al., 2012).

18.4 Reverse vaccinology

18.3 Synthetic vaccines Synthetic vaccines are designed based on antigens, for example, synthetic peptides and carbohydrates. Synthetic vaccines are proposed to be safer than the conventional vaccines derived from cultures. In synthetic vaccinology, advancement of nucleic acid-based vaccines exhibits several advantages such as opportunities of in situ antigen expression and associated safety of subunit and inactivated vaccines (Skwarczynski and Toth, 2016). Higher rate of production, simple manufacturing method, and inexpensiveness are the other major advantages associated with synthetic vaccines that allow us to combat humanitarian emergencies. Recently, DNA-based vaccines have illustrated to be very promising in animals whereas immune response was poor in humans as compared to conventional vaccines. In order to increase the efficiency of DNA-based vaccines in humans, various strategies such as electroporation-mediated DNA delivery and use of genetic adjuvants for amplified immunity have been applied clinical trials with satisfactory preliminary responses (Villarreal et al., 2013). RNA vaccines are the substitute of DNA vaccines and are primarily comprised of mRNA and RNA replicons having self-amplifying capabilities. The direct translation of the RNA in the cytoplasm results in the desired peptides or antigens whereas the integration of the gene into the host genome has been completely abolished (Ulmer et al., 2012). The stability and effectiveness of RNA-based vaccines have been improved through the utilization of engineered viral-particle expressing nonhomologous antigens rather than the viral specific genes. Conquering the limitations of DNA vaccines and viral delivery technology, the self-amplifying RNA replicons have been shown to be really promising (Lundstrom, 2018). Novel synthetic delivery methods that unite the efficacy of live attenuated vaccines, enhanced safety profile than plasmid DNA vaccines and methods of manufacturing may define the future of improved RNA vaccines. Significant research is required for the advancement of a synthetic vaccine that offers a unique tool of vaccine availability during various pandemics (Vogel et al., 2018).

18.4 Reverse vaccinology Reverse vaccinology is the most recent approach of antigen discovery for the design of newer vaccines by using genome sequencing data of microorganisms and bioinformatics. Reverse vaccinology changes the perspective of vaccine design by permitting the detection of broad spectrum vaccine candidates and immunogenicity during infection (Bidmos et al., 2018). The simultaneous application of protein arrays genomics, proteomics, and bioinformatics can significantly increase the detection of vaccine targets and vaccine development process

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(Galassie and Link, 2015). Meningococcal B are the first microorganisms for which reverse vaccinology was performed (Giuliani et al., 2006). Now this approach has also been useful to various bacterial pathogens such as Staphylococcus. aureus, Streptococcus. pneumoniae, Chlamydia, Streptococcus agalactiae, group A, and B streptococcus, Escherichia coli, and Leishmania major (Sette and Rappuoli, 2010). Reverse vaccinology involves sequencing the genome of an infectious organism and considering the whole antigenic repertoire for the identification of vaccine targets which can be evaluated for their appropriateness as vaccine candidate. Thus, the genome-based reverse vaccinology approaches can offer novel strategies for the design of vaccines, which was not possible to develop using conventional methods (Kanampalliwar et al., 2013). Other than reverse vaccinology, genomic-based antigen discovery can be increased by novel approaches enabling the investigation of entire antigenic repertoire with the help of antigenome analysis, that is, investigating the immunogenicity of proteins during infection and making the libraries of genetically expressed antigens (Furman and Davis, 2015). Furthermore, advancement in mass spectrometry has empowered the screening of existing and the amount of antigens present on surface of the pathogens (Sharma et al., 2018). Such advancements in antigen discovery technologies allowed identification of antigens for vaccine candidates that stimulate neutralizing antibody responses and assist the generation of vaccines based on T-lymphocytes (Koff et al., 2013) (Table 18.1).

18.5 Next-generation vaccine adjuvants Vaccine adjuvants are the substances that can advance the efficacy of vaccines by inducing vigorous immune responses in immunocompromised individuals, newborns, or the elderly. Adjuvants are usually required for subunit vaccines and not needed for live attenuated vaccines (Pe´rez et al., 2013). In vaccinology, adjuvants have various applications such as augmentation of immune response of the antigens by administering in native form, which diminishes the multiple immunization protocols to get protective immunity and to enhance the immune response in vaccinated individuals (Del Giudice et al., 2018). For vaccine formulation, currently, various categories of adjuvants with different mechanisms are used named as virosomes, oil emulsions, liposomes, mineral salts, immunestimulating complexes (ISCOM), virus-like particles, carbohydrate adjuvants, polymeric microparticle adjuvants, cytokines, and some bacterial derivatives (Aposto´licoJde et al., 2016).

18.5.1 Aluminum salts (Alum) Aluminum salts are commonly known as booster of Th2 immunity and they are extensively used adjuvants for human vaccines (Brewer, 2006). Aluminum is licensed for various human vaccines including human papilloma virus,

18.5 Next-generation vaccine adjuvants

Table 18.1 Merits and demerits of vaccine development technologies. Technology

Merits

Demerits

Empirical approach

• Activates all phases of the

• Secondary mutation can

•

• • •

• Recombinant DNA vaccines

• • • • • •

Glycoconjugation

• • •

•

Reverse vaccinology

immune system. Provides more durable immunity, boosters are required less frequently. Low cost. Quick immunity. Some are easy to administer, for instance, polio can be taken orally. Vaccines have strong beneficial nonspecific effects. Rapid generation. Safe and long lived immunity. No need for protein expression and purification. Potentially generic and low-cost manufacturing processes. Thermostability. Leading technology for T cell induction. Vaccines are cost effectives. Prevents asymptomatic carriage of disease. Pathogens that remain protected by encapsulation are destroying, so vaccination is possible against encapsulated bacteria. Elicits long-term protection.

• Fast and efficient in silico approach.

• For detection of antibodies

Next generation technologies

• • •

•

produced as a result of infections, allergies, autoimmune diseases, or cancers. Induce both humoral and cellular immune responses. High degree of adaptability production does not require amplification in bacteria or cell cultures. Improved safety, efficiency, and stability.

cause a reversion to virulence. • Causes severe complications in immunocompromised patients. • Required special storage conditions.

• Affected by maternal antibody.

• Limited to protein immunogen only.

• Induction of immunologic tolerance.

• It is expensive.

• Loss of immunogenicity during conjugation.

• Immunogenic response is restricted to selected antigens. • Vaccine may cause mild side effects, these includes slight fever, allergic reaction, tenderness, swelling, and redness at the site of the shot. • Only proteins can be targeted using this process, other biomolecules like polysaccharide are not targeted.

• Required alternate delivery system.

• Vaccine uptake can be limited due to the presence of enzymes like RNases.

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meningococcal, hepatitis B virus, tetanus, hepatitis A virus, diphtheria, and Haemophilus influenza type b. Aluminum has a lower toxicity profile than other vaccine adjuvants and is widely applicable for vaccine formulations to confirm antigen stabilization, high safety, and increase of high and long-lasting antibody response (Kanampalliwar et al., 2013).

18.5.2 Oil-in-water emulsions Augmentation of antibody titer and antigen dose-sparing are the main advantages associated with use of emulsion in vaccine formulations. Oil-in-water (o/w) emulsion based on squalene (AS03 and MF59) has been licensed for influenza vaccines both H1N1and H5N1in Europe. The efficacy of MF59 has been tested for various vaccine trials in conjunction with human immunodeficiency virus, cytomegalovirus, and herpes simplex virus (O’Hagan et al., 2012). Similarly, AS03 contains vitamin E (α-tocopherol) and is widely used for induction of nonspecific activation of the immune system (Morel et al., 2011).

18.5.3 Virosomes Virosomes are semisynthetic complexes derived from nucleic acid-free viral particles. Virosomes consist of two viral envelope glycoproteins: neuraminidase and influenza virus hemagglutinin are introduced between the phospholipids bilayers’ membrane. The various routes through which a virosomes can deliver an antigen in the biological system are intravenous, intramuscular, intradermal, and intranasal and depends upon intend of immunization with neglected side effects (Lee and Nguyen, 2015). Virosomes-based vaccine (Inflexal V) for influenza virus has been licensed for all age groups in Europe. Similarly, hepatitis A virus vaccine (Epaxal) has been licensed in South America, Europe, and Asia. The use of virosomes as vaccine adjuvants has various advantages like appropriateness to a wide range of population; improved antigen stability, excellent safety profile, and long-lasting antibody responses (Moser et al., 2007).

18.5.4 Monophosphoryl lipid and adjuvant System 04 Monophosphoryl lipid (MPL) is a toll-like receptor-4 (TLR4) receptor agonist and acts by increasing the expression of proinflammatory cytokines of Th1 immune responses specifically interferon gamma (IFN-γ) and interleukin-2 (IL-2). MPL, an immunostimulatory adjuvant is a detoxified form of bacterial lipopolysaccharide (LPS) obtained from Salmonella. minnesota R595 strain and one of the most promising adjuvant approved for human vaccines. Adjuvant System 04 (AS04) is made up of MPL impregnated on aluminum salts and now it became a second choice of adjuvant for human use after MF59. Currently, two AS04 containing vaccines, that is, human papilloma virus and hepatitis B virus, have been licensed for human use mainly in hemodialized patients (Ko et al., 2017).

18.5 Next-generation vaccine adjuvants

18.5.5 Carbohydrate adjuvants Various carbohydrates from natural sources can trigger the cells of immune system. γ-inulin is a potent carbohydrate adjuvant obtained from Compositae family and known for inducing cellular and humoral immunity with no side effects. Together with other adjuvants such as aluminum hydroxide, γ-inulin produces different types of adjuvants with a broad range of Th1 and Th2 activity. Acemannan is a natural polysaccharide carbohydrate adjuvant derived from mucilaginous gel of Aloe barbadensis and acts by stimulating the cytotoxic activity of natural killer cells and generation of cytotoxic T-lymphocytes-mediated responses (CTLs). The other mannose- and glucose-based polysaccharides that have adjuvant properties include galactomannans, lentanans, glucans, glucomannans, and dextrans (Mukherjee et al., 2013).

18.5.6 Cytokines adjuvants Various cytokines have been investigated for their ability to increase antigenspecific immune responses. To induce antigen-specific serum/mucosal antibody and cell-mediated immunity, a large number of cytokines have been studied. The most prominent cytokines adjuvants that have been evaluated to date for vaccine design include IL-1, IL-2, IL-6, IL-12, IL-15, IL-18, IFN, and granulocyte/macrophage colony stimulating factor (Taylor, 1995).

18.5.7 Nucleic acid-based mucosal adjuvants The mucosal adjuvants are the most promising strategy for vaccine design and are mainly obtained from toll-like receptor (TLR) ligands, novel small molecules, non-TLR immunostimulants, and bacterial toxins. Cholera toxin and Escherichia coli heat-labile enterotoxin are the most widely used mucosal adjuvants in animals. Various TLR agonists’ mucosal adjuvant such as AS04, MPL, TLR9 ligand CpG, and flagellin have been widely used for the development of newer vaccines (Chen et al., 2010).

18.5.8 Nanomaterial as adjuvants Implementation of nanotechnology in the field of vaccinology has reduced the dose and frequency of required immunizations due to the antigen-depot effect of nanocarriers. Various fascinating approaches such as the use of polycationic nanoparticles, cell-penetrating peptide-modified nanoparticles, and pH-dependent nanoparticles have been shown as promising for targeting the antibody and specific CTL responses during disease conditions and delivery of antigens into the cytosol. By controlling the physicochemical properties of these nonmaterials, they can use for antigen delivery that have high bioavailability, good targeting and imaging properties with sustained and controlled release profiles which are thought to be an advantage in the immune outcomes of vaccination (Shen et al., 2017) (Table 18.2).

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Table 18.2 Adjuvants approved for human vaccines. Adjuvant type

General description

Mechanism of action

1. Particulate delivery vehicle Liposomes

Contains synthetic phospholipids. Liposomesbased hepatitis A vaccine approved in Europe.

Immunestimulating complexes (ISCOM)

It contains a triterpenoid saponins obtained from Quillaia saponins, asterol and optionally a phospholipid. The saponins are Quil A or QS-21. It is made by biodegradable polymers. Antigens encapsulated inside the microparticles. It is considered as next generation of adjuvants. Potential for single shot vaccines.

Polymeric microparticle

Fuse with cell membrane of macrophages, enable antigen into the cytoplasm, enter major histocompatibility complex (MHC) class I path way and activate CD8 cytotoxic Tlymphocytes-mediated responses (CTL) response Generate CTL response, induce cytokines. Directly phagocytosed by macrophages.

Long-term depot effect from weeks to months. Pulsatile release of antigens. Target to antigen presenting cells.

2. Mineral salts Aluminum salts

Calcium salts

Licensed and approved by USFDA for human use. Misreferred as alum. It is widely used as human and veterinary vaccines. Aluminum hydroxide is more potent than aluminum phosphate due to their adsorption property. Considered as the safest adjuvant. Calcium salts in the form of calcium phosphate has been used as human vaccine adjuvant especially DTP, polio, yellow fever, and Bacillus CalmetteGuerin (BCG) vaccines. Approved for human use in European countries.

Short-term depot effect and Induction of cytokine network. Complement activation. Delivery of antigens to different antigen presenting cell (APC). Strong Th2 response.

Short-term depot effect. Adsorbs soluble antigens and presents them in a particulate form to the immune system.

3. Oil emulsion Freund’s complete adjuvant (FCA) Freund’s incomplete adjuvant (FIA)

W/o type of emulsion adjuvant using paraffin oil mixed with killed mycobacteria. W/o type of emulsion adjuvant using paraffin oil mixed without killed mycobacteria.

Short-term depot effect. Strong Th1 and Th2 response. Short-term depot effect. It induces weak Th1 and Th2 response. (Continued)

18.6 Vaccine delivery technologies

Table 18.2 Adjuvants approved for human vaccines. Continued Adjuvant type

General description

Mechanism of action

MF59

o/w type emulsion contains 4.3% of squalene oil, Tween 80, and span 85. Licensed for human use in European countries.

Inducing local immune stimulatory effect at the site of injection, regulates cytokines, chemokines, recruitment of CD11b 1 , MHC II 1 cells, and enhance antigen uptake by dendritic cells.

4. Microbial derivatives Adjuvant System 04 (ASO4)

It is 3-O-desacyl-40monophosphoryl lipid A obtained from the cell wall lipopolysaccharide (LPS) of Gram-negative Salmonella minnesota R595. Licensed for human use in European countries.

Local activation of NF-kB activity, cytokine production, optimal activation of APC, and generation of Th1 response.

18.6 Vaccine delivery technologies Advancements in vaccine delivery methods can provide great opportunity to develop the controlled release and targeted delivery of therapeutic agents against the broad range of pathogens. Delivery of vaccine via particulate carriers is a promising strategy for peptide and protein vaccines. Particulate delivery system mainly includes synthetic polymeric particles, lipid-based particles, and other colloidal structures for the delivery peptide and protein antigens. Delivering particulate antigens have various advantages over soluble antigens like internalization, processing, uptake via antigen presenting cells (APCs), and they can also mimic the particulate nature of pathogens (Liang et al., 2006). Discovery of a simple and efficient method to administer vaccines is another active area of research that improves immunization and extends lives (Saroja et al., 2011). Various novel vaccine delivery methods such as needle-free technologies which include edible vaccines, patches, and sprays have been investigated for their suitability in different age groups of individuals especially in children. Similarly, microscopic nanoparticles can serve as a transport mechanism for antigens for the targeted delivery into the immune cells. For the delivery of several types of vaccine-like DNA vaccines, viral vectors-based vaccines, inactivated and live attenuated vaccines, current nanotechnology-based delivery methods such as polymeric nanoparticles, liposomes, virosomes, dendrimer, micellar systems, and plant-derived viruses have been explored as vaccine delivery systems for humans and animals (Cordeiro and Alonso, 2016). Liposomes that can be used both as a delivery system and immunomodulator, offer a number of advantages in the development of novel hostdirected therapies and vaccines for various pathogens.

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18.7 Conclusion Vaccination is the most efficient medical intervention that reduces both morbidity and mortality caused by various diseases. The recent outbreaks of Zika and Ebola have increased the global alertness to human health caused by newly emerging and reemerging pathogens that can also provide the impetus to get ready against future pandemics by encouraging the improvement of vaccine platforms ready to use in humanitarian emergencies. Development of novel adjuvants may allow the design of newer vaccines for the management of various infectious diseases during humanitarian emergencies. Currently, a few vaccine adjuvants are approved and several are in clinical trial for future human use. Vaccinology has been developed from empirical to next-generation vaccines in the last three decades. Newer technologies such as, reverse vaccinology, recombinant DNA, structural vaccinology, polysaccharide chemistry, and synthetic RNA vaccines have significantly improved the effectiveness of target detection, selection, and designing of next-generation vaccines. Advancements in vaccine delivery methods can provide great opportunity to develop the controlled release and targeted delivery of therapeutic agents against the broad range of pathogens. Persistent advances should be made in the 21st century to develop novel vaccines that have a potential to save lives, contributing extensively to provide an improved quality of life.

18.8 Future perspectives The attempts required to accomplish the extensive demands for next-generation vaccines designed for emerging pathogens are only achieved by sustainable research development models. Increasing the international cooperation and modernization of technologies to encourage design, development, and manufacturing will permit an ongoing shift to a more efficient and cost- effective production of vaccines. This will altogether add to the worldwide efforts to anticipate infectious diseases, prevent vulnerable populations, and acquire a more rapid response to future outbreaks, forming human health.

Acknowledgments The authors are grateful to the Vice Chancellor, King George’s Medical University, Lucknow for the encouragement and support for this work. S.K. Saxena is also supported by CCRH, Government of India, and US NIH grants: R37DA025576 and R01MH085259. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

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Liljeroos, L., Malito, E., Ferlenghi, I., Bottomley, M.J., 2015. Structural and computational biology in the design of immunogenic vaccine antigens. J. Immunol. Res. 2015, 1 17. Loomis, R.J., Johnson, P.R., 2015. Emerging vaccine technologies. Vaccines (Basel) 3 (2), 429 447. Lundstrom, K., 2018. Latest development on RNA-based drugs and vaccines. Future Sci. OA 4 (5), FSO300. McLellan, J.S., Ray, W.C., Peeples, M.E., 2013. Structure and function of respiratory syncytial virus surface glycoproteins. Curr. Top. Microbiol. Immunol. 372, 83 104. Morel, S., Didierlaurent, A., Bourguignon, P., Delhaye, S., Baras, B., Jacob, V., et al., 2011. Adjuvant system AS03 containing α-tocopherol modulates innate immune response and leads to improved adaptive immunity. Vaccine 29 (13), 2461 2473. Moser, C., Amacker, M., Kammer, A.R., Rasi, S., Westerfeld, N., Zurbriggen, R., 2007. Influenza virosomes as a combined vaccine carrier and adjuvant system for prophylactic and therapeutic immunizations. Expert Rev. Vaccines 6 (5), 711 721. Mukherjee, C., Ma¨kinen, K., Savolainen, J., Leino, R., 2013. Chemistry and biology of oligovalent β-(1-2)-linked oligomannosides: new insights into carbohydrate-based adjuvants in immunotherapy. Chemistry 19, 7961 7974. O’Hagan, D.T., Ott, G.S., De Gregorio, E., Seubert, A., 2012. The mechanism of action of MF59 an innately attractive adjuvant formulation. Vaccine 30 (29), 4341 4348. Pe´rez O., Romeu B., Cabrera O., Gonza´lez E., Batista-Duharte A., Labrada A., et al. Adjuvants are key factors for the development of future vaccines: lessons from the finlay adjuvant platform. Front Immunol. 2013; 4:407. Rappuoli, R., Pizza, M., Del Giudice, G., De Gregorio, E., 2014. Vaccines, new opportunities for a new society. Proc. Natl. Acad. Sci. U S A. 111 (34), 12288 12293. Saroja, C.H., Lakshmi, P., Bhaskaran, S., 2011. Recent trends in vaccine delivery systems: a review. Int. J. Pharm. Invest 1 (2), 64 74. Servı´n-Blanco, R., Zamora-Alvarado, R., Gevorkian, G., Manoutcharian, K., 2016. Antigenic variability: obstacles on the road to vaccines against traditionally difficult targets. Hum. Vaccin. Immunother 12 (10), 2640 2648. Sette, A., Rappuoli, R., 2010. Reverse vaccinology: developing vaccines in the era of genomics. Immunity. 33 (4), 530 541. Sharma, V.K., Sharma, I., Glick, J., 2018. The expanding role of mass spectrometry in thefield of vaccine development. Mass Spectrom. Rev. 1 22. Shen, Y., Hao, T., Ou, S., Hu, C., Chen, L., 2017. Applications and perspectives of nanomaterials in novel vaccine development. Medchemcomm. 9 (2), 226 238. Skwarczynski, M., Toth, I., 2016. Peptide-based synthetic vaccines. Chem. Sci. 7 (2), 842 854. Taylor, C.E., 1995. Cytokines as adjuvants for vaccines: antigen-specific responses differ from polyclonal responses. Infect. Immun. 63, 3241 3244. Ulmer, J.B., Mason, P.W., Geall, A., Mandl, C.W., 2012. RNA-based vaccines. Vaccine 30 (30), 4414 4418. Villarreal, D.O., Talbott, K.T., Choo, D.K., Shedlock, D.J., Weiner, D.B., 2013. Synthetic DNA vaccine strategies against persistent viral infections. Expert Rev. Vaccines 12 (5), 537 554. Vogel, A.B., Lambert, L., Kinnear, E., Busse, D., Erbar, S., Reuter, K.C., et al., 2018. Self-amplifying RNA vaccines give equivalent protection against influenza to mRNA vaccines but at much lower doses. Mol. Ther. 26 (2), 446 455.

Further reading

Further reading HogenEsch, H., O’Hagan, D.T., Fox, C.B., 2018. Optimizing the utilization of aluminium adjuvants in vaccines: you might just get what you want. NPJ Vaccines 3 (51), 1 10.

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Advances in structureassisted antiviral discovery for animal viral diseases

19

Shailly Tomar, Supreeti Mahajan and Ravi Kumar Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, India

19.1 Introduction Antiviral molecules interfering with viral replication and multiplication in host come from diverse sources ranging from natural sources like plants, bacteria, and fungi or are produced by chemical synthesis (Kulkarni and Sanghai, 2014). Chemically synthesized antiviral drugs can either be designed or prepared by random synthesis. Latest advancements in drug development are structure-assisted identification with the help of computers, design and the synthesis of targetspecific antiviral. The action mechanism of antiviral is diverse ranging from the viral entry and budding steps to the targeting of virus-specific enzymes. The prevalence of animal viruses poses a major potential threat to animal health and also puts the human population at risk as viruses like influenza virus and encephalitis viruses belonging to the genus alphavirus are reemerging (Feldmann et al., 2002). Structural virology of animal viruses has increased our understanding of viruses, viral replication, their evolution, and interaction with the host. Structure
function relation studies are definitely the need of the hour for rational design of drugs and vaccines to effectively treat animal viral diseases. These studies are important for economical, veterinary, and human medical perspectives. In this chapter, briefly structural techniques and advances made in animal virology toward structure-based identification, and development of antiviral against animal viruses is described.

19.1.1 General strategies for identifying viral drug and vaccine targets The utmost important step in the drug discovery process is the identification of a drug target. In general, particular in vivo binding site of a drug through which the drug exerts its action is known as a drug target. From the therapeutic point of view, understanding the mechanism of drug binding inhibition and regulation of the target protein activity is very important to combat the viral diseases. Once the drug target has been validated, the next step is to identify, characterize, and Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00019-9 © 2020 Elsevier Inc. All rights reserved.

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design inhibitory molecules. Various viral targets and the strategies that are used to identify these viral and vaccine targets are summarized below: 1. Viral surface proteins and the strategies used to target these proteins: This includes viral surface glycoprotein and viral receptors. These are potential candidate for antiviral and vaccine development. Strategies used to target these include receptor-based and ligand-based drug designing. In receptorbased drug designing, mapping of ligands is done. Ligand molecules are engineered by assembling little pieces in a stepwise manner within the binding pocket constraint. In ligand-based approach, ligands for a specific receptor are determined using biophysical simulations and construction of chemical libraries. Availability of structural information is expanding the possibilities of identifying vaccine candidate by screening and mapping epitopes using bioinformatics tools such as Epivax, Epimatrix (Tan et al., 2012; De Groot and Moise, 2007). 2. Targeting the viral proteins/enzymes: Here, peptidomimetic drugs are designed against various viral enzymes such as proteases. Various techniques such as X-ray crystallography, nuclear magnetic resonance (NMR), and computational studies have also enabled for the successful designing of antiviral drugs (Tsantrizos, 2008; Wei and Zhou, 2010). 3. Targeting protein
protein interactions: Mutational and cell-based, yeast two hybrids and various biophysical methods such as surface plasmonresonance, isothermal calorimetry, fluorescence energy resonance transfer (FRET)-based, differential scanning calorimetry have helped in studying essential protein
protein interactions that are potential antivirals. Mutational studies at protein interfaces result in the increase or decrease of binding affinity of one viral protein with other protein, hence implying their beneficial role in viral life cycle (Brito and Pinney, 2017). 4. Targeting host factors: Various cellular proteins of the signal transduction pathway are a potential drug target. Antiviral molecules against host factors involved in viral replication are being targeted to prevent the hijack of host system by virus. Strategies like rapid immunoaffinity purification targeting a virus/host protein followed by mass spectrometry to identify associated protein can be implied (Rowles et al., 2013). 5. Targeting RNA-protein interactions: Using riboproteomics approach that profiles RNA-protein interactions and RNA coimmunoprecipitation which helps to identify all the proteins interacting with the viral RNA (Salim et al., 2016; Yeh et al., 2016; Figs. 19.1 and 19.2).

19.1.2 Structure determination techniques 19.1.2.1 X-ray crystallography X-ray crystallography is the most powerful and reliable method to obtain a macromolecular structure. It is used to determine structures of viruses and viral

19.1 Introduction

FIGURE 19.1 Antiviral drug targets. Viral surface proteins, enzymes, various interactions such as protein
protein, protein
RNA, and virus
host are potential antiviral targets.

FIGURE 19.2 General strategies used to identify drug targets or vaccine targets. Various strategies targeting essential virus proteins and interactions are depicted.

proteins by growing crystals (Ilari and Savino, 2008). The three components of X-ray crystallography are protein crystals, source of X-rays, and detector. Crystallographers aim high-powered X-rays at a tiny crystal and molecules arranged in the crystal lattice scatter the X-rays onto an electronic detector.

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The intensity of each diffracted ray is detected and fed into a computer, which uses a mathematical equation to calculate the position of every atom in the crystallized molecule. The result is a three-dimensional digital image of the molecule (Rhodes, 2010). X-ray crystallography is helpful in revealing the detailed threedimensional structures of thousands of proteins. Many advances in drug discovery and medicine are due to the X-ray crystallography by identifying the drug targets in many diseases. Macromolecular crystallography is a very powerful technique for drug discovery. It has a prominent role in finding drug targets in many diseases that are major challenges today, for example: HIV protease structure was identified by using Xray crystallography (Klei et al., 2007). Knowledge of the structure led to the identification of various antiviral compounds that interacted with the active site of the enzyme (Munshi et al., 1998). In a similar way, the structure of many proteins of the viruses which can be targeted to stop the viral invasion can be computationally predicted with the help of three-dimensional crystal structures of targeted virus or viral protein. This invites the structures-based drug designing against a particular targeted viral protein. Not only in drug discovery, but X-ray crystallography has also paved a way for making the drugs more effective as molecular details of drug and with targeted site are revealed at the atomic level. Adding to this, atomic structure of the potential viral target in complex drug also elucidates the mechanism of inhibition of the drug, how it interacts at molecular level, what makes it work, and so on (Munshi et al., 1998).

19.1.2.2 Nuclear magnetic resonance Another powerful biophysical technique is NMR spectroscopy which gives information not only about the structure but also the dynamics of viral proteins and their complexes (Marion, 2013). Nuclei of single atoms absorb different radio frequencies according to the environment of protein. These adsorption signals may be perturbed by adjacent nuclei. This determines the distance between nuclei. These distances, in turn, can be used to determine the overall structure of the protein. NMR is a successful technique used to find out how proteins interact with each other and with small molecules or drugs that can inhibit the interaction (Bakail and Ochsenbein, 2016). It can also be helpful in identifying possible hits for pharmaceutical use. Structures generated from the NMR structure analysis indicate the number of conformations for the protein in solution that are useful in designing small antiviral molecules against the targeted protein active site (Li et al., 2017).

19.1.2.3 Cryo-electron microscopy In recent years, major advancements have been made in cryo-electron microscopy (cryo-EM) technique and it has emerged as one of the most useful and powerful structural biology techniques that enable the characterization of complex biological systems. cryo-EM has evolved as a powerful tool in structure determination of macromolecular complexes that are not suitable for crystallographic and NMR

19.1 Introduction

studies (Murata and Wolf, 2018). Structure prediction of protein or virus models is done using the electron density of cryo-EM maps. Hence, cryo-EM is a reliable method for structure determination of macromolecules. Macromolecular complexes such as drug bound to viral enzyme active site or neutralizing antibody bound to virus surface epitope can be studied using cryo-EM technique (Ripoll et al., 2016). This availability of structural knowledge leads to rational design and synthesis of more potent drug molecules and vaccines for viral diseases.

19.1.3 Computational structure prediction and drug design Drug design and drug discovery are of extreme importance in animal and human health care. Computational (computer-based) approaches serve as an important role in structure-based drug design. Structure-based drug design utilizes the threedimensional structure (3D) of a protein target to design the potential candidate drugs that bind selectively with high affinity to the drug target (Anderson, 2003). Computational approaches utilize various methods for structure-assisted designing of drug molecules. The objective of designing a drug based on the availability of the 3D of protein is to invent or advance a molecule that binds tightly to the drugable site by competing with natural substrates of the protein and further moderating its inhibition function for viral therapy. Such a structure-picked drug molecule found on the basis of the protein structure is more effective and less toxic. Molecular docking methods use the spatial shape of the protein active site to which the drug is expected to bind for selecting a suitable compound that has the potential of being rationally designed as effective antiviral drugs (de Ruyck et al., 2016). For some proteins whose crystallization is difficult to perform, homology modeling can be put to use. It constructs a 3D model of a given protein on the basis of related similar or known structures (homologous structures). However, when the crystal structure of a protein is already known, then the knowledge regarding its active site residues involved in catalysis becomes important followed by computational virtual screening of compound libraries of small molecules. Small molecules are selected by docking them to the target protein. Molecular docking and simulation predicts the binding orientations of potential molecules (drug candidates) to protein targets so as to predict the affinity and activity of such small molecules (Katsila et al., 2016). A number of powerful software programs, for example: AutoDock, HEX, GOLD, FlexX, DOCK, Glide, Surflex, and LigandFit, are being used to predict the docking calculations. Selected in silico potential drug candidates are then tested in vitro for their antiviral effects or so. The use of computers and computational methods forms the core of computational drug design. Availability of protein 3D structures, high-performance computing, etc. is enhancing the modern day drug discovery process. Computational tools offer the advantage in a way that the new drug candidates are more quick and cheap. Various structure-based approaches in identifying and designing antiviral drugs for some animal viruses are discussed below (Table 19.1).

439

Table 19.1 Antiviral drug targets with structure-assisted inhibitors and their advantage in antiviral discovery. S. no.

Targets

Inhibitors

Advantage

References

1

Foot and mouth disease virus (FMDV) RNA-dependent RNA polymerase (RdRp) (3D polymerase) FMDV 3Dpol-novel binding pocket

Nucleotide analogs (ribavirin)

Crystal structure of the FMDV 3D polymerase in both the form-unliganded and bound to a template-primer RNA decanucleotide

Ferrer-Orta et al. (2004, 2010)

Noncompetitive inhibitors (1A8, 3A11, 4H6, 5D9, and 7F8) 4-Oxo-dihydroquinolines

They can be used for future structure-based drug design studies and antiviral drugs

Durk et al. (2010)

Identify a series of nonnucleosidic viral polymerase inhibitors The discovery of a single agent with clinical potential against existing and possible future emerging CoVrelated diseases They can be used as a basis for future anti-coronaviral inhibitor discovery experiments Potent inhibitor against CHIKV and will be used as antiviral drug in future. Help in designing other potent broad-spectrum antivirals Its usefulness for further investigations towards the development of piperazine based antialphaviral drugs Serve as the basis for antiviral development against alphaviruses

Liu et al. (2006)

Basis for the structure-based design of inhibitors for a range of paramyxovirus-induced diseases

Crennell et al. (2000)

New strategies in small-molecule drug development to overcome influenza A virus resistance Basis for the structure-based drug design Crystal structures of the bovine viral diarrhea virus 1 (BVDV1) glycoprotein E2 at neutral and low pH providing structural insight into the pre and postfusion state of the protein.

Shen et al. (2015)

2

3

5

Herpes simplex virus type 1 DNA polymerase Severe acute respiratory syndrome coronavirus (SARSCoV) main protease (Mpro) Coronavirus 3CLpro enzyme

19 ligands

6

Alphavirus nsP2 protease

Pep-I and Pep-II

7

Alphavirus nsP4

Favipiravir

8

Aura virus capsid protein

9

Alphavirus capsid protease hydrophobic pocket

10

Newcastle disease virus (paramyxovirus) hemagglutininneuraminidase (HN) Influenza virus protein NA

Piperazine (small heterocyclic molecule) (S)-(1)-Mandelic acid and ethyl 3aminobenzoate Neu5Ac2en and the β-anomer of sialic acid

4

11 12 13

Bovine viral diarrhea virus RdRp Pestivirus envelope glycoprotein E2

N1

Zanamivir, oseltamivir, and peramivir VP32947 and 1453 PTC12

Yang et al. (2005)

Berry et al. (2015) Singh et al. (2018) Delang et al. (2014) Aggarwal et al. (2017) Sharma et al. (2018)

Choi et al. (2004) El Omari et al. (2013), Pascual et al. (2018)

19.2 Animal viruses and viral diseases

19.2 Animal viruses and viral diseases 19.2.1 Foot and mouth disease virus Foot and mouth disease virus (FMDV) belongs to the Picornaviridae family of viruses. It causes foot and mouth disease (FMD) in cloven-hoofed animals. FMDV can be transmitted by close contact of animals, long-distance aerosol spread, inanimate objects like fodder and motor vehicles. It is highly contagious in cattle, pigs, buffaloes, goats, sheep, etc. It affects every part of the world where livestock are kept and more than 100 countries are still affected by FMDV. It affects wild and domesticated ruminants and therefore is a major concern in trade of livestock and animal products. It can cause acute and prolonged, asymptomatic but persistent infection. FMDV proliferates rapidly in infected species and causes vesicular disease in the feet and mouth. Seven serotypes each including a wide range of variants has been defined for FMDV. FMDV virion consists of nucleic acid and capsid enclosing the genomic positive-strand RNA. The virus genome encodes a single, long open reading frame (ORF) flanked by 50 -untranslated region (50 -UTR) and 30 -untranslated region (30 -UTR). The viral ORF upon translation and processing gives rise to four structural proteins, 10 nonstructural proteins (nsPs), and some cleavage intermediates (Forss et al., 1984). FMDV takes over host control by repressing host translation machinery and innate immune response to infection like many other viruses, by cleaving cellular proteins associated with signaling pathway and blocking protein secretion. A critical role of nsPs and noncoding elements of FMDV regulates these biological processes. Like other viruses, FMDV virus undergoes evolution and mutation, thus one of the hurdles in designing vaccines is between and within the serotypes of FMDV.

19.2.1.1 Clinical signs of foot and mouth disease virus The incubation period for FMDV usually ranges between 1
12 days. Symptoms include high fever for 2
3 days, blisters inside the mouth leading to foamy saliva, blisters on the feet, swelling in testicles of mature males, and decline in milk production in cows. The disease can also lead to myocarditis (inflammation of the heart muscles) and death in newborn animals. Some asymptomatic-infected domestic animals may also serve as carriers except pig (Jamal and Belsham, 2013).

19.2.1.2 Serotypes of foot and mouth disease virus FMDV has seven distinct serotypes—O, A, C, Southern African Territories 1, 2, 3 (SAT1, SAT2, SAT3) and Asia-1. Serotypes O and A were discovered by Vallee and Carre. Serotype C was discovered by Waldmann and Trautwein. Later another three serotypes were identified in samples from South Africa. The last serotype was identified from a sample that was collected at Okara, Punjab, and Pakistan from a water buffalo (Longjam et al., 2011).

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19.2.1.3 Structure and genome of foot and mouth disease virus FMDV is a 25
30 nm spherically shaped particle. FMDV virion has a symmetric protein shell called capsid enclosing the nucleic acid. Capsid consists of 60 copies of capsomers and each capsomer is composed of four structural polypeptides namely VP1, VP2, VP3, and VP4. FMDV genome consists of single-stranded positive-sense RNA about 8.3 kb in length. RNA encodes a single but long, ORF about 7 kb. The viral ORF is flanked by a long 50 -UTR and a short 30 -UTR. Viral genome has 30 poly-A tail. A small protein around 24
25 residues long known as VPg (or 3B), encoded by 3B region of the viral genome, is covalently attached to the 50 end of the genome. This VPg protein is released into the infected cell and plays no role in translation initiation. Viral ORF is translated into a polyprotein of around 250 kDa which is cleaved by two virus-encoded proteases namely leader (Lpro) and 3Cpro to form structural and nonstructural proteins. Generally, ORF is divided into four areas due to the different functions of mature polypeptides. These regions are—L region, P region, P2 region, and P3 region. L region located at 50 end to the capsid and encodes for Lpro. P region which encodes for precursor capsid polypeptide and generates 4 capsid proteins—VP4, VP2, VP3, and VP1 is cleaved by viral protease. P2 region codes for three viral proteins—2A, 2B and 2C. P3 region codes for four viral proteins—3A, 3B, 3Cpro, and 3Dpol. 3C is a viral protease and 3D acts as RNA-dependent RNA polymerase (RdRp). Viral nonstructural proteins play an important role in virus-mediated host evasion (Longjam et al., 2011).

19.2.1.4 Foot and mouth disease virus nonstructural proteins The first FMDV nsP that is translated is Lpro whose region lies in the polyprotein preceding the capsid precursor protein. This protein has two alternative forms, namely, Labpro and Lbpro (in vitro and in vivo forms). Lpro is a papain-like protease that releases itself from the polyprotein via cleavage between its C-terminus and the N-terminus of VP4. Lpro is major virulence factor and not required for viral replication. Lpro cleaves host translation initiation factor eIF4G and thus represses host-cell translation. This shuts off the host cap-dependent mRNA translation. Lpro also suppresses the host innate immune reaction to viral infection by blocking the interferon activity. FMDV 2A is 18 amino acid peptides and lacks protease motifs. However, it has characteristic C-terminal motif Glu(x)AsnProGly(2A)/Pro(2B). 2A is cleaved from P1-2A precursor by 3Cpro or by 3CDpro 2A cleavage event occurs only during polypeptide synthesis in such a way that 2A peptide remains connected to P1 structural protein precursor (P1-2A). 2A-2B cleavage is not a proteolytic event but a modification of the translational machinery by 2A peptide. This helps in the release of 2A protein while allowing the synthesis of the downstream proteins to proceed. 2B protein is viroporin that is hydrophobic transmembrane low molecular weight protein. FMDV 2B codes for a 154 aa peptide, slightly longer than other viroporins and contains two predicted putative transmembrane domains

19.2 Animal viruses and viral diseases

located at positions 83
104 aa and 119
137 aa. The transmembrane hydrophobic domains interact with phospholipid bilayer to increase membrane permeability and facilitate the release of viral particles. 2B is crucial for viral pathogenicity. 2C protein is 318 amino acid long proteins consisting of amphipathic helix in its N-terminal. This nsP is involved in many biological functions linked to membrane targeting. 3A protein, a 153 aa peptide is conserved in most FMDV strains. Most of the coding region in N-terminus (1
75 position) encodes a hydrophilic as well as a hydrophobic domain capable of binding to membranes. That is why FMDV 3A has membrane binding activity based on this hydrophobic motif. 3B protein, known as VPg is covalently bound to 50 -terminus of the genome. The first step in the replication of the picornavirus genome is the uridylylation of the VPg peptide primer. The genome has a 50 terminal feature of VPgU(pU) covalently linked, which allows the use of VPg as a peptide primer to synthesize viral RNA. 3Cpro is a chymotrypsin-like cysteine protease that is responsible for most cleavages of viral polyprotein. 3Dpol is RdRp and synthesizes positive and negative strand viral RNAs. It has a catalytic component of RNA replication and plays an important role in the life cycle of FMDV. 3Dpol sequences are highly conserved among different serotypes as well as subtypes of FMDV (Gao et al., 2016).

19.2.1.5 Vaccination Vaccines limited the spread of the virus during epidemics in FMD-free countries as well as in endemic regions thus playing a vital role in FMD control. Recently, vaccines are typically produced by the inactivation of the whole virus. Such vaccines have the quantity and stability of the intact viral capsids in the final preparation. First promising novel FMD vaccine was licensed for manufacture and to be used in the United States (Park, 2013). This adenovirus-vectored FMD vaccine causes in vivo expression of viral capsids in vaccinated animals. Another promising vaccine is composed of stabilized and empty FMDV capsids that are produced in vitro in a baculovirus expression system (Cao et al., 2016). Other areas under research include enhanced adjuvants, vaccine quality control procedures, vaccine protection, and immune correlation.

19.2.1.6 Structure-based drug development against foot and mouth disease virus Various proteins of FMDV have been targeted to design antivirals against them. Structural-based and computational approaches have been used to find out potent inhibitor molecules against FMDV. In 1994, the first crystal structure of FMDV in reduced form was reported. In 2004, crystal structure of FMDV RdRp (3D polymerase) has been determined in ligand-free and in complex with a templateprimer RNA (Ferrer-Orta et al., 2004; Fig. 19.3). Some conserved amino acid side chains bind to the template-primer in the complex which helps in mediating the initiation of RNA synthesis. This crystal

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FIGURE 19.3 Complex of foot and mouth disease virus (FMDV) RNA-dependent RNA polymerase (RdRp) with bound RNA at 3 A˚. The bound RNA is represented as stick. Protein is represented as alpha helix and beta sheets. Interaction between catalytic active site residues Asp338 and Asp339 and Asp238 and Asp240 of RNA is also shown (PDB ID: 1WNE) (Ferrer-Orta et al., 2004).

structure sheds light on important information for studies on viral RNA replication and the concept of designing antiviral compounds. In 2006, the crystal structure of FMDV 3C protease was reported. It revealed new insights into the structural-functional aspects of the viral replication enzyme. The crystal structure of FMDV 3Cpro confirmed that it belongs to family of chymotrypsin-like cysteine proteases (Barrett and Rawlings, 2001). The protein comprises of two six-strand barrels and between these barrels, on one face of the protein, lies the peptide binding cleft that has the active site of the enzyme. This FMDV 3C protease crystal structure paved the way for structural-based drug design (Curry et al., 2007; Fig. 19.4). In 2010, several inhibitor molecules against the crystal structure of FMDV 3Dpol were identified. These targeted a novel binding pocket on 3Dpol which could be used for future structure-based drug design studies (Durk et al., 2010; Fig. 19.5). Knowledge of the 3D structure of viral capsids allowed for the engineering of the thermostable capsids. However capsid stability and effectiveness of the viral vaccines was a concern. So in 2015, a molecular dynamics (MD)-based strategy for the evaluation of mutations which is designed to increase the stability of capsid through increased noncovalent interactions was developed. Therefore this

19.2 Animal viruses and viral diseases

FIGURE 19.4 Structure of foot and mouth disease virus (FMDV) protease at 1.9 A˚. FMDV protease showing two β-barrels. Between these barrels, lies the protein active site. Active site residues include catalytic triad of Cys163, His46, and Asp84. Alpha helices and beta sheets are shown (PDB ID: 2BHG) (Curry et al., 2007).

FIGURE 19.5 Structure of foot and mouth disease virus (FMDV) 3D polymerase at 3 A˚. Surface of the protein is shown. Inhibitor binding pocket on the surface of 3D pol of FMDV showing active site catalytic residues K59, R168, R179, and K177 (in circle) (PDB ID: 2E9Z) (Durk et al., 2010).

designed MD protocol allowed derivation of structural-based design of stabilized FMDV viruses and empty capsids which allowed for the development of stable vaccines (Kotecha et al., 2015). In 2018, the crystal structure of mutant viral polymerases with less sensitivity to ribavirin was reported (Ferrer-Orta et al, 2010).

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19.2.2 Herpesviruses Herpesviruses (HSV) are double-stranded (ds) DNA viruses of animals which belong to the Herpesviridae family. Their natural host range includes molluscs, fishes, amphibians, birds, reptiles, and mammals including human beings. It causes mucocutaneous lesions inside the oral cavity as well as genital infections in humans. Nine species of herpes virus take humans as their primary host. These cause diseases ranging from mild lesions to serious malignancies. The serologic prevalence percentage is higher in populations of developing countries than in developed countries for many human herpes virus. This widespread of human herpes virus is due to the fact that most virus infections are asymptomatic, and have mild, unnoticed symptoms. Also, the virus is capable of establishing latent, recurrent infections in their hosts. Herpes virus infection is a serious concern and especially dangerous for immunocompromised patients (Jiang et al., 2016). Herpes viruses are grouped into three subfamilies: alpha, beta, and gamma herpesvirus.

19.2.2.1 Structure of herpesvirus HSV is an enveloped DNA virus that has 150
200 nm diameter and a linear ds DNA around 120 to 230 kbp. The virus has an icosadeltahedral capsid consisting of 162 capsomers. It has a large segment containing viral proteins and an external trilaminar lipid envelope constituting at least 12 glycoproteins. The genome is composed of two regions namely unique long region, UL and unique short region, US linked covalently to each other and flanked by three segments. HSV has three origins of replication (ori)—one copy of oriL and two copies of oriS (Vadlapudi et al., 2013).

19.2.2.2 Herpesviruses lytic and latent cycle HSV lytic cycle is divided into three steps: viral entry, viral replication, and viral assembly and exit. Viral entry depends on the type of cell. Initially, viral glycoproteins bind to the host-cell receptors and after that viral envelope either fuses with plasma membrane or undergo endocytosis. Only five of the total twelve viral envelope proteins are essential for viral infection such as glycoprotein C (gC), gB, gD, gH, and gL. gB acts as a homooligomer whereas gH and gL form a functional heterooligomer. The binding of gC to heparan sulfate initiates virus contact with the host cell. Postinfection into nucleus, host RNA polymerase II initiates viral gene expression — early and late genes. HSV DNA replicates via a sigma or rolling-circle mechanism. After that, post-DNA replication genes are transcribed forming viral structural components and required for capsid assembly. These are transported into nucleus via nuclear localization sequences. The procapsid is then packaged along with viral DNA to form a mature capsid after being assembled. Inside the cytoplasm, capsids are enveloped by budding into Golgi compartment and are finally secreted out from the infected cells. Herpes viruses have the ability to undergo latency in the hosts for lifetime. During latency, viral transcription is shut off except for 8.3 kb transcript which is associated with

19.2 Animal viruses and viral diseases

latency and thus called latency associate transcript. This is unstable polyadenylated primary transcript and is further processed into two stable introns with extended half-lives. When reactivated by proper stimuli such as ultraviolet, stress, immune-suppression, these viruses get activated and entered lytic cycle resulting in spreading various diseases (Boehmer and Nimonkar, 2003).

19.2.2.3 Antivirals against herpesviruses A number of antiviral molecules that target HSV-1 and HSV-2 are present. All are nucleoside analogs except foscarnet and cidofovir, while acyclovir, famciclovir, and valaciclovir are used to treat the majority of cases of HSV-1 and HSV-2 (Bacon et al., 2003). Other medications such as foscarnet, valganciclovir, ganciclovir, and cidofovir have activity against the alpha herpesviruses and are recommended in certain circumstances, such as the treatment of some acyclovirresistant HSV. Antiviral drugs for the treatment against HSV infections have been developed over the past 40 years. However, most drug-resistant HSV isolates have been reported such as resistance to acyclovir, etc. So this demands the need of highly effective low toxicity drugs in HSV-resistant isolates. Here is the whole antiviral designing process based on the structural findings of HSV (Jiang et al., 2016). Viruses of the family Herpesviridae are cause innumerable human diseases. Up to the year 2000, the available treatments were largely ineffective, with the exception of a few drugs for treatment of herpes simplex virus (HSV) infections. However, for some DNA viruses of this family, advancement was made for biochemistry and structural biology of the enzyme viral protease, revealing common features that can be exploited in the development of a new class of antiherpesvirus drugs. So, herpesvirus proteases were identified as a unique class of serine protease with a Ser-His-His catalytic triad. A new, single domain protein fold was determined by X-ray crystallography for the proteases of HSV. It was shown that dimerization is unique for serine proteases and is required for activity of the HSV proteases. With this known fact of dimerization, there was a serious impact on functional analysis and inhibitor discovery. The conserved functional and catalytic properties of the herpesvirus protease enzyme lead to common considerations in the process of inhibitor discovery. Crystal structures of the herpesvirus proteases allowed more direct interpretation of ligand structure
activity relationships (Waxman and Darke, 2000; Fig. 19.6). In addition, screening of chemical libraries provided some novel structures as starting points for drug development. In 2000, human herpesvirus (HHV) capsids using log-phase cultures of body cavity-based lymphoma 1 cells induced with 12O-tetradecanoylphorbol-13-acetate were obtained for electroncryo microscopy and computer reconstruction. The 3D structure of the HHV-8 capsids revealed that a capsid shell is composed of 12 pentons, 150 hexons, and 320 triplexes arranged on aT516 icosahedral lattice. This structure is similar to those of herpes simplex virus type 1 (HSV-1) and human cytomegalovirus, which are prototypical members of alpha and beta herpesviruses, respectively (Wu et al., 2000). Till 2006, herpesviruses were the second leading cause of human viral diseases,

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FIGURE 19.6 Structure of herpesvirus protease at 2.1 A˚. Structure of Herpes virus protease solved through X-ray crystallography at 2.1 A˚. Catalytic triad of Ser114, His46, and His134 is located on the solvent exposed surface of the protein in the active site. Catalytic site residues are shown in the circle (PDB ID: 1FL1) (Waxman and Darke, 2000).

especially dangerous in immunocompromised individuals. Common therapies for herpes viral infections used nucleoside analogs, such as Acyclovir and target the viral DNA polymerase, essential for viral DNA replication. But the problem is that this class of drugs exhibits a narrow antiviral spectrum, and resistance to these agents was an emerging problem. So the need of the hour was a better understanding of herpes virus replication that could help in the development of safe and effective broad-spectrum antiherpetic. In 2006, the first crystal structure of a herpesvirus polymerase, the herpes simplex virus type 1 DNA polymerase, at ˚ resolution was reported (Liu et al., 2006; Fig. 19.7). 2.7 A The structural similarity of this polymerase to other polymerases allowed constructing high confidence models of a replication complex (RC) of the polymerase and of Acyclovir as a DNA chain terminator. A novel inhibition mechanism was established in which a representative of a series of nonnucleosidic viral polymerase inhibitors, the 4-oxo-dihydroquinolines, bound at the polymerase active site as well as interacting noncovalently with both the polymerase and the DNA duplex. Most viruses need cell-entry proteins called fusogens in order to get into the host cell. It was known that herpes virus fusogen does not act alone but needs a complex of other viral cell-entry proteins. In 2010, this complex structure was determined. Then it turned out that this protein complex is not a fusogen but it regulates fusogen. It was also established that certain antibodies interfere with this complex so that it cannot regulate fusogen. This gave a clue that certain antiviral can be designed which target this interaction can in turn, prevent viral

19.2 Animal viruses and viral diseases

FIGURE 19.7 Herpes simplex virus type 1 DNA polymerase structure at 2.68 A˚ DNA Pol is consisting of six domains. Pre-NH2
terminal domain is from NH2 -terminal domain up to residue 140, NH2 terminal domain is from 141-362 and 594-639 residues, 3’-5’ exonuclease domain is from 363-593 residues, palm domain consisting of residues 701-766 and 826-956, finger domain is from residues 767-825 and thumb domain is from 957-1197 (PDB ID: 2GV9) (Liu et al., 2006).

infection (Mesri et al., 2010). The nonnucleoside inhibitors of HSV DNA polymerase target the site that is less important for the binding of a natural nucleoside or nucleoside inhibitor. In 2012, using crystal structure of HSV DNA polymerase, a possibility of new lead molecule based on a-pyrone analogs as nonnucleoside inhibitors came into light using structure-based modeling approach (Karampuri et al., 2012). In 2012, different in silico approaches were applied to virtually screen for potential inhibitors targeting glycoproteins gB
gH
gL complex formation interface of HSV. Using structure-based virtual screening on gB and gH
gL glycoproteins, many potent inhibitor molecules separately target the active residues involved in their binding activity (Hussain Basha and Naresh Kumar, 2012). In 2016, nature products and new antivirals mechanisms were suggested to target the HSV proteins like DNA helicase/primase complex to fight the drug resistance of HSV. New types of molecules are anti-HSV agents such as flavonoids, sugarcontaining compounds, and peptides (Jiang et al., 2016). New antiviral mechanism included the lethal mutagenesis was proposed as a novel chemotherapeutic

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strategy for drug resistance. The high frequency of mutations in the viral genome leads to a large danger of genetic mutations causing reduction in viral infective activities. Therefore lethal mutagenesis may be effective in weakening the capacity of the virus for drug resistance. Only one nucleoside analog, ribavirin, exhibits a broad spectrum of antiviral activity against DNA and RNA-based viruses. By 2018, structure of the HSV portal-vertex at subnanometer resolution, solved by cryo-EM and single-particle 3D reconstruction has been reported (McElwee et al., 2018). This led to a number of new discoveries, including the presence of two previously unknown portal-associated structures. Moreover, 3D reconstruction revealed that the viral DNA is packaged within the capsid as a left-handed spool that is arranged in concentric shells. Adding to this, data has also shown a molecular machine that plays a critical role in the replication cycle of an important family of human pathogens. So this can be targeted and can be used in designing antivirals.

19.2.3 Coronavirus (severe acute respiratory syndrome) Coronaviruses belong to the subfamily Coronavirinae under family Coronavorodae. Coronaviruses are enveloped viruses containing single-stranded positive-sense RNA as their genetic material. RNA is surrounded by a nucleocapsid of helical symmetry. The genome size of coronaviruses varies between 26
32 kb, being the largest for a RNA virus. Many proteins contribute to the overall structure of all coronaviruses namely spike (S), envelope (E), membrane (M), and nucleocapsid (N). Coronavirus mainly infects mammals and birds (Brian and Baric, 2005). Humans can be infected by six known strains of coronaviruses. In mammals and birds, coronaviruses infect the upper respiratory and gastrointestinal tract. In humans, coronaviruses cause cold, fever, throat congestion, pneumonia, and bronchitis. A human coronavirus called Severe acute respiratory syndrome coronavirus (SARS-CoV) causes severe acute respiratory syndrome (SARS) and can cause unique pathogenesis ranging from lower and upper respiratory tract infections (Cheng et al., 2007). In the case of SARS coronavirus, a defined receptor-binding domain mediates the attachment of virus to its cellular receptor, angiotensin-converting enzyme 2.

19.2.3.1 Replication of coronavirus Replication of coronavirus occurs in the cytoplasm. Upon viral entry in the host cell, it uncoats and RNA genome is deposited into the cytoplasm. RNA has 50 methylated cap and a 30 polyadenylated tail. This allows the RNA to attach to ribosomes for translation. Because of the enzyme replicase encoded by the viral genome, the RNA transcribes into new copies of RNA. A protease, nonstructural protein of coronavirus (CoV) cleaves off each nonstructural protein from the long polyprotein (Fehr and Perlman, 2015). There are following types of human coronaviruses: (1) human coronavirus 229E, (2) human coronavirus OC43, (3) SARS-CoV, (4) human coronavirus NL63 (HCoV-NL63, New Haven coronavirus),

19.2 Animal viruses and viral diseases

(5) human coronavirus HKU1, (6) Middle East respiratory syndrome coronavirus (MERS-CoV), previously known as novel coronavirus 2012 and HCoV-EMC.

19.2.3.2 Structure-based antivirals against coronavirus Unfortunately there are no effective antivirals against coronavirus. There is an urgent need to develop new strategies to prevent and to control coronavirus infections, and to understand their biology, replication, and pathogenesis of these viruses. Better understanding of the function of CoV proteins in the virus replication and transcription mechanism may lead to the development of pioneering antivirals. In 2003, structural and functional properties of spike protein of coronavirus were characterized (Bosch et al., 2003). The function the viral spike glycoprotein is to mediate entry of coronavirus. In 2003, the crystal structure of SAR-CoVMpro (main protease) was also solved (Xue et al., 2007). It revealed that the main protease structure forms a homodimer with three domains in each monomer. The antiparallel aˆ-barrel structure of domains I and II is similar to other coronavirus proteases. It forms a chymotrypsin-like fold responsible for catalytic reactions. In 2005, native structure of coronavirus 3CLpro was reported which was solved by X-ray crystallography (Bacha, 2008). SARS-CoV main protease (Mpro), is a protein that is required for the maturation of SARS-CoV and is vital for its life cycle, thus, making it an attractive target for structure-based drug design of antiSARS drugs (Lu et al., 2006). Crystal structure of SARS-CoVMpro with inhibitor N1 was also reported (Yang et al., 2005; Fig. 19.8). In 2006, the structure-based virtual screening on a chemical database containing 58,855 compounds based on the 3D structure of SARS-CoVMpro was performed. Active compounds selected from this virtual screening approach (also confirmed by the bioassay), were taken as the templates to build the core structure for analog search. Finally, the complex structures of potent inhibitors with SARSCoVMpro were solved by X-ray crystallography. It helped to further study the ˚ crysSARS-CoVMpro inhibition mechanisms of these compounds. In 2012, 2.6-A tal structure of the feline coronavirus Nsp7:Nsp8 complex solved by X-ray crystallography was reported (Xiao, 2013). In 2015, many broad spectrum inhibitors against 3CLpro enzyme of coronavirus were identified based on crystal structure of this enzyme and virtual screening methods (Berry et al., 2015). The 3CLpro of coronavirus proved to be an effective drug discovery target. It has even been termed as “the Achilles heel of coronaviruses”. CoV helicase is one of the most evolutionary conserved proteins in nidoviruses, and hence making it an important target for drug development (Hao et al., 2017). In 2018, first full-length crystal structure of the MERS-CoV helicase was reported. CoV helicase has an Nterminal Cys/His-rich domain (CH) with three zincs, a beta-barrel domain, and a C-terminal SF1 helicase core (Durai et al., 2015). These findings are very helpful to provide novel structural information essential for structure-based drug design against CoV.

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FIGURE 19.8 Structure of severe acute respiratory syndrome coronavirus (SARS-CoV) main protease (Mpro) and inhibitor complex at 2 A˚. Inhibitor N1 binding in the active site of SARS-CoV Mpro. Active site residues involved in interaction with N1 constitutes Cys145, Met145, Phe185, and Gln192 are shown in the circle (PDB ID: 1WOF) (Yang et al., 2005).

19.2.4 Alphaviruses Alphaviruses belong to the Togaviridae family of viruses. They are enveloped, positive sense, single-stranded RNA viruses. Alphavirus particles have a 70 nm diameter, tend to be spherical (although slightly pleomorphic), and have a 40 nm isometric nucleocapsid. There are 30 alphaviruses that can infect various vertebrates like humans, rodents, birds, fish, as well as invertebrates. Their transmission between species and individuals occurs mainly through mosquitoes. Hence, they are also called arthropod-born. Alphaviruses are divided into Old World viruses and New World viruses. Chikungunya virus (CHIKV), o’nyong’nyong virus, and sindbis virus (SINV) are the Old World alphaviruses which cause rash, polyarthralgia, and chronic arthritis. New World alphaviruses such as Eastern equine encephalitis virus (EEEV) and Venezuelan equine encephalitis virus (VEEV) are mostly associated with neurological disease. During the Cold War, both the United States biological weapons program and the Soviet biological weapons program researched and weaponized VEEV (Croddy et al., 2008). It causes only moderate morbidity and low mortality in humans but severe morbidity and mortality in animals. EEEV and VEEV caused 70%
90% and 20%
80% mortality in horses, respectively as compared to mortality in humans (Zacks and Paessler, 2010). Salmonid alphavirus (SAV) is a unique group of viruses that causes pancreas disease and severe infection in fish. It is a big problem and

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economically important due to high mortality rate from 5% to 60% and poor growth performance in the recovered fish. SAV infection causes the massive loss of biomass in commercial fish farming also (Herath et al., 2016). The alphavirus genome is almost 12 kb in length which exhibits two ORFs—one encoding the nonstructural polyprotein and another encoding the structural polyprotein. nsP1, nsP2, nsP3, and nsP4 are formed as a result of cleavage from the nonstructural polyprotein. These are necessary for the regulatory functions such as transcription and translation of viral mRNA in host. Two nsP precursors (P123 or P1234) are produced by viral RNA using host-cell translational machinery. The carboxylterminal protease domain of nsP2 cleaved these precursor polyproteins. At P3/4 junction, cleavage occurs in trans or cis whereas cis at P1/2 junction. Final cleavage at P2/3 junction results in the formation of completely mature nsPs. These nsPs along with host proteins form the positive strand RC. This allows the RNA template to synthesize positive-sense genomic (49S) and subgenomic (26S) RNAs. The structural polyprotein translates into five structural proteins: the capsid (C), E1, E2 (major envelope glycoproteins), E3, and 6K proteins (Shin et al., 2012).

19.2.4.1 Functions of nonstructural proteins The nsP1 protein, an mRNA capping enzyme, has both guanine-7methyltransferase (MTase) and guanylyltransferase (GTase) activities. So, nsP1 mediates the methylation and capping functions of viral (Abu Bakar and Ng, 2018). SINV nsP1 protein does not require membrane association for its enzymatic function. The GTase activity of SINV nsP1 is metal-ion dependent, whereas metal is not required for MTase enzymatic activity of nsP1 (Tomar et al., 2011). nsP1 protein, the alphavirus capping enzyme, is a potential drug target because it has a distinct molecular mechanism of capping the viral RNAs than the conventional capping mechanism of host. nsP1 catalyzes the methylation of guanosine triphosphate (GTP) by transferring the methyl group from S-adenosylmethionine to a GTP molecule at its N7 position with the help of nsP1 MTase followed by guanylylation reaction which involves the formation of m7GMP
nsP1 covalent complex by nsP1 guanylyltransferase (GTase) (Kaur et al., 2018). The alphavirus nonstructural protein nsP2 possesses various enzymatic activities. The N-terminal region contains a helicase domain that has seven signature motifs of superfamily 1 (SF1) helicases. It functions as RNA triphosphatase that performs the viral RNA capping reactions. It also functions as nucleotide triphosphatase and thus facilitating the RNA helicase activity. The C-terminal region of nsP2 contains a papain-like cysteine protease, which is responsible for processing the viral nonstructural polyprotein (Abu Bakar and Ng, 2018; Narwal et al., 2018; Singh et al., 2018). The crystal structure of the nsP2 cysteine protease of VEEV was reported in the free and E64d-bound states which are the first report of an inhibitor bound alphaviral nsP2 protease structure. The structures and identified active site residues in this study may assist the discovery of potential protease inhibitors against VEEV (Hu et al., 2016). The exact role of alphavirus nsP3 protein is not much

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clear in the RC. The nsP3 protein has the N-terminal macro domain (phosphatase activity or nucleic acid binding ability), the alphavirus unique domain, and the Cterminal hypervariable domain. Recently, it has been shown that nsP3 has a role in pathogenicity (Abu Bakar and Ng, 2018). The nsP4 polymerase is the most highly conserved protein in alphaviruses with .50% identity in amino acid. A core RNA-dependent RNA polymerase (RdRp) domain is at the C-terminal end which has RNA synthetic properties of the viral RC. It has TATase activity that suggests a novel function of the alphavirus RdRp in the maintenance and repair of the poly(A) tail, an element required for replication of the viral genome (Tomar et al., 2006).

19.2.4.2 Viral target proteins for drug development Over the last decade in Asia, Europe, and the Americas, the alphaviruses reemergence has focused on the need of selective inhibitors. At present, no antiviral treatment is available. Viral targets within alphavirus RC include various molecular determinants. Structural and functional analysis of these targets can make the structure-based drug design and development of antivirals against alphaviruses possible. nsP1
nsP1, nsP1
nsP2, nsP1
nsP3, nsP1
nsP4, nsP2
nsP4, and nsP4
nsP4 interactions are identified in CHIKV. Many of these interactions are shown similarly in SINV and Semliki forest virus (SFV) (Abu Bakar and Ng, 2018). nsP1 recruits other nsPs as discussed in literature and is crucial for SFV replication by membrane association (Lampio et al., 2000). nsP1’s interaction with all other nsPs is very important and is an attractive target for drug development. Inhibition of nsP1 anchoring in the spherules occurs when its affinity for the cell membrane is perturbed. This will inhibit the recruitment of the other nsPs and thus preventing the initiation of RC formation. nsP2 protein has viral replication and host evasion strategies which can be targeted for the viral inhibition. nsP2 has RNA helicase, RNA triphosphatase, nucleoside triphosphatase and autoprotease activities. It is an important cofactor for the maturation of viral RC (Kappes, 2014). The nsP2 protease is a good drug target because many viruses are targeted in the same way [human immunodeficiency virus (HIV) and hepatitis C virus]. This has led to the development of many FDA-approved inhibitors (Lenz et al., 2010; Weber and Agniswamy, 2009). nsP4 is virus-specific and is RNA polymerase. It can be a good target to inhibit the viral DNA duplication and its survival. For in silico drug designing, the crystal structure of nsP2 protease is of great interest. nsP2 protease domain crystal structures of the VEEV, CHIKV, and SINV are also present with protein data bank (PDB) entries 2HWK, 5EZQ, 3TRK, and 4GUA respectively. In 2015, crystal structure of nsP2 protease from ˚ was submitted (PDB ID 4ZTB). Using this crystal structure, varCHIKV at 2.5 A ious peptidomimetic inhibitors were designed against nsP2pro in 2016 (Dhindwal et al., 2017). In 2018, a FRET-based protease assay was used to analyze the proteolytic activity of CHIKV nsP2 protease. This protease assay was used to assess the inhibitory activity of these peptidomimetic compounds identified. It was

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concluded that two peptidomimetic compounds, Pep-I (MMsINC database ID MMs03131094) and Pep-II (MMsINC database ID MMs03927237) inhibited CHIKV nsP2 protease activity and can be potential antiviral drugs (Singh et al., 2018). To find out a few potential inhibitors against CHIKV nsp2 protease, homology modeling and computer-aided drug design strategies have been implemented for the first time (Bassetto et al., 2013). Potential inhibitors based on structural studies and molecular simulations have been reported (Nguyen et al., 2015; Singh et al., 2012). Lopinavir and Nelfinavir, potent HIV protease inhibitors, are FDA-approved inhibitors against CHIKV. A number of highly selective CHIKV and VEEV nsP1 capping enzyme inhibitors are reported recently which interrupt the nsP1-GTase activity (Delang et al., 2016; Gigante et al., 2014, 2017). Nucleoside analogs are also shown to be effective against several alphaviruses, such as ribavirin, which inhibit the CHIKV and SFV genome replication by depleting GTP pools (Briolant et al., 2004). Moreover, the inhibition of nsP4 RdRp by ribavirin through its interaction with Cys483 residue increases the replication fidelity (Coffey et al., 2011). D-N4-hydroxycytidine (NHC), another nucleoside analog is the most potent inhibitor of VEEV. It can serve as a substitute for ribavirin because it cannot develop NHC resistant mutants. Favipiravir is another nsP4 inhibitor and is a potent antiviral against CHIKV infection by inhibiting its interaction with Lys291 residue (Delang et al., 2014). Compound-A could potentially inhibit RdRp’s ribonucleotide selection function by targeting Met2295 (Wada et al., 2017). However, it is believed that by chemical modification compound-A may be a starting point for reducing its toxicity. In 2017, 3D structure of capsid protein of aura virus in com˚ . Piperazine is a small heterocyclic plex with piperazine was reported at 2.2 A molecule and docking studies have shown that it binds to hydrophobic pocket of CHIKV capsid protein. It can be effective as an antialphaviral drug (Aggarwal et al., 2017). In 2018, crystal structure of CHIKV capsid protease domain was ˚ . It was found that small molecules such as (S)-(1)-mandelic determined at 2.2 A acid and ethyl 3-aminobenzoate target capsid hydrophobic pocket. These bind to the conserved hydrophobic pocket of CP (Sharma et al., 2018). This may serve as a basis for the development of antivirals against CHIKV infections. In 2018, crys˚ , which revealed that the protein tal structure of nsP2pro was determined at 2.59 A consists of two subdomains: an N-terminal protease subdomain and a C-terminal methyltransferase subdomain. Additionally, structure insights revealed that access to the active site and substrate binding cleft is blocked by a flexible interdomain loop in CHIKV nsP2pro. This may serve beneficial for structure-based drug design and optimization of CHIKV protease inhibitors (Narwal et al., 2018). Recently, a high-throughput ELISA-based assay was developed to screen inhibitors against divalent metal-ion-dependent alphavirus capping enzyme (Kaur et al., 2018). Various inhibitors such as sinefungin, aurintricarboxylic acid, and ribavirin were assessed and their inhibitory effect against nsP1 was reported. In addition, nsP2 protease-based cell-free high-throughput screening assay for evaluation of

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inhibitors against emerging CHIKV has been developed (Saha et al., 2018). These successful methods for identifying antiprotease molecules together with a highthroughput screening assay can lead to the development of industrial level largescale screening platform for identification of protease inhibitors against emerging and reemerging viruses.

19.2.5 Paramyxovirus The Paramyxoviridae family is divided into two subfamilies: Paramyxovirinar and Pneumovirinae. The Paramyxovirinae subfamily has five genera namely Respirovirus, Rubulavirus, Avulavirus, Morbillivirus, and Henipavirus. This subfamily comprises of various viruses like measles, mumps, Newcastle disease, parainfluenza, Hendra, and Nipah viruses (NiVs). The second subfamily, the Pneumovirinae, comprises of two genera: Pneumovirus and Metapneumovirus. This subfamily also includes new human and animal pathogens, such as the human and bovine respiratory syncytial viruses that specifically affect bovine, caprine, and ovine species, and the human and avian metapneumoviruses. Paramyxoviruses include major disease causing pathogens that cause significant health hazards (Aguilar and Lee, 2011). They have enveloped RNA and infected the host with the help of two surface glycoproteins that fuse their lipid membranes with the host-cell plasma membrane. These viruses have an attachment and a fusion (F) protein. Membrane fusion is probably due to receptor-induced conformational changes within the attachment protein that leads to the activation and folding of fusion protein (Plattet and Plemper, 2013).

19.2.5.1 Antivirals against paramyxovirus Paramyxoviruses are the main cause of respiratory disease in children. One of two viral surface glycoproteins, the hemagglutinin-neuraminidase (HN) has various functions in addition to being the major surface antigen that induces neutralizing antibodies. In 2000, the crystal structure of multifunctional Newcastle disease virus (Paramyxovirus 1) HN alone and in complex with inhibitor was reported (Fig. 19.9). The structure provides the basis for the structure-based design of inhibitors for a range of paramyxovirus-induced diseases (Crennell et al., 2000). In 2005, the crystal structure of the secreted, uncleaved ectodomain of the paramyxovirus was presented (Yin et al., 2005). In 2012, combined X-ray crystallography and cryoelectron tomography were done to show the structure of matrix protein of Newcastle disease virus, a paramyxovirus. Structure and sequence conservation imply that other paramyxovirus matrix proteins function similarly (Habchi and Longhi, 2012). In 2012, it was established that favipiravir may serve as antiviral against these viruses. Hendra virus (HeV) and NiV are bat-born paramyxoviruses. In 2013, experimental findings in animals have demonstrated that a human

19.2 Animal viruses and viral diseases

FIGURE 19.9 Crystal structure of multifunctional Newcastle disease virus (Paramyxovirus 1) neuraminidase (NA) at 2.5 A˚. Inhibitor binding pocket on the surface of neuraminidase active site. Active site residues in the protein that are involved in interaction are A125, A182, S181, A242 and T243. These are shown in the circle on the surface of the protein. (PDB ID 1E8T) (Crennell et al., 2000).

monoclonal antibody targeting the viral G glycoprotein is an effective postexposure treatment against Hendra and NiV infection. Also, a subunit vaccine based on the G glycoprotein of HeV affords protection against Hendra and NiV. The vaccine has been developed for use in horses in Australia. It is the first vaccine against a biosafety level-4 (BSL-4) agent to be licensed and commercially deployed. HeV is one of the members of the Henipavirus genus of paramyxoviruses, which are designated BSL-4 organisms because of the high mortality rate of NiV and HeV in humans. Paramyxovirus cell entry is mediated by the fusion protein, F, and this is in response to binding of a host receptor by the attachment protein. During posttranslational processing, the fusion peptide of F is released. Upon receptor-induced triggering, it is inserted into the host-cell membrane. F undergoes a dramatic refolding from its prefusion to postfusion conformation. This brings the host and viral membranes together, allowing entry of the viral RNA. In 2015, the crystal structure of the prefusion form of the HeV F ectodomain was reported. The structure shows great similarity with the structure of prefusion parainfluenzavirus 5 fusion protein (Wong et al., 2016). In 2018, first successful treatment of henipavirus infection in vivo with a small-molecule drug suggests that favipiravir should be evaluated as an antiviral treatment option for henipavirus infections (Dawes et al., 2018).

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19.2.6 Avian influenza virus Avian influenza virus belongs to the family Orthomyxoviridae. These are negative sense single-stranded viruses. These are classified into three types: A, B, and C based on the antigenic difference in their nucleoproteins (NPs) and matrix proteins. Influenza A is the major pathogen that causes epidemic influenza. The influenza A genome is composed of eight RNA segments. Five segments code for one protein each and the other three code for two proteins each. The proteins are: hemagglutinin (HA), NA, matrix protein 1 (M1), M2 proton channel, NP, nonstructural protein 1 (NS1), nuclear export protein (NEP; also known as NS2), polymerase acid protein (PA), polymerase basic proteins (PB1 and PB2), and a protein named PB1-F2 which is expressed from a second reading frame (11) of the PB1 gene. PB1, PB2, and PA form the RNA polymerase. The surface glycoproteins HA and NA provide distinct antigenic properties to the influenza virus. Influenza A viruses are further organized according to HA and NA subtypes (Du et al., 2012). Sixteen HA subtypes (H1
H16) and nine NA subtypes (N1
N9) have been identified. The subtypes of the 1997 bird flu and the 2009 swine flu viruses have been indicated above. In 2001, crystal structure of H5 avian hemagglutinin was reported. In 2008, crystal structure for the NS1 effector domain of avian influenza virus A was reported (Hale et al., 2008). In 2009, crystal structure of an avian influenza polymerase was reported (Fig. 19.10). This unbolted many possibilities to target the viral protein for designing of new antiinfluenza therapeutics (Yuan et al., 2009). In 2015, three inhibitors against protein NA were approved by FDA namely zanamivir (Relenza, Glaxo Smith Kline), oseltamivir (Tamiflu, Roche), and peramivir (Rapivab, BioCryst). Zanamivir was the first approved inhibitor among these (Shen et al., 2015). In 2017, structure-based drug discovery studies were done by targeting the PA
PB1 interaction. Many potent antiinfluenza drugs were reported with the help of in silico simulation studies (Watanabe et al., 2017).

19.2.7 Pestivirus The Flaviviridae family consists of three genera which include—Flavivirus genus (type species, yellow fever virus) as the largest genus, the Hepacivirus (type species, hepatitis C virus), and the Pestivirus (type species, bovine virus diarrhea). Pestivirus belonging to the family Flaviviridae is not arthropod-borne and mainly infects mammals. These cause diseases like hemorrhagic syndromes, abortion, and fatal mucosal disease. These are single-stranded viruses with positive-sense RNA. The genome is around 12.5 kb long. There is no poly-A tail at 30 -end of the genome. Hence these viruses have no posttranscriptional modifications, and have simple RNA genomes. The genome contains RNA to encode both structural and

19.2 Animal viruses and viral diseases

FIGURE 19.10 Crystal structure of avian influenza polymerase at 2.2 A˚. Pol consisting of four polypeptide chains, each containing alpha helices and beta-sheets in presence of magnesium ions (spheres form). Mg ion is coordinated by the acidic residues E80 and D108, HH41, E119, L106, and P107 (in the circle). These amino acids except P107 are conserved in Influenza virus A, B, and C (PDB ID: 3EBJ) (Yuan et al., 2009).

nsPs. These are enveloped viruses. These are icosahedral-like particles with a linear genomic arrangement (Kumar et al., 2015). Their entry into the host-cell is mediated by clathrin-coated endocytosis and is achieved by the attachment of viral envelope protein E to host receptor.

19.2.7.1 Vaccine and structure-based drug design There are vaccines against pestiviruses and the correct vaccine strain should be given, depending on the herd’s location and the endemic strain in that region. The vaccination must be given regularly to maintain immunity. There are various species in the genus pestivirus-border disease virus, bovine viral diarrhea virus (BVDV), classical swine fever virus, etc. (Kumar et al., 2014). In 2004, crystal structure of RdRp from BVDV was reported (PDB ID 1S4F). This structure explained many possibilities where inhibitor molecules could bind. This shed light on various inhibitors binding sited on the protein leading to opportunity of designing many structure-based inhibitor molecule (Choi et al., 2004) (Fig. 19.11).

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FIGURE 19.11 Crystal structure of bovine viral diarrhea virus (BVDV) RNA-dependent RNA polymerase (RdRp) at 3 A˚. Alpha helices and beta-sheets are shown. Active site residues in protein that are involved in catalysis are I261, K263, R285, D350, D448, and D449 (in the circle) (PDB ID: 1S4F) (Choi et al., 2004).

In 2012, crystal structure of bovine viral diarrhea virus 1 (BVDV1) envelope glycoprotein E2 was reported (PDB ID 2YQ2) (El Omari et al., 2013). On the basis of the crystal structure of BVDV E2 protein, small-molecule high-throughput docking was performed to identify the molecules that likely bind to the envelope protein E2 of BVDV. Several structurally different compounds were purchased as well as synthesized. These were then assayed for antiviral activity against BVDV. Their possible binding determinants were characterized by MD simulations. A common pattern of interactions was observed between active molecules and amino acid residues in the binding site in E2. These findings are believed to offer a better understanding of the interaction of BVDV E2 with these inhibitors, as well as benefit the discovery of novel and more potent BVDV antivirals (El Omari et al., 2013). In 2015, crystal structure of the NS3 helicase of the pestivirus was reported (PDB ID 4CBG) (Tortorici et al., 2015). In 2017, the closed-conformation crystal structure of the full-length pestivirus NS3 with its NS4A protease cofactor segment was established (PDB ID 5WX1) (Zheng et al., 2017) (Fig. 19.12).

19.3 Conclusion

FIGURE 19.12 Crystal structure of bovine viral diarrhea virus 1 (BVDV1) envelope glycoprotein E2, pH 8 at 2.58 A˚. Dimeric structure consisting of two monomeric units: A and B. Domains of monomer A starting from the N terminus are labeled. Domain A is starting from residues 1-87, domain B from 88-164, domain C residues from 165-271 and domain D residues from 272-333 (PDB ID: 2YQ2) (El Omari et al., 2013).

19.3 Conclusion In this chapter, various viral proteins that are necessary for the survival of virus inside host are discussed such as viral polymerase, viral helicase, protease, etc. The approaches used in predicting the structure of these necessary proteins are elaborated. Various drug molecules or antivirals designed on the basis of structure of these proteins are highlighted. The details of the available inhibitors and the potential antiviral candidates are disclosed. Vaccination status against animal viruses is discussed. Further improvement in these and the need of novel structure-based drugs and their clinical testing is emphasized. Protein structure-based drug design has been contributing to the drug discovery process since the early 1990s. This structural knowledge of interaction between drugs and the target protein has been applied mainly to predict potency changes of chemically modified lead compounds. With the help of 3D-structural information, additional aspects of the drug discovery process have become predictable. Selectivity of compounds between homologous or orthologous proteins can be predicted. This provides new possibilities to design selective compounds or predict the suitability of animal models for pharmacodynamic studies. Antivirals display a variety of mechanisms of action. Antivirals may enhance the animal immune system or block a specific enzyme or a particular step in the viral replication cycle. As viruses are obligate intracellular parasites that use the host’s cellular machinery to survive and multiply, it is essential that antivirals do not harm the host. However, a major concern is that viruses are continually developing new antiviral resistant strains due to their high mutation rate. This demands for mandatory search or development of new antiviral compounds. With the help of structure-based drug designing, there is progress in preclinical drug discovery.

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There is a deficit of information between identified hits and the many criteria that must be fulfilled side by side to convert these hits into preclinical candidates that have a real chance to become a drug. This gap can be bridged by investigating and understanding the interactions between the ligands and their receptors. Accurate calculations of the free energy of binding are still elusive. Better knowledge of all these will help in finding good drug candidates to combat the diseases caused by animal viruses.

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CHAPTER

Vaccines the tugboat for prevention-based animal production

20

Ramadevi Nimmanapalli and Vikas Gupta Department of Veterinary Microbiology, Faculty of Veterinary and Animals Sciences, IAS, RGSC, Banaras Hindu University, Mirzapur, India

20.1 Introduction Livestock farming is the most important sector of Indian agriculture and contributes in India’s economy in terms of livelihood security. A total of 512.05 million livestock are present in India and most of these are reared by landless and marginal farmers and are the main source of their livelihood. Besides, organized sectors have also started showing interest in livestock farming; mainly in poultry and dairy sectors but their numbers are very small at the present. As livestock sector is a live market, they are very susceptible to different infectious diseases such as viral, bacterial, fungal, and parasitic diseases. These infectious diseases are posing a threat to the livestock production performance due to morbidity and mortality. Therefore keeping the livestock healthy to get better production is of paramount importance to agricultural economy. Vaccination of the animals against various infectious diseases prevailing in different geographical regions is the key measure of good husbandry practices and contributes a major role in maintaining animal health and minimizing economic losses due to production losses from infectious diseases. The term vaccine (“vacca,” meaning cow) was coined by Luis Pasteur in honor of Edward Jenner who used cowpox lesion as a substitute of smallpox scab to protect from smallpox infection in humans and establish the concept of vaccination. Around a century after him, Luis Pasteur made three vaccines for rabies, fowl cholera, and anthrax through the process of attenuation. Further, in 1886 Daniel Elmer Salmon and Theobald Smith gave the concept of inactivated or killed vaccine. Presently most of the vaccines used for immunization of animals or humans are either live or inactivated in nature. The conventional veterinary vaccines protect animals against the potential dangers of many infectious diseases. It stimulates the animal’s immune system and prepares them to resist the infections caused by pathogenic microorganisms. Vaccination is the most effective way to prevent the transmission and the spread of animal disease epidemics which subsequently provide full security and public health. Genomics and Biotechnological Advances in Veterinary, Poultry, and Fisheries. DOI: https://doi.org/10.1016/B978-0-12-816352-8.00020-5 © 2020 Elsevier Inc. All rights reserved.

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The impact of veterinary vaccines is witnessed by the success of the Global Rinderpest Eradication Program which was a large-scale international collaboration involving vaccination, trade restrictions, and disease surveillance. This has been a great achievement in the animal health area and rinderpest is the second disease after smallpox eradicated globally. Vaccines against other diseases like brucellosis, rabies, foot and mouth disease (FMD) are being used as the main instruments in the eradication program of the respective diseases globally. In addition to assisting in the eradication program of animal diseases, vaccines also combat the emergence of drug-resistant pathogens and emergence of new diseases. Since the first use of a vaccine, the research for vaccinology in the past 200 years has generated continuous technical breakthroughs and led to substantial improvements in human and animal health. Various developments have taken place with regard to types of vaccines and methods of immunization. However, it is only during the last two decades where the veterinary world has observed significant development of novel prophylactics which are facilitated by the advent of biotechnological tools and techniques, and discovery of antigen/gene delivery systems or recombinant vaccines developed using biotechnological tools or genetic engineering represents an alternative strategy by which the limitations of conventional vaccines are taken care of. A number of genetically-engineered vaccines which are rationally designed such as, live flavivirus chimera vaccine (WN-FV) (PreveNile), live double-gene deleted [deleted glycoprotein E (gE2) and deleted thymidine kinase (tk2)] bovine herpesirus type 1 strain (Hiprabovis IBR Marker), and feline immune deficiency vaccine (Fel-O-Vax) have already been introduced in the veterinary market. The infectious animal diseases outbreaks are generally due to viruses, bacteria, or parasites. The vaccine against all pathogens is not available as there are some existing limitations in developed vaccines or difficulties in vaccine development. Reasons for unavailability of vaccines for certain diseases include that either it is not technically possible to develop vaccines that provide adequate protection against the etiological agent or it is not possible to develop safe and effective vaccines, or it may become ineffective in a short duration as pathogens change their characteristics. The vaccination cannot always be a universal option for control of animal epidemics. Many countries have vaccines against most viral or bacterial diseases but lack vaccines against parasitic diseases. Although there are difficulties in developing commercial vaccines against parasites, a few vaccines against parasitic infestation like coccidiosis in poultry and parasitic bronchitis in cattle caused by the nematode Dictyocaulus viviparous are available.

20.2 Vaccines and one health For better public health food security, disease-free body and healthy ecosystems are the main tenets. These can only be achieved by coordinated approaches to

20.2 Vaccines and one health

produce safe food, access to interdisciplinary medicine, and evaluation and reduced use of hazardous chemical and physical agents on the ecosystem. The World Health Organization (WHO), Food and Agriculture Organization, and the World Organization for Animal Health (OIE) together promoted a comprehensive approach for better public health called “one health” (McConnell, 2014). The objective of “one health” is to promote multisectoral response to food safety hazards, the risk from zoonotic diseases and its control (disease that can spread between animals and humans, e.g., rabies, West Nile fever, salmonella, and flu) (Vandersmissen and Welburn, 2014; Buttigieg, 2015). One health activity also includes public health threats at the human-animalecosystem interface (antibiotic resistance) and provides guidance on how to reduce these risks (Vandersmissen and Welburn, 2014; Hoelzer et al., 2018). The world population is growing at a faster pace and is expected to reach more than 9 billion in 2050. Although, meat and egg production has increased between 1961 and 2007, there is a further need to increase production by 70% in order to fulfill the food security demand of projected world population of more than 9 billion people by 2050. Safe meats, eggs, and milk are essential to achieving the food security of a growing human population in the world and it is not possible without healthy livestock. Animals and poultry are susceptible to many infectious diseases and some cause food-born zoonoses in human beings. Veterinary vaccines contribute immensely to the maintenance of health and productivity of animals. The effective use of vaccines against various diseases could be an important component to meet present and future food demands. In addition to preventing diseases against food animals, it is also important in preventing disease in companion animals and wildlife which subsequently has an important impact on reducing the incidence of zoonotic diseases in human. Vaccines for diseases of companion animals and horses have helped humans to keep animals in the household and enhanced human-animal bonding to enrich the lives of both animals and humans. Without rabies vaccines, it is unlikely that humans would have been willing to keep cats and dogs as pets. Antibiotic resistance is the major concern in both veterinary and human medicine and arose due to excessive and indiscriminate application of antibiotics in treatment and as a feed additive in food animals. There are limited numbers of effective antibiotics for treatment against bacterial diseases but the possibilities of getting resistant against these are still a concern. Vaccine acts on the bacteria by eliciting host-immune response either through humoral or cellmediated immunity and there is no possibility to get resistance against host immunity (Vandersmissen and Welburn, 2014; Hoelzer et al., 2018). Therefore vaccination is a safe and effective method to prevent bacterial infection in animals and humans. Bacterial vaccines minimize the treatment cost in food producing and companion animals which also involves the least use of antibiotics. Availability of inexpensive vaccines may reduce reliance on antibiotics for animal health.

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20.3 Types of vaccines 20.3.1 Conventional vaccines 20.3.1.1 Live-attenuated vaccines Live-attenuated vaccine is a live microorganism with very little or no pathogenicity which cannot cause disease but has the ability to induce protective immunity. Generally, they are produced by serial passages of agents in unnatural or heterologous hosts or cell lines and sometimes distant relative of pathogenic microorganisms which are not pathogenic to the target host (Jorge and Dellagostin, 2017). Viruses acquire random mutations in their genome after multiple serial passages in heterologous systems leading to loss of pathogenicity without compromise in immunogenicity (Meeusen et al., 2007). They are able to replicate in the host and induce both cellular and humoral immunity. The immunity generated by live vaccines persist a longer duration and there is no need for adjuvant (van Gelder and Makoschey, 2012; Jorge and Dellagostin, 2017). The limitations of live vaccines are adverse reaction and reversion of virulence inside the host. Besides, they have less shelf-life and are sensitive to high temperature. Therefore they require cold chain or refrigeration for storage and transportation. Examples: Brucella abortus S-19, Peste-des-petits ruminants virus vaccine, sheeppox vaccine, canine parvovirus vaccine, canine distemper vaccine, Newcastle vaccine, etc.

20.3.1.2 Inactivated vaccines Inactivated vaccines consist of killed bacteria or virus of one or more species or serotypes, mixed with an appropriate adjuvant (Jorge and Dellagostin, 2017). The vaccine microorganism is usually grown in bulk in a suitable system (cell culture, egg embryo, or bacterial media) and inactivated by physical (heat and ultravioletrays) or chemical means (formaldehyde, beta-propiolactone, and binary ethyleneimine) which denature either surface proteins (surface effect) or damage the nucleic acid of vaccine virus (Meeusen et al., 2007). The inactivated microorganism may be further purified and mixed with a suitable adjuvant (van Gelder and Makoschey, 2012). These vaccines are comparatively easy to produce than live vaccines but provide a shorter duration of immunity. Further, most of the viruses have multiple serotypes or continuously changing antigenic structures (e.g., FMD virus and influenza viruses) and one serotype does not provide protection to other serotypes, therefore, vaccine candidates for inactivated vaccines should be continuously evaluated to provide coverage against the outbreaks. Examples of such include FMD vaccine, bluetongue virus vaccine, bovine viral diarrhea virus vaccine, rabies virus vaccines, etc.

20.3.1.3 Toxoids The diseases caused by bacterial toxins are also controlled by vaccination. The vaccines against diseases of toxins’ origin are produced by inactivating native toxins by physical or chemical means and mixed with adjuvants (Jorge and

20.3 Types of vaccines

Dellagostin, 2017). However, they also possess some limitation of biological safety. The use of recombinant DNA technology can overcome these limitations, and can produce toxoid in bulk with safety. For example, the production of recombinant toxins does not require many biosafety precautions because the toxic domain of the protein is removed by biotechnological tool (Arimitsu et al., 2004). Examples of such include tetanus toxoid, anthrax protective antigen toxoid, and clostridium type A toxoid, etc.

20.3.2 Genetically-engineered vaccine 20.3.2.1 Subunit vaccine Subunit vaccines contain one or more fragments or full-length proteins of a pathogen instead of the whole pathogen to elicit protective immunity in the host (Jorge and Dellagostin, 2017). Compared to conventional vaccines, these vaccines are safe to administer, nonreplicating, easy to produce, cost effective, and have no deleterious effect due to unwanted antigenic materials. They can be made by isolating antigenic protein(s) from any infectious organism after its disruption. This type of strategy is common in aviral subunit vaccine called split vaccine. The recombinant subunit vaccines are made by identification and selection of protective antigen gene coding region followed by their cloning in suitable vector and expression in a heterologous host system such as bacteria, yeast, mammalian and insect. Escherichia coli is used extensively for protein expression as heterologous host besides limitation in the form of yield, posttranslational modification and folding of expressed recombinant proteins. The limitations of E. coli expression system were improved by the introduction of methylotropic yeast (Pichia pastoris) which has the capacity of posttranslational modification and folding of expressed recombinant proteins. Since the subunit vaccines induce less immunity in comparison to whole bacteria or viral vaccine, they are used with a suitable adjuvant. Examples of such include Newcastle disease virus (NDV) subunit vaccine using hemagglutinin-neuraminidase (HN) gene, FMDV subunit vaccine using VP-1 gene, porcine circovirus type-2 (PCV-2) subunit vaccine based on open reading frame-2 (commercialized) and prM, and E envelope proteinbased subunit vaccine of Japanese encephalitis.

20.3.2.2 Virus-like particle vaccines Multiprotein structures that mimic the conformation and authentic structure of an empty viral capsid but are devoid of genetic material are called virus-like particles (VLPs). They are nonreplicating and but contain an array of antigens similar to the outer structure of virion (Jennings and Bachmann, 2008). Since their structure and antigenic surface resemble virion, they are able to elicit both humoral and cell-mediated immune responses without the need of adjuvant (Jorge and Dellagostin, 2017). Further, they are safe due to the absence of genome and provide a high degree of protection without the possibility of reversion of virulence.

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They could provide a promising differentiation of infected from vaccinated animals (DIVA) strategy during serosurveillance and eradication program of disease. Therefore they may be a better substitute for inactivated and live vaccines. VLP has been successfully employed in two licensed vaccines, hepatitis B and human papilloma virus but there is no report of licensed veterinary vaccine until now.

20.3.2.3 Vectored vaccines The live vector, having the foreign protective antigen coding gene of a bacteria or virus used for eliciting the immune response against the protective antigen is called a vectored vaccine. The live vectors are attenuated virus or bacteria which act as a backbone to deliver large amounts of exogenous gene inside the host (Jorge and Dellagostin, 2017). A number of viruses (vaccinia virus, canary pox, fowlpox, and adenovirus) and bacteria [Bacille Calmette-Gue´rin (BCG), Listeria monocytogenes, Salmonella spp., and Shigellae spp.] have been tested for their capability to carry the heterologous genes and their expression inside the host (Rizzi et al., 2012). They provide long lasting immunity due to being viable in nature and because they do not need any adjuvant. Currently, the canary poxvirus vector system has been used for vaccines against rabies virus, canine distemper virus, feline leukemia virus, and equine influenza virus. The bacterial recombinant BCG has significant potential to express a large number of antigens and can induce solid immunity. The use of transgenic plants engineered to produce and deliver immunogenic antigens via food sources has potential perspective in vaccine industries. In veterinary vaccinology, transgenic plants are able to produce and deliver antigens through animal feed. Plant-based vaccine trials have been conducted for various parasitic diseases including poultry coccidiosis, schistosomosis, porcine cysticercosis, and ascariosis. Plant-derived rabies G protein expressed in tomato, tobacco, and spinach on oral administration in mice mount local and systemic immune response (Shams, 2005). Besides, in attempt to form edible vaccine for rabies, vaccinia, canarypox, adenovirus, and yeast; they were employed for expression of neutralizing G protein of rabies and used as a delivery system. Rabies vaccine in the form of consumable bait (edible vaccine; raboral V-RG coated in fishmeal and fish oil) was successfully used for vaccination of wildlife such as raccoon, fox, etc.

20.3.2.4 DNA vaccine Naked DNA plasmid having the protein coding gene of viral, bacteria, or parasites and that can express it in mammalian cells are defined as DNA vaccine (Paludan and Bowie, 2013). In addition to a desired exogenous antigenic gene, the plasmid contains a strong eukaryotic promoter, polyA tail, multiple cloning sites, and suitable selective marker. The basic aim of DNA vaccine system is that the antigen can be expressed directly by the cells of the host in a way similar to that occurring during viral infection and expressed antigen after processing will be represented either via major histocompatibility complex-I or major histocompatibility complex-II leading to cellular and humoral immune responses

20.5 Diversity of vaccine

(Shi et al., 2014; Meeusen et al., 2007). They are easy to manufacture, have low cost, and do not require cold chain facility. DNA vaccines were administrated either by intramuscular (I/M) injection or using a DNA particle delivery system called gene gun. Immunization of animals with DNA vaccine is comparatively safer than the use of other conventional vaccines as later unnecessarily expose the host to a wide variety of antigen (Jorge and Dellagostin, 2017). However, there is concern regarding possible integration of DNA in the host genome and might be inactivation of a tumor suppressor gene. A few examples of DNA vaccines are West Nile virus vaccine (first approved DNA vaccine), influenza virus DNA vaccine (passed clinical trial for ponies), and feline immune deficiency virus.

20.4 Developments in veterinary vaccinology Since, the discovery of the smallpox vaccine by Jenner in the 19th century, various forms of vaccines have been developed by using advanced recombination technology (Jorge and Dellagostin, 2017). Around two to three decades ago the veterinary vaccines used were mostly live attenuated, inactivated vaccines and toxoid but with recent advances in immunology and molecular biology, and sophisticated forms of genetically-engineered vaccines have been introduced. Although, live-attenuated vaccines are able to induce both cellular and humoral immune responses, they also can produce some side effects. Killed/inactivated vaccines are typically safer but may be less effective than attenuated vaccines whereas, commercial vaccines based on toxoids are difficult to produce. The side reactions, safety issues, effectiveness, etc. are certain issues of aforementioned vaccine and warranted the requirement of better and safer vaccines which can help in the prevention and control of animal diseases. A genetically-engineered vaccine has the potential to alleviate limitations of conventional vaccines. Efforts to develop more effective vaccines against a large number of diseases using genetic engineering are in progress around the world. Genetically-engineered or recombinant vaccines are developed based on rationally designed recombinant and highly purified antigens through epitopes mapping and their prediction. Currently, a number of subunit or vectored veterinary vaccines using biotechnological tool have been commercialized (Table 20.1).

20.5 Diversity of vaccine 20.5.1 Bacterial diseases 20.5.1.1 Hemorrhagic septicemia Hemorrhagic septicemia (HS), an acute and highly fatal disease of cattle and buffalo, is caused by Pasteurella multocida. HS occurs as catastrophic epizootics in

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Table 20.1 A list of recombinant veterinary vaccine. Animal species

Pathogens

Vaccine type

Cats

Feline leukemia virus

Cats

Rabies virus

Cattle

Ripcephalus (Boophilus) microplus Canine distemper virus Canine distemper virus Influenza virus and tetanus toxin Influenza virus West Nile virus West Nile virus Infectious laryngotracheitis Avian influenza

Canarypox virus Vector Newcastle disease virus (NDV) and canarypox vector Babesia bovis

Dog Ferrets Horse Horse Horse Horse Poultry Poultry Poultry Poultry Poultry Racoon/ coyotes Sheep/ goat Swine Swine Swine Swine Swine

Marek’s disease Newcastle disease Mycoplasma gallisepticum Rabies virus Echinococcus granulosus Classical swine fever virus Porcine circovirus Actinobacillus pleuropneumoniae Porcine circovirus Porcine circovirus

Canarypox vector Canarypox vector Canarypox vector Canarypox vector Canarypox/ALVAC vector DNA vaccine Fowlpox vector Fowlpox virus, NDV, herpes virus of turkey, duck enteritis herpes vector Herpes virus of turkey vector Modified NDV Fowlpox vector Racoon poxvirus vector Subunit Recombinant adenovirus vector Subunit Subunit Swinepox vector Subunit

From Jorge, S., Dellagostin, O.A., 2017. The development of veterinary vaccines: a review of traditional methods and modern biotechnology approaches. Biotechnol. Res. Innov. 1, 6 13.

many Asian and African countries, resulting in high mortality and morbidity (De Alwis, 1992; Verma and Jaiswal, 1998). Although, antibiotics is the main therapeutic to treat the disease and control the incidence of such microbial infection, remains of antibiotics in animal products and antibiotic resistance are the drawback of antibiotics use. The other alternative to control and prevention of HS is by vaccination of animals in endemic areas prior to the expected outbreak of HS.

20.5 Diversity of vaccine

Immunity generated in HS is serotype-specific therefore selection of vaccine candidates depend upon circulating serotypes in that geographical regions. Various strategies have been used to develop HS vaccines such as killed vaccines (bacterins), live-attenuated, cellular vaccines, and genetically-engineered vaccines (Myint et al., 1987; Verma and Jaiswal, 1998; Hodgson et al., 2005). But killed vaccines are used commonly for the vaccination against HS. Bacterins used against HS include formalized bacterin, aluminum hydroxide gel, and oil adjuvant vaccines (OIE, 2017). Among these, aluminum hydroxide gel vaccine and oil adjuvant vaccines elicit a good immune response in the studies conducted in many Asian countries including India during the last few years, and are the vaccine of choice.

20.5.1.2 Brucellosis Brucellosis is one of the most important bacterial zoonoses worldwide and characterized with significant economic losses in terms of reproductive performance of dairy animals and posing a continuous threat for human community (OIE, 2017). Disease has wide host range and it is primarily caused by Brucella abortus and Brucella melitensis in large (cattle) and small ruminant (goat) respectively (OIE, 2017). Abortions in late gestation, placentitis, epididymitis, and orchitis are the most common consequences. Direct or indirect contact and consumption of products from infected animals act as a source of human brucellosis. Therefore WHO, OIE, and other agencies collectively set a plan under one health program to control the brucellosis. Animal brucellosis can be prevented by applying good managemental and hygienic practices. Countries having a low prevalence of brucellosis are following the test and slaughter policy while it is not economical in highly endemic counties and vaccination is the only option. Currently, liveattenuated B. abortus strain 19 and RB-51 are used for immunization of cattle while B. melitensis Rev 1 is used for sheep and goat (Moriyo´n et al., 2004; Corbel, 2006). Most of the countries are using B. abortus strain 19 to immunize cattle because of its high protective efficacy, although it induces abortion in pregnant animals and is not capable of DIVA. While RB-51 is not abortogenic and capable of DIVA strategy due to lack of O-antigen of LPS and has similar protective efficacy. New generation vaccine strains based on attenuation organism, protein subunit, and DNA fragments were also tested experimentally to get safer vaccines (Golshani and Buozari, 2017) but none of them are yet commercialized for immunization purpose. Further, killed B. abortus 45/20 and B. melitensis H38 are also available but are less protective (Schurig et al., 2002; Plommet et al., 1970).

20.5.1.3 Anthrax Anthrax organism is a dreaded pathogen of animals and humans characterized by septicemia, sudden death, and oozing of blood from natural orifices of animals. It is caused by a gram-positive, nonmotile and spore-forming bacteria Bacillus anthracis (Kaur et al., 2013). The morbidity and mortality are very high and the affected animals or their remains are a constant threat to humans and other susceptible animals. The animals can be protected by vaccination with a single dose

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of sterne spore vaccine which is an attenuated noncapsulated spore-forming anthrax bacilli (Grabenstein, 2003). Besides, the protective antigen of B. anthracis is also used to immunize the animals in toxoids form (Kaur et al., 2013). Further, E. coli expressed protective antigen of anthrax bacillus (cap1 Tox1) was also evaluated in New Zealand white and rhesus macaques but until today there was no commercialized recombinant vaccine for field use (Chawla et al., 2009; Kaur et al., 2013).

20.5.1.4 Black quarter Black quarter is a fatal infectious disease of cattle, and some other ruminants characterized by fever, myonecrosis of active muscles, edema, lameness, and death. The disease is caused by gram-positive, endospore-forming, histotrophic anaerobic bacteria Clostridium chauvoei (Abreu et al., 2017). It generally affects unvaccinated healthy cattle of 6 24 months of age causing high mortality and significant economic loss. Blackleg is a preventable disease and formalin-treated culture of C. chauvoei formulated with alum as an adjuvant and chemically toxoid culture supernatant are used worldwide for immunization of susceptible groups (Uzal, 2012). Additionally, purified flagellin, crude cell wall proteins, and recombinant CctA were also shown to be promising antigens to induce protective immunity (Frey and Falquet, 2015).

20.5.1.5 Leptospirosis Leptospirosis is a neglected zoonotic disease of humans and animals, caused by Leptospira spp. (Bharti et al., 2003). The disease is characterized by fever, icterus, vomiting, dysentery, dehydration, petechiae of pleura, hemoglobinuria, and grayish white focal necrotic lesions of kidneys. Leptospirosis is a major public health important disease in developing, improvised countries and causes huge production loss in animal husbandry. Current vaccines used for immunization are based on whole cell killed preparation (bacterin), cell membrane extract, and purified outer envelope (Bolin et al., 1991; Cullen et al., 2002; Bharti et al., 2003). Most killed vaccines are of animal use while very few are licensed for human use. The immunity of Leptospira is serovar-specific and there are so many types of serovars present worldwide therefore multivalent bacterin formulations having locally prevalent serovar are used for immunization of cattle, pigs, and dogs worldwide (Bolin et al., 1991). Some recombinant vaccines based on outer membrane proteins, leptospira immunoglobuline-like proteins, and lipoproteins of leptospira were also experimentally evaluated but none of them are available for immunization purpose (Silveira et al., 2017; Faine et al., 1999; Levett, 2001).

20.5.1.6 Mycobacterium infection in cattle Tuberculosis and paratuberculosis are chronic diseases of ruminants caused by Mycobacterium bovis and M. avium subsp. paratuberculosis respectively (Palmer et al., 2011). Bovine paratuberculosis is an infectious, granulomatous disease leading to loss of animal health while paratuberculosis (Johne’s disease) is

20.5 Diversity of vaccine

clinically characterized with chronic shooting diarrhea and emaciation (Gilardoni et al., 2012). Both diseases are collectively causing huge economic loss of the dairy industry worldwide. M. bovis also causes infection in human beings and is one of the major zoonosis concerns of the present time (Grang, 2001). Besides, M. tuberculosis, a pathogen of humans may also infect domestic animals and these infected animals become the source of its further transmission to other susceptible animals and human beings. This phenomenon is called reverse zoonosis. Mycobacterial infection shows synergism with human immunodeficiency virus (HIV) infection in human beings and HIV/M. tuberculosis (dangerous couple model) copandemic is occurring and claiming million of lives each year (Shankar et al., 2014). Crohn’s disease, a chronic inflammatory intestinal condition of human beings is considered to be caused by M. avium subsp. paratuberculosis. Good management practices and test and slaughter policy are used for the control and prevention of these diseases in bovines. The efficacy of a live vaccine made from the attenuated strain of M. bovis, BCG has proven variable and use of this vaccine might hinder the interpretation of current diagnostic tests (Balseiro et al., 2017; Cousins, 2001).

20.5.1.7 Salmonellosis Salmonella organisms are an infectious pathogen that infects animals and humans both (Kemal, 2014). In bovine, the disease is characterized by septicemia, acute or chronic enteritis and abortions (Kemal, 2014). Bovine salmonellosis is caused by Salmonella Dublin and Salmonella typhimurium. Salmonellosis has a significant economic impact on dairy and beef farming due to poor quality of milk and meat (McEvoy et al., 2003). Besides, it also possesses human health concerns due to the consumption of contaminated meat and milk, and close contact of the animal’s handlers and veterinarians. Further, Salmonella isolates particularly S. typhimurium definitive type 104 and have develops resistance to multiple antibiotics and can act as donor for resistant determinant to other opportunistic bacteria found as commensal in intestine (Piddock, 2002). Therefore to avoid possibility of evolution of new resistant bacteria and disease manifestation in animals and humans, vaccination is the most important tool along with good animal husbandry practices. Both inactivated and modified live (MLV) Salmonella vaccines are licensed for immunization of cattle (Danielle, 2006; Adem and Bushra, 2016). Most of the inactivated commercial vaccines are bivalent in nature and have S. Dublin and S. typhimurium formulated with suitable adjuvant (aluminum hydroxide). A genetically- altered aroA mutant S. Dublin live vaccine is also used for immunization of farm animals in different developed countries (Duncan et al., 1987). Further, gram-negative core antigen bacterins such as ENDOVAC-Bovi (S. typhimurium lacking polysaccharide repeat of LPS) and J5 or J5-VAC (LPS core antigen made from mutant strain of E. coli) are commercially available, and it is claiming that they can cross protect animals from other endotoxin-producing bacteria such as E. coli, Salmonella, P. multocida, and Manheimia hemolytica. Besides, autogenous vaccines are also recommended to protect animals on the basis of prevalent Salmonella types on farms.

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20.5.1.8 Escherichia coli infection E. coli is a gram-negative bacilli found as normal intestinal flora of animals and humans, and very few are pathogenic in nature which can cause illness in animals and humans. Generally, healthy animals act as a reservoir of E. coli and asymptomatically shed the E. coli in the environment. Pathogenic E. coli of animals (cattle, sheep, pig, and goat, etc.) are diarrheagenic E. coli (DEC), uropathogenic E. coli, septicemic E. coli, [includes avian-pathogenic E. coli], and the mammarypathogenic E. coli. Enterotoxigenic E. coli (ETEC) producing enterotoxins in pigs and ruminants leads to hyper-secretary diarrhea and electrolytes loss, and the enteropathogenic E. coli causes attaching and effacing (A/E) lesions in most mammals come under the DEC (Hebbelstrup Jensen et al., 2014). E. coli producing Shiga toxin STx2e (Shiga toxin-producing E. coli, STEC, verotoxigenic E. coli or VTEC), is the cause of edema disease in pigs, whereas cattle that produce STx and A/E lesions cause subclinical or nonclinical infections in ruminants (Smith, 2014). Enterohaemorrhagic E. coli (EHEC) causes severe illness in children and the elderly. Clinical sings depend upon types of infections in animals. Clinical manifestations caused by E. coli infection are enteric colibacilosis, colisepticaemia, edema disease, and coliform mastitis, etc. in young (calves, lambs, chicks, and piglets) and adult animals, leading to economic losses (Stein and Katz, 2017). Besides, infected animals are also a potential source for human infections. ETEC infection is a noninvasive type of gastrointestinal infection, and mucosal immunity plays an important role in colonization of these bacteria. Therefore killed bacteria with fimbria or extracted fimbria with or without LT toxoid (heat labile enterotoxin) are used for immunization of dams before parturition. Commercial vaccines for cows include E. coli F5 isolates or F5 adhesin while purified F4, F5, F6, or F41 fimbria or killed E. coli expressing these fimbriae with or without LT toxoid are used for immunization of sows. Further, liveattenuated, oral subunit vaccine having purified fimbria and poly (lactide co glycolide) (PLGA)-encapsulated fimbria or live vaccine was also evaluated for prevention of colonization (Edelman et al., 1993). For prevention of EHEC, whole bacteria, adhesin-intimin, fimbria, type III secretion system were tried (Smith, 2014) and the most successful vaccine is live recombinant Salmonella Dublin expressing E. coli O157:H7 intimin (Khare et al., 2010). Recently, SPR vaccine (bacterial extract siderophore receptor and porin, SRP technology) targeting E. coli O157 serotype is licensed for use in cattle to reduce the amount of E. coli O157 pathogen (Fox et al., 2009).

20.5.2 Viral diseases 20.5.2.1 Foot and mouth disease A very infectious and contagious disease of cloven-hoofed mammals caused by FMDV has seven serotypes (O, A. Asia-1, C and SAT-1, 2, 3) and each serotype has different variants (Jamal and Belsham, 2013; Poonsuk et al., 2018). The

20.5 Diversity of vaccine

disease is characterized by high fever, lameness, formation of blister on mucosa of mouth, tongue, teats, and hoof. The disease has high morbidity and low mortality and affects all age groups of cattle. Due to high morbidity, the disease causes massive production loss and is considered as an economically important disease and a threat to livestock production worldwide. For the control of disease, virus (harvested from BHK-21 cell line) inactivated with binary-ethyleneimine is formulated with saponin/aluminum hydroxide or various oil-based adjuvant is used to potentiate the protective immune response in susceptible animals (Grubman and Baxt, 2004). Immunity induces by one serotype or subtype does not protect animals from other serotypes or subtypes of FMDV infection. Therefore which serotype or subtype is used as a vaccine candidate depends on the circulating FMDV type in that geographical areas/ countries. In India, trivalent vaccine having “O, Asia-1 and A” serotypes are being used for vaccination of cattle and buffaloes (Jamal and Belsham, 2013). Different FMDV eradication programs were launched in various countries, and disease was successfully eliminated from Western Europe and part of South America. But, the disease is still circulating in most parts of the world and posing a constant threat to dairy husbandry. Further, low quality vaccines, the simultaneous presence of various circulating types of FMDV in different countries and wildlife reservoir (African buffalo) are the main constraints in the control and eradication of this dreaded disease.

20.5.2.2 Rabies Rabies is a neglected zoonotic fatal disease of warm-blooded animals including human beings, and caused by rabies virus. It is associated with exposure of rabid animals, and incubation period of the disease depends on the extent of bite, site of bite from the brain, and quantum of virus entered by saliva at the bite wound (Blanton et al., 2009). The disease is reported from all the geographical areas of the world except Antarctica and has around 100% mortality in humans and animals. Over the last 100 years, a number of vaccines such as inactivated, MLV, and recombinant have been developed for the control and prevention of disease (Muller et al., 2001; Xiang et al., 2003; Singh et al., 2017). The neural origin vaccines have been discontinued due to their adverse effects and use of animals for the propagation of the virus. Nowadays modern vaccines, cell culture, and embryonated egg-based inactivated vaccines (Beta-propiolactone) are being used prophylactically (preexposure) and therapeutically (postexposure) to protect humans and animals against rabies (Singh et al., 2017). These modern vaccines are now available in most developing countries and have been successful to minimize the number of human exposures. Further, recombinant vaccines lack residual pathogenicity caused by rabies because they contain only single nonvirulent gene products. Various vectors such as animal poxvirus, human and canine adenoviruses encoding rabies virus glycoprotein G have been tested in different targets (dog, cat, fox, and raccoon) and nontarget wild animals via oral route (rabbit, deer, etc.). Among these vaccines, a vaccinia-based recombinant vaccine is used for immunization of wild animals as edible bait and are playing an important role in

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the prevention of rabies virus from wild animals to other domestic animals and humans (Yang et al., 2013). The oral vaccines are Raboral V-RG (vaccinia recombinant virus expressing G protein) and with Rabigen SAG2 (double mutant avirulent strain SAG2).

20.5.2.3 Peste-des-petits ruminants It is an acute, highly contagious viral disease of small ruminants characterized by fever, loss of appetite, stomatitis, gastroenteritis, and pneumonia (Muthuchelvan et al., 2015). The disease is markedly evident in goats. Goats are more susceptible to PPR compared to sheep. Transmission occurs by direct contact of infected goats and sheep, through contaminated food, water, beddings, and feces. The disease may spread in a flock through the introduction of newly purchased sick animals from the market. The disease has a serious economic impact in terms of high morbidity and mortality as well as reduces production ability. Vaccination is the most effective way to control PPR. An earlier practice to control the disease was to immunize the animals with Plowright’s live-attenuated tissue culture rinderpest vaccine (heterologous vaccine) but its use was stopped due to hindrance in the serosurveillance of rinderpest (Muthuchelvan et al., 2015). Further, homologous PPR virus was used after passage in vero cell line. Presently, Nigeria75/1 strain of Africa, Sungri-96 strain isolated from goats developed by IVRI, Mukteswar or Arasur-87 strain of peste-des-petits ruminants (PPRV) isolated from sheep by TANUVAS are used for immunization of goat and sheep (Diallo et al., 1989, 2007; Palaniswami et al., 2005; Singh et al., 2004). These vaccines are efficacious, safe, and provide a long-term protection to small ruminants.

20.5.2.4 Bluetongue It is an acute but noncontagious disease of sheep characterized by fever, inflammation, and ulceration of buccal mucosa and tongue (Chand et al., 2015; Mayo et al., 2017). It is caused by the bluetongue virus (BTV) which has at least 27 different serotypes worldwide. The disease is transmitted by Culicoides species and affects mostly sheep, goats, and rarely cattle (Chand et al., 2015; Mayo et al., 2017). The disease is prevalent in rainy seasons. For the control of disease besides management practice as well as vector control, immunization of susceptible animals is a more effective strategy. Presently, MLV vaccine and inactivated vaccines are used for the control of the disease in various continents of the world (Bhanuprakash et al., 2009; Chand et al., 2015). As the immunity in BTV is serotype-specific and there are so many circulating serotypes in a geographical area at a time, the vaccine formulation is very difficult and challenging. Because of this reason, multivalent vaccines are used for immunization of animals. MLV vaccine produces viremia in animals which leads to further transmission of the virus and causes abortion, therefore this vaccine is generally not recommended for vaccination. Most of the countries are using inactivated BTV virus using BEI and hydroxylamine (Ramakrishnan et al., 2006). Presently in India pentavalent vaccine having BTV-1, 2, 10, 16, and 23 serotypes are used for the vaccination

20.5 Diversity of vaccine

(Reddy et al., 2010). VLP-based genetically-engineered vaccine was also attempted but due to serotype-specific immunity and genetic drift in serotypes, this strategy was not successful (Chand et al., 2015).

20.5.2.5 Sheep pox and goat pox Sheep pox and goat pox are diseases of sheep and goats caused by sheep pox (SPV) and goat pox virus (GPV) of the genus Capripoxvirus and characterized with pyrexia, generalized lesion, internal pox lesion, and lymphadenopathy (Bhanuprakash et al., 2011; Madhavan et al., 2016). In a susceptible herd, morbidity and mortality are 75% 100% and 10% 85% respectively depending upon virulence of infecting virus strains. Most strains are host-specific and cause severe clinical manifestations in sheep or goat while some strains are equally virulent in both sheep and goats. For the prevention of disease both live-attenuated and inactivated vaccines are available, however inactivated vaccine provides a short duration of protection (Bhanuprakash et al., 2012; Boumart et al., 2016). Liveattenuated vaccine elicits long-term protection against SPV and GPV but its use is limited due to stimulation of pock lesion or death for some animals. Usually, the homologous vaccination strategy is useful for the protection of animals and locally prevalent strains are used as vaccine strains for immunization of sheep and goats (Rao and Bandyopadhyay, 2000). In India, live-attenuated vaccine incorporated with RM-65 strain for sheep pox and Uttarkashi strain of goat pox is currently used for immunization of sheep and goats, respectively (Madhavan et al., 2016).

20.5.2.6 Classical swine fever It is an acute, highly infectious viral disease of swine of all ages characterized by rapid and sudden onset, high morbidity, mortality, and generalized hemorrhages (Blome et al., 2017). It has a massive impact on pig industries and is therefore notifiable to the OIE (2017). For prevention of disease live-attenuated vaccines are used. Currently, live-attenuated vaccine strains such as Chinese strain, Weybridge strain, Thiverval and lapinized virus, produced by the repeated passage of virus in tissue culture of porcine origin (PK-15) and rabbit are used for immunization of pigs (Blome et al., 2017). Additionally, E2 protein- based marker vaccine is also used for differentiation between infected and vaccinated animals (Huang et al., 2014). Recently a chimeric pestivirus vaccine “CP7_E2alf” was found safe and efficacious following oral administration and licensed for the oral immunization of pig (Eble´ et al., 2014a,b).

20.5.2.7 Japanese encephalitis virus Japanese encephalitis (JV) is a zoonotic viral encephalitic disease with high morbidity and mortality in human and livestock. The causative agent of this vectorborn disease is Japanese encephalitis virus (JEV), a member of genus Flavivirus and transmitted by thebite of Culex mosquitoes (Basu and Dutta, 2017). Generally, JEV maintained in a natural cycle between mosquitoes and water bird,

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and pig acts as an amplifying host (Yun and Lee, 2014). Accidentally, at peak of mosquitoes’ prevalence in rainy seasons, the virus also infects dead-end hosts; human and horse due to mosquitoes’ bite. Infections in pigs lead to significant reproductive problems causing abortion, still-birth, and birth defects while horses suffer from pyrexia and neurological manifestations leading to death (Lindahl et al., 2013). Both inactivated and live-attenuated vaccines are available for pigs, horses, and humans. MLV [produced in hamster or swine kidney tissue culture or hamster lung (HmLu) cell line] and inactivated (prepared in mouse brain, chicken embryo eggs, or cell lines, e.g., vero cells) are used for immunization of pigs and horses (Basu and Dutta, 2017). A genetically-engineered JE vaccine that combines the attenuated JEV strain and yellow fever vaccine virus is also available for humans (Janewongwirot et al., 2016).

20.5.2.8 Bovine viral diarrhea It is an economically important infectious disease that affects a wide range of animals belonging to order Artyodactyla, including cattle, sheep, goat, camel, pig, and other domestic and wild ruminants, manifested with reproductive, respiratory, and gastrointestinal alignments. The disease is enlisted in the OIE. The disease is caused by bovine viral diarrhea virus-1 (BVDV-1) and bovine viral diarrhea virus-2 (BVDV-2) belong to genus Pestivirus of Flaviviridae family. At present at least 21 (BVDV-1a 1u) and 4 (BVDV2a 2d) subgenotypes of BVDV-1 and BVDV-2, respectively have been identified. On the basis of cytopathic effect on cell culture, each genotype is further classified into cytopathic (CP) and noncytopathic (NCP) biotypes. The clinical infections are of, acute and transient infection in immune-compromised cattle and persistent infection in new born calf when the virus infects the dam in the first trimester of gestation (before the development of the immune system) and chronic infection due to the invasion of virus in immune-privileged sites. The persistently infected (PI) animals act as a major source of disease transmission in the herd due to constant shedding of virus from all secretions. Economic impact in terms of reduction in milk yield, loss of fetus due abortion, still-birth, mummification, and low body score, led various countries to start a control program to curtail and eradicate the disease from the livestock population. In order to control and prevent, prophylactic vaccination of susceptible animals and test and culling strategy depending upon seroprevalence of disease, cattle density, and trade has been adopted in European countries and results were quite convincing. The primary goal of prophylactic vaccination against BVDV is to protect the fetus from in-utero infection to avoid birth of new PI calves. Both MLV and inactivated vaccines are used for immunization of animals (Beer et al., 2000). It is considered that inactivated vaccine is safer than MLV and therefore MLV vaccine is not recommended to pregnant animals in their first 6 months. In contrast to NCP biotypes, most modern modified-live vaccines use CP biotypes of BVDV as these types of virus are not able to establish persistent infection in fetus. A Npro gene deleted and endoribonuclease activity inactivated NCP BVDV mutant was developed to deal with safety concerns which

20.5 Diversity of vaccine

is not able to cross the placenta and provides immunity similar to field type BVDV. Marker vaccines based on glycoprotein E-2 expressed in baculovirus or transgenic plant and BVDV E-2 DNA vaccines have also been evaluated for immune response (Thomas et al., 2013). Recently, truncated glycoprotein E-2 fused with single chain antibody (APCH) subunit vaccine (Pecora et al., 2015) (Vedevax) expressed in baculovirus was commercialized for field use. Protective immune response in BVDV is genotype-specific and is not effective in conferring cross- protection to heterologous genotypes. Therefore vaccine formulations require either one or both genotypes depending upon the prevalence of BVDV in a particular continent or geographical strata. To address this aforementioned problem, a novel mosaic polypeptide chimeras having three protective determinants; of BVDV-1a, BVDV-1b and BVDV-2 genotypes using adenovirus vector construct (adBVDV prototype vaccine) was evaluated and found better immunogenic with heterologous protection (Lokhandwala et al., 2017).

20.5.2.9 Infectious bovine rhinotracheitis It is one of the agents of bovine respiratory disease complex characterized by inflammation of nose and trachea of cattle. Bovine herpesvirus-1 (BoHV-1), a member of alphaherpesevirus is the etiological agent of IBR. This virus also causes infectious pustular vulvovaginitis in cows and infectious pustular balanoposthitis in bulls. Latency inside sensory ganglion is the most unique feature of BoHV-1 which is also seen in other herpes viral infections. Latent animals become clinically infected once again due to recrudescence of virus by stressful stimuli and subsequent reexcretion of infectious virus acts as a source of infection for other susceptible animals of the herd. It is a major economic problem in dairy and beef industries of the world due to huge production losses in the form of reduced milk yield, abortion, and less weight gain. The biosecurity, test and culling, and prophylactic immunization are used for control of the disease. Around 200 vaccines have been licensed for immunization against IBR worldwide. Among these most are conventional types (nonmarker) while very few are marker types. Conventional vaccines include either live or inactivated BoHV-1 strain while marker vaccines are gene deleted type mutants. Glycoprotein E, thymidine kinase (tk) gene or both, nonessential genes for virus replication, are targets of deletion from BoHV-1 virus and are well suited for DIVA. A double-gene deleted (gE2 and tk2) Bovine Herpes Virus type 1 vaccine is commercially available (van Engelenburg et al., 1994). These vaccines are effective in preventing clinical disease and reducing virus transmission but are not able to prevent infection from field virus. Most of the European countries have banned the use of conventional live vaccine and are strictly using marker vaccines for effective protection and serosurveillance with the aim of disease eradication. Majority of these vaccines are licensed for immunization of pregnant animals. Most of these vaccines are licensed for use in the United States or European countries and manufactured by Zoetis UK, MSD Animal Health and Laboratorios Hipra, Spain.

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20.5.2.10 Influenza (flu) Influenza in domestic animals is caused by members of genus influenza virus A and most of have a zoonotic impact worldwide. Influenza virus A infects bird (both domestic and wild), pig, horse, dog, seal, whales, including human (Webster et al., 1992). Avian influenza, swine influenza, equine influenza, and canine influenza are the most common types of influenza virus A infection in birds, pigs, equines, and canines, respectively (Webster et al., 1992; Yoo et al., 2018). At present 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes have been recognized, while 2 additional HA and NA subtypes have been identified in bats and these subtypes can form thousands of antigenic combination (Gamblin and Skehel, 2010; Ciminski et al., 2017). Influenza virus A subtypes; H7N7and H3N8 cause respiratory disease in horses, H1N1, H1N2 and H3N8 cause influenza in swine, H3N8 and H3N2 cause respiratory implications in dogs (Yoo et al., 2018). Generally, all genetic combinations are reported from the domestic or wild bird that causes respiratory and systemic implication, but detection of H5 and H7 are prime importance due to their high virulence (Harfoot and Webby, 2017). In humans historically endemic H1N1, H2N2, H3N2, and more recently sporadic or limited H5N1, H7N3, H7N7, and H9N2 viruses caused respiratory diseases while H3N2 and H1N1 are currently circulating subtypes (Yoo et al., 2018). Avian influenza on the basis of the presence of single or several basic amino acids at the cleavage site in haemagglutinin are classified into low pathogenicity avian influenza (LPAI) and highly pathogenicity avian influenza (HPAI) virus (Lee et al., 2004). Both types affect different avian species but wild and migratory birds act as a reservoir of LPAI. OIE has defined avian influenza as “an infection of poultry caused by any influenza A virus with HPAI and by H5 and H7 subtypes with low pathogenicity (H5/H7 LPAI).” Affected birds exhibit varying clinical manifestations from mild to severe respiratory, nervous, gastrointestinal, and reproductive system disease and sometimes birds are dead without any clinical appearance (Horby, 2014). Further, LPAI viruses also cause a considerable loss due to anorexia, respiratory signs, reduce egg production, and less weight gain. OIE recommended eradication of HPAI virus from poultry due to severe economic consequences in poultry industries in terms of reduced egg production, low quality of eggs, mortality, and evolution of new antigenic mutants via antigenic shift and drift posing a threat to humans. In addition to the controlled elimination of infected poultry, strict biosecurity, restriction on movement and purchase of birds, and good hygiene in the poultry farm, vaccination of birds is also followed. Currently inactivated mono and bivalent vaccines having H5 and H7 strains and live recombinant vaccines (fowlpox-H5) are available for immunization of poultry (Swayne, 2012). The reverse genetic-based recombinant H5N1 vaccine was also evaluated in mice and found to be a promising candidate vaccine against HPAI in poultry (Sedova et al., 2012; Lee and Song, 2013). Swine influenza is another economically important disease caused by influenza virus A and affects the pork industry due to the significant reduction in

20.5 Diversity of vaccine

growth rate and public health misperception about eating of pork. Besides, H1N1 is the most common flu which affects humans worldwide and causes mortality. Good management practices in swine farming and use of vaccines can limit the swine influenza and consequently the possibility of human transmission. Most of the available licensed vaccines have inactivated whole virus of H1 or H3 subtypes. For immunization of pigs bivalent vaccine having H1N1 and H3N2 are used through I/M route and are protective to antigenically identical or similar strains. Recently intranasal poly I: C adjuvanted vaccine was found more protective compared to conventional vaccines (Kim et al., 2015). Further polyvalent vaccines containing multiple H1 and H3 clusters were commercialized with the goal of protection from new emerging antigenic cluster within subtypes of H1 and H3 (MaxiVac Excell 5.0, Merck Animal Health, Summit, NJ, USA; FluSure XP, Zoetis, Florham Park, NJ, USA). New generation vaccines based on reverse genetic strategy to make attenuated- live vaccine, DNA vaccine, subunit vaccine, and vectored 5 based vaccine were also evaluated experimentally for immunization of pigs but only alphavirus-like replicon particles (RP) having gene encoding the HA of a cluster IV H3N2 virus was licensed for pig use (“Swine Influenza Vaccine, RNA”; Harris vaccines, Ames, IA, USA) (Abente et al., 2019). Equine influenza, a highly contagious respiratory disease of equine characterized with high temperature, nasal discharge, coughing, with high morbidity, and occasional mortality in foal and donkeys is one of most important infectious respiratory disease of equine worldwide. The disease has an impact on racing horse industries and tourism in hilly tracts due to inability to move. Besides, equine influenza is known to infect humans and dogs, and have the potential to generate pandemic virus. Vaccination is the most effective strategy in addition to isolation, restriction in movement, biosecurity measures to prevent disease, and its consequence on public health. Three different types; inactivated whole virus/ subunit-ISCOM-matrix or ISCOM, live- attenuated and vector-based equine influenza vaccines are available commercially (Dilai et al., 2018). The currently licensed inactivated vaccines contain H3N8 and H7N7 strains while live- attenuated vaccines have cold-adopted H3N8 strain. Subunit vaccines have either HA or both HA and NA proteins formulated with a suitable adjuvant. Canary pox virus vector is used for expression of HA gene expression after injection in the host. Currently inactivated and recombinant vaccines are used most frequently for immunization of horses.

20.5.2.11 Winter dysentery Winter dysentery is an infectious and contagious gastroenteric disorder of adult cattle, often reported in winter season characterized by profuse watery diarrhea with fresh blood, significant loss in milk production, and disturbs health conditions. The causative agent of this highly morbid disease is bovine coronavirus (BCoV) (Saif, 1990). In addition to gastric infection, BCoV also affects the respiratory system of calves and feedlot cattle. The disease on sets is sudden and within a few days, most of the animals of the herd suffer from diarrhea. Milk

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production may not return to full capacity even after a long time of animal recovery or of that lactation. Due to rapid onset, high morbidity, and huge reduction in milk loss the disease is worrisome to dairy industries and farmers. For prevention of winter dysentery, there is no dedicated vaccine. However MLV coronavirus vaccine (BOVILIS Coronavirus, Intervet/Merck Animal Health) which is recommended for calves’ diarrhea caused by bovine coronavirus. Further, a solubilized antigen from BCoV-infected cells combined with an oil adjuvant was tested as a prototype vaccine to be used against winter dysentery (Takamura et al., 2000, 2002).

20.5.2.12 Rotavirus gastroenteritis Rotaviruses (RV) affect young ones of cattle, pigs, sheep, goats, horses, and poultry, including humans. It is one of the major concerns of neonatal diarrhea in domestic animals and mostly caused by group A RV (total 9 group A I). Bovine rotavirus (BRV) affects calves of 2 8 weeks of age and its susceptibility decreases as age progresses. Clinical manifestation in each species is similar ranging from asymptomatic subclinical condition to severe enteritis. Clinically calves suffer from acute, watery, dehydrating diarrhea and may succumb to infection. The morbidity and mortality are very high, leading to huge economical losses in dairy and beef industries worldwide. Mucosal immunity plays a major role to inhibit intestinal infections by any infectious pathogen and it is transferred from the dam to new young ones via colostrum feeding. Therefore if the pregnant dam is immunized with a rotavirus vaccine sufficiently, she can transfer antirotavirus maternal antibodies in surplus to protect calves sufficiently long duration. Both conventional and new generation vaccine such as subunit, DNA vaccine, VLP, plant-based edible vaccine (used VP-4, VP-6, or capsid protein), reverse geneticbased vaccine and recombinant BCG expressing VP-6 gene were evaluated to generate protective immunity (Poelaert et al., 2018; Chen et al., 2012). Commercially attenuated strains of BRV and coronavirus (Galf Guard, Zoetis, USA, PBS animal health, United States) are used to immunize calves and adult cattle. Inactivated BRV (serotypes G6 and G10) and coronavirus propagated on established cell lines and a K99 E. coli bacterin formulated with adjuvant (scourGuard, Zoetis, USA, MSD animal health) are used to immunize pregnant cattle and heifers. Further, some vaccines have Clostridium perfringens type C and type D toxoid, E. coli K99 along with inactivated bovine coronavirus type 1 and 3, inactivated BRV type G6 and G10 (Cooper).

20.5.2.13 Parasitic vaccines Livestock is susceptible for so many parasitic infestations such as nematodes, protozoa, and insects. The parasitic infestations lead to poor animal’s performance and their productions. For the control of parasites, different antimicrobials are available but due to the evolution of antimicrobials resistance, they are becoming ineffective against most of the parasites. Further, various vaccination strategies were also employed to develop parasitic vaccines but very few vaccines are

20.5 Diversity of vaccine

commercially available for immunization, probably due to difficulties in vaccines development, poor immune responses, and very high cost of production. The vaccines which are used for immunization of livestock are described here.

20.5.2.14 Theileriosis A tick-born apicomplexan parasitic disease affects domestic and wild ruminant worldwide. The disease is caused by Theileria species, most notably T. parva and T. annulata in cattle and T. lestoquardi in sheep. Transmission of T. parva and T. annulata are through Rhipicephalus appendiculatus ticks, occurs in eastern and southern Africa, and by Hyalomma ticks, occurs around the Mediterranean basin, north-east Africa, the Middle East, India, and southern Asia, respectively. African buffalo and Asian buffalo are also susceptible to T. parva and T. annulata, respectively. Theileria species cause acute lymphoproliferative disease with a high level of morbidity and mortality and economic losses (Sivakumar et al., 2014). For control of the disease, acaricides and buparvaquone (therapeutic compound) are used but due to regular use of acaricides and the high cost of buparvaquone, the overall control and treatment are very expensive. Besides, drug resistant T. annulata is also reported recently. Vaccination is the only sustainable alternative of these limitations (Nene and Morrison, 2016). Immunization with T. parva and T. annulata infected cell line as live vaccines were attempted but found to be noneconomical. A live vaccine having infectious sporozoites was developed. Because of limitations in live vaccines, other alternatives were searched to develop subunit and viral-vectored vaccines based on the use of defined antigens of sporozoite (Knight et al., 1996) and schizont (Goh et al., 2016) developmental stages. But at present only live vaccines against both T. parva and T. annulata based on sporozoites are used to immunize the animals.

20.5.2.15 Coccidiosis Avian coccidiosis is responsible for huge economic losses in the poultry sector incurred by parasitic diseases and is caused by different Eimeria species. Among them, Eimeria tenella is the most pathogenic one and can develop resistance rapidly against anticoccidial drugs. For control of coccidia in poultry farm, prophylactic use of anticoccidial drugs were followed since long and still are the preferred method. But the problem is the quick development of resistance against available anticoccidial drugs and requirement of new drugs. In addition to chemotherapy, vaccination is also used to protect chickens (Tewari and Maharana, 2011). Most commonly used vaccines for immunization of chickens are live oocysts either from attenuated or nonattenuated strains of coccidian (Chapman and Jeffers, 2014). Nonattenuated live vaccines have variable numbers of wild coccidian strains depending upon their use in broiler breeders (up to eight strains, Coccivac-D, and Immucox-C2), or broiler industries (up to four strains Coccivac-B, Immucox-C1) but the main risk is development of severe reaction in vaccinated poultry. The live-attenuated oocysts vaccine strains (Paracox and HatchPak CocciIII) have fewer vaccines-induced risks (Price, 2012). Indigenous live-attenuated

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quadrivalent coccidia vaccine having Eimeria tenella, E. acervulina, E. maxima, and E. necatrix (LivacoxQ, Hester) is available in India. DNA recombinant technology was also used to develop a recombinant vaccine based on an immunodominant portion of proteins of various stages either of sporozoites or merozoites or gametes of Eimeria species (Tewari and Maharana, 2011). At present only one commercialized subunit vaccine is available for coccidiosis (CoxAbic) based on purified native protein extracted from gametocytes of Eimeria.

20.5.2.16 Parasitic bronchitis Parasitic bronchitis is primarily a disease of cattle caused by D. viviparous which is also called lung worm. The disease is characterized with extension of neck, open mouth breathing, and coughing which is consider as “hoose or husk.” Morbidity is high but mortality is less and in less severely affected animals recover by self-cure phenomenon after several months. For control of lung worm anthelmintic drugs and prophylactic vaccines are used. Prophylactic vaccination is done by commercially-available live-attenuated vaccine incorporated with gamma irradiated third stage larva (L3). Though this vaccine is used successfully in different developed countries, it has some limitations such short shelf-life, requirement of booster, and high cost. Recently, a recombinant subunit vaccine based on parasites’ muscle protein paramyosin expressed in E. coli was evaluated to control lung worm burden in cattle in comparison to irradiated D. viviparous vaccine (Bovilis Dictol live vaccine) and found to be a promising strategy to develop recombinant vaccine against lungworm infestation in cattle.

20.6 Combined vaccination Inoculation of more than one vaccine by single shot is called combined vaccination. The first combined vaccination was done since long back in 1948 to vaccinate infants with combination of diphtheria, tetnus, and pertusis (DPT) vaccines in a single shot. Inoculation of multiple vaccines in single volume at a time reduces the multiple injections, time to vaccinate the animals, suffering of animals, cost and visit of veterinarian. It provides protection against multiple pathogens simultaneously and minimizes chances of missing vaccination schedule and time. There are so many available combined vaccines for companion and domesticated animals. For vaccination of pups core vaccine; canine parvovirus, canine distemper virus, canine adenovirus, canine parainfluenza, canine corona virus, rabies, and leptospira are given in combined form by single shot. Bovine respiratory syncytial virus, bovine viral diarrhea virus types 1 and 2 and Mannheimia haemolytica are inoculated simultaneously in cattle or buffalo while sheep pox either with PPRV or bluetongue vaccines are used for vaccination of sheep (Table 20.2).

20.7 Poultry vaccines

Table 20.2 Recommended vaccines for cattle. Name of disease

Age at first dose

Booster dose

4 months and above

Hemorrhagic septicemia Black quarter (BQ) Brucellosis

6 months and above

Annually in endemic areas

6 months and above

Annually in endemic areas

4 8 months of age (only female calves) 3 months of age and above

Once in a lifetime

Theileriosis

Anthrax IBR

4 months and above 3 months and above

Rabies (post bite therapy)

Immediately after suspected bite

1 month after first dose

Subsequent dose

Foot and mouth disease (FMD)

1 month after first dose Fourth day

Six monthly

Once in a lifetime. Only required for crossbred animals Annually in endemic areas Six monthly

7, 14, 28, and 90 (optional) days after first dose

20.7 Poultry vaccines The poultry market is the biggest market in livestock sectors, as poultry farming such as chickens require less time to attain marketable age, produce nearly one egg each day, and require less investment. Chickens are reared mainly for broiler (meat) and layer (eggs) purposes. Along with nutritional and housing management, good health is of paramount importance to achieve better growth rates in broilers and to get good quality eggs from layers throughout year. Poultry are susceptible to many infectious diseases such as infectious bursal disease, infectious bronchitis, infectious laryngotracheitis, Marek’s disease, Newcastle disease, Fowl pox, avian influenza, fowl cholera, fowl typhoid, bacillary white diarrhea, chronic respiratory disease, and coccidiosis, etc. (Deshmukh et al., 2015; Jordan, 2017; Garcı´a, 2017; Yuan et al., 2018; Alkie and Rautenschlein, 2016; Wua et al., 2011; Reddy et al., 2016). Morbidity and mortality caused by the pathogens are very high leading to negative impact on production and human welfare due to shortage of food supply. These diseases could be controlled by immunization of poultry flocks with negligible expense on each bird. For immunization of poultry conventional (inactivated and live) and biotechnological or genetic engineering (subunit, vectored, DNA, and VLP) tools have been employed to develop effective vaccines. But, availability of recombinant (biotechnological based) vaccines for field use are very limited as most of them are in different phases of clinical trials or have some quality control issues. At present either inactivated or

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live-attenuated vaccines are being used for mass immunization of poultry flocks. The susceptibility toward different diseases depends on the age of birds. Therefore two types of vaccination schedule are recommended for poultry flocks, namely for boilers and layers (Table 20.3). Table 20.3 Commercialized and candidate vaccines for poultry. Pathogens

Inactivated

Live

Recombinant

Newcastle disease

Different strains such mesogenic (R2b,) and lentogenic (Lasota, B1, F) are used after chemical inactivation

Attenuated strains such mesogenic (R2b,) and lentogenic (Lasota, B1, F)

Infectious bronchitis

Formaldehyde inactivated Massachusetts (Mass) serotype IBV (most common) and other serotypes such as; Arkansas (Ark), Connecticut (Conn), Delaware (Del), Georgia98 (GA98), Georgia 08 (GA08), and Georgia 13 (GA13)

Massachusetts (Mass) serotype IBV by serial passage or both passage and mild heat treatment

Vectored vaccine 1 Fowl pox vectored vaccine expressing hemagglutininneuraminidase (HN) or F gene 2 Herpes virus of turkey expressing F gene 3 Recombinant Marek’s disease virus vaccine of serotype 1 (Rispens strain) expressing the protein encoded by the VP2 gene of IBDV with a rHVTND 4 Recombinant Infectious bursal disease virus (IBDV) containing the HN of NDV Vectored vaccine 1 HVT and Fowl pox virus encoding S-1 gene 2 Viral backbones, such as NDV, duck enteritis virus, and avian metapneumovirus encoding S-1 and S-2 Recombinant live virus Reverse geneticbased recombinant virus coding spike gene from avirulent virus (Continued)

20.7 Poultry vaccines

Table 20.3 Commercialized and candidate vaccines for poultry. Continued Pathogens

Inactivated

Live

Recombinant

Infectious laryngotracheitis

Chicken embryo origin (CEO) SA2, A20 and tissue culture origin (TCO) GaHV-1 vaccines

Infectious bursal disease

Mild, intermediate, or intermediate plus strains

Vectored vaccine 1 FPV vector having the GaHV-1 glycoprotein B and UL32 genes 2 HVT vector coding GaHV-1 glycoproteins I, B, and D 3 Bivalent HVT or FPV vaccine for GaHV-1 and MD 4 LaSota strain expressing GaHV1 glycoproteins vaccine 5 Modified very virulent (vv) serotype I Marek disease virus (MDV) expressing GaHV-1 glycoproteins Vector vaccine 1 Fowl pox and Marek’s disease vector vaccine expressing VP-2 gene 2 Bacterial delivery VP-2 gene of IBDV by Salmonella typhimurium Subunit vaccine 1 Hypervariable region of VP-2 expressed in Pichia pastoris or E. coli DNA vaccine Immunodominant VP2 gene fragment (VP252 417), VP2 and HSP70 (fused and expressed in one plasmid), (Continued)

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Table 20.3 Commercialized and candidate vaccines for poultry. Continued Pathogens

Inactivated

Marek’s disease

Infectious coryza

Salmonella

Whole cell Avibacterium paragallinarum serovars A-1, B-1, C1, or C-2 killed with thimerosal or formalin (most widely used) Inactivated whole cell S. Enteritidis

Live

Recombinant

Serotype 3 vaccines: MDV-3: most commonly used strain is FC126. Serotype 2 vaccines: MDV-2: CVI988 strain or Rispens (most efficient vaccine) Serotype 1 vaccines: MDV-1: HPRS-16/att Live-attenuated strains of A. paragallinarum serovars A-1, B-1, C-1, or C-2

Vectored vaccine 1 FPV expressing MDV1-gB

Live-attenuated mutant or genedeleted salmonella Such Salmonella Enteritidis ΔaroA,

Subunit vaccine: S. Enteritidis protein extract or protein, FliC, Type I fimbriae and SPI-1 and SPI-2 proteins DNA vaccine: Bacterial plasmid encoding SopB, a Salmonella SPI-1 effector protein

S. typhimurium Δcya/crp, Ts S. Enteritidis mutant, S. Enteritidis ΔphoP/fliC

Fowl cholera

Killed serotypes A-1, A-3, and A-4 of Pasteurella multocida strain

Live-attenuated serotype of P. multocida

MDV-1 Gene deletion vaccines: MD virus having pp38 deletion, or vIL8 deletion or vTR deletion/mutation or Meq deletion (ΔMeq) Subunit vaccine Hypervariable region in the HA proteins of A. paragallinarum serovars A and C expressed in E. coli

Vectored vaccine: Live-attenuated Salmonella itself acts as vector for delivery of other antigen and induce immunity against itself Subunit vaccine Outer membrane protein H (rOmpH) expressed in E. coli

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20.8 Adverse effect of vaccines Though, vaccines are considered excellent in preventing infectious diseases, they have some adverse effects on the host. Adverse effects caused by vaccines may be transient or for longer duration and can be caused either by antigens or adjuvants present in vaccines. Generally the side reaction is associated with live vaccine but killed vaccine also in some cases can cause a reaction. Latent infections can be caused by a certain vaccine virus, that is, herpesvirus vaccines. In some cases animal may fail to respond to vaccine or it may be excreting vaccine virus or bacteria in their secretion and excretion such as BVDV and Brucella vaccines. Sometimes MLV BTV vaccines regain virulence inside the host/vectors leading to development of clinical manifestations and raising the concern about possibilities of genetic assortment between vaccine and wild viruses. Feline leukemia virus vaccine at the injection site in leg causes development of a lump which regresses within few days but sometimes cats suffer with a lethal cancerous condition called fibrosarcoma. Rabies vaccines also lead fibrosarcoma development at inoculation site in cats similar to feline leukemia vaccines. Other common side effects include: transient swelling at the site of injection, coughing, fever, anaphylaxis, respiratory distress, salivation, vomiting, diarrhea, urticaria, reduced fertility, abortion, and fetal abnormalities. No doubt some vaccines have adverse effects but overall advantages of vaccination outweight the adverse effects. Immunizations of livestock against different infectious agents are playing a pivotal role in keeping animals healthy, sustaining animal production and food security. This is only due to vaccination, the world has become free from two dreaded diseases such as small pox of human beings and rinderpest of cattle. Besides, some other infectious diseases also has been eradicated from different countries such as African horse sickness, FMD, swine vesicular disease, and rabies, etc., and many more are in the line of eradication.

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van Gelder, P., Makoschey, B., 2012. Production of viral vaccines for veterinary use. Berl. Mu¨nch. Tiera¨rzt. Wochenschr. 125 (3 4), 103 109. Available from: https://doi.org/ 10.2376/0005-9366-125-103. Vandersmissen, A., Welburn, S., 2014. Current initiatives in One Health: consolidating the One Heatlh Global Network. Sci. Tech. Rev. 33 (2), 421 432. Verma, R., Jaiswal, T.N., 1998. Haemorrhagic septicaemia vaccines. Vaccine 16 (11-12), 1184 1192. Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M., Kawaoka, Y., 1992. Evolution and ecology of influenza A viruses. Microbiol. Rev. 56 (1), 152 179. Wua, J., Wub, Y., Shienb, J., Hsuc, Y., Chend, C., Shiehb, H., et al., 2011. Recombinant proteins containing the hypervariable region of the haemagglutinin protect chickens against challenge with Avibacterium paragallinarum. Vaccine 29, 660 667. Xiang, Z.Q., Gao, G.P., Reyes-Sandoval, A., Li, Y., Wilson, J.M., Ertl, H.C., 2003. Oral vaccination of mice with adenoviral vectors is not impaired by preexisting immunity to the vaccine carrier. J. Virol. 77, 10780 10789. Yang, D.K., Kim, H.H., Lee, K.W., Song, J.Y., 2013. The present and future of rabies vaccine in animals. Clin. Exp. Vaccine Res. 2, 19 25. Yoo, S.J., Kwon, T., Lyoo, Y.S., 2018. Challenges of influenza A viruses in humans and animals and current animal vaccines as an effective control measure. Clin. Exp. Vaccine Res. 7 (1), 1 15. Yuan, Y., Zhang, Z.P., He, Y.N., Fan, W.S., Dong, Z.H., Zhang, L.H., et al., 2018. Protection against virulent infectious bronchitis virus challenge conferred by a recombinant baculovirus co-expressing S1 and N proteins. Virus 10 (7), E347. Available from: https://doi.org/10.3390/v10070347. Yun, S.I., Lee, Y.M., 2014. Japanese encephalitis: the virus and vaccines. Human Vaccines and Immunotherapeutics 10 (2), 263 279. Available from: https://doi.org/10.4161/ hv.26902. Epub 2013. Zhang, N., Huang, D., Wu, W., Liu, J., Liang, F., Zhou, B., et al., 2018. Animal brucellosis control or eradication programs worldwide: a systematic review of experiences and lessons learned. Prev. Vet. Med. Available from: https://doi.org/10.1016/j. prevetmed.2018.10.002.

Further reading Chen, H., Cui, P., Cui, B., Li, H., Jiao, X., Zheng, L., et al., 2011. Immune responses of chickens inoculated with a recombinant fowlpox vaccine coexpressing glycoprotein B of infectious laryngotracheitis virus and chicken IL-18. FEMS Immunol. Med. Microbiol. 63, 289 295. Dellagostin, O.A., Grassmann, A.A., Rizzi, C., Schuch, R.A., Jorge, S., Oliveira, T.L., et al., 2017. Reverse vaccinology: an approach for identifying leptospiral vaccine candidates. Int. J. Mol. Sci. 18, 158 174. Desin, T.S., Ko¨ster, W., Potter, A.A., 2013. Salmonella vaccines in poultry: past, present and future. Expert Rev. Vaccines 12 (1), 87 96. Dhama, K., Chauhan, R.S., Mahendran, M., Malik, S.V.S., 2009. Rotavirus diarrhea in bovines and other domestic animals. Vet. Res. Commun. 33, 1 23.

Further reading

Diego, E., Gomez, J., Weese, S., 2017. Viral enteritis in calves. Can. Vet. J. 58, 1267 1274. Dimitrova, K.M., Afonsoa, C.L., Yub, Q., Millera, P.J., 2017. Newcastle disease vaccines—a solved problem or a continuous challenge. Vet. Microbiol. 206, 126 136. Available from: https://doi.org/10.3390/ijms18010158. Go´mez, L., Alvarez, F., Betancur, D., On˜ate, A., 2018. Brucellosis vaccines based on the open reading frames from genomic island 3 of Brucella abortus. Vaccine 36, 2928 2936. Idrees, M.A., Younus, M., Farooqi, S.H., Khan, A.U., 2018. Blackleg in cattle: current understanding and future research perspectives—a review. Microb. Pathog. 120, 176 180. Available from: https://doi.org/10.1016/j.micpath.2018.04.047. James, A.R., 2011. Veterinary vaccines and their importance to animal health and public health. Procedia Vaccinol. 5, 127 136. ˚ ., Martonb, S., Coskunc, N., Ba´nyaib, K., Alkana, F., 2017. Karayela, I., Fehe´rb, E.A Putative vaccine breakthrough event associated with heterotypic rotavirus infection in newborn calves, Turkey. Vet. Microbiol. 201, 7 13. Kiril, M., Dimitrova, C.L., Afonsoa, Q.Y., Patti, J., 2017. Miller Newcastle disease vaccines—a solved problem or a continuous challenge. Vet. Microbiol. 206, 126 136. Kirkwood, C.D., Ma, L., Carey, M.E., Steele, A.D., 2017. The rotavirus vaccine development pipeline. Vaccine S0264-410X (17), 30410 30413. Lee, N., Lee, J., Park, S., Song, C., Choi, I., Lee, J., 2012. A review of vaccine development and research for industry animals in Korea. Clin. Exp. Vaccine Res. 1, 18 34. Lubroth, J., Rweyemamu, M.M., Viljoen, G., Diallo, A., Dungu, B., Amanfu, W., 2007. Veterinary vaccines and their use in developing countries. Rev. Sci. Tech. (Int. Off. Epizoot.) 26 (1), 179 201. Morales-Erasto, V., Maruri-Esteban, E., Trujillo-Ruı´z, H.H., Talavera-Rojas, M., Blackall, P.J., Soriano-Vargas, E., 2014. Protection conferred by infectious coryza vaccines against emergent Avibacterium paragallinarum Serovar C-1. Avian Dis. 59 (1), 162 164. Na, W., Yeom, M., Yuk, H., Moon, H., Kang, B., Song, D., 2016. Influenza virus vaccine for neglected hosts: horses and dogs. Clin. Exp. Vaccine Res. 5, 117 124. Nascimento, I.P., Leite, L.C.C., 2012. Recombinant vaccines and the development of new vaccine strategies. Braz. J. Med. Biol. Res. 45 (12), 1102 1111. Natalie, A.P., Bryce, M.B., 2015. Immunity and vaccination against tuberculosis in cattle. Curr. Clin. Microbiol. Rep. 2, 44 53. Nettleton, P., Russell, G., 2017. Update on infectious bovine rhinotracheitis. In Practice 39, 255 272. Paillot, R., 2014. A systematic review of recent advances in equine influenza vaccination. Vaccines 2, 797 831. Pourasgari, F., Kaplon, J., Karimi-Naghlani, S., Fremy, C., Otarod, V., Ambert-Balay, K., et al., 2016. The molecular epidemiology of bovine rotaviruses circulating in Iran: a two-year study. Arch. Virol. 161 (12), 3483 3494. Rajendran, P., Kang, G., 2014. Molecular epidemiology of rotavirus in children and animals and characterization of an unusual G10P[15] strain associated with bovine diarrhea in south India. Vaccine 32S, A89 A94. Rao, T.S., Arora, R., Khera, A., Tate, J.E., Parashar, U., Kang, G., 2014. Insights from global data for use of rotavirus vaccines in India. Vaccine 32 (Suppl 1), A171 A178.

503

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CHAPTER 20 Vaccines the tugboat for prevention-based

Redding, L., Weiner, D.B., 2009. DNA vaccines in veterinary use. Expert Rev. Vaccines 8 (9), 1251 1276. Sandbulte, M.R., Spickler, A.R., Zaabel, P.K., Roth, J.A., 2015. Optimal use of vaccines for control of influenza A virus in swine. Vaccines 3, 22 73. Shahid, N., Rao, A.Q., Kristen, P.E., Ali, M.A., Tabassum, B., Umar, S., et al., 2017. A concise review of poultry vaccination and future implementation of plant-based vaccines. World’s Poult. Sci. J. 73, 1 12. Tate, J.E., Arora, R., Bhan, M.K., Yewale, V., Parashar, U.D., Kang, G., 2014. Rotavirus disease and vaccines in India: a tremendous public health opportunity. Vaccine 32S, 7 12. Umthong, S., Buaklin, A., Jacquet, A., Sangjun, N., Kerdkaew, R., Patarakul, K., et al., 2015. Immunogenicity of a DNA and recombinant protein vaccine combining LipL32 and Loa22 for leptospirosis using chitosan as a delivery system. J. Microbiol. Biotechnol. 25 (4), 526 536. Wahren, B., Margaret, A.L., 2014. ). DNA vaccines: recent developments and the future. Vaccines 2, 785 796. Wray, C., 1994. Mamalian salmonellosis. In: Beran, G.W. (Ed.), Hand Book of Zoonosis, second ed. CRC Press, New York, pp. 291 300. Zamri-Saad, M., Kamarudin, M.I., 2016. Control of animal brucellosis: the Malaysian experience. Asian Pac. J. Trop. Med. 9 (12), 1136 1140.

Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A AAV. See Adeno-associated virus (AAV) ABC. See Activated B-cell (ABC) ABCA-1. See ATP-binding cassette transporter subfamily A number 1 (ABCA-1) ABCG2 gene, 111
113 aCGH. See Array-comparative genomic hybridization (aCGH) Acquired immune system, 176
177 ACT. See Adoptive cell therapy (ACT) Activated B-cell (ABC), 393
394 Acute hepatopancreatic necrosis disease (AHPND), 315
319 Acute lymphoblastic leukemia (ALL), 89, 391 Acute lymphocytic leukemia. See Acute lymphoblastic leukemia (ALL) Ad serotype 5 (AdHu5), 371 Adeno-associated virus (AAV), 230
232 Adenoviridae, 50, 364, 370t Adenovirus, 299, 367
368 adenovirus-vectored FMD vaccine, 443 E3-coded proteins, 364 vectors, 364
365 ADHIS. See Australian Dairy Herd Improvement Scheme (ADHIS) AdHu5. See Ad serotype 5 (AdHu5) Adjuvant System 04 (AS04), 426 Adjuvants, 407, 424 approving for human vaccines, 428t nanomaterial as, 427
428 Adoptive cell therapy (ACT), 89 Adoptive T-cell Transfer, 89 Advanced molecular development, 107 Adverse effect of vaccines, 495 Aer. salmonicida, 312
313, 322
323, 340, 343
344 Aer. salmonicida subsp. salmonicida, 325, 327 Aeromonas spp., 312
313 A. viridans, 312
313 A. caviae, 312
313 A. hydrophila, 312
313, 325, 327, 348
350, 408
412 A. jandaei, 312
313 A. sobria, 312
313 A. veronii, 312
313 Aeromonasis, 312
313 AFLP. See Amplified fragment length polymorphism assays (AFLP)

African swine fever (ASF), 87 Agar, 280 Agaropectin, 280 Agarose, 279
280 Agglutination tests, 302 AGOs. See Argonaute proteins (AGOs) Agriculture system, 211
212 AHPND. See Acute hepatopancreatic necrosis disease (AHPND) AHSG. See Alpha-2-HS glycoprotein (AHSG) AI. See Avian influenza (AI) Aichivirus B, 46 AIV. See Avian influenza virus (AIV) Akap4. See Anchor protein 4 (Akap4) Alginate, 279
280 Ali. salmonicida, 312
313 Aliivibrio salmonicida, 312
313 Alkylating agents, 223
224 ALL. See Acute lymphoblastic leukemia (ALL) Alloherpesviridae, 55 Aloe barbadensis, 427 Alpha Jects 1000 vaccines, 277
278 Alpha Jects Micro 1 ISA, 277
278 Alpha-2-HS glycoprotein (AHSG), 114
115 Alphaherpesevirus, 485 Alphaviruses, 452
456 expression system, 411
412 functions of nonstructural proteins, 453
454 viral target proteins for drug development, 454
456 Aluminum hydroxide gel vaccine, 475
477 Aluminum salts, 424
426 Alzheimer’s disease-like phenotype, 186 Ambiphyra, 312 Ameiurus nebulosus (Brown bullhead), 57 Amelogenin (AMEL), 117 Amino acid residues, 251
252 Amplification-based methods, 291
292 isothermal amplification methods, 291
292 PCR and variants, 291 Amplified fragment length polymorphism assays (AFLP), 338
339 Amplified rRNA gene restriction analysis (ARDRA), 341 Anaerobic bacteria, 312
313 Anaplasma spp., 302 Anchor protein 4 (Akap4), 114
115 Anguillid herpesvirus (AngHV), 56

505

506

Index

Animal agriculture, 85
93 Animal husbandry, 211
212 Animal models, 183 biotechnological approaches for generating, 192
196 of diseases, 184
187, 186f genetically-modified models, 187 induced models, 184
186 negative models, 187 orphan models, 187 species employed in biomedical studies, 185t spontaneous models, 187 engineering, 189
190 ethical and regulatory issues, 199
202 gnotobiotic animals, 192 mimicking clinical conditions, 187
189 pharmacological considerations, 199 physiological considerations, 198 SPF animals, 190
192 translational significance, 196
197 Animal viruses, 435, 441
460 Ankyrin repeat domain 1 (ANKRD1), 117
118 Annelida, 279 Anopheles gambiae (Female mosquitoes), 91
92 Anopheles stephensi, 91
92 Anthrax, 469, 477
478 organism, 477
478 Anti-CD20, 215
216 Antibiotics, 288
289 resistance, 471 Antibody-based technologies, 319
320 Antichaperon therapy, 90 Antigen detection, 290, 319
320 Antigen presenting cells (APCs), 429 Antihypertensive bioactive peptides, 279 Antiinfluenza drugs, 458 Antimicrobial drugs, 199 Antioxidants, 280
281 Antiseptics, 315
319 Antiviral drug targets, 437f, 440t Antivirals, 440t, 461
462 against coronavirus, 451 against HSV, 447
450 against paramyxovirus, 456
457 AP-PCR. See Arbitrarily primed PCR (AP-PCR) APCs. See Antigen presenting cells (APCs) Apex-IHN DNA vaccine, 344
345 Aphanomyces species, 338 A. astaci, 314 A. invadans, 313 Apiosoma, 312 Apoptosis-stimulating of p53 protein 2 (ASPP2), 114
115 Aptamers, 293

Aquaculture, 272, 287
289, 311
312. See also Mariculture genetic advances in, 275 GMOs in, 276
277 metagenomics revealing new virus species in, 55
58 next generation vaccines in, 409t, 410f polyploidy in, 276 Arabidopsis, 80 Arbitrarily primed PCR (AP-PCR), 337
338 Arbitrary PCR assays, 108 Arcanobacterium pluranimalium, 299 ARDRA. See Amplified rRNA gene restriction analysis (ARDRA) Argonaute proteins (AGOs), 166
168 Argulous, 312 Array-comparative genomic hybridization (aCGH), 384
385 ArrayExpress, 386, 387t Arthropoda, 279 Artificial selection breeding, 223 Artyodactyla, 484
485 AS04. See Adjuvant System 04 (AS04) ASF. See African swine fever (ASF) Asian sea bass. See Lates calcarifer (Asian sea bass) Aspergillus fumigatus, 302 ASPP2. See Apoptosis-stimulating of p53 protein 2 (ASPP2) Association for Assessment and Accreditation of Laboratory Animal Care International program, 200
201 Astacus astaci, 338 Astaxanthin, 281 Astroviridae, 50, 52 Astrovirus, 53, 299 Asymptomatic-infected domestic animals, 441 ATP synthase H1 transporting mitochondrial F1 complex beta subunit (ATP5B), 114
115 ATP-binding cassette transporter subfamily A number 1 (ABCA-1), 195
196 ATP5B. See ATP synthase H1 transporting mitochondrial F1 complex beta subunit (ATP5B) Attenuated virus vaccines, 277
278 Australia, achievements and status of cattle genome sequencing in, 14
15 Australian Dairy Herd Improvement Scheme (ADHIS), 19 Avastrovirus, 48 Avian influenza (AI), 368 Avian influenza virus (AIV), 365, 368, 458, 459f Avian zygotes, 233 Avipoxviruses, novel clade of, 48

Index

Avulavirus, 456 Aydin/04 strain, 47

B Babesia bigemina, 300 Babesia bovis, 300 BAC. See Bacterial artificial chromosome (BAC) Bacille Calmette-Gue´rin (BCG), 474 Bacillus amyloliquefaciens CCF7 probiotic, 348
350 Bacillus anthracis, 477
478 Bacillus subtilis BT23, 348
350 Bacterial artificial chromosome (BAC), 4
5, 368 Bacterial diseases anthrax, 477
478 black quarter, 478 brucellosis, 477 E. coli infection, 480 HS, 475
477 leptospirosis, 478 mycobacterium infection in cattle, 478
479 salmonellosis, 479 Bacterial gill disease, 312
313 Bacterial kidney disease (BKD), 312
313, 319
320 Bacterial pathogens, 312
313 Baculovirus, 411
412 Bama pigs, 156 Baylor College of Medicine (BCM4), 5 BCG. See Bacille Calmette-Gue´rin (BCG) BCM4. See Baylor College of Medicine (BCM4) BCoV. See Bovine coronavirus (BCoV) Beef cattle, genomic selection in, 19
20 Beijing Genomics Institute (BGI), 156 Beta casein, 109
111 Beta polyomavirus, 50 Beta-propiolactone, 481
482 Beta-subunit of RNA polymerase (rpoB), 342
343 β1,4-gal-actosyltransferase 1 (β4GALT1), 235
236 BEV. See Bovine enterovirus (BEV) BGI. See Beijing Genomics Institute (BGI) Bifidobacterium spp., 340 Bioactive compounds, 278
280 carotenoids, 280
281 Bioactive molecules, marine sources of, 279
280 Bioinformatics, 37
39 for animal diseases canine cancers using omics data and bioinformatics methods, 388
395 genomics, 384
385, 395
396

and omics data in cancers of domestic animals, 395 and omics data in studying animal diseases, 396
397 proteomics, 386
388, 395
396 transcriptomics, 385
386, 395
396 methods, 388
395 Biomarkers, 116, 394 Biomedical community, 197 Biomedical studies, 188
189 Bionumerics software version 4.0, 342
343 Bioprospecting for food bioactive compounds in farming, 280
281 functional foods and nutraceuticals from marine organisms, 278
281 marine sources of bioactive molecules, 279
280 Biosafety level-4 (BSL-4), 456
457 Biosecurity, 191 Biosensors, 293
294, 300
301, 335 Biotechnological approaches for development of animal models, 194, 195f to fish vaccine biotechnology in developing new generation vaccines, 407
413 fish vaccine, 413
414 for generating animal models nuclease editors, 192
194 pronuclear microinjection, 194
195 RNA interference, 195
196 SCNT, 194 Biotechnological innovations, 294 diagnosis of pathogens, 289
298 in farm and companion animal’s disease diagnosis, 299
301 infectious diseases’ impact, 288
289 Biotechnological tools in disease diagnosis, 311
312 disease problems in fish culture, 312
315 diseases in shrimp, 315
341 DNA sequence analysis, 341
342 multilocus sequence typing analysis, 342
343 preventive and control measures immunostimulants, 345, 346t vaccines for fish diseases, 323t, 343
345 probiotics, 345
350, 349t therapeutics in fish diseases, 350
352, 351t Biotin, 328 BISC. See Bowenoid in situ carcinoma (BISC) BKD. See Bacterial kidney disease (BKD) Black hole quencher (BHQ-1), 327 Black quarter, 478 Blackleg, 478 BLASTn algorithm, 342
343

507

508

Index

Blastomyces dermatitidis, 302 Bluetongue, 482
483 vaccines, 490 BMP. See Bone morphogenetic protein (BMP) BoAstV-BH89/14, 46 Bocaparvoviruses, 50 BoHV-1. See Bovine herpesvirus-1 (BoHV-1) Bone morphogenetic protein (BMP), 116
117 Booster of Th2 immunity. See Aluminum salts Bordetella, 191 Bordetella bronchoseptica, 301 BoRV-CH15, 46 Bos indicus, 7, 15
16 Bos taurus (Cattle), 5, 118 new viruses in, 46 Bovilis Dictol live vaccine, 490 Bovine genes, 114
115 papillomavirus type, 46 respiratory syncytial virus, 300 single nucleotide polymorphism (SNP) arrays, 6
8 viral diarrhea, 484
485 Bovine coronavirus (BCoV), 487
488 Bovine enterovirus (BEV), 46, 300
301 Bovine Genome Sequencing and Analysis Consortium, 4
5 Bovine herpesvirus-1 (BoHV-1), 485 Bovine polyomavirus species (BPyV2-SF), 46 Bovine rotavirus (BRV), 488 Bovine spongiform encephalopathy (BSE), 299 Bovine viral diarrhea virus (BVDV), 293
294, 459, 460f BVDV1, 460, 461f, 484
485 BVDV-2, 484
485 BovineHD BeadChip, 7
8 BovineSNP50 chip, 7 BovineSNP50 Genotyping Beadchip, 6
7 Bowenoid in situ carcinoma (BISC), 53 BPyV2-SF. See Bovine polyomavirus species (BPyV2-SF) Brazil, achievements and status of cattle genome sequencing in, 15
16 BRB-ArrayTools, 386 Broiler breeders, 489
490 Brown bullhead. See Ameiurus nebulosus (Brown bullhead) Brucella abortus, 477 Brucella canis, 301 Brucella melitensis, 477 Brucella vaccines, 495 Brucellosis, 477 BRV. See Bovine rotavirus (BRV) Bryozoa, 279

BSE. See Bovine spongiform encephalopathy (BSE) BSL-4. See Biosafety level-4 (BSL-4) Bst DNA polymerase, 329
330 Bungarus multicinctus, 293 Burdur/05 strain, 47 BVDV. See Bovine viral diarrhea virus (BVDV)

C C-C chemokine receptor type 5 gene (CCR5 gene), 259 C2c2. See Class 2 type VI-A CRISPR-Cas effector (C2c2) Caenorhabditis elegans, 165
166, 196 Caesarian section technique, 190
191 Caliciviridae, 50 Caliciviruses, 51
52 Calpastatin (CAST), 116 Campylobacter, 300 Canada, cattle genome projects in, 11
12 Cancer, 89
91, 187, 396
397 adoptive T-cell Transfer, 89 antichaperon therapy, 90 of domestic animals, 395 dysregulation of Notch signaling, 90
91 harnessing CAR T cells, 89 studying synthetic lethal interactions, 90 Candidate genes, 116
117 Canine cancers genomics studies, 391
392 using omics data and bioinformatics methods, 388
395 proteomics studies, 394
395 transcriptomics studies, 392
394 types, 390
391 Canine circovirus, 50 Canine hepacivirus, 52 Canine kobuvirus, 49
52 Canine parvovirus (CPV), 301 Canine transmissible venereal tumor (CTVT), 391 Canis familiaris (Domestic dog), 391 Capripoxvirus, 369 Capsid enclosing nucleic acid, 442 Carbohydrate adjuvants, 427 Carotenoids, 280
281 Carp. See Cyprinus carpio (Carp) Carp edema virus (CEV), 314
315 “Carp pox”, 314
315 Carrageenans, 280 CARs. See Chimeric antigen receptors (CARs) Cas system. See CRISPR-CISPR associated system (Cas system)

Index

Cas9 activity, 80 Cas9-HF1, 82 nickase, 81 Cas9 ribonucleoprotein (Cas9 RNP), 89 Casein gene, 116
117 CAST. See Calpastatin (CAST) Cation channel of sperm 1 (Catsper1), 114
115 Cats, novel viruses in, 52
54 Catsper1. See Cation channel of sperm 1 (Catsper1) Cattle. See Bos taurus (Cattle) Cattle genome projects, 11
13 status and attainments, 11
13 in Australia, 14
15 in Brazil, 15
16 in Canada, 11
12 European countries, 12
13 Cattle genomics bovine single nucleotide polymorphism arrays, 6
8 genome size and reported SNPs, 9t genome-wide association studies in dairy cattle, 8
10 INTERBULL concept for genetic evaluation of breeding bulls, 13 MAS, 10
11 releases of bovine genome assemblies and technical detail, 5t sequencing, 4
5 status of genomic selection across world in bovine, 16
20 Causes recombination (Cre), 238 CCD camera. See Charge-coupled device camera (CCD camera) CCR5 gene. See C-C chemokine receptor type 5 gene (CCR5 gene) cDNA. See Complementary DNA (cDNA) Cell culture systems, 299 Cell therapeutics, 89
91 cancer, 89
91 diabetes, 91 Cellular biology, 382
384 Cellular functions, 189
190 Cellular immunity, 361, 472 Cellular transcription activities, 385
386 Central dogma of molecular biology, 163
164 CEV. See Carp edema virus (CEV) CFTR. See Cystic fibrosis transmembrane conductance regulator (CFTR) CFU. See Colony-forming units (CFU) CH. See Cys/His-rich domain (CH) Chain-termination sequencing technique, 31
32 Charge-coupled device camera (CCD camera), 33

Charolais breed, 11 cHDA. See Circular helicase-dependent amplification (cHDA) Chemical mutagenesis, 164
165 Chemical sequencing, 31
32 Chemical-induced model, 184
185 Chemically synthesized antiviral drugs, 435 Chemotherapy, 350 Chicken, 225 eggs, 213
215 embryos lacking eggshells, 227
229 genome manipulation, 225
226 meat quality in, 119 novel viruses in, 47
48 Chikungunya virus (CHIKV), 452
453 Chimeric antigen receptors (CARs), 89 harnessing CAR T cells, 89 Chimerism, 83 ChiSCV. See Circular ssDNA virus (ChiSCV) Chlamydia spp., 312
313 Cholera toxin, 427 Cholesterol, 213
215 Cholinergic system, 184
185 Chordopoxvirinae, 57 Chromogenic in situ hybridization (CISH), 291 Chromosomal locus, 392 Chronic lymphocytic leukemia (CLL), 391 Chronic mild stress, 186 Chymotrypsin-like cysteine proteases, 443
444 Circular helicase-dependent amplification (cHDA), 291
292 Circular replication-associated protein encoding single-stranded (CRESS), 56 Circular ssDNA virus (ChiSCV), 45 CISH. See Chromogenic in situ hybridization (CISH) Class 2 type VI-A CRISPR-Cas effector (C2c2), 136 Classical animal breeding, 211
212 Classical swine fever, 483 Classical swine fever virus (CSFV), 369 Clathrin-coated endocytosis, 458
459 CLL. See Chronic lymphocytic leukemia (CLL) Clostridium botulinum, 312
313 Clostridium chauvoei, 478 Clostridium perfringens, 300 detection, 301 type C, 488 Clustered regularly interspaced short palindromic repeats (CRISPR), 76, 151, 169
170, 189
190, 224
225, 237
238, 251 arrays, 255 systems, 78

509

510

Index

Clustered regularly-interspaced short palindromic repeat/Cas9 system (CRISPR-Cas9 system), 84
85, 131
132, 153, 174
177, 193, 255
257, 261
262 development, 132
134, 134t in farm animals, 139
141, 140t gene/genome editing technology, 368 diagnostics development, 91 disease control, 87
91 fighting antimicrobial resistance, 92 food production, 86
87 producing disease models, 92
93 study of developmental biology, 85
86 vector control, 91
92 mechanism of action, 137
139, 139f molecular structure, 135
136 premises and promises of genome editing, 142 technical challenges, 142 “Clynav”, 344
345 CMV. See Cytomegalovirus (CMV) CNAs. See Copy number alterations (CNAs) Cnidaria, 279 CNVs. See Copy number variants (CNVs) Coccidioides immitis, 302 Coccidiosis, 489
490 Colibacillosis, 300
301 Colony hybridization, 328
329 Colony-forming units (CFU), 325 Combined vaccination, 490 recommended vaccines for cattle, 491t Committee for Purpose of Control and Supervision on Experiments on Animals (CPCSEA), 199
200 Community genome arrays, 331 Companion animals biotechnological tools in companion animals disease diagnosis, 301
303 and gene editing, 154
158 micro pigs, 156 pet animals as disease model, 157 prospects of gene editing in pets, 157
158 super muscular dogs, 155
156 Complementary DNA (cDNA), 385
386 encoding for ZFNs, 259
260 Computational structure prediction and drug design, 439
440 Conventional selective breeding, 107
108 Conventional vaccines inactivated vaccines, 472 live-attenuated vaccines, 472 toxoids, 472
473 Copy number alterations (CNAs), 384
385 Copy number variants (CNVs), 384
385 Coronaviridae, 50, 450

Coronavirus (CoV), 450
451 replication, 450
451 structure-based antivirals against, 451 Coronavorodae, 450 Corynebacterium, 191 Coupling nonspecific endonuclease, 151
152 CoV. See Coronavirus (CoV) Cowpox lesion, 469 COX3. See Cytochrome oxidase (COX3) CP. See Cytopathic biotype (CP) CPCSEA. See Committee for Purpose of Control and Supervision on Experiments on Animals (CPCSEA) Cpf1, 82 CPV. See Canine parvovirus (CPV) Cre. See Causes recombination (Cre) Cre/Lox-P system, 238
239, 239f CRESS. See Circular replication-associated protein encoding single-stranded (CRESS) CRISP2. See Cysteine-rich secretory protein 2 (CRISP2) CRISPR. See Clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR RNA (crRNA), 78, 255 CRISPR-Cas9 system. See Clustered regularlyinterspaced short palindromic repeat/Cas9 system (CRISPR-Cas9 system) CRISPR-CISPR associated system (Cas system), 76, 151
152 Cas9, 174
175, 251 CRISPR/Cas system, 78
80, 151
152 type II CRISPR/Cas9 system for genome editing, 79
80 Crop plants, 153
154 crRNA. See CRISPR RNA (crRNA) Crustaceans, 273 diseases, 314
315 Cryo-electron microscopy (Cryo-EM), 438
439 Cryptosporidium parvum, 293 CSFV. See Classical swine fever virus (CSFV) CTL. See Cytotoxic T-lymphocyte (CTL) CTVT. See Canine transmissible venereal tumor (CTVT) Culex mosquitoes, 483
484 Cultured aquatic animals, 312 Cumate-inducible promoter, 238
239 Customization of pets, 94 Cyclization recombinase. See Causes recombination (Cre) Cyprinid herpesviruses (CyHV), 56 Cyprinivirus, 56 Cyprinus carpio (Carp), 57, 314
315 Cys/His-rich domain (CH), 451

Index

Cysteine-rich secretory protein 2 (CRISP2), 114
115 Cysteine-rich secretory protein 3 (CRISP3), 119 Cystic fibrosis transmembrane conductance regulator (CFTR), 194 Cytochrome oxidase (COX3), 114
115 Cytokines adjuvants, 427 Cytomegalovirus (CMV), 366 Cytopathic biotype (CP), 484
485 Cytotoxic T-lymphocyte (CTL), 361, 427

D D-N4-hydroxycytidine (NHC), 455
456 Dacylogyrous sp., 312 Dairy cattle, genomic selection in, 16
19 Dairy cattle, GWAS in, 8
10 Dairy production traits, markers for, 109
114 DBD. See DNA-binding domain (DBD) dCas9. See Nuclease-deficient Cas9 model (dCas9) DDB2. See DNA binding protein 2 (DDB2) ddNTPs. See Dideoxynucleotides (ddNTPs) DEC. See Diarrheagenic E. coli (DEC) Deciphering of DNA structure, 249 Deep sequencing of nucleic acids, 49 Deextinction, 94 Deleted glycoprotein E (gE2), 470 Delivery methods, 83 Demand of food fish, 311
312 Density gradient ultracentrifugation, 30
31 DEPs. See Differentially expressed proteins (DEPs) Diabetes mellitus, 91, 185
187 Diacylglycerol acyltransferase gene (DGAT1 gene), 111
113 Diagnostics development, 91 Diarrheagenic E. coli (DEC), 480 Dicipivirus, 51 Dictyocaulus viviparous, 470 Dictyostelium cells, 80 Dideoxy method, 31
32 Dideoxynucleotides (ddNTPs), 31
32 Difference gel electrophoresis (DIGE), 115 Differentially expressed proteins (DEPs), 115 Differentiation of infected from vaccinated animals (DIVA), 473
474 DIGE. See Difference gel electrophoresis (DIGE) Diphtheria, tetanus, and pertusis (DPT), 490 Direct immunofluorescence assay, 302 “Disease-in-a-dish” in vitro model, 92
93 Disease-resistant animals, producing, 87
88 Disposable gear, 191 DIVA. See Differentiation of infected from vaccinated animals (DIVA)

DMD. See Duchenne muscular dystrophy (DMD) DNA, 249, 328
329 double helix, 151 fingerprinting, 108 microarrays, 331
332 sequence, 31
32, 385t analysis, 341
342 vaccines, 412
413, 423, 474
475 DNA binding protein 2 (DDB2), 395 DNA-based immunization, 412
413 DNA-binding domain (DBD), 173
174, 193
194, 251
253 Dogs, 157 novel viruses in, 49 Domestic animals, 211
212 bioinformatics and omics data in cancers of, 395 Domestic dog. See Canis familiaris (Domestic dog) Dot-ELISA, 290 Double-strand breaks (DSBs), 76, 131
132, 249
250, 256 Double-stranded DNA (dsDNA), 165, 193
194, 255, 326 viruses, 446 DPP. See Dual Path Platform (DPP) DPT. See Diphtheria, tetanus, and pertusis (DPT) Draught power, genes related to, 116 Drosophila, 193
194, 196 Drosophila melanogaster, 166
168 Drug design, 439
440 Drug discovery, 94
95, 189, 196 DSBs. See Double-strand breaks (DSBs) dsDNA. See Double-stranded DNA (dsDNA) Dual Path Platform (DPP), 295 Duchenne muscular dystrophy (DMD), 83 Dyokappa papillomavirus, 46

E EAAP. See European Association for Animal Production (EAAP) Early mortality syndrome (EMS), 315
319 Eastern equine encephalitis virus (EEEV), 452
453 Ebola virus, 294 Echinodermata, 279 Echinoderms, 273 Ecosystem disequilibrium, 93 Ectopistes migratorius (Passenger pigeon), 94 Edwardsiella anguillarum, 312
313 Edwardsiella ictaluri, 312
313, 343
344, 412 Edwardsiella piscicida, 312
313 Edwardsiella tarda, 312
313, 412 Edwardsiellosis, 312
313

511

512

Index

EEEV. See Eastern equine encephalitis virus (EEEV) EGF. See Enhanced green fluorescent (EGF) Egg-laying chickens, 212
213 Eggs, 119
120, 211
212 EHEC. See Enterohaemorrhagic E. coli (EHEC) Ehrlichia canis, 302 Ehrlichia spp., 301 EHV1. See Equine herpes virus (EHV1) Eimeria, 300 E. acervulina, 489
490 E. maxima, 489
490 E. necatrix, 489
490 E. tenella, 489
490 Electroporation, 137 electroporation-mediated DNA delivery, 423 ELISA. See Enzyme-linked immunosorbent assay (ELISA) EMA. See European Medicines Agency (EMA) Embryo culture, 226
229 Embryonic cells, 233 Embryonic stem cells (ESC), 132
134, 233, 392
393 EMS. See Early mortality syndrome (EMS) Engineered nucleases, 92
93, 151, 251 Engineered piggyBac vectors, 232 Enhanced green fluorescent (EGF), 392 Enhanced Specificity SpCas9 (eSpCas9), 81
82 Enolase 1 (ENO1), 114
115 Enterobacterial repetitive intergenic consensusPCR (ERIC-PCR), 339
340 Enterococcus faecalis, 301 Enterococcus faecium, 348
350 Enterohaemorrhagic E. coli (EHEC), 480 Enterotoxigenic E. coli (ETEC), 480 infection, 480 Enterovirus (EV), 46, 51 Enterovirus G (EVG), 45 Environment management, 278 Enzyme-linked immunosorbent assay (ELISA), 289
290, 300, 319
320 Enzymes, 271
272, 319
320 Equine genetic markers in, 119 influenza, 487 Equine herpes virus (EHV1), 369 EHV-1-vectored vaccines, 369 Ergasilus sp., 312 ERIC-PCR. See Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) ES ionization, 332
333 ESC. See Embryonic stem cells (ESC) Escherichia coli, 92, 132
134, 226, 300
301, 339
340, 408
411, 423
424, 473

heat-labile enterotoxin, 427 infection, 480 eSpCas9. See Enhanced Specificity SpCas9 (eSpCas9) EST. See Expressed sequence tag (EST) ETEC. See Enterotoxigenic E. coli (ETEC) Ethical issues, 93
94, 199
202 ecosystem disequilibrium, 93 genetic enhancement, 94 regulatory hurdles, 93 Ethyl methanesulfonate, 223
224 European Association for Animal Production (EAAP), 13 European Medicines Agency (EMA), 344
345 EV. See Enterovirus (EV) EVG. See Enterovirus G (EVG) Exogenous DNA, 230 Exotic pathogens, 311
312 Expressed sequence tag (EST), 14
15

F F-protein, 366
367 FACS. See Fluorescence-activated cell sorting (FACS) FAO. See Food and Agricultural Organization (FAO) Farfantepenaeus duorarum (Northern pink shrimp), 58 Farfantepenaeus duorarum nodavirus (FdNV), 58 Farm animals biotechnological tools in disease diagnosis, 299
301 farm animals-associated pathogens, 301 gene editing in, 139
141, 140t genetic markers related to productivity equine, genetic markers in, 119 large ruminants, genetic markers in, 109
116 poultry, genetic markers in, 119
120 in small ruminants, genetic markers in, 116
117 swine, genetic markers in, 117
118 Farmed whiteleg shrimp. See Litopenaeus vannamei (Farmed whiteleg shrimp) Farming bioactive compounds in, 280
281 future, 95
96 Fast-growing fishes production, 263 FAT. See Fluorescent antibody test (FAT) Fatty acids, 213
215 FDA. See Food and Drug Administration (FDA) FdNV. See Farfantepenaeus duorarum nodavirus (FdNV)

Index

Feline immune deficiency vaccine (Fel-O-Vax), 470 Feline immunodeficiency virus (FIV), 54 FIV-based vectors, 366 Feline papillomavirus type-2, 53 Feline sakobuvirus A, 52 Female mosquitoes. See Anopheles gambiae (Female mosquitoes) Ferrets, 157 Fibrosarcoma, 495 Fighting antimicrobial resistance, 92 First-generation sequencing, 31
32 FISH. See Fluorescence in situ hybridization (FISH) Fish culture, disease problems in, 312
315 crustacean diseases, 314
315 emerging diseases of fish and shellfish, 316t fish diseases, 313
314 Fish diseases, 313
314 therapeutics in, 350
352, 351t vaccines for, 323t, 343
345 Fish health management, 277
278 Fish vaccine, 413
414 Fishery science, 262 FIV. See Feline immunodeficiency virus (FIV) Flaviviridae, 47, 370t, 458
459, 484
485 Flaviviruses, 369, 458
459 Flavobacteriosis, 312
313 Flavobacterium, 340 F. branchiophilum, 312
313 F. columnare, 312
313, 325, 343
344, 412 F. psychrophilum, 312
313, 325, 343
344 Fluorescence chemistries, 291 Fluorescence energy resonance transfer (FRET), 436 Fluorescence in situ hybridization (FISH), 291, 328, 340 Fluorescence melting curve analysis (FMCA), 302
303 Fluorescence-activated cell sorting (FACS), 235
236 Fluorescent antibody test (FAT), 319
320 Fluorescent dyes, 319
320, 326
327 fluorescent dye-labeled ddNTPs and process parallelization, 32 Fluorescent microarray detector, 331
332 Fluorescent PNA probes, 302
303 FMCA. See Fluorescence melting curve analysis (FMCA) FMD. See Foot and mouth disease (FMD) FMDV. See Foot and mouth disease virus (FMDV) FokI endonuclease, 172
173 Follistatin (FST), 117

Food bioprospecting for, 278
281 ingredients, 280 mariculture technologies, 274
275 from marine sources marine algae, 273
274 marine fish, 272
273 molluscs, echinoderms, and crustaceans, 273 production, 86
87 Food and Agricultural Organization (FAO), 272 Food and Drug Administration (FDA), 196, 350
351 Foot and mouth disease (FMD), 292, 441, 470, 480
481 Foot and mouth disease virus (FMDV), 293
294, 365, 369, 441
445, 444f, 445f clinical signs, 441 nonstructural proteins, 442
443 serotypes, 441 structure and genome, 442 structure-based drug development against, 443
445 vaccination, 443 Forkhead box L 2 (FOXL2), 117 Fowl cholera, 469 Fowlpox virus (FPV), 367
368 Francisella noatunensis, 312
313 Francisella tularensis, 301 FRET. See Fluorescence energy resonance transfer (FRET) FST. See Follistatin (FST) Fulmar. See Fulmarus glacialis (Fulmar) Fulmarus glacialis (Fulmar), 49 Functional characterization of genes, 264 Functional foods, 278
281 Functional gene arrays, 331 Functional genomics, 261
262 Functional proteomics, 388 Fusogens, 448
449

G Gallus gallus, 225 gapA. See Glyceraldehyde 3-phosphate dehydrogenase (gapA) Gastrointestinal infection, 480 gC. See Glycoprotein C (gC) gE2. See Deleted glycoprotein E (gE2) GEBV. See Genomic estimated breeding value (GEBV) Gel electrophoresis, 341 Gene drive, 93, 95
96 Gene editing, 249
251 applications

513

514

Index

Gene editing (Continued) fast-growing fishes production, 263 fishery science, 262 functional characterization of genes, 264 functional genomics, 261
262 ornamental fishes production, 263
264 pollution markers development, 263 production of mono-sex population, 262 research and development, 261 sterility, 263 treatment of diseases, 261 companion animals and, 154
158 comparison of genome editing platforms, 257
259, 258t efficiency, 257
259 specificity, 259 CRISPR-Cas9, 255
257 delivery system, 259
260 ease of designing, 260 multiplexing, 260
261 in pets, 157
158 TALENs, 253
254 targeted nucleases, 250f tools, 152t CRISPR-Cas9 system, 153 TALENs, 153 ZFNs, 152 ZFNs, 251
253 Gene expression omnibus (GEO), 381, 386, 387t Gene expression profiling (GEP), 385
386, 393
394 Gene of interest (GOI), 411
412 Genetic/gene(s) associated with meat production, 116 engineering techniques, 183
184, 249, 361
363 enhancement, 94 evaluation program, 107 fingerprint, 337 functional characterization, 264 gene-based immunotherapy for HIV-AIDS, 197 gene-by-gene analysis, 293
294 gene-edited mammalian model, 190 manipulation, 249, 275
277 polyploidy, 276 selective breeding, 275 transgenics, 276
277 method, 164
165 modification, 131, 211
212 related to draught power, 116 therapy, 361, 364 Genetic markers, 105 candidate genes influencing economic traits in cattle and buffaloes, 110t

in goat and sheep, 112t in pig, equine, and poultry, 113t related to farm animal productivity, 109
120 Genetically attenuated pathogens, 412 Genetically engineered animal models, 142 Genetically-engineered vaccines, 470, 475 DNA vaccine, 474
475 subunit vaccine, 473 vectored vaccines, 474 VLPs vaccines, 473
474 Genetically-modified animals, 261 Genetically-modified models, 187 Genetically-modified organism (GMO), 412
413 Genome of FMDV, 442 genome wide siRNA screening experiment, 168 genome-edited birds, 226
227, 228f genome-edited cells, 86
87 genome-manipulated progeny, 233 manipulation, 223 Sequencer, 48 Genome Analyzer, 33, 35 Genome editing, 131
132, 153
154, 224
225 in animals CRISPR/Cas9 genome editing technology, 85
93 CRISPR/Cas9 system vs. zinc finger nucleases, 84
85 customization of pets, 94 deextinction, 94 delivery methods, 83 drug discovery, 94
95 ethical issues, 93
94 future farming, 95
96 incidence of HDR, 84 off-target effects, 81
83 RNA-guided endonucleases, 78 TALENs, 77
78 types of CRISPR/Cas system, 78
80 ZFNs, 77 CRISPR/Cas9 system development, 132
134 in farm animals, 139
141, 140t mechanism of action, 137
139, 139f molecular structure of, 135
136 delivery and expression system, 136
137, 138f in pet world, 155 companion animals and gene editing, 154
158 gene editing tools, 152
153 scope of genome editing, 153
154 premises and promises by CRISPR/Cas9, 142 technical challenges of CRISPR/Cas9 genome editing, 142

Index

tools, 263
264 type II CRISPR/Cas9 system for, 79
80 Genome-wide association studies (GWAS), 3
4, 109, 118
119 in dairy cattle, 8
10 Genome-wide selection (GWS), 11
12 Genomic estimated breeding value (GEBV), 6 Genomic selection (GS), 7
8, 10
11 in beef cattle, 19
20 in dairy cattle, 16
19 in multibreed cattle populations, 20 Genomics, 384
385, 395
396 studies in canine cancers, 391
392 Genotype-Tissue Expression (GTEx), 386, 387t Genotyping techniques in pathogens characterization, 335
341 amplified fragment length polymorphism assays, 338
339 PCR-based strain typing techniques, 337
341 PFGE, 336
337 GEO. See Gene expression omnibus (GEO) GEP. See Gene expression profiling (GEP) GHR. See Growth hormone receptor (GHR) GHs. See Growth hormones (GHs) Giant river prawn. See Macrobrachium rosenbergii (Giant river prawn) Glancing angle deposition (GLAD), 335 Global Rinderpest Eradication Program, 470 Glucagon-like peptide 1 (GLP-1), 91 Glutathione peroxidase 1 (GPX1), 116 Glutathione peroxidase 4 (GPX4), 114
115 Glyceraldehyde 3-phosphate dehydrogenase (gapA), 342
343 Glycoconjugation, 425t Glycoprotein (G), 366
367, 408
411 Glycoprotein C (gC), 446
447 Glycosylation, 90
91 GMO. See Genetically-modified organism (GMO) GNNV. See Grouper nervous necrosis virus (GNNV) Gnotobionts, 192 Gnotobiotes, 192 Gnotobiotic animals, 192 GNPs. See Gold nanoparticles (GNPs) Goat pox virus (GPV), 483 GOI. See Gene of interest (GOI) Gold nanoparticles (GNPs), 334
335 Goldfish haematopoietic necrosis herpes virus, 314
315 GPV. See Goat pox virus (GPV) GPX1. See Glutathione peroxidase 1 (GPX1) Gram negative bacteria, 312
313 Green alga. See Haematococcus pluvialis (Green alga)

gRNA. See Guide RNA (gRNA) Grouper nervous necrosis virus (GNNV), 408
411 Growth hormone receptor (GHR), 116
117 Growth hormones (GHs), 276 transgenesis, 212
213 GS. See Genomic selection (GS) 454 GS-FLX technology, 48 GTase. See Guanylyltransferase (GTase) GTEx. See Genotype-Tissue Expression (GTEx) Guanosine triphosphate (GTP), 453
454 Guanylyltransferase (GTase), 453
454 Guide RNA (gRNA), 153, 175
176, 237, 255 GWAS. See Genome-wide association studies (GWAS) GWS. See Genome-wide selection (GWS) Gyrodactylus salaris, 313 Gyrovirus (GyV8), 49

H HA. See Hemagglutinin (HA) Haematococcus pluvialis (Green alga), 281 Haemophilus influenza type b, 424
426 Hamster lung cell line (HmLu cell line), 483
484 Haploblocks, 7
8 Haplotype blocks, 17 Hatcheries, 275 HBV. See Hepatitis B virus (HBV) HCMV. See Human CMV (HCMV) HCoV-EMC, 450
451 HDA. See Helicase-dependent amplification (HDA) HDR. See Homology-directed repair (HDR) Heat shock proteins (HSPs), 116 Helicase-dependent amplification (HDA), 291
292 Helicobacer spp., 301 Hemagglutination inhibition, 302 Hemagglutinin (HA), 368, 458, 486 Hemagglutinin-neuraminidase (HN), 456, 473 Hemoglobinopathies, 94
95 Hemorrhagic septicemia (HS), 475
477 Hendra virus (HeV), 456
457 Henipavirus, 456, 458
459 Hepadnaviridae, 54 Hepatitis B virus (HBV), 54 Hepatitis C virus, 365 Hepatobacter penaei, 312
314 Hepatopancreatic parvo virus (HPV), 322
323 Hereditary traits, 163
164 Herpes simplex virus (HSV), 447 HSV-1, 371, 447
448, 449f Herpes virus infection, 446 Herpes virus of turkey (HVT), 367
368

515

516

Index

Herpes virus of turkey (HVT) (Continued) HVT-BAC clone recombinant vaccines, 368 Herpesviridae, 370t, 447 Herpesviruses (HSV), 446
450 antivirals against HSV, 447
450 lytic and latent cycle, 446
447 structure, 446 of turkey, 368 challenges in vectored veterinary vaccine, 371 vectored veterinary vaccines, 369
370 Heteropolaria/Epistylis, 312 HeV. See Hendra virus (HeV) HHV. See Human herpesvirus (HHV) High throughput assays, 292
298 High throughput sequencing, 249 High-density SNPs, 8
9 High-performance liquid chromatography, 387
388 High-quality protein, 213
215 Highly pathogenicity avian influenza (HPAI), 486 HIV. See Human immunodeficiency virus (HIV) HmLu cell line. See Hamster lung cell line (HmLu cell line) HN. See Hemagglutinin-neuraminidase (HN) HN protein, 366
367 Homologous recombination (HR), 170
172, 224, 249
250 repair, 76 Homology-directed repair (HDR), 75, 235
236, 251 incidence, 84 pathway, 132
134 Homozygosity, 188 Horseradish peroxidase enzyme (HRP), 319
320 Horses, 157 Host immunity, 408
411, 471 Hot-start PCR technique, 322
323 HPAI. See Highly pathogenicity avian influenza (HPAI) hPIV-1 virus. See Human parainfluenza type1 virus (hPIV-1 virus) HPV. See Hepatopancreatic parvo virus (HPV) HR. See Homologous recombination (HR) HRP. See Horseradish peroxidase enzyme (HRP) HS. See Hemorrhagic septicemia (HS) HSPs. See Heat shock proteins (HSPs) HSV. See Herpes simplex virus (HSV) Herpesviruses (HSV) Human adenovirus serotype 5, 364 Human atherosclerotic cardiovascular disease, 157 Human CMV (HCMV), 366 Human herpesvirus (HHV), 447
448 Human herpesvirus type 5. See Cytomegalovirus (CMV)

Human immunodeficiency virus (HIV), 230
232, 366, 454
455, 478
479 HIV-1, 365 Human nutrition, poultry transgenesis and, 213
215 Human parainfluenza type1 virus (hPIV-1 virus), 366
367 Humanized (chimeric) models, 190 Humoral immunity, 472 Hungarian flocks of turkeys, 48 HVT. See Herpes virus of turkey (HVT) Hybridization stringency, 329 techniques, 328
329 Hybridization-based methods, 291, 302
303 Hyperaccurate Cas9 variant (HypaCas9), 82
83

I I/M injection. See Intramuscular injection (I/M injection) IAASTD. See International Assessment of Agricultural Knowledge Science and Technology for Development (IAASTD) IARS syndrome. See Isoleucyl-tRNA synthetase syndrome (IARS syndrome) IBD. See Infectious bursal disease (IBD) IBDV. See Infectious bursal disease virus (IBDV) IBISS database. See Interactive bovine in silico SNP database (IBISS database) Ichthyophthirius multifiliis, 408
411 Icosadeltahedral capsid, 446 ICT. See Immunochromatography test (ICT) Ideal immunoassay, 319
320 Idiopathic pulmonary fibrosis, 396 IEF. See Isoelectric focusing (IEF) IFN-γ. See Interferon gamma (IFN-γ) IGF. See Insulin-like growth factor (IGF) IHHN. See Infectious hepatopancreas and haematopoietic necrosis (IHHN) IHHNV. See Infectious hypodermic and haematopoeitic necrosis virus (IHHNV) IHN. See Infectious haematopoietic necrosis (IHN) IHNV. See Infectious hematopoeitic necrosis virus (IHNV) IL-2. See Interleukin-2 (IL-2) Illumina BovineLD BeadChip, 7 Illumina/solexa sequencing, 33
35 IMDA. See Isothermal multiple displacement amplification (IMDA) IMF. See Intramuscular fat (IMF) Immune system, 469 Immune-stimulating, 428t Immune-stimulating complexes (ISCOM), 424

Index

Immunization, 470, 483 of livestock, 495 of susceptible animals, 482
483 Immuno-PCR, 290 Immunoassays, 299, 319
321 Immunoblotting, 319
320 Immunochromatography test (ICT), 289
290 strip tests, 294
295 Immunodiffusion testing, 302 Immunogenic genes, 412
413 Immunoproteomics, 408
411 Immunoregulators, 280
281 Immunostimulants, 345, 346t IMNV. See Infectious myonecrosis virus (IMNV) IMTA. See Integrated multitrophic aquaculture (IMTA) In situ hybridization, 328 In vitro DNA synthesis, 151 fertilization, 189
190, 226
227 hybridization, 328 mutagenesis, 164
165 Inactivated rabies viruses, 369 Inactivated vaccines, 472 Inactivated virus vaccines, 277
278 InDels. See Insertions or deletions (InDels) Induced models, 184
186 induction of disease through biological molecules, 186 lesion-induced models, 185
186 pharmacological or chemical-induced models, 184
185 stress-induced models, 186 Induced pluripotent stem (iPS), 92
93 infB. See Translation initiation factor 2 (infB) Infectious animal diseases, 470 Infectious bovine rhinotracheitis, 485 Infectious bursal disease (IBD), 368 Infectious bursal disease virus (IBDV), 368 Infectious diseases, 469 impacts, 288
289 Infectious haematopoietic necrosis (IHN), 313 Infectious hematopoeitic necrosis virus (IHNV), 319
320, 408
411 Infectious hepatopancreas and haematopoietic necrosis (IHHN), 315 Infectious hypodermic and haematopoeitic necrosis virus (IHHNV), 319
320 Infectious myonecrosis virus (IMNV), 315 Infectious pancreatic necrosis, 313 Infectious pancreatic necrosis virus (IPNV), 319
320, 408
411 Infectious salmon anemia virus (ISAV), 408
411

Infectious spleen, and kidney necrosis virus (ISKNV), 56 Infectious viral disease agents, 299 Influenza (flu), 486
487 Informatics techniques, 381 Injection-site-associated sarcoma (ISAS), 395 Insertion sequence-PCR (IS-PCR), 339
340 Insertions or deletions (InDels), 132
134 Insulin, 196
197. See also Diabetes mellitus Insulin-like growth factor (IGF), 115 Integrated multitrophic aquaculture (IMTA), 275 Interactive bovine in silico SNP database (IBISS database), 14
15 INTERBULL concept for genetic evaluation of breeding bulls, 13 Interferon gamma (IFN-γ), 426 Interleukin-2 (IL-2), 426 International Assessment of Agricultural Knowledge Science and Technology for Development (IAASTD), 211
212 Intracellular bacteria, 312
313 Intramuscular fat (IMF), 117
118 Intramuscular injection (I/M injection), 412
413, 474
475 Invasive urothelial carcinoma (iUC), 392
393 Ion Torrent semiconductor sequencing, 35
36 IPNV. See Infectious pancreatic necrosis virus (IPNV) iPS. See Induced pluripotent stem (iPS) Iridoviral infection, 343
344 Iridoviridae, 55 IS-PCR. See Insertion sequence-PCR (IS-PCR) ISAS. See Injection-site-associated sarcoma (ISAS) ISAV. See Infectious salmon anemia virus (ISAV) ISCOM. See Immune-stimulating complexes (ISCOM) ISKNV. See Infectious spleen, and kidney necrosis virus (ISKNV) Isoelectric focusing (IEF), 387 Isoelectric point (pI), 387 Isoleucyl-tRNA synthetase syndrome (IARS syndrome), 177
178 Isothermal amplification methods, 291
292 Isothermal multiple displacement amplification (IMDA), 291
292 iUC. See Invasive urothelial carcinoma (iUC)

J Japanese encephalitis (JV), 483
484 Japanese encephalitis virus (JEV), 483
484 JV. See Japanese encephalitis (JV)

517

518

Index

K K. pneumoniae MLST database, 342
343 Kappa casein, 109
111 Keratin-associated protein (KAP), 117 Khurdun virus genome, 49 KHV. See Koi herpesvirus (KHV) KIRV. See Koi ranavirus (KIRV) Knock-out animals (KO animals), 183, 224 models, 77 Knock-out transgenic chicken, 235
236 KO animals. See Knock-out animals (KO animals) KO chicken embryos. See Korean Oge chicken embryos (KO chicken embryos) Kobuvirus, 52 Koi herpesvirus (KHV), 56 Koi ranavirus (KIRV), 314
315 Korean Oge chicken embryos (KO chicken embryos), 235
236

L LA. See Latex agglutination (LA) Label-free quantitation (LFQ), 115 Labeo rohita, 348
350 toll-like receptor-22, 264 Lacking polysaccharide repeat (LPS), 479 Lactobacillus plantarum MRO3.12, 348
350 Lactococcus garvieae, 312
313, 325, 343
344 LAMP. See Loop-mediated isothermal amplification (LAMP) Large ruminants, genetic markers in genes associated with meat production, 116 genes related to draught power, 116 genetic markers related to reproductive performance, 114
115 markers for dairy production traits, 109
114 Latency associate transcript, 446
447 Latent cycle, 446
447 Lateral flow diffusion method, 320
321 Lates calcarifer (Asian sea bass), 314
315 Latex agglutination (LA), 289
290 LCDV. See Lymphocystis disease virus (LCDV) LD. See Linkage disequilibrium (LD) Leishmania major, 423
424 Lentivirus vectors, 365
366 Leptin (LEP), 109
111 Leptospira spp., 478 Leptospirosis, 478 Lesion-induced models, 185
186 Lethal mutagenesis, 448
449 LFQ. See Label-free quantitation (LFQ) Ligand molecules, 436 Light turkey syndrome (LTS), 48 Linkage disequilibrium (LD), 7
8

Linoleic acid, 213
215 Lipopolysaccharide (LPS), 426 Liposome transfection, 230 Liposomes, 428t, 429 Liquid
solid extraction methods, 30
31 Listeria monocytogenes, 332, 474 Litopenaeus vannamei (Farmed whiteleg shrimp), 58, 315 Live flavivirus chimera vaccine, 470 Live-attenuated vaccines, 472 Livestock, 287
288, 488
489 diseases, 158 farming, 469 production, 469 sector, 469 species, 155, 155t Long line farming of bivalves, 275 Long terminal repeats (LTRs), 365 Loop-mediated isothermal amplification (LAMP), 291
292, 300, 329
331 Loopamp Realtime turbidmeter, 330 Lopinavir, 455
456 Low pathogenicity avian influenza (LPAI), 486 LPAI. See Low pathogenicity avian influenza (LPAI) LPS. See Lipopolysaccharide (LPS) LTRs. See Long terminal repeats (LTRs) LTS. See Light turkey syndrome (LTS) Lymphocystis, 313 Lymphocystis disease virus (LCDV), 57
58 LCDV-Sa, 57
58 Lysis buffer, 30
31

M MACE. See Multiple across country evaluation (MACE) Macroalgae. See Seaweed Macrobrachium rosenbergii (Giant river prawn), 58, 314 Macrobrachium rosenbergii nodavirus (MrNV), 315 Macromolecular crystallography, 438 MAF. See Minor allele frequency (MAF) Main protease (Mpro), 451 Malate dehydrogenase (mdh), 342
343 MALDI-TOF. See Matrix-assisted laser desorption/ionization time-of-flight tandem (MALDI-TOF) MALDI-TOF MS. See Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) Mammalian cells, 474
475 Mammalian models, 185t

Index

Mammuthus primigenius (Woolly mammoths), 94 Manheimia hemolytica, 479 Mannheimia haemolytica, 490 Marek’s disease virus (MDV), 367
368 Marek’s diseases (MD), 212
213 Mariculture biotechnology in environment management, 278 genetic manipulation, 275
277 health management, 277
278 for food, 274
275 Marine algae, 273
274 Marine biotechnology, 271
272 bioprospecting for food, 278
281 biotechnology in mariculture, 275
278 food from marine sources, 272
274 mariculture technologies for food, 274
275 Marine ponds, 274 Marker-assisted selection (MAS), 10
11 Mass spectrometry (MS), 387
388 Mast-cell tumors (MCTs), 394
395 Maternally-derived antibodies (MDA), 367
368 Matrix protein 1 (M1), 458 Matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS), 113
114, 332
334 Matrix-assisted laser desorption/ionization time-offlight tandem (MALDI-TOF), 394 Maxam-Gilbert sequencing, 31
32 MBV. See Molecular breeding value (MBV) Monodon baculo virus (MBV) MC4R. See Melanocortin-4 receptor (MC4R) MCTs. See Mast-cell tumors (MCTs) MD. See Marek’s diseases (MD)Molecular dynamics (MD) MDA. See Maternally-derived antibodies (MDA) mdh. See Malate dehydrogenase (mdh) MDV. See Marek’s disease virus (MDV) Meat, 119, 211
212 genes associated with, 116 production, 116
117 quality traits, 117
118 Megalocytiviris, 314
315 Meganucleases, 170
172 Melanocortin-4 receptor (MC4R), 113
114 Melatonin receptor 1A (MTNR1A), 117 Melegrivirus A, 47 Membrane (M), 450 MERS-CoV. See Middle East respiratory syndrome coronavirus (MERS-CoV) Mesivirus-1 and-2, 48
49 Metagenomics, 29 arrays, 331 complete genome sequencing by NGS, 56
57

discovery of novel viruses, 57
58 practical aspects of viral, 39
44 revealing new virus species in aquaculture, 55
58 sequencing technologies, 31
37 technical aspects of viral, 30 viral metagenomics and discovery of new viruses in livestock, 45
49 in pets, 49
54 virome characterization, 55
56 virus enrichment and nucleic acid amplification, 30
31 Metapneumovirus, 456 Methicillin resistance gene, 92 Methionine synthase (MTR), 117 Methyltransferase (MTase), 453
454 Micro pigs, 156, 157f Micro-RNA (miRNA), 166 Microalgae, 280 Microarray, 249, 292
293, 331
332 Microinjection, 137, 194
195 Microsatellite loci, 119
120 Midcrop mortality syndrome, 315 Middle East respiratory syndrome coronavirus (MERS-CoV), 450
451 Milk, 211
212 production, 116
117 proteins, 109
111 Mimicking clinical conditions in animals, 187
189 Mimiviridae, 49
50 MiniLab, 295 Mining animal databases, 109
111 MinION, 37, 58
59, 294, 303 Minnesota turkeys, 48 Minor allele frequency (MAF), 7 miRNA. See Micro-RNA (miRNA) MLEE. See Multilocus enzyme electrophoresis (MLEE) MLST analysis. See Multilocus sequence typing analysis (MLST analysis) Modern diagnostic assays, 319 Modified live (MLV), 479 Modified vaccinia (MVA), 369 Modulating animal health, 177
178 Molecular docking and simulation, 439 Molecular breeding value (MBV), 6 Molecular detection-based methods, 289 Molecular diagnostics, 249 for fish diseases, 321
332 hybridization techniques, 328
329 LAMP, 329
331 microarrays, 331
332

519

520

Index

Molecular diagnostics (Continued) PCR, 322
325, 323t qPCR, 325
327 Molecular dynamics (MD), 444
445 Molecular scissors, 131
132, 133t, 136
137 Mollusca, 279 Molluscs, 273 Moloney murine leukemia virus, 366 Mono-sex population, 262 Monoclonal antibodies, 215
216 Monodon baculo virus (MBV), 315 Monophosphoryl lipid (MPL), 426 Morbidity, 491
492 Morbilliviruses, 53
54, 456 Mortality, 491
492 mPCR. See Multiplex polymerase chain reaction (mPCR) MPL. See Monophosphoryl lipid (MPL) Mpro. See Main protease (Mpro) MrNV. See Macrobrachium rosenbergii nodavirus (MrNV) MS. See Mass spectrometry (MS) MSTN. See Myostatin (MSTN) MTase. See Methyltransferase (MTase) MTNR1A. See Melatonin receptor 1A (MTNR1A) MTR. See Methionine synthase (MTR) “Mucin-type” glycosylation, 90
91 Mucosal immunity, 480, 488 Multibreed cattle populations, genomic selection in, 20 Multilocus enzyme electrophoresis (MLEE), 342
343 Multilocus sequence typing analysis (MLST analysis), 342
343 Multiple across country evaluation (MACE), 13 Multiple gene editing, 80 Multiple serotypes, 472 Multiplex polymerase chain reaction (mPCR), 291, 325 Multiplexing, 80, 260
261 Mussel farming, 274
275 Mutagenesis, 224 Mutagenic chain reaction, 95
96 Mutant models, 187 Mutation, 164
165 induction, 223
224 MVA. See Modified vaccinia (MVA) Mycobacteriosis, 312
313 Mycobacterium, 191, 291, 302
303 infection in cattle, 478
479 M. bovis, 88, 177
178, 293, 300, 369, 478
479 M. fortuitum, 312
313 M. marinum, 312
313 M. tuberculosis, 88, 302
303, 365

Mycodnaviridae, 49
50 Mycoplasma, 191 Myostatin (MSTN), 86
87, 116
117, 155
156, 178 Myxobolous, 312

N n-3 polyunsaturated fatty acids (PUFA), 213
215 NA. See Neuraminidase (NA) Naked DNA, vaccines based on, 412
413 Nanomaterial as adjuvants, 427
428 Nanoparticle amplified immune PCR (NPA-IPCR), 290 Nanoparticle technology, 334
335 Nanopore sequencing, 37 technology, 294 Nanosensors, 334
335 Nanotechnology, 334
335 NASBA. See Nucleic acid sequence-based amplification (NASBA) Natural animal models, 183
184 Natural resistance-associated macrophage protein 1 gene (NRAMP1 gene), 177
178 NCP. See Noncytopathic biotype (NCP) ND. See Newcastle disease (ND) NDV. See Newcastle disease virus (NDV) Needle-free technologies, 429 Negative models, 187 Neisseria meningitides, 259
260, 342
343 Nelfinavir, 455
456 NEP. See Nuclear export protein (NEP) Nervous necrosis virus (NNV), 412
413 Nested polymerase chain reaction (Nested PCR), 324
325 Neuraminidase (NA), 486 Neurodegenerative diseases, 187 New generation vaccines, 407
413, 409t based on naked DNA, 412
413 genetically attenuated pathogens, 412 recombinant vaccines, 408
411 reverse vaccinology, 413 vector technology, 411
412 Newcastle disease (ND), 368 Newcastle disease virus (NDV), 367
368, 456, 457f, 473 Next-generation technologies, 425t Next-generation vaccine adjuvants, 424
428 aluminum salts, 424
426 carbohydrate adjuvants, 427 cytokines adjuvants, 427 monophosphoryl lipid and AS04, 426 nanomaterial as adjuvants, 427
428 nucleic acid-based mucosal adjuvants, 427

Index

o/w emulsion, 426 virosomes, 426 Next-generation sequencing (NGS), 31, 294, 300
303, 384 genome sequencing by, 56
57 principle, 34f NGS. See Next-generation sequencing (NGS) NHC. See D-N4-hydroxycytidine (NHC) NHEJ. See Nonhomologous end joining (NHEJ) NHL. See Non-Hodgkin’s lymphomas (NHL) Nipah viruses (NiVs), 456 Nitric oxide (NO), 177
178 Nitric oxide synthase (NOS), 392
393 NiVs. See Nipah viruses (NiVs) NMR. See Nuclear magnetic resonance (NMR) NNV. See Nervous necrosis virus (NNV) NO. See Nitric oxide (NO) Nocardia crassostreae (ostreae), 312
313 Nocardia seriolae, 312
313 Nocardiaasteroides, 312
313 Nodaviridae, 49
50, 344
345 Nodoviridae, 55 Non-A rotaviruses, 52
53 Non-Hodgkin’s lymphomas (NHL), 390 Noncytopathic biotype (NCP), 484
485 Nonhomologous end joining (NHEJ), 76, 76f, 131
132, 170
172 repair, 137 Nonhuman primate models, 185t Nonmammalian models, 185t Nonnucleoside inhibitors, 448
449 Nonpathogenic PCV1-vectored vaccine, 369 Nonprimate hepacivirus (NPHV), 52 Nonrepetitive sequences, 174
175 Nonstructural protein 1 (NS1), 458 Nonstructural proteins (nsPs), 441 functions, 453
454 Northern pink shrimp. See Farfantepenaeus duorarum (Northern pink shrimp) NOS. See Nitric oxide synthase (NOS) Notch signaling, dysregulation of, 90
91 Novartis. See Alpha Jects Micro 1 ISA Novel avian gammacoronavirus, 49 Novel bovine astroviruses (BoAstV-NeuroS1), 46 Novel canine parvoviruses, 50 Novel coronavirus 2012, 450
451 Novel flavivirus, 52 Novel picornavirus, 45 Novel Sunguru virus, 47 Novel viruses in cats, 52
54 in chickens, 47
48 discovery of, 57
58 in other birds, 48
49

in turkeys, 48 NPA-IPCR. See Nanoparticle amplified immune PCR (NPA-IPCR) NPHV. See Nonprimate hepacivirus (NPHV) NPs. See Nucleoproteins (NPs) NRAMP1 gene. See Natural resistance-associated macrophage protein 1 gene (NRAMP1 gene) NS1. See Nonstructural protein 1 (NS1) nsPs. See Nonstructural proteins (nsPs) Nuclear export protein (NEP), 458 Nuclear magnetic resonance (NMR), 436, 438 Nuclease editors, 192
194 CRISPR/Cas9, 193 ZFNs, 193
194 Nuclease-based gene editing tools in modulating animal health, 177
178 Nuclease-based reverse genetics tools CRISPR/Cas9, 174
177 TALEN, 173
174 ZFN, 172
173 Nuclease-deficient Cas9 model (dCas9), 256 Nucleic acid, 163
164, 328 amplification, 30
31, 291 nucleic acid-based mucosal adjuvants, 427 nucleic acid-based vaccines, 423 Nucleic acid sequence-based amplification (NASBA), 291
292 Nucleic acid-based diagnostic assays, 290
292 amplification-based methods, 291
292 hybridization-based methods, 291 novel and high throughput assays, 292
298 biosensors, 293
294 microarray, 292
293 NGS, 294 patented diagnostic technologies, 295
298, 298t PNAs and aptamers, 293 POCD, 294
295, 296t Nuclein, 163
164 Nucleocapsid (N), 450 Nucleoproteins (NPs), 458 Nutraceuticals from marine organisms, 278
281

O o/w emulsion. See Oil-in-water emulsion (o/w emulsion) Obesity, 91 Odds ratio, 9
10 Off-target cleavage, 77 effects, 81
83 Cas9 nickase, 81

521

522

Index

Off-target (Continued) Cas9-HF1, 82 Cpf1, 82 eSpCas9, 81
82 HypaCas9, 82
83 SgRNAs design, 81 Oil-in-water emulsion (o/w emulsion), 426 Oligonucleotide oligonucleotide-directed mutagenesis, 165 probes, 331
332 Omics data, 388
395 in cancers of domestic animals, 395 in studying animal diseases, 396
397 Omics technology, 383 Oncorhynchus masou virus, 313 Oncorhynchus mykiss, 408
411 “One health”, 470
471 Open reading frame (ORF), 37
39, 170, 441 OPN. See Osteopontin (OPN) Optimizing stringency, 329 OPXVs. See Orthopoxviruses (OPXVs) Oreochromis niloticus (Tilapia), 314
315 ORF. See Open reading frame (ORF) Organ transplantation, 94 Ornamental fishes production, 263
264 Orphan models, 184, 187 Orthomyxoviridae, 344
345, 458 Orthopoxviruses (OPXVs), 54 Osteopontin (OPN), 114
115, 118 Ovalbumin (OV), 235
236 Oxford Nanopore Technologies, 37

P P-value, 9
10 PA. See Polymerase acid protein (PA) PacBio RS II, 36 Pacific Biosciences, 36 PAM. See Protospacer adjacent motif (PAM) Papillomaviridae, 50 Papillomaviruses (PVs), 50
51, 53 Paramyxoviridae, 49
50, 366
367, 370t, 456 Paramyxovirinae, 456 Paramyxovirus, 456
457 antivirals against paramyxovirus, 456
457 Parasitic bronchitis, 490 Parasitic vaccines, 488
489 Paratuberculosis, 478
479 Particulate delivery system, 429 Parvoviridae, 49
50, 52 Parvovirus (PPV7), 45 Passenger pigeon. See Ectopistes migratorius (Passenger pigeon) Pasteurella, 191 Pasteurella multocida, 475
477

Patented diagnostic technologies, 295
298, 298t Pathogen-free test animals, 277 Pathogenic bacteria, 348 Pathogens, 407
408 characterization, genotyping techniques in, 335
341 diagnosis, 289
298 nucleic acid-based diagnostic assays, 290
292 serological diagnostic assays, 289
290 evolution, 288 genetic sequences, 331
332 PCR. See Polymerase chain reaction (PCR) PCR-based strain typing techniques, 337
341. See also Polymerase chain reaction (PCR) AFLP, 338
339 AP-PCR and RAPD, 337
338 ARDRA, 341 ERIC-PCR, 339
340 IS-PCR, 339
340 Rep-PCR, 339
340 ribotyping, 340
341 VNTR-PCR, 339
340 PCR-RFLP-Ribotyping, 341 PCV-2. See Porcine circovirus type-2 (PCV-2) PDB. See Protein data bank (PDB) PEBP1. See Phosphatidylethanolamine binding protein 1 (PEBP1) Penaeus monodon, 348
350 Penicillin, 196
197 Peptide mass fingerprint, 332
333 Peptide nucleic acids (PNAs), 293 Persistently infected animals (PI animals), 484
485 Personal Genome Machine (PGM), 35
36 PERV. See Porcine endogenous retrovirus (PERV) Peste-des-petits ruminants (PPRV), 482 Pestepetits-ruminants virus (PPRV), 369 Pestivirus, 458
460, 484
485 vaccine and structure-based drug design, 459
460 Pet animals as disease model, 157 PFGE. See Pulsed-field gel electrophoresis (PFGE) PGCs. See Primordial germ cells (PGCs) pgi. See Phosphoglucose isomerase (pgi) PGM. See Personal Genome Machine (PGM) Phaffia rhodozyma (Red yeast), 281 Pharmaceutical drugs, 183
184 Pharmacological model, 184
185 Phenotypic effect, 164 Φ29 DNA polymerase-based method, 31 phoE. See Phosphorine E (phoE) Phosphatase and tensin homolog (PTEN), 392
393

Index

Phosphatidylethanolamine binding protein 1 (PEBP1), 114
115 Phosphoglucose isomerase (pgi), 342
343 Phosphorine E (phoE), 342
343 Phosphorus, 212
213 Photobacterium damselae, 312
313, 413 Photobacterium damselae subsp. Piscicida, 343
344 Photobacterium damselaedamselae, 312
313 Phycodnaviridae, 55 Phylogenetic oligonucleotide arrays, 331 Phytase enzyme, 212
213 pI. See Isoelectric point (pI) PI animals. See Persistently infected animals (PI animals) Pichia pastoris, 473 Picornaviridae, 49
50, 52 Picornaviruses, 46
48, 51 PicoTiterPlates, 33 Pig stool-associated circular ssDNA virus (PigSCV), 45 Pigeons, 48
49 PiggyBac vectors, 232 Pigs, 156 new viruses in, 45 PigSCV. See Pig stool-associated circular ssDNA virus (PigSCV) piRNA. See Piwi RNA (piRNA) Piscirickettsia salmonis, 312
313, 343
344, 408
411 Pituitary-specific transcription factor-1 (POU1F1), 116
117 Piwi RNA (piRNA), 166 PKM2. See Pyruvate kinase (PKM2) Plant-based vaccine trials, 474 Plasmodium falciparum, 365 PLGA. See Poly (lactide-co-glycolide) (PLGA) Pluripotent stem cells, 234 “Plus and minus” method, 31
32 PNAs. See Peptide nucleic acids (PNAs) Pneumovirus, 456 Point-of-care (POC), 289 Point-of-care diagnostics (POCD), 294
295, 296t Pollution markers development, 263 Polony sequencing. See Sequencing by oligonucleotide ligation and detection (SOLiD) Poly (lactide-co-glycolide) (PLGA), 480 Polyacrylamide-urea gel electrophoresis, 31
32 Polycomb targets, 392
393 Polyculture, 275 Polymerase acid protein (PA), 458 Polymerase chain reaction (PCR), 30, 32, 108, 163
164, 249, 289, 300, 311
312,

322
325. See also PCR-based strain typing techniques mPCR, 325 nested PCR, 324
325 PCR-mediated mutagenesis, 164
165 RT-PCR, 324 and variants, 291 Polymerase spiral reaction (PSR), 300 Polyploidy, 276 Polysaccharides, 271
272, 279, 421 Porcine circovirus type-2 (PCV-2), 369, 473 Porcine endogenous retrovirus (PERV), 169 Porcine parvovirus 2 (PPV2), 45 Porcine reproductive and respiratory syndrome (PRRS), 87
88, 155 Porcine reproductive and respiratory syndrome virus (PRRSV), 369 Porifera, 279 Positive predictive value (PPV), 394 Posttranslational modifications (PTMs), 386
387 POU1F1. See Pituitary-specific transcription factor-1 (POU1F1) Poultry, 287
288, 471 and environment protection, 212
213 genetic markers eggs, 119
120 meat, 119 meat consumption, 213
215 production, 212
213 science, 211
212 transgenesis and human nutrition, 213
215 and medicine, 215
216 vaccines, 491
494, 492t vectors for, 367
368 Poxviridae, 54, 57, 363
364, 370t Poxviruses, 363
364, 369 vectors, 361
363 PPRV. See Peste-des-petits ruminants (PPRV) Pestepetits-ruminants virus (PPRV) PPV. See Positive predictive value (PPV) PPV2. See Porcine parvovirus 2 (PPV2) PPV7. See Parvovirus (PPV7) Precise genome editing, 234
239 Cre/Lox-P, 238
239 CRISPR, 237
238 TALE, 235
237 ZFNs, 234
235 pri-miRNA. See Primary miRNA (pri-miRNA) PRIDE. See PRoteomics IDEntifications database (PRIDE) Primary miRNA (pri-miRNA), 166
168 Primordial germ cells (PGCs), 232 Primordial germ culture, 233

523

524

Index

PRKA. See Protamine 1 & 2, A kinase (PRKA) PRL. See Prolactin (PRL) Pro-vitamin A, 280
281 Probiotics, 345
350, 349t Progeny testing (PT), 10
11 Programmable genome editing systems, 234 Programmable nuclease-based genome editing technologies, 132
134 Prokaryotes, 224 Prolactin (PRL), 109
111 Pronuclear microinjection, 194
195 Protamine 1 & 2, A kinase (PRKA), 114
115 Protective immunity, 366, 369 Protein data bank (PDB), 383, 454
455 Protein glycosylation, 90
91 ProteomeXchange (PX), 388 Proteomics, 386
388, 389t, 395
396 analysis, 117
118 studies in canine cancers, 394
395 PRoteomics IDEntifications database (PRIDE), 388 Proteus mirabilis, 332 Proteus vulgaris, 332 Protospacer adjacent motif (PAM), 78, 255 sequence, 136
139 PRRS. See Porcine reproductive and respiratory syndrome (PRRS) PRRSV. See Porcine reproductive and respiratory syndrome virus (PRRSV) PRV. See Pseudorabies virus (PRV) Pseudomonas aeruginosa, 325 Pseudomonas anguilliseptica, 312
313 Pseudomonas fluorescens, 312
313 Pseudomonas putida, 325 Pseudomonasis, 312
313 Pseudorabies, 88 Pseudorabies virus (PRV), 88 PSR. See Polymerase spiral reaction (PSR) PT. See Progeny testing (PT) PTEN. See Phosphatase and tensin homolog (PTEN) PTMs. See Posttranslational modifications (PTMs) PUFA. See n-3 polyunsaturated fatty acids (PUFA) Pulsed-field gel electrophoresis (PFGE), 336
337, 341
342 Puromycin, 236
237 Putative novel rotavirus C VP6 genotype, 45 PVs. See Papillomaviruses (PVs) PX. See ProteomeXchange (PX) Pyrococcus furiosus, 256
257 Pyrosequencing, 33 Pyruvate kinase (PKM2), 114
115

Q q-PCR. See Quantitative PCR (q-PCR) QCM. See Quartz crystal microbalance (QCM) QIA01 strain, 47 qNASBA system. See Real-time Nucleic acid sequence-based amplification system (qNASBA system) qPCR. See Real-time polymerase chain reaction (qPCR) QTL-MAS. See Quantitative trait loci-marker aided selection (QTL-MAS) QTLs. See Quantitative trait loci (QTLs) Quality of beef, 116 Quantification cycle (Cq), 325
326 Quantitative PCR (q-PCR), 249 Quantitative trait loci (QTLs), 109
111, 117
119 Quantitative trait loci-marker aided selection (QTL-MAS), 3
4 Quartz crystal microbalance (QCM), 293
294 nanoimmunosensors, 335

R Rabies, 469, 481
482 vaccine, 474 Raceway farming, 275 Radioimmunoassay, 319
320 Radioisotopes, 319
320, 328 Ranaviruses of fish, 57 Random amplified polymorphic DNA (RAPD), 108, 337
338, 341
342 Random PCR, 31 RAPD. See Random amplified polymorphic DNA (RAPD) RAS. See Recirculating aquaculture systems (RAS) RC. See Replication complex (RC) RCA. See Rolling circle amplification (RCA) RdRp. See RNA-dependent RNA polymerase (RdRp) REAL. See Revised European-American classification of lymphoid neoplasms (REAL) Real-time nucleic acid sequence-based amplification system (qNASBA system), 292 Real-time polymerase chain reaction (qPCR), 291, 295, 300, 325
327 Recirculating aquaculture systems (RAS), 275, 278 Recombinant adenoviral vectors, 365 Recombinant Cas9 protein, 137
139 Recombinant DNA, 230, 408
411, 421, 472
473 Recombinant DNA vaccines, 277
278, 425t Recombinant HVT (rHVT), 368

Index

Recombinant vaccines, 408
411 Recombinant vaccinia virus, 361
363 Recombinant vector vaccine production process, 361
363, 362f Recombinase polymerase amplification (RPA), 292 Red yeast. See Phaffia rhodozyma (Red yeast) Regulatory hurdles, 93 Regulatory issues, 199
202 Renibacteriosis, 312
313 Renibacterium salmoninarum (Rs), 312
313, 340, 343
344 Reoviridae, 50
52 Repeat variable diresidue (RVD), 173
174, 235, 253 Repetitive element sequence-based PCR (RepPCR), 336
337, 339
342 Replication complex (RC), 448
449 Replicon particles (RP), 486
487 Reproductive homeobox 5 (Rhox5), 114
115 Reproductive traits, 117
118 Respiratory syncytial virus (RSV), 422 Respirovirus, 456 Restriction fragment length polymorphism (RFLP), 108 Retroviral vector-based vaccines, 365 Retroviridae, 55 Retrovirus vectors, 365 Reverse genetics, 164 nuclease-based gene editing tools in modulating animal health, 177
178 nuclease-based reverse genetics tools, 172
177 RNAi, 165
170 targeted genome modification by HR, 170
172 in vitro mutagenesis, 164
165 Reverse transcriptase (RT), 324 Reverse transcription LAMP assay (RT-LAMP assay), 330
331 Reverse vaccinology, 421, 423
424, 425t Reverse-transcription polymerase chain reaction (RT-PCR), 52, 291, 324 Reversible chain-terminating nucleotides, 33 Revised European-American classification of lymphoid neoplasms (REAL), 388
389 RFLP. See Restriction fragment length polymorphism (RFLP) RGENs. See RNA-guided endonucleases (RGENs) Rhabdoviridae, 47, 344
345 Rhabdoviridae DNA vaccines, 344
345 Rhesus CMV (RhCMV), 366 Rhipicephalus appendiculatus, 489 Rhox5. See Reproductive homeobox 5 (Rhox5) rHVT. See Recombinant HVT (rHVT) rHVT-AIV vaccines, 368

Ribavirin, 449
450 Riboproteomics approach, 436 Ribosomal DNA, 341 16S ribosomal DNA (16S rDNA), 332 genes, 340
342 Ribotyping, 340
341 Rickettsia rickettsia, 302 Rickettsia spp., 301 Rituximab, 215
216 RNA interference (RNAi), 165
170, 167f, 249 RNA sequencing (RNA-seq), 381, 386 RNA-dependent RNA polymerase (RdRp), 442, 453
454 RNA-guided endonucleases (RGENs), 76, 78 RNA-seq. See RNA sequencing (RNA-seq) RNAi. See RNA interference (RNAi) Roche 454 sequencer, 33 Rodent models, 185t Rolling circle amplification (RCA), 31, 291
292 Roman cropping techniques, 223 Ropporin-1, 114
115 Rotavirus A (RVA), 46 Rotavirus B (RVB), 46 Rotaviruses (RV), 299, 487
488 gastroenteritis, 488 RP. See Replicon particles (RP) RPA. See Recombinase polymerase amplification (RPA) rpoB. See Beta-subunit of RNA polymerase (rpoB) RSV. See Respiratory syncytial virus (RSV) RT. See Reverse transcriptase (RT) RT-LAMP assay. See Reverse transcription LAMP assay (RT-LAMP assay) RT-PCR. See Reverse-transcription polymerase chain reaction (RT-PCR) Rubulavirus, 456 Ruminant meat, 116 RV. See Rotaviruses (RV) RVA. See Rotavirus A (RVA) RVB. See Rotavirus B (RVB) RVD. See Repeat variable diresidue (RVD)

S Saccharomyces cerevisiae, 170 SalHV3, 277
278 Salmo salar (Salmon), 57, 411
412 Salmonella, 191, 292, 300
301, 332, 335, 474 Dublin, 479 Salmonella typhimurium, 339
340, 479 Salmonellosis, 479 Salmonid alphavirus (SAV), 411
412, 452
453 Salmonid Rickettsial Syndrome (SRS), 411

525

526

Index

Sapelovirus, 51
52 Sapovirus, 49 SARS. See Severe acute respiratory syndrome (SARS) SARS-CoV. See Severe acute respiratory syndrome coronavirus (SARS-CoV) SAV. See Salmonid alphavirus (SAV) Scavenger receptor cysteine-rich (SRCR), 87
88 Scavenger receptor cysteine-rich domain 5 (SRCR5), 177 Schmallenberg virus, 46 SCID-X1. See X-linked severe combined immunodeficiency (SCID-X1) SCNT. See Somatic cell nuclear transfer (SCNT) Scopolamine-induced amnesia, 184
185 SDA. See Strand displacement amplification (SDA) SDS-PAGE. See Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) Sea cage farming, 275 Seaweed, 273
274, 279 polysaccharides, 280 Second-generation sequencing, 33
36 illumina/solexa sequencing, 33
35 pyrosequencing, 33 semiconductor sequencing, 35
36 SOLiD, 35 Selective breeding, 275 Self-inactivating vectors (SIN vectors), 365 Semiconductor sequencing, 35
36 Semliki forest virus (SFV), 454
455 Sendai virus vectors (SeV vectors), 366
367 Sequel System, 36 Sequence-independent single primer amplification (SISPA), 31 Sequence-targeted PCR assays, 108 Sequencing by oligonucleotide ligation and detection (SOLiD), 35 Sequencing technologies, 31
37 first-generation sequencing, 31
32 second-generation sequencing, 33
36 third-generation sequencing, 36
37 Serological proteome analysis (SERPA), 388 Serological/serology assays, 299 diagnostic assays, 289
290 methods, 289, 319
320 Serotype-specific immunity, 482
483 SERPA. See Serological proteome analysis (SERPA) SeV vectors. See Sendai virus vectors (SeV vectors)

Severe acute respiratory syndrome (SARS), 288
289, 450
451 Severe acute respiratory syndrome coronavirus (SARS-CoV), 450, 452f Sex determination region of Y chromosome (SRY), 117 Sexual dimorphism, 262 SF1. See Superfamily 1 (SF1) SFV. See Semliki forest virus (SFV) SgRNA. See Single guide RNA (SgRNA) Sheep pox virus (SPV), 483 SHERLOCK. See Specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) Shigella, 300, 332 SHIV. See Shrimp hemocyte iridescent virus (SHIV) Short hairpin RNA (shRNA), 166
168 Shrimp, diseases in, 315
341 diagnostic methods, 319
341, 321t genotyping techniques in characterization of pathogens, 335
341 immunoassays, 319
321 MALDI-TOF MS, 332
334 molecular diagnostics for fish diseases, 321
332 nanotechnology and nanosensors, 334
335 Shrimp hemocyte iridescent virus (SHIV), 58 shRNA. See Short hairpin RNA (shRNA) SIBA. See Strand invasion-based amplification (SIBA) Sicinivirus, 47 Signal-mediated amplification of RNA technology (SMART), 291
292 Silurus glanis (Wels catfish), 57 Simian immunodeficiency virus (SIV), 366 Simmental breed, 11 Simple nucleotide variations (SNVs), 384 SIN vectors. See Self-inactivating vectors (SIN vectors) Sindbis virus (SINV), 452
453 Single guide RNA (SgRNA), 79, 81, 92, 136
139, 175
176 Single molecule real-time sequencing (SMRT sequencing), 36 Single nucleotide polymorphism (SNP), 3
4, 6, 108, 384 information, 114
115 Single primer isothermal amplification (SPIA), 291
292 Single sea bream. See Sparus aurata (Single sea bream) Single-molecule Forster resonance energy transfer (smFRET), 82
83

Index

Single-nucleotide polymorphism animal models, 193 SINV. See Sindbis virus (SINV) siRNA. See Small interfering RNA (siRNA) SISPA. See Sequence-independent single primer amplification (SISPA) SIT. See Sterile insect technique (SIT) SIV. See Simian immunodeficiency virus (SIV) Slc9a10. See Sperm-specific NHE (Slc9a10) Small interfering RNA (siRNA), 166, 195
196 Small ruminants, genetic markers in meat and milk production, 116
117 reproductive traits, 117 wool production, 117 Small ruminants, new viruses in, 47 Smallpox vaccine, 475 SMART. See Signal-mediated amplification of RNA technology (SMART) smFRET. See Single-molecule Forster resonance energy transfer (smFRET) SMRT sequencing. See Single molecule real-time sequencing (SMRT sequencing) SNP. See Single nucleotide polymorphism (SNP) SNVs. See Simple nucleotide variations (SNVs) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 388 SOLiD. See Sequencing by oligonucleotide ligation and detection (SOLiD) Somatic cell hybrid of ovine, 3
4 Somatic cell nuclear transfer (SCNT), 88, 139
141, 194 Sparus aurata (Single sea bream), 57
58 SPaV1. See Swine pasivirus 1 (SPaV1) SpCas9 high-fidelity variant number 1 (SpCas9HF1), 82 SpCas9-HF1. See SpCas9 high-fidelity variant number 1 (SpCas9-HF1) SpCas9. See Streptococcus pyogenes Cas9 (SpCas9) spCas9. See Type II CRISPR/Cas9 system from Streptococcus pyogenes (spCas9) Specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), 91 Specific pathogen-free animals (SPF animals), 190
192 production methodology, 190
191 in research, 191
192 SpectroSensTM optical microchip sensors, 293
294 Sperm fertilizing, 114 Sperm-assisted genome editing, 230 Sperm-specific NHE (Slc9a10), 114
115 Spermatozoa, 230

SPF animals. See Specific pathogen-free animals (SPF animals) SPIA. See Single primer isothermal amplification (SPIA) Spontaneous models, 187 Spring viraemia of carp (SVC), 313 SPV. See Sheep pox virus (SPV) SRCR. See Scavenger receptor cysteine-rich (SRCR) SRCR domain-5 (SRCR-5), 87
88 SRCR-5. See SRCR domain-5 (SRCR-5) SRCR5. See Scavenger receptor cysteine-rich domain 5 (SRCR5) SRS. See Salmonid Rickettsial Syndrome (SRS) SRY. See Sex determination region of Y chromosome (SRY) SRY gene in nonpseudoautosomal region of X chromosome (XSRY), 86
87 Staphylococcus aureus, 92, 226, 301, 332, 423
424 Sterile insect technique (SIT), 169 Sterility, 263 Strand displacement amplification (SDA), 291
292 Strand invasion-based amplification (SIBA), 291
292 Streptococcosis, 312
313 Streptococcus pyogenes Cas9 (SpCas9), 79
80, 255
256 Streptococcus spp., 312
313 S. agalactiae, 312
313, 413, 423
424 S. iniae, 312
313, 343
344, 412 S. phocae, 348
350 S. pneumoniae, 423
424 S. pyogenes, 332 S. thermophilus, 174
175, 255 S. uberis, 341 Streptomycin, 196
197 Streptozotocin-induced destruction of pancreatic beta-cells, 184
185 Stress-induced models, 186 Stringency, 329 Structural bioinformatics, 383 Structural vaccinology, 421
422 Structural variations (SVs), 384 Structural virology, 435 Structure determination techniques cryo-EM, 438
439 NMR, 438 X-ray crystallography, 436
438 Structure-assisted antiviral discovery for animal viral diseases animal viruses and viral diseases, 441
460

527

528

Index

Structure-assisted antiviral discovery for animal viral diseases (Continued) computational structure prediction and drug design, 439
440 structure determination techniques, 436
439 viral drug and vaccine targets, 435
436 Structure-based antivirals against CoV, 451 Structure-based drug development against FMDV, 443
445 Subunit vaccine, 473 Super muscular dogs, 155
156, 156f Superfamily 1 (SF1), 453
454 Surface-enhanced laser desorption
ionization time-of-flight MS, 394 Surrogate eggshells, 229 SVC. See Spring viraemia of carp (SVC) SVs. See Structural variations (SVs) Swine genetic markers meat quality traits, 117
118 reproductive traits, 118 influenza, 486
487 Swine pasivirus 1 (SPaV1), 45 SYBR Green I, 326, 330 Synthetic biology, 300 lethal interactions, 90 peptides, 290 RNA vaccines, 421 sgRNA, 137
139 vaccines, 423 Systems biology, 383

T T-cell receptor constant (TRAC), 89 T7 endonuclease I (T7E1), 235
236 TALE. See Transcription activator-like effectors (TALE) TALENs. See Transcriptional activator-like effector nucleases (TALENs) Tanks, 274 TaqMan Probe, 326
327 TaqMan real-time RT-PCR assay, 291 Targeted genome capture (TGC), 54 Targeted genome modification by HR, 170
172 nuclease-based genome-editing technologies, 171f Targeted nuclease-based mutagenesis, 177 Targeting protein
protein interactions, 436 Targeting RNA-protein interactions, 436 Targets nucleic acid base-pairing, 84
85 Taura syndrome Virus (TSV), 315, 319
320 Taurine genome, 4
5

TB. See Tuberculosis (TB) TCC. See Transitional cell carcinoma (TCC) TCGA. See The Cancer Genome Atlas (TCGA) Tenacibaculum maritinum, 312
313 Tetrahymena, 312 TF. See Transcription factor (TF) TGC. See Targeted genome capture (TGC) The Cancer Genome Atlas (TCGA), 386, 387t Theileria annulata, 489 Theileria lestoquardi, 489 Theileria parva, 489 Theileriosis, 489 Third-generation sequencing, 36
37 bioinformatics, 37
39 nanopore sequencing, 37 selection of novel animal viruses, 40t SMRT sequencing, 36 Three-dimensional structure (3D structure), 439 Thunnus obesus, 272
273 Thymidine kinase (tk), 485 Tick-borne infectious diseases, 302 Tilapia. See Oreochromis niloticus (Tilapia) Tilapia lake virus (TiLV), 57
58, 314
315 TiLV. See Tilapia lake virus (TiLV) Time-specific induction of mutations, 198 Tissue inhibitor of metalloproteinase (TIMP2), 114
115 tk. See Thymidine kinase (tk) TLR. See Toll-like receptor (TLR) TMA. See Transcription-mediated amplification (TMA) Togaviridae, 344
345, 452
453 Toll-like receptor (TLR), 427 TLR4, 426 Tombusviridae, 47 tonB (periplasmic energy transducer), 342
343 Totiviridae, 49
50 Totivirus, 57 Touchdown PCR, 322
323 Toxoids, 472
473 Toxoplasma gondii, 302 TRAC. See T-cell receptor constant (TRAC) tracrRNA. See Transactivating CRISPR RNA (tracrRNA) Trade Related Aspects of Intellectual Property Rights (TRIPS), 295 Transactivating CRISPR RNA (tracrRNA), 175
176, 256 Transcription activator-like effectors (TALE), 151
152, 173, 224
225, 235
237, 253 Transcription factor (TF), 172 Transcription-mediated amplification (TMA), 291
292

Index

Transcriptional activator-like effector nucleases (TALENs), 75, 77
78, 84
85, 131
132, 151
153, 169
170, 173
174, 251, 253
254, 259
260 Transcriptomics, 385
386, 387t, 395
396 studies in canine cancers, 392
394 Transformative technology, 87 Transgenesis, 211
212 and genome editing in chickens chicken genome manipulation, 225
226 delivery of transgenes, 229
232, 231f embryo culture, 226
229 precise genome editing, 234
239 primordial germ culture, 233 usage for poultry industry and environment protection, 212
213 Transgenic chicken/poultry birds poultry transgenesis and human nutrition, 213
215 and medicine, 215
216 transgenesis usage for poultry industry and environment protection, 212
213 Transgenic(s), 276
277 animal production, 211
212 birds, 226
227 disease-resistant chicken, 212
213 livestock production, 177 mammals, 227
229 models, 190, 198 of Alzheimer’s disease, 197 technology, 198, 211
213 Transitional cell carcinoma (TCC), 391 Translation initiation factor 2 (infB), 342
343 Translational significance of animal models, 196
197 Transmembrane hydrophobic domains, 442
443 Transposon-based vectors, 232 Transposons, 339
340 Trichodina, 312 Trichodinids, 312 Trichoplusia ni, 232 Tripartiella, 312 TRIPS. See Trade Related Aspects of Intellectual Property Rights (TRIPS) TSV. See Taura syndrome Virus (TSV) Tuberculosis (TB), 88, 478
479 Tumorigenesis, 90, 365
366 Tunesian sheep pestiviruses strains, 47 Turkey, herpesvirus of, 368 Turkey stool-associated circular virus, 48 Turkeys, novel viruses in, 48 2-dimensional electrophoresis (2-DE), 381 Type I CRISPR-Cas9 system, 256
257 Type II CRISPR/Cas9 system

for genome editing, 79
80, 79f Cas9 activity, 80 multiple gene editing, 80 from Streptococcus pyogenes (spCas9), 175
176 Type III CRISPR-Cas systems, 256
257

U Ubiquinolcytochrome-c reductase complex core protein 2 (UQCRC2), 114
115 UMD2. See University of Maryland (UMD2) UMD3 bovine genome assembly, 7
8 United States Department of Agriculture (USDA), 18 United States Department of AgricultureAgricultural Research Service (USDAARS), 6
7 University of Maryland (UMD2), 5 30 -Untranslated region (30 -UTR), 441 50 -Untranslated region (50 -UTR), 441 UQCRC2. See Ubiquinolcytochrome-c reductase complex core protein 2 (UQCRC2) Uronema, 312 USDA. See United States Department of Agriculture (USDA) USDA-ARS. See United States Department of Agriculture-Agricultural Research Service (USDA-ARS)

V Vaccination, 277, 343
344, 361, 407, 443, 471 Vaccines, 196
197, 277
278, 421, 470
471 adverse effect, 495 based on naked DNA, 412
413 combined vaccination, 490 conventional vaccines, 472
473 development technologies, 422f, 425t in veterinary vaccinology, 475 diversity, 475
490 bacterial diseases, 475
480 viral diseases, 480
490 for fish diseases, 323t, 343
345 genetically-engineered vaccine, 473
475 and “one health”, 470
471 poultry vaccines, 491
494 and structure-based drug design, 459
460 targets, 435
436 types, 472
475 vectors used for vaccine delivery adenovirus, 364
365 CMV vectors, 366 lentivirus vectors, 365
366

529

530

Index

Vaccines (Continued) poxvirus, 363
364 retrovirus vectors, 365 technologies, 429 Vaccinology, 421 structural vaccinology, 421
422 reverse vaccinology, 413, 421, 423
424, 425t veterinary vaccinology, 471, 475, 476t Variable number tandem repeat-PCR (VNTRPCR), 339
340 Vasa, 235
236 VDAC2. See Voltage-dependent anion channel 2 (VDAC2) Vector control, 91
92 Vector technology, 411
412 Vectored vaccines, 474 herpesvirus of turkey, 368 multivalent vector vaccines, 363f recombinant vector vaccine production process, 362f SeV vectors, 366
367 vectors for poultry vaccines, 367
368 vectors used for vaccine delivery, 363
367 Vectored veterinary vaccines, 369
370, 370t challenges in, 371 Vectored viral vaccines, 363 VEEV. See Venezuelan equine encephalitis virus (VEEV) Venezuelan equine encephalitis virus (VEEV), 452
453 VER. See Viral encephalopathy and retinopathy (VER) Verotoxigenic E. coli (VTEC), 480 Vesicular stomatitis virus (VSV), 366 Veterinary vaccinology, 471, 475, 476t VHS. See Viral haemorrhagic septicaemia (VHS) VHSV. See Viral haemorrhagic septicaemia virus (VHSV) Vibrio spp., 312
313, 348 V. anguillarum, 312
313, 340, 343
344, 412 V. cholerae, 332 V. harveyi, 312
313, 348
350 V. parahaemolyticus, 312
313, 332, 348
350 V. penaeicida, 322
323 V. salmonicida, 312
313, 343
344 V. vulnificus, 312
313, 332 Vibriosis, 312
313 Vigaviridae, 49
50 Viral Assembly Pipeline (VrAP), 37
39 Viral diseases, 313, 315 alphaviruses, 452
456 avian influenza virus, 458 bluetongue, 482
483 bovine viral diarrhea, 484
485

classical swine fever, 483 coccidiosis, 489
490 CoV, 450
451 FMDV, 441
445 foot and mouth disease, 480
481 HSV, 446
450 infectious bovine rhinotracheitis, 485 influenza, 486
487 JEV, 483
484 paramyxovirus, 456
457 parasitic bronchitis, 490 parasitic vaccines, 488
489 pestivirus, 458
460 PPRV, 482 rabies, 481
482 rotavirus gastroenteritis, 488 sheep pox and goat pox, 483 theileriosis, 489 winter dysentery, 487
488 Viral drug targets, 435
436, 437f Viral encephalopathy and retinopathy (VER), 313 Viral haemorrhagic septicaemia (VHS), 313 Viral haemorrhagic septicaemia virus (VHSV), 319
320, 408
411 Viral metagenomics, 29 and discovery of new viruses in livestock, 45
49 in pets, 49
54 practical aspects, 39
44 technical aspects, 30 workflow for, 38f Viral nervous necrosis virus (VNNV), 314
315, 319
320 Viral pathogens like cyprinid herpesvius-2, 314
315 Viral surface proteins, 436 Viral target proteins for drug development, 454
456 Viral vectors, 137, 230
232, 259
260, 361 Virome characterization, 55
56 Virosomes, 426 virosomes-based vaccine, 426 Virus enrichment, 30
31 Virus-like particles (VLPs), 411, 473
474 vaccines, 473
474 Virus-specific enzymes, 435 VLPs. See Virus-like particles (VLPs) VNNV. See Viral nervous necrosis virus (VNNV) VNTR-PCR. See Variable number tandem repeatPCR (VNTR-PCR) Voltage-dependent anion channel 2 (VDAC2), 114
115 VP6 protein, 290 VrAP. See Viral Assembly Pipeline (VrAP)

Index

VSV. See Vesicular stomatitis virus (VSV) VTEC. See Verotoxigenic E. coli (VTEC)

W Watson-Crick base-pairing interaction, 79 Wels catfish. See Silurus glanis (Wels catfish) West Nile virus (WNV), 369 Western blotting, 319
320 WGS. See Whole-genome sequencing (WGS) White Leghorn chickens (WL chickens), 235
236 White muscle disease (WMD), 315 White spot disease (WSD), 315 White spot syndrome virus (WSSV), 319
320 WHO. See World Health Organization (WHO) Whole genome amplification methods, 31 Whole-genome sequencing (WGS), 381 Winter dysentery, 487
488 WL chickens. See White Leghorn chickens (WL chickens) WMD. See White muscle disease (WMD) WNV. See West Nile virus (WNV) Wool production, 117 Woolly mammoths. See Mammuthus primigenius (Woolly mammoths) World Health Organization (WHO), 470
471 World Organization for Animal Health (OIE), 314
315, 470
471 WSD. See White spot disease (WSD) WSSV. See White spot syndrome virus (WSSV)

X X-linked severe combined immunodeficiency (SCID-X1), 365 X-ray crystallography, 163
164, 436
438, 448f, 451 Xanthomonas, 253 Xanthomonas genus, 173 Xanthomonas oryzae, 154 XSRY. See SRY gene in nonpseudoautosomal region of X chromosome (XSRY)

Y Yellow head virus (YHV), 315, 319
320 Yellow-head disease (YHD), 315 Yersinia enterocolitica, 332 Yersinia ruckeri, 312
313, 325, 340, 343
344

Z Zero-mode waveguides (ZMW), 36 Zika virus, 183 Zinc finger (ZF), 172 Zinc finger nucleases (ZFNs), 75, 77, 84
85, 131
132, 151
152, 169
170, 172
173, 193
194, 224
225, 234
235, 250f, 251
253, 252f ZMW. See Zero-mode waveguides (ZMW) Zoonotic diseases, 211
212 Zygotes, 155
156

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