Anti-parasitic Drug Resistance in Veterinary Practice 1800622783, 9781800622784

Table of contents :
Cover
Dedication
Antiparasitic Drug Resistance in Veterinary Practice
Copyright
Contents
About the Editors
Contributors
About the Book
Preface
1 Physiological Basis of Parasitism
Abstract
1.1 Introduction
1.2 Classification of Parasites
1.3 Ecology and Distribution of Parasites
1.3.1 Host specificity
1.3.2 Transmission
1.3.3 Environmental factors
1.3.4 Human impact
1.3.5 Diversity of parasites
1.3.6 Impact of environment
1.4 Physiological Features
1.4.1 Temperature tolerance
1.4.2 Anaerobic metabolism
1.5 Nutritional Requirement of Parasites
1.6 Establishment and Growth of Parasites
1.6.1 Genetics of parasites
1.6.2 Neuromuscular physiology of parasite
1.6.3 Invasion
1.6.4 Nutrient acquisition
1.6.5 Reproduction
1.7 Hormonal Influence on Parasite Development
1.8 Host–Parasite Interaction
1.9 Stress Tolerance
1.10 Immune Evasion and the Host Immune System
1.11 Host–Parasite Adjustment
1.12 Role of Parasitic Physiology in the Development of Drug Resistance
1.13 Future Directions for Research on the Physiological Basis of Parasitism
1.14 Conclusions
References
2 Antiprotozoal Resistance
Abstract
2.1 Introduction
2.2 Drug Resistance in Babesiosis
2.2.1 Diminazene aceturate resistance
2.2.2 Imidocarb dipropionate resistance
2.2.3 Atovaquone–azithromycin resistance
2.2.4 Buparvaquone and azithromycin resistance
2.3 Drug Resistance in Theileriosis
2.4 Drug Resistance in Trypanosomiasis
2.4.1 Diminazine aceturate resistance
2.4.2 Phenanthridine resistance
2.4.3 Quinapyramine resistance
2.4.4 Melarsomine dihydrochloride resistance
2.5 Drug Resistance in Coccidiosis
2.5.1 Anticoccidial drugs resistance
2.6 Conclusion
References
3 Anthelmintic Resistance
Abstract
3.1 Introduction
3.2 Global Parasitic Challenges and Strategies
3.3 Anthelmintics and their Mode of Action
3.3.1 Benzimidazoles
3.3.2 Levamisole/imidazothiazole
3.3.3 Macrocyclic lactones
3.3.4 Derivatives of amino acetonitrile
3.3.5 Piperazine
3.4 Factors Involved in the Development of Anthelmintic Resistance
3.4.1 Mechanism of development of anthelmintic resistance
Single nucleotide polymorphisms (SNPs)
Multidrug resistance (MDR)
Antioxidant enzymes
3.4.2 Frequency of anthelmintic use
3.4.3 Dose rates and routes of anthelmintic administration
3.4.4 Targeting and mass treatment
3.4.5 Single drug regimens
3.5 Detection and Monitoring of Anthelmintic Resistance
3.6 Strategies for Anthelmintic Resistance Management
3.6.1 Integrated approaches to AR management
3.6.2 Preventing AR development in farm animals
3.6.3 Synergistic approach to AR prevention
3.7 Alternative Strategies to Manage AR
3.7.1 Genetic improvement
3.7.2 Nutrition
3.7.3 Pasture management
3.7.4 Nematode-trapping fungi
3.7.5 Antiparasitic vaccines
3.7.6 Botanical dewormers
3.8 Conclusions and Future Trends
References
4 Insecticide Resistance
Abstract
4.1 Introduction
4.2 Classification and Mode of Action of Insecticides
4.2.1 Classification of insecticides
4.2.2 Modes of actions of insecticides
4.3 Genomics and Genetics of Insecticidal Resistance
4.3.1 Gene expression target sites
Ligand-gated ion channels
Voltage-gated ion channels
Acetylcholinesterase
4.3.2 Origin and spread of resistance-associated mutations
4.3.3 Types of mutations associated with the insecticidal resistance
4.4 Quantification and Impacts of Insecticide Resistance
4.4.1 Cross resistance
4.4.2 Genetic resistance
Mosquitoes
Bed bugs
4.4.3 Multiple resistance
4.4.4 Selection pressure
4.5 Insecticide Resistance against Different Classes of Arthropods
4.5.1 Phthiraptera
4.5.2 Siphonaptera
4.5.3 Hemiptera
4.5.4 Diptera
4.6 Mechanisms of Insecticidal Resistance
4.6.1 Metabolic insecticidal resistance
Esterase
Cytochrome P450 monooxygenases
Glutathione S transferase
4.6.2 Target site resistance
Knockdown resistance mechanism
Modified acetylcholinesterase
Insensitive gamma amino butyric acid (GABA) receptor
4.7 Insecticide Resistance Detection Methods
4.7.1 Phenotypic assays
4.7.2 Genotypic assay
4.8 Methods for Overcoming Insecticide Resistance
4.8.1 CRISPR-Cas gene technology
4.8.2 De-potentiating agents
4.8.3 Capacity strengthening on WHO guidelines
4.9 Role of the Environmental Protection Agency and the Insecticide Resistance Action Committee
4.10 Insecticide Resistance Management Strategies
References
5 Navigating Acaricidal Resistance through Implications in Veterinary Practice
Abstract
5.1 Introduction
5.2 Resistance Types
5.2.1 Acquired resistance
5.2.2 Multiple resistance
5.2.3 Cross resistance
5.3 Possible Factors Playing a Role in Resistance Development
5.3.1 Genetic factors
5.3.2 Operational factors
5.3.3 Biological factors
5.4 Acaricidal Resistance Mechanisms
5.4.1 Organochlorine resistance mechanism
5.4.2 Organophosphate and carbamate resistance mechanism
5.4.3 Amitraz resistance mechanism
5.4.4 Pyrethroid/pyrethrin resistance mechanism
5.4.5 Mechanism of macrocyclic lactone resistance
5.5 Current Status of Acaricidal Resistance
5.6 Alternative Control Methods to Overcome Acaricidal Resistance In Ticks
5.6.1 Phytotherapy
5.6.2 Biological control
5.6.3 Nanoparticles
5.6.4 Genetic and immunological control
5.7 Conclusions
References
6 Prevalence of Antiparasitic Drug Resistance in Various Areas of the World
Abstract
6.1 Introduction
6.2 Mechanisms of Antiparasitic Drug Resistance
6.3 Global Surveillance of Drug Resistance
6.4 Influencing Factors on Drug Resistance in Parasites
6.4.1 Frequency of treatment
6.4.2 Targeting and timing of mass treatment
6.4.3 Anthelmintic dose rate
6.4.4 Genetic factors
6.5 Methodologies for Assessing Drug Resistance
6.6 Prevalence of Drug Resistance in Specific Geographic Areas
6.7 Comparative Analysis of
6.8 Emerging Trends in Antiparasitic Resistance
6.9 Impact on Parasite Control Programmes
6.10 Challenges in Combatting Drug Resistance
6.11 Strategies for Prevention and Control
6.11.1 Accurate and effective parasiticide treatment
6.11.2 Co-administration of antiparasitic drugs
6.11.3 Refugia (resistance nests)
6.11.4 Integrated parasite management
6.11.5 Vaccine development (immunogenicity exploration)
6.12 Future Prospects and Research Directions
6.13 Conclusions
References
7 Molecular Methods for Detecting Antiparasitic Resistance
Abstract
7.1 Introduction
7.2 Molecular Methods for the Detection of Resistance
7.2.1 Polymerase chain reaction
7.2.2 Sequencing techniques
7.2.3 Genotyping assays
7.2.4 Loop-mediated isothermal amplification
7.2.5 Microarray technology
7.2.6 CRISPR-Cas9 technology
7.2.7 Miscellaneous molecular techniques
7.3 Successes in the Detection of Antiparasitic Resistance through Molecular Approaches
7.4 Limitations in the Molecular Detection of Antiparasitic Resistance
7.4.1 Challenges in sample collection
7.4.2 Variability in resistance mechanisms
7.4.3 Cost and accessibility of molecular techniques
7.4.4 Emerging technologies and standardization issues
7.5 Future Prospects for the Molecular Detection of Antiparasitic Resistance
7.5.1 Advancements in high-throughput technologies
7.5.2 Integration of artificial intelligence in data analysis
7.5.3 Personalized medicine approaches
7.5.4 Global surveillance programmes
7.5.5 Overcoming current limitations
7.6 Conclusions
References
8 Phenotypic Methods for Determining Antiparasitic Resistance In Vitro and In Vivo
Abstract
8.1 Introduction
8.2 In Vivo Phenotypic Methods
8.2.1 Faecal egg count reduction test
8.2.2 Drench-and-move assay
8.2.3 Resistance refugia monitoring
8.3 In Vitro Phenotypic Methods
8.3.1 In vitro assays for detection of anthelmintic resistance
Egg hatch assay
Motility assays
Feeding inhibition assay
Tubulin binding assay
Adult development assay
Larval development assay
8.3.2 In vitro assays for detection of acaricidal resistance
Larval packet test
Adult immersion test
Adult contact test
Detoxification enzyme assays
Membrane feeding assay
Histopathological examination
8.3.3 In vitro assays for detection of antiprotozoal resistance
Counting intracellular amastigotes and promastigotes: direct approach
Acid-phosphatase (APTase) assay
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
Resazuring (Alamar-Blue dye) assay
8.4 Challenges and Future Directions
8.4.1 Chemical library screening through high-throughput screening
8.4.2 General approach
8.4.3 Drug-repurposing approach
8.4.4 Enzymatic approach
8.5 Conclusions
References
9 Role of Stewardship in Mitigating Antiparasitic Resistance
Abstract
9.1 Introduction
9.2 Stewardship
9.2.1 Ensuring appropriate drug use and dosage
9.2.2 Minimize the use of unnecessary drugs
9.2.3 Monitoring drug efficacy
9.3 Development of Antiparasitic Resistance
9.4 Strategies for Mitigating Antiparasitic Resistance
9.4.1 Drug combination therapy
9.4.2 Drug rotation therapy
9.4.3 Vaccine development in antiparasitic resistance
9.5 A Comprehensive Approach to Stewardship
9.6 Sustainable Practices in Modern Veterinary Care
9.7 Conclusions
References
Index
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Antiparasitic Drug Resistance in Veterinary Practice

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I, Dr Hafiz Muhammad Rizwan, am honored to present this book titled Antiparasitic Drug Resistance in Veterinary Practice. With heartfelt reverence and deep respect, I dedicate this book to my late father, Mr Mashooq Ali. His unwavering support, boundless wisdom, and relentless encouragement have been the guiding force behind my academic and professional journey. Though he is no longer with us, his memory continues to inspire and motivate me in every endeavor. May Allah grant my father the highest place in Jannah and shower his soul with eternal peace and blessings. His legacy of kindness, integrity, and dedication to family and community remains a beacon of light for all who knew him. I pray that his soul finds comfort and that he is rewarded abundantly for all the goodness he brought into this world. Ameen.

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Antiparasitic Drug Resistance in Veterinary Practice

Edited by

Hafiz Muhammad Rizwan Department of Pathobiology (Parasitology Section) University of Veterinary and Animal Sciences, Lahore (Narowal Campus), Pakistan

Muhammad Ahsan Naeem Department of Basic Sciences (Pharmacology Section) University of Veterinary and Animal Sciences, Lahore (Narowal Campus), Pakistan

Muhammad Younus Department of Pathobiology (Pathology Section) University of Veterinary and Animal Sciences, Lahore, Pakistan

Muhammad Sohail Sajid Department of Parasitology, Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan

Xi Chen College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China

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CABI is a trading name of CAB International CABI Nosworthy Way Wallingford Oxfordshire OX10 8DE UK Tel: +44 (0)1491 832111 E-mail: [email protected] Website: www.cabi.org

CABI 200 Portland Street Boston MA 02114 USA Tel: +1 (617)682-9015 E-mail: [email protected]

© CAB International 2024. All rights reserved. No part of this publication may be reproduced in any form or by any means, electronically, mechanically, by photocopying, recording or otherwise, without the prior permission of the copyright owners. The views expressed in this publication are those of the author(s) and do not necessarily represent those of, and should not be attributed to, CAB International (CABI). Any images, figures and tables not otherwise attributed are the author(s)’ own. References to internet websites (URLs) were accurate at the time of writing. CAB International and, where different, the copyright owner shall not be liable for technical or other errors or omissions contained herein. The information is supplied without obligation and on the understanding that any person who acts upon it, or otherwise changes their position in reliance thereon, does so entirely at their own risk. Information supplied is neither intended nor implied to be a substitute for professional advice. The reader/user accepts all risks and responsibility for losses, damages, costs and other consequences resulting directly or indirectly from using this information. CABI’s Terms and Conditions, including its full disclaimer, may be found at https://www.cabi.org/terms-and-conditions/. A catalogue record for this book is available from the British Library, London, UK. ISBN-13: 9781800622784 (hardback) 9781800622791 (ePDF) 9781800622807 (ePub) DOI: 10.1079/9781800622807.0000 Commissioning Editor: Alexandra Lainsbury Editorial Assistant: Helen Elliott Production Editor: Rosie Hayden Typeset by Straive, Pondicherry, India Printed and bound in the UK by CPI Group (UK) Ltd, Croydon, CR0 4YY

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Contents

About the Editors

vii

Contributors

ix

About the Book

xiii

Preface

xv

1

Physiological Basis of Parasitism Mohsin Raza, Muhammad Muneeb Rauf, Haroon Rashid, Fakhar un Nisa, Abdullah Arif Saeed and Hizqeel Ahmed Muzaffar

2

Antiprotozoal Resistance Haider Abbas, Muhammad Younus, Zahid Fareed, Mian Mubashar Saleem, Malcolm K. Jones, HazratUllah Raheem, Adil Ijaz and Muhammad Nadeem Saleem

19

3

Anthelmintic Resistance Aayesha Riaz, Faiza Bano, Manuela Marescotti, Evelyn Saba and Zahid Manzoor

41

4

Insecticide Resistance Shumaila Naz, Rida Fatima Saeed, Mahvish Rajput, Sumra Wajid Abbasi and Ian Daniel

58

5 Navigating Acaricidal Resistance through Implications in Veterinary Practice Mahvish Maqbool, Muhammad Sohail Sajid, Hafz Muhammad Rizwan, Muhammad Younus, Kashif Kamran, Muhammad Zeeshan and Muhammad Usman

78

6

Prevalence of Antiparasitic Drug Resistance in Various Areas of the World Zubaria Shahid Amin, Nadia Nazish, Qaiser Akram, Muhammad Rizwan Saeed, Tooba Abbas, Waqas Ahmad and Aiman Maqsood

94

7

Molecular Methods for Detecting Antiparasitic Resistance Muhammad Sohail Sajid, Sadaf Faiz, Muhammad Qasim, Ibadullah Jan, Sibtain Ahmad, Dalia Fouad and Farid Shokry Ataya

1

110

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vi

8

9

Contents

Phenotypic Methods for Determining Antiparasitic Resistance In Vitro and In Vivo HazratUllah Raheemi, Zobia Afsheen, Muhammad Ahsan Naeem, Shamshad Fareed, Xi Chen, Rohit Tyagi, Muhammad Umar Farid and Adeel Ahmad Role of Stewardship in Mitigating Antiparasitic Resistance Amir Munir, Hafz Muhammad Rizwan, Urfa Bin Tahir, Ibadullah Jan, Muhammad Younus, Sadia Ghazanfar and Muhammad Abdullah Malik

Index

124

139

155

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About the Editors

Dr Hafiz Muhammad Rizwan is an accomplished researcher and academician in the field of parasitology. He has made significant contributions to the understanding and management of parasitic diseases through his research and publications. Dr Rizwan’s expertise lies in the areas of molecular parasitology, host–parasite interactions, omics-based approaches for diagnosing and managing parasitic diseases, and parasitic control strategies. Dr Rizwan is currently affiliated with the University of Veterinary and Animal Sciences (UVAS), Narowal Campus, Pakistan, where he serves as an Assistant Professor of Parasitology in the Department of Pathobiology. He is actively involved in teaching and mentoring students, sharing his knowledge and passion for the subject. With a strong academic background, Dr Rizwan holds a PhD in Parasitology. He has published numerous research articles in reputable journals, contributing to the scientific literature on parasitic infections. Dr Rizwan’s dedication and commitment to his field have earned him recognition and respect from his peers. He continues to make valuable contributions to the field of parasitology through his research, teaching and academic endeavours. Dr Muhammad Ahsan Naeem, born in Faisalabad, Pakistan in 1988, excelled academically, earning a gold medal in Doctor of Veterinary Medicine (DVM) from the University of Agriculture, Faisalabad (UAF), in 2012. He began his teaching career at UAF in 2012 and later earned an MPhil Pharmacology in 2015. Dr Ahsan worked in Dubai as Veterinary Doctor of diagnostics before receiving the Chinese Government Scholarship for his PhD at Huazhong Agricultural University, focusing on Mycobacterium tuberculosis. Joining the University of Veterinary and Animal Sciences, Lahore, Pakistan (Narowal Campus) after PhD, he actively contributes to teaching and research. His achievements include international publications, the Bill and Melinda Gates Foundation Professional Development Award 2022, II OIC youth congress award, Kazan, Russia, 2023, and an ISPPD-13 meeting early career research award in 2024. Meritorious Prof. Dr Muhammad Younus is a distinguished academician currently serving as the Vice Chancellor of the University of Veterinary and Animal Sciences, Lahore, Pakistan. He holds a Post Doctorate from the University of Minnesota, USA, and a Doctorate in Pathology and Public Health from the University of Veterinary and Animal Sciences, Lahore, Pakistan. Dr Younus has garnered numerous accolades throughout his illustrious career, including at least six academic merit scholarships and the prestigious Presidential Civil Award Tamgha-e-Imtiaz in 2023 by the Government of Pakistan. His exceptional leadership and academic contributions have been recognized internationally, with the Distinguished Leadership Award bestowed upon him by the President of the University of Minnesota, USA, in 2016. He has also received the Best University Teacher Award in vii Downloaded from https://cabidigitallibrary.org by Ivanov Ivan, on 11/04/24. Subject to the CABI Digital Library Terms & Conditions, available at https://cabidigitallibrary.org/terms-and-conditions

viii

About the Editors

2014 from the Higher Education Commission (HEC) in Islamabad, and multiple Excellence Awards from the Pakistan Veterinary Medical Council (PVMC) and the Pakistan Veterinary Medical Association (PVMA). He has been instrumental in the establishment and development of new institutions such as CVAS Jhang and CVAS Narowal, for which he has received appreciation letters from esteemed organizations including the Vice Chancellor of UVAS, Lahore, and the Planning and Development Department, Government of Punjab. Dr Younus is a prolific author with 14 books, 32 book chapters and more than 270 research publications to his name. He has also successfully secured and executed 16 projects from various national and international funding agencies, totaling more than Rs8000.00 million. Additionally, he has mentored and supervised 60 MPhil and 18 PhD scholars, contributing significantly to the academic and research landscape in veterinary and animal sciences. Prof. Dr Muhammad Sohail Sajid achieved his early education from the Kamalia and Khanewal and Higher Secondary education from Govt. Emerson College, Multan, Punjab, Pakistan. He secured silver medal in his Doctor of Veterinary Medicine (DVM) degree from the University of Agriculture, Faisalabad (UAF), Pakistan in 2002. He started his professional career as a Lecturer in the Department of Parasitology, UAF, while continuing in an MSc (Hons) and a PhD in Parasitology. He served as Assistant Professor, Associate Professor, Chairman, and Member, Climate Change Chair of the US-Pakistan Center for Advanced Studies in Agriculture and Food Security established through the USAID in the UAF, Pakistan, and finally as Professor tenured. His thematic research area is molecular epidemiology and control of arthropods and arthropod-borne diseases of One Health significance. His competitive successes include: PhD fellowship sponsored by the Higher Education Commission (HEC), Islamabad, Pakistan, HEC-funded post-doc. fellowship from the University of Southern Mississippi (USM), Hattiesburg, MS, USA, USAID-funded project through the HEC Pak-US S&T Cooperation Program (Phase IV), and the Fulbright Post-Doc. fellowship at the UC Davis, USA. So far, he has secured 10 grants as PI/ Co-PI and mentored >50 MPhil. and 12 PhD graduate students as Chairman/ co-supervisor/member. The Ministry of Science and Technology, Islamabad, Pakistan has awarded him competitive Research Productivity Awards consecutively for 6 years (2010 to 2015). So far, he has published ~90 research papers in ISI indexed journals having IF of ~100, presented at various national and international conferences, one textbook, two chapters in an international book and 150 abstracts in proceedings. He has been serving as Section Editor of the UAF’s Official Journal PJAS (IF-0.785) for more than a decade and has edited a book published by CABI, Oxfordshire, UK. He is the co-author of the first guidelines of Institutional Animal Care and Use at the UAF. His hard work and commitment enabled him to achieve the top ranked academic position of the Tenured Professor of Parasitology in the Department of Parasitology, UAF, Pakistan. Dr Xi Chen is working as Associate Professor at the College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, P.R. China. He completed his Post-Doctorate from Huazhong Agricultural University, Wuhan, China in 2019 and Doctorate in Veterinary Medicine from Huazhong Agricultural University, Wuhan, China in 2015. In 2013, he won a Chinese Government Scholarship for exchange student and was trained for a PhD at the College of Veterinary Medicine, University of Georgia, Athens, USA. During his doctoral study, he worked on the pathogenicity of CNS tuberculosis. Now he focuses on the interaction of host and Mycobacterium tuberculosis, the role of mycobacterial secretory proteins, and the immune escape of Mycobacterium tuberculosis. He has so far secured (completed/ ongoing) eight research grants from the National Natural Science Foundation of China as Principal investigator/Co-Principal investigator. He is the reviewer of six journals. He has several publications in peer-reviewed SCI journals, nine patents and a chapter in the book of Biosafety of Veterinary Medicine, and one national standard.

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Contributors

Abbas H., Department of Pathobiology (Parasitology Section), KBCMA College of Veterinary & Animal Sciences, 51600-Narowal, Sub-campus, UVAS Lahore, Pakistan. E-mail: haider.abbas@ uvas.edu.pk Abbas T., Department of Zoology, University of Narowal, 51600, Narowal, Pakistan. E-mail: tubaabas9@ gmail.com Abbasi S.W., Department of Biological Sciences, National University of Medical Sciences, Rawalpindi, 46000, Pakistan. E-mail: [email protected] Afsheen Z., Department of Health and Biological Sciences, Faculty of Life Sciences, Abasyn University, Peshawar, Pakistan. E-mail: [email protected] Ahmad A., Mobile Veterinary Dispensary, Vehari, Livestock and Dairy Development Department, Punjab, Pakistan. E-mail: [email protected] Ahmad S., Institute of Animal and Dairy Sciences, Faculty of Animal Husbandry, University of Agriculture, Faisalabad, Pakistan. and Key Lab of Agricultural Animal Genetics, Breeding and Reproduction Science, Huazhong Agricultural University, Wuhan-PR China. E-mail: [email protected] Ahmad W., Department of Clinical Sciences (Epidemiology Section), KBCMA College of Veterinary & Animal Sciences, 51600-Narowal, Sub-campus, UVAS, Lahore, Pakistan. E-mail: waqas. [email protected] Akram Q., Department of Pathobiology (Microbiology Section), KBCMA College of Veterinary & Animal Sciences, 51600-Narowal, Sub-campus, UVAS, Lahore, Pakistan. E-mail: qaiser.akram@ uvas.edu.pk Amin Z.S., Animal Sciences Institute, Livestock & Dairy Development Department, Quetta, Baluchistan, Pakistan. E-mail: [email protected] Ataya F.S., Department of Biochemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia. E-mail: [email protected] Bano F., Department of Parasitology and Microbiology, Faculty of Veterinary and Animal Sciences, PMAS-Arid Agriculture University Rawalpindi, Pakistan. E-mail: [email protected] Chen X., College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. E-mail: [email protected] Daniel I., Department of Veterinary Pathobiology, School of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, Texas, USA. E-mail: [email protected] Faiz S., Department of Pathology, University of Agriculture, Faisalabad-38040, Pakistan. E-mail: [email protected] Fareed S., Faculty of Veterinary Sciences, KBCMA College of Veterinary and Animal Sciences, Narowal, Sub Campus UVAS, Lahore, Pakistan. ix Downloaded from https://cabidigitallibrary.org by Ivanov Ivan, on 11/04/24. Subject to the CABI Digital Library Terms & Conditions, available at https://cabidigitallibrary.org/terms-and-conditions

x

Contributors

Fareed Z., Foot and Mouth Disease Research Center, Lahore, Pakistan. E-mail: drzahidfareed11@ gmail.com Farid M.U., Department of Animal Sciences, KBCMA College of Veterinary and Animal Sciences, Narowal, Sub Campus UVAS, Lahore 54000, Pakistan. E-mail: [email protected] Fouad D., Department of Zoology, College of Science, King Saud University, Riyadh 11459, Saudi Arabia. E-mail: [email protected] Ghazanfar S., Department of Parasitology, Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan. E-mail: [email protected] Ijaz A., Department of Biomolecular Health Sciences, Division of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht, The Netherlands. E-mail: [email protected] Imran M., Department of Animal Sciences (Animal Nutrition), KBCMA College of Veterinary & Animal Sciences, 51600-Narowal, Sub-campus, UVAS Lahore, Pakistan. E-mail: imran.mughal@ uvas.edu.pk Jan I., College of Veterinary Sciences, The University of Agriculture Peshawar, 25130, Pakistan. E-mail: [email protected] Jones M.K., School of Veterinary Science, The University of Queensland, Gatton Queensland, 4343, Australia. E-mail: [email protected] Kamran K., Department of Zoology, University of Balochistan, Pakistan. Malik M.A., Department of Parasitology, Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan. E-mail: [email protected] Manzoor Z., Department of Parasitology and Microbiology, Faculty of Veterinary and Animal Sciences, PMAS-Arid Agriculture University Rawalpindi, Pakistan. E-mail: zahidmanzoor@uaar. edu.pk Maqbool M., Department of Parasitology, Faculty of Veterinary Sciences, University of Agriculture, Faisalabad, Pakistan and Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan. E-mail: [email protected] Maqsood A., Department of Zoology, University of Narowal, Narowal. E-mail: aimanmaqsood1@ gmail.com Marescotti M., University of Edinburgh Centre for Discovery Brain Sciences, Edinburgh, UK and Brain Communications Editorial Offce, University of Edinburgh, Edinburgh, UK. E-mail: m.marescotti@ ed.ac.uk Munir A., Tharb Camel Hospital, Doha, Qatar. E-mail: [email protected] Muzaffar H.A., Faculty of Veterinary Sciences, KBCMA College of Veterinary and Animal Sciences, Narowal, Sub Campus UVAS, Lahore, Pakistan. E-mail: [email protected] Naz S., Department of Biological Sciences, National University of Medical Sciences, Rawalpindi, 46000, Pakistan. E-mail: [email protected] Nazish N., Department of Zoology, University of Sialkot, Pakistan. E-mail: [email protected] Nisa F.U., Department of Animal Breeding and Genetics, Faculty of Animal Production and Technology, University of Veterinary and Animal Sciences, Lahore (Ravi Campus), Pakistan. E-mail: [email protected] Qasim M., Department of Parasitology, University of Agriculture, Faisalabad-38040, Pakistan. E-mail: [email protected] Raheemi H., Department of Health and Biological Sciences, Faculty of Life Sciences, Abasyn University, Peshawar, Pakistan. E-mail: [email protected] Rajput M., Department of Parasitology, University of Agriculture, Faisalabad, 38040, Pakistan. E-mail: [email protected] Rashid H., Department of Physiology, Faculty of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Pakistan. E-mail: [email protected] Rauf M.M., Department of Basic Sciences (Anatomy Section), KBCMA College of Veterinary and Animal Sciences, Narowal, Sub Campus UVAS, Lahore, Pakistan. E-mail: muneeb.rauf@uvas. edu.pk

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Contributors

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Raza M., Department of Basic Science (Physiology Section), KBCMA College of Veterinary and Animal Sciences, Narowal, Sub Campus UVAS, Lahore, Pakistan. E-mail: [email protected] Riaz A., Department of Parasitology and Microbiology, Faculty of Veterinary and Animal Sciences, PMAS-Arid Agriculture University Rawalpindi, Pakistan. E-mail: [email protected] Rizwan H.M., Department of Pathobiology (Parasitology Section), KBCMA College of Veterinary & Animal Sciences, 51600-Narowal, Sub-campus, UVAS, Lahore, Pakistan. E-mail: hm.rizwan@ uvas.edu.pk Saba E., Department of Biomedical Sciences, Faculty of Veterinary and Animal Sciences, PMAS-Arid Agriculture University Rawalpindi, Pakistan. E-mail: [email protected] Saeed A.A., Department of Physiology, University of Veterinary and Animal Sciences, Lahore, Pakistan. E-mail: [email protected] Saeed M.R., Department of Pathobiology (Microbiology Section), KBCMA College of Veterinary & Animal Sciences, 51600-Narowal, Sub-campus, UVAS, Lahore, Pakistan. E-mail: rizwan.saeed@ uvas.edu.pk Saeed R.F., Department of Biological Sciences, National University of Medical Sciences, Rawalpindi, 46000, Pakistan. E-mail: [email protected] Sajid M.S., Department of Parasitology, Faculty of Veterinary Sciences, University of Agriculture, Faisalabad, Pakistan. E-mail: [email protected] Saleem M.M., Department of Animal Sciences (Poultry Section), KBCMA College of Veterinary & Animal Sciences, 51600-Narowal, Sub-campus, UVAS Lahore, Pakistan. E-mail: mubashar.saleem@ uvas.edu.pk Saleem M.N., Department of Animal Sciences (Animal Breeding and Genetics), KBCMA College of Veterinary & Animal Sciences, 51600-Narowal, Sub-campus, UVAS Lahore, Pakistan. E-mail: [email protected] Tahir U.B., Molecular Parasitology and One Health Lab, Department of Parasitology, Faculty of Veterinary Science, University of Agriculture, Faisalabad, Punjab, Islamic Republic of Pakistan. E-mail: [email protected] Tyagi R., College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. E-mail: [email protected] Usman M., Section of Histology, Department of Basic Sciences, KBCMA College of Veterinary and Animal Science, Narowal, Sub-campus UVAS, Lahore, Pakistan. E-mail: [email protected] Younus M., Department of Pathobiology (Pathology Section), KBCMA College of Veterinary & Animal Sciences, 51600-Narowal, Sub-campus, UVAS Lahore, Pakistan. E-mail: younusrana@uvas. edu.pk Zeeshan M., Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan. E-mail: [email protected]

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About the Book

Antiparasitic Drug Resistance in Veterinary Practice explores the complicated dominion of combatting drug resistance in various parasitic organisms that afflict animals. Across nine comprehensive chapters, the book circumnavigates through critical topics indispensable for understanding and addressing this pressing issue. Beginning with an exploration of the physiological basis of parasitism, the book progresses to dissect specific forms of resistance such as antiprotozoal, anthelmintic, insecticidal and acaricidal resistance. Through meticulous examination, it sheds light on the prevalence of antiparasitic drug resistance across different regions worldwide, offering insights into the global scope of this challenge. Moreover, the book probes into leading-edge molecular diagnostic methods indispensable for detecting antiparasitic resistance, alongside in-vivo and in-vitro phenotypic methods for comprehensive evaluation. As stewardship and mitigation strategies take centre stage in the battle against drug resistance, the book concludes in a discussion on strategies to mitigate the development of resistance and ensure responsible drug usage in veterinary practice. By meticulously addressing these aspects, Antiparasitic Drug Resistance in Veterinary Practice serves as a vital resource for veterinarians, researchers and policy makers striving to combat drug resistance effectively. With a focus on understanding the underlying mechanisms, evaluating alternative therapies and formulating stringent policies, this book aims to catalyse multi-sectorial action towards accomplishing sustainable development goals (SDGs) in veterinary medicine while promoting animal health and welfare.

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Preface

As veterinarians and researchers in the field of veterinary medicine, we are intensely aware of the growing threat posed by drug resistance in parasitic organisms that distresses animals. The emergence and blowout of resistance to antiparasitic drugs present a polygonal challenge that demands our immediate attention and vigorous efforts to address effectively. In this book, Antiparasitic Drug Resistance in Veterinary Practice, we embark on a journey to discover the complex area of battling drug resistance in various parasitic organisms. Throughout the book, we shine a light on the prevalence of antiparasitic drug resistance across different regions of the world, offering acumens into the global scope of this challenge. We also examine stewardship and mitigation strategies that take centre stage in the battle against drug resistance. Our goal with this book is not only to deepen our understanding of the complexities of antiparasitic drug resistance but also to catalyse multi-sectoral action towards achieving justifiable development goals in veterinary medicine. We sincerely hope that this book serves as a spur for meaningful change in veterinary practice, ultimately contributing to the safeguarding of animal health and the wellbeing of our planet. With contributions from leading experts in the field, this book reconciles the molecular mechanisms underlying drug resistance, the epidemiology of resistant parasites, and the impacts of resistance on animal health and welfare. Furthermore, we explore innovative approaches to drug development and deployment, including combination therapies, rotation strategies, and the use of alternative treatments such as vaccines and biocontrol agents. By fostering a deeper understanding of the drivers and consequences of antiparasitic drug resistance, we aim to empower veterinarians, researchers, policy makers and other stakeholders to take collaborative action to mitigate this demanding global threat. Together, we can safeguard the efficacy of antiparasitic drugs, protect animal welfare, and preserve the health of our communal environment for future generations.

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1

Physiological Basis of Parasitism

Mohsin Raza1*, Muhammad Muneeb Rauf2, Haroon Rashid3, Fakhar un Nisa4, Abdullah Arif Saeed5 and Hizqeel Ahmed Muzaffar6 1 Department of Basic Science (Physiology Section), KBCMA College of Veterinary and Animal Sciences, Narowal, Sub-Campus UVAS, Lahore, Pakistan; 2 Department of Basic Sciences (Anatomy Section), KBCMA College of Veterinary and Animal Sciences, Narowal, Sub-Campus UVAS, Lahore, Pakistan; 3Department of Physiology, Faculty of Veterinary and Animal Sciences,The Islamia University of Bahawalpur, Pakistan; 4Department of Animal Breeding and Genetics, Faculty of Animal Production and Technology, University of Veterinary and Animal Sciences, Lahore (Ravi Campus), Pakistan; 5Department of Physiology, University of Veterinary and Animal Sciences, Lahore, Pakistan; 6Faculty of Veterinary Sciences, KBCMA College of Veterinary and Animal Sciences, Narowal, Sub-Campus UVAS, Lahore, Pakistan

Abstract The relationship between a parasite and its host depends upon biochemical, genetic and physiological processes. Both the hosts and the parasites play a role in these processes. With the passage of time, parasites have evolved to utilize the host’s machinery more effectively. Parasites can be classifed on the basis of their life cycles, modes of transmission and host specifcity. Host specifcity of parasites is determined by both the hosts and the parasites. Parasite distribution is affected by their patterns of transmission among the host’s populations. Animals and humans are carriers of parasites highly sensitive to environmental changes. The survival of parasites in the environment depends upon their physiological capabilities. The major factor for the abundance of the parasites is that they can adapt to changing environments. In order to control parasites, an understanding of their physiology and nutrient requirements is essential. Parasites also affect the hormonal system of the hosts, leading to the alteration of normal reproductive behaviour. To discern and manage drug resistance in parasites, it is essential to have a comprehensive understanding of parasite physiology.

1.1

Introduction

Parasitism is a captivating biological phenomenon that complexly intertwines the survival strategies of two distinct organisms: the parasite and its host. This relationship, characterized by one organism living on or within another, is marked by a dynamic interplay of physiological, genetic and biochemical mechanisms. The term

‘parasitism’ encapsulates a symbiotic bond wherein the parasite thrives at the expense of its host, introducing a complex web of interactions that shape the lives of both parties involved (Overstreet and Lotz, 2016). The host, in this biological drama, is akin to an island besieged by intruders, each with unique needs, specific food requirements, and varying preferences for locations to raise their progeny (Campbell et al., 1983).

*Corresponding author: [email protected] © CAB International 2024. Antiparasitic Drug Resistance in Veterinary Practice (eds H.M. Rizwan, M.A. Naeem, M. Younus, M.S. Sajid and X.Chen) DOI: 10.1079/9781800622807.0001

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The invaded host becomes a landscape of opportunities and challenges for the parasite, as it navigates the delicate balance between extracting sustenance and avoiding the potential defences of its host. Physiological adaptations are central to the success of parasitism (Fincher and Thornhill, 2012). Parasites evolve mechanisms that allow them to exploit the host’s resources efficiently. These adaptations range from specialized structures for attachment and nutrient absorption to the ability to evade the host’s immune responses (Danilova, 2006). The intricate dance between the parasite and its host unfolds on a molecular level, with genetic modifications paving the way for the parasite’s survival within the intricate environment of the host organism. Moreover, biochemical strategies employed by parasites underscore the multifaceted nature of this relationship. Parasites produce an arsenal of molecules that manipulate host physiology, ensuring a conducive environment for their survival. These biochemical tools may influence host behaviour, alter immune responses, or even modulate the host’s metabolism to meet the parasite’s specific needs (Douglas, 2018). Parasitism is not a one-size-fits-all concept, as the outcomes of this intricate relationship can vary widely. Some parasites cause harm to their hosts, leading to diseases and physiological disruptions. Others coexist more harmoniously, establishing a delicate equilibrium where the parasite extracts nutrients without causing severe harm. The spectrum of parasitic interactions ranges from parasitoids that eventually kill their hosts to mutualistic parasites that confer benefits to their hosts in exchange for resources (Leung and Poulin, 2008). Understanding parasitism is crucial not only for unravelling the complexities of ecological systems but also for addressing the health implications for humans and other organisms (Rigaud et al., 2010). Parasites play a significant role in the transmission of diseases, and a deeper comprehension of their biology is essential for devising effective strategies for disease prevention and control (Anonymous, 2019). Hence, parasitism stands as a testament to the intricate tapestry of life, where organisms intertwine in a dance of survival. The host becomes a battleground, an ecosystem shaped by the presence of intruders with diverse needs. Physiological adaptations, genetic intricacies and biochemical

manipulations define the saga of parasitism. The remarkable strategies employed by parasites unfold across multiple dimensions. These organisms thrive in a world that constantly challenges the delicate balance of life (Olano et al., 2011). Figure 1.1. shows the relationship between various ecological, anthropogenic and biological factors affecting interactions between the host and the parasites.

1.2

Classification of Parasites

Because of the diverse nature of parasites and their interactions with hosts, various criteria are employed in the classification process, including morphological features, life cycle characteristics, host specificity and genetic information (Roberts et al., 2009). The field of taxonomy plays a crucial role in organizing and categorizing parasites into hierarchical groups. The hierarchical classification system typically follows the structure of kingdom, phylum, class, order, family, genus and species. Each level of classification represents a specific rank based on shared characteristics and evolutionary relationships. It is important to note, however, that the classification of parasites is not always straightforward because some organisms may exhibit unique features or complex life cycles that challenge traditional taxonomic boundaries (Garcia, 2009). Parasites belong to various kingdoms, with the most prominent being Animalia, Plantae, Fungi and Protista. Within the kingdom Animalia, the phyla Platyhelminths (flatworms) and Nemathelminths (roundworms) include many parasitic species (Roberts et al., 2009). The phylum Platyhelminths encompasses flatworms, including important parasitic groups such as Trematoda (flukes) and Cestoda (tapeworms). Nemathelminths, another phylum, includes parasitic roundworms. Within the phylum Platyhelminthes, classes such as Trematoda and Cestoda further classify parasites on the basis of morphological and life-cycle characteristics (Collins, 2017). In Nematoda, classes such as Secernentea and Adenophorea include various parasitic species. Orders within each class represent additional subdivisions based on specific characteristics. For example, within the class Trematoda, orders such as Strigeida and Echinostomida include various

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Behaviour Population Ecological Predation

Diet

Climate

Migration

Host

Parasite

Sex Translocation Biological

Health

Anthropogenic Zoonosis Conservation

Age Fig. 1.1. The relationship between ecological, biological and environmental factors affecting host–parasite interactions. (Author’s own figure.)

fluke species. Further classification occurs at the family level, where groups of related genera are categorized (Olson et al., 2003). Families such as Schistosomatidae (blood flukes) and Taeniidae (tapeworms) exemplify this level of classification. At the genus and species levels, parasites are precisely identified (Roberts et al., 2009). Protists are single-celled eukaryotes that include parasitic protozoans. They share characteristics such as being heterotrophic, motile during certain life phases and dependent on a host for survival. Protozoans belong to diverse evolutionary groups classified into at least five eukaryotic kingdoms or supergroups: Harosa, Excavata, Amoebozoa, Archaeplastida and Ophistokonta. The first three supergroups encompass protozoan parasites affecting pets and agricultural animals. These organisms can significantly impact the health and mortality rates of domestic animals, leading to substantial financial losses in livestock production or causing concerns for pet owners. Additionally, some protozoans have zoonotic implications, posing public health risks (Schnittger and Florin-Christensen, 2018).

Arthropods, the largest phylum of animals on Earth, play crucial roles in medical and veterinary contexts in terrestrial environments. Some arthropods are vital in transmitting infections, posing threats to a wide range of vertebrates, including humans, companion animals and livestock. Terrestrial parasitic arthropods can be categorized into three groups: (i) trophically transmitted parasites (pentastomids); (ii) directly transmitted parasites (sucking lice, chewing lice, skin mites and itch mites); and (iii) micropredators. The micropredator category further distinguishes between short-term (blood-sucking or secretophagous flies, kissing bugs, bed bugs, ticks and fleas) and long-term (chigoe fleas and myiasiscausing larvae) micropredators (di-Giovanni et al., 2021). Parasites can also be classified based on their life cycles, distinguishing between direct life cycles (involving a single host) and indirect life cycles (involving multiple hosts). Additionally, host specificity is a crucial factor, with parasites categorized as host-specific or generalists based on the range of hosts they can infect (Hoberg and Brooks, 2008). Advancements in molecular

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biology and genetic sequencing have enhanced parasite classification by providing insights into evolutionary relationships. DNA analysis and phylogenetics contribute valuable data for refining and revising existing classifications (Olson and Tkach, 2005). Thus, the classification of parasites is a dynamic and evolving field, with taxonomists utilizing a combination of morphological, life cycle and genetic information to organize these diverse organisms into meaningful categories. As research continues, our understanding of parasite classification will probably become more refined and reflective of the intricate relationships between parasites and their hosts (Hoberg and Brooks, 2008). The general classification of parasites of veterinary and medical importance is shown in Fig. 1.2.

1.3

Ecology and Distribution of Parasites

Parasites have a wide range of ecological roles and can be found in virtually every environment on Earth. Some general features of the ecology and distribution of parasites are described here.

1.3.1 Host specificity Parasites are typically highly specialized to their host species or groups of closely related hosts. This can limit their distribution and may influence their evolution. Both the host and the parasites are involved in maintaining host specificity (Cacciò et al., 2018). Theories that determine the host specificity conclude that there are many factors involved, including physiology, biochemistry, ecology, evolution, genetics and molecular interactions of parasites, that may alone or in combination affect the host specificity of parasites (Wolinska and King, 2009). Despite the limited knowledge of the basis of host specificity, physiological factors that have a role in host specificity include microbe-associated molecular patterns, chemotactic sensors, transcriptional regulators and lipopolysaccharides (Jacques et al., 2016). Host genotypes that are resistant to just a subset of parasite genotypes and parasite genotypes that are infectious on a subset of host genotypes can be used to characterize the specificity of parasitic interactions (Little et al., 2006).

Amoeba e.g. Entamoeba histolytica Flagellates e.g. Trichomonas vaginalis Protozoa Sporozoa e.g. Toxoplasma species e.g. Plasmodium species Ciliata e.g. Balantidium coli Ticks Parasites

Arthropods

Arachnids Mites Cestoda e.g. Taenia species

Helminths

Nematoda e.g. Roundworms

Trematoda e.g. Fasciola hepatica

Fig. 1.2. General classification of parasites of veterinary and medical importance. (Author’s own figure.)

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1.3.2 Transmission

1.3.5 Diversity of parasites

The mode of transmission can also affect the distribution of parasites. Some parasites are transmitted directly from host to host, while others require intermediate hosts or vectors such as mosquitoes or ticks. The parasites with complex life cycles can manipulate the behaviour of intermediate hosts in such a way that their transmission to final hosts is paced. The physiological principle behind this lies in the fact that there are phenotypic alterations in the hosts that affect their ability to transmit the parasite to other organisms (Franceschi et al., 2007).

Parasites constitute one of the most diverse groups of organisms on Earth, boasting an estimated 5–10 million species that inhabit an extensive array of ecosystems, ranging from the depths of deep-sea vents to the pinnacles of mountainous landscapes (Carlson et al., 2020). Remarkably, a significant portion of parasite species remains undiscovered by science, highlighting the complexity and enigmatic nature of these organisms (Okamura et al., 2018). Carlson et al. (2020) suggested that accurately estimating the population of parasites may require hundreds of years of dedicated research. Among the myriad examples of parasites, malaria stands out as a globally significant health concern (Breman et al., 2004).

1.3.3 Environmental factors Environmental factors such as temperature, moisture and pH can also influence the distribution of parasites. For example, some parasites are adapted to thrive in the acidic environment of the stomach or the anaerobic conditions of the intestines (Garcia et al., 2018). They are highly sensitive to the changes in the environment. Soil characteristics also play an important role in the transmission of parasites (Cable et al., 2017). An increase in temperature facilitates survival and dispersion of cysts. On the other hand, cysts of some species can’t survive at high environments, they require low temperatures for their survival (Cociancic et al., 2021).

1.3.4

Human impact

Human activities such as travel, trade and land use can also influence the distribution of parasites. Humans are currently modifying habitats all over the world, frequently with disastrous effects for host–parasite interactions and parasite prevalence (Budria and Candolin, 2014). As the human population increases, the distribution of parasites also increases. The main reason is that most of the parasites have some part of their life cycle in the gastrointestinal tract, leading to the excretion of faeces containing infective forms of parasites. So, the transmission of parasites by the faeco-oral route increases among the human and animal populations (Nunn et al., 2011).

1.3.6 Impact of environment A study conducted by Shafiq et al. (2023) investigated the prevalence, diversity and mean intensity of parasites in nine freshwater fish species, with 45 samples per species. Ecto-parasites were examined on the skin, gills and fins using various techniques, including microscopy and artificial digestion. A total of 26 parasite species were identified, including protozoans, trematodes and monogeneans. The diversity of parasites was found to be highest at the Okara site, and Labeo rohita had the highest parasite load. Interestingly, two fish species, Notopterus notopterus and Sperata seenghala, appeared to be resistant to parasitic infection. The study also concluded that the prevalence of parasites increased with the size, length and age of the fish. Another study by Kornyychuk et al. (2023) focused on parasites in hypersaline waters. They identified 85 parasite species and forms belonging to five phyla. Platyhelminths was the most diverse, with the highest species richness in class Cestoda. The study found that the total number of parasitic species decreased exponentially with increasing salinity, and free-living crustaceans, particularly Artemia spp., served as hosts for most parasite species. Adlard et al. (2015) explored the interactions of parasites in aquatic wildlife, emphasizing the need for interdisciplinary approaches to understand

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and mitigate disease impacts in freshwater and marine systems. The study highlighted the complexities arising from environmental changes, anthropogenic translocation of hosts and parasites, and the interconnectedness of diseases in wildlife, humans and domestic animals. Titcomb et al. (2021) investigated the influence of water sources on the transmission of fecal–oral parasites in an East African savannah. They found that water sources increased the density of faeces from wild and domestic herbivores, acting as disease hotspots. This effect was more pronounced in drier areas and periods, with varying impacts on different herbivore species, including elephants and cattle.

1.4

Physiological Features

The survival and distribution of parasites in various environmental systems are intricately linked to their physiological features. Factors such as host specificity, reproductive strategies and life-cycle adaptations contribute to the success and persistence of parasites in diverse habitats (Poulin, 2010). Additionally, the interactions between parasites and their hosts, as well as environmental conditions, play pivotal roles in shaping the ecology of parasitic organisms (Martinez and Merino, 2011). Thus, parasites, with their staggering diversity and widespread presence, represent a fascinating area of study in biology. Understanding their distribution and survival in diverse ecosystems requires a multidisciplinary approach that considers both ecological and physiological factors. Continued research is crucial for unveiling the mysteries surrounding unknown parasite species and comprehensively assessing their population dynamics. Some examples of physiological features that are involved in the distribution and survival of parasites in different environments are discussed here.

1.4.1 Temperature tolerance The survival and distribution of parasites are influenced by temperature, with different species exhibiting varying thermal tolerances. While some parasites thrive in warmer climates, others

are adapted to cooler environments (Hance et al., 2007). Current models predicting the impact of climate change on mosquito-borne diseases often overlook thermal adaptation. A study on Aedes aegypti, the primary vector for diseases like dengue and Zika, reveals significant phenotypic variation in thermal tolerance among populations. The research demonstrates that thermal responses can differ based on geographical origin, challenging the efficacy of one-size-fits-all models in predicting disease transmission under changing temperatures (Dennington et al., 2024). Mosquitoes play a crucial role as disease vectors, and a study in the Nile Delta, Egypt, correlates physicochemical parameters of breeding habitats with mosquito diversity. Breeding places such as unused wells and pools were found to be vital sources, and their larval density correlated positively with temperature, salinity, nitrate and conductivity. The study emphasizes the importance of understanding and managing mosquito breeding habitats for disease prevention (Baz et al., 2024).

1.4.2 Anaerobic metabolism Mucosal-associated parasites, including Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis, are clinically significant due to their association with high incidences of gastroenteritis and sexually transmitted infections. The treatment of these infections relies on drugs targeting the anaerobic metabolism of these parasites, such as nitroimidazole and benzimidazole derivatives (Carvalho-de-Araújo et al., 2023). The ability of parasites to produce ATP under anaerobic conditions, a feature absent in vertebrate hosts, makes them attractive therapeutic targets (Avilán et al., 2011). In the realm of protists, which include microbial eukaryotes, hypoxic environments have led to the evolution of mitochondrion-related organelles (MROs). While these organelles share some metabolic features, their independent evolution is evident across diverse protist lineages. Recent deepsequencing studies of free-living anaerobic protists have unveiled unique configurations of metabolic pathways adapted to low-oxygen conditions. This article provides examples of anaerobic metabolism in both free-living and parasitic

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Physiological Basis of Parasitism

protists, shedding light on the evolutionary origins of these pathways (Stairs et al., 2015). Helminths, parasitic worms infecting millions of animals and humans globally, release excretory– secretory products (ESPs) containing various molecules. Despite the lack of a comprehensive review, a recent study by Wangchuk et al. (2023) analysed helminth metabolomics, focusing on small molecules from ESPs and somatic tissue extracts. The review assessed 29 studies, highlighting challenges such as the limited number of identified metabolites and the absence of standardized software and databases in helminth metabolomics. Saccà (2013) emphasizes the key role of energy metabolism in all organisms, particularly anaerobic metabolism prevalent in Protozoa. The chapter critically reviews anaerobic energy metabolism in Protozoa, considering the debated evolution of eukaryotes and their MROs. Mathur et al. (2021) explore gregarines, early-diverging apicomplexan parasites, providing insights into their mitochondrial metabolic repertoire. Unlike well-studied parasites, gregarine trophozoites exhibit reduced energy metabolism, with parallel reductions in respiratory complexes and the tricarboxylic acid (TCA) cycle. This parallel reduction emphasizes the diversity of MROs in apicomplexan evolution. Ascetosporea, marine invertebrate endo-parasites, are poorly studied owing to limited molecular data. Onuț-Brännström et al. (2023) sequenced the genome and transcriptome of Paramikrocytos canceri, revealing a MROs with a unique metabolic profile. This organelle, resembling mitosomes, produces ATP through a partial glycolytic pathway and synthesizes phospholipids de novo via the CDP–DAG (cytidine diphosphate diacylglycerol) pathway. This combination of metabolic pathways represents a novel aspect in MROs, with potential implications for host invasion strategies.

1.5

Nutritional Requirement of Parasites

Parasites have diverse nutritional requirements, which are essential for their survival, growth and reproduction. Parasites obtain nutrients from their host or the environment, depending on the type of parasite and its life-cycle stage

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(Chappell, 1979). Understanding the nutritional requirements of parasites is important for developing strategies to control parasitic infections. For example, targeting the nutrient requirements of parasites could potentially lead to the development of new therapies or vaccines (Coop and Kyriazakis, 1999). The metabolic pathways involved in the nutritional dependence of parasites on their host can vary depending on the parasite and the host–parasite interaction. However, some general principles can be applied to understand how parasites obtain nutrients from their host and how this contributes to pathogenesis (Kloehn et al., 2016). Amino acids are the building blocks of proteins, and parasites require them for protein synthesis. Some parasites are unable to synthesize certain amino acids and must obtain them from their host or the environment or synthesize them de novo (Dietzen, 2018). Some parasites, such as Plasmodium spp., are unable to synthesize certain amino acids and must obtain them from their host (Krishnan and Soldati-Favre, 2021). Amino acid metabolism can also contribute to pathogenesis by generating toxic by-products. For example, the conversion of arginine to ornithine by Trypanosoma cruzi generates toxic reactive oxygen species that contribute to tissue damage (Comini and Flohé, 2013). Carbohydrates provide energy for the metabolism of parasites. Some parasites can break down complex carbohydrates, such as cellulose, using enzymes produced by symbiotic bacteria in their gut (Brune, 2014). Many parasites rely on glycolysis as their primary source of energy. For example, the malaria parasite Plasmodium falciparum relies on glycolysis for ATP production and survival within erythrocytes (Olszewski and Llinás, 2011). Glycolysis is also important for the survival of other parasites, such as Trypanosoma cruzi and Leishmania spp. (Saunders et al., 2010). Lipids are important for the structure and function of cell membranes, as well as for energy storage. Parasites may obtain lipids from their host or their environment (Toledo et al., 2016). The metabolism of fatty acids can also contribute to pathogenesis by generating toxic by-products. For example, the metabolism of fatty acids by the parasite Schistosoma mansoni generates toxic oxylipins that contribute to inflammation and tissue damage (Friesen et al., 2022). Parasites require vitamins for various metabolic functions,

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such as enzyme catalysis and redox reactions. Some parasites, such as P. falciparum, are unable to synthesize certain vitamins and must obtain them from their host (Krishnan et al., 2020). Iron is essential for the survival and growth of many parasites, including Plasmodium spp., which cause malaria. These parasites require iron for the synthesis of haem, a component of haemoglobin (Clark et al., 2014). Iron metabolism can also contribute to pathogenesis by generating toxic by-products. For example, the metabolism of iron by the parasite Trichomonas vaginalis generates toxic reactive oxygen species that contribute to inflammation and tissue damage (Vallières et al., 2023). Some parasites have evolved to exploit specific nutrients in their host’s diet. For example, some parasitic flatworms obtain nutrients from the blood of their host, while others feed on host tissues or fluids (Halton, 1997). Figure 1.3 gives an overview of the metabolism of parasites.

1.6

Establishment and Growth of Parasites

The establishment and growth of parasites within their hosts is a complex process that can

be influenced by a variety of factors, including host immunity, parasite virulence factors and environmental conditions (Mabbott, 2018). Understanding the establishment and growth of parasites within their hosts is important for developing strategies to control parasitic infections. For example, targeting parasite invasion mechanisms, immune evasion strategies or nutrient acquisition pathways could potentially lead to the development of new therapies or vaccines (Coop and Kyriazakis, 1999). Some general principles that can be applied to understand the establishment and growth of parasites within their hosts follow here. 1.6.1 Genetics of parasites Parasites have a unique genetic and neuromuscular physiology that enables them to adapt and survive in their hosts. Parasites can rapidly adapt to changing environmental conditions within their host (Drew et al., 2021). This is often achieved through genetic recombination and mutation, which allows the parasite to evolve quickly in response to selective pressures (Wielgoss et al., 2016). For example, the protozoan parasite NH H2N

N H

O OH NH2

Amino acid Parasites

Protein

Cellulose Glycolysis

Fe ATP

Haemoglobin

Toxins production

Iron

Fatty acid Cell membrane maintenance Fig. 1.3. Overview of the metabolism of parasites. (Author’s own figure.)

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Physiological Basis of Parasitism

P. falciparum can quickly evolve resistance to antimalarial drugs (Cowell and Winzeler, 2018). Many parasites have reduced genome sizes compared to their free-living relatives (Ogburn, 2019). This reduction is thought to be due to a process of genome reduction that has occurred over time as parasites adapt to life within their hosts (Derilus et al., 2021). For example, the parasitic tapeworms have a genomic size of 106–147 Mb as compared to free-living planarian Schmidtea which is about 700 Mb (Poulin and Randhawa, 2015).

1.6.2

Neuromuscular physiology of parasite

Parasites often have complex neuromuscular systems that allow them to move, attach to host tissues and avoid host immune defences (Pax and Bennett, 1992). For example, the nematode parasite Trichinella spiralis has a complex neuromuscular system that includes sensory neurons, interneurons and motor neurons. Some parasites can manipulate host behaviour through their actions on the host nervous system. For example, the protozoan parasite Toxoplasma gondii can alter the behaviour of infected rodents, making them more likely to be eaten by cats, which are the definitive host for the parasite (Webster, 2007).

1.6.3

Invasion

Parasites must first invade their host and establish themselves within host tissues. The mechanisms of invasion can vary depending on the parasite and host–parasite interaction. For example, some parasites, such as the protozoan Toxoplasma gondii, can actively invade host cells (Sibley, 2011) while others, such as the hookworm Necator americanus, penetrate the host skin through their mouthparts (Kalousova et al., 2016).

1.6.4 Nutrient acquisition Parasites must obtain nutrients from their host to support their growth and reproduction. This can be achieved by a variety of mechanisms, including the use of host-derived nutrients, the

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production of virulence factors that modify host metabolism, and the manipulation of host signalling pathways (Coop and Kyriazakis, 1999). For example, the protozoan Leishmania spp. can modulate host macrophage metabolism to obtain nutrients and evade immune responses (Bodhale et al., 2021).

1.6.5 Reproduction Parasites must be able to reproduce within their host to establish and maintain an infection. Parasites can reproduce asexually or sexually, depending on the parasite species (Meeûs et al., 2009). For example, the protozoan Plasmodium spp. reproduce asexually within erythrocytes during the blood stage of infection (Venugopal et al., 2020), while the helminth Schistosoma mansoni reproduces sexually within the human host (Buddenborg et al., 2023).

1.7

Hormonal Influence on Parasite Development

Different hormones, such as oestrogen, progesterone and androgens, play critical roles in the metabolism and reproduction of vertebrates; however, in invertebrates, the influence and occurrence of steroid hormones have received less attention (Lafont, 2000). The interplay between the parasite and the host defines the intensity of parasite infections. In many cases, the presence of parasites in the host changes its endocrine equilibrium due to the activation of the immune system response, which finally affects the endocrine system through the influence of cytokines and growth factors released by the immune cells. It is now widely accepted that corticosteroids and sex-related hormones influence the immune response (Reyes-Hernandez et al., 2013); thereafter any endocrine perturbation initiated by an infection will change the neuroendocrine equilibrium. These hormonal changes resulting from a spontaneous or experimental infection affect the parasitic charge, the course of the infection and the parasite’s survival (Barthelemy et al., 2004). Alternatively, some parasite infections disrupt the host endocrine system. In a noteworthy

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example, the dromedary bull, Camelus dromedarius, parasitized with Trypanosoma evansi, presents changes in the sexual steroid plasmatic concentrations, as well as in the semen characteristics (Al-Qarawi, 2005). Furthermore, the male fence lizard (Sceloporus occidentalis) infected with the malarial parasite Plasmodium mexicanum shows several reproductive pathologies, such as fewer courtship behaviours and decreased testosterone levels (Dunlap and Schall, 1995) and Toxoplasma gondii infection enhances testicular steroidogenesis in rats (Lim et al., 2013). Interestingly, it has been shown that the host hormonal environment determines the susceptibility, course and severity of many parasite infections, and therefore a clear dichotomy in infection susceptibility between males and females had been observed (Morales-Montor et al., 2004). Figure1.4 represents the effect of parasites on the host’s endocrine system.

1.8 Host–Parasite Interaction Host–parasite interactions involve complex processes that occur within the body of the infected host. Parasites must first invade and colonize their host’s tissues to establish an infection. This

Endocrine disruption

Helminth

GnRH

Serotonin and dopamine

Oestradiol DHT Testosterone

Testosterone Oestrogen Aromatase

Host cell

may involve physical penetration of host barriers or active invasion of host cells (Olano et al., 2011). For example, the protozoan parasite P. falciparum invades red blood cells (Tilley et al., 2011) and the helminth parasite Schistosoma mansoni actively penetrates the skin of its host (Haas, 2003). Once a parasite has established an infection, the host’s immune system will typically mount a response to try to eliminate the parasite. This response may involve the activation of immune cells such as macrophages, T cells and B cells, and the production of antibodies and other immune molecules (Kellie and Al-Mansour, 2016). However, many parasites have developed strategies to evade or suppress the host’s immune response, allowing them to persist in the host for extended periods (Schmid-Hempel, 2009). Parasitic infections can cause tissue damage and inflammation in the host. This may occur as a result of the direct effects of the parasite on host cells or as a result of the host’s immune response. For example, liver damage caused by a liver fluke can lead to the development of liver cancer (Watanapa and Watanapa, 2002). Parasites may compete with their hosts for nutrients, which can lead to malnutrition and other health problems in the host. For example, the tapeworm Hymenolepis diminuta can absorb large amounts

Miscarriage Feminization, Th1 to Th2 shift, Weight of seminal vesicles decreases Testicular failure, Prostate abnormalities

Endocrine disruption Oestrogen Progesterone Oestrogen Progesterone

Protozoa

Testosterone Oestradiol

Reproductive dysfunction Haemorrage, Apoptosis of placental cells, IFN-Y secretion

Host cell

Reproductive tract development ceases

Dopamine Norepinephrine

No fear perception

Fig. 1.4. Effect of parasites on the host’s endocrine system. (Author’s own figure.)

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of vitamin B12 from its host, leading to deficiencies in the host (Platzer and Roberts, 1970). Understanding parasite–host interactions is critical for developing effective strategies to control parasitic infections.

1.9

Stress Tolerance

Parasites are exposed to various environmental stresses during their life cycle, including temperature fluctuations, osmotic stress, oxidative stress and exposure to host immune responses. To survive and thrive in these stressful conditions, parasites have developed a variety of stress tolerance mechanisms (Singh et al., 2011). Heat shock proteins (HSPs) are a family of proteins that help to protect cells from heat stress and other environmental stresses. Many parasites produce HSPs to protect themselves from the high temperatures encountered during their life cycle. For example, the protozoan parasite Leishmania donovani produces a specific HSP (HSP70) to protect itself from heat stress (Hombach et al., 2014). Parasites are exposed to oxidative stress when they encounter reactive oxygen species (ROS), which are produced by host immune cells and other sources (Nimmanee et al., 2014). To protect themselves from oxidative damage, parasites produce antioxidant enzymes such as superoxide dismutase (SOD), catalase and glutathione peroxidase (Drews et al., 2010). For example, the helminth parasite Haemonchus contortus produces high levels of SOD to protect itself from oxidative stress (Alam et al., 2020). Parasites encounter changes in osmotic pressure during their life cycle, such as when they move between different host environments. To survive these changes, parasites have developed osmoregulation mechanisms to maintain their internal water balance. For example, the protozoan parasite Trypanosoma cruzi has a complex osmoregulation system that allows it to adapt to different osmotic environments (Dave et al., 2021).

1.10 Immune Evasion and the Host Immune System Many parasites have developed strategies to evade the host immune system by creating a

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protective covering that helps them to avoid detection or destruction by the host’s immune system. Many parasites have surface proteins that can mimic host proteins or that can bind to host cells, allowing the parasite to enter and establish an infection without being recognized by the host immune system (Zambrano-Villa et al., 2002). For example, the malaria parasite P. falciparum uses a protein called PfEMP1 to bind to host cells and avoid detection by the host immune system (Hviid and Jensen, 2015). The glycocalyx is a layer of polysaccharides that surrounds some parasites and helps to protect them from host immune cells. For example, the parasitic protozoan Leishmania spp. produces a glycocalyx that helps it to evade detection and destruction by host macrophages (de-Morais et al., 2015). The survival of parasites can be significantly challenged by the immune system of the host. To survive the host defence mechanism, parasites have developed adaptive mechanisms, using a few different strategies (Camus et al., 1995). Indeed, some parasites can modify their surface antigens to escape the host immune system. For example, the African trypanosome Trypanosoma brucei (causative agent of sleeping sickness) can undergo antigenic variation, to alter its membranous glycoprotein to resist the immunity of the host (Horn, 2014; Steverding, 2017). Some parasites can inhibit the host’s immune system to prevent detection and eradication. For example, Heligmosomoides polygyrus can release proteins that alter the immune system, enabling the parasite to survive in the host for longer (Reynolds et al., 2012). Some parasites can alter the physiology of the host according to their needs. For example, Schistosoma mansoni, a trematode, can stimulate the granuloma formation in the liver of the host, which protects it from the attack of the host defence system and enables it to get nourishment from the host blood (Chai and Jung, 2022). Some parasites can modulate their own development and life cycle in response to stress induced by the host immune system. For example, the nematode Strongyloides stercoralis can transform between free-living and parasitic mode depending upon the immune status of the host (Lok, 2014). To create new therapeutics to treat parasitic diseases, it is essential to understand the techniques parasites employ to avoid or inhibit the host’s immune response. Researchers may

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be able to create novel medications or vaccinations that boost host immunity and aid in parasite eradication by focusing on these pathways.

1.11

Host–Parasite Adjustment

Host–parasite adjustment is the co-evolutionary process in which the parasite and the host gradually adjust to one another. This process may involve adaptations to the physiology, behaviour and morphology of both the host and the parasite, and it may lead to the emergence of mutualistic or commensal relationships (Zinner et al., 2023), for example, the mutualistic relationship between the termites and the microbiota living in their digestive system, in which termites need microorganisms to break down ingested cellulose. In this relationship, the gut microorganisms live in a stable environment and have a surplus of nutrients in the primary food of termites. This relationship is the result of a co-evolutionary adaptation of physiology and morphology of the termites’ gut and the microorganisms of the gut (Rosengaus et al., 2011). Another example of host–parasite adjustment is seen in the evolution of virulence in pathogenic bacteria. Pathogenic bacteria can evolve to become more virulent over time, as they adapt to the host environment and become more efficient at evading the host immune response (Didelot et al., 2016). High levels of virulence can, however, be counterproductive for the parasite, potentially resulting in the death of the host and the elimination of the parasite’s source of nutrients. As a result, there is often a trade-off between virulence and transmission in pathogenic bacteria (Drew et al., 2021). Understanding host–parasite adjustment is important for developing effective strategies to control parasitic infections. By studying the co-evolutionary dynamics between hosts and parasites, researchers may be able to identify new targets for therapeutic intervention or develop vaccines that can exploit mutualistic or commensal relationships between hosts and parasites. In addition to evading host defences, parasites may also modify host cell behaviour and physiology to promote their survival (Adamo, 2002). For example, some parasites can induce the host to produce cytokines and other immune

molecules that create an environment favourable for their growth and replication. One well known example of a parasite that becomes part of the host, eventually, is the bacterium Wolbachia, which infects a wide range of arthropods and nematodes. Wolbachia has evolved a range of strategies to manipulate the reproductive biology and behaviour of its host, to increase its transmission and persistence within host populations (Werren et al., 2008).

1.12

Role of Parasitic Physiology in the Development of Drug Resistance

The physiology of a parasite plays a crucial role in the development of resistance against various drugs. Parasites are living organisms that have complex life cycles, and their ability to adapt and evolve is influenced by their physiological characteristics. When exposed to drugs, parasites can develop resistance through several mechanisms (Gregson and Plowe, 2005). Parasites have a high reproductive rate and produce a large number of offspring. Parasites experiencing a high fluctuation due to host health, the immune system or drugs can undergo mutation rapidly (Blanquart et al., 2017). This genetic diversity allows for the existence of rare mutants that might possess resistance-conferring genetic mutations (Greenspoon and Mideo, 2017). If a drug is applied to the parasite population, these mutants can survive and pass on their resistant traits to subsequent generations, leading to the spread of drug-resistant parasites (Wilson et al., 2016). Many parasites have short generation times, which means they can go through several generations in a short period. This accelerated reproduction enhances the chances of mutation and increases the speed at which drug resistance can emerge within the parasite population (Jennings and Calow, 1975). Some parasites can develop resistance by overexpressing efflux pumps that actively remove drugs from their cells. This reduces the drug concentration inside the parasite, making it less effective in killing or controlling the infection (Ray et al., 2017). Parasites may develop resistance by altering the target sites of drugs. For example, in the case of antiparasitic drugs that act on specific enzymes or proteins, mutations

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can occur in these targets, rendering the drug less effective (Pramanik et al., 2019). Some parasites can develop alternative metabolic pathways to bypass the actions of drugs, allowing them to continue to thrive despite the presence of the drug (Guidi et al., 2020). Some drugs require activation within the parasite to exert their effects. Parasites may develop mechanisms to reduce or block this activation, making the drug less potent (Capela et al., 2019). Some parasites may enhance their ability to detoxify or break down drugs, reducing their harmful effects (Herraiz et al., 2019). It is important to note that the development of drug resistance is a natural evolutionary process and can occur with any type of pathogen, including bacteria, viruses and parasites. To combat drug resistance, it is essential to use drugs judiciously and in combination with other control measures (Laxminarayan et al., 2013). This helps to minimize the selection pressure on the parasites and reduce the likelihood of resistance development. Additionally, ongoing research and the development of new drugs and treatment strategies are crucial in staying ahead of evolving drug-resistant parasites.

1.13 Future Directions for Research on the Physiological Basis of Parasitism Future research on the physiological basis of parasitism should aim to deepen our understanding of the complex interactions between parasites and their hosts. The availability of whole-genome sequences of many parasites provides an opportunity to understand the molecular mechanisms of parasite–host interactions. Further, proteomic analysis can help identify novel therapeutic targets (Guo et al., 2013). Research on the mechanisms used by parasites to evade host immune defences can provide insights into new therapies for parasitic infections (Gomes et al., 2016). Studying the long-term interactions between parasites and their hosts can shed light on how parasites have evolved to adapt to different host environments (Penczykowski et al., 2016). Investigating the protective coverings and other strategies used by parasites to evade host

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immune systems can help identify new therapeutic targets (Elmahallawy et al., 2021). Further research into the metabolic pathways and nutritional requirements of parasites can help develop more targeted therapies for parasitic infections (Mamoun et al., 2010). Understanding the physiological changes that parasites undergo to manage the stress of host expulsion can provide insights into new treatments for parasitic infections (Rynkiewicz et al., 2015). The development of new and innovative therapeutic approaches, such as vaccines, gene editing or nanotechnology-based drug delivery systems, could revolutionize the treatment of parasitic infections (Kirtane et al., 2021). By exploring these future directions, researchers can deepen their understanding of the physiological basis of parasitism and develop new and more effective approaches to prevent and treat parasitic infections.

1.14

Conclusions

In conclusion, parasites are a diverse group of organisms that have unique physiological adaptations to survive and thrive in their hosts. They have developed various mechanisms to evade host defences, obtain nutrients, and reproduce outside and within their hosts. The ecological and distributional aspects of parasites are important for understanding their transmission and pathogenesis. The study of parasite–host interactions is crucial for understanding the pathogenesis of parasitic diseases and developing effective prevention and treatment strategies. Stress tolerance is an important characteristic of parasites that allows them to survive in adverse conditions within their hosts. Furthermore, the establishment and growth of parasites within their hosts involve complex physiological changes that can ultimately influence the host’s physiology. To understand these interactions, it is necessary to study the genetic and neuromuscular physiology of parasites. Finally, host–parasite adjustment is a crucial factor in the long-term survival of parasites within their hosts. By studying these physiological and ecological aspects of parasites, we can gain a better understanding of parasitic diseases and develop more effective methods for their prevention and control.

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Antiprotozoal Resistance

Haider Abbas1*, Muhammad Younus2, Zahid Fareed3, Mian Mubashar Saleem4, Malcolm K. Jones5, HazratUllah Raheemi6, Adil Ijaz7 and Muhammad Nadeem Saleem8 1 Department of Pathobiology (Parasitology Section), KBCMA College of Veterinary & Animal Sciences, Narowal, Sub-Campus UVAS, Lahore, Pakistan; 2Department of Pathobiology (Pathology Section), KBCMA College of Veterinary & Animal Sciences, Sub-Campus UVAS, Lahore, Pakistan; 3Foot and Mouth Disease Research Center, Lahore, Pakistan; 4Department of Animal Sciences (Poultry Section), KBCMA College of Veterinary & Animal Sciences, Narowal, Sub-Campus UVAS, Lahore, Pakistan; 5School of Veterinary Science,The University of Queensland, Gatton Queensland,Australia; 6 Department of Health and Biological Sciences, Faculty of Life Sciences,Abasyn University, Peshawar, Pakistan; 7Department of Biomolecular Health Sciences, Division of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht,The Netherlands; 8Department of Animal Sciences (Animal Breeding and Genetics), KBCMA College of Veterinary & Animal Sciences, Narowal, Sub-Campus UVAS, Lahore, Pakistan

Abstract Antiprotozoal drug resistance is a major challenge in the treatment and control of protozoal infections. Protozoan parasites (members of the phylum Protozoa), which are among the major livestock pathogens, e.g., Theileria, Babesia and Trypanosoma, have developed various mechanisms to evade the effects of antiprotozoal drugs, including reduced drug uptake, enhanced drug effux and target site modifcations. This has been presumed to lead to the emergence of drug-resistant strains of protozoans. This resistance can spread rapidly and pose a serious threat to the health of animals. This chapter highlights the modes of action and the mechanisms of the development of resistance in protozoa against drugs used against enteric and haemo-protozoal infections. Understanding these mechanisms provides a way forward for the discovery of new drugs to ensure animal welfare, health and sustainable livestock production and can  aid in the prevention and management of antiprotozoal resistance. The development of new antiprotozoal drugs, combination therapy, and improved surveillance and monitoring are among the essential strategies to combat antiprotozoal drug resistance and ensure effective treatment of protozoal infections.

2.1

Introduction

Protozoa (kingdom: Animalia; phylum: Protozoa), unicellular organisms with a cosmopolitan distribution comprising more than 65,000 species, exist

as free-living or/and parasitic forms, many of which have notable veterinary and public  health significance (Ashley et al., 2018; de Koning, 2020). Risk factors such as inadequate control strategies for arthropod vectors and suboptimal hygiene

*Corresponding author: [email protected] © CAB International 2024. Antiparasitic Drug Resistance in Veterinary Practice (eds H.M. Rizwan, M.A. Naeem, M. Younus, M.S. Sajid and X. Chen) DOI: 10.1079/9781800622807.0002

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conditions make protozoal infections more prevalent in the tropical and subtropical regions of the globe (Sebaa et al., 2021). Protozoal infections of animals are divided into two categories: (i) haemo-parasitic infections, e.g. those caused by Theileria spp., Babesia spp. and Trypanosoma spp. in mammals; and (ii) enteric infections, e.g. those caused by Giardia spp., Toxoplasma spp., and the coccidians traditionally assigned to the genera Eimeria and Isospora (Biobaku et al., 2010; Ababu et al., 2021). In developing countries, antimicrobials, including antivirals, antifungals, antiprotozoals and antibiotics, are often available over the counter. Farmers, animal handlers and the general public can access these drugs without a prescription from a medical or veterinary professional. This unrestricted access may result in inappropriate usage. Consequently, it contributes to the development of resistance in protozoa against these drugs (Adamu et al., 2020). The usage of antiprotozoal agents, particularly against malaria, has been reported to have increased very rapidly during the past few decades (Beargie et al., 2019;Tisnerat et al., 2022). In addition, the use of antiprotozoal drugs, including antibabesial, antitrypanosomal and anticoccidial drugs, in veterinary practice has similarly expanded (van Boeckel et al., 2015). Although new antiprotozoal agents in the treatment of protozoal infections have been introduced, the emergence of resistance to antiprotozoal drugs has been on the  rise (Lee et  al., 2020; Rizwan et  al., 2021). For example, in  1967, antiprotozoal resistance (APR) was reported in Nigeria (Na’Isa, 1967). In 1981, isometamidium and homidium resistance were also reported in cows (Jones-Davies, 1968). Subsequently, various studies have reported antiprotozoal resistance in animals (Dagnachew et al., 2017; Capela et al., 2019). In Cameroon, trypanosomes that became resistant to diminazene aceturate were identified in their vectors (Simo et  al., 2020). Moreover, multi-drug-resistant trypanosomes were also identified (Obi et al., 2021). Natural selection and selection pressure are two general mechanisms through which resistance develops against antiprotozoal drugs owing to the irrational use of these drugs (Simo et al., 2020). Natural selection attributes the mutations in the genes of parasites leading to the emergence of drug resistance in succeeding generations, while

selection pressure correlates to the poor diagnosis of the infections and inappropriate use of the drugs (Clement et al., 2019). One possible explanation for the emergence of resistance mutations in parasites is the selective sweep model (Shaukat et al., 2019). Antiparasitic drugs can create positive selection pressure for adaptive mutations in the parasite’s candidate loci, leading to the rise of resistance haplotypes. Selective sweeps can be hard or soft, with the former characterized by a single resistance haplotype rising from a recent mutation and the latter by multiple resistance haplotypes derived from recurrent or pre-existing mutations. It is important to understand the origin and spread of adaptive mutations in response to selection, which phylogenetic models suggest could arise from a single or multiple origins and spread through parasites via migration due to the gene flow of drug resistance alleles (Chaudhry et al., 2021). The current chapter provides insights into the modes of action and mechanisms involved in the development of resistance against drugs used in haemo- and enteric protozoal infections, which can provide potential alternative and/or augmentative ways for the discovery of new drugs to ensure animal welfare, health and sustainable livestock production.

2.2

Drug Resistance in Babesiosis

Babesiosis, a tick-borne disease, is caused by malaria-like apicomplexan parasites of the genus Babesia (Krause, 2019), which target the red blood cells of the host (Schwartz and Mawhorter, 2013). Anti-babesial drugs, vaccines and tick control are the tools for controlling babesiosis (Mosqueda et al., 2012). In babesiosis-endemic regions, disease is controlled by live vaccines, but these vaccines are not used on a wide scale due to issues such as incompetency against variable strains and the chances of other haemo-pathogen contamination (Brown et al., 2006). To lower the economic burden due to babesiosis, chemotherapy is considered the primary option for the control of the disease, but these parasites have been reported to develop resistance against most of the antibabesial agents (Hwang et al., 2010; Yamasaki et al., 2017). Examples of some of the antiprotozoal agents and the development of resistance in protozoa against them are described below.

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Antiprotozoal Resistance

2.2.1 Diminazene aceturate resistance The drug most commonly used for treating babesiosis in animals is diminazene aceturate (DA), an aromatic diamidine that is available on the market in the form of diaceturate salt (45%), along with a 55% stabilizer, antipyrine (Mosqueda et al., 2012; Baneth, 2018). It is reported that DA attaches to various blood proteins and may bind haemoglobin after crossing the cell membrane of red blood cells (Miller et al., 2005). The mode of action (MoA) of DA is not clear, but it is proposed that it interferes with the aerobic glycolysis and DNA synthesis of Babesia, leading to death (Peregrine, 1994; Plumb, 2015). If the relapse of babesiosis occurs after treatment with DA, it is supposedly due to the emergence of DA-resistant Babesia spp., as reported in the case of canine babesiosis (Sakuma et al., 2009). The development of resistance in Babesia gibsoni against low to high doses of DA during in vitro trials has been reported (Hwang et al., 2010). Furthermore, drug-resistant Babesia bovis may possibly emerge, because DA has been documented to be less effective in cattle against B. bovis in some cases (Mosqueda et al., 2012). The mechanisms of development of DA resistance have not been clearly described in Babesia spp. (Kumara, 2016), but they are reported in Leishmania (Sereno et al., 1997) and Trypanosoma spp. very precisely. Transporters (P2 nucleoside type) in Trypanosoma brucei use diamidine as a substrate (Teka et al., 2011). The resistance to diamidine has been reported after the disruption of the role of pentamidine and P2 nucleoside transporters in African trypanosomiases (Carter et al., 1995). Furthermore, a decline in the enzymes (such as F1F0-ATPase and dehydrogenases) in mitochondria has been observed in parasites that developed resistance against diamidine (Basselin et al., 1998). The mechanism of the development of resistance in Babesia spp. against DA is not clearly described (Kumara, 2016). The drugs effective against apicomplexan parasites are found to act primarily on the mitochondrial membrane of these parasites. Therefore, it is considered that DA resistance is associated with changes in mitochondrial molecules (de Souza et al., 2009). These modifications involve modifications in mitochondrial DNA owing to mutations in cytochrome c oxidase subunit III or I and cytochrome b (CYTb)

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genes (Sakuma et al., 2009). A later study conducted on B. gibsoni, which developed resistance against DA, revealed that there was neither any modification in the nucleotide sequence nor in the amino acid profile of these genes, however, indicating no correlation of DA resistance with mutation in these genes (Rajapakshage et al., 2012a). Most apicomplexan parasites, like B. gibsoni and Plasmodium spp., depend on their glycolytic pathway to gain energy needed in favourable physiological settings (Mogi and Kita, 2009). Changes in the glycolytic system have been found in different protozoans during resistance (Rajapakshage et al., 2012b). Therefore, higher glucose and ATP levels have been reported in DA-resistant parasites in comparison to susceptible strains (Rajapakshage et al., 2012b). Heat shock proteins (HSPs) have been found to develop resistance against stress and help in survival in the environment (Hu et al., 2022). Moreover, in drug resistance mechanisms, HSP90 and HSP27 heat shock proteins are found to be involved (Lindquist, 1986). The lower levels of HSP70 were expressed in DA-resistant B. gibsoni (Hwang et al., 2010).

2.2.2 Imidocarb dipropionate resistance Imidocarb dipropionate (aromatic diamidine) works in a number of ways to control babesiosis. It is proposed that imidocarb dipropionate blocks cellular repair and replication and damages the nucleic acid by integrating into the DNA of susceptible Babesia parasites (Plumb, 2015). Imidocarb interferes with the inositol incorporation in Babesia-infected red blood cells, leading to the death of parasites due to starvation (McHardy et al., 1986). In addition, it inhibits the ability of parasites to synthesize or utilize polyamines, as reported for T. brucei (Bacchi et al., 1981). This drug is eliminated from the body through the kidney and liver (Baneth, 2018). Imidocarb is an FDA-approved drug for the therapy of canine babesiosis caused by B. canis in the USA and is found to be more efficacious for large forms of canine Babesia spp. (Checa et al., 2017). There are no data available describing the mechanism of the development of imidocarb dipropionate drug resistance in Babesia in animals.

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2.2.3 Atovaquone–azithromycin resistance

2.2.4 Buparvaquone and azithromycin resistance

Atovaquone (hydroxy-1,4-naphthoquinone) is a synthetic antiprotozoal drug. The mode of action of atovaquone (AQ) includes the suppression of pyrimidine and ATP production through the selective inhibition of the electron transport complex in the mitochondria of the protozoan (de Oliveira Silva et al., 2016), while azithromycin (AZ) interrupts protein translation in the apicoplasts (Birth et al., 2014). The combined AQ/ AZ treament proved efficacious against the small forms of canine Babesia such as B. conradae and B. gibsoni (Baggish and Hill, 2002; di Cicco et al., 2012). This combination is also used for human babesiosis treatment (Wormser et al., 2006). In Japan and Taiwan, AQ therapy in canine babesiosis due to B. gibsoni was unresponsive and resulted in resistance to this drug, which was considered to be due to interference with the site of action of the drug after mutation at the cytochrome b gene of the protozoan (Sakuma et al., 2009; Liu et al., 2016). The mutation in the same gene was reported in the development of AQresistant Plasmodium falciparum and Plasmodium berghei, causing malaria in humans and rodents, respectively (Korsinczky et al., 2000; Blake et al., 2017). In addition, this gene is related to the electron transport complex of the mitochondria of the parasite (Siregar et al., 2008). The M121I mutation was reported in the cytochrome b gene of B. gibsoni, which developed resistance to AQ (Iguchi et al., 2020). This mutation was suggested to be due to the replacement of methionine with isoleucine in this gene (Lemieux et al., 2016). The recommended therapy is a combination of AQ and AZ in human babesiosis caused by Babesia microti, but resistance to the AQ–AZ combination was observed, resulting in the persistence of parasitaemia in humans despite the therapy. The mechanism of AQ–AZ combination resistance has been considered to be due to mutations in RPL4 and CYTb genes (Lemieux et al., 2016). An adenine-to-guanine modification and a tyrosine-to-cysteine shift at 815 and 272 positions are two-point mutations reported in the CYTb gene, respectively. Similarly, an arginineto-cysteine shift has been reported at position 86 in the RPL4 gene (Simon et al., 2017).

Buparvaquone (BQ), like AQ, is a hydroxynaphthoquinone drug. BQ is used in bovines (Wilkie et al., 1998) and equines (Zaugg and Lane, 1992) to treat theileriosis. But this drug, in combination with AZ, is also used in canine babesiosis resulting from Babesia vulpes (Checa et al., 2017). The mode of action and mechanism of development of resistance to BQ are considered the same as in the case of AQ for B. gibsoni. In addition, BQ resistance to Theileria annulata in bovine theileriosis has been reported to be due to point mutations in the cytochorome b gene (Mhadhbi et al., 2015).

2.3

Drug Resistance in Theileriosis

The protozoan Theileria spp. is an intracellular haemo-parasite that is responsible for tick-borne disease in livestock, resulting in a significant socio-economic burden on farming. Bovine tropical theileriosis is caused by Theileria annulata, while Theileria parva causes East Coast Fever (Irvin, 1985; Morrison, 2009). The pathogenesis of this parasite involves cell immortalization, rapid multiplication, and propagation of infected white blood cells, phenotypes similar to cancer resulting in transformation of the host cells (Morrison, 2009). The molecular mechanisms through which the transformation of the mammalian host cells occurs in theileriosis consist of modifications in cell signalling pathways. These changes result in fluctuations in the host cell gene expression or activation resulting from the effect of Theileria parasites on the host epigenome (Cheeseman and Weitzman, 2015; Morrison, 2009). The changes in the oncogenic signalling pathways (such as JNK and IKK) and modulation of transcription factors (such as AP-1, HIF-1α and c-Myc) in the host are induced by the infected white blood cells in theileriosis (Metheni et al., 2015). The typical pathological findings include lymph node enlargement, spleen enlargement, pulmonary emphysema, and subcutaneous and intramuscular haemorrhages (Osman and Al-Gaabary, 2007).

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Antiprotozoal Resistance

2.3.1

Buparvaquone resistance

The most commonly used drugs for the treatment of theileriosis are hydroxynaphthoquinone compounds such as buparvaquone (bq) (McHardy and Morgan, 1985; McHardy et al., 1985). Buparvaquone is an effective drug used for the treatment and control of theileriosis in livestock. It is a hydroxynaphthoquinone that is highly effective against the acute form of theileriosis caused by T. parva. It works by inhibiting the electron transport chain in the parasite, leading to its death (Fry and Pudney, 1992). It has been reported that peptidyl prolyl isomerase Pin1 (TaPin1), an enzyme secreted by the parasite T. annulata, interacts with host cell proteins to regulate oncogenic signalling pathways (Marsolier et al., 2015). Specifically, in theileriosis, the transformation of the mammalian host cells is enhanced by c-Jun, which is established by the degradation of host ubiquitin ligase (Fbw7) by TaPin1. TaPin1 also stabilizes the host pyruvate kinase isoform M2 (PKM2), resulting in HIF-1α-dependent metabolic reprogramming (Marsolier et al., 2019). Theileriainduced host cell transformation can be reversed by bq, an anti-theilerial drug that destroys both stages (schizont and piroplasm) of T. annulata, resulting in low or undetectable parasitaemia within seven days. It is hypothesized that bq inhibits TaPin1 isomerase activity as one of its mechanisms of action (McHardy et al., 1985; Marsolier et al., 2015; Villares et al., 2022). The resistance to bq has been reported to lead to treatment failure, increased morbidity and mortality rates, and production losses in farming (Chatanga et al., 2019; Valente et al., 2022). The impact of theileriosis on socioeconomic factors has been worsened by the emergence of drug resistance to bq. Theileria annulata isolates that developed resistance to bq have been reported in Iran, Turkey, Sudan and Tunisia (Sharifiyazdi et al., 2012; Chatanga et al., 2019). The mechanism of bq resistance in Theileria is not fully understood (Mhadhbi et al., 2015); however, it is suggested that it may involve a combination of genetic mutations and metabolic changes in the parasite (Mhadhbi et al., 2015). Resistance to bq was associated with mutations in a protein called cytochrome b, which is involved in energy production in the parasite (Mhadhbi et al., 2015; Chatanga et al.,

23

2019; Ali et al., 2022). Changes in the expression of genes involved in drug metabolism and detoxification may also play a role in bq resistance (Valente et al., 2022). In Tunisia, mutations in the TaPin1 gene were detected in a bq-resistant parasite strain. An alanine-to-proline substitution was identified at residue 53 (A53P) in the catalytic loop of the TaPin1 gene. This one specific mutation was found to cause a loss of sensitivity to bq (in recombinant TaPin1 protein). The mutant TaPin1 protein was found both in in vitro and in vivo trials not to be inhibited by Pin1 inhibitors, including bq (Marsolier et al., 2015). A study was conducted to understand better the role of mutations in developing bq-resistant T. annulata and the impact of selection pressure on the emergence and dissemination of resistance (Ali et al., 2022). Genetic analysis of the cytochrome b locus was conducted on seven resistant and ten susceptible T. annulata isolates, resulting in the identification of a mutation (129G-GGC) in the binding pocket Q01 and two mutations (253S-TCT and 262S-TCA) within the binding pocket Q02, all of which were associated with bq resistance. Further investigation of field isolates identified cytochrome b mutations in 21 out of 75 buffalo-derived and 19 out of 119 cattle-derived T. annulata isolates. Based on the selective sweep model, both hard and soft selective sweeps occurred with significant variations between isolates. The resistance haplotypes, including 253S (TCT) and 129G (GGC), were observed at high frequency in some strains isolated from cattle and buffalo, suggesting the involvement of a single mutation in the development of resistance. The resistance haplotypes with the same levels of high frequencies, including 129G (GGC)/253S (TCT), 253S (TCT), 129G (GGC), and 262S (TCA), were identified in other isolates, indicating an association of repeated mutations with the development of resistance (Table 2.1) and susceptible isolates (Table 2.2). Furthermore, it was found through phylogenetic analysis that there is a single origin of bq resistance based on the presence of certain haplotypes in a single lineage. Transportation of animals between farms in endemic areas was identified as a major factor in the dissemination of bq resistance. To investigate bq resistance in Sudan, the study found three non-synonymous mutations

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24

Table 2.1. Buparvaquone-resistant Theileria annulata isolates that have mutations in their cytochrome b gene. (Authors’ own table.) Resistant isolates

Q01 (130–148)

Codon

739

ST2/19

881III

8307

ST2/13

BC2T

5911

385 (AGC/GGC) 398 (GCT/GTT) 403 (GTC/TTC) 406 (ATA/CTA) 413 (GGT/GTT) 418 (TTG/TTT) 420 (TTG/TTT) 423 (AAA/AAC) 427 (GGA/GTA) 431 (GGA/GTA) 436 (ACT/GCT) 757 (CCT/TCT) 785 (TTA/TCA)

S129G A133V V135F I136L G138V L139F L140F K141N F143L G144V T146A P253S L262S

S/G A V/F I G/V L/F L/F K F G/V T P/S L/S

S/G A/V V/F I/L G/V L/F L/F K F G/V T P/S L/S

S/G A V I G/V L L/F K F G/V T P/S L

S/G A V/F I G/V L L/F K F G/V T P/S L

S A V/F I G/V L/F L/F K/N F G/V T/A P/S L/S

S/G A V I G/V L L/F K F G/V T P/S L

S/G A V/F I G L/F L/F K F G/V T/A P/S L

Abbas et al.

Q02 (244–266)

Nucleotide

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Table 2.2. Buparvaquone-susceptible T. annulata isolates having mutations in their cytochrome b gene. (Authors’ own table.) Susceptible isolates

Q02 (244–266)

Codon

Jed4

674

TA-HAS

Battan C

TA-BUT

Battan P4

TA-ANK

Chargui P5

Jed4P10

TA-MARR

385 (AGC/GGC) 398 (GCT/GTT) 403 (GTC/TTC) 406 (ATA/CTA) 413 (GGT/GTT) 418 (TTG/TTT) 420 (TTG/TTT) 423 (AAA/AAC) 427 (GGA/GTA) 431 (GGA/GTA) 436 (ACT/GCT) 757 (CCT/TCT) 785 (TTA/TCA)

S129G A133V V135F I136L G138V L139F L140F K141N F143L G144V T146A P253S L262S

S A V I G L L/F K F/L G/V T P L

S A V I G/V L/F L/F K F G/V T P L

S A V I G L L K F G T P L

S A V/F I G/V L/F L/F K/N F/L G/V T P L

S A V I G L L K F G T/A P L

S A/V V/F I/L G/V L/F L/F K/N F G/V T P L

S A V I G L L K F G T/A P L

S A/V V/F I/L G L/F L/F K/N F/L G/V T P L

S A V/F I/L G/V L/F L/F K F/L G/V T P L

S A V I G L L K F G T P L

Antiprotozoal Resistance

Q01 (130–148)

Nucleotide

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26

Abbas et al.

in the Q01 (130–148) and Q02 (244–266) binding sites of bq, including a mutation at codon 146 (alanine to threonine) in all 50 isolates and a mutation at codon 129 (serine to glycine) in 18 isolates. Another mutation was identified at position 227 (valine to methionine) in three isolates with the codon 129 mutation. The study suggests that these mutations may be associated with bq treatment failure in Sudan (Chatanga et al., 2019). To combat drug resistance in the case of theileriosis, a variety of approaches are being explored. These include the development of new drugs with novel mechanisms of action (Barman et al., 2021), the use of drug combinations to prevent or delay the emergence of resistance (El-Saber Batiha et al., 2020), and the development of vaccines to prevent infection in the first place (Goh et al., 2021). Additionally, improved management practices, such as the use of tick control measures, can reduce the burden of disease and limit the spread of drug-resistant strains (Dzemo et al., 2022).

2.4 Drug Resistance in Trypanosomiasis Trypanosomiasis is an illness caused by the Trypanosoma parasite that can affect both humans and animals (Abbas et al., 2021). This disease is transmitted cyclically by tsetse flies and mechanically by biting flies (Degneh et al., 2019; Abbas et al., 2023). This disease is predominantly found in developing countries in Asia, Africa and America, and it poses a significant threat to wildlife (Kasozi et al., 2021) and livestock, including various small and large ruminants, horses, camels and equines (Giordani et al., 2016). This disease exerts a substantial economic burden on livestock owners and significantly impacts animal health and production in livestock (Degneh et al., 2019). African animal trypanosomiasis (AAT), which is caused by T. vivax, T. brucei, T. congolense and T. evansi, is the most common form of animal trypanosomiasis. The disease in humans is generally categorized as American trypanosomiasis (Chagas disease) or human African trypanosomiasis (sleeping sickness) based on the protozoan species involved. Despite the implementation of strategic control measures in some countries to stop the

transmission of the disease, determining the actual incidence of the disease is challenging owing to inadequate monitoring and diagnostic practices, as well as difficult access and unstable socio-economic conditions in affected regions (Isaac et al., 2017; Kasozi et al., 2022). This is a significant concern because humans and animals interact closely in the Tsetse Belt region, contributing to the socio-economic growth of the affected communities (Dagnachew et al., 2015). The trypanocidal and trypanostatic drugs that are routinely used in veterinary practice are shown in Table 2.3. The resistance against these trypanocidal drugs has been reported in 18 countries (Degneh et al., 2019). 2.4.1 Diminazine aceturate resistance Diminazene aceturate (DA) has been found to be more efficacious against the T. brucei group than T. congolense. This might be due to the efficient absorption allowed by the P2-type purine transporter (TbAT1). Diminazene aceturate is an ideal therapeutic drug rather than a prophylactic drug because it is readily metabolized and excreted from the body (Giordani et al., 2016). The mechanism of action of DA involves the binding of the drug to the minor groove present on the DNA (AT-rich sites) of the protozoan. Kinetoplast DNA (kDNA) biosynthesis inhibition and its loss occur owing to the drug targeting the kDNA. Furthermore, this drug also leads to the disintegration of the membrane transport protein in the mitochondria as a result of the suppression of type II topoisomerase (Lu et al., 2022). This DA drug is believed to disrupt host immune reactions by significantly stimulating the immune response and reducing pro-inflammatory cytokines. These actions ultimately lead to modifications in its in vivo properties (Kuriakose et al., 2012). Only specific transporters in cell membranes allow the absorption of DA. This suggests the drug is ineffective in nervous-related infections because it cannot cross the blood–brain barrier. Induced resistance may develop owing to the loss of membrane transporters in response to the activity of variant surface glycoproteins. Additionally, mutations in the genes encoding carrier proteins of the parasite contribute to this resistance (Davkharbayar et al., 2020; Kasozi et al., 2022).

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Antiprotozoal Resistance

27

Table 2.3. Trypanocidal and trypanostatic drugs used for animal trypanosomiasis. (Authors’ own table.) Drug class

Drug

Diamidine

Diminazine

Disease form

Molecular targets

Powder (reconstituted with fluid); administration routes: IM or IV Phenanthridine Homidium, Powder (reconstituted Isometamedium with fluid); administration route: IM Aminoquanaldine Quinapyramine Powder (reconstituted with fluid); administration routes: IM or IV

Treatment of T. evansi

Inhibition of kinetoplast DNA biosynthesis

Treatment of T. vivax, T. congolense, and T. evansi Treatment of T. vivax, T. congolense, T. evansi and T. brucei

Powder (reconstituted with fluid); administration routes: IM or IV

Treatment of T. evansi

Inhibition of topoisomerase II during DNA biosynthesis Trypanostatic; inhibition of kinetoplast DNA; biosynthesis and loss of ribosomal function Inhibition of trypanothine reductase

Melaminophenyl arsenical

Melarsomine

Drug form

IM, intramuscular, IV, intravenous.

There are several factors that play a crucial role in determining the rate at which drug resistance evolves in a parasite population. These include drug usage level and pattern, the parasitaemia within the host (exposure of parasites to the drug), and the rate of mutation from wildtype to resistant genotype (Hastings, 2001). It has been reported that T. vivax is inherently resistant to DA (Dagnachew et al., 2015), while T. congolense is resistant to homidium (Okello et al., 2023). During in vitro studies, it has been found that dyskinetoplastic lines of trypanosomes presented drug resistance against DA (diamidines), homidium and isometamedium (phenanthridines) (Gould and Schnaufer, 2014). In T. brucei, the adenosine transporter (TbAT1) gene encodes a P2-type purine transporter, which plays a key role in the uptake of DA, and polymorphisms (six-point mutations) in this gene have been implicated in resistance to DA (Kulohoma et al., 2020). In contrast, in an orthologous gene of the T. brucei P2-type purine transporter (TcoAT1), a single-point mutation has been correlated to the resistance to DA in T. congolense (Delespaux et al., 2006). The drug resistance has been thought to be linked to these genetic mutations, which occur randomly and dissipate upon the exposure of a population of parasites to the drug, ultimately giving a selective advantage over the wild type (O’Meara et al., 2006). However, these

mutations may reduce the parasite’s fitness, leading to progressive destruction when no drug pressure is present, while survival rates are higher in the presence of drugs (Babiker et al., 2009). Despite this, these mutations will still prevail in the parasite population at low frequencies (Hastings, 2004).

2.4.2

Phenanthridine resistance

Isometamidium chloride can be used as a curative as well as a prophylactic drug in the case of African trypanosomiasis (Tihon et al., 2017). It belongs to the phenanthridine group, has amphiphilic and cationic properties, and is used in the initial stages of AAT. Unfortunately, this drug is not efficacious in T. evansi-related trypanosomiasis (CDC, 2020). The mechanism of action of this drug involves the inhibition of kDNA type II topoisomerase, which prevents kDNA replication, resulting in cell death in the trypanosome (Chowdhury et al., 2010; Tihon et al., 2017). Isometamidium can be used as a curative drug in combination with DA to minimize the risk of developing resistance. Trypanosomes, including T. congolense, T. vivax and T. brucei, have been reported to become resistant to isometamidium

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Abbas et al.

and sporadically cross-resistant to DA (Gould and Schnaufer, 2014; Baker et al., 2015; Mekonnen et al., 2018). The mechanism of resistance to phenanthridines (isometamidium and homidium bromide) has been reported to involve mutation in F1F0-ATP synthase because the mitochondrial membrane potential in this protozoan was not disturbed by the disruption of the kinetoplast (Dean et al., 2013). In T. congolense, the development of resistance was found to be linked with reduced mitochondrial membrane potential, resulting in poor absorption of the drug (Tihon et al., 2017). Moreover, a membrane-embedded transporter’s role in active drug elimination has also been described as another method of resistance development (Wilkes et al., 1997). Ethidium bromide is another name for homidium bromide, and it belongs to the phenanthridine group used in T. congolense and T. vivax infections in animals (Assefa and Shibeshi, 2018). It is also available as a chloride salt (Wainwright, 2010). Drugs related to the phenanthridine group have been reported to develop cross-resistances; therefore, homidium is recommended as a curative drug in combination with DA rather than isometamidium (Peregrine et al., 1997; Dina et al., 2002). The mode of action of this drug is similar to that of DA and isometamidium, which involve dyskinetoplasty and modifications in gene activity (Shapiro and Englund, 1990). In T. brucei, this drug disrupts the biosynthesis of nuclear and kinetoplastic DNA through twisting and modifying its structure (Chowdhury et al., 2010; Venturelli et al., 2022). The studies conducted on T. brucei indicated that this drug is thoroughly absorbed into the nucleus and kinetoplast of this protozoan. But still, the mechanism of the development of resistance against this drug, homidium, is not clearly known, although it is thought to be similar to isometamidium (Chowdhury et al., 2010).

2.4.3 Quinapyramine resistance Quinapyramine sulfate was produced from Surfen C (Curd et al., 1945). The mode of action of this drug is not clearly described except for a few hypotheses, which state that this drug interrupts DNA biosynthesis and reduces cytoplasmic ribosome activity, leading to the inhibition of

protein synthesis. Similar to DA, homidium and isometamedium, dicationic characteristics of the drug suggest its accumulation in the mitochondria (Giordani et al., 2016). In a study conducted on horses, it was suggested that quinapyramine sulfate in combination with DA has more therapeutic potential in treating dourine (Davkharbayar et al., 2020). Until 1976, the drug was prohibited for the treatment of animal trypanosomiasis due to massive resistance development (Connor, 1992). Then, from 1984 to present, it was again continued as a therapeutic drug for trypanosomiasis in horses and camels caused by T. evansi. Owing to the cross-resistance of this drug to phenanthridines and diamidines in T. congolense infections, it is not recommended in livestock (Peregrine et al., 1997). The mechanism of resistance of this drug is not clearly known; however, as mitochondria are the main target of this drug, it is presumed to develop because of modifications in the mitochondrial membrane potential, or multi-drug resistance might result from the loss of cation carriers of the plasma membrane in the mitochondria (Kasozi et al., 2022). 2.4.4 Melarsomine dihydrochloride resistance Melarsomine dihydrochloride (MDCl) is a melamino-phenylarsine in nature, and it is a highly hydrophilic drug compared to melarsoprol (Giordani et al., 2016). It is used as a trypanocidal drug for T. evansi infection (Mdachi et al., 2023). In T. brucei, the drug was reported to be absorbed by the trypanosome adenosine transporter (P2/ TbAT1) and the TbAQP2 transporter, and this mechanism is similar to that of DA and melaminophenyl arsenicals (Munday et al., 2014). The cross-resistance with DA and melarsoprol was reported to be due to the decreased activity of P2/TbAT1 transporters. It has also been observed in T. brucei infections that the strains that developed resistance to MDCl have been found to be susceptible to suramin but not to DA and melarsoprol (Zhang et al., 1991). This drug has low efficacy against T. congolense and T. vivax, which do not have TbAT1 and TbAQP2 orthologous genes. A study concluded that MDCl should not be used for treating trypanosomiasis in equines (Raftery et al., 2019).

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Antiprotozoal Resistance

2.5 Drug Resistance in Coccidiosis Coccidiosis is an infectious disease that is caused by various protozoa, including Eimeria and Isospora of the phylum Apicomplexa, affecting the intestinal tract of poultry and livestock. These parasites are cosmopolitan in distribution and cause weight loss, poor performance, production loss and death in livestock, also putting the animals at risk of developing secondary infections (Nagi and Mathey, 1972). At least seven Eimeria spp. have been found to infect various parts of the intestine, causing avian coccidiosis, while a number of species have been reported in ruminants (Shirley and Lillehoj, 2012). This pathogen spreads through the faeco-oral route. The oocysts of this parasite shed in the faeces of the infected hosts and become sporulated oocysts (infective stages) in the environment (Shapiro et al., 2019). The economic losses owing to avian coccidiosis have been considered to be more than US$3 billion per annum, affecting the poultry industry globally (McDougald et al., 2020). The control of avian coccidiosis is necessary to meet the requirement of protein for humans all over the world, as poultry meat production is expected to increase (> 2 times the current rate) by the year 2050 (Alexandratos and Bruinsma, 2012). For the sustainable availability of poultry protein sources, drug usage is essential for controlling this coccidiosis (Kadykalo et al., 2018). The drugs approved for use in the poultry sector and livestock sector for the control of avian coccidiosis and livestock coccidiosis are given in Tables 2.4 and 2.5, respectively. These drugs have been used for decades as no new drugs have been developed, which might have led to the emergence of resistance against them in fowl that has spread widely (Peek and Landman, 2011). In order to mitigate the resistance against anti-coccidials, the drugs are being used in combination to enhance their efficacy through their potential synergistic effects and/or to slow down the resistance process (Chapman et al., 2010; Chapman and Rathinam, 2022). Different combinations of anticoccidial drugs reported to be administered in Aves are given in Table 2.6. 2.5.1 Anticoccidial drugs resistance The modes of action of different anticoccidial drugs have been described clearly (Kant et al.,

29

2013), but the information related to the mechanisms involved in their resistance development is very limited or unknown. Quinolones (methyl benzoquate) inhibit the electron transport chain in the cytochrome system of the mitochondria of these protozoa. The resistance to quinolones has been reported, but information on the exact mechanism of this resistance development is not available (Wang, 1975). It is proposed that this parasite concentrates the quinolones from their surroundings, and the development of resistance could be due to the selection of the parasites, which leads to their inability to absorb the drug (Abbas et al., 2011). Amprolium blocks thiamine transport in the plasma membranes of the coccidia. The development of resistance is thought to be due to a change in the target receptor, which leads to a reduction in the sensitivity to block thiamine transport in the plasma membranes. Pyrimethamine (sulphonamides) block the activity of the dihydrofolate reductase involved in the folic acid pathway, and a similar mechanism of resistance as described for amprolium has been suggested for this drug (Kant et al., 2013). Ionophores can control the delivery of cations across the cell membranes of the parasite; therefore, they can impact a variety of processes in the cell that are based on ion transport (Chapman et al., 2010). These drugs are absorbed in the sporozoites of the parasite prior to their entry into the host cells, which leads to the death of the parasite before or after entering the host cells (Smith and Galloway, 1983). The sporozoites of Eimeria have lipid rafts and flotillin-1 protein, which they use to invade the host cells, and monensin is found to disintegrate the flotillin-1 protein, which leads to the loss of parasites’ ability to attack the host cells (Chapman and Rathinam, 2022). Monensin has been found to accumulate more in sensitive sporozoites than in resistant ones. The dose of the drug has been reported to increase 20 to 40 times for the control of resistant sporozoites compared to sensitive forms (Augustine et al., 1987). Thus, variation in the aggregation of ionophore might be related to the development of resistance. The resistance to different anticoccidial drugs has been given in Table 2.7, and a summary of the mode of action and mechanism of resistance of antiprotozoal drugs used in veterinary practice is given in Table 2.8.

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Table 2.4. Anticoccidial drugs approved for use in the poultry industry in different countries. (Authors’ own table.) Europe

USA

USA (cont’d)

Australia

Australia (cont’d)

Asia

Semduramicin Diclazuril

Clopidol Diclazuril

Sulfadimethoxine Sulfachloropyrazine

3,5-Dinitro-O-toluamide

Monensin, Lasalocid

Robenidine

Lasalocid

Narasin + nicarbazin

Lasalocid A Toltrazuril Monensin Halofuginone Narasin + nicarbazin Nicarbazin Narasin Salinomycin

Monensin Nicarbazin Robenidine Semduramicin Salinomycin Narasin Maduramicin Halofuginone

Ormetoprim + sulfadimethoxine Sulfamethazine Sulfamethazine + sulfaquinoxaline Zoalene Sulfaquinoxaline Amprolium + ethopabate – –

Toltrazuril – – – – – – –

Amprolium Sulphadimidine, Sulphaquinoxaline, Sulphadimethoxine, Sulphanitran Sulphaguanidine

Decoquinate Maduramicin

Decoquinate Amprolium

Methyl benzoquate + clopidol Amprolium Decoquinate Lasalocid Monensin Maduramicin Narasin Robenidine Maduramicin + nicarbazin Salinomycin Amprolium + ethopabate

Semduramicin Diaveridine + sulfaquinoxaline Sulfaquinoxaline

Abbas et al.

– –

Salinomycin

– –

Sources: Europe, https://www.vmd.defra.gov.uk/ProductInformationDatabase; USA, https://www.fda.gov/AnimalVeterinary/default.htm; Australia, https://apvma.gov.au/node/427 (accessed 9 April 2024); Asia, Kant et al., 2013.

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Antiprotozoal Resistance

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Table 2.5. Anticoccidial drugs approved for use in the livestock industry in different countries. (Authors’ own table.) USA

USA (cont’d)

Europe

Australia

Amprolium Diclazuril Maduramicin Ormetoprim Sulfachloropyrazine Sulfaquinoxaline Clopidol Ethopabate Monensin Robenidine Sulfadimethoxine Tylosin

Zoalene Dexamethasone Lasalocid Nicarbazin Semduramicin Sulfamethazine Decoquinate Halofuginone Narasin Salinomycin Sulfamerazine

Decoquinate Lasalocid Toltrazuril Monensin Diclazuril – – – – – – –

Toltrazuril Lasalocid Salinomycin Monensin Narasin – – – – – – –

Sources: USA, https://www.fda.gov/AnimalVeterinary/default.htm; Europe, www.EMA.Europa.eu; Australia, https://apvma. gov.au (accessed 9 April 2024).

Table 2.6. Combination anti-coccidials administered in avian coccidiosis for mitigating the resistance. (Authors own table.) Drug combination

Route of administration

Amprolium + sulfaquinoxaline + ethopabate Maduramicin + diclazuril Sulfaquinoxaline + diaveridine Narasin + nicarbazin Sulfanitran + roxarsone + aklomide Monensin + nicarbazin Sulfaquinoxaline + sulfamezathine + sulfamerazine Methyl benzoquate + clopidol Sulfanitran + roxarsone + nitromide Maduramicin + nicarbazin Sulfaquinoxaline + pyrimethamine Amprolium + sulfaquinoxaline + ethopabate + pyrimethamine Sulfadimethoxine + ormetoprim Amprolium + ethopabate

Feed Feed Water Feed Feed Feed Water Feed Feed Feed Feed Feed Feed Feed

Table 2.7. Anticoccidial drug resistance reported against field strains of Eimeria in fowls. (Authors’ own table.) Drug

Eimeria species

Resistance described by/study year

Sulphaquinoxaline Nitrofurazone Nicarbazin Dinitolmide Amprolium Clopidol

E. tenella Not available E. tenella (severe) E. tenella and E. necatrix E. tenella (moderate) E. acervuline, E. maxima and E. tenella (moderate) Not available E. tenella E. tenella E. maxima E. maxima E. tenella E. acervuline

Abbas et al., 2011; Ojimelukwe et al., 2018 Sundar et al., 2017 Ferdji et al., 2022; Sun et al., 2023a Sundar et al., 2017 Ojimelukwe et al., 2018; Sun et al., 2023a Sun et al., 2023a

Buquinolate Methyl benzoquate Decoquinate Monensin Robenidine Halofuginone Lasalocid

Noack et al., 2019 Noack et al., 2019 Tan et al., 2017; Hao et al., 2023 Djemai et al., 2016 Ferdji et al., 2022 Sun et al., 2023b Györke et al., 2012 Continued

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Table 2.7. Continued. Drug

Eimeria species

Resistance described by/study year

Arprinocid Salinomycin Narasin

E. tenella E. tenella (severe) E acervuline, E. maxima and E. tenella E. tenella E. acervuline, E. maxima and E. tenella E. tenella

Shirley and Harvey, 2000 Sun et al., 2023a Djemai et al., 2016

Maduramicin Diclazuril Toltrazuril

Tan et al., 2017 Györke et al., 2011 Ojimelukwe et al., 2018

Table 2.8. Summary of mode of action and mechanism of resistance of antiprotozoal drugs used in veterinary practice. (Authors’ own table.) Drug/ protozoal disease (references)

Mode of action

Diminazene aceturate/ Interference with the aerobic Babesiosis glycolysis and DNA synthesis of (Lindquist, 1986; Peregrine, 1994; the Babesia Sakuma et al., 2009; Rajapakshage et al., 2012b; Plumb, 2015; Kumara, 2016)

Imidocarb dipropionate/ Babesiosis (Bacchi et al., 1981; Plumb, 2015)

Atovaquone–azithromycin/ Babesiosis (Sakuma et al., 2009; Birth et al., 2014; de Oliveira Silva et al., 2016; Liu et al., 2016; Lemieux et al., 2016) Buparvaquone and azithromycin/ Babesiosis (Mhadhbi et al., 2015) Buparvaquone/ Theileriosis (Fry and Pudney, 1992; Chatanga et al., 2019; Ali et al., 2022)

Blocking of the cellular repair and replication and damage to the nucleic acid by integrating in the DNA of susceptible Babesia parasite Inhibiting the ability of parasites to synthesize or utilize the polyamines Atovaquone suppresses the pyrimidine and ATP production through the selective inhibition of the electron transport complex in mitochondria Azithromycin interrupts the protein translation in the apicoplast Same as for atovaquone– azithromycin combination

Inhibition of the electron transport chain in the parasite Inhibition of TaPin1 isomerase activity

Resistance (Yes/No) and its mechanisms Yes/ Not been clearly described in Babesia spp. except a few theories: mitochondrial DNA due to mutation in cytochrome c oxidase subunit III or I and cytochrome b (CYTb) genes Changes in the glycolytic pathway Involvement of HSP90 and HSP27 heat shock proteins No/ Not available

Yes/ Atovaquone resistance due to mutation at cytochrome b gene of the protozoan Resistance to this combination of drugs due to mutations in RPL4 and CYTb genes Yes/ Same as for atovaquone – azithromycin combination Yes/ Not been clearly described Mutations in mitochondrial cytochrome B gene Mutation (129G-GGC) in the binding pocket (Q01) and two mutations (253S-TCT and 262S-TCA) within the binding pocket (Q02) Continued

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Table 2.8. Continued. Drug/ protozoal disease (references)

Resistance (Yes/No) and its mechanisms

Mode of action

Diminazene aceturate/ Inhibition of kinetoplastic Trypanosomiasis DNA biosynthesis (Delespaux et al., 2006; Kulohoma et al., 2020)

Isometamidium chloride/ Trypanosomiasis (Dean et al., 2013)

Binding to the kinetoplast DNA of trypanosome

Homidium bromide/ Trypanosomiasis (Shapiro and Englund, 1990; Chowdhury et al., 2010) Quinapyramine sulfate/Trypanosomiasis (Giordani et al., 2016; Kasozi et al., 2022)

Dyskinetoplasty and modification in gene activity

Melarsomine dihydrochloride/ Trypanosomiasis (Zhang, et al., 1991; Munday et al., 2014) Anticoccidial drugs/ Coccidiosis (Smith and Galloway, 1983; Abbas et al., 2011; Ferdji et al., 2022; Sun et al., 2023a)

2.6

Not clearly known but two hypotheses: Inhibition of protein synthesis Accumulation of drug in the mitochondria

Yes/ Polymorphism in trypanosome adenosine transporter (TbAT1) gene in T. brucei Single point mutation in T. brucei P2-type purine transporter (TcoAT1) in T. congolense Yes/ Mutation in F1F0-ATP synthase subunit Reduced mitochondrial membrane potential resulting in poor absorption Yes/ Not clearly known Mutation in F1F0-ATP synthase subunit Yes/ Not clearly known, and it is supposed: Due to modification in the mitochondrial membrane potential Multi-drug resistance might happen due to loss of cation carrier of the mitochondrial plasma membrane Yes/ Due to decreased activity of P2/TbAT1 transporters

Selective absorption of drug by trypanosome adenosine transporter (P2/TbAT1) and TbAQP2 transporter Quinolones (methyl benzoquate) Yes/ No clear mechanisms inhibit electron transport in the available mitochondria Amprolium blocks the thiamine transport in the plasma membranes Pyrimethamine (sulphonamides) blocks the activity of the dihydrofolate reductase involved in the folic acid pathway Ionophores can control the delivery of cations across the cell membranes

Conclusion

Protozoal parasites, such as Theileria, Babesia and Trypanosoma, have developed various mechanisms including mutations in different genes to evade the effects of antiprotozoal drugs. This has

led to the emergence of drug-resistant strains, which can spread rapidly and pose a serious threat to both animal and human health. The current chapter highlighted the mode of action and mechanisms involved in the development of resistance against drugs used in haemo- and

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enteric protozoal infections to provide a way forward for the discovery of new drugs to ensure the animal welfare and health and sustainable livestock production. Therefore, drugs with new

molecular mechanisms of action and novel targets are in dire need to overcome the emergence of resistance and to control protozoal infections efficiently.

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3

Anthelmintic Resistance

Aayesha Riaz1*, Faiza Bano1, Manuela Marescotti2, Evelyn Saba3 and Zahid Manzoor1 1 Department of Parasitology and Microbiology, Faculty of Veterinary and Animal Sciences, PMAS-Arid Agriculture University Rawalpindi, Pakistan; 2University of Edinburgh Centre for Discovery Brain Sciences, Edinburgh, UK and Brain Communications Editorial Office, University of Edinburgh, Edinburgh, UK; 3 Department of Biomedical Sciences, Faculty of Veterinary and Animal Sciences, PMAS-Arid Agriculture University Rawalpindi, Pakistan

Abstract Helminths, including cestodes (tapeworms), trematodes (fukes) and nematodes (roundworms), are parasites that cause severe health-related illnesses in both livestock and humans worldwide. Various anthelmintics are employed to control infections caused by these helminths. Anthelmintic resistance (AR) refers to the genetic impairment of parasite sensitivity to anthelmintics that were previously effective against similar parasites. Several helminths of veterinary signifcance have developed AR, which is transmitted to subsequent generations. In livestock, AR results in substantial economic losses due to decreased production and increased treatment costs. This resistance poses a signifcant threat to the future welfare and productivity of animals. This summary highlights potential predisposing and contributing factors towards the development of AR, such as the frequent use of the same anthelmintic, the administration of anthelmintics at suboptimal dosages, prophylactic measures in animals, and the continuous and frequent use of a single medication. Anthelmintic resistance is a global concern, and the selection of suitable anthelmintics coupled with a reduction in dependency on these drugs is essential to mitigate this challenge. Implementation of rigorous quarantine and hygiene measures, along with the adoption of combination drug therapy, are crucial approaches to prevent the development of AR.

3.1 Introduction Helminths constitute a diverse group of parasites, encompassing nematodes, cestodes and trematodes, causing severe diseases in both animals and humans globally (Thaenkham et al., 2022). While pasture control for domestic animals can mitigate the impact of parasites, such measures are sometimes insufficient for complete parasite elimination (Sargison, 2020). Pharmaceutical anthelmintics play a crucial role in controlling

helminthiasis (Mortensen et al., 2003), gaining significant importance owing to the absence of parasitic vaccines (Gilleard et al., 2021). In the past, anthelmintic control was highly effective in animals, attributed to the drugs’ efficiency, safety, broad-spectrum effect and cost-effectiveness (Lanusse et al., 2014). The irrational use and overuse of anthelmintics over time have, however, led to a substantial increase in anthelmintic resistance (AR), particularly in gastrointestinal parasites affecting sheep, cattle, equines and goats

*Corresponding author: [email protected] © CAB International 2024. Antiparasitic Drug Resistance in Veterinary Practice (eds H.M. Rizwan, M.A. Naeem, M. Younus, M.S. Sajid and X.Chen) DOI: 10.1079/9781800622807.0003

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(Gerwert et al., 2002). The rising levels of AR pose significant challenges to animal health and production on a global scale. Currently, three families of anthelmintics are in use: benzimidazoles, nicotinic receptor agonists and macrocyclic lactones. The use of these anthelmintic drugs at the commercial level has been linked to an increase in AR (Mederos et al., 2018). Overuse of anthelmintics stands out as the primary reason for the escalating AR. The development of anthelmintic resistance is a highly intricate process influenced by various factors, including host species, parasite species, type of anthelmintic, dosage, animal management and climatic variations (Potârniche et al., 2021). Addressing these factors, alongside challenges in developing new anthelmintics and overcoming issues with resistant strains, is crucial in tackling the problem of AR (Sargison, 2016). Effective alternative strategies for controlling helminth infections have not been identified thus far. Researchers are dedicating significant efforts to consistently recognize predisposing factors, elucidate potential mechanisms of AR and develop novel detection methods (van Wyk et al., 1999). The key objective of this chapter is to highlight major groups of helminths affecting livestock, delve into AR, examine predisposing and contributing factors, explore potential mechanisms of AR, discuss detection methods, and present strategies for mitigating or postponing AR.

3.2 Global Parasitic Challenges and Strategies The demand for meat production and dairy products is on the rise due to a growing global population (Van Kernebeek et al., 2016). Livestock production plays a critical role in meeting this demand, particularly in areas where land is unsuitable for crop cultivation (Nieuwhof and Bishop, 2005). Helminths cause severe illnesses in their hosts, posing a threat not only to animal and human health but also to overall welfare (Overgaauw et al., 2020). Helminth infections present a massive global challenge due to the stable nature of their eggs and larvae in the environment (Baulcombe et al., 2009). These diseases, often neglected by both livestock and healthcare organizations, can result in long-lasting and

asymptomatic infections if not addressed in the initial stages (Alvar et al., 2020), leading to debilitation, economic losses, and morbidity in both livestock and humans (Rist et al., 2015). Common helminths include filarial worms, soil-transmitted helminths, schistosomes, abdominal nematodes and onchocerciasis (Bethony et al., 2006; Holzhauer et al., 2011). Parasites exhibit complex life cycles with multiple phases, hosts and adaptations to various environments. The life cycle of certain helminths, such as soil-borne nematodes, hookworms and strongyloides, involves free-living phases like larvae, spanning different environments and hosts (Chiotti and Johnston, 1995). In cattle, infections from gastrointestinal (GI) nematodes, liver flukes and lungworms significantly impact production efficiency, with GI nematodes and liver flukes often prevailing in subclinical forms (Morgan et al., 2013). Lungworm infections are more severe, imposing an extraordinary economic burden on farms owing to deaths and sharp reductions in milk yield. The general rise in prices has severely impacted the economy of dairy farms, with small changes in production efficiency making significant differences in profits and losses (Lopes et al., 2016). It is crucial to assess carefully the economic damages caused by these parasites and implement precautionary measures to reduce losses (Holzhauer et al., 2011). Most animals are exposed to helminth infections in pastures during grazing, causing challenges in achieving national and international trade goals (Rehbein et al., 2013). Currently, there is a restricted and uneven understanding of costs associated with parasitism and the economy linked with parasite control. Future policies should be devised to control the described losses, ensuring realistic implementation and easy adoption by farming systems (Charlier et al., 2012). Global warming and environmental changes contribute to increased parasite survival, leading to prolonged pasture contamination with different infectious stages of these parasites (Fig. 3.1). These global changes act as drivers for a change in parasitic behaviour to adapt to the evolving environment. For instance, changes in the periodic stages of nematode and liver fluke infections have been reported in northern areas of the UK (de la Rocque et al., 2008). In Switzerland, transmission of Haemonchus contortus is occurring at higher altitudes than previously observed (Schweizer et al., 2005), and in

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Impact of global changes Climate change

Husbandry practices

Land use

Farm economy

Farming practices

Parasitic contamination Increase in: parasitic diseases, treatment cost, use of anthelmintics

Production losses

Anthelmintic resistance

Fig. 3.1. Impact of global changes on pasture contamination with helminth parasites. (Author's own figure.)

Sweden, transmission occurs near the Arctic sphere (Lindqvist et al., 2001). The quantity and transmission of helminth infections in animals present multifarious and global issues (Laurenson et al., 2013). Global changes can affect factors such as climate change, land use, husbandry and farming practices, and farm economy (Fig. 3.1), causing an increase in parasitic contamination in pastures, leading to parasitic diseases in grazing animals, economic losses and anthelmintic resistance. According to the World Organization for Animal Health (Office International des Epizooties, OIE), AR is a One Health issue and a significant component of antimicrobial resistance (AMR) affecting both human and animal health (Picot et al., 2022). It is also considered an escalating danger for the livestock industry. The issue of AR has been faced for many years but gained global attention in 2019 (OIE, 2021), prompting the initiation of strategies to devise guidelines for the responsible and prudent use of anthelmintic chemicals to control AR in grazing livestock species (OIE, 2021, 2022). The control of AR is essential for the livestock industry, food industry, and the general population relying on animal products such as milk, meat, hides, etc. (Sargison, 2020). AR can also

affect human health as some of these parasites are zoonotic and can cause severe infections. The development of AR can strengthen zoonotic pathogens pathogenically and lead to prolonged survival (Picot et al., 2022). To control or limit AR in helminths, a strategy similar to AMR control should be employed. However, there is limited information available on the risk of AR in animals, its transmission and its impact on human health (Ferreira et al., 2022).

3.3 Anthelmintics and their Mode of Action One key prerequisite for an effective anthelmintic is selective toxicity, ensuring the ability to eliminate parasites without harming the host (Nixon et al., 2020). This is typically achieved through mechanisms such as inhibiting vital metabolic processes specific to parasites or employing pharmacokinetic properties that enhance anthelmintic concentrations in parasites compared to host cells (Alavi and Shahmabadi, 2021). Anthelmintic activity relies on diverse pharmacokinetic processes that influence drug concentration at the site of action and subsequent pharmacological effects (Fig. 3.2).

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Concentration of the drug interacting with parasites on/in host’s body The fate of the drug within the target parasites (influx and efflux balance)

Anthelmintic activity depends on Drug’s interaction with parasite’s physiological mechanisms Time of exposure of drug on the basis of parasite’s life cycle

Fig. 3.2. Diverse pharmacokinetic processes of anthelmintics. (Author's own figure.)

While the precise mode of action for many anthelmintics remains unclear, their sites of action and biochemical mechanisms are generally known (Jayawardene et al., 2021). The mode of action primarily depends on the parasite’s ability to produce proteins, cations, neuromuscular receptors and anions, as well as the anthelmintic drug class (Choudhary et al., 2022). Pharmacologically, these drugs should disrupt parasitic cellular processes, such as benzimidazoles inhibiting tubulin polymerization, interfering with neuromuscular coordination by inhibiting or mimicking neurotransmitters (organophosphates and imidazothiazoles, etc.) (Woodgate et al., 2017). They bind to specific sites, inducing unsteadiness and convulsive paralysis, ultimately leading to the parasite’s death. It has been reported, however, that certain parasitic strains develop resistance to anthelmintics (Prichard, 2008).

3.3.1

Benzimidazoles

Benzimidazoles (BZs), introduced commercially in 1961, faced resistance by 1964 (James et al., 2009). The BZ group includes oxfendazole, albendazole, mebendazole, thiabendazole, thiophanate, febantel and netobimin (Taylor et al., 2007). With low or no absorption from the GI tract, direct administration into the abomasum enhances efficacy against nematodes, increases excretion rates and decreases GI tract absorption (Lanusse et al., 2016). Belonging to the class of anthelmintic drugs, BZs bind to the β-tubulin subunit of cytoskeletal microtubules in parasitic worms. Microtubules are crucial for maintaining cell shape, division and movement. By binding to β-tubulin, BZs inhibit new microtubule formation, disrupting existing ones, causing cytoskeletal collapse, and leading to cell death (Lanusse et al., 2016).

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Thiabendazole (TBZ), a BZ variant, inhibits fumarate reductase activity, disrupting the parasite’s energy metabolism and causing its death (Aremu et al., 2012). Benzimidazoles, besides affecting the cytoskeleton, interfere with other cellular processes such as glucose uptake and energy metabolism. This broad-spectrum impact contributes to their efficacy against various parasitic worms, encompassing intestinal and tissue-dwelling roundworms, tapeworms and flukes (Friedman, 1979).

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7–8 years (James et al., 2009). Macrocyclic lactones, including ivermectin, disrupt gamma-aminobutyric acid (GABA) neurotransmission by interfering with glutamate-gated chloride channels (GluCl) in parasites, which include α7 nicotinic acetylcholine receptors (nACh) (Wolstenholme and Rogers, 2005). GluCl exhibits a high affinity for ivermectin, contributing to the anthelmintic efficacy of the drug (Holden-Dye and Walker, 2007).

3.3.4 Derivatives of amino acetonitrile 3.3.2

Levamisole/imidazothiazole

Levamisole (LEV), also known as imidazothiazole, became commercially available in 1970, with the first report of AR surfacing in 1979 (James et al., 2009). Levamisole targets nicotinic acetylcholine receptors, acting as an agonist at the neuromuscular receptor, and inhibiting the enzyme acetylcholinesterase, responsible for acetylcholine breakdown (Izquierdo et al., 2023). By blocking this enzyme, LEV accumulates acetylcholine in the neuromuscular junctions of the parasite, inducing spastic paralysis, reducing egg laying and ultimately causing death (Holden-Dye and Walker, 2007). Levamisole anthelmintic activity primarily involves stimulating ganglion activity, leading to sustained muscle contractions and subsequent neuromuscular depolarizing blockade, resulting in paralysis. Subsequently, peristalsis facilitates the expulsion of intestinal parasites (Malik et al., 2022). Effective against various nematode parasites, including intestinal worms and lungworms in animals, LEV is also utilized in humans for treating specific parasitic infections such as ascariasis and hookworm infection (Gilleard et al., 2021). While LEV demonstrates efficacy against parasitic worms, its use requires careful management to prevent drug resistance. Notably, LEV can induce side effects in both animals and humans, emphasizing the importance of its use under the guidance of a veterinarian or healthcare professional (Caudell et al., 2022).

Amino acetonitrile derivatives (AADs), considered modern synthetic anthelmintics, are currently in experimental use exclusively in animals (Rufener et al., 2009). Synthesized through Strecker synthesis and amine acylation via aroyl chlorides, as well as phenol alkylation using chloroacetone, AADs exhibit tolerance to diverse aroyl moieties and aryloxy on an amine acetonitrile core (Kaminsky et al., 2008; Rufener et al., 2009; Aremu et al., 2012). Functionally, AADs inhibit the unique clade of acr-23 nicotinic acetylcholine receptor subunits, inducing paralysis in the targeted parasites (Rufener et al., 2009; Aremu et al., 2012). Monepantel, specifically AAD 1566, demonstrates selective action against nematodes, positioning it as a promising candidate for future anthelmintic applications against nematodes (Tritten et al., 2011).

3.3.5 Piperazine Piperazine, introduced in the 1950s, emerged as a valuable tool for the management of filariasis and threadworms (Idris et al., 2019). Its efficacy as a GABA receptor antagonist results in reversible flaccid paralysis of body walls and muscles in helminths, such as Ascaris suum (Holden-Dye and Walker, 2007).

3.3.3 Macrocyclic lactones

3.4 Factors Involved in the Development of Anthelmintic Resistance

Ivermectin, classified under macrocyclic lactones, was introduced in 1981, yet AR emerged within

AR poses a significant threat to the sustainability and efficacy of animal production. Various

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methods, including chemical, immunological, nutritional and biological approaches, are employed to control helminths in animals. However, the current practical control relies heavily on effective anthelmintic drugs, which, while having broad-spectrum actions against common helminths, require specific and frequent treatments each year (Demeler et al., 2009). Initially highly effective, the regular, widespread use, and misuse, of these drugs may have contributed to the development of resistant parasitic populations (El-Abdellati et al., 2010). The reported effectiveness of anthelmintics stands at approximately 99% against sensitive strains (Le Jambre et al., 1999); however, a small number of parasites exhibit resistance, transmitting into the environment and contaminating it. This contamination leads to the proliferation of resistant populations, contributing to the development of AR owing to selection pressure (Fissiha and Kinde, 2021). The expansion of AR is influenced by numerous factors responsible for its occurrence and spread (Jabbar et al., 2006). The potential factors contributing to the development of anthelmintic resistance are outlined below. 3.4.1 Mechanism of development of anthelmintic resistance AR in helminths is a complex phenomenon driven by various mechanisms. Understanding these mechanisms is crucial for devising effective strategies to combat resistance. Several key mechanisms are implicated in the development of AR.

are directly linked to AR, and their impact may vary among different parasites (Doyle and Cotton, 2019). Multidrug resistance (MDR) Certain genes, such as P-glycoproteins belonging to the ATP-binding cassette (ABC) transporter subfamily (ABCB1), mediate AR by actively transporting drugs out of the cell. This active transport, fuelled by ATP hydrolysis, leads to multidrug resistance (MDR). The increased expression of P-glycoproteins, observed in parasites resistant to drugs such as praziquantel, triclabendazole and ivermectin, indicates their role in AR development (Lage, 2003; Reed et al., 1998; Smith and Prichard, 2002). Antioxidant enzymes Parasites rely on a balance between oxidation and antioxidation for survival. Anthelmintics generating free radicals can be detoxified by the natural antioxidant defence enzymes of parasitic cells. Changes in these defence systems or enzymes can contribute to the development of AR against various anthelmintics. The sensitivity of immature schistosomes compared to mature or adult parasites may be attributed to differences in the detoxification enzymes (Hewitson et al., 2009). Understanding these mechanisms provides valuable insights for the development of targeted interventions to curb the emergence and spread of anthelmintic resistance. Mechanisms of resistance development in commonly used anthelmintic classes are given in Fig. 3.3.

Single nucleotide polymorphisms (SNPs)

3.4.2 Frequency of anthelmintic use

AR is often associated with modifications in the cellular receptor binding sites of drugs, resulting from changes in the genome arrangement. SNPs, unique genetic variances arising from gene mutations post-drug interaction, play a crucial role. Examples include mutations in the β-tubulin gene causing BZ resistance in H. contortus (Roos et al., 1990; Avramenko et al., 2020). Deep amplicon sequencing (DAS) has proven effective in detecting and quantifying SNPs linked to BZ resistance (Avramenko et al., 2019; 2020). It is important, however, to note that not all SNPs

The extensive use of similar anthelmintics types may lead to the development of AR (Picot et al., 2022). Resistance tends to develop more efficiently in areas where animals undergo frequent deworming. The frequency of treatment is identified as a crucial factor in the development of AR, allowing resistant parasites a reproductive and replication advantage for two to three weeks post-treatment (Fissiha and Kinde, 2021). In the case of H. contortus, instances of AR have been observed in certain tropical regions where animals received 10–15 doses each year to treat parasitic

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Anthelmintic class

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Mechanism of resistance formation

Benzimidazoles, probenzimidazoles

Mutation in the gene coding for ˜tubulin isotype 1

Imidazothaizoles, tetrahydropyrimidines

Decrease in the number of nicotinic acetylcholinesterase receptors or by a reduced affinity of these receptors for the drug

Amino-acetonitrile derivatives

Loss of part of nAChR superfamily member ACR-23

Macrocyclic lactones: avermectins milbemycins spinosyns

Changes in the P-glycoproteins that are involved in export of the drug, leading to reduced concentration of the drug inside the cell

Fig. 3.3. Mechanisms of resistance development in commonly used anthelmintic classes. (Author's own figure.)

infections. The development of AR is closely tied to the treatment frequency, providing better-fit parasites with increased chances of survival compared to susceptible parasites (Coles et al., 1995), as shown in Fig. 3.4. 3.4.3 Dose rates and routes of anthelmintic administration Underdosing is a significant factor in the development of AR, as sub-therapeutic dosages may allow the existence of heterozygous-resistant parasites (Fissiha and Kinde, 2021). Laboratory trials have also revealed that doses less than the dosage calculated on the basis of body weight can lead to the development of resistant strains (Leathwick and Luo, 2017). Furthermore, differences in bioavailability across diverse host classes are critical when determining precise dosages (Hoekstra et al., 1997). Prolonged use, improper management and variable anthelmintic dosages are major factors

contributing to the development of AR. Body weight is commonly used to determine anthelmintic dosage in veterinary medicine, but it can be unsuitable at times, leading to underdosing (Hennessy, 1994). Various administration routes, including oral drenches, topical or injectable routes, are commonly used in animals. The route of administration significantly affects the time to reach the target site, parasitic population and drug efficacy (Leathwick and Luo, 2017). Administering the same drug by different routes against the same worm populations results in differences in the delivery of the active drug to the target worms (Leathwick et al., 2020). This variability in active drug dose reaching the parasite can lead to suboptimal dosing, contributing to the development of AR (Walker et al., 2021). Not only frequent dosing but also underdosing over an extended period increases the probability of AR and enhances the selection of a resistant parasite population (Verma et al., 2018). Leathwick and Luo (2017) used a model

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P Population of bacteria rresistant to antibiotic Frequent anthelmintic c treatment Acquisition of anthelmintic resistance

Resistant parasites

Reproductive and replication advantage by resistant parasites

Sensitive parasites Fig. 3.4. The frequency of treatment contributes to an increased population of resistant parasites, owing to their reproductive and replication advantage. (Author's own figure.)

framework to estimate AR in animals given variable dose rates. The study results indicated that lower concentrations or variable concentrations of the drug reaching the target parasites are more likely to select for resistance. 3.4.4 Targeting and mass treatment Mass treatment of animals is another factor contributing to the development of AR in helminths. Computer programs have indicated that the development of AR can cease when 20% of the animal herd is left untreated; however, clinical and laboratory verification is necessary. While leaving some animals with parasitic infections untreated may not significantly impact the treatment, it can affect the delay of AR (van Wyk, 2001). In routine practice to control parasitic infections in animals, it is common to treat them with anthelmintic drugs before moving them to clean pastures. These activities can contribute to the development of resistant groups of parasites (Smith, 1990). 3.4.5

Single drug regimens

The routine and frequent use of a single medicine can lead to the development of AR, as discussed

previously. For example, a particular drug that is initially active and effective may lose its effectiveness after constant use (Shalaby, 2013). Reports indicate that nearly 50% of farmers continue using a single drug until it becomes ineffective (Waller, 1997). Prolonged use of levamisole in cows has been linked to the development of AR (Geerts et al., 1987). Additionally, the frequent and single use of the ivermectin drug has resulted in the development of AR in H. contortus in some regions of the world (Shoop, 1993).

3.5 Detection and Monitoring of Anthelmintic Resistance AR should be monitored and diagnosed with extreme care using different standards. It is crucial to note that various conditions may cause clinical signs similar to those associated with parasitism. Additionally, factors such as underdosing, expiration of drugs and improper administration can lead to the failure of anthelmintics to control parasites, aside from AR itself (Avramenko et al., 2019). Various methods are employed for the detection and monitoring of AR. The most widely used method for detecting AR in nematodes is the faecal egg count reduction test (FECRT),

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which is suitable for all types of anthelmintics, including those that undergo metabolism in the host (Papadopoulos et al., 2012). In vitro assays that measure the effects of anthelmintics on the development, growth or movement of nematode stages have also been developed as alternative methods of detection (Zajac et al., 2021). The irrational use of anthelmintics is often linked to problems with AR (Jabbar et al., 2006). Care should be taken during the administration of anthelmintics, considering factors such as the live weight of the animal for correct dose calculation, information regarding storage conditions, and the correct method of administration (pour on, drenching and/or injections). Additionally, measures should be in place to avoid contamination, such as using clean utensils for administration (drenching equipment, etc.; Ihler, 2010). Various methods and strategies, including in vivo methods (FECRT), in vitro methods (egg hatch assay, larval development assay, larval motility test, tubulin binding assay, esterase activity test; for details of in vivo and in vitro methods, see Chapter 8), molecular techniques (for details, see Chapter 7), and other reliable and uniform detection systems, are being utilized to detect, diagnose and monitor AR (Gilleard, 2006; Avramenko et al., 2019).

3.6 Strategies for Anthelmintic Resistance Management Anthelmintic resistance poses a significant challenge in managing parasite infections among livestock globally. Early studies in Denmark identified levamisole resistance in Ostertagia circumcincta sheep nematodes (Sangster et al., 1979). Subsequent reports highlighted resistance to ivermectin, levamisole and BZ in Trichostrongyles among goat flocks (Chartier et al., 1998). Sweden reported BZ resistance in four out of 26 flocks, indicating a complex scenario (Lind et al., 2007). Controlling animal movement emerges as a key strategy to mitigate resistance transmission (Sih et al., 2018). 3.6.1

Integrated approaches to AR management

Addressing AR requires strategic, integrated management approaches. Delaying resistance

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onset involves maintaining sufficient parasites in refugia, although the efficacy of using multiple products diminishes over time. Methods such as mixing susceptible and resistant worms and non-chemical controls are in early development stages (Bartram et al., 2012). Maintaining an acceptable refugia level is crucial for delaying resistance, with refugia representing a population sensitive to anthelmintics but devoid of AR (Hodgkinson et al., 2019). Scientists traditionally advocated using one anthelmintic at a time, a strategy challenged by the emergence of AR (Sangster and Dobson, 2002).

3.6.2 Preventing AR development in farm animals Effective management policies are futile if farmers acquire animals carrying resistant parasites. Stringent quarantine measures for new additions become imperative. Recent cases revealed farms with superior breeding stock forming discrete groups with resistance to BZ and moxidectin (Fontenot et al., 2003). Preventing the spread of resistance necessitates precautions, including suspending feed before treatment, administering multiple anthelmintics and conducting thorough assessments post-treatment (Mphahlele et al., 2021).

3.6.3

Synergistic approach to AR prevention

Combining anthelmintics from different groups concurrently is a preventive method against AR. This strategy, involving dissimilar biochemical classes, aims for a synergistic effect to enhance treatment efficacy, especially when resistance to both drugs is low (Lanusse et al., 2018). If resistance significantly increases for both drugs, however, the synergistic effect may diminish (Barnes et al., 1995). Traditional beliefs in favour of annual rotation to reduce AR have been challenged, and current recommendations suggest changing anthelmintics only when resistance has developed (van Wyk, 2001; Bartram et al., 2012). Combination therapies exhibit efficacy against various parasitic infections. A praziquantel and oxamniquine combination effectively

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combats Schistosoma infections (Utzinger et al., 2003). Synergism is observed with ivermectin and albendazole against shistosomiasis and soiltransmitted helminthiasis (Utzinger and Keiser, 2004). Levamisole and mebendazole show synergistic action against H. contortus in sheep (Bennet et al., 1980). Such combinations are crucial in delaying and controlling AR development in nematodes at the farm level (Lind et al., 2007; Bartram et al., 2012). Combination drugs play a pivotal role in controlling liver flukes, especially in cases of resistance. Triclabendazole (TCBZ) resistance, initially identified in Australia, spread across Europe. The combination of TCBZ and luxabendazole has proven effective against liver flukes (Fairweather, 2005). Studies also explore combinations of natural and synthetic anthelmintics, with promising outcomes. For instance, a combination of ivermectin and Nigella sativa oil demonstrated greater anthelmintic potential in vitro than either alone (Nielsen et al., 2006).

3.7 Alternative Strategies to Manage AR Decreasing the frequency of anthelmintic use is valuable in reducing the rate of AR development (Fissiha and Kinde, 2021). Implementing better feed management is a potential and supportive method to diminish the frequency of anthelmintic use (Bartram et al., 2012). Biotic control is also a useful method to reduce anthelmintic use (Larsen, 2006). These management strategies aim not only to eradicate free-living larval phases but also to kill adult parasites (Waller et al., 2004).

3.7.1 Genetic improvement Genetics plays a significant role in variations in resistance/sensitivity to helminth infection. Resistance to disease depends on immunity-related genes in the host (Sarai et al., 2014). Some animal breeds are relatively unaffected by infection, showcasing better resistance to parasitic infection. Experiments in New Zealand for a decade on breeding sheep have shown successful results, reducing the effects of roundworms on the

health and productivity of sheep and the use of anthelmintics (Bisset et al., 2001). 3.7.2 Nutrition The relationship between parasitism and nutrition is exemplified by protein consumption and resistance to gastrointestinal infection (Besier et al., 2016). Immunity is associated with protein repletion, and gastrointestinal parasites increase the need for amino acids in animals. Animals infected with gastrointestinal nematodes will willingly select advanced protein regimes compared to uninfected animals (Silva et al., 2022). Animals infected with gastrointestinal nematodes may initially decline feed consumption. Supplementation with phosphorus has been shown to stop parasite formation, and cobalt insufficiency has been linked to concentrated immunity to gastrointestinal nematodes. Adequate copper standards are essential for improving immunity to gastrointestinal nematodes (Sykes and Coop, 2001).

3.7.3 Pasture management The objective of pasture management is to offer safe meadows for feeding. A safe pasture is one where animals have not grazed for six months during cool weather and three months during the hot season (Steiner et al., 2019). Grazing areas should be sectioned into smaller lots to permit an extended period before feeding. Pasture that is severely affected by parasites due to mishandling can be disinfected and reseeded (Shalaby et al., 2012). Pasture management is crucial during spring and rainy weathers to reduce pasture contamination, and deworming of animals during these seasons will provide an added effect in clearing the parasites (Athanasiadou et al., 2001). Allowing animals to graze in larger areas can also reduce acquiring larvae because most infective larvae are found within 2 inches of the soil surface (Niezen et al., 2002).

3.7.4 Nematode-trapping fungi Biological control of nematodes can be achieved by certain fungi, such as Duddingtonia flagrans,

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recognized as biological control mediators against nematodes in the environment. These fungi live in the soil and feed on various nematodes present. Fungi like Duddingtonia flagrans arrest nematodes by making sticky traps on their developing hyphae. After passing through the gastrointestinal tract, spores grow and form hyphae, trapping and releasing larval phases in faeces. To achieve satisfactory control of nematode larvae, spores of these fungi must be added to feed and fed for no less than 60 days (Larsen, 2000).

3.7.5 Antiparasitic vaccines Efforts are being made to develop efficient vaccines because of the significance of AR. Currently, only one helminth vaccine is available in the market against lung nematodes of cows (Dictyocaulus viviparous) called Bovilis lungworm. This vaccine consists of irradiated third larvae that cannot grow into the adult phase. The growing drug resistance of gastrointestinal nematodes has made them important candidates for emerging vaccines (Knox et al., 2003). A hidden gut antigen of H. contortus has shown high immunogenicity and is used for vaccine development. This antigen, derived from the parasitic gut, induces the production of antibodies in the host’s body. When parasites suck blood, they ingest these antibodies that attack the parasite’s gut cells, disrupting the parasite’s capability to process nutrients necessary for proper growth and maintenance. This vaccine has been successfully verified only in sheep under laboratory conditions (Deghiedy et al., 2008). Another effective vaccine can be developed using essential enzymes such as glutathione S-transferase (GST), which plays a key role in cellular detoxification. GSTs in helminths act as immune protection proteins (Massoud et al., 2012).

3.7.6 Botanical dewormers Plants have been used since ancient times to treat diseases in humans and animals. Plants provide a rich source of botanical anthelmintics, insecticides and antibacterials (Akhtar et al., 2000). Plants with anthelmintic activity include Acacia spp., Allium sativum, Nigella sativa, Artemisia

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spp., Balanites aegyptiaca, cucurbits, Commiphora molmol, Peganum harmala, turmeric and Artemisia spp. (Akhtar et al., 2000; Massoud et al., 2012; Santos et al., 2019). Several factors, singly or in combination, are assumed to be involved in the antiparasitic activity of tanniferous plants. The compounds (tannins) present in tanniferous plants are released in the stomach (rumen and abomasum) due to its low pH, improving the host’s metabolism, immunity against parasitic infection, and making the host more resistant to parasites (Niezen et al., 1996). Tannins can also kill internal parasites as a result of their anthelmintic activity, and compounds or metabolites from tanniferous plants have a harmful effect on parasite populations in dung/manure (Shalaby et al., 2012).

3.8

Conclusions and Future Trends

Anthelmintic resistance and global climate change are pivotal factors influencing the current and future developments in parasitic infections in animals. The warming environment impacts immature parasite stages outside the primary host and could alter the magnitude and efficacy of the maximum infection pressure. The interpretation of this phenomenon into different disease patterns is influenced by various factors such as host immunity, grazing practices and other farm management strategies. Managing increased infection intensity through more frequent administration of anthelmintic drugs is unsustainable owing to the rapid emergence of resistance in nematode and potentially trematode populations. Therefore, new strategies are imperative. Increasingly, treatment targeting at both the group and individual levels seems to be a practical approach to advancing sustainable helminth control on farms. It is crucial to be well-informed about optimal approaches and the implementation of such strategies in different settings. We have identified some challenges in this regard and highlighted key areas where advances in science and technology can contribute to the development of practical and effective policies to sustain efficiency in the face of significant future challenges. The adoption of improved parasite control practices is essential for sustainable and efficient animal production on pasturelands.

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The factors considered most crucial include excessive treatment frequency and administration of inadequate doses, with the latter being particularly relevant for emerging regions. It may be determined that sustainable control policies for helminthiasis require a holistic approach incorporating ecological management

and necessitate a diversified drug policy to minimize pressure for parasite adaptation. The chapter also encompasses potential mitigation and adaptation strategies to counter the development of anthelmintic resistance, along with exploring alternative/augmentative control strategies.

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4

Insecticide Resistance

Shumaila Naz1*, Rida Fatima Saeed1, Mahvish Rajput2, Sumra Wajid Abbasi1 and Ian Daniel3 1 Department of Biological Sciences, National University of Medical Sciences, Rawalpindi, Pakistan; 2Department of Parasitology, University of Agriculture Faisalabad, Pakistan; 3Department of Veterinary Pathobiology, School of Veterinary Medicine & Biomedical Sciences,Texas A&M University, College Station,Texas, USA

Abstract This chapter focuses on resistance in different insect vectors, against various drug classes and insecticides, their modes of action, the role of genetics and genomics in the development of insecticide resistance, and the impact of insecticidal resistance on insects. It includes a comprehensive summary of the mechanisms of the insecticide resistance, detection targets, techniques and insecticide resistance management strategies, placing them in the context of the role of the Environmental Protection Agency (EPA) and the Insecticide Resistance Action Committee (IRAC) on insecticide regulations.

4.1

Introduction

The hematophagous nature of a wide range of insects makes them important vectors of veterinary and zoonotic infectious agents. For instance, mosquitoes belonging to three main genera, Aedes, Anopheles and Culex, transmit different diseases when taking blood meals. Culex mosquitoes act as vectors of filariasis and Japanese encephalitis. Mosquito vectors of the genera Aedes and Anopheles transmit human malaria, which is responsible for approximately 400,000 deaths annually, especially in children (WHO, 2020). Successful malaria control efforts are reliant on insecticide deployment in endemic areas, making insecticide resistance (IR) a tremendous global threat to malaria treatment and control. These challenges call for integrated management approaches and collaborative efforts from various stakeholders.

Insect vectors are responsible for the transmission of human and veterinary infectious diseases. Therefore, the utilization of insecticides in pest control is a significant tool in the public health management of human, animal and plant pests (WHO, 2017). Insecticide resistance in some insects is a leading cause of their resurgence as important veterinary and public health pests. During the past decade, just like antimicrobial resistance (AMR) in bacterial pathogens, IR reports in approximately 450 species of arthropods have been growing, posing a threat to human and animal welfare (Denholm et al., 2002), environmental health, including economic burdens, increased workers’ exposure to toxins, environmental pollution, and damage to other species (Soderlund and Bloomquist, 1990). The insects and hosts co-evolved under mutual pressure from multiplex host–insect

*Corresponding author: [email protected]

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© CAB International 2024. Antiparasitic Drug Resistance in Veterinary Practice (eds H.M. Rizwan, M.A. Naeem, M. Younus, M.S. Sajid and X.Chen) DOI: 10.1079/9781800622807.0004 Downloaded from https://cabidigitallibrary.org by Ivanov Ivan, on 11/04/24. Subject to the CABI Digital Library Terms & Conditions, available at https://cabidigitallibrary.org/terms-and-conditions

Insecticide Resistance

interactions (Dang et al., 2017; Su et al., 2020), and hosts mounted effective defence mechanisms to eliminate insects during an infection. However, host specificity depends on the parasite’s attempt to evade host defences by modifying its genome and signalling pathways. In other host–insect interactions, insects and parasitoids utilize chemical cues for species recognition, complex relationships and identifying host locations (Speer, 2022). Host–insect interactions strongly influence host health, parasitic survival mechanisms, IR and ultimately the success of effectively managing infectious diseases. Insecticide resistance involves a set of multifactorial mechanisms, including gene mutants on targeted polypeptides, inhibited insecticide penetration resulting from cuticular changes and overexpression of enzymatic detoxification genes (Hemingway and Ranson, 2000). Recent advances in genomic research have enabled scientists to identify the mechanisms that govern IR in vectors (Dang et al., 2017). As a result, much is known regarding drug resistance in most insect-borne diseases; however, there is still a rudimentary understanding of vectorial competence and insecticidal interaction with insect vectors for other important diseases.

4.2 Classification and Mode of Action of Insecticides Insecticides are categorized according to their structure and mode of operation. Most insecticides are not highly selective and may be toxic to non-target species including humans and have the potential to significantly alter ecosystem components.

4.2.1

Classification of insecticides

Insecticides can be classified into organic and inorganic categories based on chemical make-up that can be further divided into four categories: contact poisons, fumigant poisons, stomach poisons and systemic poisons, depending on how the poison enters the insect (Nauen et al., 2002). They can also be divided as physical poisons, nerve poisons, respiratory poisons, protoplasmic

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poisons, general poisons and chitin inhibitors based on the mode of action. These are further divided into four categories according to toxicity levels: (i) extremely toxic – red colour, skull and poison symbol, oral LD50: 1–50; (ii) highly toxic – yellow colour and poison symbol, oral LD50: 51–500; (iii) moderately toxic – blue, danger symbol, oral LD50: 501–5000; and (iv) least toxic – green colour, caution symbol, oral LD50: >5000. They can also be classified on the basis of the stage of specificity, including ovicides, pupicides, larvicides and adulticides, which are further divided into four groups based on their chemical make-up: (i) biological insecticides; (ii) artificial insecticides; (iii) natural insecticides; and (iv) unrelated substances. 4.2.2 Modes of actions of insecticides Different insecticides target different parts of any insect. Most insecticides used by pest management professionals can be categorized as neurotoxins because they kill the organisms by targeting their nervous system. Sodium and chloride channels as well as various acetylcholine system components have specific neurological target sites (Nauen et al., 2001). Insecticides that lack neurotoxic effects can also be classified according to their modes of actions and target sites; these include desiccants, insect growth regulators (IGRs), energy-production inhibitors, non-specific cellular disruptors and muscular calcium channel disruptors (exoskeleton). A small number of widely used insecticides, including IGRs, such as juvenile hormone analogues and chitin synthesis inhibitors, and a few other active substances such as borates, energy inhibitors and dehydrating dusts do not affect the nervous system (Nauen and Bretschneider, 2002). Stomach poisons are most effective against insects with biting or chewing mouthparts. Stomach poisons are toxic only if ingested through the mouth (Goel and Aggarwal, 2007). Arsenicals, such as Paris green (copper acetoarsenite), lead arsenate and calcium arsenate, as well as fluorine compounds, including sodium fluoride and cryolite, are the main stomach irritants (Hassan, 2019). Synthetic insecticides have gradually replaced the stomach poisons because they pose a lesser threat to people and other mammals (Nauen et al., 2002).

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Contact poisons penetrate through the pest’s outer layer and can be used to control insects. The two main categories of contact insecticides are organic synthetic compounds and naturally occurring compounds (Biondi et al., 2012). Nicotine, derived from tobacco (Cwalina et al., 2021), pyrethrum obtained from the flowers of Chrysanthemum, Cineraria folium and Tanacetum coccineum (Dhangar and Choudhury, 2021), rotenone derived from the roots of Derris sp. and related plants (Zubairi et al., 2016), and oils derived from petroleum are some of the naturally occurring contact insecticides (Kim et al., 2003). Fumigants are poisonous substances that enter an insect’s respiratory system through its spiracles or breathing openings (Boopathy et al., 2022). They primarily consist of nicotine, naphthalene, hydrogen cyanide and methyl bromide, and are used to kill insect pests on stored or to fumigate nursery stock (Jayaprakas et al., 2023). The mode of action of various insecticides is given in Table 4.1.

causes rapid paralysis and insect death (Daborn et al., 2002). The cloning of these resistance genes has enabled us to address dependence of resistance phenotypes on a single gene or multiple genes, its independent origins, and internal mutations in resistance genes. The majority of initiatives to deal with these fundamental problems, however, have centred on target site resistance, but current advancements in insect genomics allow the analysis of more complex metabolic systems (Tijet et al., 2001), as shown in Fig. 4.1.

4.3.1 Gene expression target sites The analysis of insecticide target-site mutants, which were isolated in field studies, provides evidence for the importance of single genes that have a significant impact. There are three primary targets for conventional insecticides: ligand-gated ion channels, voltage-gated ion channels and acetylcholinesterase.

4.3 Genomics and Genetics of Insecticidal Resistance Insecticide resistance is a crucial example of natural selection instigated by humans, and the variables governing the emergence and spread of mutations associated with resistance are both of academic and practical significance. Most of the target genes of small-molecule insecticides have recently been discovered and cloned in the genetic models of Drosophila melanogaster. Most of these targets are crucial receptors or enzymes in the nervous system of insects, whose poisoning

Ligand-gated ion channels Neurotransmitters such as acetylcholine or gamma-amino butyric acid (GABA) are taken up by ligand-gated ion channels, which then convert those signals into electrical signals by opening their integral ion channels. Cyclodiene insecticides and more recently developed phenylpyrazoles like fipronil act on the GABA receptors of insects. Resistance to di-eldrin, or Rdl, is the name given to a field-isolated Drosophila mutant that was resistant to the cyclodiene insecticide dieldrin. This gene encodes for the

Table 4.1. Insecticide examples and their mode of action. (Author’s own table.) No.

Insecticide

Mode of action

References

1

Pyrethroids

Sodium channel modulator

2

Neonicotinoids, fipronil

3 4

Organophosphates Bt (Bacillus thuringiensis)

5

Insect growth regulators (IGRs)

Nicotinic acetylcholine receptor agonists Acetylcholinesterase inhibitors Disrupts insect gut by producing crystal proteins that bind to gut receptors, causing gut damage Mimic insect hormones or interfere with hormone regulation

Hemingway and Ranson, 2000 Jeschke et al., 2011 Lionetto et al., 2013 Gassmann et al., 2011

Dhadialla et al., 2005

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Injudicious use of insecticides

Penetration Detoxification of toxin

Mutation at binding site

Recognition/ toxin resistance Missing codon A A G

Modification in genome

A U G U U U G G C U A A

MET

PHE

GLY

STOP

Fig. 4.1. A schematic diagram of gene mutation towards insecticidal resistance. (Author’s own figure.)

pharmacologically significant GABA-receptor subunit. Although Drosophila is not usually a pest, insecticide exposure has probably contributed to the widespread presence of IR genes in Drosophila field populations (Newcomb et al., 1997).

allele super-kdr, and subsequently, a single amino acid replacement was discovered to be associated with kdr. The analysis of a variety of pest insects also revealed equivalent mutations at comparable locations in the channel, mirroring the Rdl situation (Soderlund and Knipple, 2003).

Voltage-gated ion channels Voltage-gated ion channels, in contrast to ligandgated channels, are activated by changes in membrane voltage rather than changes in neurotransmitter concentration. Dichloro-diphenyltrichloroethane (DDT) and the pyrethroid insecticides act on insects through the voltagegated sodium channel (Field et al., 2017). In house flies, knockdown resistance (kdr) was the first pyrethroid target-site resistance phenotype identified (Claudianos et al., 1999). However, the identification of a gene from a temperaturesensitive paralytic Drosophila mutant was crucial for the cloning of the major subunit of the channel. Genetic research after the para gene was cloned in Drosophila revealed a relationship between the kdr phenotype and the para homologue in the house fly. The addition of a second replacement was found to be associated with an enhanced

Acetylcholinesterase The neurotransmitter acetylcholine is broken down by the enzyme acetylcholinesterase, which is the target site for carbamate insecticides and organophosphates. Drosophila was used to clone the first insect acetylcholinesterase (ace) gene. Multiple resistant Drosophila strains were examined, and scientists have discovered a variety of single amino acid replacements, either individually or collectively, and increased levels of enzyme insensitivity (Foster et al., 2007). However, mosquitos have two distinct Ace proteins, whereas Drosophila only has one ace gene (Weill et al., 2002). Only one of these two genes, ace-1, is associated with resistance in Culex pipiens. The two Ace proteins are encoded by two distinct genes, ace-1 and ace-2, the presence of which were confirmed within the full genomic sequence

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of the malarial mosquito Anopheles gambiae. Finally, it should be noted that all three of the main insecticide target sites contain naturally occurring single amino acid replacements that confer resistance (Weill et al., 2003).

upcoming insecticide or other xenobiotic challenges (Russell et al., 2011).

4.3.2 Origin and spread of resistance-associated mutations

The impacts of IR on different insects varies depending on several factors that include the mode of transmission, the type of insect vector involved and the specific mechanism of IR. The following are some examples of the impact of IR.

Understanding the number of independent origins of resistance-associated mutations is necessary to evaluate the relative importance of mutation rates and migrations in the spread of resistance alleles (Andreev et al., 1999). Few studies have been conducted on a single population to provide researchers with enough data to make informed judgements (Oksas et al., 2022). Humans may also have a sizable impact on migration rates, which will subsequently affect the spread of resistance alleles (Hernando-Amado et al., 2019). A resistance allele may have a single global origin, according to research on amplified esterase genes in C. pipiens (Raymond et al., 1991).

4.3.3 Types of mutations associated with the insecticidal resistance Early research on target-based resistance suggested that point mutations were the main cause of resistance-linked mutations, and the rate of neutral substitutions per site influenced how often new mutations occurred in natural populations (Andreev et al., 1999). The use of sprays that degrade such as conventional insecticides allowed the survival of resistant heterozygotes, which led to the emergence of resistance to Bacillus thuringiensis (Bt) in the field (Zhang et al., 1994). The application of genomic technologies to previously unsolvable cases of IR has greatly improved our understanding of the mechanisms available to insects to evolve IR. It is now clearly seen that the rate of extinction of some insect species by multiple resistance mutations will drastically reduce the variation in related genes (Bell and Collins, 2008). Because many detoxification genes are found in tightly linked clusters, these selective sweeps will influence the genetic variation that these species have to respond to

4.4

Quantification and Impacts of Insecticide Resistance

4.4.1 Cross resistance Cross-IR in insects refers to the development of resistance to several drugs with similar modes of actions. Specifically, this may occur from a mutation rendering pests resistant to all insecticides with similar mechanisms of actions. For example, if a mosquito population has developed resistance to one class of insecticides that is commonly used to control malaria vectors, the pests are likely to develop resistant alleles to anti-malarial drugs with similar modes of action (Bryant and Reay-Jones, 2020). The IR to dichlorodiphenyltrichloroethane (DDT) in Anopheles mosquitoes, which transmit malaria, has been shown to confer some level of cross-IR to other insecticides such as pyrethroids (Tchigossou et al., 2020). Similarly, cross-IR has been reported in insectborne parasites such as Leishmania, vectored by sandflies. Furthermore, cross-IR in sandflies has been reported to compromise their susceptibility to available insecticides (van den Berg et al., 2021). It is obvious that cross-IR creates significant implications for control and transmission of insects by limiting the range of available insecticides (Moyes et al., 2021). The impact of cross-IR varies, however, depending on target insects and the modes of transmission (Chapman, 2003). In some cases, cross-IR limits the effectiveness of insecticide-based control of insect vectors, e.g., making it more difficult to control mosquito populations and ultimately leading to the increased transmission of malaria (Tungu et al., 2023). Cross-IR is an important consideration in the development and implementation of insecticide-based control strategies (Zhu et al., 2016). Careful pre-planned strategies including

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insecticide rotations with drugs of varied modes of action will help to manage cross-IR while maintaining effectiveness (Dusfour et al., 2019). 4.4.2

Genetic resistance

Genetic IR is a well-known phenomenon in which the insects can develop resistance to insecticides through genetic changes that allow them to survive exposure to the chemicals that would normally kill them (Zhu, 2008). This resistance can be passed down from generation to generation and over time can lead to a significant reduction in the effectiveness of insecticides (South and Hastings, 2018). One of the ways that genetic IR can occur is through mutations in the genes that control the targets of insecticides. For example, insecticides may target enzymes in the nervous system of insects, and mutations that alter these enzymes confer resistance to the chemicals (Zhu et al., 2016). Insects may also develop resistance to overcome insecticides by increasing the production of detoxifying enzymes that degrade the chemical before causing harm (Kumar et al., 2023). The development of genetic IR is a major concern for the effectiveness of insecticides (Tungu et al., 2023). To combat this problem, the rotation of insecticides with different modes of action is recommended (Zhu et al., 2016). Additionally, it is important to use insecticides only as a last resort and to focus on non-chemical methods of pest control whenever possible. There are many examples of genetic IR arising as a result of frequent or inappropriate exposure of insects to the insecticides (Denholm et al., 2002). Mosquitoes Mosquitoes (Insecta: Nematocera) are major vectors of diseases such as malaria, dengue fever and Zika virus (Socha et al., 2022). The knowledge of IR metabolic mechanisms involving detoxification genes such as cytochrome P450 genes, carboxylesterases and glutathione S-transferases is important (Cheng et al., 2022). Identifying genetic markers associated with these regulatory alleles is a critical next step that would significantly improve IR surveillance and population genetic studies in this important vector species (Main et al., 2018).

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Bed bugs Bed bugs (Insecta: Hemiptera) have become increasingly resistant to insecticides in recent years. Bed bugs can detoxify many insecticides by producing enzymes that break down the chemicals (Gonzalez-Morales and Romero, 2019). This has made it difficult to control bed-bug infestations, and there is currently no single insecticide that is effective against all bed-bug populations (Dang et al., 2017). Bed-bug resistance to insecticides is contributing to a resurgence of the pest. There have been reports of increased resistance to pyrethroids all over the world (Gonzalez-Morales and Romero, 2019). Among bed bugs, IR may be mediated by genetic mutations on target proteins, by overexpression of detoxifying enzymes (cytochrome P450 monooxygenases and esterases), or by thickening of the insect cuticle, with reduced insecticide penetration. The existence of multiple resistance pathways portends doom for ever-increasing problems with infestation eradication (Romero et al., 2007).

4.4.3

Multiple resistance

Multiple IR is a phenomenon in which a single insect population has developed resistance to multiple insecticides, when exposed to different types of insecticides (Siddiqui et al., 2023). This creates a serious threat to insect control and management and requires prompt attention (Hardy, 2014). Insect vectors that survive one type of insecticide exposure are more likely to survive exposure to other types of insecticides (Alyokhin et al., 2008). Two major mechanisms contributing to multiple IR are: (i) the presence of multiple resistance genes, each of which provides resistance to a different insecticide; and (ii) cross-IR, where resistance to one insecticide provides partial or complete resistance to another insecticide that targets a similar biological pathway or mechanism of action (Mackinnon and Hastings, 1998). Multiple IR is a major concern in many insect species, including mosquitoes, bed bugs and agricultural pests. For example, the malariacarrying mosquito A. gambiae has developed multiple IR to all four classes of insecticides used for vector control (Soko et al., 2015). Similarly, bed bug resistance to multiple classes of insecticides

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has increased, making it difficult to control infestations (Yu et al., 2023). To combat multiple IR, it is important to use integrated pest management strategies that rely on a combination of chemical and non-chemical methods of control. These include rotating insecticides with different modes of action, using insecticides in combination with other control methods, and reducing the overall use of insecticides to minimize selection pressure on the insect populations. It is also important to monitor the insect populations for resistance and to adjust control strategies accordingly (Kisinza et al., 2017).

4.4.4

Selection pressure

Selection pressure is the process by which the use of insecticides creates an environment where insects with genetic IR are more likely to survive and reproduce. This pressure leads to the evolution and spread of IR in insect populations (Hawkins et al., 2019). The use of insecticides creates a selective advantage for insects that have genetic mutations or traits that make them resistant to the chemical (Siddiqui et al., 2023). Selection pressure is increased by indiscriminate use of insecticides, including overuse and dose misuse; these practices result in the rapid development and spread of resistance (Miller et al., 2022). In addition to the direct selection pressure exerted by insecticides, various others are involved in selection pressures and the evolution of IR in insects. For example, insects that are resistant to one type of insecticide may also be cross-resistant to other insecticides that have a similar mode of action (Bryant and Reay-Jones, 2020). Overall, the evolution of IR in insects is a complex process that involves multiple genetic and environmental factors. Understanding these factors is critical for developing effective management and control strategies for insect populations and for reducing the risk of IR (Hawkins et al., 2019).

4.5 Insecticide Resistance against Different Classes of Arthropods Insecticide resistance is a common problem in many insect species, including lice, fleas, bugs

and flies (Siddiqui et al., 2023). Resistance occurs through a variety of mechanisms, such as changes in the target site of the insecticide, reduced penetration of the insecticide into the insect, and increased metabolism or detoxification of the insecticide by the insect (Hemingway, 2000). Different insects may exhibit different levels and types of resistance to different insecticides. It is important to consider the specific insect species and the insecticide being used when developing a management plan for IR (Siddiqui et al., 2023). 4.5.1 Phthiraptera Insecticide resistance is thought to be a major factor in the rising number of lice infestations. Because of the high insecticide selection pressure induced by conventional topical pediculicides, resistance has emerged and spread in many parts of the world. Resistance mechanisms include accelerated detoxification of insecticides via enzyme-mediated reduction, esterification, oxidation that can be overcome by synergistic agents such as piperonyl butoxide, binding site alteration, such as altered acetylcholinesterase or altered nerve voltage-gated sodium channels, and kdr (Marcombe et al., 2009). Clinical, parasitological and molecular data on resistance to conventional topical pediculicides show that neurotoxic insecticide treatments have lost a significant amount of activity globally. The molecular events that govern pyrethroid resistance have been partially elucidated. Early reports suggested that the recessive kdr trait was primarily responsible for permethrin resistance in lice. Three point mutations in the voltage-gated sodium channel (VGSC) α-subunit gene (M815I, T917I and L920F) associated with permethrinresistant phenotypes were proposed to be responsible for kdr-type resistance (Durand et al., 2012). When converted to its oxon form, malathion, a neurotoxic organophosphorus insecticide, binds irreversibly to acetylcholinesterase, inhibiting its function and causing spastic paralysis and lice death. Malathion resistance mechanisms in head lice have not been formally reported (Amanzougaghene et al., 2020). Malathion resistance is primarily attributed to elevated esterases in a variety of insects. Esterases may contribute to resistance by rapidly converting insecticides to inactive forms or by sequestering

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them. Esterases, specifically a carboxylesterase, were discovered to be involved in the metabolism of malathion in a head louse strain collected from Bristol, UK, and may be involved in resistance (Gogoi et al., 2016). 4.5.2

Siphonaptera

Fleas are the most common insects of cats, dogs and rodents worldwide, and potential disease vectors. During the past two decades, most new control treatments have been applied topically or orally to the host. The majority of reports on the development of IR concern the cat flea, Ctenocephalides felis (f.) felis (Rust, 2016). Many insecticides historically used to control fleas in the environment, including carbamates, organophosphates and pyrethroids, have developed IR. Some of the new topical treatments have experienced product failures, but actual resistance has yet to be demonstrated (Abdullah et al., 2019). Ctenocephalides f. felis is the focus of a historical account of IR in fleas. Although resistance ratios (RR50) were typically less than 20-fold, C. f. felis clearly demonstrated target-site resistance to multiple insecticides as well as some crossresistance between carbaryl and organophosphates (Erkunt et al., 2020). Ctenocephalides f. felis, Pulex irritans and Xenopsylla cheopis have been reported to be resistant to organochlorine, carbamates, organophosphates, pyrethroids and pyrethrins (Malik et al., 2010). The availability of a standard susceptible strain has been a problem in testing IR in cat flea populations. Most laboratory ‘susceptible’ strains have resistant alleles for Rdl and knockdown alleles, according to molecular studies (Rust et al., 2015). A collaborative effort is required to identify populations of C. f. felis that are homozygous susceptible to all the known suspected resistant alleles and mutations. The strain must be preserved and made accessible to researchers and industry (Rust, 2016). 4.5.3

Hemiptera

Bed bugs may have developed resistance to DDT within 5 years of its initial use, and the rapid rate of resistance was most likely due to the insecticide’s excessive and continuous use (Dang et al., 2017). Pyrethroids are a class of highly

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effective and extremely efficient neurotoxic insecticides that are synthetic analogues of pyrethrin found in pyrethrum, an extract of the Chrysanthemum cinerariaefolium flower. Resistance to pyrethroids has been documented in many parts of the world for both Cimex lectularius and Cimex hemipterus, owing to the worldwide resurgence of bed bugs during the past two decades (Matsuo, 2019). Neonicotinoids are now widely used to control a wide range of chewing and sucking pests, including bed bugs. In recent years, neonicotinoids have been combined with pyrethroids in formulated products such as Temprid ® SC (beta-cyfluthrin + imidacloprid), Transport® Mikron (bifenthrin + acetamiprid) and Tandem® (lambda-cyhalothrin + thiamethoxam), as well as with diatomaceous earth (Dang et al., 2017). 4.5.4 Diptera With new insecticides for pest control on the market, IR management in homes has become increasingly important. Resistance to permethrin, deltamethrin and beta-cypermethrin was widespread among the field strains (Wang et al., 2019). In 2017, all fly strains tested positive for permethrin and deltamethrin resistance. Resistance to deltamethrin increased significantly between 2011 and 2014, reaching an all-time high in 2017. Although some field strains remained sensitive to beta-cypermethrin, very high-level resistance emerged in 2017 (Silalahi et al., 2022). According to researchers, house fly resistance to some insecticides is unstable, and reversion may occur after several generations, but not to all insecticides. Certain IR strains had lower fecundity, hatchability, number of nextgeneration larvae and net reproductive rate when compared to susceptible strains, according to some researchers (Khan et al., 2014). Because resistance alleles may be disadvantageous under natural selection, the level of resistance may be reduced in the absence of insecticide selection pressure (Wang et al., 2019).

4.6

Mechanisms of Insecticidal Resistance

Insecticidal resistance is a crucial element to considered while developing approaches for

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minimizing the usage of insecticides whilst conserving insecticide efficacy (Siddiqui et al., 2023). Despite the wide variety of insect species and insecticide chemicals, different pathways are known to result in IR, which include metabolic resistance, in which higher levels of enzymes cause an increase in metabolic detoxification activities, and target site insensitivity, in which modifications to the sensitivity of the target site prevent insecticide (Siddiqui et al., 2023), as shown in Table 4.2.

4.6.1 Metabolic insecticidal resistance Metabolic insecticidal resistance, enabling the insect to break down or sequester the insecticide more rapidly before releasing their poisonous impact, is among the most prevalent forms of resistance in insects (Siddiqui et al., 2023). It has been extensively covered in previous work that metabolic resistance is regarded as a fundamental resistance mechanism (Hemingway et al., 2004). The main cause of this resistance mechanism is the substitution of amino acids, which enables insects to produce excessive amounts of various enzymes, primarily cytochrome P450 monooxygenases (CYP450s), glutathione S transferases (GSTs) and carboxylesterases (CarEs), to counteract the harmful effects of insecticides (Hemingway and Ranson, 2000). Amplification of the transferase gene has been shown to have an insecticide-sequestering and detoxifying effect on a variety of endogenous and xenobiotic substances, including insecticides. Detoxification occurs in two stages: phase I, which includes oxidation or hydrolysis, and phase II being the conjugation of the products of phase I (Berenbaum and Johnson, 2015). Esterase Esterases are an immense class of phase I metabolic enzymes that metabolize a range of substrates. The esterases E4 and FE4 respond to specific types of insecticide and result in IR by destroying the ester bonds present in insecticides (Siddiqui et al., 2023). Two basic processes by which esterases mediate metabolic resistance include: (i) gene upregulation and (ii) mutations in gene-coding sequences. The initial mechanism

causes insects to overproduce non-specific esterases or CarEs by up-regulating genes (e.g. carbamates) and it has been observed in a wide variety of insect species, such as ticks, cockroaches, mosquitoes and aphids (Hemingway et al., 2002). The second mechanism increases esterase-mediated metabolism by altering the sequences of coding genes, such as through point or substitution mutations in structural genes (Dang et al., 2017). A CarE, for instance, is encoded by the LcαE7 gene in the sheep blowfly Lucilia cuprina. When glycine at residue site 137 of the LcαE7 gene is changed to aspartic acid, the carboxylesterase is transformed into an organophosphorus hydrolase, which provides resistance to organophosphorus (Claudianos et al., 1999). Cytochrome P450 monooxygenases Microsomal oxidases, also known as cytochrome P450 monooxygenases (CYP450s) are crucial in the breakdown of chemical carcinogens, endogenous substances and insecticides (Bergé et al., 1998). They can facilitate a variety of chemical processes, including hydroxylation, N-dealkylation and O-dealkylation. As a result, CYP450s are crucial for the metabolism of insecticide (Philippou et al., 2010). Genes from the CYP families have been associated with IR, and more than 600 insect CYP450 genes have been categorized (Feyereisen, 2015). The multiple duplication of the single cytochrome P450 gene (CYP6CY3) is mostly linked to an aphid’s resistance. The resistant aphid contains 18 copies of this gene, whereas susceptible individuals only have two copies. Several CYP450 genes, including CYP6P3, CYP6M2, CYP6P9a and CYP6Z3, have been demonstrated to be involved in resistance to pyrethroids in mosquito species, the vectors of lymphatic filariasis and malaria (Rai et al., 2019). In pests that are resistant to treatment, cytochrome p450 overexpression has shown mutation. Overexpressed CYP450 genes are found in most common pests, including Myzus persicae, a neonicotinoid resistance-causing organism (Puinean et al., 2010). These investigations have demonstrated that the type of insecticide, its toxicity and concentration have an impact on the overexpression of a particular CYP450 gene.

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Table 4.2. Different mechanisms of insecticide resistance. (Author’s own table.) Enzyme/molecular mechanism

Mutation

Vector

Reference

Metabolic resistance

Cytochrome P450 monooxygenases Glutathione S transferases Carboxylesterases Knockdown resistance mechanism Acetylcholinesterase (AChE)

CYP6P3 and CYP6M2, CYP6P9a, and CYP6Z3 Leu119 to Phe Gly137 to Asp Leu1014 to Phe

Lymphatic filariasis, Culex quinquefasciatus Anopheles funestus Lucilia cuprina Musca domestica

Rai et al., 2019 Sandeu et al., 2020 Claudianos et al., 1999 Rust et al., 2015

Mutations in the AChE gene (Val180 to Leu, Gly262 to Ala, Gly262 to Val, Phe327 to Tyr and Gly365 to Ala) Ala302 to Ser

Musca domestica

Chen et al., 2001

Drosophila melanogaster

Gassel et al., 2014

Target site mutation

Insensitive gamma amino butyric acid (GABA) receptor

Insecticide Resistance

Resistance mechanism

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Glutathione S transferase The glutathione S transferases (GSTs) belong to a multi-functional enzyme superfamily that also contribute to IR. These enzymes convert the insecticides into water-soluble, readily execrable metabolites by a conjugation reaction with reduced glutathione and hydrophobic xenobiotic (Hayes and Wolf, 1988). The GST-based resistance typically results from an excess of the enzyme, which can be caused by either gene amplification or overexpression (Vontas et al., 2002). By securing the insecticide, GSTs might also offer protection against pyrethroid toxicity in insects. Anopheles mosquitoes, which have the GSTs that give resistance to DDT, and A. funestus mosquitoes, which have the GSTs that confer pyrethroid resistance, have been observed in Uganda and Kenya (Sandeu et al., 2020). The GST genes in D. melanogaster have been discovered and the overexpression of these genes led to the development of DDT resistance (Gonzalez et al., 2018).

4.6.2 Target site resistance It is possible to genetically modify the insecticide target site of action. By preventing the insecticide from binding or interacting at its site of action, the insecticide efficacy is decreased (Bass et al., 2011). Insecticides such as organophosphates, pyrethroids and carbamates affect specific target sites (e.g. acetylcholinesterase (AChE), GABA receptors and VGSCs) associated with the nervous system of the insect (Liu, 2015). The target site for insecticide action may be genetically altered, preventing insecticide binding or contact at its site of action, and thereby decreasing the potency of the insecticide. Knockdown resistance mechanism The primary mechanism behind resistance to all pyrethrins, DDT, pyrethroids and analogues is kdr (Soderlund and Knipple, 2003). The kdr/ super-kdr was initially discovered in the house fly, caused by a point mutation in the vssc1 gene. Different mutations (point) in VGSCs lead to kdr (Siddiqui et al., 2023). Numerous investigations revealed that the coding sequences of VGSCs in a variety of insects, pests and bed bugs harbour a single mutation or multiple point mutations

(often referred to as kdr mutations) that confer kdr-type resistance to DDT and pyrethroids. Modified acetylcholinesterase Acetylcholinesterase (AChE), a serine protease, is necessary for controlling the neurotransmitter acetylcholine (ACh). The AChE enzyme is a neurotransmitter that transmits impulses produced by pests and causes insecticides to degrade, which ultimately results in death by paralysis (Siegfried and Scharf, 2001). For instance, the insecticide pirimicarb predominantly targets and inhibits AChE. The first substantial insect study to identify the Ace locus at the molecular level was conducted on D. melanogaster, and genomic sequencing work showed that Ace encodes the AChE. Many insects, including Musca domestica (Walsh et al., 2001), L. cuprina (Chen et al., 2001), Pieris rapae (Lu et al., 2012) and Aphis gossypii (Li and Han, 2002), have been shown to contain the ace gene and have the same genomic structure. Insensitive gamma amino butyric acid (GABA) receptor Phenylpyrazole has been shown to have a proven target in the GABA receptor, also known as the GABA-gated chloride channel. Inhibiting GABA-stimulated chloride absorption causes insects to produce more neuronal impulses, which causes them to die (Wafford et al., 1989). Insecticidal activity is, however, impacted by changes or mutations in the molecular structure of the GABA ion channel. The dieldrin (Rdl) gene, which largely encodes GABA receptors with five subunits arranged around a core gated ion channel, was shown to be resistant in most of the parasites. Drosophila was the first to exhibit such a resistant mutation. The amino acid alanine 302 in the GABA-gated chloride channel, encoded by Rdl, was changed to the amino acid serine, according to studies on resistance-associated mutations in resistant D. melanogaster strains (Gassel et al., 2014). Further research on the single base-pair replacement mutation (Ala302 to Ser) in the GABA-gated chloride channel gene Rdl in the insect orders Diptera, Dictyoptera and Coleoptera, reveals that these mutations are highly conserved (Thompson et al., 1993).

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4.7

Insecticide Resistance Detection Methods

The detection of IR is aided by many bioassays, including those that employ phenotypic and genotypic information as described below (Kaura et al., 2022).

4.7.1 Phenotypic assays The lifespan of currently used vector control equipment is shortened by the evolution of IR, which also lowers the efficacy of newly developed products for vector control. In order to significantly reduce malaria morbidity and mortality rates, the World Health Organization’s (WHO) Global Plan for IR Management (GPIRM) developed strategies to maintain the effectiveness of current vector control tools while concurrently constructing futuristic vector control tools that are new and inventive (WHO, 2012). For more effective management of resistance in the face of the current issue, it is critical to understand the molecular changes creating IR in resistant populations (Ranson et al., 2011). Effective vector control is necessary for controlling and eliminating malaria. Nets and long-lasting insecticidal indoor residual spraying are now the two main strategies for reducing vector populations (Nalinya et al., 2022). To improve the effectiveness of vector control interventions, bioassays using WHO-defined diagnostic doses of insecticide, biochemical and dose response bioassays, to check for the activity of enzymes linked to IR, and molecular assays to check for known resistance alleles have all been developed (Sahu et al., 2019). A study in the years 2009 and 2010 reported multiple resistance to DDT, malathion and deltamethrin in A. culicifacies from different districts of India (Bhatt et al., 2012; Raghavendra et al., 2017). The Mosquito Contamination Device (MCD) bottle bioassay is an additional bioassay for identifying phenotypic resistance in malarial vectors. These bioassays are valuable and significant because they showed how insecticide exposure altered the behaviour of Anopheles (Sternberg et al., 2014). Some other studies have been conducted to study and detect filarial vectors insecticide resistance in Egypt

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(Zayed et al., 2006) and target sited mutations in the C. pipiens complex and their hybrids in Morocco (Tmimi et al., 2018). The main vector of Leishmania major, the cause of cutaneous leishmaniasis in Sudan, is the sand fly Phlebotomus papatasi (Scopoli) (El-Hassan and Zijlstra, 2001). Insect populations with resistant genotypes have been identified by using biochemical and molecular methods in investigations on IR (Surendran et al., 2005), although very few studies across the globe have indicated that enzymes might have a role in sand fly IR (Surendran et al., 2005). More recently, however, upon exposure to malathion, deltamethrin, DDT and propoxur, mutations in kdr and enhanced activity of resistance metabolic enzymes were found in Phlebotomus argentipes in Sri Lanka using the WHO bioassays (Pathirage et al., 2020).

4.7.2 Genotypic assay Insecticidal resistance has been linked to only a small number of gene alterations, according to genetic and molecular investigations (FfrenchConstant, 2013). The ability to manage resistance effectively depends on the issue being discovered quickly, and national vector control programmes analyse thousands of mosquito specimens each year globally. However, TaqMan genotyping of individual mosquitoes has a rather high running cost (Hemingway et al., 2013). The main causes of resistance in Anopheles are overexpression of detoxification or sequestration enzymes and mutations at the insecticide target site that alter the sensitivity of the insecticide. The most used test is a PCR method that may identify target site alterations caused by resistance to various insecticides including pyrethroids, carbamates and organophosphates in several Anopheles spp. (Soma et al., 2020). The technique aids in the detection of kdr mutations in many Anopheles spp. owing to the use of pyrethroids and DDT (Irving and Wondji, 2017). Multiplexing was also used to profile gene expression, and qRT-PCR was then used to detect IR in vectors, according to the results of this research. Additionally, research has been conducted on the pyrethroid resistance of the

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P450 genes CYP6M7, CYP6Z1 and CYP6Z3 in A. funestus. Quantitative trait loci (QTL)-driven changes in amino acids were revealed in the study, highlighting the function of these polymorphic genes in the emergence of IR (Irving et al., 2012). As a result, genetic profiling techniques and characterizations can help us better understand target site alterations and metabolic resistance in vectors. Additionally, resistance genes have been found in Culex species through transcriptome profiling using whole-transcriptome microarrays. Pyrosequencing was used to pinpoint the mutations and metabolic IR induced by deltamethrin in C. quinquefasciatus from Zanzibar (Rai et al., 2019).

4.8

Methods for Overcoming Insecticide Resistance

Researchers and subject-matter experts have investigated several strategies for combating IR. Some of these techniques are described here.

4.8.1

CRISPR-Cas gene technology

CRISPR is a genetic engineering tool that allows the manipulation of an organism’s genetic material rapidly and efficiently (Bhowmik and Islam, 2020). In the case of IR, researchers have successfully reversed the resistance phenotype by editing the genes that cause resistance and the ubiquity of kdr mutations with CRISPR-Cas technology. This strategy has shown promise as a potential means of overcoming IR after being tested on a variety of insect species, including mosquitoes (Taning et al., 2017). Using CRISPR-Cas9 technology, biologists at the University of California, San Diego, have devised a mechanism for reversing pesticide resistance. According to Nature Communications, researchers from the Tata Institute for Genetics and Society (TIGS) and their colleagues employed the genomic editing tool and changed an insecticideresistant gene in fruit flies with a non-resistant variant, a feat that could significantly reduce the number of insecticides used (Kaduskar et al., 2022).

4.8.2 De-potentiating agents De-potentiating agents act as inhibitors of IR by lessening the activity of the enzymes needed to metabolize them (Araújo et al., 2013). Like CRISPR-Cas, when tested on a range of insect species, including mosquitoes, this tactic represents a solution to overcoming IR (Taning et al., 2017). For instance, the four most cutting-edge antimalarial drugs, Kae609, KAF156, DSM265 and MMV048, have recently been reported and are currently in phase II trials. The effectiveness of a dispersible tablet containing a fixed dose of piperaquine phosphate (187.5 mg) and artemether-lumefantrine (37.5 mg) in paediatric patients with P. falciparum infection was compared in a two-group multicentre, randomized comparative study. The efficacy of the combination was compared to that of artemether-lumefantrine, and both therapies were deemed to be safe with good tolerance (Chaves et al., 2022).

4.8.3 Capacity strengthening on WHO guidelines In order to implement the WHO’s recommendations for managing IR, capacity strengthening strategy entails a build-up of public health programmes. This involves enhancing source reduction and larvicidals as alternative control strategies, creating and implementing plans to manage resistance, and enhancing surveillance systems to detect resistance (WHO, 2012). Overall, both laboratory and field studies have indicated that these alternative approaches to overcoming IR are promising strategies, particularly for malaria and dengue (WHO, 2003). More research is required, however, to ascertain their efficacy in various situations and to devise plans for enhancing their use in public health initiatives.

4.9 Role of the Environmental Protection Agency and the Insecticide Resistance Action Committee The Environmental Protection Agency (EPA) is an independent federal government agency in the USA charged with guarding the environment and protecting public health. Its main objectives

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are to ensure that federal environmental laws are upheld and to control various industries that might have an impact on the environment. To safeguard both human health and the environment, the EPA regulates the use of pesticides and insecticides. The organization registers these chemicals, governs their use, and places restrictions on how much of them can be used where. The EPA also conducts studies on the effects of pesticides and insecticides on the environment and human health, while offering advice and training to farmers, homeowners, and other chemical users (Pickard et al., 2015). The Insecticide Resistance Action Committee (IRAC) is a global organization formed to address the rising problem of IR. When IR develops, the chemicals become ineffective and information on the management of IR can be coordinated and shared using the IRAC framework. To develop and advance best practices for managing resistance, the committee assembles academics, policy makers and representatives from the business community. IRAC offers recommendations on how to use insecticides effectively and apply effective integrated pest management, and on the development of novel insecticides with less likelihood of creating resistance (Sparks et al., 2021). In safeguarding the environment and humankind from the adverse effects of pesticides and insecticides, both the EPA and IRAC are playing critical roles. While IRAC recommends how to use these chemicals effectively to avoid resistance, the EPA regulates their use to ensure their safety.

4.10 Insecticide Resistance Management Strategies Insect control efforts around the world are significantly hampered by IR. Several integrated pest management–insecticide resistance management programme elements, including selective and

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biorational insecticides, rotation of insecticides with various modes of action, and non-chemical control techniques, are among the counter measures for managing insecticide resistance for insects (Horowitz et al., 2020). Overall, to effectively manage IR, a variety of different management strategies is required. Some methods for managing IR that can be used to prevent the emergence of resistance while maintaining the efficacy of insecticides include: 1. Rotate insecticides to prevent pests from developing resistance to a single chemical, use insecticides with various modes of action and alternate when you apply them. This tactic aids in lowering the pressure for selecting resistance (Sudo et al., 2017). 2. Monitor insect populations regularly, which can aid in the early detection of IR. Making informed decisions about the use of insecticides and adapting control methods in accordance with this information (Horowitz et al., 2020). 3. Reduce the use of insecticides to slow down the development of IR. The use of nonchemical techniques, such as biological control, cultural control and physical control, can accomplish this (Jepson et al., 2020). 4. Use insecticides at the right time to increase their effcacy and lessen the selection pressure for IR. Insecticide applications, for instance, may not be necessary and can be avoided when insect populations are low (Jepson et al., 2020). 5. Use synergists, which are the substances that increase the effciency of insecticides by preventing ability of the insects to detoxify them (Norris et al., 2019). They can be applied to strengthen effectiveness and lessen the possibility of IR (Claude and Bernard, 1993). 6. Implement integrated pest management, which is a comprehensive approach to pest management that employs a variety of control techniques to lower pest populations (Karlsson et al., 2020).

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5

Navigating Acaricidal Resistance through Implications in Veterinary Practice Mahvish Maqbool1, Muhammad Sohail Sajid2, Hafiz Muhammad Rizwan3*, Muhammad Younus4, Kashif Kamran5, Muhammad Zeeshan2 and Muhammad Usman6 1 Department of Entomology, College of Agriculture and Life Sciences,Virginia Tech, Blacksburg, Virginia, USA; 2Department of Parasitology, Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan; 3Section of Parasitology, Department of Pathobiology, KBCMA College of Veterinary and Animal Science, Narowal, Sub-Campus UVAS, Lahore, Pakistan; 4Section of Pathology, Department of Pathobiology, KBCMA College of Veterinary and Animal Science, Narowal, SubCampus UVAS, Lahore, Pakistan; 5Department of Zoology, University of Balochistan, Pakistan; 6Section of Histology, Department of Basic Sciences, KBCMA College of Veterinary and Animal Science, Narowal, Sub-Campus UVAS, Lahore, Pakistan

Abstract Tick infestations cause signifcant fnancial losses for the livestock industry, especially in subtropical and tropical areas. Environmental contamination and acaricidal resistance are results of traditional chemical control, which includes ivermectin and arsenic. Three essential requirements for using acaricides are that the product must only harm ticks, contain no drug residues and be environmentally friendly. Investigating other management strategies is necessary because of the emergence of resistant tick strains, which are harmful to One Health principles. The development of resistance is infuenced by biological, operational and genetic variables. Biological factors include behaviours and breeding patterns, whereas operational factors include drug quality, dosage and length of usage. Acaricidal resistance mechanisms affect amitraz, pyrethroids, organochlorines, organophosphates, macrocyclic lactones, metabolic detoxifcation and target site mutations. As assessed by phenotypic, genotypic and enzymatic techniques, the present resistance status highlights the need for alternative control approaches. Sustainable tick control requires an integrated strategy that incorporates both modern scientifc discoveries and conventional ecological knowledge.

5.1

Introduction

In the livestock sector, the primary cause of economic loss is the tick population, especially in subtropic and tropical areas, which infest approximately 80% of the cattle population (Yessinou et  al., 2018a). Ticks can transmit a wide range of

pathogens, such as Babesia bigemina, Anaplasma marginale, Babesia bovis, Theileria parva and Ehrlichia ruminantium. Economic losses caused by ticks through their bite, toxin and pathogen transmission are estimated to be US$22–30 billion annually (Sungirai et  al., 2018). The irrational and frequent use of synthetic acaricidal compounds

*Corresponding author: [email protected]

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Acaricidal Resistance

to control these ticks has led to the development of acaricidal resistance (Abbas et al., 2014; Rodriguez-Vivas et  al., 2018). Acaricidal resistance, an evolutionary adaptation, is a heritable trait in acaricides in which tick population exposure results in a higher survival rate (RodriguezVivas et al., 2018). During the last 75 years, chemical control for ticks and most pest problems has been widely adopted (George et al., 2004). Firstly, arsenic was used for tick control, which was then replaced by organochlorines in the late 1940s owing to certain drawbacks. Compounds such as organophosphates, synthetic pyrethroids, carbamates and amitraz showed better results in later developments. Ivermectin has also been a useful aid in the control of ticks. All the aforementioned drugs, however, gave rise to tick-resistant development along with drug residues in products and environmental contaminations, which, in turn, urged scientists to discover new approaches to counter this issue (Sagar et al., 2020). The basics for the use of acaricides are that they should be only harmful to ticks, not to the animals and persons applying them, there should be no drug residue in treated animal products, and they should be environmentally friendly. In conventional methods of controlling ticks, acaricides are showing some efficacy but

Synthetic pyrethroids

Chemical methods

Macrocyclic lactones

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with some major drawbacks such as the development of drug resistance and the presence of drug residue in treated animals’ products (Reck et  al., 2014). In turn, the blind use of these chemicals has led to the development of resistant tick strains, which are known to have detrimental effects on One Health concepts (Garcıa-Garcıa ́ ́ et al., 2000). Various groups of insecticides and acaricides have been found to have significant effectiveness for tick control, including arsenic, pyrethroids, avermectins, organophosphates, organochlorines, carbamates and insect growth regulators, summarized in Fig. 5.1 (Sagar et al., 2020). Mostly, these drugs target the tick nervous system: e.g. organochlorines act as antagonists for GABA-gated chloride channels; carbamates inhibit cholinesterase; pyrethroids are sodium channel modulators; organophosphates block the acetylcholine esterase; formamidines play a role as octopamine agonists; and macrocyclic lactones are known as activators of chlorine channels (Abbas et al., 2014).

5.2

Resistance Types

There are three main types of resistance, i.e. acquired resistance, cross-resistance and multiple resistance. Cypermethrin permethrin cyhalothrin

Avermctins

Milbemycin

• • • •

Abamectin Ivermectin Eprinomectin Dormectin

Milbemycin oxime moxidectin

Carbamates

Benzene hexachloride

Lindane

Organophosphates Fig. 5.1. Commonly used acaricides against ticks. (Author’s own figure.)

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5.2.1 Acquired resistance

5.3.1 Genetic factors

Over time, heritable decreases in drug sensitivity, known as acquired resistance, have been observed. For example, Tetranychus cinnabarinus demonstrated resistance in the laboratory against fenpropathrin and abamectin (Meyer et al., 2013). A direct relationship is observed between drug concentration and resistance. Similarly, a strain controlled by one dose of the drug may exhibit resistance at low concentrations of the same drug (Lees et al., 2014), enhancing the selection of resistant strains. Periodic exposure to acaricides results in a concomitant increase in resistant strains and the removal of susceptible members from the population (Faza et al., 2013).

Genetic factors involved in resistance development include population genetic diversity, resistance allele dominance, resistant organism fitness, genetic recombination and genes associated with resistance. Resistance genesis consists of three steps: resistance establishment, development and emergence (Sutherst et al., 1979). There might be a possibility that a very low population of genes responsible for resistance development is already present in the tick population before acaricidal use (Alonso-Diaz et al., 2013). The time required for the establishment of resistant alleles and tick control depends on certain factors, including mutation frequency before treatment, inheritance mode, acaricidal treatment frequency and acaricide concentrations (Bardosh et  al., 2013). The frequency of resistant genes was slow initially, but with time and continued exposure to treatment, there was a higher rate of heterozygous and homozygous single allele mutation in the population (Stützer et al., 2013).

5.2.2

Multiple resistance

Resistance to more than one drug is known as multiple resistance, even though the modes of action of these drugs are different. Multiple resistance is reported in the cattle tick population against pyrethroids, amitraz, organophosphates and chlorinated hydrocarbons (Bielza et al., 2007).

5.2.3

Cross resistance

Resistance to different acaricides that have the same mode of action is known as cross-resistance. Cross-resistance patterns have been reported for coumaphos, diazinon (organophosphates) and carbaryl (carbamate) acaricides in Rhipicephalus microplus strains. Both acaricides inhibit acetylcholinesterase, a crucial enzyme in the insect nervous system (Pérez-González et  al., 2014). Acetylcholinesterase insensitivity is considered a key mechanism for organophosphate and carbamate resistance, reducing the selection pressure by the rotation of different acaricidal groups (Dawkar et al., 2013).

5.3

Possible Factors Playing a Role in Resistance Development

Three main factors play a role in resistance development: genetic factors, biological factors and operational factors.

5.3.2

Operational factors

The main components in this category include the chemical composition of the drug, persistence and clearance kinetics in the host, drug application, host life cycle stages selected, treatment duration and frequency. The main contributing factors to resistance development are dosing and the use of poor-quality drugs (Rezende et  al., 2013). The irrational use of the same drug for a long duration also contributes to resistance development. The degree of resistance against various acaricides exhibits variation in different regions based on differences in their frequent use (Alonso-Diaz et  al., 2013). Managing operational factors is easier and more manageable than genetic factors, by educating farmers about the proper use of acaricides, the correct method of application and important points to note while selecting the drug.

5.3.3 Biological factors These factors are classified into two categories: behavioural and biotic factors. Among the

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Acaricidal Resistance

behavioural factors are those related to gene flow and selection processes, such as mobility, host range, isolation and survival. Among the biotic factors are breeding patterns, the number of offspring per generation and generation time. Biological parameters are related to host–parasite interaction and also affect the resistance development process, e.g. effective immunity-inducing parasites are considered under weaker selection pressure of resistance. The golden rule regarding resistance development is that the higher the refugia population, the lower their selection for resistance (Abdullah et al., 2012). The mode of action of commonly used acaricides is included in Table 5.1.

5.4 Acaricidal Resistance Mechanisms Mechanisms of acaricidal resistance are categorized into three groups: metabolic detoxification, target site mutations and reduction in cuticular penetration (Agwunobi et  al., 2021). The most prevalent resistance mechanisms are either target site mutation or metabolic detoxification. During target site resistance, single nucleotide substitution occurs at the gene coding for the acaricidal target molecules, leading to amino acid mutation and decreasing the efficacy of acaricides (Guerrero et al., 2012). This type of resistance is mostly reported in pyrethroids, where four single nucleotides mutations occur: i.e. in Mexican ticks, a mutation at the III domain of the sodium channel is observed that results in an amino acid substitution of phenylalanine to isoleucine (He et al., 1999); a mutation at domain II results in the substitution of leucine to isoleucine; also a mutation at domain II of the sodium channel results in the substitution of glycine to valine (Jonsson et al., 2010); and lastly, another

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mutation at II domain results in the substitution of methionine to threonine (Sager et al., 2018). It needs to be noted that the molecular and metabolic mechanisms for resistance development are defined in ticks against pyrethroid.

5.4.1

Organochlorine resistance mechanism

The organochlorine group of acaricides has been used since 1946 and they were the first synthetic acaricides that were marketed. The mechanism of action of this group is through binding at the picrotoxin site in the gamma amino-butyric acid (GABA) chloride ionophore complex (Hope et al., 2010), which results in the inhibition of chloride ion flux to the nerves. This GABA inhibition impairs nerves, causing hyperexcitation and ultimately death (Corley et al., 2012). The resistance mechanism in organochlorine is reported to be due to enhanced metabolism and a reduction in the chemical absorption rate (Brown, 1969).

5.4.2 Organophosphate and carbamate resistance mechanism The first chemical group used for the control of arachnids was organophosphates, and the mode of action of carbamates and organophosphates is through the inhibition of the acetylcholinesterase (AChE) enzyme, which plays an important role in the normal functioning of the nervous system. The inhibition of AChE leads to the unavailability of cholinesterase to the arachnids for the breakdown of acetylcholine, resulting in overstimulation of the nervous system, consequently causing death (Faza et  al., 2013). The

Table 5.1. Mode of action of common acaricides. (Author’s own table.) Acaricides

Mode of action

Reference

Carbamates Formamidines Pyrethrins/pyrethroids Organochlorides Macrocyclic lactones Organophosphates

Inhibition of cholinesterase Agonists of octopamine Modulators of sodium channel Antagonists of GABA-gated chloride channel Activation of chloride channel Inhibition of acetylcholine esterase

Li et al., 2005 Chen et al., 2007 Coats, 2012 Van Leeuwen et al., 2015 Kearns et al., 2015 Li et al., 2003

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first case of resistance against organophosphates was reported in the early 1950s, and hitherto resistance has been reported in more than 30 organophosphates and carbamates (Arivalagan et  al., 2013). The most common reason for resistance is considered to be the insensitivity of the target site (Pérez-González et  al., 2014). Among the molecular mechanisms, point mutations have been identified to play a significant role in resistance development (Temeyer et  al., 2007; Carvalho et  al., 2013). Temeyer et  al. (2007) reported six mutations in the organophosphate-resistant strain of Rhipicephalus microplus, and the most common mutation was the substitution of glutamine for arginine at position 86 in the acetylcholinesterase BmAChE3.

5.4.3 Amitraz resistance mechanism A member of the class amidine, amitraz has been used against cattle ticks for the past 30 years (Jonsson and Hope, 2007), but resistance against this compound has also been reported from time to time (Mendes et al., 2013). Amitraz causes a mutation in the octopamine receptor, resulting in differences in the amino acid profile of resistant and susceptible ticks (Corley et al., 2012). The presence of these mutations in resistant ticks provides the possibility of an altered target site for the resistance mechanism (Pohl et al., 2012).

5.4.4 Pyrethroid/pyrethrin resistance mechanism Pyrethrins are derivatives of the chrysanthemum family and have a quick knockdown effect, so they are unstable in the environment. Synthetic adaptations of pyrethrins are pyrethroids, which are considered to be more stable with long-lasting effects. The mechanism of action is neurotoxin production through acting on sodium ion channels, which leads to nerve excitation and a change in nerve permeability for potassium and sodium ions (Weston et  al., 2013). Resistance against pyrethroids is reported to be due to the involvement of glutathione-S-transferases and esterases, and p450s enzymes. Target site mutation resistance against pyrethroids is on the sodium ion channel, resulting in decreased sensitivity of the channel to pyrethroids (Oliveira et al., 2013).

Another metabolic esterase enzyme with permethrin-hydrolysing activity, i.e. CzEst9, is reportedly linked to resistance development in Mexican ticks (Miller et al., 2013).

5.4.5 Mechanism of macrocyclic lactone resistance Members of this group are milbenycins and avermectins, which are fermentation products of actinomycetes. Avermectins and milbemycin are produced from Streptomyces avermitilis and Streptomyces hygroscopicus, respectively, and are structurally similar to each other except milbemycins lack disaccharide at C13 (Campbell et al., 1984). The mechanism of action of macrocyclic lactones is by blocking the transmission of electrical activity in nerves and muscle cells by stimulating the release and binding of GABA at the nerve endings. This binding of GABA leads to chloride ion influx into cells, resulting in hyperpolarization and paralysis of the neuromuscular system (Martin et  al., 2012). This group is effectively used against cattle ticks and partial resistance is reported against this group that questions its frequent use, but the resistance mechanism in ticks is still not determined. Based on the resistance of nematodes to macrocyclic lactones, it is hypothesized that, in ticks, resistance is developed as a result of target site insensitivity, e.g. GABA and glutamate-gated chloride ion channel insensitivity (Lovis et al., 2013).

5.5

Current Status of Acaricidal Resistance

Resistance is an important issue related to the use of chemical control methods, and almost all chemical groups are reported to show resistance (Janer et  al., 2019). Multiple acaricidal tick strains are identified in different areas of the world (Fular et al., 2018). Resistance emergence is a combination of intrinsic and operational factors (Rodríguez-Hidalgo et  al., 2017). To check the current acaricidal resistance status, determination of enzymatic, phenotypic and genotypic resistance status through bioassays, molecular tests and biochemical tests are necessary (Yessinou et al., 2018a). For phenotypic resistance, bioassays, e.g. adult immersion test, larval immersion test,

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larval tarsal test and larval packet test, are employed for susceptible samples to determine resistance factors or resistance ratio (Shakya et al., 2020). The effectiveness of phenotypic assays is limited, so further confirmation through enzymatic and genotypic resistance status is necessary (Sungirai et al., 2018). Molecular assays have provided immense help in checking resistance status through the detection of resistant single nucleotide mutations in the field (Kumar, 2019). The most commonly used molecular techniques are polymerase chain reaction, transcriptomic analysis, probe-based techniques, sequencing and target gene amplification (Sungirai et  al., 2018). Among the biochemical assays, the determination of the activity of cytochrome P450 monooxygenases, total protein, esterases and glutathione-S-transferases are included (Yessinou et al., 2018b). The current status of acaricidal resistance in ixodid ticks is summarized in Table 5.2.

5.6 Alternative Control Methods to Overcome Acaricidal Resistance In Ticks Different control methods are being used for the control of ticks and are divided into two groups, namely conventional and modern. Pasture spelling and rotational grazing are the main grazing strategies used for the control of ticks. Pasture spelling is commonly used for one-host tick species rather than multi-host tick species. Changing the pasture after every 2–3 months in summer and 3–4 months in winter, depending upon the climatic conditions of the geographic area, helps in controlling the tick population. Habitat modification is also a tick control method, as various tick stages can attach themselves to grass blades and vegetation from where they can attach to the cattle. Changing the habitat through firing and ploughing may help in causing damage to the developmental stages of the ticks, but they may cause ecosystem damage due to soil erosion (Eisen, 2020).

5.6.1

Phytotherapy

Ethnoveterinary practices are still in use in many developing countries where several plants and their extracts are being used to control ticks. Different plants and their extracts, i.e. Melia

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azadarach (fruit), Phytolaca dodecandra, Lepidium sativum, Vernonia amygdalina, Capsicum sp., Gynandropsis gynandra, Margaritaria discoidea, Andropogon gayanus and Acalypha fruticosa, have a toxic effect on tick life-cycle stages, causing tick mortality, tick repellence and tick immobilization (Borges et  al., 2003). Surveillance studies have been conducted in various parts of the world (Jeyathilakan et al., 2019; Pereira et al., 2019) and in Pakistan (Zaman et al., 2018; Khan et al., 2019) to develop inventories of ethnoveterinary practices adopted against parasitic infections in general and/or tick infestation specifically. 5.6.2 Biological control Scientists are moving towards alternative control methods after ticks have developed resistance to chemical acaricides. Natural control is biological control, with its origins in ancient China, where it was discovered that ants feed on citrus pests and increase their population by moving their nest to orchards. Different biological agents were found, ideas were created and history was considered for the creation of biological control agents in the late 19th century. Biological agents such as nematodes, fungi and crystalliferous bacteria have been used to control mosquitoes; some of them were also used against ticks. The parasitic wasp Ixodiphagus hookeri was recognized for its ability to control ticks in the 20th century (Samish et  al., 2008). Predators such as birds, ants, rodents, shrews and spiders have been used to control ticks. Poultry birds also play a role in removing infested ticks directly from animals as well as from housing, which ultimately decreases tick burdens on animals. But mixed practices with dairy and poultry are reduced owing to the transmission of diseases such as salmonellosis and cryptococcosis. As well as harbouring diseases, poultry birds utilize cattle feed and create a mess on feed ingredients, reducing dairy feed consumption (Ghosh et al., 2007). Entomopathogenic fungi (EPF) have been used as a biological control of arthropods since the 1880s, and currently many examples of successful fungus-based insect control programmes are seen worldwide. Beauveria bassiana and Metarhizium anisopliae have been reported to have potential against resistant ticks (Salas et  al., 2019). EPFs are found to be quite efficient in

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Table 5.2. Current status of acaricidal resistance in ixodid ticks. (Author’s own table.) Parameter used for analysis

Amitraz

Resistance ratio, resistance Larval immersion test, R. microplus, factor, % larval mortality, % larval packet test, adult R. appendiculatus acaricide efficacy, % immersion test reproductive estimate, factor for resistance, % egg laying

Deltamethrin

Resistance factor, resistance ratio, % larval mortality, % resistance, % acaricide efficacy

Cypermethrin Resistance ratio, % reproductive estimate, % egg laying, % acaricide efficacy Ivermectin Resistance ratio, LC50 and LC99, % resistance, % larval mortality, resistance factor Fipronil

Resistance ratio, % larval mortality

Diazinon

Resistance factor, % larval mortality

Bioassay

Tick species

Larval packet test, adult immersion test

Larval immersion test, adult immersion test, larval tarsal test Larval immersion test, adult immersion test, larval packet test

Country

References

Ducornez et al., 2005; South Africa, Uganda, Lovis et al., 2013; Brazil, Argentina, Rodriguez-Hidalgo Mexico, New Caledonia, et al., 2017; Bardosh Ecuador et al., 2013; Mendes et al., 2001; Miller et al., 2013 Bardosh et al., 2013; R. annulatus, Benin, Uganda, Brazil, Mendes et al., 2001; R. appendiculatus, New Caledonia, India Abdullah et al., 2012; R. microplus, Hyalomma Yessinou et al., anatolicum, R. decoloratus, 2018a,b; Ducornez R. annulatus et al., 2005; Lovis et al., 2013; Sagar R. microplus, H. anatolicum, South Africa, Brazil, et al., 2020; Faza et al., R. annulatus Argentina, Mexico, India 2013; Miller et al., 2013 R. microplus

Larval immersion test, R. microplus larval tarsal test, larval packet test Adult immersion test, R. microplus, H. anatolicum larval packet test

Brazil, Ecuador, India

Brazil, Mexico

Mexico, India

Maqbool et al.

Acaricide

Rodriguez-Hidalgo et al., 2017; Sagar et al., 2020; Mendes et al., 2013 Miller et al., 2013; Faza et al., 2013 Alonso-Diaz et al., 2013; Sagar et al., 2020

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other parts of the world, including Italy (Fernández-Salas et al., 2019), Mexico (Rodríguez-Casas et  al., 2018), Malaysia (Dominic et  al., 2019), Brazil (Webster et al., 2018) and the USA (Yoder et al., 2017). EPF has been reported to have efficacy against plant insecticides in Pakistan (Yasin et  al., 2019). It attacks the tick cuticles in the three following phases. The entomopathogenic attaches its spore to the ticks’ cuticle where it germinates, penetrates the cuticle and causes disseminates within its host (Mora et al., 2017). In the first part, the adhesion of conidia is performed by hydrophobic and electrostatic interactions to the tick cuticle. In the second part, enzymatic and mechanical degradation facilitate the penetration of fungi into the cuticle of ticks. In the third phase, after the passing through the cuticle, the EPF propagates in hemolymph, ingests food, induces toxins, kills cells and tissue, and ultimately destroys the tick (Ribeiro et al., 2014). Entomopathogenic nematodes (EPNs) had been commonly used against crops, but their efficacy against ticks has been unexplored for many years in cattle. Later studies revealed that they have the potential to control ticks as well J3 infective juveniles enter the blood system of host through body openings

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as insects (invertebrates) in crops. These nematodes have been used in various countries across the world for various insect control measures (Dolinski et  al., 2006) and, for a few decades, these nematodes have been used against ticks (Samish et al., 2008). The efficacy of the EPNs has been tested against R. microplus and Rhipicephalus annulatus (Carvalho et al., 2010a). Species used as EPNs for the biological control of pests belong to two families (Steinernematidae and Heterorhabitidae). The aforementioned families are facultative parasites, and they have symbiotic relationships with the bacteria of the genus Xenorhabdus (Monteiro et al., 2020). The third stage of the nematode (juvenile) is infective and it carries the bacteria in the vesicle inside the foregut. Infective juveniles search for the host through a chemotactic response and then penetrate the host (Gaugler and Molloy, 1981). They then transfer bacteria from their intestine into the bloodstream, where they multiply and kill the host by inducing septicaemia, which typically takes two days (Monteiro et  al., 2013). The mode of action of EPNs is shown in Fig. 5.2.

Symbiotic bacteria released by nematodes

Search for new host

Infective juveniles leave host

First and second generation nematodes develop on the dead host

Fig. 5.2. Mechanism of action of entomopathogenic nematodes for tick control. (Author’s own figure.)

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5.6.3 Nanoparticles Nanoparticles are novel pesticides for arthropod pests and vectors (Benelli and Lukehart, 2017). Copper, zinc, sulphur, silver and many other particles are used for the control of ticks and they provide efficient results (Gandhi et  al., 2017). Various nanoparticles and their mode of action are described in Fig. 5.3.

5.6.4

Genetic and immunological control

Tick-resistant livestock breeds may be useful in terms of producing high-lactating tick-resistant animals. In response to the selection for greater tick resistance in the Bos taurus line, Frisch et al. (2000) reported genetic alterations in tick resistance over 15 years. The northern Australian beef industry has been controlling ticks mostly through non-chemical methods during the past 20 years by improving host resistance via increasing Bos indicus content (Sutherst et al., 1979). Robbertse et al. (2018) compared the levels of T and B lymphocytes in the skin and lymph nodes of three different breeds of cattle (resistant and susceptible). They reported that tick-resistant breeds showed increased levels of T lymphocytes compared to susceptible breeds, which showed

decreased levels followed by an elevated level of B lymphocytes. The research suggested that resistant breeds can manipulate B lymphocytes compared to susceptible animals. However, it has been reported that higher levels of IgG1 in resistant animals did not play a role in tick resistance while causing an increase in susceptibility to tick infestation (Piper et al., 2017). A study examined the concentration of cytokines and chemokines in the skin of tickresistant and susceptible animals, suggesting that, upon tick biting, resistant animals produce pro-inflammatory chemokines and cytokines, which, in turn, recruit granulocytes and T lymphocytes immediately through the delayed response to tick bite in susceptible individuals (Franzin et  al., 2017). In South Africa, it is reported that genes responsible for variation in tick counts in different breeds of cattle could be used as markers for genetic manipulation of cattle for tick resistance. These potential candidate genes could be identified through heritability estimates in different high-producing local breeds of cattle (Mapholi et al., 2016). Similarly, in Brazil and Australia, heritability has been estimated for different crossbred, purebred and mixed cattle breeds. These heritability estimates show that the genes responsible for tick resistance are highly heritable to the next generation for the lifetime and may help in controlling tick burden,

Graphite oxide

Cell death

Polystyrene

Reduced acetylcholinesterase Activity

Oxidative stress

Inhibit CY450 isoenzyme Impact on antioxidant enzymes

Disrupted reproduction and development

Silica & alumina

Bind to cuticle Sorbing cuticular lipids and waxes

Insect dehydration

Trypsin inhibitors

Bind with S and P proteins Silver

Developmental damage Toxicity, fitness reduction

Gold

Fig. 5.3. Mechanism of action of various nanoparticles for tick control. (Author’s own figure.)

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ultimately elevating the production of animals (Shyma et al., 2015). It has been reported that lipid metabolism plays a key role in the control of inflammation and impairment of tick infestation in resistant animals, while a similar kind of response has been observed in susceptible animals at the acute phase of tick infestation (Ferreira et al., 2014). The role of inflammatory cytokines and chemokines CD25, IL-10, CXCL10 and CXCL8 has also been observed through gene expression analyses, which showed that the expression level of these genes altered 48 hours post-infestation. Moreover, a positive correlation has been found between Tγδ cell activity and the immune response of tick-resistant animals (Domingues et al., 2014). It has been reported that genes coding for the heavy chain of IgG2 have different genotypes associated with tick phenotypes, creating a hindrance in the normal function of tick immunoglobulin G binding proteins (IGBPs) in tick-resistant animals (Carvalho et al., 2016). The genes responsible for the inflammatory and immune response in Holstein Friesian (B. taurus) cows to tick bite and infestation have been compared with those in Brahman (B. indicus) cattle. These studies have shown the greater upregulation of genes responsible for the immune response in Holstein Friesian rather than Brahman cattle (Piper et al., 2010). Histological examinations have shown that skin reactions of resistant breeds have higher levels of basophils and eosinophils than susceptible individuals. The expression of adhesion molecules, e.g. intracellular adhesion molecule-1 (ICAM-1), P-selectin, and vascular cell adhesion molecule-1 (VCAM-1), has been found to be higher in susceptible animals than in tick-resistant breeds. Contrarily, in resistant animals, the level of E-selectin, which plays a role in the adhesion of T memory cells, was observed to be higher than in susceptible animals (Carvalho et al., 2010b). The role of epidermal permeability barriers in the skin of resistant and susceptible breeds of cattle has also been observed. The increased concentrations of KRT5 and KRT14 (basal epidermal keratins), LCN9 (lipid processing protein), TGM-1 (catalysing epidermal enzyme) and Blimp1 (B lymphocyte maturation protein) in the skin of resistant breeds have been reported to be responsible for mediating resistance against tick infestation (Kongsuwan et  al., 2010). In a

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study by Piper et al. (2009), the establishment of a stabilized response of T cells has been observed in B. taurus and B. indicus. It has been reported that B. indicus has a consistent and stabilized T-cell-mediated response compared to B. taurus, which shows only an innate immune response. However, higher levels of IgG1 have also been observed in B. taurus. Genes associated with the Ca2+ channel signalling, i.e. IL2, CASQ, PLCG1 and AHNAK, could play a role in developing tick resistance in the host (Bagnall et  al., 2009). Investigating how tick infestation affects the expression levels of various proteins, including keratin, mitochondrial proteins, odorant-binding protein and the B. taurus main allergen BAD20, could aid the identification of prospective candidates for selection in resistant breeds of animals (Wang et al., 2007). Martinez et al. (2006) have reported that bovine lymphocytic antigen BoLA-DRB 3.2 alleles could be used to select animals with tick-resistance capability. The use of an immunological approach for tick control results in a decreased number of tick populations on animals and leads to vaccine formulation against a wide range of ticks (Fuente and Kocan, 2006). Two basic types of antigens are employed in ticks, i.e. concealed and exposed (Kiss et al., 2012). Antigen selection is a major hurdle in the development of anti-tick vaccines. Concealed antigens can be accessed by host antibodies taken during blood feeding. The metagenomic study of ticks with the help of bioinformatics tools, mutagenesis, immunomapping, transcriptomic studies, RNA interference (RNAi) and expression library immunization (ELI) has enabled new pathways toward vaccine development against ticks (Fuente and Kocan, 2006). There have been various trials of the tick vaccine. Recently, cattle were immunized using Hd86 antigens with Bm86 antigens of Hyalomma scupense and R. microplus, respectively (Galaï et al., 2012). In Australia and America, the first anti-tick vaccines used for immunizing cattle are Tick GARD, TickGARDPLUS™ and Gavac™, and results show a 59.19% reduction in H. scupense nymphs using Hd86 antigens, whereas Bm86 antigens show no response against H. scupense nymphs, although, in the case of adult ticks, both antigens (Hd86 and Bm86) were unable to protect the cattle (Kasaija et  al., 2022). Rhipicephalus microplus antigens (de Vos et al., 2001), Bm86 antigens (Rodriguez-Valle et  al., 2012),

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and the orthologue of Bm86, rHaa86, can be employed to control Hyalomma anatolicum (Jeyabal et  al., 2010). Rhipicephalus microplus antigens protect the host against Hyalomma dromedarii as well. Figure 5.4 shows how vaccinations for tick protection function. The use of advanced molecular techniques (RNAi) for tick control results in the suppression of targeted genes, i.e. calreticulin, subolesin and cathepsin-L-like cysteine proteinase of H. anatolicum, resulting in a reduction of the engorgement rate of ticks and providing potential candidates for anti-tick vaccine development (Kumar et al., 2017).

5.7 Conclusions

Immunization of animal populations at risk and reservoir hosts

This chapter emphasizes the critical need for diverse and long-lasting tick control strategies in the fight against acaricidal resistance. A paradigm change away from traditional chemical controls is required owing to the economic consequences of tick infestations on the cattle industry, which are exacerbated by the estimated yearly losses of billions of dollars. The previous over-reliance on acaricides – from ivermectin to arsenic – has not only resulted in the emergence of resistant tick strains but has also left drug residues in animal products, which has contaminated the environment.

The classification of resistance types – acquired, multiple and cross-resistance – offers a thorough grasp of the difficulties encountered. Because of its biological, operational and genetic roots, acaricidal resistance necessitates a comprehensive strategy. Dosing accuracy, resistance allele dominance, genetic diversity and the function of biological parameters highlight the complex interactions affecting the development of resistance. The weaknesses in regularly used acaricides are highlighted by the mechanisms of acaricidal resistance that have been elucidated: metabolic detoxification and target site mutations. Designing substitute tactics that obstruct or avoid these resistance channels requires an understanding of these systems. The current state of acaricidal resistance is a clear call to innovation, in which resistance can be assessed using complex methods such as phenotypic, genotypic and enzymatic analysis. Bioassays, molecular testing and biochemical assays work together to provide a more nuanced picture of resistance prevalence to help formulate targeted control strategies. Conventional and contemporary alternative control methods offer a range of options. To reduce the risk of harm to the ecosystem, conventional methods such as pasture spelling, rotational grazing and habitat management must be carefully considered. Phytotherapy, biological management Commercially available vaccine

Reduction in tick infestation

TickGARDPLUS˜ BM86

VBP VBP VBP VBP

VBP

Reduction in: infected tick level vector borne pathogen (VBP) infection level vector capacity

Number of infected Ticks Weight Reproductive capacity

Fig. 5.4. Mode of action of vaccines against tick control. (Author’s own figure.)

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using nematodes and fungi, nanoparticle-based therapies and genetic/immunological techniques are at the forefront of contemporary strategies.

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The next step in tick management is combining these various approaches into a unified, longlasting framework.

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Prevalence of Antiparasitic Drug Resistance in Various Areas of the World Zubaria Shahid Amin1, Nadia Nazish2, Qaiser Akram3*, Muhammad Rizwan Saeed3, Tooba Abbas4, Waqas Ahmad5 and Aiman Maqsood4 1 Animal Sciences Institute, Livestock & Dairy Development Department, Quetta, Baluchistan, Pakistan; 2Department of Zoology, University of Sialkot, Pakistan; 3 Department of Pathobiology (Microbiology Section), KBCMA College of Veterinary & Animal Sciences, Narowal, Sub-Campus UVAS, Lahore, Pakistan; 4Department of Zoology, University of Narowal, Narowal, Pakistan; 5Department of Clinical Sciences (Epidemiology Section), KBCMA College of Veterinary & Animal Sciences, Narowal, Sub-Campus UVAS, Lahore, Pakistan

Abstract Antiparasitic drug resistance is an ever-growing global problem for animal health, welfare and productivity. This chapter briefy examines the prevalence of antiparasitic drug resistance in different parts of the world and explores the mechanisms behind the emergence and development of resistance to assist in the advancement of effcient and long-lasting drug resistance management. Techniques for detecting and monitoring antiparasitic drug resistance are also covered, demonstrating the variety of ways used to measure the effcacy of antiparasitic medications and the international efforts to develop cooperative surveillance programmes. The chapter also highlights the implications of drug resistance on parasite control programmes, underscoring the necessity for concerted efforts in the areas of drug development, research and management to counteract this escalating threat. The chapter ends by discussing the need for continued research, especially in diagnostic tools, vaccine development, selective breeding and other strategic interventions, to improve control choices for drugs resistance and to attain a more viable and holistic resistance management strategy in support of animal productivity and wellbeing.

6.1

Introduction

On Earth, parasites are a continual threat to life, and they can spread like a chain reaction. To lessen the risk of infection, a pathogen must be controlled at the local, national and international levels. It is evident that problems related to parasitosis are returning despite tremendous progress in our knowledge of parasites, our attempts to eradicate them, and the state of cleanliness (Dziduch et al., 2022).

Worldwide, helminths are the main root cause of endemic animal diseases that limit productivity, others being insects, arachnids and protozoans (Vercruysse et al., 2018). As a result, many farmers are dependent upon the use of antiparasitic agents to maintain their livestock health. Anthelmintics, insecticides/acaricides and antiprotozoals are three types of antiparasitic agents. The invention and availability of extremely effective antiparasitic medications has greatly lessened the

*Corresponding author: [email protected]

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© CAB International 2024. Antiparasitic Drug Resistance in Veterinary Practice (eds H.M. Rizwan, M.A. Naeem, M. Younus, M.S. Sajid and X.Chen) DOI: 10.1079/9781800622807.0006 Downloaded from https://cabidigitallibrary.org by Ivanov Ivan, on 11/04/24. Subject to the CABI Digital Library Terms & Conditions, available at https://cabidigitallibrary.org/terms-and-conditions

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monetary cost of parasitism. But resistance has developed to practically all antiparasitic medication classes used to treat helminths, protozoans and arthropod parasites and, as a result, this has become a major global economic issue for the livestock sector (Sazmand et  al., 2020; Alvi et  al., 2023). Antiparasitic resistance to medications is defined as the ability of a particular parasitic species to survive, despite being exposed to an antiparasitic medicine at the appropriate dose. The most common causes of resistance include mass treatment of animals, repeated usage of the same antiparasitic medication, and use of the medication at suboptimal dosages (Alvi et al., 2023). It is challenging to create efficient management strategies owing to the multitude of elements that contribute to parasitism. Therefore, depending on the prevalence of the disease and antiparasitic drug resistance, regionally based methods for controlling the spread of parasitic diseases that do not exclusively depend on antiparasitic medicines need to be devised (Alvi et al., 2023). Geographic variations in the emergence and spread of resistance have been attributed to a number of variables, including weather patterns, parasite species, therapeutic modalities, medication usage patterns and more. In the temperate areas of the Northern Hemisphere, the resistance rate is marginally lower (Malik et  al., 2022). Examining the occurrence of resistance to drugs in several worldwide regions such as the South Pacific, Australia, Latin America, North America, Africa, the Eastern Union and South-east Asia gives us an international outlook on this emerging issue (Malik et  al., 2022). Unfortunately, there is currently a lack of worldwide data about drug resistance, necessitating more surveillance, The Starworms Project is one initiative attempting to close this information gap (Vlaminck et al., 2018), as is the Australian survey on anthelmintic resistance in horses (Abbas et al., 2024). In this chapter, the prevalence of drug resistance in different parts of the world is thoroughly examined, covering important aspects such as resistance mechanisms, surveillance programmes, influencing factors and emerging trends. We investigate novel approaches to counter this growing threat, such as developing novel analogues, utilizing bioactive forage and embracing advances in diagnostics. The chapter ends with a call to action that emphasizes the necessity for international cooperation, cooperative research,

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and the development of molecular assays in order to protect the efficacy of antiparasitic drugs, as well as the development of non-chemotherapeutic agents against parasites in the future.

6.2

Mechanisms of Antiparasitic Drug Resistance

In addition to being a tool for researching parasite biology and potential treatment targets, an understanding of resistance mechanisms can aid researchers in better forecasting the rate at which resistance will develop (Fissiha and Kinde, 2021). An increase in drug metabolism, a modification to drug receptor sites that lessens drug binding or the functional effects of drug binding, an increase in cellular efflux mechanisms, or a decrease in drug receptor abundance through decreased expression or another downregulation mechanism are common mechanisms of antiparasitic resistance (Fig. 6.1). P-glycoproteins, in particular, are protein transporters that function as an efflux mechanism to move substances across the cell membrane and reduce their intracellular concentration. As a result, the medication cannot reach its intended location. It has been reported that Haemonchus contortus has ivermectin resistance linked to P-glycoproteins (Reyes-Guerrero et  al., 2020). The relationship between the aforementioned changes and resistance varies between parasite species (Sarai et al., 2014). It has been suggested that mutations in the genes encoding ligand-gated chloride channels may impart antiparasitic resistance (AR); these channels are the most potential targets of macrocyclic lactone therapy. Early publications on the mechanism of ivermectin resistance in parasitic worms mentioned the prevalence of mutations in glutamate-gated chloride channel receptors (GluClRs), as described in Fig. 6.2. An allele of a GluCl α-subunit gene was detected more frequently in isolates of H. contortus that were resistant to ivermectin and moxidectin, suggesting that a mutation in this gene was associated with macrocyclic lactone resistance (Kotze et al., 2014). Changes in β-tubulin have been conclusively related to the mechanism of benzimidazole (BZ) resistance. The phenylalanine to tyrosine alteration at position 200 in isotype I β-tubulin may be the source of resistance to BZ (Shayan et al., 2006). In resistant worms, BZ binding is inhibited by even a

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Drug a: Disturbance in drug metabolism b: Modification to drug receptor site c

c: Increase in cellular efflux d

d: Decrease in drug receptor abundance

b

a

Fig. 6.1. Possible mechanisms of drug resistance against antiparasitic drugs. (Author’s own figure.)

GluCIR gene

Mutation

Resistance to ivermectin

Tubulin gene

Mutation

Resistance to benzimidazoles

nACh gene

Mutation

Resistance to midothiazoles and tetrahydropyrimidines

Fig. 6.2. Genomics of resistance against ivermectin, benzimidazoles and midothiazoles. (Author’s own figure.)

single amino acid mutation in the tubulin protein. The isotype 1-tubulin gene has three nonsynonymous single nucleotide polymorphisms (SNPs) that have been connected to BZ-resistant gastrointestinal nematode species. The phenylalanine to tyrosine substitution at position 200 (F200Y) is caused by the most common SNP; a glutamic acid to alanine substitution at position 198 (G198Y) (E198A) and a phenylalanine to tyrosine substitution at position 167 (F167Y) are caused by the other two SNPs (Chaudhry et al., 2015). Research on the mechanisms underlying resistance to nicotinic agonist medications has been focused on the role of nicotinic acetylcholine receptors (nAChRs), specifically the L-type subset of these receptors that is preferentially activated by levamisole

and pyrantel (Martin et al., 2012). The activation of these L-nAChRs results in spastic paralysis and neuromuscular depolarization. Changes to the nematode’s target site seem to be the most likely route of resistance to levamisole and pyrantel in a variety of Trichostrongylid species (Sarai et al., 2015).

6.3

Global Surveillance of Drug Resistance

Faecal egg count reduction, egg reappearance period, in vitro worm motility assessment, larval development assays, DNA extraction, PCR-based next-generation sequencing and Nemabiome

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analysis are some of the techniques used to evaluate the effectiveness of antiparasitic medications. Regretfully, worldwide data on drug resistance against parasites are scarce (Abbas et al., 2024). Cyathostomins showed resistance to a range of anthelmintics, including combination products (oxfendazole and pyrantel) and single actives such as abamectin, ivermectin, moxidectin, and oxfendazole, according to a national survey conducted in Australia on anthelmintic resistance in horses. Similarly, resistance of Parascaris spp. to ivermectin, abamectin, oxfendazole and moxidectin was noted (Abbas et al., 2024). The Starworms Project was funded by the Gates Foundation to track anthelmintic resistance in human soil-transmitted nematodes. Despite the establishment of an international resistance screening network by the World Health Organization (WHO’s) Tropical Diseases Research network (TDR), technique limitations have resulted in low- to medium-throughput screening methods (Smout et  al., 2010). Smout et  al. (2010) presented a novel application of the real-time cell assay device (xCELLigence), overcoming the limitations of existing techniques and enabling the objective and effective assessment of anthelmintic drug effectiveness in real time by measuring motility across different developmental stages of human and livestock helminth parasites. The WHO introduced a new protocol in 1996 to evaluate antimalarial medication efficacy in areas where disease transmission is high. Since then, based on feedback from nations and scientific input, the WHO has regularly revised the therapeutic efficacy protocol for such hightransmission areas and validated a different protocol for regions with low-to-moderate transmission. The most recent version of the efficacy test protocol was approved in 2001 (WHO, 2003). It is necessary, however, to incorporate the changes that the Technical Expert Group on Malaria Chemotherapy, which met in 2005 and 2008 to discuss the guidelines for the treatment of malaria, recommended (WHO, 2006). Recent research by Emsley et al. (2023) reveals a worrying trend of AR against gastrointestinal parasites in sheep and goats across several South African provinces. Resistance was revealed by in vivo evaluations, which showed a faecal egg count reduction percentage greater than 95% or a lower confidence limit of 90%. The results of the egg hatching assay and the

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larval motility assay confirmed the emergence of resistance by showing that there was no obstacle to egg development or larval mortality. Positively, acaricide resistance against fluazuron–flumethrin, a frequently prescribed medication in South Africa, was not found (Emsley et al., 2023). Prior studies that attempted to address AR at the national level or through projects that concentrated on particular aspects of the AR challenge have not been very successful. According to Entrican et  al. (2020), these efforts have had particular difficulty in developing diagnostics for AR detection, taking socio-economic factors into account, and integrating a variety of control tools. The establishment of the COST Action Combatting Anthelmintic Resistance in Ruminants (COMBAR) was motivated by the realization that cooperation and the sharing of knowledge were essential on a European and worldwide level. The main goal of COMBAR was to create a network of recognized authorities on the topic of long-term parasite control in animals from Europe and other regions (Charlier et al., 2023). The creation of the STAR-IDAZ IRC research roadmaps for helminths and AR is a noteworthy achievement of COMBAR. These roadmaps provide a thorough framework for coordinating international research projects and accelerating the delivery of necessary control instruments (Entrican et al., 2020).

6.4

Influencing Factors on Drug Resistance in Parasites

Modern antiparasitic drugs are effective against susceptible strains to a degree of about 99% (Fissiha and Kinde, 2021). Still, a small percentage of hardy parasites make up the hardiest subset of the population. After being discharged into the environment, these parasites that have survived contaminate pastures and encourage the emergence of resistant generations in the future. As a result of continuing selection pressures, AR eventually emerges (Jabbar et al., 2006).

6.4.1 Frequency of treatment One important factor that accelerates the development of AR is the frequency of anthelmintic

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treatment. Resistance develops more quickly when anthelmintic administration occurs more often. The underlying idea of AR selection is that, about two to three weeks after the anthelmintic is administered, therapy gives surviving parasites a reproductive and replicative advantage over susceptible parasites (Vercruysse et al., 2011).

Interestingly, when resistance is a recessive feature, only homozygous parasites can tolerate an appropriate dose of antiparasitic, which eradicate heterozygous parasites (Shalaby, 2013).

6.4.2 Targeting and timing of mass treatment

Several factors must be considered before an AR diagnosis can be made. First, it is important to understand that a number of diseases might present with clinical symptoms that are similar to those of parasitism. Furthermore, resistance may not be the reason why antiparasitic therapies fail to control parasites. Instances such as defective soaking equipment or erroneous dosing due to incorrect body weight calculations are common causes of failures in these situations. The increasing ubiquity of AR has increased the need for consistent and trustworthy detection techniques. AR is detected and tracked using both in vitro (egg hatch assays, larva development test, larval motility test) and in vivo (faecal egg count reduction test) methods (Ihler, 2010). The detail of various in vitro methods for assessing antiparasitic drug resistance are shown in Fig. 6.3.

The development of AR has been linked to mass preventive therapy. However, treating about 80% of the flock can postpone the development of resistance, adding a layer of strategic management to the control process (Jabbar et al., 2006).

6.4.3 Anthelmintic dose rate One of the main factors contributing to the development of AR is the use of a drug at wrong or unsuitable dosages. Visual weight estimation is a frequently used method for calculating dosage rates, yet it is not always precise and can result in underdosing. According to Nielsen et al. (2010), underdosing promotes the survival of heterozygous resistant worms and aids in the selection of resistance strains.

6.4.4

Genetic factors

As a pre-adaptive process, resistant parasites are already present in the parasite population. When resistance alleles exist in the parasite population prior to exposure to the particular antiparasitic drug, this situation is known as antiparasitic resistance. In the absence of antiparasitic drugs, natural selection preserves resistance alleles at low frequencies since they make the parasite carrying them less adapted for life than the parasites that are entirely susceptible. When antiparasitic drugs are introduced and used consistently, resistant parasites gain a survival advantage that allows them to proliferate more quickly than susceptible parasites. As a result, until antiparasitic resistance is established, the frequency of parasites exhibiting a resistance phenotype rises.

6.5

6.6

Methodologies for Assessing Drug Resistance

Prevalence of Drug Resistance in Specific Geographic Areas

AR is now a prominent problem in many European countries, as a result of the heavy use of antiparasitic drugs to control parasites. It has frequently been observed that certain parasite strains are resistant to imidazothiazoles, benzimidazoles and/or macrocyclic lactones. This is especially the case for the three most significant genera: Haemonchus, Teladorsagia and Trichostrongylus. Furthermore, it has been discovered that these parasites have several drug-resistant populations (Papadopoulos et al., 2012). The widespread dissemination of parasite populations resistant to one or more parasiticide classes has resulted from the extensive use of antiparasitic drugs in the treatment and management of parasites that affect animals. This condition, which is a serious issue in the Southern Hemisphere, has now started to pose a threat in Europe as well (Traversa and von SamsonHimmelstjerna, 2016).

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The egg hatch assay is a technique for assessing benzimidazole resistance by counting the remaining eggs and hatched larvae and calculating LD50 values

Egg Hatch Assays (EHAs)

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The larva development test reveals resistance to key anthelmintic families and it measures the larvae’s ability to survive and develop in response to different anthelmintic concentrations

The larval motility test stimulates nonparalysed larvae by exposing them to light for 20 minutes after a 24-hour incubation period at 25°C in various medication doses

Using a PCR-based technique, mutations on ˜tubulin residue (phenylalanine to tyrosine) linked to benzimidazole resistance can be identified in adult worms or larvae

Larva Development Test (LDT)

Larval Motility Test (LMT)

Molecular Characterization (PCR)

Fig. 6.3. In vitro methods for assessing antiparasitic drug resistance. (Author’s own figure.)

About 10 years ago, the USA saw the first known instance of cattle gastrointestinal nematodes developing resistance to macrocyclic lactones. Since then, there has been a worrying increase in the rate at which anthelmintic resistance has grown. Now, more than half of the procedures studied have shown evidence of parasites belonging to the genera Cooperia and Haemonchus that are resistant to macrocyclic lactones (Gasbarre, 2014). There is also strong evidence that anthelmintic resistance is a very serious problem in some regions of the USA especially in the south-east (Howell et al., 2008). There is a burden of antimalarial drug resistance in Africa, resulting in malaria being endemic (Takala-Harrison and Laufer, 2015). About 90% of worldwide malaria deaths and morbidities occur in Africa, where the disease is still a major problem. Plasmodium falciparum, the most virulent human malaria parasite and the one most likely to develop drug resistance, is the  primary cause of malaria in most of sub-Saharan Africa (Conrad and Rosenthal, 2019). Diseases associated with protozoan parasites and common drug resistances are given in Table 6.1. In endemic areas, managing P. falciparum malaria is severely hampered by the establishment and dissemination of drug-resistant parasites. This is particularly evident in South-east Asia, where the failure of mefloquine monotherapy in the 1980s worsened multi-drug resistance

to sulfadoxine–pyrimethamine and chloroquine (Uhlemann and Krishna, 2005). Haemonchus contortus has a remarkable capacity to develop resistance to anthelmintic medications. The majority of the anthelmintic medication families, including closantel, imidazothiazoles, macrocyclic lactones and benzimidazoles, are said to be resistant to H. contortus in Asian field populations (Arsenopoulos et al., 2021). There is evidence that equine cyathostomins are resistant to various anthelmintic drugs in some countries of Europe such as Italy, Germany and the UK (Traversa et  al., 2009). In sheep parasitic nematodes, anthelmintic resistance is frequent in New Zealand. New Zealand sheep farmers and advisors should reconsider how they handle parasites (Waghorn et  al., 2006). Resistance to ivermectin was more widespread than anticipated, in addition to resistance to albendazole and levamisole. In Australia, AR has been found in some regions such as Western Victoria, while there is no evidence of AR in other regions (Preston et al., 2019).

6.7 Comparative Analysis of Resistance Patterns Benzimidazoles (BZs) are the most often utilized class of anthelmintics for managing parasitic

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Table 6.1. Diseases associated with protozoan parasites and common drug resistances. (Author’s own table.) Parasite

Disease

Drug resistance

Toxoplasma gondii Trypanosoma spp. Entamoeba spp. Plasmodium spp. Leishmania spp.

Toxoplasmosis Trypanosomiasis Amoebiasis Malaria Leishmaniasis

Artemisinin, atovaquone, sulfadiazine Melarsoprol, suramin, nifurtimox, nitrofuran, benznidazole Metronidazole, emetine Chloroquine, pyrimethamine, artemisnin, piperaquine Paromomycin, amphotericin B, pentamidine, antimonials

infections in animals because of their high therapeutic index and ratio, lack of drug residue in milk and meat, and economic viability. Owing to the ongoing use of BZs, trichostrongylid parasitic worms have developed resistance to BZ medications globally. The BZ resistance of strongyle nematodes is primarily associated with mutations in the β-tubulin isotype 1 gene (Bhinsara et al., 2018). For certain nematodes, the processes underlying BZ resistance are well known. Helminths develop BZ resistance because of distinctive SNPs at codons 167, 198 and 200 of the β-tubulin gene. There is no known SNP that can account for the failure of BZ treatment in hookworm and other nematode-associated human disorders (Furtado et al., 2016). Significant attention is being paid to reports of loss-of-efficacy (LOE) events in dogs infected with Dirofilaria immitis who adhered to recommended prophylactic regimens using a macrocyclic lactone anthelmintic. The theory that parasites derived from LOE cases underwent a strong selection event is supported by molecular evidence. These populations are distinguished by very high frequencies of SNPs in a D. immitis gene encoding a P-glycoprotein transporter, which is composed of homozygous guanosine residues at two locations (known as the ‘GG-GG’ genotype). Moreover, a microfilarial population afflicted dog brought to Canada from the southern USA was resistant to extremely high dosages of macrocyclic lactones and exhibited a high frequency of the GG-GG genotype linked to LOE cases (Geary et al., 2011). There is some type of resistance of parasites to ivermectin but no complete cross-resistance against moxidectin (Bygarski et al., 2014). Among the macrocyclic lactones, abamectin is the most susceptible drug against which parasites are developing resistance (Kotze et al., 2014). Although the exact mechanisms of resistance in  H. contortus are unknown, it is possible that nematode ATP-binding cassette transporters such as P-glycoproteins are responsible for the

removal of macrocyclic lactones. Ivermectin and abamectin significantly and saturably decrease the capacity of H. contortus P-glycoprotein 2 to transport several fluorophore substrates. The profile of transport inhibition for moxidectin is noticeably different. The expression of H. contortus P-glycoprotein 2 in the pharynx, the first section of the worm’s intestine, and possibly in nearby nervous tissue suggests that this gene may have a function in controlling ivermectin uptake and shielding nematode tissues from the harmful effects of macrocyclic lactone anthelmintic medications. Thus, P-glycoprotein 2 in H.  contortus may be involved in the organism’s resistance to these medications (Godoy et  al., 2015). If a parasite becomes resistant to any class of macrocyclic lactones like ivermectin, then other members of the group can be used, e.g. moxidectin and abamectin, but resistance can also develop against these drugs (Leathwick et al., 2019).

6.8 Emerging Trends in Antiparasitic Resistance Antiparasitic resistance has become so severe in recent decades that it is now unavoidable as a significant concern in animal parasite management. Multiple-resistant parasites are quite widespread worldwide, and it is no longer unusual to discover cattle ranches where all existing antiparasitic medications are ineffective against the parasites (Fissiha and Kinde, 2021). Earlier, farm animals with parasite diseases were successfully treated with antiparasitic chemotherapeutics, also known as anthelmintics, ectoparasiticides (insecticides and acaricides) and antiprotozoals (Malik et  al., 2022). But for a number of reasons, the parasite population has become resistant to the majority of antiparasitic treatments that have been deployed (Fissiha and

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Kinde, 2021). For example, phenothiazine was the very first anthelmintic to be introduced in the 1940s, and by 1957 resistance to the drug had been documented in the USA. In South Africa, it was discovered that ivermectin, one of the miracle medications of the 1980s, proved useless against gastrointestinal nematodes (Malik et al., 2022). In some regions of the world, it has also been observed that recently introduced anthelmintics such as derquantel and monepantel are inefficient at controlling roundworms, which are frequently seen in small ruminants (Kaminsky et al., 2011). The great susceptibility of these medications is demonstrated by the establishment of resistance to anthelmintic agents within 10 years after their introduction. Globally, the overall incidences of resistance have grown, and this trend is predicted to continue owing to widespread medication administration, which creates a pressure to select for resistance alleles. Anthelmintic resistance surveillance should be implemented to postpone the beginning of treatment failure (Shalaby, 2013). Resistance against monepantel which has emerged in less than 4 years after its launch is very concerning and needs to be considered a caution (Sales and Love, 2016). Thus, during the coming years, the availability of novel anthelmintic treatment will be crucial. Additionally, significant work must be invested in creating novel chemical entities, particularly those that have stronger effects on parasites that are resistant to traditional antiparasitic medications (Lanusse et  al., 2018). While new compounds and medications have traditionally been found by empirical means, chances for target-based discovery of novel therapeutics have been greatly increased by  developments in genomic, proteomic and metabolomics research. Modern drug discovery methods, which often depend on the mechanism-based screening of novel compounds, make advantage of the recently identified mechanisms of pharmacological effects and their targets (Geary, 2016). The use of plants as medicines is another new emerging trend for controlling parasitic diseases in the age of antiparasitic medication resistance (Siddique et al., 2023). The development and accessibility of synthetic medications has stifled the application of plant-based prevention and therapy for parasite infections, a collection of diverse methods utilized for millennia. However,

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plants that contain a vast array of different active  compounds, such as flavonoids, terpenes or  condensed tannins, have historically been the  main source of new medicinal discoveries (Acevedo-Ramírez et  al., 2019). Recently, it has been discovered that Azadirachta indica extract from leaves is useful in treating gastrointestinal helminthiasis (Srivastava et al., 2020). Furthermore, research on the biological management of parasites with nematophagous or predatory fungus has increased in the era of AR. Microorganisms called nematophagous fungus have the ability to lower the number of parasites without endangering the host animals. When controlling nematode larvae, Duddingtonia flagrans is seen to be the most effective fungal species (Rodrigues et al., 2018). It has also been reported that fungi such as Monacrosporium thaumasium and Arthrobotrys robusta are effective parasite control agents. Their capacity to adhere to, pierce and demolish helminth eggshells means some fungal species (Pochonia chlamydosporia, Mucor circinelloides, Purpureocillium lilacinum and Trichoderma spp.) have demonstrated action against helminth eggs and even coccidian oocysts (Hernández et al., 2018; AhuirBaraja et al., 2021). In short, there is a critical need for international multi-actor projects owing to the emergence of new difficulties and solutions resulting from the ongoing expansion of AR and increased research efforts, respectively. To establish objectives, encourage best practices, and create indicators of infection and sustainable control, these projects should engage every relevant stakeholder.

6.9

Impact on Parasite Control Programmes

Chemotherapy is still the first line of defence against parasites that cause a wide range of infections as there are no reliable vaccinations to prevent them. Unfortunately, most commonly used medications to treat such diseases have evolved resistance in the corresponding parasites (Auld and Tinsley, 2015). Because the mechanisms underlying drug resistance in parasites are varied in relation to their life cycle and nature, combatting these parasites is made more difficult (Pramanik et al., 2019). Antiparasitic drug

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resistance has put parasite control techniques in a dire situation (Molento, 2009). The most important impact exerted by parasiticide resistance on parasitic controls is the genetic susceptibility of the parasite. The dominant role of resistant alleles, the number of genes that are involved, the initial frequency of resistant genes, the diverse genetic makeup of the population, the relative viability of resistant organisms, the likelihood of linkage disequilibrium, and the potential for the recombination of genes are among the parasite genetic factors that influence the development of resistance (Sangster, 2001). All of these factors make the selection pressure on the parasite population more intense and provide resistant parasites with a competitive edge for survival. This makes it possible for them to multiply more quickly than susceptible ones, thus raising the frequency of resistance-phenotype parasites in the population and reducing the effectiveness of parasitic control programmes that are mostly chemotherapeutically based (Fissiha and Kinde, 2021). Complexities in the host–parasite relation are another effect that parasiticide resistance has on parasitic controls. Through molecular modifications that alter host gene expression and suppress the reaction of the immune system, parasites thrive inside or outside of their host (Coakley et  al., 2016). By initially imitating the host’s immunological antigen, silencing the host detection mechanisms that would otherwise set off the host’s own immune response and thus suppressing immune system activity, parasites take control over the response of the host immune system, which ultimately leads to failure in parasitic control measures (Idris et al., 2019). The control of parasites is additionally affected by the fact that several types of antiparasitic medication exhibit cross-resistance. For example, numerous antiparasitic medications focus on diverse nicotinic acetylcholine receptors (nAChRs). It is crucial to delve deeper into the precision of the different nAChR-targeting antiparasitic drugs and uncover potential interactions. This exploration holds significance because it could impact the decision to modify antiparasitic medications acting on these receptors. Previous findings have shown evidence of cross-resistance between levamisole and the anthelmintics morantel/pyrantel/oxantel in this scenario. This cross-resistance worsens chemotherapeutic parasite control because parasite species that are

resistant to one class may also become resistant to other related classes (Fissiha and Kinde, 2021). Consequently, resistance to antiparasitic drugs is now changing the standards for parasite control. It is now evident that integrated innovative non-chemical treatments, better husbandry, pasture management techniques and refugia management strategies must become an important part of effective parasite control programmes if economical livestock farming is to be sustained for the long term (Kaplan and Vidyashankar, 2012).

6.10

Challenges in Combatting Drug Resistance

To combat the possibility of resistance, new chemical classes with unique modes of action are needed, but the creation of new substances for all parasites has lagged despite the pressing need for innovation (Nixon et al., 2020). For the expensive process of drug discovery, which is expected to cost between US$50 and $100 million for veterinary medicines and more than US$2.5 billion for human drugs, there must be strong economic forces (DiMasi et  al., 2016). During the procedure of developing antiparasitic drugs, considerations including the total market value, therapeutic advantages and ultimate costs of products need to be made (Moffat et al., 2017). As a result, the effectiveness of new antiparasitic drug discoveries has been and remains to be greatly influenced by the financial factors of the animal healthcare sector. The development of affordable, useful and long-acting medications, together with the need for greater doses of medication per animal, are major obstacles for animal antiparasitic drugs (Nixon et al., 2020). The capacity to make use of diagnostic techniques for detecting AR at an early stage is an essential component of controlling it. As resistance becomes increasingly common in host–parasite systems as a whole, diagnostics may play a significant role in selecting the antiparasitic drugs that seem to be the most efficient (Kotze et al., 2020). Nevertheless, no molecular-based testing is readily available for determining the presence of resistance in the field, despite years of investigation on the molecular foundation of antiparasitic drug resistance and the necessity for tools for detecting and monitoring drug resistance (Hassan and Ghazy, 2022). Therefore, it is also a challenge in combatting AR. Neither existing AR diagnostic

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problems nor the expectation that only one test will work in every situation can be met by molecular diagnostic testing. Rather, a phased strategy that introduces molecular diagnostics in a tailored manner to offer advantages in certain circumstances is required. If this proves to be productive, more evidence for cases will appear, enabling a shift in perceptions on the use of molecular diagnostics for the alleviation of AR (Hassan and Ghazy, 2022). At present, the main diagnostic test is the faecal egg count reduction test (FECRT). Nevertheless, due to its high price, laborious sample operations and limited sensitiveness, it is not generally employed. Currently, in vitro phenotypic assays have limitations to use in laboratories and are not useful for various types of drugs or parasites (Kotze et al., 2020; AhuirBaraja et al., 2021). Management approaches typically try to conserve a section of the parasite population in ‘refugia’, not exposed to treatment, with the objective to reduce the pressure to selection towards resistance while preserving the efficacy of therapy (Leathwick et al., 2019). Refugia insertion in parasiticide resistance treatment plans typically necessitates a shift in farming techniques and farmer attitudes. More resources are always needed, regardless of whether they be in the form of time, effort or money spent on technology, when refugia are considered (Greer et al., 2020). The data supporting the effectiveness of refugia-based techniques in slowing the development of drug resistance in parasites are still limited, despite their convincing rationale and widespread advocacy as best practices. This presents a challenge in the fight against drug resistance (Kenyon et al., 2009). Moreover, a range of variables that could be important for refuges exist in different parasite–host systems. These variables include the cost of drug resistance, the degree of integration between subpopulations of parasitic organisms that are selected through treatment or not, and the impact of environment, genetics and parasite life history on the dynamics of the resistance population (Hodgkinson et al., 2019).

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against parasites. As a result, creating novel parasiticides to control resistance is an expensive and time-consuming task. Therefore, it is important to employ currently available antiparasitic drugs in a manner that reduces the likelihood of resistance, as described in Fig. 6.4 (Ahuir-Baraja et al., 2021). Some of the strategies to control or prevent antiparasitic drug resistance are described here.

6.11.1 Accurate and effective parasiticide treatment The emergence of parasiticide resistance is thought to occur when frequent parasiticide exposure occurs in populations of parasites. Remarkably, the likelihood of developing resistance escalates with underdosing and the repeated administration of antiparasitic drugs belonging to the same class (Nixon et al., 2020). Weighing animals prior to medication therefore becomes essential to prevent underdosing and overdosing. It has been proposed that changing the parasiticide classes might delay the emergence of resistance. Important tactics to postpone parasiticide resistance include using a trustworthy diagnostic to identify the type of parasite, using parasiticides that are appropriate for the diagnosis, and adhering to the instruction label for the proper dose and administration (Fissiha and Kinde, 2021). 6.11.2 Co-administration of antiparasitic drugs Because combined antiparasitic drugs greatly decrease the proportion of the heterozygous genes that are resistant in a parasite population, antiparasitic drugs with different modes of action but related spectra of activity can be used to control AR. This prolongs the half-life of the drug (Molento, 2009; Fissiha and Kinde, 2021). For example, it has been proposed that ivermectin and albendazole work in concert to suppress resistant parasites to both substances in sheep (Entrocasso et al., 2008).

6.11 Strategies for Prevention and Control

6.11.3 Refugia (resistance nests)

Most antiparasitic therapies develop resistance because of the use of chemotherapy treatments

Slowing down the build-up of resistance alleles in parasitic populations is the main goal of

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Reliable diagnosis and correct usage of antiparasitic drugs

Use of vaccines to increase immunity of hosts

Strategies for prevention and control of antiparasitic drug resistance Treatment with combination of antiparasitic drugs having related mode of actions

Refugia reduce selection pressure and delay resistance

Integrated Parasite Management, control of parasites with chemicals usage Fig. 6.4. Strategies for the prevention and control of antiparasitic drug resistance. (Author’s own figure.)

resistance prevention. The earliest stage in the resistance stage is when efforts to limit the emergence of resistance are required (Hodgkinson et al., 2019). The easiest way to do this is to follow the rules that guarantee a high enough degree of refugium. The resistance of a parasite population to antiparasitic drugs is inherited and, once it has been developed, it cannot be lost or reverted. Refugia prevent the spread of resistance by allowing the conservation of vulnerable individuals to reduce the number of resistant parasite progeny that survive treatment (Greer et al., 2020; Fissiha and Kinde, 2021).

6.11.4

Integrated parasite management

Another strategy to control antiparasitic drug resistance is integrated parasite management. It involves understanding the connection between the host and the parasite, monitoring infection and resistance more closely, and using a range of tactics to boost host resistance while controlling parasites without the need for medication. This approach takes time, however (Sangster, 2001;

Burke and Miller, 2020). One of the most intriguing approaches to integrated management involves the selection of parasite-resistant hosts. Host selection can be influenced by immunological responses (genotypic) or by choosing those resistant to infections (phenotypic). Appropriate genetic markers are being found and, if the host population is given the genotypic–phenotypic data, they might provide a low-cost method of controlling parasites (Malik et al., 2022).

6.11.5 Vaccine development (immunogenicity exploration) Because vaccines do not leave residues of chemicals in animal products or the environment, they are thought to be a good alternative for protecting animals against antiparasitic drug resistance (Claerebout and Geldhof, 2020). Now, efforts are underway to pinpoint targets on, or released by, the parasite that may be amenable to high titre antibody responses. A number of target molecules on the intestinal surface of the parasite have been discovered for blood-feeding

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species. Molecules on the luminal surface of intestinal cells are often regarded as ‘hidden’ antigens because the host does not ordinarily identify them during infection. For H. contortus in sheep, a number of vaccines utilizing ‘hidden’ antigens were created; they offered 94% protection when assessing eggs per gram, and when worm burdens were examined, their effectiveness was 90% (Claerebout and Geldhof, 2020). Using an ‘homologous’ antigen – an antigen that was initially demonstrated to be effective against a different helminth species – has also been proven to provide protection. For example, the glutathione-S transferases (GSTs) for Fasciola hepatica were selected as potential vaccine antigens because of the protective effects of homologous proteins from Schistosoma mansoni and Schistosoma japonicum in in vitro infection models (Kaplan et al., 2004).

6.12 Future Prospects and Research Directions The escalation of antiparasitic drug resistance persists, expanding its geographic reach, the diversity of affected species, and the spectrum of implicated drugs. Infection patterns are also changing owing to changes in farming techniques, land use and climate. Scientific discoveries must be incorporated into farming practices in the future. The forthcoming research trajectory combines the use of novel control approaches such as vaccinations and selective breeding with the development of diagnostic instruments and parasiticides (Vercruysse et al., 2018). Numerous research teams are actively searching for antiparasitic medications that demonstrate high efficacy while minimizing side effects. Investigating existing drugs, known for their antiparasitic properties in different disease contexts, and exploring novel chemical structures such as analogues of well-established drugs are potential avenues for developing effective treatments for parasitic diseases. For instance, six novel analogues of linezolid, an antibiotic used to treat nosocomial infections, have been discovered. These compounds show promise and may be useful in treating Hymenolepis nana tapeworm infections in vitro. Thus, more research into the mechanisms of action of

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all types of potential analogue is necessary to prevent antiparasitic medication resistance continuing to spread (Alcántar-Zavala et al., 2020; Dziduch et al. 2022). The intrinsic anthelmintic qualities of bioactive forage, such as plant cysteine proteinases, flavonoids and condensed tannins, have been validated both in laboratory settings and within living organisms such as small ruminants and cattle. These results indicate a potential avenue for enhancing anthelmintic efficacy by combining plant materials rich in condensed tannins with those containing quercetin or luteolin flavonoids, or by selecting plants with elevated levels of both tannins and quercetin or luteolin. Further investigation is needed to understand the antiparasitic potential of bioactive forage to combat resistance (Singh et al., 2020). The progress in developing diagnostic instruments holds importance in directing the management of parasitic infections and addressing AR effectively. The digital transformation should lead to a fresh approach, focusing on swift, cost-effective and precise point-of-care diagnostics, coupled with proper data storage to identify specific animals or situations warranting management intervention (Pomari et  al., 2019). Although not yet applied to parasite diseases in animals, this advancement has already produced novel methodologies for the entire genome and metagenome analysis available for routine diagnosis in a variety of medical sectors. The use of machine learning and artificial intelligence has recently brought a dramatic change in the way that parasite diseases are diagnosed, treated and understood (Ezenwaka and Nwalozie, 2023). Furthermore, the CRISPR-Cas12/13 system and other genetic technologies might be investigated for their use in the detection and development of therapeutics for treating parasitic infections. More investigations in this area are needed to combat antiparasitic drug resistance effectively (Ezenwaka and Nwalozie, 2023; Mishra et al., 2023). In summary, research on anthelmintic resistance has mostly remained at the descriptive stage, with little progress made in understanding the underlying mechanisms of resistance. Developing molecular assays and improving diagnostic tests for antiparasitic drug resistance should be the main goals of future research. Furthermore, a  potential direction for researching parasite

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biology is to consider resistance as a selective marker for genetic transfection in parasites.

6.13

Conclusions

AR is an ever-growing global issue in the livestock sector. The thorough investigation of the prevalence of AR in different geographical areas has illuminated the complex network of variables affecting this worldwide issue. Considering our knowledge on different factors such as underdosing, treating all animals at the same time on the same farm, continued administration of the same anthelmintic, substandard quality, frequent use of drugs and the biology of the parasites that are responsible for AR, an effective strategy for controlling parasitic diseases and parasitic drug resistance must be recommended. This strategy must not be entirely dependent on chemotherapy or include the proper use of chemotherapeutics. Rather, it must consider emerging trends in antiparasitic drug resistance, which reveal potential threats for the future and require better awareness of challenges in combatting drug resistance, the pre-adoption of strategies for prevention, and the epidemiology or prevalence of resistance to each parasitic drug. These studies must be conducted at the regional level. The use of prototype molecular tests in managing specific parasite/drug class resistances in the field provides valuable information on

practical aspects regarding resistance management. Thus, genetic, molecular evolutionary and machine learning technologies (proteomics and biosensing) for understanding the evolution and spread of parasite drug resistance, the improvement and development of diagnostic tools, the development of more efficacious antiparasitic drugs, exploring novel analogues of wellestablished drugs and the sustainable utilization of novel antiparasitic drugs need to be prioritized for the management of AR. A faultless set of strategies that would make the best practice of parasite control is not the goal, but harmony and collaboration in monitoring and addressing this issue among the global health community is needed to combat AR effectively. So, AR is redefining best practice livestock parasite control. The collaborative dedication of researchers, medical professionals, legislators and institutions can open doors for the cross-border transfer of information, assets and optimal methodologies. In addition, a cooperative approach promotes the creation of novel remedies and long-lasting therapies, as well as deepening our understanding of the complex mechanisms of drug resistance. The livestock healthcare sector can continue to make great progress in reducing the effects of drug resistance and supporting the long-term viability of antiparasitic control efforts by encouraging an inclusive and coordinated global response.

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Haemonchus contortus isolates: Susceptible and resistant to ivermectin. Molecular and Biochemical Parasitology 238, 111281. Rodrigues, J.V.F., Braga, F.R., Campos, A.K., de Carvalho, L.M., Araujo, J.M. et al. (2018) Duddingtonia flagrans formulated in rice bran in the control of Oesophagostomum spp. intestinal parasite of swine. Experimental Parasitology 184, 11–15. Sales, N. and Love, S. (2016) Resistance of Haemonchus sp. to monepantel and reduced efficacy of a derquantel/abamectin combination confirmed in sheep in NSW, Australia. Veterinary Parasitology 228, 193–196. Sangster, N.C. (2001) Managing parasiticide resistance. Veterinary Parasitology 98(1-3), 89–109. Sarai, R.S., Kopp, S.R., Coleman, G.T. and Kotze, A.C. (2014) Drug-efflux and target-site gene expression patterns in Haemonchus contortus larvae able to survive increasing concentrations of levamisole in vitro. International Journal for Parasitology: Drugs and Drug Resistance 4(2), 77–84. Sarai, R.S., Kopp, S., Knox, M., Coleman, G. and Kotze, A.C. (2015) In vitro levamisole selection pressure on larval stages of Haemonchus contortus over nine generations gives rise to drug resistance and target site gene expression changes specific to the early larval stages only. Veterinary Parasitology 211(1-2), 45–53. Sazmand, A., Alipoor, G., Zafari, S., Zolhavarieh, S.M., Alanazi, A.D. et al. (2020) Assessment of knowledge, attitudes and practices relating to parasitic diseases and anthelmintic resistance among livestock farmers in Hamedan, Iran. Frontiers in Veterinary Science 7, 584323. Shalaby, H.A. (2013) Anthelmintics resistance; how to overcome it? Iranian Journal of Parasitology 8(1), 18. Shayan, P., Eslami, A. and Borji, H. (2006) Innovative restriction site created PCR-RFLP for detection of benzimidazole resistance in Teladorsagia circumcincta. Parasitology Research 100(5), 1063–1068. Siddique, R.M., Iqbal, A., Nisa, F.U., Arijo, A.G. and Liaqat, H.A. (2023) Parasite control strategies: Biological materials. In: Rizwan H.M. and Sajid M.S. (eds) Parasitism and Parasitic Control in Animals: Strategies for the Developing World. CAB International, UK, pp. 183–200. Singh, A., Mishra, A., Chaudhary, R. and Kumar, V. (2020) Role of herbal plants in prevention and treatment of parasitic diseases. Journal of Scientific Research 64(1), 50–58. Smout, M.J., Kotze, A.C., McCarthy, J.S. and Loukas, A. (2010) A novel high throughput assay for anthelmintic drug screening and resistance diagnosis by real-time monitoring of parasite motility. PLoS Neglected Tropical Diseases 4(11), e885. Srivastava, S.K., Agrawal, B., Kumar, A. and Pandey, A. (2020) Phytochemicals of Azadirachta indica source of active medicinal constituent used for cure of various diseases: A review. Journal of Scientific Research 64(1), 385–390. Takala-Harrison, S. and Laufer, M.K.. (2015) Antimalarial drug resistance in Africa: key lessons for the future. Annals of the New York Academy of Sciences 1342, 62–67. Traversa, D. and von Samson-Himmelstjerna, G. (2016) Anthelmintic resistance in sheep gastro-intestinal strongyles in Europe. Small Ruminant Research 135, 75–80. Traversa, D., von Samson-Himmelstjerna, G., Demeler, J., Milillo, P., Schürmann, S. et al. (2009) Anthelmintic resistance in cyathostomin populations from horse yards in Italy, United Kingdom and Germany. Parasites and Vectors 2(2), 1–7. Uhlemann, A.C. and Krishna, S. (2005) Antimalarial multi-drug resistance in Asia: Mechanisms and assessment. Malaria: Drugs, Disease and Post-genomic Biology 295, 39–53. Vercruysse, J., Albonico, M., Behnke, J.M., Kotze, A.C., Prichard, R.K. et al. (2011) Is anthelmintic resistance a concern for the control of human soil-transmitted helminths? International Journal for Parasitology: Drugs and Drug Resistance 1(1), 14–27. Vercruysse, J., Charlier, J., Van Dijk, J., Morgan, E.R., Geary, T. et al. (2018) Control of helminth ruminant infections by 2030. Parasitology 145(13), 1655–1664. Vlaminck, J., Cools, P., Albonico, M., Ame, S., Ayana, M. et al. (2018) Comprehensive evaluation of stoolbased diagnostic methods and benzimidazole resistance markers to assess drug efficacy and detect the emergence of anthelmintic resistance: A Starworms study protocol. PLoS Neglected Tropical Diseases 12(11), e0006912. Waghorn, T.S., Leathwick, D.M., Rhodes, A.P., Lawrence, K.E., Jackson, R. et al. (2006) Prevalence of anthelmintic resistance on sheep farms in New Zealand. New Zealand Veterinary Journal 54(6), 271–277. WHO (2003) Assessment and Monitoring of Antimalarial Drug Efficacy for the Treatment of Uncomplicated Falciparum Malaria. World Health Organization, Geneva (WHO/HTM/RBM/2003.50). WHO (2006) Guidelines for the Treatment of Malaria. World Health Organization, Geneva.

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7

Molecular Methods for Detecting Antiparasitic Resistance

Muhammad Sohail Sajid1*, Sadaf Faiz2, Muhammad Qasim1, Ibadullah Jan3, Sibtain Ahmad4,5, Dalia Fouad6 and Farid Shokry Ataya7 1 Department of Parasitology, University of Agriculture, Faisalabad, Pakistan; 2 Department of Pathology, University of Agriculture, Faisalabad, Pakistan; 3College of Veterinary Sciences,The University of Agriculture Peshawar, Pakistan; 4Institute of Animal and Dairy Sciences, University of Agriculture, Faisalabad, Pakistan; 5 Key Lab of Agricultural Animal Genetics, Breeding and Reproduction Science, Huazhong Agricultural University, Wuhan-PR, China; 6Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia; 7Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia

Abstract Antiparasitic resistance (AR) poses a substantial threat to the global health and the veterinary sectors. While conventional methods for detecting AR have limitations, molecular techniques have emerged as powerful tools for assessing effcacy and understanding the underlying genetic markers associated with APR. This chapter explores the principles of molecular diagnostics, elucidating the various genetic markers indicative of resistance mutations. Techniques such as polymerase chain reaction (PCR), real-time PCR and loop-mediated isothermal amplifcation (LAMP), along with genotypic assays and sequencing techniques, are critically analysed for their ability to amplify specifc DNA fragments linked to resistance. Molecular diagnostics offer a formidable weapon in the fght against AR, allowing a glimpse into the genetic secrets of parasites. This deeper understanding enables the development of smarter, faster and more effective strategies to protect human health. Furthermore, the chapter delves into the ongoing development of novel molecular tools, including next-generation sequencing and CRISPR-based assays, designed to enhance the detection and characterization of AR. The advantages and limitations of each method are scrutinized, providing a comprehensive understanding of their applicability in different contexts. This comprehensive exploration aims to contribute to the evolving landscape of AR detection, fostering advancements that are pivotal for global health and veterinary practices.

7.1

Introduction

Currently, parasites are controlled by using various antiparasitic drugs; however, the widespread use of antiparasitic drugs has led to the development of resistance in these parasites globally (Sangster

et al., 2002). Drug resistance poses the strongest barrier in the control of parasites such as protozoa, helminths and arthropods that affect both humans and animal species (Francesconi et al., 2021). Antiparasitic resistance (AR) has also been observed in beneficial parasites used for

*Corresponding author: [email protected]

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biological control of phytophagous mites and other micro-arthropods, such as Phytoseiulus persimilis, Amblyseius swirskii and Neoseiulus californicus (Van-Lenteren et al., 2018), indicating extensive use in some regions of the world (Bajda et al., 2022). The development of AR is a global issue, causing failures in parasite management and resulting in economic losses through crop destruction, livestock death and the occurrence of diseases in animals and humans (RodriguezVivas et al., 2018). To address this issue, compounds with different modes of action (MoA) are needed as alternatives. However, challenges such as the cost of producing compounds with different MoAs, and stringent legal approval processes for eco-friendly compounds are major constraints in meeting the market demands (Sparks and Lorsbach, 2017). Parasitic diseases pose significant challenges in different parts of the world and, owing to the widespread development of resistance against various antiparasitic drugs, treating parasitic infections have become a serious problem (Capela et al., 2019). Scientists have classified resistance mechanisms into two groups: (i) toxicodynamic changes (modifications occurring at the target sites); and (ii) toxicokinetic changes (involving decreased penetration, increased detoxification, excretion or sequestration). Toxicodynamic changes include point mutations at the target site affecting antiparasitic binding ability, rare gene amplification or knock-out mechanisms influencing target-site protein availability (Augustin et al., 2022). In contrast, toxicokinetic resistance is associated with differential expression of regulatory elements (cis and trans) of detoxification genes or the addition of duplicate genes (Kurlovs et al., 2022). Molecular diagnostic techniques analyse key components of organisms, such as DNA, RNA and various proteins, helping to understand the disease properties and the organism’s response to specific drugs (Shen, 2023). Various molecular approaches, including genome and transcriptome sequencing, mapping of quantitative trait loci (QTL) and bulked segregate analysis (BSA), have been developed and employed to identify resistance mechanisms (Li et al., 2022). Functional validation of resistant loci and mutations are carried out through enzyme inhibition assays, recombinant expression, promoter analysis, electrophysiology and reverse genetics using RNAi and gene editing (Rouck et al., 2023). Progress is

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being made in associating resistance development with genetic mapping of mutations and genomic loci linking with chromosome physical assemblies (Sugimoto et al., 2020). High-resolution mapping of the resistant genes has been made possible by combining population-level BSA and next-generation sequencing (NGS) (Kurlovs et al., 2019). Transcriptome sequencing is an effective approach for identifying differences in genetic expressions between resistant and susceptible populations on a genome-wide scale. Combining transcriptome sequencing and QTL mapping has proven to be an influential approach for uncovering MoAs of resistance (De-Beer et al., 2022). Methods of functional validation of resistance genes have also increased the understanding of resistance, e.g. gene expression in model organisms such as bacteria, yeasts and Xenopus oocytes along with voltage-clamp electrophysiology and enzyme assays, and in vivo methods including reverse genetics and marker-assisted back-crossing where interaction between proteins of interest and antiparasitic compounds are being investigated. These methods have recently identified the role of target-site and metabolic resistance in major resistance cases (Xue et al., 2021; Papapostolou et al., 2022). Thus, owing to the availability of whole genome sequences of economically important parasitic species and reverse genetic tools, our knowledge about molecular mechanisms for the development of resistance has increased in recent years. Evolved molecular techniques provide appropriate tools for comprehending biological problems, and molecular methods are applied to address these parasitological issues. These molecular diagnostic strategies play a central role in treatment strategies for imminent problems (Gunawardena and Karunaweera, 2015).

7.2 Molecular Methods for the Detection of Resistance The field of infectious disease research has undergone a radical transformation with the advent of molecular diagnostic methods (Schmitz et al., 2022). In studies on AR, molecular diagnostics offer a unique insight into the genetic and molecular mechanisms responsible for resistance (Capela et al., 2019). One of the major advantages of

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molecular diagnostics lies in their heightened sensitivity and specificity compared to traditional methods (Ndao, 2009). Some molecular techniques and/or tools used for the diagnosis of AR are detailed below.

7.2.1 Polymerase chain reaction The polymerase chain reaction (PCR), a widely used technique in molecular diagnostics, has gained currency over time presumably because of its evolution (Kadri, 2019). PCR amplifies specific DNA sequences, facilitating the detection of resistance genes and mutations associated with AR. It utilizes a thermostable DNA polymerase enzyme to copy the target DNA in the presence of nucleotides and primers (Zhu et al., 2020). By using PCR, the amount of genetic material in the starting sample can be doubled, and multiple cycles can yield millions to billions of copies, which can be helpful for the detection of specific genes such as drug-resistance-associated genes in the target parasite (Feyziazar et al., 2022). Recent advancements, such as quantitative PCR (qPCR), have improved the precision and sensitivity of PCR-based diagnostics, allowing the detection of low-level AR (Zhang et al., 2022). The ability to produce rapid and specific quantitative results is one of the key advantages of qPCR, crucial for the detection of resistance mutations. PCR is a highly sensitive and specific technique with a moderate cost, frequently used for quantification. It serves as a valuable tool for guiding and monitoring treatments, with a turnaround time of less than 1 hour (Kurkela and Brown, 2009). PCR can also be used to help reduce the chance of developing drug resistance by testing for multi-drug resistance and guiding specific antimicrobial medication (Waseem et al., 2019). For instance, real-time PCR, a simple and quantitative technique in DNA sequencing, employs fluorescently labelled probes to analyse, authenticate and quantify PCR products generated in real time (Loftis and Reeves, 2012). The reverse transcription polymerase chain reaction (RT-PCR) is a slight modification of the typical PCR, focusing on the amplification of RNA molecules and enabling the study of gene expression levels in response to antiparasitic drugs (Mo et al., 2012). This information is vital for

understanding the dynamics of AR development and the mechanisms behind altered gene expression (Mo et al., 2012). The key principle of RT-PCR is based on the conversion of specific RNA sequences to complementary DNA (cDNA) using a reverse transcriptase enzyme. This is followed by the amplification of cDNA through PCR. The entire process can be carried out in one step, where both reverse transcription and PCR are performed in a single reaction, or in two steps, where reverse transcription and PCR are conducted separately (Nazeer et al., 2013).

7.2.2 Sequencing techniques Sanger sequencing, also known as the ‘chain termination method’, is a traditional but still invaluable technique. It provides accurate information about the sequence of DNA fragments and is considered the ‘gold standard’ for validating DNA sequences (Heather and Chain, 2016). The principle of Sanger sequencing involves the use of a DNA primer that is complementary to the template DNA, serving as a starting point for DNA synthesis. The polymerase enzyme extends the primer by adding complementary deoxynucleotide triphosphates (dNTPs) to the template DNA strand in the presence of the four dNTPs: A, G, C and T (Dey, 2023). To determine the nucleotide included in the chain of nucleotides, four dideoxynucleotide triphosphates (ddNTPs) are added. These ddNTPs lack a 3’-hydroxyl group, necessary for the addition of the next nucleotide, resulting in chain termination. Gel electrophoresis is then used to separate the resulting DNA fragments, and the size of these fragments determines the sequence (Crossley et al., 2020). In automated Sanger sequencing, the sequencing machine conducts single-capillary gel electrophoresis for all oligonucleotides, and the sequence is determined using specific fluorescent dyes (Locher et al., 2022). Alternatively, next-generation sequencing (NGS) revolutionizes our ability to analyse entire genomes rapidly. The mechanism of NGS involves library preparation in which DNA or RNA samples are fragmented, and adapters are ligated. These fragments are then amplified and sequenced in a massively parallel fashion. The raw sequencing data are cleansed of adapter sequences and

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low-quality reads, and the reads are aligned to a reference genome or assembled de novo (Hu et al., 2021). This allows the identification of novel mutations and a comprehensive understanding of the genetic basis of AR (Capela et al., 2019).

7.2.3 Genotyping assays These are the experimental procedures to identify differences in DNA sequences among individuals. Genotyping assays, including single nucleotide polymorphism (SNP) analysis and microsatellite genotyping, play a vital role in characterizing genetic diversity within parasitic populations (Kwok, 2001). These techniques contribute to the identification of resistanceassociated mutations, guiding clinicians in selecting appropriate therapeutic interventions. Some of the genotyping assays used for detection of AR are as follows:

•

•

Single nucleotide polymorphism analysis is the process that identifies differences in nucleotides at specific positions in the genome. Most DNA sequence variants in the human genome are SNPs, which are germline substitutions of a single nucleotide at a specific position in the genome (Johnson, 2009). These SNP markers have made it possible to explore the genetic basis of complex diseases through population approaches (Heidema et al., 2006). The principle behind many genotyping techniques, including SNP analysis, is the hybridization between genomic DNA and matching probes. The SNP analysis plays a crucial role in understanding the relationship between genotype and phenotype, and it can help explain differences in susceptibility to a variety of diseases throughout a population (Kockum et al., 2023). Microsatellite genotyping involves the identifcation of the variation in microsatellites, which are short tandem repeat sequences consisting of one to six nucleotides (Guichoux et al., 2011). It is based on the PCR amplification of the microsatellite region using specifc primers. The amplifed product is then separated by capillary electrophoresis, and the microsatellite variation is identifed by the size of the PCR

•

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product (Lepais et al., 2020). These assays aid in identifying specifc genetic markers linked to AR, offering insights into the spread of resistant strains (Tan, 2008). Allelic discrimination assays are genotyping assays that specifically focus on discriminating between different alleles of genetic variation (Malkki and Petersdorf, 2012). TaqMan assays and high-resolution melt analysis (HRMA) are becoming increasingly popular due to their high precision in allele discrimination (Heissl et al., 2017). The TaqMan assay, also called the 5’-nuclease allelic discrimination assay, is based on PCR for genotyping SNPs. In this method, the region fanking the polymorphism, typically 100 to 150 bp, is amplifed in the presence of two allele-specifc fuorescent probes. The probes do not fuoresce when free in the solution because of a quencher at the 3’ end that absorbs the fuorescence from the reporter fuor present at the 5’ end (Malkki and Petersdorf, 2012). During PCR, the Taq DNA polymerase cleaves the probe that is base-paired specifcally with its target and releases the reporter fuor from the quencher, increasing the net fuorescence. Hence, whenever each probe binds to a specifc allele, it is detected by fuorescence in this technique. The two probes are labelled with different fuorophores, allowing the detection of two alleles in a single tube (Hui et al., 2008). Another advantage of this technique is that, unlike other genotyping techniques, it does not require post-PCR processing because probes are included in the PCR (Fedick et al., 2012). HRMA is, however, a post-PCR method for identifying genetic variation. The principle of HRMA is based on the quantitative analysis of the melt curves of the resulting DNA fragments following PCR amplifcation (Twist et al., 2013). 7.2.4 Loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a molecular test capable of generating billions of DNA copies within an hour. The crucial aspect of this technique is that all steps can be performed

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under isothermal conditions at an optimal temperature of 65°C. Multiple primers are employed in this technique. Bst DNA polymerase (isolated from Bacillus stearothermophilus) serves as the enzyme for DNA replication and exhibits high displacement activity (Hadersdorfer et al., 2011). The LAMP uses 4–6 primers that recognize 6–8 distinct regions of the target DNA, ensuring a highly specific amplification reaction (Wong et al., 2018). This technique is more efficient and faster than PCR. The presence of Bst DNA polymerase allows all steps to be performed at the same temperature. Therefore, this technique lacks a DNA denaturation stage and involves three steps: initiation, amplification and LAMP acceleration (Notomi et al., 2000). In the initiation step, primers attach to the DNA template at 65°C and anneal to a specific sequence of nucleotides. DNA polymerase recognizes the primer attachment and initiates synthesis (Nzelu et al., 2019). The forward inner primer, in particular, hybridizes with the original target sequence, initiating the synthesis of a new strand from the 3’ end. This step marks the initiation of the synthesis of the complementary strand. The forward outer primer provides template regions for the backward primers, aiding in elongation. It releases single-stranded DNA (ssDNA) as a template for DNA synthesis by the backward inner primers, and elongation continues towards the 5’ end (Panno et al., 2020). During amplification, a stem-loop structure of the strand is formed, contributing to the exponential amplification of the DNA. Reverse primers initiate subsequent elongation using the DNA strands as templates. At the end of the first replication, the complementary strand and the double-elongated stems act as templates for further amplification, resulting in additional exponential amplification. After multiple amplifications, stem-loop DNAs of various sizes and DNA structures with multiple loops are produced (Mori et al., 2006). Two additional primers, i.e. loop forward and backward primer, are usually employed to further expedite the amplification. These primers hybridize with the stem-loops, increasing the efficiency and sensitivity of the reaction while reducing the reaction time by almost 50% (Nagamine et al., 2002). The application of this technique in detecting drug-resistance-associated DNA sequences enhances our ability to diagnose AR (Castellanos-Gonzalez et al., 2018).

7.2.5 Microarray technology The ability of microarrays to probe a sample for hundreds to millions of different molecules simultaneously has enabled the analysis of various genes concurrently. This makes microarray technology useful in identifying potential therapeutic targets by pinpointing gene expression patterns associated with AR with precision and in less time (Murphy, 2002). The basic principle of microarrays is the hybridization of complementary DNA strands (Wiltgen and Tilz, 2007). DNA microarrays, also called DNA chips, are characterized by the binding of several known DNA sequences, i.e. probes, on planar solid surfaces such as a chip (Bier et al., 2008). The five steps involved in microarray technology are as follows: (i) target or sample preparation, in which the nucleic acid, such as DNA, is isolated from the sample and fluorescently labelled; (ii) the labelled DNA is then allowed to hybridize with the complementary sequence on the DNA microarray; (iii) washing the microarray removes the unpaired sequences, leaving only the paired strands on the microarray; (iv) following the hybridization of fluorescently labelled DNA and probe, the microarray scanner collects image data based on the fluorescence of the DNA; and finally (v) the image data are analysed to detect the presence of specific DNA sequences in our sample or the level of gene expression (Marzancola et al., 2016).

7.2.6

CRISPR-Cas9 technology

CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) is a genome editing tool that provides precise control over genome manipulation (Savić and Schwank, 2016). In Fig. 7.1, the CRISPRCas9 gene-editing process is explained. It is a precise and versatile tool for manipulating DNA. The mechanism of genome editing through CRISPR-Cas9 involves three main steps: recognition, cleavage and repair. It consists of two main components, namely guide RNA (gRNA) and the CRISPR-associated protein 9 (Cas9) (Barman et al., 2020). The guide RNA recognizes the target DNA sequence through complementary base pairing at the 5’ end and forms a complex with

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sgRNA Non-homologous end joining Disrupts gene of interest

PAM sequence

Homology-directed repair Corrects gene of interest

Double-stranded break

Nucleotide deletion

Donor DNA Disrupted DNA

Repaired DNA

Nucleotide addition

Plasmodium spp.

Anopheles spp.

Disrupted DNA Mendelian

dCas9 Sir2A

Gcn5

Gene drive

Cas9 DSB NHEJ/HDR

Leishmania spp. T7 RNAP

5’ HR

Barcode

Drug

3’ HR

HDR donor

Cas9

50% inheritance

T7

>50% inheritance

sgRNA sgRNA

Fig. 7.1. CRISPR-Cas9 gene editing: a precise and versatile tool for manipulating DNA. (Author’s own figure.)

the Cas9 nuclease. The previously inactive Cas9 protein, in the absence of gRNA, becomes active and cleaves the target sequence at specific positions, creating double-stranded breaks (DSBs). At this step, the existing genetic sequence can be removed or new sequences can be introduced. Finally, the DSBs are repaired by the cell’s DNA repair mechanisms (Asmamaw and Zawdie, 2021). In AR studies, this technology facilitates the creation of isogenic strains with known resistance-associated mutations. Its ability to manipulate the genome enables the validation of identified genetic markers and their functional significance (Tao et al., 2022). Thus, it plays a promising role in the detection and control of AR.

7.2.7

Miscellaneous molecular techniques

There are many other techniques such as reverse transcription loop-mediated isothermal amplification (RT-LAMP), biosensors, field-effect transistors (FETs), localized surface plasmon resonance

(LSPR) sensors, and cell-based potentiometric biosensors that are now employed (Kreitmann et al., 2023). The RT-LAMP technique can be used for the detection of parasitic RNA and AR associated with it (Garg et al., 2022). The development of electrochemical biosensors provides rapid results with high sensitivity in the detection of AR. The basic principle of biosensors involves the binding of an analyte (a substance to be detected such as an AR biomarker) with a bioreceptor. Upon binding, a transducer converts this biological response to an electrical signal that is then displayed on screen. Researchers at Imperial College, London, have developed DNA-based biosensors to detect AR markers in schistosomes, called SNAILS (specific nucleic acid ligation for the detection of schistosomes). Such biosensors can detect and differentiate between parasites by using species-specific DNA sequences (Webb et al., 2022). The FETs are also utilized in the development of biosensors and can be used for the detection of AR in similar fashion. The FET biosensors have been used in the detection of AR by recognition of a single nucleotide related to drug-resistant strains of malaria, which showcases their potential

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in this field (Sadighbayan et al., 2020). The LSPR sensors and cell-based potentiometric biosensors are other types of biosensors that have different mechanisms. LSPR sensors are, however, more commonly used than cell-based potentiometric biosensors for detecting AR (Kim et al., 2021).

7.3 Successes in the Detection of Antiparasitic Resistance through Molecular Approaches Molecular diagnostic techniques have played a pivotal role in detecting issues related to resistance against various drugs during the past few decades. With advancements in the field of medicine and the emergence of drug resistances, molecular diagnostic techniques have seen significant improvements across various domains (Serban, 2019). The pathogenicity of parasites in animals makes them important subjects for studying microbial genomic products (Fierer et al., 2017). These molecular approaches play a crucial role in identifying even low levels of resistance resulting from mutations. This addresses a key limitation of conventional methods, ultimately aiding in the prevention of the escalation of resistance strains (Sanchini, 2022). Real-time molecular diagnostic methods facilitate the continuous monitoring of the development of AR. These techniques have transformed the dynamics of diagnostics, bringing insightful changes to the research and treatment of diseases, revolutionizing basic healthcare (Dwivedi et al., 2017). The real-time data generated through these methods enable the observation of continuous changes adopted by parasites, aiding in the development of appropriate treatment goals and strategies to overcome these resistances. Real-time monitoring of molecular diagnostics helps researchers establish suitable schemes to address emerging problems and stay current with recent studies in this field (Sangster et al., 2002). Molecular diagnostic techniques have proven to be highly successful in identifying AR in various case studies. These historical cases underscore the clinically important impact of molecular approaches in guiding treatment decisions. For example, Plasmodium falciparum developed resistance to artemisinin, which was one of the most potent and fast-acting antimalarial drugs.

Whole-genome sequencing of an artemisininresistant strain revealed the association of its resistance with mutations in the PF3D7_1343700 kelch propeller domain (K13-propeller). Because the K13-propeller was identified as a molecular marker for artemisinin resistance, it holds great potential for containing the artemisinin-resistant strains of the malaria-causing parasite P. falciparum through molecular surveillance, preventing global spread (Ariey et al., 2014). Chloroquine, a 4-aminoquinoline, was also widely used as a treatment for malaria during the 1960s and 1970s. Chloroquinolone acts by inhibiting hemozoin formation in the digestive vacuole during the trophozoite stage. The emergence of P. falciparum resistant to chloroquine in Southern Asia was a significant setback in the treatment of malaria during that period. (Capela et al., 2019). Nested PCR, in which different pairs of primers are used in two sequential amplification reactions, was used to detect the chloroquine drug resistance genes, i.e. pfcrt-o (P. falciparum chloroquine resistance transporter-o) and pfmdr-1 (P. falciparum multidrug resistance-1) of P. falciparum (Singh et al., 2016). Molecular assays have also been helpful in the detection of anthelmintic resistance. The SNP genotyping mechanism has enabled us to understand benzimidazole resistance in gastrointestinal nematode species, which has been linked to an alteration in β-tubulin (Fissiha et al., 2021). Hence, molecular methods have succeeded in the past and will be the leading tools for the detection of AR in the future, with much better advancements.

7.4 Limitations in the Molecular Detection of Antiparasitic Resistance Although there are many advantages of the use of  molecular tools for the detection of AR, some limitations have been comprehensively described below.

7.4.1 Challenges in sample collection The quality of the samples collected is crucial for the success of molecular diagnostics. A wide variety of samples, such as serum, whole blood, urine, cerebrospinal fluid, genital swabs, respiratory

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swabs, stool and various tissue biopsies are analysed through molecular assays for AR. The good quality of these samples depends on appropriate collection, handling and transport to the lab to avoid false-negative results (Kurkela and Brown, 2009). Labelling of collected samples is also of prime importance; otherwise, it can lead to a mixture of results that are of no use and may cause serious consequences in molecular diagnostic testing (Debnath et al., 2010). Incorrect timing of sample collection may also result in false results, such as false-negative outcomes in PCR testing of stool and respiratory samples collected late in the disease when nucleic acid has been cleared (Charlton et al., 2018). Samples should be representative of the targeted population, and challenges in obtaining representative samples from diverse settings may impact the accuracy of resistance assessments.

7.4.2 Variability in resistance mechanisms The development of novel resistance mechanisms demands advancements and changes in molecular diagnostic tools (Fluit et al., 2001). Parasites exhibit variability in resistance mechanisms, presenting several challenges for molecular biologists (Kotze et al., 2020). For example, while the loss of drug activation is the main mechanism for metronidazole resistance in Trichomonas and Giardia spp., it is not the same for the chloroquine resistance in Plasmodium spp., which involves the efflux of the drug (Borst and Ouellette, 1995). These challenges include limited drug options, the complexity of resistance mechanisms, and variability in host–parasite interactions. Laboratory-based resistance models have been employed for a long time to study drug resistance; however, due to ongoing mutations and evolution in resistance strains, their clinical relevance is now less reliable (Santos and Rebello, 2022). The diversity of resistance markers and pathways necessitates a comprehensive understanding to develop effective diagnostic tools. With ever-emerging new resistances, there is a need for specific diagnostic tools for the rapid detection of resistance markers, and molecular diagnostics are the leading tools for that purpose (Anas et al., 2019).

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7.4.3 Cost and accessibility of molecular techniques The high costs of molecular techniques such as PCR, sequencing and gene expression analysis mean they are not accessible to all researchers, particularly in resource-limited settings (Capela et al., 2019). For instance, the cost of PCR-based tests can range from US$22 to US$60 per sample, while the most widely used long-read sequencing platform costs about US$1000 per Gigabyte (Gb) (Apinjoh et al., 2019). The evolving resistant strains of parasites require these tests to be repeated with the change of time and region, which adds extra cost to the detection of AR globally. Limited access to instruments such as thermocyclers and sequencers can hinder researchers from conducting research on drug resistance in parasites. Additionally, many settings lack trained personnel in molecular techniques and require training workshops for molecular diagnostic techniques to enable independent work. Therefore, ensuring the widespread availability of these instruments and techniques is crucial for monitoring AR through global research funding, particularly in underdeveloped countries (Pramanik et al., 2019). 7.4.4 Emerging technologies and standardization issues Although significant progress has been made in molecular diagnostics, and new techniques continue to be developed, it is crucial to emphasize the need for ongoing validation and standardized checks. A notable example is the establishment of thresholds for the positive results in molecular assays. Researchers have established thresholds for the detection of AR in gastrointestinal nematodes of cattle such as Cooperia spp. through a droplet digital PCR (ddPCR) approach (Baltrušis et al., 2019). There is, however, a need for the establishment of thresholds for other cost-effective molecular assays as well. The swift evolution of molecular technologies poses challenges in terms of standardization and validation. Additionally, novel drug targets must undergo validation to confirm their relevance in combatting drug resistance in parasites (Müller and Hemphill, 2013). The standardization of diagnostic technologies is imperative for identifying these targets (Dubey

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et al., 2015). Collaborative efforts will be essential for the development of standardized molecular techniques. Such endeavours will not only facilitate comparability, but also enhance the reliability of results across different research studies, including those related to AR.

7.5 Future Prospects for the Molecular Detection of Antiparasitic Resistance In light of the above limitations, the following considerations could be the way to progress in the arena of detection of AR through molecular tools. By adhering to the correct guidelines for sample collection, the full potential of molecular diagnostics can be utilized to minimize challenges in the identification of drug resistance (Swets, 1988). 7.5.1 Advancements in high-throughput technologies High-throughput technologies and omics-based methods have the potential to revolutionize the detection of novel drug resistance markers and the understanding of drug resistance mechanisms in parasites. Omics-based methods, such as genomics, transcriptomics, proteomics and metabolomics, may provide detailed information about the genetics and metabolic basis of drug resistance, enabling the identification of targets for novel antiparasitic drugs (Cowell and Winzeler, 2019). For example, the omics-based methods have unravelled the mode of resistance in antikinetoplastid drugs and have allowed the identification of novel or repurposed entities that show promising anti-kinetoplastid activity (Kerkhof et al., 2020). High-throughput screening assays have been employed in the development of novel drugs to combat AR. Continued advancements in high-throughput sequencing and amplification technologies will further enhance the precision, speed and efficiency of molecular diagnostics for AR (Siqueira-Neto et al., 2010). 7.5.2

Integration of artificial intelligence in data analysis

Artificial intelligence (AI) and machine learning algorithms can be utilized to analyse and interpret

large-scale molecular data. The integration of AI in the identification of resistance patterns is an emerging area of research that holds the potential to improve the accuracy of resistance predictions and identify novel drug targets for AR development (Sakagianni et al., 2023). When designed perfectly, AI facilitates data analysis that exceeds human capabilities and is free from behavioural constraints, including irrational deviations from guidelines and fatigue. Highthroughput screening assays generate a large amount of data, which has been used to predict resistant and susceptible mutations using AI and machine learning in pathogens. The use of AI and machine learning in the identification of AR needs validation but has a bright future (Jamal et al., 2020).

7.5.3 Personalized medicine approaches The major cause of drug resistance is the excessive use of self-prescribed or unnecessary medications and prolonged exposure to low doses (Bungau et al., 2021). One way to combat the development of drug resistance is through specific prescribed medications. Molecular diagnostics play a crucial role in identifying specific drug targets, facilitating a shift towards personalized medicine approaches. By utilizing molecular technologies to identify individual resistance profiles, treatment strategies can be tailored for optimized therapeutic outcomes (Jain, 2021). Molecular diagnostics are also valuable in the early detection of genetic disorders and conditions, such as breast cancer, which can be treated more effectively in the early stages without severe consequences (Cheung et al., 2019). Additionally, they can be employed to identify genetic mutations associated with the development of AR, helping to prevent its spread (Rizwan et al., 2021).

7.5.4 Global surveillance programmes The lack of reliable epidemiological data is one of the major constraints in the control of drug resistance. Although various surveillance campaigns for infectious diseases, including parasitic diseases, are carried out each year

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worldwide, there is a significant gap in the surveillance of resistance development, especially in developing countries and regions with limited resources (Elelu et al., 2022). Screening for specific pathogens through molecular diagnostics leads to the early identification of the causative agent and enables specific treatment, followed by the recovery of the patient. Conversely, if not detected early, it can lead to the development of drug resistance in patients resulting from the undesired use of drug for an extended period. Molecular surveillance of antimalarial drug resistance has provided great insights into the resistance mechanisms in protozoa (Iskandar et  al., 2021). There is a need for more such global surveillance programmes leveraging molecular diagnostics for a comprehensive understanding of AR on a worldwide scale. This information is crucial for informed public-health decision making and the control of AR (Capela et al., 2019).

7.5.5

Overcoming current limitations

Ongoing research aims to overcome current limitations in molecular diagnostics, such as challenges in sample collection, variability in resistance mechanisms, cost and standardization (MacLean et al., 2020). Collaborative efforts are essential between developed and developing countries to establish standardized molecular diagnostic techniques and utilize them in the surveillance of AR. Addressing antiparasitic resistance falls

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under the One-Health concept, requiring alignment among veterinarians, physicians and researchers. Researchers advise that veterinarians and physicians refrain from prescribing antiparasitic drugs that are at risk from developing resistance (Velazquez-Meza et al., 2022). Identifying resistance obstacles at the molecular level aids in developing proper strategic goals and therapies (Gasser, 2006). This approach significantly magnifies efficacy and helps prevent treatment failures. In addition to all this, nanotechnology has been pushing the limits of molecular diagnostics in the upcoming era. Applications such as magnetic nanoparticles, magnetic immunoassay and nanopore technology are all making a positive impact on the domain of molecular diagnostic techniques (Jain, 2003).

7.6

Conclusions

In conclusion, molecular diagnostic methods have played a significant role in enhancing our understanding of AR mechanisms. There is a dire need for the development of strategies to control AR. Molecular techniques can be effectively utilized for the detection of resistance markers, as well as for identifying novel drug targets in parasites. This ultimately aids in the identification of AR and the development of novel antiparasitic drugs. With continued advancements in molecular diagnostic technologies, these techniques hold a bright future in combatting AR.

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Phenotypic Methods for Determining Antiparasitic Resistance In Vitro and In Vivo HazratUllah Raheemi1*, Zobia Afsheen1, Muhammad Ahsan Naeem2, Shamshad Fareed3, Xi Chen4, Rohit Tyagi4, Muhammad Umar Farid5 and Adeel Ahmad6 1 Department of Health and Biological Sciences, Faculty of Life Sciences,Abasyn University, Peshawar, Pakistan; 2Department of Basic Sciences (Pharmacology Section), University of Veterinary and Animal Sciences, Lahore (Narowal Campus), Pakistan; 3Faculty of Veterinary Sciences, KBCMA College of Veterinary and Animal Sciences, Narowal, Sub-Campus UVAS, Lahore, Pakistan; 4College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China; 5Department of Animal Sciences, KBCMA College of Veterinary and Animal Sciences, Narowal, Sub-Campus UVAS, Lahore, Pakistan; 6Mobile Veterinary Dispensary,Vehari, Livestock and Dairy Development Department, Punjab, Pakistan

Abstract This chapter explores the detailed methods for assessing anti-parasitic resistance utilizing phenotypic approaches in vivo and in vitro. Using in vivo testing, such as the faecal egg count reduction test (FECRT), is crucial to determining how well anthelmintic drugs work against gastrointestinal nematodes. By mimicking actual grazing animal environments, the drench-and-move assay integrates environmental dynamics into resistance studies. Resistance refugia monitoring purposefully slows down the development of resistance by concentrating on animal subgroups that have not received treatment. Scientists can investigate resistance mechanisms and learn about the underlying biological and biochemical systems in the lab by employing in vitro approaches. The chapter navigates the diffculties of combining both in vivo and in vitro approaches to provide a thorough understanding of antiparasitic resistance. For scientists, veterinarians and other practitioners developing effective parasite management strategies for animal health, this collaborative study is an important resource.

8.1

Introduction

Globally, antiparasitic resistance poses a major threat to public health, agriculture and veterinary medicine (Picot et al., 2022). Effective control strategies require an awareness of resistance and an understanding of its mechanisms (Zamanian and Andersen, 2016). In this instance, antiparasitic resistance determination requires the use of

phenotypic approaches, both in vitro and in vivo (Rao et al., 2023). The ability of bacteria, viruses, hosts or living cells to develop resistance is referred to as ‘drug resistance’. This reduces the effectiveness of a drug in treating a disease or increases the ability of parasites to multiply and survive in the presence of a drug (Cortez-Maya et al., 2020). According to Babják et al. (2018), the main cause of serious and economically

*Corresponding author: [email protected]

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Phenotypic Methods for Determining Antiparasitic Resistance

significant illnesses in ruminants is gastrointestinal parasites. Diseases caused by protozoan parasites, including giardiasis, trypanosomiasis, leishmaniasis, toxoplasmosis and malaria, considerably raise the global mortality toll from infections. Unfortunately, in areas where protozoan illnesses are common, drug resistance has made it more difficult to treat effectively using antiparasitic medications. Drug resistance is currently rising to dangerously high levels throughout the world, jeopardizing our ability to treat even common illnesses (Rizwan et al., 2021). The shift in modern medicine towards chemically based pharmaceuticals was largely influenced by herbal medicine. These drugs can be used with protein-, lipid-, DNA- and RNA-based treatments (Kwon et al., 2018). Drug resistance raises serious health issues and may have detrimental social and economic effects. The majority of individuals concur that the emergence of global drug resistance has been linked to the extensive use of drugs in both humans and farm animals (Idris et al., 2019). A considerable degree of resistance to previous medications aids in the creation of innovative treatments that can be applied to the treatment of a variety of illnesses, including infections. Most parasites are now resistant to practically all of the drugs used against them, or if medication is not given in the right dosage, they will eventually develop resistance (Levy, 2013). Combining two or more medications is a useful tactic to stop resistance from developing. This strategy has proven to be especially effective against malaria and is increasingly advised for other parasites as well, while the limited number of medications on the market frequently makes it  difficult to create a combination that works effectively. Documenting medication resistance in parasitic infections received from patients, in addition to combination therapy, aids in maintaining the efficacy of currently available molecules and aids in the initial therapeutic decision-making process (Guerrant et al., 2001). The procedure for testing for parasite resistance is not widely available in clinical laboratories, especially in low-income countries, owing to its labour-intensive and non-standardized nature. Antiparasitic resistance poses a major risk to animal health and has a substantial impact on the world economy in terms of lost output and financial damage (Sharma et al., 2015). Despite the alarming claim and the significant impact that parasites inflict, research on parasitic dis-

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orders is still progressing slowly in comparison to many other infectious diseases. Innovative and developing technologies may soon have a major impact on parasite management and diagnostics as a result of our expanding understanding of treatment resistance (Légaré and Ouellette, 2017).

8.2

In Vivo Phenotypic Methods

The effectiveness of antiparasitic medications is evaluated in vivo, or on a living host. One often used tactic is standardizing therapeutic efficacy assays, which assess the parasite’s response to therapy in laboratory animals. For example, the World Health Organization (WHO) provides guidelines for in vivo studies to assess the efficacy of antimalarial drugs (WHO, 2015). In veterinary medicine, closely watched experiments involving sick animals are used to evaluate the effectiveness of anthelmintic drugs against parasitic worms. These studies assess factors such the decrease in parasite burden, recovery rates and the overall health of the host (Kaplan and Vidyashankar, 2012). Although in vivo studies are beneficial, they can have disadvantages, including costs, the possibility of host-specific variation and moral dilemmas.

8.2.1 Faecal egg count reduction test Worm egg counts of the animal before and after treatment can be used to assess the anthelmintic efficacy of a drug. The extensive uniformity of the test has allowed its widespread adoption. The faecal egg count reduction test (FECRT) defines resistance as the occurrence of each of the following two conditions: the decline in egg count must be less than 95% and the lower limit of the 95% confidence interval must equal or be less than 90% (Levecke et al., 2018; Salgado et al., 2019). In a Pakistani study, three popular anthelmintics – oxfendazole, levamisole and ivermectin – were evaluated for their efficacy in treating three breeds of dairy goats kept at the Government Livestock Farm: Dera Din Panah, Pak Angora and Beetal. The result showed resistance to the oxfendazole formulation; however, Haemonchus contortus decreased (P < 0.05) in response to ivermectin in all breeds and to levamisole in two breeds (Jabbar et al., 2008). Conversely, H. contortus was demonstrated to be resistant to albendazole,

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levamisole and ivermectin in FECRT by Dey et al. (2020) from Bangladesh, according to the percentage decline and 95% confidence intervals. In an attempt to assess the effectiveness of anthelmintic drugs, small ruminant farms in Malaysia were also investigated; regrettably, all anthelmintic groups demonstrated complete incapacity to suppress H. contortus (Chandrawathani et al., 2004).

measuring the inhibitory effect on their growth or survival. Antimalarial medication susceptibility has been evaluated using this approach on a large scale (Desjardins et al., 1979). Larval migration inhibition assays for worms provide another in vitro technique, used to measure the capacity of larvae to move in the presence of anthelmintic medicines (Kaplan and Vidyashankar, 2012).

8.2.2 Drench-and-move assay

8.3.1 In vitro assays for detection of anthelmintic resistance

The drench-and-move assay evaluates the mobility and survival of treated parasites when hosts are transferred to fresh pastures, simulating real-world circumstances (Waghorn et al., 2009). Because this assay considers the environmental parameters that influence parasite resistance, it is especially pertinent for grazing animals. Through the observation of treated parasites adapting to novel environments, scientists are able to have a thorough grasp of the advantages and possible drawbacks of anthelmintic medication (Höglund and Gustafsson, 2023).

8.2.3 Resistance refugia monitoring This is an in vivo tactic that entails deworming some animals while not treating others. By doing this, a reservoir of vulnerable parasites is produced, deliberately delaying the emergence of resistance (Greer et al., 2020). Veterinarians can use this information to develop sustainable parasite control strategies by observing both the treated and untreated animals. It also provides important information on the dynamics and prevalence of antiparasitic resistance (Burke and Miller, 2020).

8.3

In Vitro Phenotypic Methods

Examining the susceptibility of a parasite to medications outside of its host organism is done in vitro. Cost-effectiveness, repeatability and the capacity to assess a large number of drug candidates in a controlled setting are some benefits of these techniques. The microdilution method is a popular in vitro technique that involves exposing parasites to different medication doses and

Egg hatch assay The egg hatch assay serves as a pivotal tool in the assessment and monitoring of anthelmintic drug resistance in nematode parasites. This assay is instrumental in detecting and quantifying resistance to anthelmintic drugs by exposing parasite eggs to varying drug concentrations, enabling the observation of potential resistance through a reduced hatching rate. Researchers can quantitatively measure drug efficacy, determining the drug concentration required to inhibit a specific percentage of egg hatching (Demeler et al., 2012). This information is critical for evaluating resistance levels and devising effective treatment strategies. Continuous monitoring of egg hatch rates offers an early warning system for potential drug resistance development. A gradual decrease in susceptibility over time signals the need for alternative treatment approaches. The egg hatch assay allows species-specific analysis, aiding in understanding the effectiveness of drugs against specific parasites and identifying those losing efficacy due to resistance. In the development of new anthelmintic drugs, this assay is valuable for screening potential compounds, helping identify drugs with high efficacy and assessing their potential to delay or prevent resistance development (Calvete et al., 2014). For accurate results, fresh eggs should be utilized within three hours of shedding from the host. While effective for nematodes because of their short egg-hatching time, the assay is not extensively used in field surveys owing to challenges in result interpretation (Dobson et al., 1986). Motility assays Motility assays offer a direct and observable means of measuring the impact of anthelmintic

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Phenotypic Methods for Determining Antiparasitic Resistance

drugs on parasites. Reduced or altered motility can serve as an indicator of resistance, as susceptible parasites typically exhibit paralysis or death in response to effective treatment. These assays provide a quantitative evaluation of motility inhibition caused by anthelmintic drugs, enabling researchers to assess the extent of drug resistance within a population. By comparing the motility of treated and untreated parasites, the degree of resistance can be determined (Martin and Le Jambre, 1979). Motility assays contribute to the early detection of resistance development, allowing the observation of changes in motility before other clinical signs manifest. Early identification is crucial for implementing effective control measures and preventing resistance spread. These assays also aid in elucidating the mechanisms of resistance, providing insights into the genetic and physiological basis through the study of parasite responses to different ̌ drugs (Várady and Corba, 1999). Regular motility assays enable continuous monitoring of treatment efficacy, allowing for the tracking of changes in susceptibility and adjustment of treatment protocols over time. They serve as a valuable tool for validating in vivo observations of drug efficacy, providing a controlled environment for testing drugs and confirming whether observed clinical responses align with expected outcomes based on laboratory testing (Geerts et al., 1989). Motility assays play a pivotal role in diagnosing and understanding anthelmintic resistance, offering practical, quantitative and species-specific information crucial for effective parasite management and the development of new treatment strategies. Feeding inhibition assay The larval feeding inhibition assay (LFIA) is utilized to identify resistance against levamisole and macrocyclic lactones in trichostrongylid infections, because these anthelmintics paralyse the pharyngeal muscles, impairing feeding. In this assay, the first larval stage (L1) is incubated at 22°C for 24 hours, followed by an additional 18 hours at 25°C in anthelmintic dilutions along with fluorescein-labelled Escherichia coli (Jackson, 1993). Larvae are then transferred to a microscopic slide and examined under a fluorescent microscope. The presence of fluorescein indicates active feeding in larvae. Control samples without

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added anthelmintics are used for comparative analysis. The dose-response curve is obtained by comparing the non-feeding larvae portion, indicating larval feeding inhibition against various logarithmic concentrations of anthelmintic, and the IC50 value is calculated. This test has limitations, however, including challenges in the identification process and the need for fully susceptible strains for comparison purposes (Álvarez -Sánchez et al., 2005). Tubulin binding assay Resistance to benzimidazole is associated with a reduced affinity of tubulin towards the anthelmintic. Benzimidazole binds to tubulin, a structural protein present in both resistant and susceptible nematodes. The interaction between this anthelmintic and tubulin continues until equilibrium is reached. After incubation, unbound drug in the test suspension is removed using charcoal, and the tubulin-bound sample is labelled, and quantified using scintillation spectrophotometry (Johansen and Waller, 1989). In resistant parasites, weak binding occurs compared to susceptible ones. The tubulin-binding test is considered robust and highly responsive to variations in the resistance status of the parasite. However, it has limitations, including the need for large quantities of larvae, making it impractical for fieldwork, and requiring expensive equipment such as high-performance liquid chromatography (Taylor et al., 2002). Adult development assay The assay involves exposing adult parasites to anthelmintic drugs and monitoring their development, survival and reproductive capacity. This direct measurement provides a real-time assessment of the effectiveness of the drug against the target parasites. In comparison to other assays, the adult development assay (ADA) comprises a complete life cycle starting from eggs to the adult stage. The assay helps to identify specific populations of parasites that are resistant to anthelmintic drugs. This information is crucial for developing targeted strategies to control and manage resistant parasite populations, preventing the further spread of resistance. Successful cultivation of H. contortus to the sexually mature stage is documented (Small and Coles, 1993).

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Similarly, Taylor et al. (2007) also documented the cultivation of nematode larvae to their adult stage. This method has successful applications for testing nematode resistance to anthelmintic drugs, but its application is limited due to some constraints, such as culture techniques. By analysing the results of the ADA, researchers can differentiate between various mechanisms of resistance. This knowledge is essential for understanding how resistance develops and for designing new drugs or treatment approaches that can overcome or prevent these resistance mechanisms. The ADA has made a significant step in detecting benzimidazole resistance in trichostrongylids and the cultivation of H. contortus (Jackson and Coop, 2000). Larval development assay The larval development assay (LDA) is a single assay used for all drugs irrespective of their mode of action. Larval development assays are highly sensitive and can detect subtle changes in the response of parasite larvae to anthelmintic drugs. This sensitivity is crucial for identifying early stages of resistance development before they become clinically evident in the host. It is used for the detection of resistance to avermectin, benzimidazole, milbemycin and levamisole for nematodes infections, e.g. Trichostrongylus colubriformis, Teladorsagia circumcincta and H. contortus. For LDA, nematode eggs are collected from faecal samples and placed in a microtitre plate, where hatching of larvae starts, until the L3 stage is obtained in the presence of anthelmintic. The concentration of anthelmintic needed to hinder larval development is directly related to the anticipated in vivo efficacy of the anthelmintic (Amarante et al., 1997). The basic principle of LDA is the cultivation of nematode eggs to the L3 stage in the presence of serially diluted anthelmintic. Fresh eggs are recommended for this test, but there is no limitation for specific sampling time. Larvae reaching the L3 stage are plotted against the anthelmintic log10 concentration, a dose–response curve is obtained, and the LD50 value is estimated, similar to the minimum inhibitory concentration (MIC) (Levine, 1978). Like adult assays, larval development assays aid in differentiating between various mechanisms of resistance. This understanding is essential for developing strategies that can

overcome or mitigate resistance effectively. By regularly conducting larval development assays on parasite populations, researchers and veterinarians can monitor trends in anthelmintic resistance over time. These longitudinal data are crucial for implementing proactive measures and adapting control strategies to changing resistance patterns. 8.3.2 In vitro assays for detection of acaricidal resistance In vitro assays for acaricidal resistance detection involve testing the susceptibility of ticks and mites to acaricides under controlled laboratory conditions. These assays are valuable tools for monitoring and managing acaricide resistance in veterinary and agricultural settings. Some common in vitro assays along with examples are described here. Larval packet test The larval packet test (LPT) assesses the susceptibility of tick larvae to acaricides, providing information on the effectiveness of different chemicals and aiding in determining the resistance status of a population based on larval mortality. Engorged adult female ticks are collected, and the resulting larvae are exposed to different concentrations of acaricides in small packets. Subsequently, larval mortality is assessed. The LPT has been employed to evaluate resistance in cattle ticks, such as Rhipicephalus microplus, against acaricides (Lovis et al., 2011), as well as in other tick species like Hyalomma anatolicum (Jyoti et al., 2019), Amblyomma mixtum (Higa et al., 2020) and Rhipicephalus appendiculatus (Chitombo et al., 2021). A flow chart showing the activities involved in the larval packet test to analyse the effect of an antiparasitic drug is shown in Fig. 8.1. Adult immersion test The adult immersion test (AIT) is a bioassay utilized to detect resistance to acaricides in adult ticks, providing an evaluation of the acaricidal activity against adult ticks and insights into potential resistance mechanisms. It serves as a valuable tool for monitoring the development of resistance in adult tick populations, allowing the

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3 2

4 5

1

Fig. 8.1. Flow chart of the larval packet test to analyse the effect of an antiparasitic drug. (Author’s own figure.) 1, Serial dilution of the anti-parasitic drug; 2, saturation of Whatman paper no 1 with the diluted anti-parasitic drug and incubation for 30 minutes at 37°C; 3, folding of saturated filter papers and the addition of larvae in packets; 4, incubation at 27±1°C and 75–85% relative humidity for 24 h; 5, counting live and dead larvae.

detection of changes in susceptibility to acaricides over time (Dzemo et al., 2022). The AIT involves exposing adult ticks directly to acaricide solutions for a specified period, simulating field exposure conditions. Subsequently, ticks are observed for mortality and other sublethal effects. This test provides a more realistic assessment of resistance compared to laboratory assays. The AIT has been applied to evaluate resistance in Rhipicephalus microplus against synthetic pyrethroids (Singh et al., 2015), assess the acaricidal activity of fipronil against Haemaphysalis bispinosa (Ravindran et al., 2012), determine resistance in Hyalomma anatoilcum against synthetic pyrethroids (Kumari and Sangwan, 2016), and evaluate acaricidal resistance in Amblyomma mixtum (Higa et al., 2020). A flow chart of the adult immersion test to analyse the effect of antiparasitic drugs on hard ticks is shown in Fig. 8.2. Adult contact test The adult contact test (ACT) evaluates the ability of acaricides to control ticks through direct contact, providing valuable insights into the chemical's effectiveness. Unlike immersion tests, the ACT

involves direct contact between adult ticks and the acaricide, simulating field conditions where ticks encounter treated surfaces (Brito et al., 2011). During the ACT, ticks are exposed to a treated surface or fabric, replicating scenarios where animals are treated with acaricide formulations. The test monitors the ticks’ contact with the treated surface. This method has been employed to assess the efficacy of a fipronil and permethrin combination against Rhipicephalus sanguineus and Ixodes ricinus (Dumont et al., 2015), and to compare the efficacy of permethrin/imidacloprid in Ehrlichia canis-infected Rhipicephalus sanguineus ticks (Jongejan et al., 2016). Detoxification enzyme assays Detoxification enzyme assays play a crucial role in the study of acaricidal resistance in ticks. The use of acaricides, chemicals used to control ticks and mites, can lead to resistance development over time. Detoxification enzymes, including cytochrome P450 monooxygenases, glutathione S-transferases and esterases, contribute to the breakdown and elimination of acaricides within the tick’s body. These assays help monitor changes

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3

2

4

1

Fig. 8.2. Flow chart of the adult immersion test to analyse the effect of antiparasitic drugs on hard ticks. (Author’s own figure.) 1, Synthesis and serial dilution of an antiparasitic drug; 2, immersion of adult ticks in the diluted antiparasitic drug and shifting to new tubes; 3, incubation at 28–30°C with 80–90% relative humidity for 15 days; 4, note the mortality % and egg mass.

in detoxification enzyme levels over time, and elevated levels can indicate the potential development of resistance in tick populations. Esterase activity measurement has been employed to investigate resistance mechanisms in Rhipicephalus microplus (Fadahunsi et al., 2023), enzymatic detoxification-mediated deltamethrin resistance in Hyalomma anatolicum (Prerna et al., 2019) and permethrin detoxification in Rhipicephalus sanguineus (Duscher et al., 2014). Membrane feeding assay The membrane feeding assay is a specialized in vitro technique designed to assess the impact of acaricides on ticks or mites during the feeding process. This assay is significant for mimicking natural feeding conditions, providing insights into the effects of acaricides on feeding behaviour, mortality and potential sublethal effects (Kröber and Guerin, 2007). In this assay, blood is mixed with varying concentrations of acaricides to create a blood meal containing the desired dosage. The blood meal is placed in a membrane feeding system, which may involve a silicone membrane or an artificial skin membrane. Ticks or mites are allowed to feed on the acaricide-containing blood through the membrane, and the feeding period is controlled to mimic natural conditions.

Engorged ticks or mites are collected after feeding and observed for mortality, reproductive effects or any other sublethal effects (Krull et al., 2017). The impact on feeding success is a crucial aspect of the assay, and the percentage of feeding success, mortality and other effects are analysed for each concentration of acaricide. The in vitro feeding assay is used to assess the acaricidal resistance of Ixodes ricinus against different concentrations of fipronil and ivermectin (Kröber and Guerin, 2007). The membrane feeding assay is a valuable tool for assessing acaricidal effects during the feeding process, offering a more realistic simulation of natural conditions. It provides essential information for understanding the dynamics of acaricide resistance, particularly in the context of feeding behaviour and potential sublethal effects on ticks and mites (Trentelman et al., 2017; Guizzo et al., 2023). Histopathological examination Histopathological examination is a microscopic analysis of tissues aimed at understanding structural changes and lesions caused by exposure to acaricides. In the context of acaricidal resistance detection in ticks, this method is significant for providing insights into the effects of acaricides at the cellular and tissue levels. It helps identify

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morphological alterations, cellular damage and potential resistance mechanisms (Kanapadinchareveetil et al., 2019). Post-exposure to acaricides, engorged ticks or mites are collected. Tissues of interest, such as the midgut, salivary glands, and reproductive organs, are dissected for histological examination. The tissue samples are fixed in appropriate fixatives (e.g. formalin) to preserve cellular structures. Fixed samples undergo processing steps, including dehydration, embedding in paraffin wax and sectioning. Sections are stained with histological dyes (e.g. hematoxylin and eosin) to enhance contrast and visualize cellular structures. Prepared slides are examined under a light microscope by a histopathologist, and cellular changes, alterations in tissue architecture and any lesions are documented (Grabowski and Kissinger, 2020). Quantitative measures, such as counting damaged cells or assessing the extent of tissue damage, may be performed. Digital imaging and computer-assisted analysis may be employed for more precise quantification (Delmonte et al., 2017). Konig et al. (2020) reported morphological changes in the ovaries of Rhipicephalus sanguineus at low concentrations of acetylcarvacrol. Histopathological changes in R. sanguineus at different concentrations of thymol are also reported (da Silva Matos et al., 2014). Different concentrations of azadirachtin neem seed oil exhibit histopathological changes in the salivary glands of R. sanguineus (Remedio et al., 2016). Histopathological examination is a powerful tool for understanding the impact of acaricides on tick tissues, revealing morphological changes and potential resistance mechanisms. By providing detailed insights at the cellular level, this method contributes significantly to the comprehensive assessment of acaricidal resistance in ticks. It complements other resistance detection techniques, offering a deeper understanding of the physiological responses of ticks to acaricides.

8.3.3 In vitro assays for detection of antiprotozoal resistance Counting intracellular amastigotes and promastigotes: direct approach Microscopic counting methods, such as haemocytometers, electronic counters and Coulter counters, are commonly employed to assess the

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effectiveness of a compound or indirectly gauge an organism’s resistance to a parasite’s vitality (Antwi et al., 2019). The use of Giemsa stain allows a microscopic examination of the average reduction in the percentage of infected macrophages with amastigotes count per cell, aiding in the determination of the IC50 value for pharmacological action. However, this approach is now infrequently utilized to evaluate therapeutic efficacy against parasites because of its slow pace and susceptibility to user bias (Zahid et al., 2019). Acid-phosphatase (APTase) assay Acid phosphatases catalyse the hydrolysis of P-nitrophenyl phosphate (PNPP), generating chromogenic P-nitrophenol, a valuable indicator for pathogenicity and parasite resistance, measured at 405 nm. Despite its reliability in screening pharmacological libraries, the phosphatase activity in avirulent parasite clones is lower than in virulent clones. Ibrar et al. (2015) used a hybridization approach to evaluate biscoumarin-iminothiazole hybrids against calf intestinal alkaline phosphatase, identifying compound M as the most effective with an IC50 of 1.50 μM. Compound N, inhibiting around 70%, showed promise in antileishmanial efficacy against Leishmania major. Molecular docking of an iminothiazole derivative library against alkaline phosphatase reveals potential ligand–protein interactions, emphasizing the role of residue interactions in enzyme inhibition. 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay Roy et al. (2008) employed the MTT assay, a quantitative and colorimetric technique, to assess the viability of metabolically active promastigotes and axenic amastigotes during the exponential phase of cultivation. Through the MTT experiment, they observed a dose-dependent inhibitory impact of various concentrations of diindolylmethane (DIM) molecules on Leishmania donovani parasites. According to their findings, DIM induced programmed cell death in the parasites by inhibiting the activity of F0F1-ATP synthase, disrupting the mitochondrial membrane potential. Resazuring (Alamar-Blue dye) assay Alamar-Blue dye is utilized as an oxidative– reductive indicator to assess the impact of potent

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medications on metabolically active promastigotes. The dye monitors the reducing environment of the cell by measuring the conversion of resazurin to pink fluorescent resorufin, which is dependent on the quantity of metabolically active cells. Tuha’s group successfully synthesized seven pyrazole derivatives with excellent yields using an aldol condensation and cyclization method (Tuha et al., 2017). The physical characterization of these compounds was confirmed through elemental analysis, nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy. In vitro testing against L. donovani and Leishmania aethiopica strains revealed superior antileishmanial activity for the synthesized compounds using the dye reduction technique. Notably, phenyl pyrazoline, designated as compound O, exhibited exceptional efficacy against L. donovani parasites, surpassing standard drugs such as amphotericin B and miltefosine with an IC50