Basic and Clinical Aspects of Veterinary Immunology [1st Edition] 9781483215778

Table of contents :
Content:
Advisory BoardPage ii
Contributors to this VolumePage ii
Front MatterPage iii
Copyright pagePage iv
ContributorsPages ix-x
PrefacePages xi-xiiB.I. OSBURN, RONALD D. SCHULTZ
Structural and Functional Characteristics of Immunoglobulins of the Common Domestic SpeciesPages 1-21P. PORTER
Primary and Secondary Immune Deficiencies of Domestic AnimalsPages 23-52LANCE E. PERRYMAN
Mechanisms of Immunity in Bacterial InfectionsPages 53-69A.J. WINTER
The Immune System and Helminth Infection in Domestic SpeciesPages 71-102E.J.L. SOULSBY
Mechanisms of Viral ImmunopathologyPages 103-136BARRY T. ROUSE, LORNE A. BABIUK
Immunology of a Persistent Retrovirus Infection—Equine Infectious AnemiaPages 137-159TRAVIS C. McGUIRE, TIMOTHY B. CRAWFORD
Immunology and Pathogenesis of African Animal TrypanosomiasisPages 161-182J.B. HENSON, JAN C. NOEL
Tumor Immunology in Domestic AnimalsPages 183-228M. ESSEX, C.K. GRANT
Applications of Transplantation Immunology in the DogPages 229-265H.M. VRIESENDORP
Effects of Environmental Contaminants on the Immune SystemPages 267-295LOREN D. KOLLER
Subject IndexPages 297-301
Contents of Previous VolumesPages 303-309

Citation preview

ADVISORY BOARD W. I. B. BEVERIDGE

C. E. HOPLA

J. H. GILLESPIE

NORMAN D. LEVINE

W. R. HINSHAW

C. A. MITCHELL

W. R. PRITCHARD

CONTRIBUTORS TO THIS VOLUME LORNE A. BABIUK

JAN C. NOEL

TIMOTHY B. CRAWFORD

LANCE E. PERRYMAN

M. ESSEX

P. PORTER

C. K. GRANT

BARRY T. ROUSE

J. B. HENSON

E. J. L. SOULSBY

LOREN D. KOLLER

H. M. VRIESENDORP

TRAVIS C. MCGUIRE

A. J. WINTER

ADVISORY BOARD W. I. B. BEVERIDGE

C. E. HOPLA

J. H. GILLESPIE

NORMAN D. LEVINE

W. R. HINSHAW

C. A. MITCHELL

W. R. PRITCHARD

CONTRIBUTORS TO THIS VOLUME LORNE A. BABIUK

JAN C. NOEL

TIMOTHY B. CRAWFORD

LANCE E. PERRYMAN

M. ESSEX

P. PORTER

C. K. GRANT

BARRY T. ROUSE

J. B. HENSON

E. J. L. SOULSBY

LOREN D. KOLLER

H. M. VRIESENDORP

TRAVIS C. MCGUIRE

A. J. WINTER

ADVANCES IN VETERINARY SCIENCE AND COMPARATIVE MEDICINE Volume 23 Edited by C.

A.

CHARLES

BRANDLY

E.

CORNELIUS

College of Veterinary Medicine University of Florida Gainesville, Florida

College of Veterinary Medicine Univers1:ty of Illinois Urbana, Illinois

BASIC AND CLINICAL ASPECTS OF VETERINARY IMMUNOLOGY Guest Editors B.

I.

RONALD

OSBURN

D.

SCHULTZ

School of Veterinary Medicine Auburn University Auburn, Alabama

School of Veterinary Medicine University of California Davis, California

1979

ACADEMIC PRESS A Subsidiary of Harcourt Brace Jovanovich, Publishers

New York

London

Toronto

Sydney

San Francisco

COPYRIGHT © 1979, BY ACADEMIC PRESS, I N C . ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

A C A D E M I C PRESS, INC. I l l Fifth Avenue, New York, New York 10003

United Kingdom Edition published by A C A D E M I C PRESS, INC. (LONDON) L T D . 24/28 Oval Road, London NW1 7DX

LIBRARY O F CONGRESS CATALOG CARD N U M B E R : 5 3 - 7 0 9 8

ISBN 0 - 1 2 - 0 3 9 2 2 3 - 2 PRINTED IN THE UNITED STATES OF AMERICA 79 80 81 82

9 8 7 6 5 4 3 2 1

CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors' contributions begin.

A. BABIUK, Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saska­ toon, Saskatchewan, Canada (103)

LORNE

B. CRAWFORD, Department of Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pull­ man, Washington 99163 (137)

TIMOTHY

M.

Department of Microbiology, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115 (183)

ESSEX,

C. K. GRANT, Department of Microbiology, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115 (183) J. B. HENSON,* International Laboratory for Research on Animal Dis­ eases, Nairobi, Kenya (161) D. KOLLER, Veterinary Medicine, University of Idaho, Moscow, Idaho 83843 (267)

LOREN

C. MCGUIRE, Department of Microbiology and Pathology, Col­ lege of Veterinary Medicine, Washington State University, Pullman, Washington 99163 (137)

TRAVIS

JAN C. NOEL, Graduate School, Washington State University, Pullman, Washington 99164 (161) E. PERRYMAN, Department of Veterinary Microbiology and Pa­ thology, Washington State University, Pullman, Washington 99163 (23)

LANCE

P.

Department of Immunology, Colworth Laboratory, Unilever Research, Bedford, United Kingdom (1)

PORTER,

* Present address : Department of Microbiology and Pathology and Graduate School, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164. ix

CONTRIBUTORS

X

T. ROUSE, Department of Microbiology, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee 37916 (103)

BARRY

E. J. L. SOULSBY,* Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 (71) H. M. VRIESENDORP, Radiobiological Institute, GO-TNO, Rijewijk, The Netherlands, and Laboratory for Experimental Surgery, Erasmus University, Rotterdam, The Netherlands (229) A. J.

WINTER, Department of Clinical Sciences, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 (23)

* Present address : Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 OES, England.

PREFACE Advances in veterinary immunology continue to shed new light on the basic and applied aspects of this speciality. As the reviews in the present volume clearly indicate, these elucidating advances in our understand­ ing of the biology of immune responses have led* to a better appreciation of disease processes in a number of animal species. Many of the im­ portant contributions made by investigators in basic and applied vet­ erinary immunology are included here with the hope that others will follow and take advantage of the unique opportunities available in veterinary medicine. In the first article, Porter reviews immunoglobulin classes observed in different animal species. Particular emphasis is placed on the role of colostral and local immunity in the neonatal ungulate. The contribution by Perryman emphasizes the importance of the immune response to the well being of an individual and highlights clinical manifestation of dis­ eases observed in primary and acquired immune deficiencies. Criteria for characterizing different types of immune deficiencies observed in domestic animals are of value to clinicians and researchers. Bacterial and helminth infections remain important causes of disease in domestic species. In the third contribution to this volume, Winter has elaborated on the mechanisms by which cellular and humoral im­ mune factors effect their action on bacteria. In addition, adverse con­ sequences of immune responsiveness are highlighted in a review of those bacteria, e.g., Campy lob act er fetus, that undergo antigenic variation in the presence of antibody, thereby allowing the bacteria to persist in the host. Soulsby, in the fourth article, has presented a comprehensive re­ view of the modulation of both host and parasite which permits parasites to survive in the host. Evidence for the immunologie basis of seasonal and self-cure phenomena is described for different species. A basic understanding of immune responses to viral infections is pro­ viding important insight into the different ways that cellular and humoral factors provide protection. In the fifth article, Rouse and Babiuk have summarized the mechanisms of immunity by different animal species as they encounter viral diseases. Their focus on the cooperative role be­ tween the immune system and accessory factors such as complement and phagocytic cells emphasizes the complexity of the host defense system. It is apparent that a number of pathways are in operation to prevent disease and/or eliminate infections. XI

Xll

PREFACE

The sixth contribution by McGuire and Crawford is a comprehensive review of a classical immunopathologic disease caused by the virus of equine infectious anemia. The authors characterize the immune response to the virus and its subcomponents and offer an explanation for the means by which the virus eludes immune responses in order to persist in the host. African animal trypanosomiasis has proved to be one of the most difficult of diseases to control. As a result, few of the improved domestic ruminants have been afcle to survive in the vast grasslands of East Africa. Intensive investigations focusing on the immunologie features necessary for developing suitable measures to contain this disease are under way. In the seventh article, Henson and Noel review the processes that allow the parasite to persist and those aspects of the host's response that may be most beneficial for preventing infection. In the eighth review, Essex and Grant have covered those significant immunologie studies performed on domestic animal species. It is evident from their review that many of the diseases have proved to be models that have contributed to a better understanding of the immune fac­ tors playing a role in tumor biology. Again, the wide varieties of re­ sponses to viral-induced neoplasms are presented. The importance of the histocompatibility system became evident during the era of organ transplants. The canine has been used as an important animal model for perfecting the surgical procedures for organ transplan­ tation. At present, the dog histocompatibility system is probably the most advanced of all domestic animal species. In the ninth article, Vriesendorp reviews the information now available on the canine sys­ tem. In addition, information is provided for effective immunosuppressive regimes and for the future application of transplantation immunology in the dog. In the tenth contribution, Koller provides insight into an important new area of veterinary immunology. The contribution includes the cri­ teria used to establish that toxic substances or environmental contam­ inants cause aberrant immune responses. Examples are given of toxic products that are known to cause alterations in immunologie responsive­ ness. It has been a pleasure to be able to work with these contributors, with the Editors, C. E. Cornelius and C. A. Brandly, and with the publisher, each of which have made this volume possible. B. I. OSBURN RONALD D.

SCHULTZ

ADVANCES I N VETERINARY SCIENCE AND COMPARATIVE MEDICINE, VOL. 2 3

Structural and Functional Characteristics of Immunoglobulins of the Common Domestic Species P. PORTER Department of Immunology, Unilever Research, Bedford, United Kingdom I. II. III. IV.

Introduction Classification of Immunoglobulins in Animal Species Immunoglobulins in Fetal and Germfree Life . . . . . . . . Immunoglobulins in the Materno-Neonatal Relationship . . . . . 1. The Regulatory Effect of Maternal Immunoglobulins in the Neonate 2. Protective Effect of Maternal Immunoglobulins in the Neonate . 3. The Role of Maternal Immunoglobulin in the Neonatal Gut . . V. Ontogeny, Transport, and Function of Intestinal Immunoglobulin . . VI. Immunoglobulins in the Nasolacrimal Fluids VII. Effector Mechanisms in Mucosal Immunity References

1 2 4 6 6 8 11 12 15 16 19

I. Introduction At the outset it is perhaps best stated that the objective in this chapter is not to provide the classical treatise on immunoglobulin structure involving the description of H-chains, L-chains, J-chains, Hinge regions, and the like. Obviously a substantial amount of effort has been devoted to the isolation and characterization of immunoglobulins in domesti­ cated animal species. This has largely served to provide confidence in a universal nomenclature for classification, and such investigations are more generally noted rather for their tedium than for their creativity. Their primary contribution must lie in assisting a more adequate ex­ ploration and comparison of immune function between species. Under­ standably, it is in the realms of functional significance that the main attractions are presented to those concerned in regulating immune mechanisms for the benefit of animal health and production. Much might be gained by exploiting the natural mechanisms of im­ munity in the way they would normally function, rather than imposing 1 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-039223-2

2

P.

PORTER

some unnatural constraints upon animals already stressed by the sys­ tems of intensive husbandry. In this respect it may be said that definition of the immune mechanisms of exocrine systems represents one of the most fundamental advances during the past decade, and this presents some exciting prospects for veterinary medicine. Immunoprophylactic measures exploiting the principle of surface immunity are already in use through the exploitation of oral and intranasal routes of immuniza­ tion. New advances are being made in ways of regulating the immune response to meet specific requirements in intensive animal rearing systems. The diverse range of domestic species can be used to advantage to select topics that demonstrate the manner of adaptation of natural mechanisms. The various physiological differences can be used to highlight such features. In this respect a range of functional variations can be adequately exemplified in the major farm species—the pig, cow, sheep, and chicken. These species are the ones mainly featured here, and with them exists an abundance of opportunity to create a broad basis of interest for those engaged in the many and varied areas of veterinary investigation. For example, in gut function alone there is obvious variety in immunobiology between monogastric, ruminant, and avian species. With current emphasis of many researchers on the central role of the gut in exocrine immunity, these species have more to offer than just comparative immunology.

II. Classification of Immunoglobulins in Animal Species The spectrum of immunoglobulins produced within the lymphoid tissues form a family of proteins having broad similarities in structure and function between animal species. Establishing homologies between classes in different species is of special interest in relating function to class. Furthermore, it is of obvious importance for the purpose of deriving a consistent nomenclature. For this reason it is essential that the reference species be man, since only in man has a formal nomen­ clature been established (World Health Organization, 1966). Criteria have been proposed by which, throughout the animal species, there can be a competent evaluation of prospective analogs of the human immunoglobulins. The first and most rigid is based on signifi­ cant similarity of primary structure; lesser-order criteria are based on biologic and physicochemical similarities. Studies of immunologie cross reactivity have facilitated the establishment of homology (Vaerman et al, 1969).

IMMUNOGLOBULINS OF COMMON DOMESTICS

3

Immunoprecipitin studies of the blood plasma from 90 species of mammals with antisera to human immunoglobulins, demonstrated the broad homology of the major immunoglobulin classes, IgG, IgA, and IgM (Neoh et al.y 1973). In addition, IgD homologs were demon­ strated in 16 nonprimates and IgE homologs in 9 nonprimates; the poor evidence of cross-reactivity in these classes is probably attributable to the low levels available for detection rather than to the lack of homology. Similarity of antigenic structure of given proteins across numerous animal species implies that they may be products of common ancestral genes, and obviously the extent of homology would be determined by the number of subsequent mutations. In this context it is interesting that several antigenic determinants can be identified on IgG from various species (Esteves and Binaghi, 1972) ; some are unique to a species whereas others are shared. Thus there are broad similarities of struc­ ture and function across the mammalian species. In the chicken, how­ ever, it is of interest that cross reactivity with mammalian IgG cannot be demonstrated even with sensitive complement-fixation techniques (Mehta et al., 1972), and the putative γ-chain differs from that of mammals in so many physicochemical parameters that Leslie and Clem (1969) were disposed to provide a different nomenclature. None­ theless on the basis of the simple properties of abundance in the avian body fluids and similarity of molecular size, it is preferable to retain the mammalian equivalent IgG in order to simplify terminology. Chicken IgM, on the other hand, shows excellent cross reactivity with human /A-chains, thereby suggesting less evolutionary change in the genes controlling the antigenic determinants in the //--chain than in the γ-chain. The possession of secretory component (SC) by IgA derived from mucous secretions of mammalian species is a universal characteristic that has been used to aid the identification and classification of a putative IgA. In the bovine, SC is present in substantial quantities in the milk whereas IgA is not; this has led to the unusual situation of SC being well characterized in advance of IgA (Butler, 1971). In the chicken, investigations of cross reactivity by hemagglutination inhibition have failed to show any structural homology between mammalian and avian α-chains (Porter and Parry, 1976) ; identification of SC is there­ fore important for demonstrating functional analogy in the avian IgA system. Chicken bile has been the main source of SIgA for studies by a number of authors (Lebacq-Verheyden et al., 1972; Bienenstock et al, 1973; Leslie and Martin, 1973). Two reports have provided evidence for the association of SC with

4

P.

PORTER

chicken IgA (Watanabe and Kobayashi, 1974; Porter and Parry, 1976). The study by the former group suggested the existence in the chicken of a peculiar secretory IgA system in which intestinal IgA appeared to possess SC, whereas IgA from bile secretions did not. This, however, conflicted with the indirect evidence of Bienenstock et al. (1973), in whose studies chicken serum IgA was demonstrated to bind human SC selectively, whereas biliary IgA did not, implying that the binding sites might already be blocked by an analog of SC. Furthermore, recent studies (Leslie et al, 1976) show that the gall bladder is an integral part of the intestinal secretory immune system, in that most of the bile IgA is derived from local synthesis. In view of the ubiquitous nature of SC in those epithelia derived embryologically from endodermal structures, it is unlikely that biliary IgA would be free of SC. The discrepancies appear to have been resolved in studies that have now identified and isolated an avian analog of mammalian SC (Porter and Parry, 1976). There appears to be nothing "peculiar" about the avian IgA system, in that SC is present upon both intestinal and biliary IgA. Studies in germfree birds confirm that the characteristics of synthesis, secretion, and assembly into SIgA compare directly with those of the mammal (Parry and Porter, 1978).

III. Immunoglobulins in Fetal and Germfree Life The lack of transfer of immunoglobulin between material and fetal tissues in pigs and cattle prepartum provides an ideal model for study of immune responses in the fetus. Furthermore, animals of these species, derived and maintained in the germfree state, would be expected to have a zero base line of immunoglobulin in their body fluids and tissues; however, they do not. In many respects this is practically the case, but some interesting features have arisen from studies of sheep and pigs. For example, a low molecular weight component of the IgG class of immunoglobulins has been demonstrated in serum from newborn and germfree pigs (Stertzl et al, 1960; Franek et al, 1961). The sedimenta­ tion coefficient of this unusual component is approximately 5 S, and it is uncertain whether there are L-chains in the structure (Franek and Riha, 1964). It seems likely that this product is synthesized in fetal life (Prokesova et al., 1970). Although it has doubtful value as an antibody, some anti E. colt activity has been detected in association with it in precolostral piglet serum (Porter and Hill, 1970). Possibly this immunoglobulin component is a product of metabolic processes re-

IMMUNOGLOBULINS OF COMMON DOMESTICS

5

lating to lymphocyte membrane receptors. It is known that lymphoid differentiation along plasma cell lines occurs in Peyer's patches of fetal pigs (Chapman et al., 1974) ; lymphoid cells bearing immunoglobulin were seen in characteristic follicular distribution as early as 55 days of gestation. In fetal sheep also, low levels of immunoglobulin IgM and IgG can be detected in the body fluids in the absence of known antigenic stimuli (Silverstein et al., 1963). In piglets (Binns, 1967) and sheep (Silverstein et al., 1963), antibody responses to antigens injected directly into the fetal tissues can be obtained after 80 days of gestation. Similarly, bovine fetal and newborn calf serum collected prior to colostrum ingestion contain both IgM and IgG. It was found that cells producing these classes of immunoglobulins were present by 90 days of gestation. The source of fetal stimulation would include fetal, maternal, and microbial antigens (Schultz et al., 1973). IgA production in the cattle is normally not observed until after birth, a time when massive stimulation with environmental antigens occurs (Schultz, 1973). Normally the intestine and lungs of the fetus would be the main route of stimulus derived from any antigen that might invade the sterile sanctity of the uterus and become part of the amniotic fluid. Recent studies with antigens introduced into the amniotic fluid of pregnant sheep have shown a capability for oral immunization of the fetus with the development of lymphocytes synthesizing IgM antibody in the intestinal tissues (Husband and McDowell, 1975). In the absence of known antigenic stimulus, the lymphoid differen­ tiation in Peyer's patches of the fetal pig occurred before immunocytes were evident elsewhere (Chapman et al., 1974). However, in the ovine and bovine fetus, lymphoid differentiation in the gut occurs very late in gestation (Silverstein et al, 1963; Schultz et al., 1973). For example, immunoglobulin-bearing cells were not evident in the thymus until 10 to 15 days later. In the neonatal pig the lymphoid cell activity of the intestinal tissues is low, and it remains at a negligible level in animals reared germfree (Fig. 1). It develops rapidly in response to bacterial colonization or the regular oral administration of bacterial antigens (Porter et al., 1974). Again the response is dominated by IgM, and it seems that IgM plays an initiating role in the onset of antibody function in the exocrine system, in much the same manner as it does in the systemic response to parenterally administered antigens. Synthesis of higher than normal levels of IgM can be an artifact of germfree life. For example, germfree mice synthesize a higher proportion of IgM relative to total serum protein than do normal mice (Vitetta et al., 1974). It was suggested that these animals lacked a population

6

P. PORTER

300f I IgA JlgM

2001-

100

CeN count

H M

* ■

Duodenum

ü-trT

Jejunum

*+ il M lleum

M Peyer's Patches

ΑΛ

M O Spleen

G

FIG. 1. Characteristics of immunoglobulin synthesis in immunocytes populating intestinal tissues and spleen of gnotobiotic and germfree pigs in response to Escherichia coli antigens. M, monocontaminated E. coli 08:K87 K88a,b, 22 days; 0 , orally administered heat-inactivated E. coli 08, 22 days; G, germfree, 35 d.O., same age as M and 0 above. [Data from Porter et al. (1974).]

of stimulated T cells that can induce a switch from IgM to IgG syn­ thesis. Similarly, in gnotobiotic chicks (Parry et al., 1977) serum IgM levels are considerably elevated compared to levels in conventional birds whereas IgA and IgG remain extremely low. Possibly, lack of antigenic stimulation by a normal gut flora may retard or interfere with the bursal switch of IgM to IgG or IgA production described by Kincade and Cooper (1973). IV. Immunoglobulins in the Materno-Neonatal Relationship 1. T H E REGULATORY EFFECT OF MATERNAL IMMUNOGLOBULINS IN THE NEONATE

Investigations of the maternal-fetal relationship provide a variety of immunologie and genetic problems. For example, the role of the fetus as a homograft (Beer and Billingham, 1974) and the genetic control of the immune response (Gasser and Silvers, 1974) have been the subjects of investigation. Maternal immunity in the neonate is a subject that has been investigated widely in veterinary science; however, one aspect of importance that has been largely neglected is the potential effect of the passive maternal immunity on the subsequent capability

IMMUNOGLOBULINS OF COMMON DOMESTICS

7

of the fetus or neonate to respond to antigenic stimuli. Two pieces of evidence signal the need for research on this topic. First, studies of the immune response in rodents have shown the potential for enhancement or depression of antibody production in the offspring of immunized mothers, and that the effect was genetically influenced (Davis and Gill, 1975). Second, it has been established that passive antibody may exert a regulatory effect on the immune response (Uhr and Möller, 1968); for example, the passive administration of antibody prior to, or shortly after, immunization with antigen greatly suppresses the primary response. It is generally held that the primary step in this effect is combination with antigen, and this is substantiated by the demonstration that suppression is immunologically specific. One is well aware that the use of vaccines in animal production has placed great emphasis on maternal immunity and the subsequent passive antibody status of young animals. While much effort has been directed to providing antigens for hyperimmunization of the dam with a view to transferring optimum amounts of antibody to the neonate, the fact that this procedure may also interfere with the development of active immunity has sometimes been neglected. Thus, although effective protection might be gained against infectious disease in neonatal life, the continuing development of active immunity thereby suffers. The mechanisms are not presently understood, but recent work on the pig (Muscoplatt et al., 1977) indicates that maternal antibody obtained through colostrum serves to regulate the immune response probably at a number of levels. In the first place, the most important mechanism is probably inhibition of background development of antibody-producing cells, most likely by a mechanism involving antigen elimination. Second, by a mechanism involving cytophilic antibody. Specific antibody of the IgG class is most effective in exerting these suppressive effects on active antibody synthesis. However, there is good evidence (Henry and Jerne, 1968) that IgM at least in certain concentrations, does not suppress the stimulation of synthesizing lymphocytes, but, on the contrary, may even contribute to the enhancement of the primary immune response. Thus, the role of IgM in maternal immunity for the neonate merits further serious consideration. The limited view of IgM fulfilling a principal function in the primary response may have to be extended, and its role in passive immunity with potent antibacterial characteristics should be seriously considered. These aspects have been recently reviewed in a novel approach to sow immunization designed to control neonatal enteric colibacillosis (Chidlow and Porter, 1978). In these studies, a synergism of oral and parenteral antigen administration was exploited whereby E. coli vaccine was

8

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PORTER

presented in the feed and subsequently by injection in such a way as to deliberately engineer production of IgM antibody. 2. PROTECTIVE EFFECT OF MATERNAL IMMUNOGLOBULIN S IN THE NEONATE

The universal role of IgG is seen to operate by its quantitative abundance in maternal immunity in all species, no matter what is the route of transport. Although there is a capability of most species to respond to antigenic stimuli during the later stages of embryonic de­ velopment, it is not normal for such stimuli to occur, and passively acquired maternal immunity must compensate for the immunologie deficiency of the neonate. The susceptibility of the neonate to enteric pathogens is readily evident in those species that exhibit chorioepithelioplacentation. In such species, exemplified by pigs and calves, studies of the blood serum indicate that there is virtually no transmission of im­ munoglobulin from maternal to fetal circulations by any route other than intestinal absorption of the colostrum. The ability to absorb substantial quantities of maternal immuno­ globulin is fundamental to survival. In spite of the short term of in­ testinal permeability in the farm animal species—no more than 12 to 36 hours whereas rodents retain this function for some 21 days—quite remarkable quantities of immunoglobulin are acquired in normal func­ tion. In the first few hours of life the piglet will acquire approximately 3 gm, and the calf may acquire twenty times more than this. Even in the chicken, approximately 60 mg of immunoglobulin are taken up from the yolk sac. The selective characteristics of the ruminant for transport of IgGi as opposed to IgG 2 into the mammary secretions have been adequately reviewed (Butler, 1971). Thus, although colostrum comprises a transudate of maternal serum immunoglobulin, owing to this selection the serum immunoglobulin profile of the calf differs substantially from that of the dam in lacking significant amounts of IgG 2 . In the pig, on the other hand, no such selective process can be demonstrated to exist in colostrum formation (Porter, 1969) ; thus the serum profile of the neo­ natal pig bears close resemblance to that for immunoglobulins in the dam. Because of its quantitative abundance, IgG has been an obvious candidate in maternal immunization schedules to affect passive im­ munity in the offspring. Brandenburg and Wilson (1973) have shown that colostrum IgG from sows vaccinated with formolized E. coli played an important role in the protection of young pigs against E. colt

IMMUNOGLOBULINS OF COMMON DOMESTICS

9

enteritis by neutralizing heat-labile enterotoxins and decreasing the rate of multiplication of the bacterium. However, as indicated earlier in this text, it may not be advisable to allow the dominant physiological role of IgG in maternal immuno­ globulin transfer to override the opportunities presented by the minor immunoglobulin components of the colostrum in any specific area of neonatal defense. Although IgM and IgA together may account for no more than 15% of the immunoglobulin content of colostrum, they may, under the right circumstances, exert functional dominance and provide the most suitable basis of passive immune protection. For ex­ ample, IgM anti-£J. coli antibody from the colostrum of sows (Chidlow and Porter, 1978) produced potent complement-mediated in vitro bac­ tericidal activity and also very efficient in vivo bacterial clearance. These potent antibacterial mechanisms provided a good basis for neonatal defense, which was fully realized in trials of protective efficiency in neonatal piglets given a lethal infection with a virulent strain of E. coli. The infection proved fatal for 76% of piglets main­ tained on control sows, whereas litters suckled on vaccinated sows were able to resist effectively the infectious challenge, with only 2% fatality. It might be argued that in many respects it is advantageous to mimic the natural processes in stimulating immune function. In porcine neo­ natal E. coli enteritis the sow is a source of infection, herself under­ going an E. coli proliferation during the stressful latter stages of gesta­ tion (Arbuckle, 1970). The characteristics of the immune response in the sow and the antibody profile in the colostrum following infection during the last 2 to 3 weeks of pregnancy again are predominantly associated with IgM (Porter et al, 1978). The protective function is comparable with that described above, whereas the protective capacity of IgG produced by a multiple inoculation schedule was substantially lower, with 36% fatality. The foregoing represents just one aspect of reappraisal of the present status of vaccination in the field of passive immunity. Certainly the means of manipulation of the maternal response invites further revision, and this can best be approached by considering some of the effector mechanisms as well as the immunologie function. For example, the calf presents some interesting physiologic features in relation to IgA that might be exploited. The calf intestine exhibits no selection in the absorption of maternal immunoglobulins, and in consequence a phenomenon occurs in which substantial levels of 11 S secretory IgA are absorbed from the colostrum and are temporarily present in the blood circulation (Porter, 1972).

10

P.

PORTER

It appears to be an express feature of the mammalian species that under normal circumstances little or no 11 S IgA ever appears intravascularly. There have been studies in man in which 11 S IgA was administered intravenously and the immunoglobulin disappeared rapidly from the circulation, exhibiting a half-life of less than 2 days (W. T. Butler et al., 1967). It may be significant that the half-life of maternal 11 S IgA in the blood serum of the calf has a similar value (Fig. 2). The immunoglobulin is steadily lost, presumably by transudation into all the external secretions of the calf, and thereby continues its normal role in external defense (Porter, 1973a). This phenomenon, whereby the calf utilizes the short-term capability of intestinal absorption to provide a temporary reservoir of maternal 11 S IgA in its blood circu­ lation, has not been adequately exploited in current vaccination programs. The relevance of this phenomenon in the calf bears examination in relation to enteric infections. Susceptibility to E. coli septicemia in the neonatal calf is attributable to deficiency in absorption of colostral antibody and probably more specifically to antibody with given class characteristics. For example, IgM has proved to have significantly better protective efficiency than IgG against E. coli septicemia induced in colostrum-deprived calves (Logan and Penhale, 1971). However, a significant feature arose from these studies; a comparison of the pro­ tective efficiency of the colostrum from which these fractions were

Lacrimal fluids 100 u

\\ Λ

\/\ \/\

C7 Blood

z\ 50

^

serum pool

\Y/AK

^

i>

^

SaNva Intestinal tract

Respiratory tract

rpassive s l g A / ^ S ^ >w^ 10

^ -» ^ 20

Active 30

Da)fs of age FIG. 2. Role of maternal IgA in the neonatal calf. Blood serum concentration (mg/100ml) following absorption at birth and decline in neonatal life.

IMMUNOGLOBULINS OF COMMON DOMESTICS

11

derived showed that its activity was unaccounted for by the propor­ tional recombination of the IgM and IgG fractions, thereby implying the presence of a further undefined factor—perhaps IgA. 3. T H E ROLE OF MATERNAL IMMUNOGLOBTJLIN IN THE NEONATAL GUT

The phenomenon of intestinal absorption of maternal immunoglobulin has been extensively examined, and the relatively short dura­ tion of the phenomenon is certainly not matched by the brevity of the literature that has been produced in its study. Investigations on the subject encompass such topics as mode of absorption, period of permea­ bility, mechanisms of closure, receptors in transport, specificity factors, antiproteolytic factors, and a variety of others. However, one theme that has tended to be neglected has been the role of lactation in bridging the temporary immunologie inadequacy of the neonate to cope with the hostile microbial challenge of its new environment. The in­ testine of the newborn is rapidly and extensively colonized, and it is in this respect that it is useful to examine the additional purpose of lactation in continuing to supply antibody to suckling offspring when intestinal closure has occurred. Unlike the calf the piglet fails to absorb significant quantities of maternal 11 S IgA from the colostrum, and antibodies of this immunoglobulin class have been shown to operate largely in the lumen of the intestine by providing a continuous bathing of the epithelium. The physiological characteristics of milk antibody secretion, suckling pat­ tern, passage through the alimentary tract, and resistance of IgA to enzyme degradation, combine to advantage in providing this function (Porter et al., 1970). In the milk of the sow, IgA is the dominant immunoglobulin. Studies of antibody function to E. coli (Porter, 1969) and transmissible gastroenteritis (TGE) (Saif et al., 1972) have demon­ strated the essential local mammary origin of the IgA antibody class. Perhaps the best example of local protection attributable to milk IgA antibody in the alimentary tract is that demonstrated for TGE infections, in which this class of antibody was essential for solid passive protection (Bohl et al, 1972). Control of E. coli infection in gnotobiotic pigs can be attained by feeding milk from immunized sows, and the essential local nature of this immune function has been emphasized by comparative studies of oral versus parenteral administration of anti­ bodies (Miniats et al., 1970). In more detailed studies of the protective effect of individual class-specific anti-E. coli antibodies against infection of germfree piglets, IgA proved to be most effective (Miler et al, 1975). In contrast, ruminants appear to differ from the majority of mam-

12

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PORTER

mais in not exhibiting an IgA-dominant immunoglobulin profile in the milk. Indeed, unlike most mammals, after the period of colostrum formation ruminants produce milk with a uniformly low content of immunoglobulin A—so little as to be of no significance in providing further passive immunity for the suckling offspring. It appears that the properties of local immunosynthesis in the mammary gland are determined by its physiologic state. Plasmacytosis occurs in the dry udder and during colostrum formation (Campbell et al., 1950). In the sheep, antigen stimulation of the gland before parturition increases the number of lymphoid cells and IgA is the dominant im­ munoglobulin class synthesized; however the lymphocyte population decreases rapidly after parturition when suckling begins (Lee and Lascelles, 1970). Similarly, organ culture studies of mammary tissue from the lactating cow have demonstrated it to be singularly deficient in immunoglobulin synthesis (J. E. Butler et al., 1972). From a teleological standpoint, it has been argued that the peculiar capability of the calf to conserve maternal colostral 11 S IgA and utilize it by passive transudation into external secretions from its serum pool is a means of compensating for the deficiency of the ruminant udder (Porter, 1973a). It would be difficult to imagine that any analogy could be drawn be­ tween events relating to the function of maternal immunoglobulin in the chick and the newborn mammal. However, recent observations on the appearance of IgA and IgM in the amniotic fluid of embryonating eggs and the chick embryo intestinal tract have drawn attention to this prospect (Rose et al., 1974). These immunoglobulins are acquired from the oviduct secretions where the wThite of the egg is laid down and appears in the embryonic gut via swallowed amniotic fluid, thus deriving a possibly similar function to that of lactation immunoglobulins in the mammal.

V. Ontogeny, Transport, and Function of Intestinal Immunoglobulin Structural investigation of SIgA from cattle and pigs (Porter, 1973a,b) and chickens (Parry and Porter, 1978) by reductive dissociation, gel filtration, and polyacrylamide electrophoresis provides evidence of es­ sentially the same peptide chain characteristics as those determined for human SIgA. Thus analogs of secretory component and J-chain are de­ tectable. The synthesis and assembly of SIgA in these species proceeds in precisely the same manner as that defined in the human. Secretory component is present in the crypt epithelium and intestinal mucin and

IMMUNOGLOBULINS OF COMMON DOMESTICS

13

is present separately and synthesized independently from α-chains, being detectable in the 80-day fetal pig whereas α-chain is invariably absent until neonatal life (Allen and Porter, 1973). The early intestinal immune response either to natural microbial colonization or oral administration of bacterial antigen in germfree life is dominated by IgM. Sequential studies on pigs have shown that dur­ ing the first days of life IgM cells outnumber those containing IgA or IgG (Fig. 3). Ultrastructural localization of μ- and α-chain in the intestinal tissues of the pig indicate that the characteristics of transport of the two immunoglobulins are essentially the same. Numerous vesicles containing the immunoglobulins were detected in the apical cytoplasm of crypt epithelial cells and also in the intercellular spaces (Allen et al., 1973, 1976). Thus pinocytosis appears to be an essential characteristic of transport from lamina to lumen. The role of IgM in intestinal surface immunity is intriguing. Studies of lower vertebrates (Portis and Coe, 1975) provide the prospect that primitive secretory immunoglobulin was related to IgM. Unlike IgA in mammals, this immunoglobulin was not secreted against a concen-

%ofc·!· synthesizing Ig

FIG. 3. Characteristics of development of immunoglobulin-bearing cells in the intestinal mucosa of the pig. [Data from Allen and Porter (1977).]

14

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PORTER

tration gradient. Nevertheless, the data implied that the presence of this primitive SIgM in secretory fluids occurred as a result of selective transport. The homology of avian and mammalian μ,-chains has been previously referred to in this text; furthermore, it is significant that a substantial degree of sequence homology exists between the C-terminal end of human μ- and a-chains. This has led to the suggestion that the phylogenetic origins of SIgA may lie with IgM, the divergence occurring when birds and mammals evolved from reptiles (Kehoe et al.} 1973). The potent protective function of maternal IgM in the alimentary tract of the piglet and calf in relation to enteropathogenic E. coli has already been discussed. From a teleologic standpoint it would appear likely that the active onset of local antibody protection might appropri­ ately be dominated by IgM. Certainly since the enteric flora has been identified as a major stimulus to lymphoid development and lymphocyte infiltration of the intestinal lamina, it would appear that IgM with its potent antibacterial characteristics might be ideally suited to estab­ lishing a favorable balance in the early host-pathogen relationship. There is one drawback, however, and that is in the relationship of IgM with secretory component. The interaction is quite weak compared with that of IgA, and this leads to interesting speculation on the role of SC in mucosal immunity. The receptor function in transport into the epithelial cell suggested by Brandtzaeg (1974) appears to have been conclusively substantiated by immunoelectron microscopy (Brown et al., 1976), which suggests that this may be the basis of pinocytotic uptake demonstrated for IgA by Allen et al. (1973). The weak binding of IgM by SC might under these circumstances be expected to militate against its transport. Nonetheless, all proceeds well in normal function, but IgM tends to be more readily released from the mucin than IgA, losing its attachment—if any—to SC, which otherwise might bind it in high local concentration. The release of the immunoglobulin from the mucous surface may, how­ ever, be advantageous, particularly in the young animal seeking to establish a satisfactory balance with the new challenges of its environ­ ment. Agglutination and release of potential pathogens with subsequent flushing by peristaltic flow of the gut contents would be an efficient means of disposal. Additionally, IgM is a potent opsonin and could act to assist the phagocytic function of neutrophils that emigrate into the lumen (Bellamy and Neilsen, 1974). The numerical order of immunocytes synthesizing IgA and IgM in the intestinal mucosa declines markedly through the small intestine from the duodenum through the jejunum and ileum (see Fig. 1). This is contrary to the numerical order of the microbial population of the

IMMUNOGLOBULINS OF COMMON DOMESTICS

15

small intestine which increased toward the ileum. It is significant, how­ ever, that Peyer's patches increase in number and size toward the distal small intestine and show a closer correlation with the increase in microbial population. It is now known that Peyer's patches are a rich source of IgA-producing immunocytes that populate the lamina propria (Cebra et al., 1977). Clearly these mechanisms combine to populate the in­ testinal tissues with committed lymphocytes, and significantly the upper small intestine is that which is most susceptible to the diarrheagenic effects of enteropathogens. It may be most suitable, therefore, that this should be the most heavily populated with antibody-producing cells.

VI. Immunoglobulins in the Nasolacrimal Fluids The paraocular glands serve to produce fluids that lubricate the eye, but in addition the presence of plasma cells in these glands and of IgA in the tears suggests a local immunologie function. Immunohistologic examination of the human lacrimal gland has demonstrated this to be a presumptive source of IgA (Franklin et al., 1973). However, the main observations on the origin and functional significance of local antibody in the oculonasal region are those made in the fowl. It is only in the chicken that detailed investigation has been carried out, the major paraocular gland being the Harderian gland, which lies within the orbital cavity. It contains very large numbers of immunocytes (Albini et al., 1974), and its secretion passes through a draining duct to a small pouch opening on the corneal surface. One function of the gland appears to be entrapment of antigen, and in this respect some similarity has been drawn to the bursa's function of antigen sampling from the alimentary tract (Aitken et al., 1976). Ocular route administration of Newcastle disease virus results in ac­ cumulation of immunocytes and stimulates lymphoid follicle develop­ ment in the gland and its duct, with consequent secretion of antibody. As judged by relative plasma cell population, the avian lacrimal gland contributes little to local immunity in comparison with the Harderian gland. However, ligation of the draining duct of the Harderian gland leads to a diversion of plasma cells to the lacrimal gland with local immunologie compensation. Excision of the lacrimal gland under these circumstances leads to loss of immunoglobulin from the lacrimal fluids (Survashe and Aitken, 1977). The use of such surgical models has also indicated the probable contribution of antibody of Harderian gland origin to the protection of the oropharynx and trachea. These concepts provide a basis for understanding the mechanisms of

16

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immunization via the oculonasal routes; however, the mechanism of protection should not be overlooked. Current studies of local immunity place emphasis on mechanisms that prevent access of invasive agents; thus it is interesting that the Harderian gland plays a role in trapping viral antigens and producing antibody locally. With this insight in the fowl, of the immunologie significance of local immunity in the oculonasal region, it was inevitable that a comparative investigation should follow in other species. The recent studies of Aitken and Survashe (1977) have demonstrated that the ruminant species possess Harderian and lacrimal glands with a moderate plasma cell infiltration, whereas only the lacri­ mal gland demonstrated any significant infiltration in the pig, horse, and dog. Thus the selective accumulation of plasma cells in the paraocular glands is a varied phenomenon between species, probably de­ termined mainly by access of antigens. The alternative source of immunoglobulin in the nasolacrimal fluids is by transudation from the serum'. In this respect the ruminant is in­ teresting in view of the established selective transport of IgGi into saliva and mammary secretions (Watson and Lascelles, 1973). IgGi constitutes about 40% of the immunoglobulin present in the nasal secre­ tions of sheep (Smith et al, 1975), and this is the main source of antibody following parenteral immunization with parainfmenza 3 virus. Radiomarker studies with IgGi and IgG 2 provide no evidence for selective transfer, both components passing from blood to nasal secretions at similar rates (Wells et al., 1977). The two subclasses appeared in nasal secretions at approximately 2% of their serum levels, so that in effect no concentration gradient was established.

VII. Effector Mechanisms in Mucosal Immunity One of the primary activities associated with mucous antibody is that of complex formation with antigen on the mucosal surface. Thus agglu­ tination is an important feature in antimicrobial immunity, facilitating dislodgement and clearance by peristalsis. AVith soluble as opposed to particulate antigens, immobilization on the mucous surface with enzymes acting in concert may effectively destroy an antigen before it is ab­ sorbed (Walker et al, 1975). Prevention of antigen transmission across the epithelium will be an effective antitoxic mechanism. This appears to be the likely antienterotoxic function in pigs orally immunized with heat-inactivated E. coli (Linggood and Ingram, 1978). In these studies, using the ligated gut (LG) test, LT enterotoxin activity mediated by cell-free extracts

IMMUNOGLOBULINS OF COMMON DOMESTICS

17

of enteropathogenic E. coli could be inhibited by passive transfer of intestinal secretions. The epithelial exclusion phenomenon has also been examined in rela­ tion to coccidial infection of chickens with Eimeria tenella. This coccidial parasite completes its life cycle in the mucosa and submucosa of the cecum, a region rich in lymphoid cells producing secretory IgA antibody. Active penetration of the host epithelial cells by sporozoites from the gut lumen is an essential feature of the infection. Secretory antibodies were observed to inhibit sporozoite penetration of epithelial cells in culture (Davis et al., 1978). Further evidence has also been pro­ vided that many of those sporozoites that manage to break through the antibody barrier to enter epithelial cells are thereafter no longer able to develop. It appears that, prior to such penetration, enzymes of the cecal fluids are able to seriously damage the invading sporozoite so that, having achieved its ideal intracellular habitat, it is still unable to develop. This is a perfect example of a thesis derived for molecular exclusion being tenable at the microbial level. Contrary to the theme of blocking and immobilization on the mucous surface, there is the prospect that surface antibody may interfere with attachment. The phenomenon of bacterial adhesion to host membranes is recognized as being of considerable significance in contributing to the virulence of bacteria, in particular enteropathogenic strains of E. coli. Antibodies in or on a membrane at the site of bacterial adhesion may be anticipated to intervene in the specialized interaction between host membrane receptor and microbial surface determinant. This mechanism has become one of the prevalent themes of current investigations of protective secretory antibody function in the hostpathogen relationship. The contribution of antibodies and the specificity of function against the surface K88 determinant of porcine enteropathogens has been demonstrated in blocking attachment to host-cell mem­ branes (Wilson and Hohman, 1974; Parry and Porter, 1978). The potential protective value of such anti-adhesins has been demonstrated "in vivo" (Rutter and Jones, 1973). These phenomena appear to present a satisfactory basis for protection. However, to take a different stance, one fundamental question seems to have been overlooked. It is quite evident that K88 and K99 are es­ sential virulence determinants of E. coli for pigs and calves, respectively; therefore, are antibodies against such antigens equally essential for host defense? Recent evidence on this point indicates that anti-K88 adhesins are not an essential parameter of host resistance (Porter et al., 1978). In an infection model for neonatal piglet enteritis, significantly better protection was obtained with sow colostrum possessing negligible

18

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PORTER

anti-K88 antibody than with those possessing high titers. The anti­ bacterial mechanisms that contribute to solid immunity are complex. In this respect, an intriguing observation was made that modification of surface antigenic characteristics of enteropathogens occurred in the presence of antibody both "in vivo" (Porter et al., 1977) and "in vitro" (Linggood and Porter, 1978). An immune mechanism inducing apparent loss of the transmissible plasmid responsible for synthesis of K88 has been identified (Fig. 4) ; it represents an "unmasking process," which can be of vital importance to the host. Normally, bacterial modification takes place to the disadvantage of the host, and it is interesting that natural antibody mechanisms have this potential to reduce the virulence of the pathogen. So far it appears that all classes of immunoglobulin participate in the phenomenon, but to date there is no clear evidence as to the nature of the antigen re­ sponsible for producing the antibody. It is interesting that anti-K88 antibodies do not appear to be able to exert this "plasmid curing" effect (Porter et al., 1978). Furthermore, the antigen appears to be heat stable, but the antibodies do not appear to require 0 antigen specificity. This phenomenon of genetic regulation in the pathogen must have been a natural feature in the host-pathogen relationship in order to maintain a successful balance throughout evolution. However, as a biologic function it has great potential significance not just for host defense, but also for the environment of the herd. As one of the tools of immunoprophylaxis, it should be of considerable value in reducing K88 HA Titer (Log 2 ) Control culture

24 20 16 12 8 4 1 Passage

2

3

4

5

FIG. 4. Antibody elimination of K88 determinant on porcine enteropathogenic Escherichia coli. Effect of passage through nutrient broth containing sow milk antibodies raised against heat-stable antigens.

IMMUNOGLOBULINS OF COMMON DOMESTICS

19

the virulence of pathogens excreted, thereby contributing to hygiene and reducing the susceptibility of other animals simultaneously and subsequently accommodated in that environment. REFERENCES Aitken, I. D., and Survashe, B. D. (1977). Int. Arch. Allergy Appl. Immunol. 53, 62-67. Aitken, I. D., Parry, S. H., Powell, J. R., and Survashe, B. D. (1976). Cong. Int. Assoc. Biol. Stand., 14th, 1975 33, 302-308. Albini, B., Wick, G., Rose, M. E., and Orlans, E. (1974). Int. Arch. Allergy Appl. Immunol. 47, 23-34. Allen, W. D., and Porter, P. (1973). Immunology 24, 493-501. Allen, W. D., and Porter, P . (1977). Immunology 32, 819-824. Allen, W. D., Smith, C. G., and Porter, P. (1973). Immunology 25, 55-70. Allen, W. D., Smith, C. G., and Porter, P. (1976). Immunology 30, 449-457. Arbuckle, J. B. R. (1970). J. Med. Microbiol. 3, 333-340. Beer, A. E., and Billingham, R. E. (1974). J. Reprod. Fertil., Suppl. 21, 59-88. Bellamy, J. E. C., and Nielsen, N . O. (1974). Infect. Immun. 9, 615-619. Bienenstock, J., Perey, D . Y. E., Gauldrie, J., and Underdown, B. J. (1973). J. Immunol. 109, 403-532. Binns, R. M. (1967). Nature (London) 214, 179-181. Bohl, E. H., Gupta, R. K. P., Olquin, M. V. F., and Saif, L. J. (1972). Infect. Immun. 6, 289-301. Brandenberg, A. C., and Wilson, M. R. (1973). Immunology 24, 119-127. Brandtzaeg, P . (1974). Immunology 26, 1101-1114. Brown, W. R., Isobe, Y., and Nakane, P. K. (1976). Gastroenterology 7 1 , 985-995. Butler, J. E. (1971). J. Dairy Sei. 54, 1315-1316. Butler, J. E., Maxwell, C. F., Pierce, C. S., Hylton, M. B., Asofsky, R., and Kiddy, C. A. (1972). J. Immunol. 109, 38-46. Butler, W. T., Rossen, R. D., and Waldman, T. A. (1967). J. Clin. Invest. 46, 18831893. Campbell, B., Porter, R. M., and Petersen, W. E. (1950). Nature (London) 166, 913-914. Cebra, J. J., Kamat, R., Gearhart, P., Robertson, S. M., and Tseng, J. (1977). Ciba Found. Symp. 46 (new ser.), 5-22. Chapman, H. A., Johnson, J. S., and Cooper, M. D. (1974). J. Immunol. 112, 555-563. Chidlow, J. W., and Porter, P . (1978). Res. Vet. Sei. 24, 254-257. Davis, B. K., and Gill, T. J. (1975). J. Immunol. 115, 1166-1168. Davis, P. J., Parry, S. H., and Porter, P. (1978). Immunology 34, 879-888. Esteves, M. B., ajid Binaghi, R. A. (1972). Immunology 23, 137-145. Franëk, F., and Riha, I. (1964). Immuno chemistry 1, 49-63. Franëk, F., Riha, I., and Stertzl, J. (1961). Nature (London) 189, 1020-10?2. Franklin, R. M., Kenyon, K. R., and Tomasi, T. B. (1973). J. Immunol. %10, 984992. Gasser, D. L., and Silvers, W. K. (1974). Adv. Immunol. 18, 1-66. Henry, C , and Jerne, N . K. (1968). J. Exp. Med. 128, 133-152. Husband, A. J., and McDowell, G. H. (1975). Immunology 29, 1019-1028. Kehoe, J. M., Chuang, C , and Capra, J. D. (1973). Fed. Proc, Fed. Am. Soc. Exv· Biol. 32, Abstr. 4207.

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Kincade, P. W., and Cooper, M. D. (1973). Science 179, 398-400. Lebacq-Verheyden, A. M., Vaerman. J. P., and Heremans, J. F. (1972). Immunology 22, 165-175. Lee, C. S., and Lascelles, A. K. (1970). Amt. J. Exp. Biol. Med. Sei. 48, 525-535. Leslie, G. A., and Clem, L. W. (1969). J. Exp. Med. 130, 1337-1352. Leslie, G. A., and Martin, L. N . (1973). J. Immunol. 110, 1-9. Leslie, G. A., Stankus, R. P., and Martin, L. N . (1976). Int. Arch. Allergy Appl. Immunol. 51, 175-185. Linggood, M. A., and Ingram, P. L. (1978). Res. Vet. Sei. 25, 113-115. Linggood, M. A., and Porter, P. (1978). Immunology 35, 125-127. Logan, E. F., and Penhale, W. J. (1971). Vet. Rec. 88, 222-228. Mehta, P. D., Reichlin, M., and Tomasi, T. B. (1972). J. Immunol. 109, 1272-1277. Miler, L, Cerna, J., Trâvnicek, J., Rejnek, J., and Kruml, J. (1975). Folia Microbiol. (Prague) 20, 433-438. Miniats, O. P., Mitchell, L , and Barnum, D. A. (1970). J. Comp. Med. 34, 269-276. Muscoplatt, C. C , Setcavage, T. M., and Kim, Y. B. (1977). Int. Arch. Allergy Appl. Immunol. 54, 165-170. Neoh, S. H., Jahoda, D. M., Rowe, D. S., and Voiler, A. (1973). Immunochemistry 10, 805-813. Parry, S. H., and Porter, P. (1978). Immunology 34, 471-478. Parry, S. H., Allen, W. D., and Porter, P. (1977). Immunology 32, 731-741. Porter, P. (1969). Biochim Biophys Acta 481, 381-392. Porter, P. (1972). Immunology 23, 225-238. Porter, P. (1973a). J. Am. Vet. Med. Assoc. 163, 789-794. Porter, P. (1973b). Immunology 24, 163-176. Porter, P., and Hill, I. R. (1970). Immunology 18, 565-573. Porter, P., and Parry, S. H. (1976). Immunology 31, 407-415. Porter, P., Noakes, D. E., and Allen, W. D. (1970). Immunology 18, 245-257. Porter, P., Kenworthy, R., Noakes, D. E., and Allen, W. D. (1974). Immunology 27, 841-853. Porter, P., Parry, S. H., and Allen, W. D. (1977). Ciba Found. Symp. (new ser.), 55-66. Porter, P., Linggood, M. A., and Chidlow, J. W. (1978). Secretory Immunity and Infection, 133-142. Portis, J. L., and Coe, J. E. (1975). Nature (London) 258, 547-548. Prokesovâ, L., Kostka, J., Rejnek, J., and Trâvnicek, J. (1970). Folia Microbiol. (Prague) 15, 337-340. Rose, M. E., Orlans, E., and Buttress, N. (1974). Eur. J. Immunol. 4, 521-523. Rutter, J. M., and Jones, G. W. (1973). Nature (London) 242, 531-533. Saif, L. J., Bohl, E. H., and Gupta, R. K. P. (1972). Infect. Immun. 6, 600-609. Schultz, R. D. (1973). J. Am. Vet. Med. Assoc. 163, 804-806. Schultz, R. D., Dunne, H. W., and Heist, C. E. (1973). Infect. Immun. 5, 981-991. Silverstein, A. M., Thorbecke, G. J., Kraner, K. L , and Lukes, R. J. (1963). J. Immunol. 91, 384-395. Smith, W. D., Dawson, A. McL., Wells, P. W., and Burrells, C. (1975). Res. Vet. Sei. 19, 189-194. Stertzl, J., Kostka, J., Riha, I., and Mandel, L. (1960). Folia Microbiol. (Prague) 5, 29-45. Survashe, B. D., and Aitken, L D. (1977). Res. Vet. Sei. 23, 217-223. Uhr, J. W., and Moller, G. (1968). Adv. Immunol. 8, 81-127.

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Vaerman, J. P., Heremans, J. F., and Kerckhoven, van G. (1969). J. Immunol. 103, 1421-1423. Vitetta, E. S., Grundke-Iqbal, I., Holmes, K. V., and Uhr, J. W. (1974). J. Exp. Med. 139, 862-876. Walker, W. A., Wu, M., Isselbacher, K. J., and Block, K. J. (1975). J. Immunol 115, 854-861. Watanabe, H., and Kobayashi, K. (1974). J. Immunol. 113, 1405-1409. Watson, D. L., and Lascelles, A. K. (1973). Aust. J. Exp. Biol. Med. Sei. 51, 247-254. Wells, P . W., Dawson, A. McL., Smith, W. D , and Smith, B. S. W. (1977). Res. Vet. Sei. Ill, 201-204. World Health Organization (1966). Bull. W.H.O. 35, 953. Wilson, M. R., and Hohman, A. W. (1974). Infect. Immun. 10, 776-784.

ADVANCES I N VETERINARY SCIENCE AND COMPARATIVE MEDICINE, VOL. 2 3

Primary and Secondary Immune Deficiencies of Domestic Animals LANCE E. PERRYMAN Department

of Vetennary Microbiology and Washington State University Pullman, Washington

Pathology

I. Introduction I I . Overview of Immune Responses I I I . Classification of Immune Deficiencies IV. Detection and Characterization of Immune Deficiency Disorders . . . 1. Characterization of Disorders Involving Lymphocytes . . . . 2. Characterization of Disorders Involving Neutrophils 3. Characterization of Complement System Disorders V. Immune Deficiencies of Domestic Animals 1. Equine 2. Bovine 3. Ovine 4. Porcine 5. Canine 6. Feline VI. Summary References

23 24 25 28 28 31 31 32 32 38 42 42 43 45 46 47

I. Introduction Domestic animals interact with an environment teaming with patho­ genic microorganisms. Resistance to and recovery from infection by these organisms is dependent upon normal host defense systems. The value of these systems is best demonstrated in those primary or sec­ ondary disease states where a particular defense mechanism is deficient. Such cases have clarified the role of the immune system in host defense and have contributed to the understanding of normal immune respon­ siveness. The purpose of this article is to review the primary and secondary immune deficiency states described in domestic animal species. 23 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-039223-2

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II. Overview of Immune Responses It is now generally accepted that immune responses are mediated by at least two major classes of lymphocytes. Both classes are derived from a common pluripotential stem cell precursor, the major location of which varies with the developmental age of the animal. Stem cells first arise in the yolk sac during fetal development (Moore and Metcalf, 1970). Subsequently, fetal liver becomes the primary site of stem cell activity before giving way to bone marrow. Postnatally, the bone mar­ row is the major source of lymphocyte precursor cells (Micklem and Loutit, 1966). Developing lymphoid precursor cells may follow one of two maturational pathways. Those cells destined to become T lymphocytes migrate to the thymus where mitotic division and maturation occurs under the influence of humoral products derived from thymic epithelium (A. L. Goldstein et al, 1966; G. Goldstein, 1974; Trainin and Small, 1970). Upon completion of certain maturational changes, T lymphocytes mi­ grate to peripheral lymphoid tissues of the body where they are found principally in periarteriolar lymphocytic sheaths of the spleen, and in paracortical regions of lymph nodes. T lymphocytes comprise the ma­ jority of lymphocytes in peripheral blood. In these sites, T cells play three major roles in immune responses: (1) They may interact with B lymphocytes in the production of specific antibody, in which case they are referred to as helper T cells; (2) they may suppress immune responses and, in those cases, are classified as suppressor T lympho­ cytes; (3) finally, these lymphocytes are responsible for cell-mediated immunity, which is important in host defense against neoplastic cells, fungi, protozoa, intracellular bacteria, and some viral infections (re­ viewed in Good, 1972; Horowitz and Hong, 1977). B lymphocytes derived from similar lymphoid stem cell precursors may require a similar intervening maturational phase as described for T cells. In avians the bursa of Fabricius, a blind outpocketing of the hindgut, provides the maturational environment for B lymphocytes. The mammalian counterpart of the avian bursa of Fabricius has never been conclusively identified. B lymphocytes express significant amounts of immunoglobulm (Ig) on their surfaces (Rabellino et al., 1971) and can synthesize and secrete IgM, IgA, or IgG. On appropriate stimulation, B cells differentiate to plasma cells which have a short life span and a remarkable capacity to synthesize and secrete Ig. Specific antibodies secreted by lymphocytes and plasma cells play an important role in host defense against extracellular bacteria and some viral infections. B lymphocytes are preferentially located within germinal centers of the

IMMUNE DEFICIENCIES OF DOMESTIC ANIMALS

25

spleen and lymph nodes. They comprise approximately 10% of periph­ eral blood lymphocytes (Winchester et al., 1975). Under normal conditions, the interaction of antigen with immunologically specific and functionally mature lymphocytes results in an immune response. The details of antigen interaction with lymphocytes and macrophages, and the mechanisms by which lymphocyte products in­ fluence other cells are beyond the scope of this article. The reader is referred to Horowitz and Hong (1977) for details and references. In brief, the result of antigen interaction with T lymphocytes is the pro­ duction and release of lymphokines which may (1) augment or suppress the activity of T and B lymphocytes, (2) chemotactically attract lymphocytes, monocytes, or granulocytes, (3) inhibit the migration of leukocytes, and (4) cause cytolysis of cells bearing new surface anti­ gens. Upon interaction with specific antigen B lymphocytes secrete antibodies. Once secreted, antibody may directly neutralize some microbial agents. More typically, antibody and components of the complement system interact to neutralize or lyse microbial agents. In addition to these modes of microbial clearance, the interaction of anti­ gen, antibody, and complement can augment the phagocytosis of in­ fectious agents by neutrophils (reviewed in Ruddy et al., 1972; MüllerEberhard, 1975). Neutrophils and macrophages are significantly involved in host de­ fense through the phagocytosis and enzymatic digestion of microorgan­ isms. This process involves the internalization of the agent, the joining of the phagosome with cytoplasmic lysosomal granules, and the release of degradative enzymes into the phagosome. The above scheme is a brief overview of the events in normal immune responses, and the manner in which the complement system and phagocytic cells may interact with products of the immune response to favor host defense. From this outline one could postulate sites at which de­ fective development or function could occur (Table I ) . All of these defects could increase susceptibility to infection and could be classified, in the broadest sense, as immune deficiency disorders. An extensive dis­ cussion of the possible mechanisms of immunodeficiency diseases of man has been presented by Horowitz and Hong (1977).

III. Classification of Immune Deficiencies For the purpose of this article, immune deficiencies will be classified in two ways (Table I I ) . Classification according to host defense system is useful because diagnostic tests are usually performed on a systems

Neutrophils

Synthesis of complement components

Zinc metabolism

B lymphocytes

Third and fourth pharyngeal pouch Thymic epithelium T lymphocytes "Bursal equivalent"

Yok sac, fetal liver, or bone marrow

Site of abnormality

TABLE I

Decreased killing of microbrial agents

(3) Inability to differentiate to plasma cells Thymic hypoplasia> impairment of cellmediated immunity (1) Decreased lysis of microbial agents (2) Decreased phagocytosis of microbial agents

(1) Delayed onset of synthesis and secretion of immunoglobin (2) Inability to synthesize and/or secrete immunoglobulin of a particular class

Inability to produce thymic hormonal factors Suppression of immune responses Failure of lymphoid precursor maturation to B lymphocytes

(1) Failure to produce lymphoid precursor cells (2) Failure of lymphoid precursor cells to differentiate to B and T lymphocytes Failure of thymic development

Manifestation on host defense

Equine Bovine

Selective IgC 2 deficiency

Cyclic neutropenia Granulocytopathy syndrome Chediak-Higashi syndrome

Canine Canine Bovine Feline

Bovine

Equine

Transient hypogammaglobulinemia Selective IgM deficiency

Lethal trait A46

Equine

Equine

Species

Agammaglobulinemia

Combined immunodeficiency

Disease

POSSIBLE M E C H A N I S M S OF I M M U N E DEFICIENCY STATES I N DOMESTIC ANIMALS

IMMUNE DEFICIENCIES OF DOMESTIC ANIMALS

27

TABLE II CLASSIFICATION OF I M M U N E DEFICIENCY DISORDERS

Basis Host defense system

Mechanism

Category (1) Lymphoid system Stem cells T lymphocytes B lymphocytes (2) Phagocytic system Neutrophils Monocytes (3) Complement system (1) Primary (2) Secondary

basis. Classification as a primary or secondary disorder implies a mechanistic basis in the characterization of the disease. A primary dis­ order is one in which a genetic basis is proven or suspected. A secondary disorder is one in which the animal was initially capable of producing normal defense responses. However, because of a mechanical problem, disease, or treatment, the response capacity of the animal is now sup­ pressed or depleted. Examples of secondary deficiencies include malnu­ trition, irradiation, failure of the newborn to absorb colostral Ig, treat­ ment with corticosteroids, neoplasia, and infection by immunosuppressive agents. A number of clinical signs have been associated with primary and secondary immune deficiency states. When observed, they should alert one to the need for specific diagnostic tests. Some of these signs include (1) onset of infections in the first 6 weeks of life, suggesting inadequate transfer of maternal Ig; (2) repeated infections that respond poorly to standard therapy; (3) increased susceptibility to low-grade patho­ gens; (4) infection with organisms rarely observed in immunocompetent animals, such as the protozoan Pnenmocystis carinii; (5) systemic ill­ ness following vaccination with live virus vaccines; and (6) marked deficiencies in leukocyte levels, particularly profound neutropenia or lymphopenia of several days duration. These signs suggest a defect in the phagocytic, lymphoid, or complement systems which needs to be verified if the disorder is to be diagnosed accurately, clinically managed, and studied.

28

LANCE E. PERRYMAN

IV. Detection and Characterization of Immune Deficiency Disorders When clinical signs, patterns of infection, and response to therapy suggest a defect in host defense, the first step is to establish the system involved. Several tests for evaluation of B and T lymphocytes have been described in the past 5 years. Tests for evaluation of the comple­ ment system and phagocytic cells are not as readily available.

1. CHARACTERIZATION OF DISORDERS INVOLVING LYMPHOCYTES

Disorders of lymphocyte function may involve B lymphocytes, T lymphocytes, or both. To determine which are involved, the absolute peripheral blood lymphocyte count is determined and tests for B- and Tlymphocyte function and quantitation are performed. a. B-Lymphocyte

Evaluation

The presence and quantities of Ig in serum reflect the functional status of B lymphocytes. Gross abnormalities of total Ig levels can be de­ tected by serum electrophoresis or immunoelectrophoresis. However, diagnosis of subtle Ig depressions, or total deficiencies of a single class or subclass, requires more specific and quantitative procedures. Classspecific quantitative data can be obtained by single radial immunodiffusion employing monospecific antiserum for each class or subclass to be measured. Antisera are commercially available on a limited basis for some species. Usually, the reagents are prepared by individual research and diagnostic laboratories, and quantitation can often be obtained by submitting samples to these places. Some commercially available antisera to human immunoglobulins will cross-react sufficiently and spe­ cifically with domestic animal immunoglobulins, and can be used for valid quantitative work (Buening et al., 1977). Response to immunization is a valuable procedure for evaluating B-lymphocyte function. The presence of specific antibody in serum provides evidence for the presence of B lymphocytes which can recognize antigen, cooperate with T lymphocytes and macrophages, and finally synthesize and secrete antibody. Immunization can be performed in a manner to yield additional valuable data. Injection can be made at a site allowing convenient biopsy of the draining lymph node. Histological examination of the node will reveal active germinal center activity and the presence of plasma cells in normal conditions. Several

IMMUNE DEFICIENCIES OF DOMESTIC ANIMALS

29

antigens may be used for these studies. Heterologous (xenogeneic) eryth­ rocytes and keyhole limpet hemocyanin have been useful antigens in our studies. B lymphocytes can be quantitated by detecting those lymphocytes with surface Ig, and receptors for the third component of complement (C3). Lymphocytes for evaluation are obtained from peripheral blood by density centrifugation (Boyum, 1968) or from surgically excised lymph nodes. B lymphocytes possess approximately 100,000 molecules of Ig on their surfaces (Rabellino et al., 1971). In those species ade­ quately studied, this Ig is of the IgM or IgD class. Detection is usually achieved by immunofluorescence employing fluorochrome-conjugated antisera to IgM of the animal species studied. Several technical para­ meters must be carefully controlled to obtain valid data. Some of these include enzymatic cleavage of the Fc portion of fluorochrome-conjugated anti-immunoglobulins, and the removal of aggregated Ig from reagents (Winchester et al., 1975). References for this procedure are listed for each species in Table III. B lymphocytes possess a surface receptor for C3 detectable by rosette assays. Erythrocytes (E) are treated with subagglutinating quantities of antierythrocyte antibodies (A), and then incubated with sublytic quantities of complement (C). This results in attachment of C3b mole­ cules to the surface of erythrocytes (EAC). B lymphocytes will bind the erythrocytes through C3b and are identified by the formation of a rosette. Monocytes and sometimes neutrophils will also bind EAC and must be removed from the cell preparation studied or in some way identified to avoid errors. Procedural details are available in references listed in Table III. b. T-Lymphocyte

Evaluation

Quantitation of T lymphocytes is usually done by E rosette assays. In several species, incubation of lymphocytes with appropriate xeno­ geneic erythrocytes results in the formation of erythrocyte clusters around T lymphocytes. Domestic species demonstrating this phe­ nomenon are indicated in Table III. T-lymphocyte function can be evaluated in vivo or in vitro utilizing phytolectins or antigens. In vivo skin testing employing dinitrochlorobenzene (DNCB) for sensitization and challenge is an accepted pro­ cedure for measuring cell-mediated immune responsiveness to specific antigen (Hodgin et al., 1978). A limitation in this procedure is the time required for sensitization and testing before an answer is obtained. In the case of DNCB, a period of 2 to 3 weeks is usually required. Re-

E rosette

Bovine

Feline

Canine

Ovine Porcine

E rosette SIg EAC rosette

Equine

—

SIg

_

+ +

SIg E rosette

-

+

-

-

+ + +

+

-

?

—

EAC rosette

-

+

+

+

+ +

+ +

—

+

_

+

—

+

B cells

Reactive cells

T cells

EAC rosette

SIg E rosette SIg E rosette

SIg

Marker

Species

TABLE I I I

Guinea pig rbc

Bind to monocytes

Human, guinea pig rbc Bind to monocytes and neutrophils

Sheep rbc

Bind to monocytes and neutrophils Sheep rbc

Guinea pig (rbc)

Comments

References

Onions (1975) ; Holmberg et al. (1977) Krakowka and Guyot (1977) Onions (1975); Holmberg et al. (1977) Holmberg et al. (1977) Cockerell et al. (1976a); Mackey et al. (1975); Taylor et al. (1975); Holmberg et al. (1976) Cockerell et al. (1976a); Taylor et al. (1975); Holmberg et al. (1976) Cockerell et al. (1976a); Taylor et al. (1975); Holmberg et al. (1976)

Grewal et al. (1976); Higgins and Stack (1977); Reeves and Renshaw (1978) Reeves and Renshaw (1978); Muscoplat et al. (1974a,b); Weiland and Straub (1975) Symons and Binns (1975); Ey (1963) Escajadillo and Binns (1975) Binns and Symons (1974) Bowles et al. (1975) Krakowka and Guyot (1977)

Tarr et al. (1977) Banks and Henson (1973) Banks et al. (1976)

MARKERS FOR T AND B LYMPHOCYTES OF DOMESTIC ANIMAL SPECIES

IMMUNE DEFICIENCIES OF DOMESTIC ANIMALS

31

cently, the intradermal injection of phytohemagglutinin (PHA) for assessment of delayed hypersensitivity has been described. In horses, it appears to be a valid test of T-lymphocyte function and no prior sensitization is required. The procedure can be performed and evaluated in 24 to 48 hr (Hodgin et al., 1978). Rejection of allogeneic skin grafts is a well accepted in vivo procedure for evaluating T-lymphocyte ac­ tivity in domestic animals (Perryman et al., 1972; Dennis et al., 1969). Lymphocytes isolated from peripheral blood or obtained from lymph nodes can be cultured in vitro. The ability of these cells to undergo DNA synthesis when stimulated with PHA or concanavalin A (Con A) is a reflection of T-lymphocyte function (Janossy and Greaves, 1971). The presence of T lymphocytes can also be determined by histological examination of lymph node biopsy specimens. T lymphocytes are located primarily in the paracortical regions of lymph nodes. Absence of cells in this region suggests a deficiency of T lymphocytes. 2. CHARACTERIZATION OF DISORDERS INVOLVING NETJTROPHILS

Neutrophil dysfunction can result in decreased phagocytic capacity, failure or delayed fusion of lysosomal granules with phagosomes, or decreased killing capacity. These abnormalities are detected by phago­ cytosis studies, leukocyte bacteriocidal assays, nitroblue tetralozium reduction tests, and ultrastructural examination of cells from suspect animals (Renshaw et al., 1974; Johnston and Newman, 1977). 3. CHARACTERIZATION OF COMPLEMENT SYSTEM DISORDERS

The availability of purified complement components or antisera to specific components of domestic species is limited. However, certain screening tests can be performed that will detect some complement de­ ficiencies. Total hemolytic complement levels can be determined and compared to normals of the same species. Hemolysis requires assembly of C5-9 on the erythrocyte membrane and decreased hemolysis indi­ cates a defect at some point of the pathway. Grant (1977) has reviewed this procedure and the optimal source of erythrocytes and antibodies for evaluating the hemolytic activity of each species' complement. Ac­ tivity of the early C components can be estimated by measuring the ability of the serum source to render antibody-coated erythrocytes suitable for EAC rosette procedures, since fixation of C through C3 is required. When antiserum is available, the levels of individual comple­ ment components can be quantitated by single radial immunodiffusion (Perryman et al, 1971).

32

LANCE E. PERRYMAN

V. Immune Deficiencies of Domestic Animals 1.

a. Combined

EQUINE

Immunodeficiency

Several immunodeficiency disorders have been described in horses. The most severe disorder is combined immunodeficiency (CID), a genetic disease resulting in failure to produce functional B and T lym­ phocytes. Although first recognized and reported in 1973 (McGuire and Poppie, 1973), descriptions highly suggestive of CID appeared in earlier publications (Johnston and Hutchins, 1967; McChesney et al., 1970). The disorder is limited to Arabian horses and is inherited as an autosomal recessive trait (Thompson et al., 1975; Poppie and McGuire, 1977). Affected foals appear normal at birth and remain healthy for up to 2 months of age. Once maternally derived Ig has been reduced to low levels through normal catabolic processes, the foals are remarkably susceptible to infections (Poppie and McGuire, 1976) and inevitably die by 5 months of age (Perryman et al., 1978b; McChesney et al., 1970, 1973, 1974; Thompson et al, 1976; Clayton, 1976; Clark et al., 1978; Snyder et al., 1978). The most common cause of death is respi­ ratory infection attributable to three groups of microorganisms: bacteria, equine adenovirus, and the protozoan Pneumocystis carinii. In one survey of 66 foals with CID, adenovirus infection was diagnosed in 44 and Pneumocystis carinii was observed in 22 foals (Perryman et al., 1978b). At necropsy, the lesions of the secondary infectious diseases are obvious. Striking changes are also apparent in lymphoid tissues. The thymus is hypoplastic and very often difficult to locate. Grossly, the tissue of the anterior mediastinum is predominantly adipose, and often, multiple sections of this area must be examined histologically to find small islands of thymic epithelial cells containing few lympho­ cytes (McGuire et al., 1976a). Lymph nodes are smaller than normal. The spleen varies from normal to subnormal size depending on the degree of vascular engorgement. Histologically, lymphoid tissues are characterized by nearly total depletion of lymphocytes. The thymus consists of nests of epithelial cells usually containing Hassall's cor­ puscles. Areas of hemorrhage, necrosis, and mineralization are occa­ sionally observed. Lymph nodes are depleted of lymphocytes and devoid of plasma cells. Germinal centers, corticomedullary differentiation, and other features of normal lymph node morphology are absent. The spleen is devoid of germinal centers and periarteriolar lymphocytic sheaths. A consistent finding has been the absence of the connective tissue stromal framework at sites where germinal centers would be expected

IMMUNE DEFICIENCIES OF DOMESTIC ANIMALS

33

(McGuire et al, 1976a). This lesion is useful in differentiating CID from other disorders in which lymphoid depletion is the result of atrophy rather than a primary lesion. Sufficient studies have been performed to classify this disease as a genetic disorder influencing B- and T-lymphocyte formation. Prospec­ tive breeding trials have substantiated the mode of inheritance and sex analysis of all cases we have diagnosed reveals essentially equal numbers of male and females affected (Poppie and McGuire, 1977; Perryman, unpublished observations). Lymphocyte function tests have shown these foals to be markedly lymphopenic ( < 1000 lymphocytes/mm 3 ) with usually no demonstrable functional lymphocytes (McGuire et al, 1974, 1975b). Mononuclear cells from foals with CID lack surface immunoglobulin and complement receptors indicative of B lymphocytes. With the exception of one case we have studied, mononuclear cells from these foals have been nonresponsive to in vitro stimulation with PHA, Con A, or pokeweed mitogen (PWM). Cells from CID foals have been evalu­ ated in one-way mixed lymphocyte reactions. Cells from all foals tested were capable of stimulating allogeneic lymphocytes from normal horses. None of the foals, however, had cells capable of responding to allogeneic stimulation (Perryman and McGuire, 1978). Affected foals do not synthesize Ig, are unable to respond to immunization, and fail to give delayed-type hypersensitivity skin reactions (Hodgin et al, 1978). The defect in combined immunodeficiency of Arabian horses is ap­ parently limited to lymphoid cells. Studies of neutrophils and monocytes from foals with CID have shown they are present in normal numbers. Furthermore, the phagocytic capacity, and the presence and quantity of surface receptors are within normal limits (Banks and McGuire, 1975). Evaluation of the complement system has revealed no abnormalities. Total hemolytic complement levels were within normal limits and functional C3 apparently exists. Furthermore, neutralization of equine viral arteritis virus, a process completed by fixation of comple­ ment through C2, is adequately mediated by serum from affected foals (McGuire et al., 1975b). Although Ig synthesis does not occur, recent studies have shown these foals produce secretory component, a nonlymphoid protein (Buening et al., 1978). Criteria for the diagnosis of CID have been defined (Perryman et al., 1978a) and adherance to these criteria is important when establishing the diagnosis. Since CID is inherited as an autosomal recessive trait, diagnosis of the condition in a foal marks the dam and sire as carriers of the trait. This significantly influences the market value and future usefulness of mares and stallions in breeding programs. For these reasons it is critical that the diagnosis be accurate. At present, three

34

LANCE E. PERRYMAN

criteria are employed: (1) lymphopenia, (2) hypoplastic alterations of lymphoid tissues, and (3) absence of IgM in serum. IgM is synthesized by the equine fetus at approximately 190 days of gestation (Perryman, unpublished observations). Therefore, at the time of birth, normal foals have IgM in their serum which can be detected by single radial immunodiffusion and gel diffusion employing monospecific antiserum (Buening et al., 1977; McGuire et al., 1974). We have found that no single criterion listed above is reliable, and that a minimum of two must be satisfied to adequately establish a diagnosis of CID. Combined immunodeficiency is an important disease from two major viewpoints. First, it is a significant disease problem in Arabian horses and is encountered by veterinarians involved in equine practice. The prevalence of the disorder is not accurately known but limited surveys have revealed that 2.3 to 2.7% of Arabian foals sampled hav° the disease (Poppie and McGuire, 1977; McChesney, personal communica­ tion). Second, CID in horses is the only animal counterpart of severe combined immunodeficiency of children (reviewed in Horowitz and Hong, 1977). It therefore provides a valuable experimental system for determining the biochemical basis of CID, for evaluating methods of immunotherapy applicable to children with immunodeficiency diseases, and for studying the mechanisms involved in normal lympho­ cyte differentiation from precursor stem cells. Attempts have been made to establish the biochemical aberration (s) responsible for CID. Absence of an enzyme (adenosine deaminase) involved in purine metabolism has been noted in some children with CID and a causal role for this enzyme defect has been suggested (Giblett et al., 1972). Evaluation of foals with CID has shown adenosine deaminase is present in normal quantities (McGuire et al., 1976b, Castles et al., 1977). Abnormalities of purine metabolism have been detected in foals suggesting a defect in this general pathway may be important in the disorder (Magnuson and Perryman, 1979). Further study will be needed to verify and extend these observations. Additional studies questioning the role of corticosteroids (Magnuson, 1978) or zinc deficiency (Perryman, unpublished ob­ servations) indicate these factors are not involved in the pathogenesis of CID in horses. Immunotherapy attempts involving transplantation of fetal tissues have been done to characterize further the basis of the disease and to evaluate potential modes of therapy for immunodeficient children lack­ ing histocompatible bone marrow donors (Ardans et al., 1977; Perry­ man et al., 1979). These studies have met with limited success, largely because of insufficient knowledge of the ontological development of equine fetal immune responses.

I M M U N E DEFICIENCIES OF DOMESTIC ANIMALS

b.

35

Agammaglobulinemia

Two cases of agammaglobulinemia occuring in a Thoroughbred and a Standardbred have been diagnosed (Banks et al, 1976; McGuire et al.y 1976c; Deem et al., 1979). Both horses were males, suggesting a sexlinked mode of inheritance. Diagnosis was established on the basis of normal T-lymphocyte function but absence of B lymphocytes. The horses lacked B lymphocytes as determined by surface Ig and C3 re­ ceptor assays. Ig levels were extremely low or absent, and antibody response following immunization did not occur. Histological examination of lymphoid tissues revealed absence of plasma cells and germinal centers. T-lymphocyte evaluations were normal. Both horses developed positive skin reactions following intradermal injection of PHA. Their lymphocytes responded normally to PHA and Con A stimulation in vitro. Repeated episodes of bacterial infections with incomplete response to standard therapy were typical of both cases. An interesting feature of this disorder is the life span of affected animals. The Thoroughbred lived 17 months and the Standardbred 18 months, with apparent total absence of B lymphocytes and inability to produce antibodies to microbial agents. Compared to foals with CID that invariably die by 5 months of age, horses with agammaglobulinemia survive considerably longer, attesting to the importance of T lymphocytes in host defense. c. Selective IgM

Deficiency

The first case of selective IgM deficiency was observed in 1974. Since that time we have diagnosed 13 cases involving Arabian and Quarterhorses. All cases had very low or absent serum IgM levels. This was the only apparent immunologie defect, as serum C3 levels, lymphocyte counts, Ig other than IgM, B and T lymphocyte numbers, and lym­ phocyte responses to phytolectin stimulation were within normal limits (Perryman et al., 1977). While the total number of lymphocytes with surface Ig was in the normal range, it is not known if there is a selective absence of IgM B cells, or an inability of B cells to secrete IgM. Two clinical manifestations have been observed in foals with IgM deficiency. Most foals had repeated respiratory infections beginning within the first months of life and resulting in death by 4 to 8 months of age. A few have lived to 2 years of age. In these cases, unthriftiness, poor growth, and repeated low-grade respiratory infections have been observed. At this time, it is not clear if selective IgM deficiency is a genetically based disorder in horses. Common pedigrees were present in two of the

36

LANCE E. PERRYMAN

Quarterhorses studied, but a limited breeding trial was uninformative. Both primary and secondary forms of IgM deficiency are recognized in children (Hobbs, 1975), and additional breeding trials are required to resolve this question. d. Transient

Hypogammaglobulinemia

Hypogammaglobulinemia resulting from delayed onset of Ig synthesis has been described in one Arabian foal (McGuire et al., 1975a). Very low levels of IgG and IgG(T) were present at 2 months of age. Signifi­ cant Ig synthesis did not occur until the foal was 3 months old. When evaluated at 3.5 months of age, total B lymphocyte levels, antibody response to immunization, in vitro lymphocyte stimulation with PHA and Con A, and skin reaction to intradermal PHA injection were all normal. During the period of hypogammaglobulinemia, the foal de­ veloped systemic adenoviral and bacterial infections resulting in pul­ monary, hepatic, renal, and sublaryngeal abscesses. The mechanism of this deficiency and its primary or secondary basis are unknown. e. Failure of Passive Transfer (FPT) of Maternal

Immunoglobulin

ΓΡΤ is the most common immunological deficit of foals. It occurs in all breeds of horses from ponies to draft breeds and is significantly correlated with susceptibility to microbial infections in the early post­ natal period. FPT is not a genetic disorder. It is classified as a secondary immunodeficiency without reservation. The process by which foals obtain maternal Ig has been characterized and reviewed in detail by Jeffcott (1972, 1974a,b,c, 1975; Jeffcott and Jeffcott, 1974). Because of the diffuse epitheliochorial type of placentation in the mare, the equine fetus receives no maternal antibody across the placenta. At birth, the foal is essentially agammaglobulinemic with the exception of measurable IgM levels and occasionally trace quantities of IgG which are synthesized by the foal in utero (Perryman, unpub­ lished observations). Therefore, all maternal Ig is obtained from in­ gested colostrum. Following colostrum ingestion, large molecular weight proteins including Ig are taken up nonselectively by villous epithelial cells throughout the small intestine. Ability to absorb Ig is time de­ pendent, occurring most efficiently in the first 6 hr after parturition, and declining to negligable levels at 24 hr of age. Termination of absorption is apparently related to. changes in intestinal epithelial cells. Rapid turnover resulting in replacement of specialized epithelial cells by more mature cells unable to absorb macromolecules is well advanced by 24 hr postpartum. Quantitative studies of maternal Ig transfer have been reported (Rouse and Ingram, 1970; McGuire and Crawford, 1973). Colostrum was found to contain levels of IgG, IgG(T), IgA, and IgM similar to

IMMUNE DEFICIENCIES OF DOMESTIC ANIMALS

37

those in the mare's serum. By 24 hr of age, under normal conditions, the foal absorbs enough Ig to achieve serum concentrations similar to those of adult horses. Maternal Ig is then catabolically eliminated with a half-life of approximately 21 days for IgG. Synthesis in significant quantities begins at 2 weeks of age. Synthesis of Ig by the foal replaces maternal Ig almost entirely by 5 months of age. The balance between synthesis of Ig by the foal, and catabolic elimination of maternal Ig has been defined (McGuire and Crawford, 1973). Assuming normal passive transfer, adult levels of IgG, and IgG(T), are present by 24 hr of age. These levels decline to reach a low point at about 2 months of age. Levels then increase steadily, reaching near adult levels by 6 months of age. It would be anticipated that foals are most susceptible to infection at 2 months of age when total Ig is lowest. The period of vulnerability can be much longer and start as early as 1 day of age if F P T occurs. The key feature is the amount of immunoglobulin initially absorbed (McGuire et al, 1975a, 1977; Crawford et al, 1978). Foals must absorb sufficient Ig to achieve a minimum of 400 mg IgG/100 ml plasma. Values from 0 to 200 mg/100 ml constitute failure of passive transfer. Concentrations from 200 to 400 mg/100 ml are considered partial failures. The transmission of maternal Ig from mare serum to colostrum, the intestinal tract, and ultimately the vascular system of the foal, is a complex process with several potential sites at which transfer could be prevented or inhibited. Some mares produce colostrum of low IgG con­ tent. McGuire et al (1977) have shown that colostrum with less than 1000 mg IgG/100 ml is often associated with substandard absorption of IgG by the foal, even when nursing is immediate and vigorous. Pre­ mature lactation can also influence colostral quality. Mares who drip colostrum for several days prior to parturition significantly deplete this material. Since colostrum is secreted only once, a steady drip from the udder over a few days reduces the amount of Ig available to the foal. Inability of the foal to ingest sufficient colostrum is an obvious cause of FPT. Some foals are too weak to nurse or are rejected by the mare. The older the foal before receiving colostrum, the poorer the chance that sufficient absorption will occur. By 24 hr, essentially no transfer will occur even if colostrum of high Ig content is provided. Finally, there are foals who nurse quickly after parturition, consume a sufficient volume of colostrum containing adequate amounts of IgG, yet fail to absorb enough. "Malabsorption" may be related to stress and perhaps mediated by adrenal hormones (Jeffcott, 1972). Of these factors, low Ig content of the colostrum is the most common cause of FPT (McGuire et al, 1977). The consequences of FPT are well recognized. Omphalophlebitis, septic arthritis, and respiratory infections are typically observed. FPT

38

LANCE E. PERRYMAN

is believed to be the underlying basis for most infections and deaths of neonatal foals (McGuire et al., 1977). In prospective studies involving two well-managed Thoroughbred brood mare operations, 10% of 87 foals tested absorbed less than 200 mg IgG/100 ml serum (FPT) and 14% absorbed from 200 to 400 mg/100 ml serum (partial failure). In­ fections requiring therapy occurred in 7 of 9 foals with FPT, 3 of 12 foals with partial FPT, and none of 66 foals absorbing 400 mg IgG/100 ml serum (McGuire et al., 1977). The prevalence of FPT in a large, well-managed Arabian brood mare operation was similar to results ob­ tained in studies of Thoroughbreds (Perryman, unpublished observa­ tions) . Management of foals with FPT requires prompt diagnosis and initiation of treatment before infections occur. Quantitative Ig data are obtained by performance of single radial immunodiffusion (McGuire et al., 1975a; Buening et al., 1977). This allows accurate quantitation of IgG concentration which correlates well with protection. Estimation of total immunoglobulin by zinc sulfate turbidity tests is a useful screening procedure in the absence of quantitative IgG data (Jeffcott, 1974c; Rumbaugh et al., 1978). Treatment of deficient foals with oral colostrum or intravenously administered plasma has been successful. Computational formulas, and methods of plasma donor selection, plasma preparation, and administration for treatment of foals have been de­ scribed (Crawford et al., 1978). /. Equine Herpes Virus I Infection of Neonatal Foals Interstitial pneumonia, lymphopenia, marked necrosis and atrophy of thymus and spleen, and increased susceptibility to bacterial infections have been observed in neonatal Thoroughbred foals. The disorder is associated with infection by herpes virus I, apparently acquired in the terminal portion of gestation (Bryans et al., 1977). It is speculated that virus-induced lymphoid necrosis results in secondary immuno­ deficiency and increased susceptibility to bacterial infections.

2.

BOVINE

a. Lethal Trait A-46 In 1970, a lethal primary immunodeficiency disorder, occurring in Black Pied Danish cattle of Friesian descent, was first described (Andresen et al., 1970; Gronborg-Pedersen, 1970). The disorder is inherited as an autosomal recessive trait (Andresen et al., 1970, 1974; Andresen, 1974). Affected calves appeared normal at birth but developed char­ acteristic skin lesions at 4 to 8 weeks of age, consisting of exanthema

IMMUNE DEFICIENCIES OF DOMESTIC ANIMALS

39

and alopecia, and parakeratosus around the mouth, eyes, and under the jaw. If not treated, calves died within 4 months of age. Gross and microscopic examination of lymphoid tissues revealed marked hypoplasia of thymus, spleen, lymph nodes, and gut-associated lymphoid tissues with depletion of lymphocytes in the thymic-dependent regions. Susceptibility to infection was greater than for nonaffected calves, sug­ gesting a defect in host defense. Immunological studies indicated that cellular immunity was significantly decreased, while the levels of each of the Ig classes, and antibody response following immunization were relatively normal (Flagstad et al., 1972; Brummerstedt et al., 1971). Affected calves formed near-normal levels of antibody to tetanus toxoid but responded weakly or negatively in delayed hypersensitivity skin tests using Mycobacterium tuberculosis and DNCB. Ability to respond to Fasciola hepatica larvae was also depressed, compared to normal calves. An intriguing feature of lethal trait A-46 is its association with zinc. Affected calves treated orally with zinc oxide recovered fully as evi­ denced by resolution of skin lesions, normal development of lymphoid tissues, recovery from infections, and ability to respond to DNCB and Fasciola hepatica larvae (Brummerstedt et al., 1971; Flagstadt et al., 1972). Based on the pattern of inheritance, impairment of cellular immunity, and response to zinc, the disorder was classified as a primary immunodeficiency disorder influencing T lymphocytes. It was speculated that calves homozygous for the recessive gene have an unusually high requirement for zinc ions which are needed to sustain normal develop­ ment and function of T lymphocytes (Andresen et al, 1973). b. Selective IgG-2

Deficiency

The major Ig classes of cattle are IgGi, IgG 2 , IgM, and IgA. Mansa (1965) described the absence of IgG 2 in cattle of the Red Danish Milk Breed. Twenty-two of 780 animals tested were deficient and an ad­ ditional 107 cattle had subnormal levels of IgG 2 . Cattle with selective IgG 2 deficiency were more susceptible to gangrenous mastitis (Kulkarni, 1971) and pyogenic bacterial infections including bronchopneumonia, peritonitis, and abomasoenteritis (Nansen, 1972). This condition appears to be a primary immunodeficiency disorder with failure or marked reduction of IgG 2 synthesis, rather than excessive catabolism of IgG 2 (Nansen, 1970). c. Failure of Passive

Transfer

The process by which maternal immunoglobulin is transferred to calves and the implications of failure of this process have been exten­ sively described and reviewed (e.g., see Simpson-Morgan and Smeaton,

40

LANCE E. PERRYMAN

1972; Jeffcott, 1972; Stormont, 1972). Many of the comments made in the section on FPT in foals were first described in calves. The fetal calf develops within a protected environment and, at birth, is exposed to massive challenge with pathogenic microorganisms. Although immunocompetent at birth (Osburn et al, 1974; Renshaw et al, 1977a), several days to weeks are required for the development of a protective immune response and, in the interim, passively acquired maternal antibody is necessary for protection. The cotyledonary epitheliochorial type of placentation of the bovine prevents transfer öf immunoglobulin in utero. At birth the calf is hypogammaglobulinemic but usually absorbs protective amounts of colostral Ig by 24 hr of age (Klaus et al, 1969; Porter, 1972; Logan, 1974). Failure to ingest and absorb sufficient colostral Ig often results in neonatal septicemia or diarrhea (Smith and Little, 1922; Gay et al, 1965; McEwan et al., 1970; Penhaie et al, 1970, 1973; Fey, 1971; Logan and Penhale, 1971; Selman et al, 1971; Boyd, 1972; Boyd et al, 1974; Logan et al, 1974; McGuire et al, 1976d; Johnston et al, 1977). Not only does colostrum provide neutralizing antibodies to microorganisms, it is also necessary for normal phagocytic activity by leukocytes of newborn calves (Renshaw et al., 1976). The mechanisms by which calves fail to obtain sufficient colostral Ig are similar to those described earlier for foals: insufficient colostrum ingested, delayed suckling, and defective intestinal absorption (Klaus et al, 1969). Management of calves with FPT depends on early diagnosis. Four procedures have been used to assess the adequacy of Ig absorption in calves. Single radial immunodiffusion, zinc sulfate turbidity, serum electrophoresis, and refractometry have been compared by Pfeiffer et al (1977), Naylor and Kronfeld (1977), and Naylor et al (1977). Both groups concluded that zinc sulfate turbidity tests and single radial immunodiffusion were valid procedures for assaying Ig absorption. The validity of total serum protein determination by refractometry for this assessment is controversial. Recently an accurate objective field test based on sodium sulfite precipitation has been recommended (Pfeiffer and McGuire, 1977). d. Immunosuppression

Secondary to Microbial

Infections

Suppression of lymphocyte function occurs in chronic bovine virus diarrhea (BVD) infection of calves. The suppression appears to influence both T and B lymphocytes since lymphocyte response to PHA stimu­ lation is inhibited by BVD virus (Muscoplat et al, 1973a) and the number of peripheral blood lymphocytes with surface immunoglobulin

IMMUNE DEFICIENCIES OF DOMESTIC ANIMALS

41

is significantly decreased (Muscoplat et al., 1973b). The action of BVD virus on the lymphoid system may favor the development of the chronic BVD syndrome. Suppression of cell-mediated but not humoral immunity in cattle with Johne's Disease {Mycobacterium johnei) was described by Davies et al. (1974). Failure to develop delayed hypersensitivity skin reactions to tuberculin and inhibition of lymphocyte response to PHA stimulation were associated with a humoral suppressive factor. The factor was able to inhibit PHA stimulation of lymphocytes from normal cattle as well. A serum suppressive factor was also detected in a bull with deficient cellular immunity and persistent papillomatosis. Bacterial septicemia apparently resulted in secondary immunodeficiency with inability to resolve multiple papillomas (Duncan et al., 1975). e. Bovine

Leukosis

Abnormal numbers of lymphocytes bearing surface immunoglobulin have been observed in cattle with adult enzootic leukosis. Contrary to the adult disease, two calves with acute lymphocytic leukemia studied by Muscoplat et al. (1974a) had fewer than 1% peripheral blood lymphocytes with surface Ig. Furthermore, lymphocytes from these calves were poorly responsive to PHA and PWM stimulation. Lympho­ cytes from cattle with the adult form of the disease have a normal lymphoproliferative response to mitogens and common viral antigens (R. D. Schultz, personal communication). The same authors (Muscoplat et al., 1974b) studied four persistent lymphocytotic cows and found that nearly two-thirds of the peripheral blood lymphocytes had surface immunoglobulin. Pierce et al. (1977) quantitated IgM and IgG levels in persistent lymphocytotic cows and found no consistent deviations from normal. Bovine leukemia virus apparently infects B lymphocytes (Paul et al., 1977). Whether this increases the number of B lympho­ cytes or the increase in the cells with surface Ig is the result of adsorp­ tion of Ig to lymphocyte surface remains to be shown. /. Chediak-Higashi

Syndrome

The disorders discussed have dealt with impairment of lymphocyte activity, or a failure of the newborn to obtain passive immunity. The Chediak-Higashi syndrome (C-HS) differs in that the deficiency in host defense involves neutrophil and monocyte dysfunction (Gallin et al., 1975; Renshaw et al., 1974). C-HS is an inherited disorder of cattle, mink, cats, mice, killer whales, and man (reviewed in Prieur and Col­ lier, 1978). The clinical manifestations include partial oculocutaneous albinism, photophobia, hemorrhagic tendency, and increased suscepti-

42

LANCE E. PERRYMAN

bility to bacterial infections. C-HS in cattle occurs in Herefords which are easily recognized by the dilution of coat color and the presence of large cytoplasmic granules within neutrophils and several other cell types (Padgett et al, 1964). The basis for the increased susceptibility to pyogenic infections in cattle with C-HS has been investigated by Renshaw et al. (1974) who found the ability of neutrophils to ingest bacteria was normal. Intracellular bacterial killing, however, was abnormal, occurring at a slower rate than in normal neutrophils. Neutrophils from C-HS cattle were able to reduce nitroblue tetrazolium dye but exhibited reduced hexose monophosphate shunt activity. There was also a delay or failure of primary granules to degranulate. The alteration of hexose monophos­ phate shunt pathway activity and delayed postphagocytic degranulation appeared to account for increased susceptibility to pyogenic bac­ terial infection (Renshaw et al., 1974). 3. OVINE

Information on primary immunodeficiency disorders of sheep is limited. A transient IgG 2 deficiency has been reported in neonatal lambs (Varela-Diaz and Soulsby, 1972). Onset of synthesis depended on whether lambs had ingested colostrum. IgG 2 synthesis was delayed to 5 to 6 weeks of age in lambs fed colostrum. In contrast to IgG 2 de­ ficiency of Red Danish Milkbreed Cattle, transient IgG 2 deficiency is apparently not associated with increased disease susceptibility in lambs. The importance of passive immunity in newborn lambs has been documented (Campbell, 1974; Findlay, 1973; Halliday, 1968; Sawyer et al., 1977). Placentation is of the cotyledonary epitheliochorial type and transplacental Ig transfer does not occur. Absorption of colostrumderived Ig is complete by 24 hr after birth (reviewed in Jeffcott, 1972). As for cattle, but in contrast to horses, there was no correlation of absorption with colostral Ig content, indicating events at the level of the neonatal intestine are the primary factors controlling passive transfer. Sawyer et al. (1977) found that 14% of lambs tested failed to absorb normal amounts of maternal Ig. Fatalities in neonatal lambs were heavily weighted toward the group with subnormal absorption. 4. PORCINE

No primary immunodeficiency disorders of pigs have been described. Secondary deficiency resulting from failure of passive transfer is a significant problem. The mechanisms of passive Ig transfer from

IMMUNE DEFICIENCIES OF DOMESTIC ANIMALS

43

colostrum and milk to the piglet, specificity and efficiency of absorption, and the relative roles played by absorbed versus local passive Ig in the gut lumen in defense against colibacillosis, transmissible gastroenteritis, and other diseases have been studied extensively (e.g., see Shreeve and Thomlinson, 1971; Scoot et al, 1972; Martinsson, 1972; Jönsonn, 1973; Brandenburg and Wilson, 1973; Martinsson and Jönsson, 1975, 1976; Stone et al, 1977; Chidlow and Porter, 1977) and reviewed (Porter and Allen, 1972; Jeffcott, 1972; Bourne, 1973; Wilson, 1974). The fetal pig receives essentially no maternal Ig in utero because of the diffuse epitheliochorial type of placentation. Colostral Ig is derived from sow serum and is nonselectively absorbed by the piglet up to 24 hr of age. As colostrum is replaced by milk, the derivation of Ig changes. The vast majority of milk Ig is produced and secreted by the mammary gland, is not absorbed, but provides significant local protection within the gut lumen.

5. CANINE

a. Primary Deficiency

Disorders

Schultz (1974) has developed a systematic approach to the diagnosis of immunologie disorders in the dog. While no primary immunodeficiency disorders have been definitively characterized, a study by Farrow et al. (1972) suggests one disorder may exist. Six male miniature Dachshunds between 9 and 12 months, of age developed fatal pneumonia caused by Pneumocystis carinii. Pedigrees available for five of the dogs indicated a close relationship. Since Pneumocytis carinii is usually limited to immunosuppressed or immunodeficient individuals and all cases were seen in closely related male dogs, it is tempting to hypothe­ size a primary immune defense disorder with a sex-linked mode of in­ heritance. Without presenting data, the authors stated that preliminary studies indicated deficient lymphocyte function in the dogs. Further re­ ports of this interesting disorder have not been seen. b. Secondary Immunodeficiency

Disorders

Secondary suppression of immune responsiveness has been reported in three groups of diseases: neoplasia (MacEwen and Hurvitz, 1977), canine distemper, and generalized demodicosis. Demodex canis is present in the skin of most dogs and is associated with localized and generalized demodectic mange. Factors which de­ termine the extent of the dermatitis and response to therapy are specu­ lative. Scott et al. (1974) hypothesized that at least one factor favoring

44

LANCE E. PERRYMAN

generalized demodicosis was suppression of T-lymphocyte function. To test this, lymphocytes from affected dogs were stimulated in vitro with PHA and the response compared with lymphocytes from normal dogs. Lymphocytes from dogs with generalized demodicosis were poorly re­ sponsive or nonresponsive to PHA if cultured in autologous serum (Hirsh et al., 1975). However, if washed and cultured in the presence of normal dog serum, full response to PHA was observed. Similarly, normal dog lymphocytes were markedly suppressed if cultured with serum from dogs with generalized demodicosis. The presence of a serum suppressive factor was also shown in nearly identical studies by Corbett et al. (1975) and Scott et al. (1976). In the study of Scott et al. (1976) only a limited number of dogs had the serum suppressive factor. Immunosuppression has been associated with canine distemper virus infection. While the basis of this suppression is not completely defined, infection of gnotobiotic dogs results in absolute lymphopenia, thymic atrophy, and generalized lymphoid depletion (McCullough et al., 1974; Krakowka et al., 1975). Krakowka and Koestner (1977) showed the lymphoid depletion was nonselective in that B lymphocytes and T lym­ phocytes were equally affected. The lack of involvement of adrenal corticosteroids (Jacoby and Griesemer, 1970) and the presence of viral antigens in lymphoid tissues and leukocytes at the time of lympho­ penia (Krakowka et al., 1975) suggest a direct effect of virus on lymphoid cells. Experiments evaluating the severity of immunosuppression in gnoto­ biotic dogs infected with canine distemper virus have been performed (Krakowka et al., 1975). Ability of peripheral blood lymphocytes to respond in vitro to PHA stimulation was compared with the ability of the same dogs to reject allogeneic skin grafts. PHA responsiveness was markedly reduced at the onset of lymphopenia and persisted well after peripheral blood lymphocytes returned to normal levels. On the other hand, no significant reduction in capacity to reject skin allografts was observed. Skin allografts represent a strong antigenic stimulus and normal rejection indicates less than complete suppression of immune reactions during the course of canine distemper virus infection. How­ ever, the level of suppression detected by in vitro PHA tests may be sufficient to increase susceptibility to secondary bacterial infections in conventional dogs with canine distemper virus infection. c. Neutrophil Disorders of Dogs Two disorders involving neutrophils and resulting in heightened sus­ ceptibility to bacterial infections have been described. Lund et al. (1967) described a condition in grey collies characterized by regular cyclic

I M M U N E DEFICIENCIES OF DOMESTIC ANIMALS

45

fluctuations of peripheral blood neutrophils and termed the disorder cyclic neutropenia after a similar disorder of man. Others have shown that the cyclic fluctuations involve all the cellular elements of blood and prefer the term cyclic hematopoiesis (Dale et al, 1972; Jones et al, 1975). The disorder is inherited as an autosomal recessive trait. While all blood cellular elements fluctuate, it is the level of neutrophils which determines the defense status of the dog. The cycles generally occur every 10 to 12 days and the neutropenia lasts 2 to 4 days during which time fever, anorexia, and life-threatening bacterial infections frequently occur. The defect appears to be operative at the level of hematopoietic stem cells. Bone marrow transplantation experiments have shown that the disorder can be corrected by marrow transplants from normal histocompatible dogs. Furthermore, cyclic neutropenia can be induced by transplantation of marrow from affected to normal dogs (Weiden et al., 1974; Jones et al, 1975). The second disorder involving neutrophil function has been termed canine granulocytopathy syndrome (CGS) (Renshaw et al., 1975, 1977b). The disorder was recognized and studied in a male Irish Setter dog with marked neutrophilia, and recurrent life-threatening bacterial infections. Increased susceptibility was associated with a defect in the ability to kill phagocytized bacteria. Preliminary breeding trials suggest a genetic basis with an autosomal mode of inheritance (Renshaw et al, 1977b).

6. FELINE

a. Primary Immunodeficiency

Disorders

Primary immunodeficiency disorders have not been recognized in domestic cats. Thymic hypoplasia and lymphopenia has been described in a Siberian tiger (DeMartini, 1974). Thymic hypoplasia in young Burmese kittens has allegedly been observed but not reported. b. Secondary Immunodeficiency

Disorders

Secondary immunosuppression associated with feline leukemia virus (FeLV) infection is a well-recognized entity (Anderson et al., 1971 ; Perryman et al, 1972; Essex et al, 1975; Cotter et al, 1975; Mackey, 1975). Natural and experimental infection of kittens results in thymic atrophy, lymphoid depletion, and increased susceptibility to infection (Anderson et al, 1971; Hoover et al, 1972). Thymic atrophy is associ­ ated with depressed cell-mediated immunity and precedes the appearance of neoplastic lymphocytes by several weeks (Perryman et al, 1972;

46

LANCE E. PERRYMAN

Hoover et al., 1973). Within 5 weeks of infection, the ability of kittens to reject allogeneic skin grafts was markedly depressed (Perryman et aL, 1972), thymic atrophy was apparent (Perryman et al., 1972; Hoover et al., 1973), and in vitro lymphocyte response to Con A was reduced (Cockerell et al, 1976b). Evidence to explain the mechanism of immunosuppression in FeLV infection has recently emerged. Olsen et al. (1977) showed that im­ munization of kittens with killed FeLV actually increased susceptibility to feline oncornavirus disease and suggested the virus may be directly suppressive. In subsequent experiments, these workers showed that inactivated FeLV inhibited Con A stimulation of feline lymphocytes in vitro (Hebebrand et al., 1977). Low-molecular weight virion polypeptides, particularly pl5e, were found to be responsible for this suppression of lymphocyte function (Mathes et al., 1979). Feline panleukopenia virus causes severe thymic atrophy and mor­ phologic changes in secondary lymphoid tissue including lymphoid hypoplasia and atrophy. Functional changes were observed which were more suppressive for T-cell functions than B cells (Schultz et al., 1976). c. Chediak-Higashi

Syndrome of Cats

Kramer et al. (1977) described six "blue smoke" Persian cats with partial oculocutaneous albinism and enlarged cytoplasmic granules with­ in neutrophils, eosinophils, and basophils. Enlarged melanin granules were observed in hair and skin. Preliminary breeding trials indicated an autosomal mode of inheritance. Affected cats have a bleeding tendency, but increased susceptibility to infection has not been observed.

VI. Summary Abnormalities of lymphocytes, phagocytic cells, and complement sys­ tem components increase susceptibility to infection and often lead to death. Frequently the focus of attention is on the diagnosis and treat­ ment of the secondary infection and the question "What underlying mechanism was responsible for this infection?" is not asked. A poten­ tially large number of host defense deficiencies are not recognized for this reason. Examination of the list of references will reveal the majority have been published since 1970. This undoubtedly reflects the explosion of knowledge in basic immunology and the greater availability of diagnostic reagents and tests. It also reflects increased awareness on the part of diagnosticians and investigators of the possibility of im­ munodeficiency disorders and the clinical findings which suggest their

IMMUNE DEFICIENCIES OF DOMESTIC ANIMALS

47

presence. In the next 5 years the list of disorders described here should increase significantly. The rate of recognition and quality of character­ ization can be increased by sharing reagents and collaborating with laboratories with established evaluation procedures until specific reagents for each of the domestic species are commercially available. ACKNOWLEDGMENT Work performed by the author was supported by N I H grant No. H D 08886 from the Institute for Child Health and Human Development.

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McGuire, T. C., and Poppie, M. J. (1973). Infect. Immun. 8, 272-277. McGuire, T. C., Poppie, M. J., and Banks, K. L. (1974). J. Am. Vet. M ed. Assoc. 164, 70-76. McGuire, T. C , Poppie, M. J., and Banks, K. L. (1975a). / . Am. Vet. Med. Assoc. 166, 71-75. McGuire, T. C., Banks, K. L., and Poppie, M. J. (1975b). Clin. Immunol. Immunopathol. 3, 555-566. McGuire, T. C., Banks, K. L., and Davis. W. C. (1976a). Am. J. Pathol. 84, 39-54. McGuire, T. C., Pollara. B., Moore, J. J., and Poppie, M. J. (1976b). Infect. Immun. 13, 995-997. McGuire, T. C., Banks, K. L., Evans, D. R., and Poppie. M. J. (1976c). Am. J. Vet. Res. 37, 41-46. McGuire, T. C., Pfeiffer, N. E., Weikel, J. M., and Bartsch, R. C. (1976d). J. Am. Vet. Med. Assoc. 169, 713-718. McGuire, T. C , Crawford, T. B , Hallowell, A. L., and Macomber, L. E. (1977). J. Am. Vet. Med. Assoc. 170, 1302-1304. Mackey, L. (1975). Vet. Rec. 96, 5-11. Mackey, L., Jarrett, W., Jarrett, O., and Wilson, L. (1975). / . Natl. Cancer Inst. 54, 1483-1487. Magnuson, N. S. (1978). Thesis, Washington State Univ., Pullman. Magnuson, N . S., and Perryman, L. E. (1979). / . Clin. Invest. 64, 89-101. Mansa, B. (1965). Acta Pathol. Microbiol. Scand. 63, 153-158. Martinsson, K. (1972). Acta Vet. Scand. 13, 191-196. Martinsson, K., and Jönsson, L. (1975). Zentralbl. Vetèrinaermed., Reihe A 22, 276-282. Martinsson, K., and Jönsson, L. (1976). Zentralbl. Vetèrinaermed., Reihe A 23, 277-282. Mathes, L. E., Olsen, R. G., Hebebrand, L. C , Hoover, E. A., and Schaller, J. P. (1978). Nature {London) 274, 687-689. Micklem, H. S., and Loutit, J. F. (1966). "Tissue Grafting and Radiation." Academic Press, New York. Moore, M. A. S., and Metcalf, D. (1970). Br. J. Hacmatol. 18, 279-296. Müller-Eberhard, H. J. (1975). Annu. Rev. Biochem. 44, 697-724. Muscoplat, C. C., Johnson, D. W., and Stevens, J. B. (1973a). Am. J. Vet. Res. 34, 753-755. Muscoplat, C. C., Johnson, D. W., and Teuscher, E. (1973b). Am. J. Vet. Res. 34, 1101-1104. Muscoplat, C. C., Johnson, D. W., Pomeroy, K. A., Olson, J. M., Larson, V. L., Stevens, J. B., and Sorensen, D. K. (1974a). Am. J. Vet. Res. 35, 1571-1573. Muscoplat, C. C , Johnson, D. W., Pomeroy, K. A., Olson, J. M., Larson, V. L., Stevens, J.B., and Sorensen, D. K. (1974b). Am. J. Vet. Res. 35, 593-595. Nansen, P. (1970). Thesis, Munksgaard, Copenhagen. Nansen, P . (1972). Acta Pathol. Microbiol. Scand., Sect. B 80, 49-54 Naylor, J. M., and Kronfeld, D. S. (1977). Am. J. Vet. Res. 38, 1331-1334. Naylor, J. M., Kronfeld, D. S., Bech-Nielsen, S., and Batholomew, R. C. (1977). / . Am. Vet. Med. Assoc. Ill, 635-638. Olsen, R. G., Hoover, E. A., Schaller, J. P., Mathes, L. E., and Wolff, L. H. (1977). Cancer Res. 37, 2082-2085. Onions, D. E. (1975). Vet. Rec. 97, 108. Osburn, B. I., Stabenfeldt, G. H., Ardans, A. A., Trees, C , and Sawyer, M. (1974). J. Am. Vet. Med. Assoc. 164, 295-298.

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Padgett, G. A., Leader, R. W., Gorham, J. R., and O'Mary, C. C. (1964). Genetics 49, 505-512. Paul, P. S., Pomeroy, K. A., Johnson, D. W., Muscoplat, C. C , Handwerger, B. S., Saper, F . F., and Sorensen, D. K. (1977). Am. J. Vet. Res. 38, 873-876. Penhale, W. J., Christie, G., McEwan, H. D., Fisher, E. W., and Salman, I. E. (1970). Br. Vet. J. 126, 30-37. Penhale, W. J., Logan, E. F., Selman, I. E., Fisher, E. W., and McEwan, A. D. (1973). Ann. Rech. Vet. 4, 223-233. Perryman, L. E.. and McGuire, T. C. (1978). Transplantation 25, 50-52. Perryman, L. E., McGuire, T. C , Banks, K. L., and Henson, J. B. (1971). J. Immunol. 106, 1074-1078. Perryman, L. E., Hoover, E. A., and Yohn, D. S. (1972). J. Natl. Cancer Inst. 49, 1357-1365. Perryman, L. E., McGuire, T. C , and Hubert, B. J. (1977). J. Am. Vet.. Med. Assoc. 170, 212-215. Perryman, L. E., McGuire, T. C , Poppie, M. J., and Banks, K. L. (1978a). Proc. Int. Conf. Equine Infect. Dis., l^ih, J. Equine Med. Surg., Suppl. 1, 279^-286. Perryman, L. E., McGuire, T. C , and Crawford, T. B. (1978b). Am. J. Vet. Res. 39, 1043-1047. Perryman, L. E., Buening, G. M., McGuire, T. C , Torbeck, R. L., and Poppie, M. J. (1979.) Clin. Immunol. Immunopathol., 12, 238-251. Pfeiffer, N . E., and McGuire, T. C. (1977). J. Am. Vet. Med. Assoc. 170, 809-811. Pfeiffer, N . E., McGuire, T. C , Bendel, R. B., and Weikel, J. M. (1977). Am. J. Vet. Res. 38, 693-698. Pierce, K. R., Young, M. F., McArthur, N . H., and Williams, J. D . (1977). Am. J. Vet. Res. 38, 771-774. Poppie, M. J., and McGuire, T. C. (1976). Vet. Rec. 99, 44-46. Poppie, M. J., and McGuire, T. C. (1977). J. Am. Vet. Med. Assoc. 170, 31-33. Porter, P . I. (1972). Immunology 23, 225-238. Porter, P., and Allen, W. D. (1972). J. Am. Vet. Med. Assoc. 160, 511-518. Prieur, D. J., and Collier, L. L. (1978). Am. J. Pathol. 90, 533-536. Rabellino, E., Colon,'S., Grey, H. M., and Unanue, E. R. (1971). J. Exp. Med. 133, 156-167. Reeves, J. H., and Renshaw, H. W. (1978). Am. J. Vet. Res. 39, 917-923. Renshaw, H. W., Davis, W. C , Fudenberg, H. H., and Padgett, G. A. (1974). Infect. Immun. 10, 928-937. Renshaw, H. W., Chatburn, C , Bryan, G. M., Bartsch, R. C , and Davis, W. C. (1975). J. Am. Vet. Med. Assoc. 166, 443-447. Renshaw, H. W., Eckblad, W. P., Thacker, D. L., and Frank, F . W. (1976). Am. J. Vet. Res. 37, 1267-1274. Renshaw, H. W., Eckblad, W. P., Everson, D. 0 „ Tassinari, P. D., and Amos, D. (1977a). Am. J. Vet. Res. 38, 1141-1150. Renshaw, H. W., Davis, W. C , and Renshaw, S. J. (1977b). Clin. Immunol. Immunopathol. 8, 385-395. Rouse, B. T., and Ingram, D. G. (1970). Immunology 19, 901-907. Ruddy, S., Gigli, I., and Austen, K. F . (1972). N. Engl. J. Med. 287, 489-495, 545549, 592-596, 642-646. Rumbaugh, G. E., Ardans, A. A., Ginno, D., and Trommershausen-Smith, A. (1978). J. Am. Vet. Med. Assoc. 172, 321-325. Sawyer M., Willadsen, C. H., Osburn, B. I., and McGuire, T. C. (1977). / . Am. Vet. Med. Assoc. 171, 1255-1259.

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Schultz, R. D. (1974). Vet. Clin. North Am. 4, 153-174. Schultz, R. D., Mendel, H., and Scott, F. W. (1976). Cornell Vet. 66, 324. Scoot, A., Owen, B. D., and Agar, J. L. (1972). J. Anim. Sei. 35, 1201-1206. Scott, D. W., Farrow, B. R., and Schultz, R. D. (1974). / . Am. Anim. Hosp. Assoc. 10, 233. Scott, D. W., Schultz, R. D., and Baker, E. (1976). «7. Am. Anim. Hosp. Assoc. 12, 203. Selman, I. E., de la Fuente, G. H , Fisher, E. W., and McEwan, A. D. (1971). Vet. Rec. 88, 460-464. Shreeve, B. J., and Thomlinson, J. R. (1971). J. Med. Microbiol. 4, 461-466. Simpson-Morgan, M. W., and Smeaton, T. C. (1972). Adv. Vet. Sei. Comp. Med. 16, 355-386. Smith, R , and Little, R. (1922). / . Exp. Med. 36, 181-198. Snyder, S. P., England, J. J., and McChesney, A. E. (1978). Vet. Pathol. 15, 12-17. Stone, S. S., Kemeny, L. J., Woods, R. D., and Jensen, M. T. (1977). J. Am. Vet. Med. Assoc. 38, 1285-1288. Stormont, C. (1972). J. Anim. Sei. 35, 1275-1279. Symons, D. B. A., and Binns. R. M. (1975). Int. Arch. Allergy Appl. Immunol. 49, 658-669. Tarr, M. J., Olsen, R. G., Krakowka, S., Cockerell, G. L., and Gabel, A. A. (1977). Am. J. Vet. Res. 38, 1775-1779. Taylor, D., Hokama, Y., and Perri, S. F. (1975). / . Immunol. 115, 862-865. Thompson, D. B., Studdert, M. J., Beilharz, R. G., and Littlejohns, I. R. (1975). Aust. Vet. J. 51, 109-113. Thompson, D. B., Spradbrow, P. B., and Studdert, M. J. (1976). Aust. Vet. J. 52, 435-437. Trainin. N., and Small, M. (1970). J. Exp. Med. 132, 885-897. Varela-Diaz, V. M , and Soulsby, E. J. L. (1972). Res. Vet. Sei. 13, 99-100. Weiden, P. L., Robinett. B., Graham, T. C.. Adamson, J., and Storb, R. (1974). J. Clin. Invest. 53, 950-953. Weiland, F., and Straub, O. C. (1975). Res. Vet. Sei. 19, 100-102. Wilson, M. R. (1974). J. Anim. Sei. 38, 1018-1021. Winchester, R. J., Fu, S. M., Hoffman, T., and Kunkel, H. G. (1975). / . Immunol 114, 1210-1212.

ADVANCES IN VETERINARY SCIENCE AND COMPARATIVE MEDICINE, VOL. 2 3

Mechanisms of Immunity in Bacterial Infections A. J. WINTER Department of Clinical Sciences New York State College of Veterinary Medicine Cornell University Ithaca, New York

I. II.

III.

Parasitism and Pathogenicity Mechanisms of Immunity 1. Opsonization 2. Cellular Immunity 3. Complement-Dependent Phenomena. Bactericidal and Bacteriolytic Reactions 4. Complement-Independent Phenomena a. Toxin neutralization b. Inhibition of adherence c. Immobilization Antigenic Variation in Bacteria—Occurrence and Possible Contribution to Disease References

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I. Parasitism and Pathogenicity A general classification of pathogens will facilitate an understanding of the diverse immunity mechanisms brought to bear upon different microbes. On the basis of their nutritional requirements, biological or­ ganisms are either autotrophic or heterotrophic, and heterotrophic prokaryotes are divisible into saprophytes and parasites. Saprophytes subsist on nonliving organic material, whereas parasites require organic matter from a living microorganism (Moulder, 1962). The essence of parasitism, therefore, is a dependency for subsistence on another living organism and is unrelated to possible harmful effects produced by the parasite upon its host (e.g., pathogenicity). Thus, a parasite may live in a mutually beneficial relationship with its host (mutualist), in a

53 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-039223-2

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relationship not harmful to the host (commensal), or in a fashion pro­ ducing harmful effects on the host (pathogen).* Theobald Smith (1934) has pointed out clearly that the natural tendency in a host-parasite relationship is toward mutual toleration, and that mutualists are the most successful parasites. Pathogenicity is therefore not an essential feature of parasitism, although in many instances disease of the host is required for propagation of the parasite.f However, a majority of fatal infectious diseases afford no survival value to the parasite and result from infections produced in unnatural hosts (rabies) or by sapro­ phytes functioning as facultative parasites (tetanus). The adaptation of pathogens to survival in phagocytes is of crucial importance in determining the kind of immune response that is decisive in conferring protection. Obligate extracellular parasites, such as Streptococcus, Meningococcus, Hemophilus, Neisseria, and Klebsiella, fail to survive in phagocytes, whereas many types of facultative or obligate intracellular parasites can do so. Representative genera in the latter category include Mycobacterium, Bruceila, Listeria, Yersinia, Francisella, and some species of Salmonella and Pasteurella. Clearly, survival of obligate extracellular pathogens requires evasion of uptake by phagocytes, whereas survival of intracellular pathogens requires evasion of killing within phagocytes. There is compelling evidence derived from individuals with spontaneously occurring immunodeficiency diseases and from experimental systems (Good, 1970; Mackaness, 1971; Allison, 1974; McGregor and Kostiala, 1976) that protective immunity toward obligate extracellular pathogens is mediated through the humoral immune sys­ tem whereas cell-mediated immunity, sometimes in conjunction with humoral immunity, is essential to combat intracellular pathogens. It is also clear that neutrophils are of crucial importance in the destruc­ tion of obligate extracellular pathogens, whereas macrophages are required to control or eliminate diseases caused by intracellular pathogens. * Usage of these terms varies, unfortunately. For example, "symbiont" has been used synonymously either with "parasite" or "mutualist" as defined above. The term "parasite" has also been employed as a synonym for "pathogen," a misleading and inappropriate usage. Another common error is the designation of mutualists or commensals as "saprophytes." In this connection it is well known that certain sapro­ phytes (Pscudomonas, Proteus, Clostridium) are facultative parasites, and in their parasitic mode may be dangerous pathogens. t For example, diseases of secretory organs, such as enteritis, rhinitis, and abortion, are commonly associated with transmission of microbes.

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II. Mechanisms of Immunity 1. OPSONIZATION

Bacteria have evolved a variety of surface structures that prevent phagocytosis, and in many obligate extracellular species, classically the streptococci, these are essential for invasiveness and disease pro­ duction. Antiphagocytic structures are generally capsules or microcapsules (Luderitz et al., 1968; Smith, 1977), although the M protein of group A streptococci is a pilus component (Swanson et al., 1969), and pili of Neisseria gonorrheae may be antiphagocytic (Ofek et al., 1974; Punsalang and Sawyer, 1973; Smith, 1977). The 0 antigens of the cell wall of gram-negative species have also been shown to possess antiphagocytic properties (Medearis et al., 1968; Cunningham et al., 1975). Most antiphagocytic structures are proteins, glycoproteins, or polysaccharides (Luderitz et al., 1968; Smith, 1977), which, by reason of their negative charge, confer upon the microbe hydrophilic surface properties. Van Oss and his colleagues (van Oss and Gillman, 1972; Cunningham et al., 1975) have demonstrated that phagocytosis of a particle fails to occur when its surface is more hydrophilic than that of the phagocyte, and they propose that surface charge has a critical role in evasion of phagocytosis. Opsonization of bacteria is mediated principally through specific IgG antibodies or complement factors C3b and C4b (Stossel, 1974; Cooper, 1976). The latter may be triggered through either the classical or the alternate pathway. It has been established that neutrophils and macro­ phages bear receptors specific for the Fc fragment of certain IgG sub­ classes and for C3b and C4b, and that opsonization requires interaction of antigen complexed to antibody or complement factors with these receptors (Huber and Fudenberg, 1968; Stossel, 1974; Cooper, 1976). IgG opsonins may also promote phagocytosis by fixing complement through the classical pathway in that it has been observed in several species that the same subclasses subserve both functions (Huber and Fudenberg, 1968). In contrast, IgM antibodies are much more effective than IgG in fixing complement on the surface of particulate antigens (Borsos and Rapp, 1965), but fail to bind to Fc receptors (Huber and Fudenberg, 1968). Opsonization of certain bacterial species (Winkelstein et al., 1972; Jasin, 1972; Forsgren and Quie, 1974) can occur without the participa­ tion of specific antibody, through activation of the alternate pathway of complement fixation. This has been termed the "heat labile opsonic"

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(HLO) system (Forsgren and Quie, 1974), and may be activated, for example, by aggregates of immunoglobulins, which may, but need not, constitute specific antibody (Winkelstein et al., 1972), or by the cell wall lipopolysaccharide of gram-negative organisms (Cooper, 1976). Of the multitude of biologic functions attributed to the complement system, opsonization is probably the single most important one. This is so because of the crucial importance of phagocytosis in control of obligate extracellular pathogens and the low degree of redundancy in fulfilling the body's requirements for opsonizing capability. It has been documented that individuals with congenital deficiencies either in the humoral immune response (Good, 1970) or in C3 (Allison, 1974; Cooper, 1976) are highly susceptible to infections with pyogenic bacteria, in­ dicating that C3 as well as antibodies constitute essential defense mechanisms against these pathogens. The mechanisms whereby opsonins promote phagocytosis are incom­ pletely understood, and it must be recognized that binding and internalization may be separate phenomena. For example, Scribner and Fahrney (1976) presented evidence that attachment of Staphylococcus aureus to human neutrophils through C3b (immune adherence) did not lead to internalization, whereas attachment through IgG antibodies (opsonic adherence) did so. Recent evidence has suggested that one essential function of opsonins, which may influence both attachment and internalization, is to increase the hydrophobic properties of the bacterial surface. Van Oss and co-workers (1974) and Stendahl et al. (1977) have demonstrated that both hydrophobicity of microbial surfaces and phagocytosis are increased by attached antibodies or complement factors. Stendahl et al. (1977) have calculated further that the minimum num­ ber of IgG antibodies required to produce phagocytosis of smooth-phase S. typhimurimn (ca. 8000 molecules per bacterium, in vitro) was about the same as tY^t required to produce a significant increase in hydro­ phobicity. r ine hydrophobic character of such cells was comparable to that of readily phagocytized rough-phase variants. 2. CELLULAR IMMUNITY

It has been established (McGregor and Kostiala, 1976) that resis­ tance to intracellular parasites capable of surviving in phagocytes hinges upon a cell-mediated immune (CMI) response in which immune T cells bring about proliferation, aggregation, and activation of macrophages. The macrophage, rather than the neutrophil, is the essential effector cell required for control of intracellular pathogens. Antibodies no doubt increase resistance to some intracellular parasites, functioning as

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opsonins and/or enhancing the killing of microbes within the macro­ phage. It should be stressed that the ability of bacteria to survive within phagocytes is not an absolute quality, in that a proportion of the popula­ tion, frequently the majority, is killed (Youmans, 1975; Collins, 1974). The survivors, however, multiply within and eventually kill the cell, resulting in release of the organism and spread of infection. Pathogens, which include viruses and fungi as well as bacteria, have evolved dif­ ferent mechanisms for evading destruction within the phagocyte. Evasion mechanisms include (Smith, 1968; Jones, 1974; Armstrong and D'Arcy Hart, 1975; Chang and Dwyer, 1976) prevention of fusion of phagosome and lysosome (M. tuberculosis, Chlamydia spp., Toxoplasma gondii), induction of phagosomal lysis with release of the pathogen into the cytoplasm (vaccinia), and resistance to destruction by lysosomal constituents (M. Icpraemurium, Leishamania donovani, Bruceila, and possibly M. tuberculosis). Tuberculosis has long stood as the prototype of a disease produced by a facultative intracellular parasite, in which an initial exposure confers some degree of resistance that is unrelated to the humoral im­ mune response, even though antibodies are produced in abundance. Lurie noted many years ago the association of infectious granulomas, or "tubercles," with increased resistance and suggested that the epithelioid cells within the tubercle had a crucial role as effector cells in antitubercular immunity. He believed that the bacterium had a direct effect in conversion of macrophages to the active state required for expression of immunity (Lurie, 1964). Dannenberg (1968) has demon­ strated that epithelioid cells do indeed correspond to activated macro­ phages, and the work of Mackaness and others has modified Lurie's conclusion, in that the macrophage is activated by the other critical cell type within the tubercle, the lymphocyte. Thus, immune T cells, after contact with specific antigen to which they are sensitized, release soluble factors (lymphokines) that confer upon macrophages the ca­ pacity to kill, or suppress the multiplication of, intracellular pathogens (Mackaness, 1971). Such macrophages are "activated" and acquire distinctive morphologic and physiologic characteristics including in­ creased size and motility, a "ruffled" membrane, higher metabolic rate, larger numbers of lysosomes, and possibly an alteration in lysosomal contents (Jones, 1974; Armstrong and D'Arcy Hart, 1975). Activated macrophages are more avidly phagocytic in addition to having enhanced powers of killing or stasis. It is important to recognize that, although a specific immune response is one important means whereby macro­ phages are activated, the function of these cells is in important measure

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nonspecific. Activated macrophages will kill intracellular pathogens other than the one that stimulated their development, although killing of the original infecting pathogen is almost always more effective (Collins, 1974; Youmans, 1975). Activated macrophages are also able to distinguish and kill transformed cells (Hibbs, 1976) and may play a major role in immunity to tumors. The properties of the T cell responsible for immunity in infections with intracellular pathogens has been studied most extensively by McGregor, North, and their co-workers (McGregor and Kostiala, 1976). T effector cells are stimulated by contact with antigen, principally in the T-dependent areas of lymph nodes and spleen. Large numbers of such cells leave their sites of origin and enter the circulation. Morphologically they are immunoblasts, and, unlike other T cells, they do not recirculate through the lymphatic system. Immunoblasts, however, display a great propensity for extravasation into foci of inflammation. Although this is a nonspecific phenomenon, it doubtless represents an essential mechanism for bringing T effector cells into contact with foreign antigens. Once in tissues, immunoblasts cease dividing and differentiate into smaller cells, which within 2 to 3 days constitute the majority of T effector cells as judged not only by production of migration inhibitory factor (MIF), but through adoptive transfer of protective immunity (Kostiala et al., 1976). The fate of these T effector cells in tissues is as yet uncertain. The exact relationship between delayed hypersensitivity (DH) and protective cellular immunity remains unresolved (Collins, 1974; You­ mans, 1975; Salvin and Neta, 1975), even though the two phenomena are normally inextricably entwined. A major basis for controversy lies in the possibility, under certain experimental conditions, of dissociating DH and cellular immunity.. However, it is now recognized that under certain conditions of immunization or desensitization D H (as measured by the skin test) may fail to occur, despite the presence in particular organs of T cellp radiating both D H and cellular immunity. Neverthe­ less, it is not yet possible to conclude that these phenomena are mediated by the same T cell subgroup. A final unresolved question concerns the nature of the essential al­ teration in activated macrophages that accounts for their enhanced capacity for killing or stasis. As noted above, it must be borne in mind that T effector cells in an inflammatory site not only cause macrophage activation, but also bring about a generalized as well as a local prolifera­ tion of macrophages and cause an attraction and retention of macro­ phages at the site of inflammation (McGregor and Kostiala, 1976). Increased numbers of macrophages, even though not activated, if de-

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ployed quickly at the site of bacterial proliferation, may suffice to control certain intracellular pathogens. The presence of antibodies on the surface of certain intracellular pathogens may allow their killing by normal macrophages by allowing fusion of phagosomes and lysosomes that is otherwise prevented. When fusion fails to occur, the pathogen either persists within the phagosome (Jones, 1974) or destroys the phagosome and is released into the cyto­ plasm (Jones, 1974; Armstrong and D'Arcy Hart, 1975). In contrast, although antibodies on the surface of M. tuberculosis permit phagoly­ sosomal fusion, the organism multiplies freely in the phagolysosomes of normal macrophages (Armstrong and D'Arcy Hart, 1975). Bacteriostasis associated with resistance in tuberculosis may therefore depend upon phagolysosomal fusion in conjunction with the effects of an increased quantity of, or novel constituents in, the lysosomes of activated macro­ phages. It is equally possible that bacteriostasis is not dependent upon effects of lysosomal components, even in immune animals, but results from undefined factors preventing multiplication in the phagosome, analogous to the finding in Toxoplasma infection (Jones, 1974). In other antibacterial immunity mechanisms mediated by antibodies, CMI reactions are not involved and phagocytes do not have a primary role. These phenomena may be divided into complement-dependent and complement-independent reactions. 3. COMPLEMENT-DEPENDENT PHENOMENA BACTERICIDAL AND BACTERIOLYTIC REACTIONS

Pfeiffer observed in 1895 that Vibrio cholerae were lysed when injected intraperitoneally into previously immunized guinea pigs. It has been established that the "Pfeiffer phenomenon" is an antibody-dependent, complement-mediated reaction affecting many species of gram-negative bacteria, and that death of bacteria may occur, and more frequently does occur, without lysis (Davis et al., 1966; Wilson and Spitznagel, 1968; Donaldson et al., 1974). The sequence of events in this reaction may be compared to that involved in complement-mediated lysis of erythrocytes. O antibodies combine witli the lipopolysaccharide (LPS) of the cell wall of gram-negative species, allowing fixation of Cl and, sequentially, the remaining complement components through C9. LPS has been shown to be a substrate for complement and is subject to lesions having essentially the same appearance as those that develop in erythrocyte membranes (Bladen et al., 1967). Such lesions allow access of complement factors and other serum factors, such as beta lysin

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(Donaldson et al., 1974), to the cytoplasmic membrane, resulting in rapid loss of its selective permeability and consequent cell death even though cell morphology is retained. If lysozyme is present, depolymerization of murein occurs, with resultant spheroplast formation (Wilson and Spitznagel, 1968; Donaldson et al., 1974). These events may also be triggered through activation of the alternate pathway of comple­ ment fixation by LPS alone (Gewürz et al., 1968). Gram-positive bacteria are refractory to complement-mediated killing not because their cytoplasmic membranes are more resistant to effects of complement, but because their cell walls do not provide a substrate for complement. Thus, the cytoplasmic membrane is not exposed to serum factors even though complement is fixed on the cell surface. Classical complement-induced lesions are readily produced in cell mem­ branes of protoplasts derived from gram-positive species (Muschel and Jackson, 1966). It should be clear, moreover, that not all gram-negative species are susceptible to complement-mediated killing (Rowley, 1968). As would be expected, sensitivity of encapsulated strains is decreased in propor­ tion to the thickness of the capsule (Glynn and Howard, 1970; Howard and Glynn, 1971). However, Campy lob acter fetus} despite strong mor­ phologic resemblance to V. cholerae, is completely refractory to comple­ ment-mediated killing even in the absence of its microcapsule and with an excess of 0 antibody in the system (Corbeil et al., 1974). The reasons for such insusceptibility are unclear. A rough variant of C. fetus was readily killed in the presence of complement (Corbeil et al., 1974), and rough variants of other gram-negative species have also been found to be much more susceptible to killing (Rowley, 1968; Chédid et al., 1968). It is likely that the interaction of complement with the diminished cell walls of rough variants, which may (Rowley, 1968; Chédid et al., 1968), but need not (Dlabac, 1968), require antibodies, produces a more rapid access of complement and beta lysin to the cell membrane and therefore more efficient killing. The demonstrated effectiveness of vaccination with Re mutants of Salmonella in protecting mice against smooth strains of K. pneumoniae and Escherichia coli (McCabe, 1972) may have resulted from a complement-mediated bactericidal effect. The exposure of at least a few R groups of the LPS on the smooth-phase bacteria, as postulated by Chédid et al. (1968), would seem to be required. The actual importance of the complement-mediated bactericidal sys­ tem in protective immunity is difficult to judge. The limited data on individuals congenitally deficient in late complement components (Alli­ son, 1974; Cooper, 1976) do not suggest that this mechanism is of crucial importance in protection against bacterial infections. Still, the bacterici-

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dal reaction may provide an important contribution to resistance in some instances. For example, bacteremias of gram-negative organisms may, at least in nascent stages, be controlled by serum bactericidal reactions initiated through either classical or alternate pathways of complement fixation. IgM antibodies, known to be by far the most effective in fixing complement to particulate antigens (Borsos and Rapp, 1965), could be present early in infection as "natural" antibodies or by in­ duction in the primary immune response. Antibodies specific for the 0 side chains, core, or backbone of the LPS may participate in comple­ ment activation. There are also findings (McCutchan et al., 1976) which suggest that the serum bactericidal effect may be of importance in development of resistance to systemic infection with Neisseria gonorrheae. Finally, there are reports that secretory IgA, in the presence of lysozyme, is able to initiate a complement-dependent bactericidal effect (Hill and Porter, 1974). However, the relevance of this reaction in secretory immunity remains to be established. 4. COMPLEMENT-INDEPENDENT PHENOMENA

a. Toxin

Neutralization

Exotoxins are powerful virulence factors in many bacterial diseases, classically those produced by clostridia, corynebacteria, streptococci, and staphylococci, as well as those due to enterotoxins of V. cholerae, E. coli, and S. aureus. Neutralization of toxins constitutes a crucial mechanism of immunity in most of. these infections. The essential interaction in toxin neutralization is a primary union between toxin and antitoxin (Hammond, W. D., 1970; Humphrey and White, 1970). Complement is not required, and phagocytes only sec­ ondarily, to ingest and degrade the antigen-antibody complexes. It has been demonstrated that the antigenic determinant that combines with antitoxin corresponds in only some instances with the site com­ bining with the target cell receptor. In either case, the mechanism of action of antitoxin is to prevent sterically an effective combination of the toxin with its target cell, and it is well known that antitoxins are ineffective once toxin has attached to its target cell, nor can they reverse damage done to target cells. Although antibodies specific for toxins may be found in each of the major classes of immunoglobulins, high-affinity IgG antibodies are by far the most effective antitoxins. In fact, it has been reported that anti­ bodies able to neutralize diphtheria toxin are exclusively of the IgG class (Ourth, 1974).

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The production of antitoxins serves as an excellent demonstration of the survival value of the anamnestic response, in which a minimal dosage of toxin stimulates the formation of a large quantity of highly efficient neutralizing antibodies. b. Inhibition

of Adherence

Mucous membranes are the site of many bacterial infections and the principal portals of entry through which pathogens invade the body and produce generalized disease. Establishment of such infections re­ quires initially a colonization of bacteria on the surface of the mucous membrane, and, as a general rule, a prerequisite for colonization is the capacity of the microbe to adhere to an epithelial surface. Adherence, therefore, constitutes an essential virulence mechanism, and its pre­ vention would contribute in important measure to protective immunity (Gibbons, 1973). Adherence has been shown to occur, in various experimental systems, by pathogens affecting each of the secretory organs (Gibbons and van Houte, 1975; Smith, 1977; Frost et al, 1977). Studies with Streptococcus spp. (Gibbons and van Houte, 1975) indicate that adherence is a specific phenomenon, in which microbial surface components interact with mammalian cell receptors. This is consistent with reports that pathogens producing infections of a particular secretory organ adhere to epithelium from that site in a more effective fashion than do nonpathogens, or strains of low virulence (Frost et al., 1977; Mardh and Weström, 1976). However, such correlations do not always hold (Smith, 1977). In most instances only in vitro systems have been used to study adherence phenomena, and the difficulty of extrapolating such results to in vivo events must not be overlooked. The nature of microbial surface components functional in adherence, termed "adhesins" by Fréter and his co-workers (Jones et al., 1976), are only partially understood. The cariogenic oral pathogen S. mut ans synthesizes a water-insoluble dextran-levan polymer, which coats the tooth enamel and to which the bacterial cell binds through a specific receptor (Mukasa and Slade, 1973). Pili have been proposed as adhesins in a variety of genera including Streptococcus, Neisseria, Escherichia, Salmonella, Proteus, and Corynebacterium (Silverblatt, 1974; Gibbons and van Houte, 1975; Smith, 1977). Ellen and Gibbons (1972) originally proposed that the M pro­ tein on pili of group A streptococci functioned as the adhesin. More recent studies, however, provide strong evidence that lipoteichoic acid is also a component of the pilus and is responsible for adherence (Beachy and Ofek, 1976).

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Adherence of V. cholerae has been the subject of several recent in­ vestigations that point up the potential complexity of bacterial ad­ herence mechanisms. There appear to be at least two adhesins on V. cholerae, one reacting with a fucose-sensitive receptor on the brush border and the other with a fucrose-resistant receptor of uncertain lo­ cation (Fréter and Jones, 1976). The role of pili in adherence is unre­ solved. Adherent cells must be motile (Jones et al., 1976), yet morphologic observations indicate that adherence does not occur via flagella (Nelson et al., 1976). Possibly the biosynthesis of adhesins is closely linked with that of flagella (Jones and Fréter, 1976). Antibodies have been shown to interfere specifically with adhesion of various pathogens (Williams and Gibbons, 1972; Fréter and Jones, 1976; Frost et al., 1977). Inhibition of adherence is brought about by IgA antibodies (Williams and Gibbons, 1972), although activity is not restricted to this class (Fréter and Jones, 1976; Frost et al., 1977). Sev­ eral ideas have been proposed for the effectiveness of antibodies in pre­ venting adhesion (Fréter and Jones, 1976), and more than one mechan­ ism may be involved. It seems reasonable, however, that primary union of antibodies would suffice to block adherence and that this function would be performed principally by IgA antibodies because of their dominance in most secretions. This may well be the most important antibacterial function of IgA antibodies. Teleologically, it is a function best served by an immunoglobulin incapable of activating complement, because it can thus be accomplished effectively without stimulation of an unnecessary, and possibly harmful, inflammatory response. c.

Immobilization

Although frequently overlooked, motility is unquestionably a require­ ment for virulence in flagellated organisms in that nonmotile variants are not found in nature, and are ineffective in experimental production of disease (Guentzel and Berry, 1975). Motility may have several roles in enhancing invasiveness, including transport of the bacterium within an organ system (Schurig et al., 1974), provision of intimate contact of the bacterium with a mucous surface, thereby facilitating adherence (Guentzel and Berry, 1975), or evasion of phagocytosis (Mims, 1977). Immobilization may be brought about by antibodies in each of the major classes. IgA antibodies in the vaginal mucus of heifers con­ valescent from C. fetus infection were, on a molar basis, manyfold more effective than IgG antibodies in mediating immobilization (Corbeil et al., 1974). In bovine vibriosis (campylobacteriosis), immobilization may be one important function of secretory IgA antibodies, preventing reinvasion of the uterus during later stages of infection.

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Antibody-mediated immobilization may be the result simply of clump­ ing of flagella on a peritrichous organism, or clumping of monotrichous cells. However, the mechanism of single-cell immobilization of mono­ trichous bacteria, as observed with C. jetas (Corbeil et al., 1974; McCoy et al., 1976), is uncertain. It may be explained in part by the observa­ tion that immobilization of C. fetus is brought about by antibodies with specificities other than for the flagellar filament, including the flagellar hook and a heat-stable somatic antigen other than the 0" antigen (McCoy et al., 1976). There is also evidence that the microcapsule of C. fetus functions to prevent immobilization (McCoy et al., 1976) as well as to prevent phagocytosis (McCoy et al., 1975).

III. Antigenic Variation in Bacteria—Occurrence and Possible Contribution to Disease Variation in the surface structure of a microorganism represents a highly effective means of thwarting the immune response, in that anti­ bodies or immune T cells produced against the infecting strain become ineffective. This means of evasion of host defense mechanisms has been best documented in influenza (Kilbourne, 1973; Laver and Downie, 1974; Stuart-Harris, 1975; Fenner and White, 1976) and in protozoan blood parasitisms, including the salivarian trypanosomes (Vickerman, 1974) and the agents of malaria (Brown, 1974). Antigenic polymorphism in bacteria, including E. coli, S. pneumoniae, and Salmonella spp., is well known (Beale and Wilkinson, 1961), yet the demonstration that antigenic variations occur in vivo in persistent bacterial infections has been made in only a few instances. While it has been clearly established, for example, that 0 serotypes of E. coli in the human intestine vary over a period of months (Robinet, 1962; Cooke et al., 1969), there is at present no evidence that such variation represents a transition of one serotype to another. In a recent study Bergan and Midtvet (1975) pro­ duced stable alterations in the 0 serotype of Pseudomonas aeruginosa isolated from the feces of monocontammated rats, following the feeding of two types of bacteriophage. The authors proposed that serotype al­ terations occurred as a result of phage conversion, and they suggest that this phenomenon may account in part for the apparent serotypic lability of this organism in natural infections, Sack and Miller (1969) demonstrated in vivo alterations of V. cholerae serotypes in the intestines of monocontammated mice. Inoculation of either Inaba or Ogawa strain produced a chronic infection in which

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alteration to the opposite smooth (S) serotype began within 2 weeks. R forms appeared subsequently and were sometimes dominant during the later stages of the 3-month experimental period. Serotypic con­ version from one to the other S form was accompanied by the appear­ ance of circulating antibodies, leading the authors to propose a causal relationship. Results of vaccination and immunosuppressive treatments were consistent with this concept. The M protein of group A streptococci constitutes a crucial virulence factor, functioning to prevent phagocytosis (Fox, 1974). Many antigenic types of M protein occur, and conversion of serotypes has been noted in vitro as well as in human populations experiencing chronic infections (Lancefield and Todd, 1928; Maxted and Valkenberg, 1969). This is consistent with the possibility that such antigenic alterations occur in chronically infected individuals under the selective pressure of specific antibodies. Bratthall and Gibbons (1975a,b) believe that antibodies on mucous membranes play an important selective role in regulating indigenous bacterial flora, based on the presence in secretions of antibodies that coat indigenous bacteria, coupled with the demonstration that antibody coating inhibits adherence and thereby limits or prevents colonization. Indirect evidence for "immunological selection pressure" on indigenous bacteria was obtained by studying agglutination patterns of salivary IgA samples, obtained from 2 subjects over a period of 5 months, on stock strains of several streptococcal species. In a subsequent study (Bratthall and Gibbons, 1975b), antigenic variation was studied in oral and fecal isolates of S. mutans serotypes b and d, obtained from monocontaminated rats over a period of several months. Alterations were detected in both strain and type antigens, and prior immunization with the homologous strain appeared to increase the number and variety of variants, taken to support a role of antibodies in selection of variants. Antigenic variation in C. fetus has been observed in heifers, in that isolates differed remarkably from the infecting strain in structure and/or proportional content of heat-labile surface antigens, as measured by agglutination reactions (Schurig et al., 1973). Comparison of titers indi­ cated that no changes had occurred in the 0 antigen. It was postulated (Schurig et al., 1973) that locally produced antibodies were responsible for the emergence of antigenic variants. It was known that alteration in either 0 antigens (von Mitscherlich and Heider, 1968) or heat-labile surface antigens (Ogg and Chang, 1972) of C. fetus could be brought about in vitro by cultivation of the organism in the presence of specific antisera. Ogg and Chang (1972) provided evidence that such changes

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resulted from loss of prophage or the selection of nonlysogenized cells. Our results were consistent with these possibilities but could have been explained equally well by other types of antibody selection. In a subsequent study by Corbeil et al. (1975), in heifers infected with a cloned strain of C. fetus venerealis, changes in surface antigens (but not the 0 antigen) were suggested by large fluctuations in the agglutinability of the infecting strain by sequential samples of cervicovaginal mucus (CVM) taken over a period of 8 months postinfection (PI). Some isolates were agglutinated less well by CVM taken at the time of isolation than at later periods, suggesting a pattern similar to that in blood-borne protozoan infections. Direct evidence for antigenic variation was obtained by comparing agglutination patterns of the infecting strain with those of isolates taken at sequential periods PI, using monospeciflc rabbit antisera. The antigenic changes in isolates appeared stable, as each was subjected to 2 or 3 subcultures between isolation and cultivation for antigen in the agglutination test. Also, in contrast to the variability of isolates from infected animals, the séro­ logie reactions of strains passaged in the laboratory are highly stable. Using similar criteria, Lawson et al. (1977) have shown evidence for antigenic variation in C. sputorum subsp. mucosalis, the proposed etiologic agent of porcine intestinal adenomatosis and related enteropathies. Although in C. fetus infections antigenic variation of the bacterium could not always be correlated with antibody response or with survival in the host (Schurig et al, 1975, 1978; Bier et al, 1977), we believe that in this, and probably many other chronic bacterial infec­ tions, population changes in vivo occur because of selective pressures due to the immune response, that variants result from genotypic altera­ tion, and that antigenic variation is an adaptive mechanism important to survival. REFERENCES Allison, A. C. (1974). Transplant. Rev. 19, 3-55. Armstrong, J. A., and D'Arcy Hart, P. (1975). J. Exp. Med. 142, 1-16. Beachey, E. H.. and Ofok, I. (1976). J. Exp. Med. 143, 759-771. Beale, G. EL. and Wilkinson. J. F. (1961). Annu. Rev. Med. 15, 263-296. Bergan, F., and Midtvet, T. (1975). Acta Pat hoi. Microbiol Scand. 83, 1-9. Bier, P. J., Hall, C. E., Duncan. J. R., and Winter, A. J. (1977). Vet. Microbiol. 2, 13-27. Bladen, H. A., Gewurz, H.. and Mergenhagen, S. E. (1967). J. Exp. Med. 125, 767-786. Borsos, T.. and Rapp. H. J. (1965). Science 150, 505-506. Bratthall, D., and Gibbons, R. J. (1975a). Infect. Immun. 11, 603-606. Bratthall, D., and Gibbons, R. J. (1975b). Inject. Immun. 12, 1231-1236.

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Brown, K. N . (1974). Ciba Found. Symp. 25 (new ser.). 35-51. Chang, K. P., and Dwyer, D. M. (1976). Science 193, 678-680. Chédid, L., Parant, M., Parant, F., and Boyer, F . (1968). J. Immunol. 100, 292-301. Collins, F . M. (1974). Bacteriol. Rev. 38, 371-402. Cooke, E. M., Ewins, S , and Shooter, R. A. (1969). Br. Med. J. 4, 593-601. Cooper, N . R. (1976). In "Basic and Clinical Immunology" (H. H. Fudenberg et al., eds.), pp. 58-69. Lang Med. Publ., Los Altos, California. Corbeil, L. B., Schurig, G. D., Duncan, J. R., Corbeil, R. R., and Winter, A. J. (1974). Inject. Immun. 10, 422-429. Corbeil, L. B., Schurig, G. G. D., Bier, P., and Winter, A. J. (1975). Inject. Immun. 11, 240-244. Cunningham, R. K., Soderstrom, T. O., Gillman, C. F., and van Oss, C. J. (1975). Immunol. Commun. 4, 429-442. Dannenberg, A. M., Jr. (1968). Bactenol. Rev. 32, 85-102. Davis, S. D., Gemsa, D., and Wedgwood, R. J. (1966). J. Immunol. 96, 570-577. Dlabac, V. (1968). Folia Microbiol. (Prague) 13, 439-449. Donaldson, D. M., Roberts, R. R., Larsen, H. S., and Tew, J. G. (1974). Infect. Immun. 10, 657-666. Ellen, R. P., and Gibbons, R. J. (1972). Infect. Immun. 5, 826-830. Fenner, F., and White, D. O. (1976). "Medical Virology," 2nd ed. Academic Press, New York. Forsgren, A., and Quie, P. G. (1974). Infect. Immun. 10, 402-404. Fox, E. (1974). Bacteriol. Rev. 38, 57-86. Fréter, R., and Jones, G. W. (1976). Infect. Immun. 14, 246-256. Frost, A. J., Wanasinghe, D. D., and Woolcock, J. B. (1977). Infect. Immun. 15, 245-253. Gewürz, H., Shin, H. S., and Mergenhagen, S. E. (1968). J. Exp. Med. 128, 1049-1057. Gibbons, R. J. (1973). Rev. Microbiol. 4, 49-60. Gibbons. R. J., and van Houte, J. (1975). Annu. Rev. Microbiol. 29, 19-44. Glynn, A. A., and Howard, C. J. (1970). Immunology 18, 331-346. Good, R. A. (1970). In "Infectious Agents and Host Reactions (S. Mudd, ed.), pp. 76-114. Saunders, Philadelphia, Pennsylvania. Guentzel, M. N., and Berry, L. J. (1975). Infect. Immun. 11, 890-897. Hammond, W. D. (1970). In "Biology of the Immune Response" (P. Abramoff and M. F. LaVia, eds.), pp. 376-378. McGraw-Hill, New York. Hibbs, J. B., Jr. (1976). / . Reticuloendothel. Soc. 20, 223-231. Hill, I. R., and Porter, P. (1974). Immunology 26, 1239-1250. Howard, C. J., and Glynn, A. A. (1971). Immunology 20, 767-777. Huber, J., and Fudenberg, H. H. (1968). Int. Arch. Allergy Appl. Immunol. 34, 18-31. Humphrey, J. H., and White, R. G. (1970). "Immunology for Students of Medi­ cine," 3rd ed. Blackwell, Oxford. Jasin, H. E. (1972). J. Immunol. 109, 26-31. Jones, G. W., and Fréter, R. (1976). Infect. Immun. 14, 240-245. Jones, G. W., Abrams, G. D., and Fréter, R. (1976). Infect. Immun. 14, 232-239. Jones, T. C. (1974). / . Reticuloendothel. Soc. 15, 439-450. Kilboume, E. D. (1973). / . Infect. Dis. 127, 478-487. Kostiala, A. I., McGregor, D. D., and Lefford, M. J. (1976). Cell. Immunol. 24, 318-327. Lancefield, R. C , and Todd, E. W. (1928). J. Exp. Med. 48, 769-790.

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Laver, W. G., and Downie, J. C. (1974). Virology 59, 230-244. Lawson, G. H. K., Rowland, A. C , and Roberts. L. (1977). Res. Vet. Sei. 23, 378-382. Luderitz, 0., Jann, K., and Wheat, R. (1968). Compr. Biochem. 26A, 105-228. Lurie, M. B. (1964). "Resistance to Tuberculosis: Experimental Studies in Native and Acquired Defense Mechanisms." Harvard Univ. Press, Cambridge, Massa­ chusetts. McCabe, W. R. (1972). J. Immunol. 108, 601-610. McCoy, E. C , Doyle, D., Burda, K., Corbeil, L. B., and Winter, A. J. (1975). Infect. Immun. 11, 517-525. McCoy, E. C , Wiltberger, H. A., and Winter, A. J. (1976). Inject. Immun. 13, 1266-1272. McCutchan, J. A., Levine, S., and Brande, A. I. (1976). J. Immunol. 116, 1652-1655. McGregor, D. D., and Kostiala, A. A. I. (1976). Contemp. Top. Immunobiol. 5, 237-266. Mackaness, G. B. (1971). J. Infect. Dis. 123, 439-445. Mardh, P. A., and Weström, L. (1976). Inject. Immun. 13, 661-666. Maxted, W. R., and Valkenberg, H. A. (1969). J. M éd. Microbiol. 2, 199-210. Medearis, D. N., Camitta, B. M., and Heath, E. C. (1968). J. Exp. Med. 128, 399-414. Mims, C. A. (1977). "The Pathogenesis of Infectious Disease." Academic Press, New York. Moulder, J. W. (1962). "The Biochemistry of Intracellular Parasitism." Univ. of Chicago Press, Chicago, Illinois. Mukasa, H., and Slade, H. D. (1973). Inject. Immun. 8, 555-562. Muschel, L. H., and Jackson, J. E. (1966). J. Immunol. 97, 46-51. Nelson, E. T., Clements, J. D., and Finkelstein, R. A. (1976). Inject. Immun. 14, 527-547. Ofek, L, Beachey, E. H., and Bisno, A. L. (1974). J. Inject. Dis. 129, 310-316. Ogg, J. E., and Chang, W. (1972). Am. J. Vet. Res. 33, 1023-1029. Ourth, D. D. (1974). Immuno chemistry 11, 223-225. Punsalang, A. P., Jr., and Sawyer, W. D. (1973). Inject. Immun. 8, 255-263. Robinet, H. G. (1962). J. Bacteriol. 84, 896-901. Rowley, D. (1968). J. Bacteriol. 95, 1647-1650. Sack, R. B., and Miller, C. E. (1969). J. Bacteriol. 99, 688-695. Salvin, S. B., and Neta, R. (1975). Am. Rev. Respir. Dis. 111, 373-377. Schurig, G. D., Hall, C. E., Burda, K., Corbeil, L. B., Duncan, J. R., and Winter, A. J. (1973). Am. J. Vet. Res. 34, 1399-1403. Schurig, G. D., Hall, C. E , Burda, K., Corbeil, L. B., Duncan, J. R., and Winter, A. J. (1974). Cornell Vet. 64, 533-548. Schurig, G. G. D., Hall, C. E., Corbeil, L. B., Duncan, J. R., and Winter, A. J. (1975). Infect. Immun. 11, 245-251. Schurig, G. D., Duncan, J. R., and Winter, A. J. (1978). / . Infect. Dû. 138, 463-472. Scribner. D. J., and Fahrney, D. (1976). J. Immunol. 116, 892-897. Silverblatt, F. J. (1974). J. Exp. Med. 140, 1696-1711. Smith, H. (1968). Bacteriol. Rev. 32, 164-184. Smith, H. (1977). Bacteriol. Rev. 41, 475-500. Smith, T. (1934). "Parasitism and Disease." Princeton Univ. Press, Princeton, New Jersey. Stendahl, O., Tagesson, C , Magnusson, K.-E., and Edebo, L. (1977). Immunology 32, 11-18. Stossel, T. P . (1974). N. Engl. J. Med. 290, 717-723.

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Stuart-Harris, C. (1975). Med. Lab. Technol. 32, 161-169. Swanson, J., Hsu, K. C , and Gotschlich, E. C. (1969). J. Exp. Med. 130, 1063-1091. van Oss. C. J., and Gillman, C. F. (1972). J. Reticuloendothel. Soc. 12, 283-292. van Oss, C. J., Gillman, C. F., and Neumann, A. W. (1974). Immunol. Commun. 3, 77-84. Vickerman, K. (1974). Ciba Found. Symp. 25 (new ser.), 53-70. von Mitscherlich, E., and Heider, R. (1968). Zentralbl. Veterinaermed., Reihe B 15, 486-493. Williams, R. C., and Gibbons, R. J. (1972). Science 177, 697-699. Wilson, L. A., and Spitznagel, J. K. (1968). J. Bacteriol. 96> 1339-1348. Winkelstein, J. A., Shin, H. S., and Wood, W. B., Jr. (1972). J. Immunol. 108, 1681-1689. Youmans, G. (1975). Am. Rev. Respir. Dis. 11, 110-118.

ADVANCES I N VETERINARY SCIENCE AND COMPARATIVE MEDICINE, VOL. 2 3

The Immune System and Helminth Infection in Domestic Species E. J. L. SOULSBY* Department of Pathobiology, School of Veterinary Medicine University of Pennsylvania, Philadelphia, Pennsylvania

I. II.

Introduction Survival Mechanisms of Helminths in Their Hosts 1. Parasite-Associated Modulations 2. Host-Associated Modulations I I I . Immunological Basis for Seasonal Variation in Gastrointestinal Nematode Burdens of Ruminants IV. Protective Mechanisms in Helminth Infections 1. Gastric Nematodes 2. Intestinal Nematodes . V. Physiological and Morphological Alterations of Nematodes Induced by Immune Reactions VI. Trematodes VII. Cestodes Tissue-Dwelling Metacestode Infections VIII. Conclusion References

·

71 72 72 75 76 80 80 83 90 91 94 94 97 97

I. Introduction Though the immune response to helminth infections is comparable, as far as is known, to that stimulated by any other type of infective agent, the antigenic complexity of the helminths, their varied and at times extended developmental cycles, and the differing locations which they inhabit imply that a wide range of responses can be expected. Nevertheless, there is ample evidence that acquired resistance occurs in helminth infections, and that this can be induced artificially and trans­ ferred passively or adoptively by serum and lymphoid cells and be * Present address: Department Cambridge, Cambridge, England.

of Clinical

Veterinary

Medicine,

University

of

71 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-039223-2

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abrogated by techniques which ablate immunity in other systems. The proportionate quantitative and qualitative contribution of immunoglobulins, lymphoid cells, myeloid cells, and nonspecific effector systems to protective immunity varies considerably for each helminth infection and, indeed, may vary for different parts of the life cycle of an individual helminth. Much of the basic information on the immune response to parasitic helminths of domestic animals has been obtained from studies of laboratory animals infected with them. This has permitted the immunological manipulations applicable to inbred and congenic strains of animals, but caution is necessary in the extrapolation of results ob­ tained in rodents, to natural hosts of the helminths such as swine and ruminants. An area where there has been little research hitherto, but is of in­ creasing importance in the understanding of host-parasite relationships, is that concerned with the mutual accommodations adopted by host and parasite in response to evolutionary pressures. Striking examples of such accommodation include the ability of many helminths to persist in a host for extended periods, up to years in some instances, and the ability to synchronize parasite development with physiological events of the host to permit infection of the fetus or the neonate with larval stages previously arrested in development in various tissues or permit maturity of the parasite leading to contamination of the environment with infection for a new generation of hosts. The immune system plays a role in at least some of these but this role is far from fully understood. Aspects of the above will be dealt with in this article to provide a basis for understanding the relationship of the immune response to parasite biology and the epidemiology of infection. II. Survival Mechanisms of Helminths in Their Hosts A consideration of how a parasite may survive for extended periods in immunologically competent hosts is pertinent to the discussion of immunity to parasites. Various mechanisms are known by which hel­ minths accomplish this; some are concerned with the abrogation of the affector or effector arms of the host response, while others are associated with adaptive changes on the part of the parasite. 1. PARASITE-ASSOCIATED MODULATIONS

Smithers and Terry (1976) have reviewed the evidence for the ex­ istence of antigens shared between schistosomes and their hosts and which are purported to be responsible for the antigenic disguise that

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protects the parasites from immunological recognition. Both host-derived and parasite-derived "shared" antigens seem to exist. Host-derived blood group antigens are acquired by schistosomules when they are grown in vitro in the presence of erythrocytes of various A, B, or 0 specificities, but not with cells of other specificities such as Rh or Mn (Dean, 1974). There is also strong evidence for shared antigens of para­ site origin. For example, Damian et al. (1973) have demonstrated an antigenic determinant on the surface of Schistosoma mansoni which crossreacts with mouse a2 microglobulin. However, recent studies of the tegument of schistosomes show that the outer membrane is continually breaking down and being reformed and evasion of the host response may be more complex than thought previously. In this respect, Wilson and Barnes (1977) report that the hepta­ laminate appearance of the schistosome tegument is contributed to by a multilaminate vesicle, the lamelate contents of which adhere to the plasma membrane. As new material is secreted, old material is displaced and flows onto the tegumental surface and off into the worm's environ­ ment. Labeling studies indicate the half-life turnover rate is 2.5 to 3 hr. Smithers and Terry (1976) suggest that host antigens of parasite origin, such as the membranocalyx above, reduce the overall immunogenicity of the parasite, while those of host origin provide the immunological disguise for the adult worm. However, Smithers and Terry (1976) make the point that there is no direct evidence that host-like antigens serve to protect the parasite from the host in natural infections. The common liver fluke of ruminants Fasciola hepatica may survive for a long time in the bile ducts, up to 11 years having been reported in sheep (Durbin, 1952). A comparable mechanism to that for schisto­ somes might be suspected but there is no evidence so far that host determinants are used to mask the parasite from the host response (Hughes and Harness, 1973). Indeed, the tegument of F. hepatica is trilaminate rather than heptalaminate though the significance of this in respect to survival in an immune host is not known. Resistance to oral challenge infection with metacercariae of F. hepatica occurs when flukes have reached and are living in the bile ducts (Hughes et al., 1977). This immunity will kill adult flukes transferred to the peritoneal cavity but is without effect on flukes located in the bile duct. This situation is similar to schistosomes in which adults persist in the venous system but developmental stages of a challenge infection are destroyed. This has been referred to as concomitant immunity (Smithers et al., 1969). Larval cestodes of the Taenia species are common in domestic animals and they may persist in a viable state for many months or years in animals immune to reinfection. In the case of the larval stages of the

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Echinococcus species (E. granulosus and E. multilocularis), they may grow or proliferate unchecked until they kill the host. Rickard (1974) has proposed a hypothesis for the long-term survival of Taenia spp. cysticerci. He suggests that when oncospheres (the in­ vasive stage) enter immunologically nai\re animals they develop rapidly and reach a stage which is resistant to antibody before antibody is pro­ duced in substantial quantities. They then become coated with specific immunoglobulin which acts as a blocking antibody, preventing attack by cell-mediated immunity. As long as the cysticercus has this protective antibody coat it is able to survive. Oncospheres of a challenge infection are not afforded such protection since they stimulate an anamnestic antibody response at a stage earlier than when the oncospheres acquire the ability to resist antibody-mediated damage. Confirmation of rapid development of resistance to the host response was reported by Musoke and AVilliams (1975) who have shown that the early, post-oncospheral, stages of T. taeniaejormis in mice are suscep­ tible to complement-dependent antibody-mediated attack, but rapidly become resistant to this mechanism. Hammerberg et al. (1976) suggested that this may be associated with the onset of production of anticomple­ mentary factors by the parasite. Studies (Hammerberg et al., 1976) using the cysticercus of T. taeniaejormis have shown that parasitederived materials inhibit complement-dependent hemolysis, generate anaphylatoxin activity in normal serum in vitro, and cause a profound depression of serum complement in vitro. The larval stages of the para­ site appear to suffer no déterminai effect from the consumption of C3 and the generation of anaphylatoxin, though Hammerberg et al. (1976) sug­ gest that the intense local cosinophilia evident around an early differen­ tiating metacestodes may be mediated by this reaction. The survival of hydatid cysts may be related to the relative permea­ bility of the hydatid cyst membrane to immunoglobulins. Varela-Diaz and Coltorti (1972) have demonstrated that the membrane of the hydatid cyst is permeable to serum proteins, including immunoglobulins, and Kassis and Tanner (1976a) confirm that IgG and IgM immuno­ globulins occur in the cysts of the two species of Echinococcus. Rau and Tanner (1976) have further shown that heat-inactivated serum from infected hosts is cytotoxic to the protoscolices of E. multilocularis in vitro, and Kassis and Tanner (1976a) hypothesize that insufficient quantities of these cytotoxic antibodies reach the parasite under natural conditions to effect a cytotoxic response. Nevertheless, a role for an anticomplementary substance of parasite origin can be envisaged for hydatid cysts also. For example, Kassis and Tanner (1976b) describe

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a novel approach to the treatment of hydatid disease in which cyst fluid is replaced by fresh autologous serum from infected hosts, resulting in eventual death of the parasite. This could be interpreted on the basis that sufficient complement was supplied in the replacement process to initiate irreparable damage to the parasite. Kassis and Tanner (1976a) demonstrated the rapid in vitro lysis of the protoscolices of both E. granulosus and E. multilocularis by complement when this was activated by surface immunoglobulins acquired by the cestode larval stages in vivo. The blood group antigens are known to be part of the normal antigenic mosaic of several parasitic helminths, including Ascaris suum of the pig (Soulsby and Coombs, 1959). Such antigens, or the carbohydrate moiety recognized by anti-blood group sera and lectins, may facilitate the provision of a surface coating of host material (e.g., antibodies to blood group determinants) and the establishment of the organism in the host. However, reduced reactivity of parasites upon their entry into a host may be an alternative mechanism for survival and Leventhal and Soulsby (1975) have shown that the early larval stages of A. suum have reduced reactivity in terms of binding immunoglobulins, activa­ tion of complement, and the attachments of phagocytic cells. This might be interpreted as a mechanism which would permit a parasite to gain a foothold, an initial advantage, which would assure its establishment in the host. 2. HOST-ASSOCIATED MODULATIONS

There is increasing evidence that the gastrointestinal nematodes of ruminants, and probably other nematodes of other host species, are affected by external environmental factors which determine the duration of the prepatent period of a parasite and its survival as a larval stage in the host. At the boundaries of geographical distribution of a para­ site, survival mechanisms may be invoked which lead to prolonged arrested development in the host, usually during a period of unfavorable external environmental conditions (Schad, 1977). Reactivation of ar­ rested developmental stages of gastrointestinal nematodes of sheep, for example, coincides with biological events in the ecosystem favorable to the progression of the parasite, such as satisfactory external con­ ditions for the development of free living larval stages, the occurrence of parturition and lactation in ewes, both associated with a relaxation of immunity, and the availability of a population of hosts (lambs) which are unable to respond with an adequate protective immune

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response until they are several months of age. These events will be considered in more detail, but together they represent a remarkable synchronization of host and parasite factors which results in an un­ hindered transmission of infection to the next generation of hosts.

III. Immunological Basis for Seasonal Variation in Gastrointestinal Nematode Burdens of Ruminants Some 40 years ago, Taylor (1935) reported the seasonal variation of helminth burdens and fecal egg output in sheep and he emphasized that worm numbers did not coincide with the period of maximum availability of infective larvae on pasture. Subsequently, the phenom­ enon was studied extensively in hill sheep in Scotland (Morgan and Sloan, 1947) and in lowland sheep in England (Crofton, 1954). The Spring Rise, as it is now known, or something very similar to it, has been observed in the majority of countries where gastrointestinal nematode populations of sheep have been studied. The phenomenon represents an adaptation of gastrointestinal nematodes to environ­ mental conditions in which arrested development of larval stages and the reproductive cycle of the host are major components and which re­ sult in synchronization of the life cycles of the parasite and the host referred to above. An important host-associated component is lactation and hence the terms 'Tostparturient Rise" and "Lactation Rise" have been used instead of Spring Rise. However, they are not synonymous with the Spring Rise; the phenomena associated with them constitute part of the Spring Rise phenomenon but they may occur at other times of the year and they are not applicable to the Spring Rise phenomenon which occurs also in barren females, castrated males, and males. Phenomena similar to the Spring Rise, synchronizing host and para­ site life cycles are seen in other ruminants, swine, horses, and rabbits under natural conditions. Increases in fecal egg output are associated with parturition and lactation and these lead to increased pasture con­ tamination with infective larvae and infection of fully susceptible young stock. The interrelationship of the resumption of development of arrested larvae and the onset of lactation is unclear at present. With Ostertagia ostertagi of cattle, for example, inhibition of developmental stages is of a fixed and predetermined length equivalent to the period of adverse conditions (Armour and Bruce, 1974) and the spontaneous maturation which occurs is characteristic of a diapause and independent of host physiology. However, the resumption of development of inhibited stages

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of other nematode species in their various hosts differs from species to species and Michel (1976) has concluded that in sheep there is a clear relationship between the physiological status of the ewe and the activa­ tion of larvae arrested in their development, these having been acquired in the previous grazing season. He has proposed that parasite popula­ tions are controlled by a biorhythm of parasite origin, in that arrested (hypobiotic) larvae in sheep of all descriptions mature at the same time, but in parturient and lactating animals parasites are retained longer than in barren animals. This explanation ris consistent with the parasitological and immunological data available on the spring rise phenomenon. Connan (1973b) has reviewed the evidence that lactation impairs acquired resistance to gastrointestinal nematodes in sheep. For example, the ability to reduce the establishment of newly acquired in­ fective larvae, to suppress egg production, and to expel developed worms is affected during lactation (Connan, 1976). O'Sullivan and Donald (1973) also demonstrated that ewes in late pregnancy behaved similarly to lactating ewes, demonstrating a reduced ability to control challenge infections. Hence, the arrival of new, fecund parasites from the reservoir of arrested (hypobiotic) larvae in the mucosae occurs at a time when host factors have minimal impact in controlling parasite populations. Further, since the postparturient rise in egg count is asynchronous, animals in the latter part of the lambing season may be exposed to an increased intake of infective larvae, without the ability to control it immunologically. As well as the failure to express an efficient effector mechanism during lactation there is also some evidence of suppression of the afferent arm of the immune response to helminths at this time. Thus lactating mice, infected with Trichinella spiralis, failed to acquire immunity to a chal­ lenge infection (Ngwenya, 1977). If a similar situation were to pertain in lactating sheep, then the antigenic stimulus provided by reactivation of larval stages and an increased intake of infective larvae from herbage would not induce protection against subsequent reinfection. The lactogenic hormone, prolactin, has come under detailed study in the spring rise phenomenon. In general, the behavior of the helminth population is related to the pattern of prolactin secretion, except that during estrus, temporary high levels of prolactin occur; however, there is no obvious increase at this time in parasite burdens or the output of eggs by existing burdens. Connan (1973a) comments that the peak levels of prolactin may be less important than elevated base levels, and further, other endocrine changes attend lactation and are .not evident at estrus. Artificial elevation of prolactin levels by the injection of diethylstilbestrol resulted in a rise in fecal egg output in sheep and

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similarly repeated injection of acepromazine maleate elevated prolactin levels and fecal egg counts in ewes (Connan, 1973b). The basic mechanism (s) of this periparturient relaxation of immunity have yet to be defined. Studies of the rat infected with the intestinal trichostrongyle Ni-ppo strong y lus brasiliensis have permitted a better understanding of the phenomenon. In this system adult worms are expelled by a a self-cure" mechanism after which the rat is immune to reinfection (Ogilvie and Love, 1974). Lactation suppresses the self-cure. Antibody production is unimpaired during lactation and adult worms continue to be damaged by antibody at this time. However, damaged worms are not expelled and it has been suggested that prolactinmediated suppression may compromise either the specific component of the rejection mechanism (antibody and lymphocyte) or the non­ specific (the bone marrow-derived myeloid elements and biogenic amines) (Kelly and Dineen, 1973). Connan (1976) has suggested that the defect is the failure of sensitized lymphocytes to differentiate to terminal effector cells and Dineen and Kelly (1972) have shown in this system that potentially reactive cells are present in lactating donors. The immunological defect in lactating rats cannot be repaired by syngeneic transfer of cells from immune barren rats, however, trans­ fer of such cells will lead to rejection of a challenge dose of infective larvae if this is given on the same day as the cells (Ogilvie et al., 1977). This illustrates the difference in responses of the different development stages of the parasite. There is evidence in several species that lymphocyte reactivity is reduced during pregnancy and lactation. For example, in sheep, antigeninduced blastogenesis of peripheral blood lymphocytes is markedly re­ duced at the onset of lactation and is associated with a significant increase in fecal egg output at that time (Chen and Soulsby, 1976). In man there is a reduced lymphocyte mitotic response to phytohemagglutinin (PHA) in pregnancy, apparently due to a plasma inhibitory factor, which is also present in the plasma of cord blood but which is no longer demonstrable in the blood of mothers of infants taken 7 days after delivery (Yu et al., 1975). Impaired in vitro lymphocyte respon­ siveness to purified protein derivative (PPD) (Smith et al., 1972) and prolonged homograft survival of skin in pregnant women (Andersen and Monroe, 1962), responses dependent on T lymphocytes, are indica­ tive of the important influence of the physiological and endocrinological events associated with parturition. These have yet to be investigated in detail in domestic animals. The reduced immunity of the ewe at parturition and lactation is mirrored by an inability of the lamb to respond immunologically to

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gastrointestinal nematodes. In the case of H. contortus, lambs are unable to respond to an X-irradiated vaccine until they are several months of age (Urquhart et al., 1966). The reasons why the lamb is unable to respond well to helminth antigens is not understood. Silverstein et al. (1963) have demonstrated that the lamb may show immunocompetency even as a fetus, but this depends on the antigen involved and the age of the animal. For example, a 41-day-old fetus inoculated with the bacteriophage φχ-174 will produce antibody, but responsiveness to diphtheria toxoid is not seen until 42 days after birth. This hierarchy in responsiveness of lambs to various antigens may apply to helminth antigens also. Silverstein et al. (1964) also showed that antigens applied before the acquisition of competence lead to tolerance. A phenomenon similar to this might explain the work of Gibson (1952) who showed that lambs exposed to excessive infections of parasites at an early age developed a lower degree of immunity than those with light infections. Other possibilities include the colostral transfer of tolerogenic factors similar to those indicated by Auerbach and Clark (1975) in which unresponsiveness to sheep red cells was induced in mice suckled on animals injected with a high-speed supernate of lysed cells. Recent studies by Halsey and Benjamin (1976) also indicate that deaggregated human γ-globulin, when injected into mice within 24 hr of delivery, enters colostrum and is absorbed intact through the intestine by nursing neonates in which it induces a specific tolerant state for at least 10 weeks. In view of the increased activity in nematode populations in sheep at parturition and lactation there is a possibility that circulating antigen might be passed via the colostrum to the neonate to induce a com­ parable tolerance to that evident in mice. Lambs would be less sensitive to tolerance than mice because of their immunologie maturity at birth. Worm proteins (antigens) have been detected in the serum of sheep infected with H. contortus (Stumberg, 1933). However, this cannot pro­ vide a complete explanation for the postnatal unresponsiveness since the phenomenon is seen in lambs born and raised under worm-free conditions. A further explanation might be the occurrence of suppressor lympho­ cytes stimulated by high doses of infective larvae. However, little is known of this phenomenon in domestic animals though it is of in­ creasing interest in parasitic infections. Eventually, the worm burden in adult sheep resulting from contami­ nated pasture is brought under control. In adult sheep, worm popula­ tions decrease abruptly, being eliminated by a self-cure reaction (see

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Section IV) in mid to late spring, while in lambs worm burdens de­ crease rapidly in late fall (Soulsby, 1966), after which animals are relatively immune to reinfection. This pattern is fairly constant year after year. It may be modified in some of its detail by climatic or husbandry conditions, but essentially it reflects the interplay of the immune response in a susceptible popu­ lation and a resistant population.

IV. Protective Mechanisms in Helminth Infections 1. GASTRIC NEMATODES

Haemonchus

contortus

Haemonchus contortus infection of the abomasum of sheep was one of the first to be studied immunologically. Stoll (1929) described the phe­ nomenon of "self-cure" in this infection, in which infected grazing lambs suddenly lost their infection and were resistant to reinfection. The phenomenon was described for several other intestinal nematode infections over the next 20 years but was not examined critically until the 1950s when Stewart demonstrated it was a hypersensitive reaction initiated by a challenge dose of infective larvae in a previously infected and sensitized animal (Stewart, 1953). Soulsby et al. (1959) identified the responsible physiological event in the parasite as the molting period between the third and fourth larval stage and postulated that a "molting fluid," physiologically and antigenically similar to exsheathing fluid (produced a few days earlier in the life cycle when infective larvae ecdyse to become parasite third stage larvae), was the putative antigen. Information on the chemical nature of molting fluid is scanty, though it is presumed to resemble the exsheathing fluid produced by infective larvae. The latter contains a leucine amino peptidase (Rogers, 1970) responsible for the elimination of the retained sheath of the second stage larva, and Rogers and Brooks (1978) hypothesize that ecdysis of all stages of nematodes involves leucine amino peptidase and a lipase. Unfortunately, there has been little detailed study of the self-cure reaction in the last two decades. Allonby and Urquhart (1973) have reported that its occurrence in sheep in Kenya is independent of ex­ posure to infective larvae and they indicated that climatic or pastoral changes were a sufficient stimulus for the induction of the response. While such results are interesting and demand explanation, there is no doubt that the "classical" self-cure is initiated by a challenge infection of larvae.

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At the time of self-cure there is a significant increase in blood histamine (Stewart, 1953) and the curative reaction can be ablated by the administration of antihistamines, though the antibody response is unaf­ fected. Administration of histamine did not induce the reaction. The abomasal reaction during self-cure is consistent with an immediate type hypersensitivity response. As such it probably causes expulsion of parasites in a nonspecific manner by an alteration in the local environ­ ment of the worm. This idea is supported by the fact that self-cure provoked against other abomasal nematodes will result in the elimina­ tion of H. contortus and vice versa. Also, the reaction is not necessarily site specific since an abomasal-induced self-cure will cause elimination of an intestinal infection with Trichostrongylus colubriformis (Stewart, 1953). The self-cure reaction in N. brasiliensis infection in the rat has been studied extensively. Although it shows many superficial differences from that in H. contortus infection, the reactions may be similar and probably there is a close analogy between the self-cure of N. brasiliensis in the rat and that of T. colubrif ormis in sheep. An obvious difference between H. contortus and N. brasiliensis self-cure is that the latter does not require a secondary dose of infective larvae to initiate it. It is the natural termination of the infection in a rat. The synchronous occurrence of mast cells, IgE antibodies, and worm antigen in the N. brasi'h'ensis-parasitized intestine of the rat has focused attention on the role of vasoactive amines in the permeability of the gut mucosa, the effect of this permeability on the passage of plasma proteins, including antibodies, to the gut lumen, and their contribution to the elimination of an adult population of N. brasiliensis from the gut. This is the "leak lesion" hypothesis of Murray (1972), which assumes that protective antibodies generally of the IgGi subclass [though Mur­ ray (1972) has suggested that both IgG and IgA are concerned] are delivered to the sites occupied by the worms by the intercellular pathway created by the pharmacological mediators. To date IgA has not been demonstrated to be a functional immunoglobulin in parasitic nematode infections, though in the case of N. brasiliensis-'mîectea rats, anti-worm IgA antibodies were detected in the intestinal contents as early as 6 days after infection (Poulain et al, 1976). Ogilvie et al. (1977) con­ sider there is no evidence that IgA antibodies have any effect on the worms. However, with larval cestode infections of neonatal rats, specific IgA of the colostrum is the protective immunoglobulin (Lloyd and Soulsby, 1978). ' Objections to the "leak lesion" hypothesis were offered by Ogilvie and co-workers (see Ogilvie and Love, 1974). For example, in lactating rats in which the self-cure reaction fails to occur, mast cell invasion

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is similar in time and extent as in nonlactating controls and degranulation is evident yet adult worms are not expelled. Additionally, lactating rats are susceptible to anaphytaxis and a "leak lesion" in the bowel without affecting the population of worms. Other evidence from infections in neonatal rats in which, for example, IgE levels are com­ parable to infected mature rats are not associated with expulsion of thg parasites from the lumen of the bowel (Ogilvie and Love, 1974). The deficiency in the immune response in these instances appears to be cellular rather than humoral. Recent studies of N. brasiliensis in mice support this concept in that Jacobson and Reed (1976) demonstrated thymus dependency of expulsion and also that expulsion occurs in mice lacking antibody production potential (Jacobson et al., 1977). In a recent review of the factors responsible for the rejection of N. brasiliensis from the rat intestine Ogilvie et al. (1977a) conclude that, following damage to worms by a humoral factor, specifically stimulated, thymusdependent immunoglobulin-negative lymphocytes are responsible for rejection. Such cells are found in mesenteric lymph nodes of infected rats by 8 days after infection or in the thoracic duct in the same time and they are still present in thoracic duct lymph 25 days after infection (Ogilvie et al., 1977b). The need for a collaborating bone marrowderived cell is no longer thought necessary (Ogilvie et al., 1977a) (see below). Nawa et al. (1978) have confirmed that surface immunoglobulin negative cells, obtained from the thoracic duct, are the effector cells in the expulsion of worms; however, Nawa and Miller (1978) failed to demonstrate the need for worms to be "damaged" by antibody to be expelled. They suggest the different susceptibilities of normal and damaged worms to adoptive protection is quantitative rather than qualitative. Self-cure in N. brasiliensis infection is followed by resistance to reinfection; the phenomenon in sheep is not necessarily followed by protection against reinfection. Indeed, the larvae which initiated the response in sheep may develop to maturity and cause severe, even fatal, infection. This is probably a function of age since self-cure can be induced in animals a few weeks old (Varela-Diaz, 1970) but resis­ tance to reinfection is not fully expressed until animals are 6-9 months of age (Manton et al., 1962). Once the ability to respond with a pro­ tective immune response is established, animals manifest this by a reduction in total parasite burden, by arrested development at the fourth larval stage, by reduced egg production, as well as by rejection of worms by the self-cure mechanism. Dineen and Wagland (1966a) have reported immunological exhaus­ tion in sheep in the presence of persisting infections and hence exposure

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to continuous antigenic insult. This was associated with the depletion of germinal follicles in the cortex of the local lymphoid centers. How­ ever, this was not evident in sheep given prolonged repeated daily in­ fection of H. contortus larvae over a period of several months (Christie et al.,. 1978). In this work, strong immunity developed and the authors concluded that "if there is an upper limit to the size of challenge which hyperimmunized animals can resist, then it is a very high one indeed." The basis of the protective immune response to H. contortus is un­ clear. In sheep exposed to multiple infections, Smith (1977) detected anti-larval antibody which was associated with IgA and IgG immunoglobulin classes. The response developed slowly and declined soon after reinfection ceased; no immunological memory was observed wrhen sheep were challenged with larvae later. Local production of the spécifie IgA was suggested whereas specific IgG was considered to be plasma derived. knti-H. contortus IgA hemagglutinins have been reported in the sera of sheep infected with this parasite and high anti-parasite antibody levels were found in the IgAi and IgA 2 fractions of colostrum of sheep sub­ jected to repeated infections up to the time of parturition (Varela-Diaz, 1970). However, maternal transfer of such antibodies does not result in protection against infection in lambs. Filmer and McClure (1951) obtained similar results. A role for cell-mediated immunity in H. contortus infection is possible. Chen (1972) demonstrated that antigen-induced blastogenesis of periph­ eral blood lymphocytes of lambs infected with H. contortus did not reach adult levels until they were several weeks of age. Chen (1972) also demonstrated the production of the lymphokine M I F by antigenstimulated peripheral blood lymphocytes of infected lambs and that the capacity of cells to produce it was related to age and to the time of a challenge infection. It is likely that in concert with helminth in­ fections in other animals, the immune response to H. contortus is thymic dependent. One report (Scott et al., 1971) indicates the successful transfer of immunity to sheep by allogeneic mesenteric lymph node cell sus­ pensions. This somewhat unusual result, if confirmed, would provide a point of departure for future studies of immunity to H. contortus.

2. INTESTINAL NEMATODES

Substantial advances have been made in the understanding of im­ munity to intestinal nematodes by studies of infections with Trichostrongylus colubrijormis (of ruminants), Trichinella spiralis (of swine), Ascaris suum (of swine), and Nippostrongylus brasiliensis (of rats).

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a. Intestinal

Tnchostrongyles

The majority of the studies have been done in laboratory rodents which allow a closer definition of the immune responses than is possible with domestic animals. Extrapolation of results obtained with rodents to normal hosts requires caution since normal and experimental in­ fections may differ substantially, but nevertheless the differences are not usually of the order that would make rodent-derived data invalid for the normal host. However, an illustration of the variance that may occur is the response at weaning of lambs to vaccination with irradiated T. colubriformis larvae (Dineen et al.y 1978). Lambs segregated into "responders" and "nonresponders" on challenge with normal infective larvae and responses in the duodenal mucosa indicated that the globule leukocyte was involved in the resistance mechanisms. The authors concluded it was unlikely that either eosinophils or neutrophils were so involved. This is in contrast to the conclusions reached by Rothwell and Dineen (1972) that in the guinea pig infected with T. colubrijorrnis, the accumulation and degranulation of eosinophils and basophils at the site of infection is correlated with rejection of the parasite. Despite such examples, the work with rodents has helped to clarify the understanding of the immune response of the intestinal tract to parasitic helminths. Ogilvie and Love (1974) have reviewed the evidence for cooperation between antibody and cells in the rejection of N. brasiliensis from the intestine of rats. The authors maintain that the first stage of the process is antibody-mediated damage to the worm: This step is complement independent, it reduces 32P uptake by worms, affects the secretion of acetylcholinesterase, and is manifest morphologically by changes in the cytoplasm of the gut cells of the worms. Such changes make the worms susceptible to the second step, which-is attributed to lymphocytes. Ab­ rogation of the rejection of antibody-damaged worms following irradia­ tion, neonatal thymectomy, or treatment with antilymphocyte serum was demonstrated by Keller and Heist (1972) and the restoration of the rejection mechanism by lymphocytes identified the need for a lymphoid (and T cell) response in the process. The need for a further cell component from the bone marrow was claimed by Dineen and Kelly (1972), but Ogilvie et al (1977b) failed to confirm the need for collaboration of bone marrow-derived cells. The claim now made by Nawa and Miller (1978) (see above) that antibody-mediated damage is not a prerequisite for rejection indicates that further clarification of the role played by antibody in N. brasiliensis infection is needed. Im­ mune serum given prior to infection with infective larvae results in a marked reduction in the number of parasites which become established

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(Jones and Ogilvie, 1967). However, when given after the establish­ ment of adult worms, antiserum alone will not cause elimination of the infection and immune cells are also required. Indeed Jacobson et al. (1977) have shown in mice that immunity can occur in the absence of immunoglobulin-forming capability and this raises the question of whether some serum factor other than antibody is concerned in the im­ mune response and responsible for the apparent success of passive transfer in some cases. Various immunoglobulin classes of antibody occur during infection. Immunoglobulin A antibodies are detectable within days of infection and IgE antibodies are recognized as a characteristic of helminth in­ fections. The accumulation of IgE at the mucosal surface has been demonstrated in recent studies by Mayrhofer et al. (1976) who have shown that IgE is not a secretory immunoglobulin but the major site of its production during infection with N. brasiliensis is the regional mesenteric lymph node, while the Peyer's patches and the spleen played a minimal role in this. In addition, these authors have demonstrated IgE both on the surface and in the cytoplasm of intestinal mast cells. Hence, IgE localization at mucosal surfaces depends on two cell types, the mast cell and the plasma cell. If mucosal mast cells concentrate passively acquired IgE in their cytoplasm in helminth infections then concentrations of IgE at mucosal surfaces could be achieved by the migration of cells to the mucosal surface, the shedding of cells from the mucosa, or by the specific degranulation of them by interaction with worm allergen. An exponential increase in mast cell numbers occurs in the intestinal mucosa of rats infected with N. brasiliensis (Murray, 1972) and this has been ascribed to a "mastoblast-stimulating" factor. With T. colubriformis infections, cellular elements appear more im­ portant in immunity than antibodies. The passive transfer of protective immunity to T. colubriformis by mesenteric lymph node cells, but not with serum, was demonstrated in the guinea pig by Wagland and Dineen (1965). Spleen and other lymph node cells were not as effective as those from the mesenteric nodes but they were still able to transfer immunity. Later, these authors (Dineen and Wagland, 1966b) demon­ strated that the susceptible developmental stage was the fourth stage larva (L4), the fifth and adult stages being insusceptible to the immunity transferred by cells. Dineen et al. (1968) demonstrated that 51Cr-labeled immune lymphoid cells from mesenteric nodes accumulated in the in­ fected gut, came into close contact with the parasite in the epithelium of the bowel, and underwent "allergic death" there. At that time they were uncertain whether the cells homed to the infection site or reached it in a random manner. Additional evidence for CMI in this infection

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was obtained by Dineen and Adams (1971) by the demonstration that mesenteric lymphatic duct drainage rendered guinea pigs incapable of mounting an immune response to T. colubriformis. The recognition of the fourth larvae as an important developmental stage led to the demonstration that homogenized L4 or adult worms or soluble products released by L4 in vitro would stimulate sufficient im­ munity to protect against a challenge infection (Rothwell and Love, 1974). These studies encouraged the idea that enzymes (e.g., acetylcholinesterase) secreted by the worm (Rothwell et al., 1973) might be the important immunogens, and, in fact, in N. brasiliensis infection antiacetylcholinesterase antibodies modulate the production of this enzyme by the parasite (Jones and Ogilvie, 1972). However, Rothwell and Merritt (1975) indicated that immunization with acetylcholinesterase in T. colubriformis failed to induce immunity in guinea pigs to challenge infection. The accumulation of lymphocytes in the intestine of parasitized ani­ mals has been studied in detail in Trichinella spiralis infection of mice. Rose et al. (1976a) have shown the enhanced accumulation of mesenteric immunoblasts in the small intestine of mice 2 and 4 days after infection with T. spiralis, but not at later times. This accumulation occurred with cells from both infected and uninfected donors, thus denoting an absence of antigen specificity in the response. These authors concluded that the parasite caused the small intestine to become more attractive or retentive for mesenteric T cell blasts during early infection, though the reason for this is still unknown. That the ability of T lymphocytes to extravasate to given sites is determined largely by the origin of the cells was demonstrated by Rose et al. (1976b), in that T blasts from mesenteric lymph nodes, but not similar cells from peripheral lymph nodes drain­ ing a site of oxazolone sensitation, migrated to the gut during a T. spiralis infection. These studies support the observations of Dobson and Soulsby (1973), who concluded that the increased number of lymphoblasts in the rapid response of the lamina propria in a Trichostrongyhis colubriformis infection (which began on the first day of infection) was due to the recruitment of lymphocytes from mesenteric lymph nodes and the spleen. A temporal relationship between the responses in the lamina propria, the Peyers patches, the mesenteric nodes, and the location of the parasitic infection strongly suggested a closely integrated response by the gut-associated lymphoid tissue. Lymphocytes which are capable of transferring immunity are gen­ erated early in infection with T. spiralis. For example, thoracic duct lymphocytes (TDL), derived from animals exposed to the first 24 to 48 hr of infection, will transfer significant immunity (Despommier, 1977).

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Crum et al. (1977) have demonstrated that specific antibody fails to immunize passively, but B lymphocytes can confer protection against the enterai phase of T. spiralis (Despommier et al., 1977). This has led to the suggestion that resistance is mediated by antibody-producing cells only after they leave the circulation and become functionally mature. This appears to be under T cell control, which cells promote the formation of protective B cells. Despommier et al. (1977) observe that the majority of the dividing B cells in rat TDL contain IgA and display IgA surface determinants. They suggest that IgA may be con­ cerned in the local mediation of immunity in the bowel to T. spiralis. There is increasing evidence that IgA is an important immunoglobulin in intestinal helminth infections. Elevated specific IgA levels were found in the intestine of mice infected with the trichostrongyle Heligosomoides polygyrus (Cypess et al., 1977), in the mucosal extract of rats in­ fected with N. brasiliensis (Sinski and Holmes, 1978) and antigenspecific IgA will confer protection in neonatal mice exposed to infection with the larval stage of Taenia taeniaef ormis (Lloyd and Soulsby, 1978). Transfer of immunity to gastrointestinal nematode infections of sheep by leukocyte lysates has been reported by Ross and Halliday (1978). Challenge infections of Trichostrongylus axei, Ostertagia circumcincta, and T. colubrijormis are modified by the prior injection of a leukocyte lysate and this is effective across different breeds of sheep. It is interest­ ing that leukocyte lysate did not effect protection in rats against N. brasiliensis or T. axei infection in calves. There has been increasing interest in the role of soluble mediators in the expulsion of helminths from the bowel. Prostaglandins, especially prostaglandin E (PGE), have come under intensive study. Dineen and Kelly (1976) have shown that during primary infection with N. brasiliensis, PGE levels increased 10-fold in intestinal tissues and peak levels occurred at the jejunal site of infection on the seventh day of infection. On reinfection, peak PGE levels occurred 3 to 4 days earlier. Dineen and Kelly (1976) suggest that the damage to worms ascribed by Ogilvie and Jones (1971) to antibodies may be due to a direct effect of PGE on the parasite. However, prostaglandins do not appear to be concerned in the immune expulsion of T. colubrij ormis from the intestine of the guinea pig, since Rothwell et al. (1977) failed to induce expulsion by the intraduodenal injection of synthetic prostaglandins, which in the case of N. brasiliensis in the rat would result in expulsion of that para­ site. Nevertheless it is obvious that the role of prostaglandins in the immune response to parasitic nematode infections requires further study. Prostaglandin activation during inflammation (produced by carrageenan) has been described by Vinegar et al. (1976), several steps

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of which involve generation of neutrophil chemotactic factor, accumula­ tion of these cells, and release of lysosomal enzymes during phago­ cytosis. Lysosomal enzymes such as phospholipase A2 liberate arachidonic acid from the phospholipid of the neutrophil which then forms prostaglandins via the prostaglandin synthetase chain in the neutrophil (Anderson et al, 1971; Zurier and Sayadoff, 1975). Larsh et al (1975) have demonstrated elevated phospholipase activity in the intestinal tract of mice during the expulsion of Trichinella spiralis at a time when nonspecific inflammatory reactions resulting from T-cell-dependent cellmediated immunity are in progress (Larsh and Weatherby, 1975). Recently, attention also has been focused on vasoactive amines such as histamine and 5-hydroxytryptamine as effector substances in the expulsion of T. colubriformis from the gut of guinea pigs. Previously, Rothwell et al. (1974) had demonstrated that the infusion of histamine into the lumen of nonimmune guinea pigs caused significant expulsion of T. colubriformis. Expulsion of L4 could be inhibited by drugs which modify the release of histamine (Rothwell et al, 1978) and Jones et al. (1978) demonstrated a temporal relationship between intestinal mucosal histamine and the onset of worm expulsion, both in vaccinated and adoptively immunized guinea pigs. In animals unable to expel an infection mucosal histamine levels were not elevated. It will be remembered that a quarter of a century ago, Stewart (1953) incriminated histamine in the self cure reaction to H. contortus infection in sheep, demonstrating that antihistamine drugs ablated the response. The cellular components present in the parasitized intestine are capable of producing the local concentrations of soluble mediators. Thus mast cells are very much in evidence, the eosinophil is similarly present, and neutrophils occur in the early stages of infection. The eosinophil has received detailed scrutiny in helminth infections recently because of its role in producing damage to various developmental stages of schistosomes (Butterworth et al, 1975; Mahmoud et al., 1975). Eosinophilia is pathognomonic of helminth infection and the inter­ action between developmental stages of helminths and eosinophils has been recognized for many years. In filarial infections Fros and Liqui Lung (1953) reported adhesion of eosinophils to microfilariae and Higashi and Chowdhury (1970) observed adherence of eosinophils to infective larvae of filarids in the presence of immune serum. Soulsby (1963a) observed adhesion and degranulation of eosinophils on the surface of ascarid larvae previously sensitized with immune serum and suggested (Soulsby, 1966) that this might release pharmacologically active substances which affect the structure or function of the cuticle. However, the role of the eosinophil in the elimination of parasites is

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unclear; McLaren et al. (1977) have demonstrated adhesion of eosinophils to and their degranulation upon parasite surfaces, with the release of enzymes such as peroxidase. However, there is little ultrastructural evidence of damage caused by eosinophils in the case of attack by these cells on T. spiralis and N. brasiliensis. The thymus dependency of eosinophilia in T. spiralis infection has been demonstrated by Basten and Beeson (1970) and Walls et al. (1971). There is now evidence that eosinophilia can be associated with cellmediated immunity responses, immediate type hypersensitivity, and the interaction of antigen-antibody complexes with complement (James and Colley, 1976). The demonstration of release of eosinophil chemotactic factor (ECF) from neutrophils during phagocytosis (Czarnetzki et al., 1975) adds a role for neutrophils which are commonly found in the early stages of parasitic lesions. Indeed, Czarnetzki (1978) has demonstrated that parasite larvae (N. brasiliensis) will induce the release of ECF from neutrophils. b. Ascarid

Infections

The immune response to the ascarid of swine, Ascaris suuni, seems less complicated compared to that to intestinal trichostrongyles. For ex­ ample, there is ample evidence from experimental investigations in swine and rodents to indicate that a strong immunity to reinfection occurs. This can be transferred, isogeneically in guinea pigs by serum or cells (Khoury et al., 1977) or by colostral transfer in swine (Kelley and Nayak, 1965). In guinea pigs, syngeneic transfer of IgE plus IgGi is particularly effective. Antigens which stimulate protective immunity are associated with viable and developing larval stages. Thus, Rhodes et al. (1965) immunized guinea pigs and swine with worm-derived malic dehydrogenase and pro­ duced both specific antibodies and a measure of protection against a challenge infection. More recent work by Stromberg and Soulsby (1977) has identified an immunogen which can be produced in defined media, in vitro, at the time of molting of third stage larvae to the fourth larval stage and which will induce a significant level of protection in guinea pigs to a challenge infection. Unusually high levels of IgE have been associated with Ascaris in­ fection (Johannson et al., 1968) and a possible advantageous aspect of IgE production may be that associated with the elimination of im­ mature and mature stages of the parasite from the bowel of swine. A marked loss of late larval stages of A. suum occurs in the pig at the time of the molt from the fourth to the fifth larval stage (Schwartz, 1959) and Taffs (1958) has described a "self-cure" phenomenon, in

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which adult worms were eliminated from pigs following an oral chal­ lenge infection with infective eggs. As with other self-cure mechanisms in helminth infections, this is probably attributable to a local immediate type hypersensitivity reaction in the intestinal tract. Khoury et al. (1977) in their work on adoptive transfer of immunity by lymphoid cells noted that cells from the spleen of immune animals did not transfer immunity well, in fact their presence in a cell suspension reduced its protective capacity. Subsequent studies have shown that the effect is due to a cell with T-cell characteristics, possible a suppressor cell, which is present in the spleen along with other cells which will transfer immunity adoptively when the suppressor T-cell population has been removed (unpublished studies).

V. Physiological and Morphological Alterations of Nematodes Induced by Immune Reactions There is relatively little information available on the physiological and morphological changes which are induced by the immune response and which result in death of the worm or its rejection from its normal site. With N. brasiliensis infections, worms become "damaged" by a humoral factor (? antibody) by the tenth day of infection (Jones et al., 1970) and show a reduced capacity to take up 32P and changes in the secretion of acetylcholinesterase (Edwards et al., 1971) ; in addition, morphological changes occur in the gut cells (Lee, 1969) and lipid droplets with an accumulation of neutral lipid are evident (Lee, 1971). However, these changes are not enough to cause expulsion since worms with these defects can persist in neonatal or lactating rats (Ogilvie et al., 1977a,b) though they are expelled when transferred to normal rats. Worms may also become "adapted" (Edwards et al, 1971) and form a residual population in an immune animal ; these also show changes in the isozyme pattern of acetylcholinesterase. Such worms can be transplanted to nonimmune rats where they reestablish and renew egg production (Chandler, 1936). Other physiological changes induced by the immune response have been reported in N. brasiliensis. For example a decrease in the oxygen consumption of infective larvae of N. brasiliensis was noted when they were placed in immune serum (Schwabe, 1957) and alterations in the behavior of adult worms in a thermal gradient have been reported fol­ lowing exposure to immune serum (McCue and Thorson, 1965). More recently Ballantyne et al. (1978) have reported a fall in the adenylate energy charge value of adults of N. brasiliensis and Nematodiras battus

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(of lambs) during the course of infection with these. They conclude that the immune response may affect the energy status of the nematodes and this could result in the expulsion of the parasites from the immune host. Interruption in the development of nematodes is a common sequel to an immune response. The point at which development is halted is a characteristic of the species, e.g., Cooperia curticei of sheep is arrested in the fourth larval stage (Sommerville, I960) while Trichostrongylus retortaeformis of the rabbit is arrested in the late third larvae stage (Michel, 1952). A specific delay in moulting, due to immunity, has been reported with Cooperia species in cattle (Stewart, 1958). In all these latter examples, the physiological basis for the inhibition of develop­ ment is unknown. The occurrence of crystals in the intestinal lumen of N. battus during the development of immunity to this nematode has been reported by Martin and Lee (1976) and Bird et al. (1978) observed similar crystals in the intestinal cells of H. contortus and in the lumen of 0. ostertagi. In the latter cases the crystals were thought to be by-products of degenerative processes in the nematodes. Further study of such struc­ tures should provide information on the changes induced by the immune responses or by senility. Antibody-mediated adherence of leukocytes (neutrophils, eosinophils, macrophages, etc.) to development stages of helminths has been reported by various authors (e.g., Leventhal and Soulsby, 1976). Adhesion is followed by degranulation of the cell as to the nonphagocytosable surface of the nematode, detectable by the reduc­ tion of nitro blue tetrazolium (Leventhal and Soulsby, 1972), but there is little evidence, even at the ultrastructural level, of damage to the para­ site. For example, McLaren et al. (1977) found no morphological evi­ dence to suggest that peroxidase" secreted by eosinophils directly onto the surface of nematodes (T. spiralis, N. brasiliensis) affected the integrity of the surface. However, Morseth and Soulsby (1969) have observed ultrastructural changes of the cuticle larvae of A. suum associated with liposomal discharge of adherent leukocytes.

VI. Trematodes Members of the genera Fasciola and Fascioloides are the cause of major morbidity and mortality in domestic animals throughout the world. In some areas (e.g., Sudan, Ethiopia, etc.) the genus Schistosoma (S. bovis, S. mattheei) is responsible for serious disease in sheep and cattle. Immune responses to the schistosomes of sheep and cattle have

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received attention recently because of the natural animal model system they provide for the study of immunity to human schistosomes (Taylor, 1975) and also because of the possibility of inducing protection to human schistosomes by cross-immunizing with an animal schistosome (Zooprophylaxis; Nelson, 1974). Partial resistance to reinfection with S. mattheei in cattle was reported by Lawrence (1973), and Preston et al. (1972) induced protection in sheep to S. mattheei by exposure to infection with heterologous schisto­ somes. Radiation-attenuated cercariae and schistosomula of S. mattheei will induce a considerable degree of protection in sheep (Taylor et al., 1976). An alternate approach to immunization has been to develop a strain of S. mattheei of reduced virulence by passage in hamsters (Dargie et al., 1977a) ; using this Dargie et al. (1977b) induced pro­ tection in sheep against acute schistosomasis caused by a virulent strain of S. mattheei. The basis of such immunity is unknown. Fasciola hepatica Domestic animals vary considerable in their susceptibility to F. hepatica and ability to resist a second infection. Ross (1967) has as­ cribed these differences to the fibrous structure of the liver. Thus, in hosts of low resistance, such as sheep, little or no obstruction occurs to the migration of immature forms and high levels of infection are usually fatal. Medium resistance, as expressed by cattle, results in limited lifespan for the parasite and marked fibrosis of the hepatic parenchyma and bile duct wall. A high degree of resistance is seen in F. hepatica infection in swine and restriction of the migration of the immature forms of the parasite is maximal, so that few if any reach the bile ducts and mature. Cell reactions in the latter instance are very marked and immature parasites are enveloped in an intense eosinophilic response. The proportionate contribution of fibrosis of the liver and specific immune responses to resistance to F. hepatica requires further evalua­ tion in view of recent studies with the parasite. Doyle (1971) and Ken­ dall et al. (1978) reported significant resistance to reinfection following an initial infection. The latter authors noted that cattle cleared of their adult flukes by a.nthelmintic treatment resisted the second infection consequently the continued presence of adult flukes was not necessary for the expression of immunity. Even more radical rethinking on immunity to F. hepatica is necessary in view of the publication by Campbell et al. (1977) which reports the

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stimulation of resistance in sheep to F. hepatica by infection with the metacestode of Taenia hydatigena {Cysticercus tenuicollis). Infection of sheep with the metacestode for 12 weeks generated a high level of protection against F. hepatica. The protective effect could not be ex­ plained on the basis of liver fibrosis. These data are especially interest­ ing since previously it has been generally thought that the sheep was unable to respond with a satisfactory protective immune response to F. hepatica and further, a priori, heterologous immunization would not be expected to lead to such a high degree of resistance. Studies of the basic aspects of acquired immunity to F. hepatica in domestic species presents obvious difficulties. However, in experimental models, passive transfer of immunity has been achieved with lymphoid cells from mice (Lang et al., 1967) and rats (Corba et al., 1971 ; Armour and Dargie, 1974) infected with F. hepatica. In addition, Armour and Dargie (1974) successfully transferred protective immunity with homologous (rat) or heterologous (sheep or bovine) serum from infected animals to rats. The extent of the protective effects was directly related to the parasite burdens in the donor animals and, to some extent, to the origin of the lymphoid cells. Armour and Dargie (1974) indicated that the protective effect of serum occurred before the parasites entered the liver (i.e., as they pene­ trated the bowel wall and crossed the peritoneal cavity), whereas syngeneic transferred cells had their effect when the parasite had entered the liver parenchyma. In this respect, Vernes et al. (1972) demonstrated delayed skin sensitivity and the production of macrophage inhibition factor in guinea pigs 15 days after infection with F. hepatica, at which time in comparable infections in mice, parasites are migrating in the liver parenchyma. The inflammatory responses to F. hepatica infection probably consti­ tute a major immunological component. These are maximal during the migration of the immature forms in the liver parenchyma and decrease in intensity after parasites enter the bile ducts. This may be associated with the loss of parasite antigen into the bile (Hayes, 1970), the occur­ rence of which is associated with a decrease in the lymphoid hyperplasia of associated lymph nodes and the hepatic parenchyma. Evidence for an immunological basis for the inflammatory responses is also obtained from studies by Lang et al. (1967) who showed that the inflammatory response in the liver was enhanced during a secondary infection, and by Sinclair (1968) who demonstrated in sheep that corticosteroid treat­ ment led to enhanced pathogenesis (anemia) and an absence of inflam­ matory cell responses.

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VII. Cestodes Adult intestinal lumen and bile duct lumen-dwelling cestodes and tissue-dwelling metacestode stages occur in domestic species. Little is known of the immune response to genera such as Stilesia, Thysanosoma, or Anoplocephala except that they tend to be more prevalent in young animals than old. An example of this is seen in sheep exposed to Moniezia spp. Lambs become infected early in life and may carry sub­ stantial burdens of tapeworms. However, by 6 months of age they have rejected their burdens and it is unusual to find more than a few tape­ worms in the intestine of adult ruminants (Soulsby, 1968). The basis of such resistance is unknown. Immune responses to the adult stages of other cestodes have been received by Gemmell and Soulsby (1968) but, with the exception of Echinococcus granulosus of the dog, are not applicable to the present consideration. Immunity to the adult stages of E. granulosus can be induced artificially by immunization with hydatid cyst material (Turner et al., 1936), but in general repeated immunization is necessary before resistance is acquired by the dog (Gemmell, 1966). Herd et al. (1975) used worm secretory antigens obtained from in vitro maintained E. granulosus to immunize dogs. In this case, the immune response reduced the fecundity of the worms rather than their numbers on challenge. Artificially activated embryos of E. granulosus, Taenia hydatigena, and Taenia ovis when given intravenously or intramuscularly to dogs induced a degree of protection against E. granulosus (Gemmell and Soulsby, 1968). TISSUE-DWELLING METACESTODE INFECTIONS

Strong immune responses occur to the metacestodes of the Taenia and Echinococcus species. The majority of such work has been with larval cestodes of rodents, but there is increasing evidence that successful artificial immunization is applicable to the economically important metacestodes of man and his domestic animals. Early work by Campbell (1938), based on passive transfer, revealed a preencystment and postencystment phase of immunity. Thus, serum obtained during the second week of infection of animals with the metacestodes of T. taeniaejormis and Taenia pisijormis caused destruc­ tion of larvae in recipient infected hosts before encystment occurred. However, serum taken several weeks after infection was responsible for thé destruction of larvae after encystment. Immunity of the preencyst­ ment type is of pivotal importance in the control of metacestode in-'

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fections, since establishment of stages less affected by the immune response is avoided. Present evidence suggests that antigens associated with and/or secreted by viable oncospheres and the early reorganizing metacestodes are responsible for this preencystment immunity. Artificial immunization against larval cestodes has been demonstrated with several species of parasites. Gemmell and his co-workers have demonstrated in sheep that strong immunity to ovine metacestodes is induced by the parenteral injection of artificially activated embryos (Gemmell and Macnamarra, 1972). Dead embryos fail to induce im­ munity (Gemmell, 1966). Active immunization against bovine cysticercosis has been induced using parenterally administered eggs (Soulsby, 1961) or activated em­ bryos of T. saginata or T. hydatigena (Wikerhauser et al., 1971 ; Sewell and Gallie, 1974). In the latter studies, the development of a focus of metacestodes at the site of injection of hatched oncospheres appeared to be necessary for good immunity, though occasionally metacestodes from the immunizing injection become generally distributed in the musculature (Sewell and Gallie, 1974). Parenteral injection of oncos­ pheres of T. saginata, attenuated by X irradiation, induced a significant degree of protective immunity and avoided the development of a postvaccinal colony of metacestodes at the site of injection (Wikerhauser et al., 1974). The immunizing capacity of antigens from early develop­ mental stages of larval cestodes is illustrated by evidence that resistance to a challenge infection with eggs is manifest by a reduction in the number of metacestodes developing within 7 to 14 days of the im­ munizing infection (Lloyd, 1975), and Rickard et al. (1976) have also reported successful vaccination of lambs against Taenia ovis with ma­ terial harvested from the in vitro culture of larvae of T. ovis. In further studies Rickard et al. (1977 ) showed that a single vaccination with culture-derived antigen stimulated a high level of immunity which persisted for at least 12 months. Similarly Rickard and Adolph (1976) reported successful immunization of calves against Taenia saginata in­ fection using antigens collected during the in vitro cultivation of larvae. In endemic areas of bovine cysticercosis extended survival of meta­ cestodes (several years) can occur. This is probably associated with the neonatal infection which leads to extended survival of metacestodes, a poor antibody response, and a failure of the animals to develop protective immunity to a second infection (Soulsby, 1963 ). Neverthe­ less, such neonatal infections do not interfere with the establishment of a primary immune response following secondary infection, even while the metacestodes from the primary neonatal infection remain viable in the tissues (Sewell and Gallie, 1974). Thus, though neonatal infection

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may result in the prolonged survival of metacestodes derived from the initial infection, there is no firm evidence that such infections induce immunological tolerance to subsequent infections. The importance of neonatal infection has emphasized the need to con­ trol this type of infection by passive immunization. Urquhart (1961) and Froyd (1964) failed to demonstrate passive transfer of immunity in naturally occurring bovine cysticèrcosis. However, Lloyd and Soulsby (1976) were able to protect neonatal calves against T. saginata infection by feeding immune serum immunoglobulins or immune colostral immunoglobulins. The latter were obtained by local injection of the mammary gland of a preparturient cow with activated onchospheres of T. saginata. Recently, Rickard et al. (1977 ) showed that neonatal calves which had received colostrum from cows vaccinated with anti­ gens obtained from cultures of T. saginata embryos showed a high level of resistance to a challenge infection with T. saginata eggs. Calves which had been protected by colostrum responded with a strong pro­ tective response when immunized with the culture-derived antigen at 8 to 10 weeks of age. Rickard and Arundel (1974) have reported colostral transfer of im­ munity of the larval state of Taenia ovis in sheep following natural infection of the ewes with the parasite. Subsequent work using an in vitro produced antigen showed that ewes immunized with it in late pregnancy conferred a high degree of transcolostral immunity to their lambs against a T. ovis challenge infection (Rickard et al., 1977 ). The immunoglobulin responsible for their protection is not known; but it may be IgA if the situation in rodents is comparable to that in cattle and sheep. Studies of passive immunization against metacestodes of tapeworms which occur in rodents (e.g., T. taeniaejormis) indicate that with colostral transfer of immunity IgA is the primary immuno­ globulin concerned (Lloyd and Soulsby, 1978). Removal of IgA from colostrum ablates the protective response, while IgA isolated by immunoabsorbant columns and fed to neonatal mice protects against T. taeniaejormis infection (Lloyd and Soulsby, 1978). Immunoglobulin A isolated from the intestinal contents is similarly protective and this capacity can be removed by absorption with specific antigen or acti­ vated onchospheres. Transfer with serum has shown that protection in the mouse is due to IgGi, while similar transfer in the rats is due to IgG 2 (Leid and Williams, 1974). An additional aspect of immunity to metacestodes occurs with larval E. multilocularis. The response parallels concomitant immunity in ex­ perimental tumor systems. Rau and Tanner (1976) have demonstrated

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that surgical removal of large primarily established subcutaneous cysts of E. multiloculans resulted in a 10-fold increase in the weight of intrathoracic métastases, though such animals were resistant to subsequent intraperitoneal challenge inoculation with cestode material. As is the case with tumors, thymectomy and anti-thymocyte serum treatment also enhanced the growth and métastases of E. multiloculans in mice (Baron and Tanner, 1976).

VIII. Conclusion Major advances have been made in the understanding of the immune mechanisms which accompany parasitic helminth infection in domestic species. It is obvious, however, that the systems are complicated and are in need of further intensive study. Nevertheless, there is every hope that effective immunoprophylactic measures can be developed for several of the more pathogenic infections. Several are under development and it is likely that the next decade will see the practical application of these. REFERENCES Allonby, E. W., and Urquhart, G. M. (1973). Parasitology 66, 43-53. Anderson, A. J., Brocklehurst, W. E., and Willis, A. L. (1971). Pharmacol. Res. Commun. 3, 13. Andresen, R. H., and Monroe, C. H. (1962). J. Am. Obstet. Gyneocol. 84, 1096-1103. Armour, J., and Bruce, R. G. (1974). Parasitology 69, 161-174. Armour, J., and Dargie, J. D. (1974). Exp. Parasitol. 35, 381-388. Auerbach, R., and Clark, S. (1975). Science 189, 811-812. Ballantyne, A. J., Sharpe, M. J., and Lee, D. L. (1978). Parasitology 76, 211-220. Baron, R. W., and Tanner, C. E. (1976). Int. J. Parasitol. 6, 37-42. Basten, A., and Beeson, P. B. (1970). / . Exp. Med. 131, 1288-1305. Bird, A. F., Waller, P. J., Dash, K. M., and Major, G. (1978). Int. J. Parasitol. 8, 69-74. Butterworth, A. E., Sturrock, R. F., Houba, V., Mahmoud, A. A. F., Sher, A., and Rees, P. H. (1975). Nature (London) 256, 727-729. Butterworth, A. E., David, J. R., Franks, D., Mahmoud, A. A. F., David, P. H., Sturrock, R., and Houba, V. (1977). J. Exp. Med. 145, 136-150. Campbell, D. H. (1938). J. Immunol. 35, 205-216. Campbell, N . J., Kelly, J. D., Townsend, R. B., and Dineen, J. K. (1977). Int. J. Parasitol. 7, 347-351. Chandler, A. C. (1936). Am. J. Hyg. 23, 4&-54. Chen, P. M. (1972). Ph.D. Thesis, Univ. of Pennsylvania, Philadelphia. Chen, P. M., and Soulsby, E. J. L. (1976). Int. J. Parasitol. 6, 135-141. Christie, M. G., Hart, R., Angus, K. W., Devoy, J., and Patterson, J. E. (1978). J. Comp. Pathol. 88, 157-165.

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ADVANCES I N VETERINARY SCIENCE AND COMPARATIVE MEDICINE, VOL. 2 3

Mechanisms of Viral Immunopathology BARRY T. ROUSE* AND LORNE A. BABIUKf * Department of Microbiology, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee, and f Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

I. Introduction I I . Inflammatory Responses Mediated by T Cells I I I . Immune Complex-Mediated Inflammatory Responses IV. Allergic Immunopathological Responses to Viruses V. Immunologie Destruction of Virus-Infected Cells 1. T Cell-Mediated Destruction 2. Destruction by Normal Killer ( N K ) Cells . . . . . . . . 3. Direct Cytotoxicity by Macrophages 4. Destruction by Antibody-Dependent Cell Cytotoxicity (ADCC) . 5. Destruction Mediated by Complement 6. Interaction of Complement and Effector Cells in Destruction of Virus-Infected Targets 7. Destruction by Cytotoxic Lymphokines VI. Initiation of Autoimmune Responses 1. Release of Sequestered Antigens 2. Altered-Self Hypothesis 3. Immunologie Cross-Reactivity 4. Aberrance of Immune Regulation VII. Initiation of Lymphoid Neoplasms VIII. Direct Effects of Viruses on the Immune System 1. Damage to Reticuloendothelial Function 2. Virus-Induced Destruction of Lymphocytes . . . . . . . 3. Suppression of Lymphocyte Function 4. Alteration of Lymphocyte Traffic 5. Suppression of Lymphocytotoxic Antibody 6. Impairment of Suppressor-Cell Control I X . Speculations References

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103 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-039223-2

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I. Introduction Viruses bear the common property of being obligate intracellular parasites, but the outcome of their relationship with the host cell varies considerably. Some, such as several herpesviruses and picornaviruses, irreversibly damage host cell metabolism and cause cell death. Disease results from the direct destruction of massive numbers of particular infected cells. Other viruses, as typified by rubella virus in man does not cause cytolysis but may affect cell metabolism such that cells divide less frequently. In the fetus this may result in abnormal development. Several noncytopathic viruses, although they may not kill cells directly, may trigger cell destruction by indirect means. This indirect destruc­ tion occurs because cells infected by viruses, including both cytolytic and nondestructive forms, may express foreign antigens at their surfaces and these antigens may form the target of a host immunologie response. As we shall discuss subsequently, the mechanisms of cell destruction by immune processes can vary markedly and can be either beneficial, lead­ ing to recovery, or harmful, resulting in damage to the host which we shall consider immunopathology. Noncytopathic viruses may persist in cells, and some such cells seem not to be rapidly destroyed by immune processes. Those infected cells may, however, stimulate an inflammatory response. Such responses are frequently harmful to the host, especially if they occur in certain "delicate" regions, such as the choroid plexus, as occurs in lymphocytic choriomeningitis (LCM) (Hotchin, 1974). Chronic inflammatory processes are considered as immunopathologic and usually involve the participation of antigen-sensitized T lymphocytes and/or the formation of immune complexes that activate complement with inflammatory consequences. Other ways by which viruses may trigger immuno­ pathologic inflammatory responses include the IgE-mediated hypersensitivity mechanism and the activation of complement by way of the alternate pathway. Under some circumstances, the inflammatory response once initiated seems to become "misdirected" and damages certain noninfected host cells, giving rise to a situation referred to as autoimmune disease or immunologically mediated disease. A "misdirected" inflammatory re­ sponse may be the mechanism of demyelination that occurs in visna and as an occasional late manifestation of canine distemper virus in­ fection (Lampert, 1978). Furthermore, the chronic inflammatory re­ sponse can on occasion undergo a neoplastic change, as seems to occur in Marek's disease of chicken. Additional disease states considered as immunopathologic can result

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from virus infections that selectively affect cells of the immune system, such as lymphocytes and macrophages. The result of replication in such cells can be immunosuppression, which in turn usually allows innocuous endogenous or exogenous agents to cause disease. An alternative effect of viruses on cells of the immune system is to cause an aberration of con­ trol mechanisms, such as the destruction or impaired efficiency of suppressor cells—a mechanism favored by many for the pathogenesis of autoimmune disease in NZB mice (Talal, 1977). The purpose of the present review is to discuss briefly the wide range of mechanisms, discovered largely from in vitro models, by which viruses and virus-infected cells can trigger an immunopathologic state. The review will not attempt to credit the original authors of all relevant work in the field, and other reviews will be referenced whenever possible. After a discussion of the philosophy of deciding whether or not a particular process can be considered as immunopathologic, the mechan­ isms of immunopathology are discussed under the five categories listed in Table I. Throughout the review, diseases will be mentioned to illustrate possi­ ble examples of particular mechanisms. The reader should never lose sight of the fact that almost invariably the exact immunopathologic process at play in a particular disease is not adequately understood, that almost certainly multiple mechanisms may be functioning, and finally that our viewpoint may not find favor with others. II. Inflammatory Responses Mediated by T Cells The typical event that follows infection with an acute cytopathic virus infection is cell death occurring in the first 2-3 days. At the site

TABLE I IMMUNOPATHOLOGIC REACTIONS AGAINST VIRAL INFECTIONS

1. Immunopathologic inflammatory responses a. mediated by T cells b. mediated by immune complexes with complement activation ("toxic" com­ plexes) c. allergic reactions against viruses 2. Immune mediated destruction of virus-infected cells (see Table II for examples) 3. Generation of autoimmune disease 4. Generation of neoplastic disease 5. Virus-induced immunodepression

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of the infection, there is a fleeting infiltration of polymorphonuclear neutrophils (PMN), followed by a marked perivascular accumulation of mononuclear cells. This mononuclear cell (MC) response is evident at around 4 days post infection, and virus concentrations rapidly diminish starting at the time of the MC infiltration and have completely disap­ peared by the time antibody in the circulation first becomes detectable at 7-10 days after infection. The MC inflammatory response is quite typical of virus infection and was formerly considered to represent a nonspecific response to virus-induced cell destruction. However, this theory has been repudiated by the experimental studies of Blanden with ectromelia virus and McFarland with Sindbis virus in mice. Their work has clearly shown that the MC inflammatory response is an expression of specific cell-mediated immunity (CMI). The experiments that led to this conclusion have been reviewed by both Blanden and McFarland, and these reviews should be consulted for details (Blanden, 1974; Blan­ den et al, 1976; McFarland and Johnson, 1975). Three basic approaches have been used to indicate the nature and importance of inflammatory responses in acute cytopathic virus infection. First, quantitative de­ terminations were made to follow the time course of appearance of in­ fectious units and the development of immunologie parameters such as antibody production, CMI as detected by both in vivo and in vitro assays, interferon production, and the appearance of macrophages in the inflamed foci. Such studies showed that viral elimination defined as recoverable infectious units coincided with the appearance of specific T-cell immunity and the development of the inflammatory response. The actual nature of the inflammatory response was investigated by pulsing animals for varying periods of time with tritiated thymidine in order to detect dividing cells visualized by autoradiography. Simul­ taneous experiments were performed to identify phagocytic cells. Such approaches indicated that the majority of cells in the inflammatory re­ sponse were rapidly dividing cells of blood-borne origin and were of the monocyte-macrophage cell lineage. The second approach used to characterize the inflammatory response was to correlate the course of viral infection with the nature of the response following procedures such as thymectomy and immunosuppression. These types of experiments showed that recovery was im­ paired when T-cell responses were selectively impaired. Finally, direct evidence for the role and nature of the MC inflam­ matory response came from adoptive-transfer experiments. The usual model involved the transfer of different cell populations to animals either acutely infected and showing necrotic foci or immunosuppressed

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to the extent that the cellular infiltration at the foci was minimal. These studies clearly showed two distinct phases. Thus although the majority of cells in the foci were classified as macrophages, these cells were recruited to the site of virus damage following the appearance of specific T lymphocytes. Only when T cells were adoptively transferred did the full inflammatory response occur and levels of virus diminish. The T cells provide only a minor but essential component of the in­ flammatory reaction, and surprisingly these T cells show only a slight tendency to specifically accumulate at the sites of virus damage. The sequence of events in the MC inflammatory response to viruses is quite typical of all CMI responses studied so far; however, the time course of events may show wide variation depending upon the virus involved. All three of the above approaches argue that, against the two cytopathic viruses investigated, the MC inflammatory response serves a protective role and contributes to recovery from the infection. How­ ever, there are at least three circumstances under which the inflam­ matory response is apparently detrimental to the host and can be considered as immunopathologic : (1) when destruction of cells infected with cytolytic viruses occurs late in the virus host interaction; (2) when destruction of cells infected by noncytolytic viruses occurs; (3) when the antiviral inflammatory response occurs in certain "delicate" areas, such as the choroid plexus. Regarding the first instance, it is considered that if the host response succeeds in destroying virus-infected cells be­ fore fully infectious virus is assembled or spread, then the celldestructive process can be considered as playing a protective role. Experimentally, T cells have been shown to destroy ectromelia virus or vaccinia virus-infected cells before fully infectious virus is formed (Blanden et al., 1976; Zinkernagel and Anthage, 1977)—a protective situation. However, if infected cells do not become susceptible to de­ struction until after infectious virus is assembled, cell destruction may serve no useful purpose. On the contrary, it could indeed be harmful to the host by releasing intracellular infectious virus prematurely, thus accelerating the dissemination of the virus. Furthermore, as discussed below, if antibody is also present, then virus-antibody complexes may be formed that together may incite an inflammatory response. Some have suggested that the destruction of measles virus-infected cells occurs only after virus assembly. Thus, measles may be an example of a viral infection where cell destruction by the inflammatory response is immunopathologic (Morgan and Rapp, 1977). The experiments de­ scribed above have been performed in vitro, and their in vivo relevance has yet to be determined.

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It is generally accepted that the inflammatory cell response against viral infections, similar to that observed in the homograft reaction and the delayed hypersensitivity reaction, results in tissue damage. Such tissue damage is acceptable in most locations in the body, especially if virus inactivation is a consequence. However, in locations such as the choroid plexus, as occurs in lymphocytic choriomenigitis (LCM) in­ fection of mice (Porter, 1975), and in the white matter of the brain, as occurs in visna of sheep (Nathanson et al., 1975), the response can be considered harmful. The final example of the T-dependent immunopathologic reaction is that which results in destroying cells that are infected by noncytopathic viruses and, in the absence of the immune response, would remain intact and viable. A large number of viruses are known that persist intracellularly and usually do not destroy the host cell. Examples include the leukemia viruses and those of visna, equine infectious anemia, aleutian disease, and LCM. In all cases however, a T-cell-mediated immune response (or other mechanisms to be discussed subsequently) may result in cell destruction. Of the viruses mentioned, by far the bulk of experimental evidence for the pathologic role of the inflammatory response concerns LCM virus in the mouse. This topic has received frequent reviews (Nathanson et al., 1975; Doherty and Zinkernagel, 1974; Hotchin, 1974) and will be dealt with only in brief. The LCM virus is an interesting one, since it provided the virus model that helped stimulate Burnet to propose the clonal selection theory of immunity. Thus infection of mice in the neonatal period gives rise to persistent infection and little or no immunologie response. In addition, there is no inflammatory response. Burnet (1959) had proposed that such mice had developed a state of immunologie tolerance. The LCM virus is also noncytocidal to mice fibroblasts in vitro. However, if mice are infected intracerebrally as adults, the mice develop a marked inflammatory response in the choroid plexus and usually die. In distinction to what happens in acute cytolytic virus infections, if the LCM-infected mice with choriomenigitis are given immunosuppressive drugs they usually survive. This observation was taken to indicate that the inflammatory response is the actual cause of death. Further proof for this notion came from adoptive transfers of immune cells into virus-infected, drug-treated recipients. Only the adoptive transfer of specific T cells could impart disease, but, some delay was required to generate an inflammatory focus that consisted largely of host macrophages. These experiments clearly showed that the disease resulted from the inflammatory response and that it was T-cell mediated. Further support for this idea was

109

MECHANISMS OF VIRAL IMMUNOPATHOLOGY

gathered from in vitro studies where it was shown that LCM-infected fibroblasts remained viable, but were rapidly destroyed in the presence of immune T cells (discussed below). Although LCM is the best-studied model to illustrate "delinquent" T-cell-mediated inflammatory responses, there are several other diseases in which an identical mechanism is suspected. These are listed in Table II. Some of these diseases are discussed elsewhere in this volume, and we shall discuss others in more detail in the section on autoimmunity. It is timely to point out here that the chronic inflammatory response in several of these diseases, although it may start as an antiviral reaction, may take on an autoimmune character and destroy normal host tissues.

III. Immune Complex-Mediated Inflammatory Responses It was Arthus who first recognized that, whereas neither antigen nor antibody alone caused inflammatory responses, complexes of the two could do so. Since then, considerable effort has been directed at determin­ ing the pathogenetic mechanism and assessing the importance of immune complex deposition in several diseases. It has clearly been shown that both the size of the complex and the ratio of antigen to antibody in the complex are important in disease production and that several effector enzyme systems are activated by such immune complexes. A diagram depicting the probable interrelated sequence of events is shown in Fig. 1, and the subject has been reviewed (Cochrane and Koffler, 1973;

TABLE II POSSIBLE EXAMPLES OF T-CELL-MEDIATED IMMUNOPATHOLOGIC INFLAMMATORY RESPONSES

Response

References

1. Postvaccinal encephalomyelitis 2. Visna

Nathanson et al., 1975 Nathanson et al., 1975

3. Certain late neurologic signs in measles and canine distemper 4. Coonhound paralysis and the Guillain-Barre syndrome 5. Marek's disease

Morgan and Rapp, 1977 Cummings, 1972; Arnason, 1975 Payne et al., 1976

110

BARRY T. ROUSE AND LORNE A. BABIUK FORMATION OF VIRUS-ANTIBODY COMPLEXES lOCALIZATION OF COMPLEXES IN VESSEL WAllS

J

+ BASOPHIL DERIVED

~

PAF

INCREASED VESSEL

+ COMPLEMENT

PERMEABILITY PLATELET CLUMPING VASOACTIVE AMINE RELEASE

~

RELEASE OF HYDROLASES

BREAKDOWN OF VESSEL WALLS I - - - - - - - - '--A_N_D_B_A_S_E_M_EN_T_M_E_M_B_R_A_NE_S---'-------..

GLOM~RJC~~~~HRlrIS ARTHRITIS

FIG. 1. Diagram depicting the probable interrelated sequence of events in immune complex-mediated inflammatory response. PAF, platelet activating factor; P M N , polymorphonuclear neutrophil.

Oldstone and Dixon, 1975; Cochrane and Dixon, 1976). Basically, it is thought that complexes, usually in moderate antigen excess, and so of small size, become trapped in the walls of small arteries or capillary beds, such as those of the glomeruli and joints. In order for trapping of circulating immune complexes to occur, increased vascular permea­ bility is required. The release of vasoactive amines, such as histamine and serotonin, are perhaps the usual mediators of the increased permea­ bility, and it has been noted that the deposition of complexes and the inflammatory response can be reduced by antagonists of histamine and serotonin (Henson, 1978). At least in rabbits, the platelets are considered to be the most important source of the serotonin. Experi­ mentally, such platelets were shown to release amines after clump­ ing, as occurs when they react with immune complexes in the presence of complement (Humphrey and Jacques, 1955). A more intriguing, complement-independent method of increasing vascular permeability was described in which basophils bearing cytophilic IgE release a mediator termed platelet-activating factor (PAF) upon reaction with the antigen portion of complexes (Henson and Cochrane, 1971). The PAF in turn triggers platelets to release amines. Platelets also contribute to the immune complex lesions by causing vessel thrombosis. Other mechanisms of vascular permeability mediation are also possible (see Fig. 1). One mechanism, shown in Fig. 1 is that by the kinins generated as a result

MECHANISMS OF VIRAL IMMUNOPATHOLOGY

111

of activation of a protein named Hageman factor (Ulevitch and Cochrane, 1977). Whatever the means of localization of immune complexes in vessel walls, the important consequences seem to be the activation of comple­ ment and the generation of an inflammatory response in which chemotactic factors, such as C 5a and CMÏ, attract predominantly polymorphonuclear cells. It is well known that complement activation can lead to the generation of several biological activities as well as inflict membrane damage as occurs when complement (C579, membrane attack unit) is activated on the surface of target cells (Müller-Eberhard, 1975). Included among the substances generated by complement activation are anaphylotoxins, which cause the release of vasoactive amines from mast cells and basophils, and chemotactic factors for neutrophils. The neutrophils bind complexes by means of their Fc and complement re­ ceptors, and this process triggers both phagocytosis and degranulation with release of cathepsins D and E as well as basic proteins that are proteolytic (Henson et al, 1977). Many of these enzymes released cause direct damage to vessel walls and the histopathologic signs, such as arteritis, glomerulonephritis, arthritis, and choriomeningitis. Both the vessel damage and some of the enzymes released, can ac­ centuate the immunopathologic lesion by activating Hageman factor. As with complement activation, Hageman factor activation can lead to a wide range of biological activities, but especially kinin generation, plasmin production as well as blood clotting by way of the intrinsic pathway (Ulevitch and Cochrane, 1977). Circulating and tissue-deposited immune complexes have been found in a variety of viral diseases but are especially prominent in persistent noncytopathic virus infections such as LCM (Oldstone and Dixon, 1975), equine infectious anemia (Henson and McGuire, 1974), aleutian mink disease (Porter, 1975), African swine fever (Pan et al., 1975), and hog cholera (Brunner and Gillespie, 1973). However, some of the lesions induced by acute cytocidal viruses, such as canine distemper and the skin rashes of several virus infections, may be immune complex mediated.

IV. Allergic Immunopathologic Responses to Viruses The type 1 hypersensitivity reaction of the Gell and Coombs classi­ fication (Gell and Coombs, 1968), in which vasoactive amines are re­ leased from cells following the reaction of antigen with surface-bound IgE or some subtypes of IgG, is a mechanism by which viruses could

112

BARRY T. ROUSE AND LORNE A. BABIUK

possibly mediate an inflammatory response. For this mechanism to occur, an initial sensitizing infection would be needed followed by a secondary exposure to a shocking dose of virus. Clinical data in man have associated upper respiratory tract viral infection with bronchial asthma, and viruses have been suspected as précipitants of asthmatic attacks in children (Berkovich et al, 1970). The studies have usually associated incidence of virus isolation with suspected allergic disease and have not directly implicated the virus as the causative antigen. Thus viruses may be associated with allergy by stimulating interferon, which in turn was shown to increase levels of histamine release from IgE-sensitized cells (Gresser, 1977). Some initial reports of IgEmediated hypersensitivity against viruses, such as the suggestion that the mechanism of bronchiolitis in infants with respiratory syncytial in­ fection was of the allergic type (Gardner et al., 1970), have been refuted (Brandt et al., 1973; J. Bienenstock, personal communication). Bienen­ stock and his colleagues were able to show the production of IgE antibody in rabbits upon experimental infection with herpes simplex virus (Day et al., 1976), but attempts to show similar antibody in man and to incriminate this as of immunopathologic significance have not been successful (J. Bienenstock, personal communication). The authors are unaware of studies in domestic animals attempting to demonstrate IgE-type antibodies to viruses, but clearly this would be an interesting avenue of research to investigate. Perhaps the shipping fever complex owes part of its pathogenesis to the IgE-mediated inflammatory re­ sponse, but this notion awaits experimental investigation.

V. Immunologie Destruction of Virus-Infected Cells As previously discussed, the destruction of virus-infected cells by the immune system can be considered either as beneficial or immunopatho­ logic according to the type of virus infection and the kinetics of the destructive process. In this section, we briefly discuss the large numbers of mechanisms, recognized from in vitro studies, by which virus-infected cells may be destroyed. These are summarized in Table III. Apart from destroying the virus-infected cell, additional tissue damage can result from the further dissemination of virus and from the release of certain metabolites from infected cells. Viral capsids released from cells may produce toxic effects (Amako and Dales, 1967). The best known other product is double-stranded RNA—an intermediate formed during

MECHANISMS

OF VIRAL

IMMUNOPATHOLOGY

113

TABLE III SOME IMMUNOLOGIC M E C H A N I S M S FOR DESTROYING VIRUS-INFECTED CELLS

Mechanisms of cytotoxicity

References

1. T cell

Blanden, 1974

2. Normal killer cell

Herberman et al, 1975; MacFarlan et al, 1977 Stott et al, 1975; Lodmell et al, 1973 Evans and Alexander, 1972 Rager-Zisman and Bloom, 1974; Shore et al, 1974; Rouse et al, 1976

3. Macrophages (nonspecific) 4. Macrophages ("armed") 5. Antibody-dependent cell cytotoxicity (ADGC) 6. Complement-facilitated ADCC 7. Complement-dependent cell cytotoxicity 8. Antibody-complement lysis 9. Alternate pathway complement activation 10. Release of cytotoxic lymphokines by T lymphocytes

Rouse et al, 1977 Grewal and Rouse, 1979 Rawls and Tompkins, 1975; Joseph et al, 1975 Welsh and Oldstone, 1977 Granger and Williams, 1968

RNA virus replication (Carter and deClercq, 1974). The multiple bio­ logie effects of double-stranded RNA have been reviewed by Carter and deClercq (1974). One of these effects is interferon production; this, as discussed subsequently, may in turn have immunopathologic consequences, since it may inhibit cell division and suppress the im­ mune response (Gresser, 1977). A further effect of interferon that may contribute to the immunopathologic scene is its ability to enhance the effector phase of the immune response. By so doing, the extent of immune destruction of virus-infected cells may increase. This enhance­ ment effect of interferon has been noted against at least two of the in vitro immunologic effector mechanisms by which virus-infected cells may be destroyed—namely, T-cell cytotoxicity and antibody-dependent, cellular cytotoxicity (Babiuk and Rouse, 1978; Gresser, 1977). Inter­ feron may also enhance some functions of macrophages (Gresser, 1977), which, as was discussed previously, are the major cell types found in antiviral inflammatory responses. There follows a discussion of some of the immunologic mechanisms, recognized from in vitro models by which virus-infected cells may be destroyed.

114

BARRY T. ROUSE AND LORNE A. BABIUK

1. CELL-MEDIATED DESTRUCTION

As first described by Brunner's group, specific T lymphocytes are extremely effective at lysing target cells in vitro (Cerottini and Brunner, 1974). To detect such lysis, usually target cells are labeled with sodium chromate, and the release of the 51Cr label is taken as an indi­ cation of cell destruction. In the mouse system, where several distinct cell markers of T cells are available, this has clearly been shown to be a T-cell-mediated mechanism. A wide range of antigens have been examined, including several cytolytic and persistent virus infections (Blanden, 1974; Doherty and Zinkernagel, 1974; Rouse and Babiuk, 1977). The T-cell-mediated destruction is specific; however, in the mouse system the antigen being recognized by T cells appears not to be solely of viral origin. Thus Doherty, Zinkernagel, Blanden, and others (Doherty and Zinkernagel, 1974) have irrefutable evidence to show that certain antigens coded by the major histocompatibility region additionally need to be recognized at least in murine antiviral T-cell cytotoxicity. The exact nature of the recognition mechanism of T cells remains one of active research; it is outside the scope of the present review and has been lucidly discussed in several re­ views (Doherty and Zinkernagel, 1974; Blanden et al., 1976; Zinker­ nagel, 1978; Shearer, 1977). It is perhaps germane to indicate that genetic restriction has not been easy to demonstrate in nonrodent species. Thus, such restriction could not easily be demonstrated in either bovine (Rouse and Babiuk, 1977), rabbit (Woan et al., 1978), or canine (Ho et al., 1978) systems, and results in man have been controversial (P. C. Doherty, personal communication).* Apart from the controversy regarding the mechanism of recognition in direct T-cell-mediated direct cytotoxicity, the actual mechanism of cell destruction needs further definition. Moreover, it has not been possible to show unequivocally that direct T-cell-mediated cytotoxicity occurs in vivo and whether or not it is an important mechanism either of recovery or of producing immunopathologic lesions. Although in vitro one T cell can destroy several target cells (Henney, 1973), the mechanism requires contact, and consequently individual immune T cells would need to come into actual contact with virus-infected cells to be effective. Since there is only a very slight tendency for immune T cells to selectively localize at the site of their specific antigen (Blanden et al., 1976), the chance of T cells contacting all target cells in vivo may not be high. For this reason, it may be that indirect T-cellmediated effects, such as macrophage recruitment, may be the more important in vivo mechanism of immunity and immunopathology. * Genetic restriction has recently been demonstrated in the dog (Shek, 1979). [Ed.]

MECHANISMS

OF VIRAL

IMMUNOPATHOLOGY

2. DESTRUCTION BY NORMAL KILLER (NK)

115

CELLS

In human and murine systems, a population of lymphocytes has re­ cently been demonstrated that can destroy, apparently nonspecifically, a wide range of cells that bear foreign antigens. (Herberman et al, 1975; MacFarlan et al, 1977; Welsh and Zinkernagel, 1977). These targets include tumor cells and cells infected with viruses. As shown by Herberman's group, NK cells are considered to be T cells but have characteristics that distinguish them from the "classical" cytotoxic T cells discussed above. In the mouse system, for example, NK cells have only a low affinity for sheep erythrocytes and, unlike cytotoxic cells, have membrane receptors for the Fc portion of IgG molecules. Several groups have described properties that distinguish NK cells, but these descriptions tend to differ among different laboratories (Kay et al., 1977; Haller et al, 1977). As to whether NK cells play any role in immunity or in the development of immunopathologic lesions in vivo needs additional investigation. However, NK cells will destroy cells infected with herpes simplex virus (Fujimiya et al., 1978), togaviruses (MacFarlan et al, 1977), LCM virus (Welsh and Zinkernagel, 1977), and probably oncornaviruses (Shellam and Hogg, 1977).

3. DIRECT CYTOTOXICITY BY MACROPHAGES

As discussed previously, macrophages are considered to be important cells in controlling virus infection following their migration to, and activation at, the sites of viral lesions. Such recruitment is accomplished by T-cells, lymphokines as well as activated inflammatory agents (Blanden et al., 1976). At the site of the lesion, monocytes from the circulation take on a different appearance and are referred to as activated. These cells may be prominent in chronic immunopathologic reactions occurring with persistent infections. Upon activation, macro­ phages may be cytotoxic for tumor cells and virus-infected cells (Hibb's et al, 1972; Stott et al, 1976; Lodmell et al, 1973; C. J. Wust and A. Brown, personal communication). This cytotoxicity is not generally specific for cells bearing the inducing antigen although normal cells are unaffected. Reports of specific so-called "armed" macrophages have been reported in some tumor systems (Lohmann-Matthes et al, 1972; Evans and Alexander, 1972). P. M. Henson (unpublished observations, 1978) has indicated that there is a sliding scale of macrophage activation from quiescent through stimulated to activated. Only the ac­ tivated macrophages express the complete spectrum of macrophage function, which includes direct cytotoxicity. One agent that can change stimulated macrophages to the fully activated form appears to be a

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BARRY T. ROUSE AND LORNE A. BABIUK

C5a—a fragment of the complement cascade (P. M. Henson, unpub­ lished observations, 1978). Whether generated by the classical, alter­ nate, or plasmin pathways, this fragment should be present at a site of inflammation, and this may be an important mechanism of generating cytotoxic macrophages in vivo. However, as with all systems of direct cytotoxicity discussed thus far, evidence that the phenomenon actually occurs in vivo had not been presented. 4. DESTRUCTION BY ANTIBODY-DEPENDENT CELL CYTOTOXICITY (ADCC)

As first reported by Shore et al. (1974) and Rager-Zisman and Bloom (1974), virus-infected cells may be destroyed by "nonspecific" effector cells provided that the infected cells have bound specific antiviral antibody. This mechanism of cytotoxicity, by design, can be demon­ strated only in vitro but some have suggested an in vivo role for ADCC in killing virus-infected cells (Rouse et al., 1976; Zisman and Allison, 1973; Allison, 1976). Certainly, the mechanism is a potentially important one since it requires only trace amounts of antibody, and any of several cells, all of which must bear Fc receptors for the sensitizing antibody, can act as effector cells (McClennan, 1972; Cerottini and Brunner, 1975). A wide range of effector cells have been reported; these include T, B, and null lymphocytes (K cells), macrophages, and granulocytes. In any particular circumstance, the relative importance of different cell types seems to vary, and explanations for these variations have not been forthcoming. Thus against IBR virus-infected cell targets, we have shown that neutrophils are more efficacious than macrophages and that lymphocytes fail to destroy cells despite the presence of the required Fc receptors (Grewal et al., 1977). Although ADCC is a dramatic and rapid means of destroying virus-infected cells in vitro, this phenomenon may not be operative in vivo, at least in some persis­ tent viral infections where immunopathologic events are suspected to occur. Thus, with several viruses, especially measles, antibody has been shown to remove viral antigens from the membranes of infected cells by modulation (Joseph and Oldstone, 1975). This modulation process would serve to protect virus-infected cells against ADCC. 5. DESTRUCTION MEDIATED BY COMPLEMENT

In vitro studies have shown with a variety of viruses that virusinfected cells can be destroyed by antibody and complement (Rawls and Tompkins, 1975; Joseph et al, 1975; King et al., 1977). The com­ plement activation may be either by the classical or alternate pathways.

MECHANISMS OF VIRAL IMMUNOPATHOLOGY

117

Furthermore, Oldstone's group has indicated that certain virus-infected cells can activate complement directly by the alternate pathway and can be destroyed without the participation of antibody (Welsh and Oldstone, 1977). Immunopathologic reactions against virus-infected cells that clearly involve complement have been shown to occur in equine infectious anemia (see review by McGuire and Crawford in this volume). In this disease, the anemia is at least partly attributable to complementmediated lysis or opsonization of erythrocytes. The involvement of complement in the immunopathologic viral infection LCM has been inferred from the fact that mice depleted of complement by cobra venom factor are more resistant to LCM disease (Rawls and Tompkins, 1975). However, as discussed under ADCC, a role for complement in the in vivo destruction of antibody-sensitized virus-infected cells may not occur in vivo because of the phenomenon of antigenic modulation. 6. INTERACTION OF COMPLEMENT AND EFFECTOR CELLS IN DESTRUCTION OF VIRUS-INFECTED TARGETS

Recently we have shown two additional ways in which complement can aid in the destruction of virus-infected cells in vitro. These have been referred to respectively as complement-facilitated ADCC (CADCC) (Rouse et al., 1977) and complement-dependent cell cytotoxicity (CDCC) (Grewal and Rouse, 1979). In the first mechanism of cytotoxicity (C-ADCC), complement was shown to enhance the speed and extent of destruction of antibody-sensitized IBR virus-infected cells. This effect was especially apparent under limiting conditions, such as short-term assays, low levels of antibody, and low effector-totarget cell ratios. The second means of cytotoxicity, CDCC, has recently been observed to occur when complement is added to IBR-infected cells in the presence of polymorphonuclear leukocytes. Since this latter cytotoxic mechanism occurs in the apparent absence of components of specific immunity, it may provide an early mechanism of recovery or immunopathology in vivo. These speculations, however, are in need of experimental verification. 7. DESTRUCTION BY CYTOTOXIC LYMPHOKINES

Finally, a mechanism of destruction of virus-infected cells could occur by means of a cytotoxic lymphokine, such as lymphotoxin re­ leased by lymphocytes following reaction with viral antigen (Granger and Williams, 1968). As with all the above-mentioned in vitro assays that detect destruction of virus-infected cells, whether or not a lymphotoxin-mediated mechanism occurs in vivo has not been definitely proved.

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BARRY T. ROUSE AND LORNE A. BABIUK

VI. Initiation of Autoimmune Responses Antibodies and autoreactive cells can be present in an apparently benign form, but in some cases they are considered to play an important role in immunopathology. These situations are referred to as autoimmune disease (AID). Immunologists delight in speculating about the etiology and pathogenesis of AID; of several hypotheses that have appeared, many have incorporated a role for infectious agents, particularly viruses. A list of some of the more common AID hypotheses is presented in Table IV, and the topic of the role of viruses in AID has been reviewed elsewhere (Levy, 1974; Talal, 1977; Hirsch and Proffitt, 1975; Lewis, 1974; Datta et al, 1978; Phillips, 1975). No single hypothesis satisfactorily explains all autoimmunity, and it seems likely that more than one mechanism could occur in a given disease, especially since these diseases present a widely varying history and clinical expression. For convenience, in this review we shall deal with some of the major hypotheses and briefly discuss diseases that in our opinion may best be explained by the particular mechanism. T A B L E IV SOME VIRUSES OF VETERINARY INTEREST T H A T CAUSE IMMUNOSUPPRESSION

Cell mediated Virus

In vitro

In vivo

Canine distemper

+

+

Rinderpest Bovine viral diarrhea

Humoral In vitro

Krakowka and Koestner, 1977; Mangi et al., 1976

+

+

Woodruff and Woodruff, 1975a

+

+

Muscoplat et al., 1973; Johnson and Musco­ plat, 1973 Porter, 1975

+

+

+ +

+ +

+ +

Avian leukosis Feline leukemia Feline panleukopenia Infectious bural agent Hog cholera

References

+

Aleutian disease Marek's disease

In vivo

+

+

+ + + +

Purchase et al., 1968; Payne et al., 1976 Purchase et al., 1968 Essex, 1975 Schultz et al., 1976 Faragher et al., 1974 Tizard, 1978

MECHANISMS

OF VIRAL

IMMUNOPATHOLOGY

119

1. RELEASE OF SEQUESTERED ANTIGENS

This hypothesis proposes that any antigen absent or physically separated from the immune system during the time when tolerance to self is being established will be treated as foreign if it subsequently becomes exposed to the immune system. Major support for this idea came from the observation that the apparently sequestered self-antigens could induce autoimmune disease upon injection into the host under appropriate conditions (Witebsky and Rose, 1956). There is clearly a link between certain virus infections and demyelinating syndromes, which apparently are of autoimmune nature (Weiner et al., 1973; Lampert, 1978). This notion receives the strongest support from the induced disease, experimental allergic encephalomyelitis (EAE), that can be produced by injecting myelin components into animals, usually in the presence of an adjuvant (Patterson, 1969). The disease EAE is characterized by a progressive development of neurologic signs and pathologically by marked infiltrâtes of mononuclear cells, myelin destruc­ tion, and ultimately Wallerian degeneration of axons. A similar syn­ drome, affecting peripheral nerves, can be induced by exposing animals to myelin components from the peripheral nervous system (Arnason, 1975). Both EAE and experimental allergic neuritis (EAN) have been shown to result largely from an autoreactive T-cell response against myelin components (Nillson, 1972). The T-cell response results in the recruitment of a mononuclear cell inflammatory response and marked tissue damage. Immunosuppressive treatment markedly alleviates the disease. Syndromes resembling EAE and EAN can occur as a sequel to infection with several enveloped virus infections (Weiner et al., 1973 ; Cummings, 1972; Arnason, 1975; Morgan and Rapp, 1977). Viruses that bud from the cell surface and incorporate host antigens in their outer envelope, such as measles and canine distemper virus (CDV), are the usual culprits. In the case of CDV, encephalomyelitis occasionally occurs some weeks or even months after acute, or in some cases inapparent, infection (Appel and Gillespie, 1972). This, however, is the exception, and encephalomyelitis generally occurs as an acute manifesta­ tion of viral infection. In the subacute or chronic disease, there is often a marked perivascular infiltration of mononuclear cells and demyelination, and the condition is usually progressive and fatal. The disease has been extensively investigated by Koestner's group, who consider that the disease occurs mainly with neurogenic strains that may either be largely defective or temperature-sensitive mutants (Koestner, 1975). Whether the disease induced occurs as a result of the release of myelin components because of viral effects on oligodendroglia cells, or whether

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BARRY T. ROUSE AND LORNE A. BABITJK

it occurs because of the well-known ability of parainfluenza viruses, such as CDV, to cause cell fusion, has not been proved. However, that the postinfectious disease could have an autoimmune nature is indicated by the predominant effects on white matter and the mononuclear cell infiltrâtes reminiscent of EAE (Wisniewski et al., 1972). There are also reports of autoantibodies against myelin (McCullough et al, 1974 ) and reports that sera from CDV-affected dogs cause demyelination in vitro (Lampert, 1978). However, whether or not circulating T cells reactive against myelin components also occur or,if immunosuppressive drugs are beneficial is in need of investigation. Since both measles virus and CDV are immunosuppressive (Morgan and Rapp, 1977; Schultz, 1975; Krakowka and Koestner, 1977), it is feasible that their effect on immunoregulation may be the primary or contributory reason for the late autoimmune complication. The immunoregulation hypothesis of AID is discussed subsequently. Other diseases of domestic animals, which apparently trigger an autoimmune state and whose etiology may be similar to postdistemper encephalomyelitis included coonhound paralysis (Cummings, 1972), and Marek's disease (Payne et al., 1976). The former disease, which resembles the Guillain-Barrc syndrome of man (Arnason, 1975; Behan et al., 1970) and affects peripheral nerves of dogs, is thought to result from a viral infection (Cummings, 1972). Evidence that the Marek's disease agent may precipitate an autoimmune demyelination is discussed by Payne et al. (1977), and the topic of the pathogenesis of MD is further discussed elsewhere in this review. 2. ALTERED-SELF HYPOTHESIS

Autoimmune immunopathologic reactions resulting from virus-induced alterations of host cell membranes -are expected to occur following in­ fection with a wide range of oncogenic and nononcogenic viruses. Thus, several viruses cause cells to express either viral antigens in their mem­ branes or the expression of new host cell coded antigens. These latter antigens may either be embryonic antigens (Baldwin et al., 1974; Coggin and Anderson, 1974) or alloantigens normally expressed by other members of the species (Garrido et al, 1976). A recent example of the latter effect was postulated by Festenstein's group, who showed that foreign H-2-like transplantation antigens were expressed following infection with vaccinia and Moloney virus (Garrido et al., 1976). These studies have yet to distinguish whether the mechanism of H-2-like antigen expression is the result of activation of an endogenous virus that happens to cross-react with the foreign H-2 antigens or the result of derepression of structural genes for foreign H-2 alleles. Although

MECHANISMS OF VIRAL IMMUNOPATHOLOGY

121

semantically only the latter circumstance, of which there is no definite evidence in eukaryotic cells, is true autoimmunity, to distinguish be­ tween the two may be academic since either may result in cell destruc­ tion by an immunopathologic response. Since the H-2 transplantation antigens elicit strong immune responses, it is easy to imagine that an autoimmune response could be directed against them. Several viruses of both the DNA and RNA groups may trigger the reexpression of embryonic antigens on cells (Coggin and Anderson, 1974). The explanation for the appearance of these antigens is not known, but derepression is considered to be a likely mechanism. Ex­ amples where these embryonic antigens form the likely target of an autoimmune response are not numerous, but the hemolytic antibodies directed against the human fetal erythrocyte antigen l a that occurs after Epstein-Barr and cytomegalovirus infections may be a case in point (Pier et al, 1974). 3. IMMUNOLOGIC CROSS-REACTIVITY

This mechanism proposes that viruses, particularly those that bud from cell surfaces and incorporate host antigen, may induce an im­ mune response that may react with antigens shared by the virus and host cell. Viruses are considered to act as carriers for host antigens and so render the weak self-antigens more immunogenic. Evidence for this mechanism was obtained with influenza virus, where it has been shown that infection of tumor cells renders the cell immunogenic in the normally unresponsive host—a phenomenon frequently referred to as the Lindenmann effect (Lindenmann, 1974). A similar explanation was put forth to explain the autoimmune hemolytic anemia that may follow leukemia virus infection of mice and perhaps cats (Cox and Keast, 1973; Scott et al., 1973). The murine viruses bud from erythrocyte cell membranes and incorporate membrane antigen that becomes im­ munogenic, since normal uninfected erythrocytes are ultimately de­ stroyed. Similarly with Moloney virus infection of mice, a cytotoxic T-cell response is generated that initially destroys cells producing virus. Eventually, however, the lymphocytes become cytotoxic only for noninfected cells (Proffitt et al., 1973) ; possibly in this case an additional phenomenon occurs with antiviral antibody blocking the cytotoxic cells as described by the Hellströms (Hellström et al., 1977). 4. ABERRANCE OF IMMUNE REGULATION

Viruses are strongly suspected as etiological agents in the well studied AID that occurs in NZB mice and their hybrids (Levy, 1974). In these models, the actual mechanism of disease pathogenesis is thought

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to occur as a result of diminished suppressor T-cell function (Talal, 1977). Considerable evidence has been generated to show that at least some examples of immunologie tolerance are maintained normally by the presence of suppressor cells that serve to prevent other cells from responding to antigen (Gershon, 1974). Consequently, unresponsiveness is regulated in an active fashion. If the suppressor cell becomes aberrant, then animals show hyperresponsiveness to foreign antigen and also responsiveness to self. This is the case in NZB mice. These mice respond more actively, especially as young adults, to a wider range of antigens than do mice of other strains (Talal, 1977). It is also very difficult to produce experimental tolerance in NZB mice. Furthermore, starting at around 2 months of age, there is a loss of tolerance to several self antigens and autoantibodies are produced. Several investi­ gators have shown that the onset of loss of tolerance coincides with the disappearance of suppressor cells, but what causes their disappearance is not known (Talal, 1977; Steinberg, 1974; Krakauer et al, 1976). Suppressor cells have been demonstrated by a variety of complex assays (reviewed by Gershon, 1974) and were identified as subsets of T cells. Of possible future therapeutic significance, it was shown that the decline in suppressor function could be delayed not only by re­ peated transfers of thymus cells from young mice, but also by thymic extracts containing thymic hormones (Talal, 1977). In line with these observations, thymectomy may speed up the development of disease. Several recent papers have also appeared that support the hypothesis that loss of suppressor cell control is involved in the pathogenesis of human systemic lupus erythematosis (SLE) (Abdou et al., 1976; Horo­ witz et al., 1977 ). The crucial question, of course, is what are the factors responsible for the loss of suppressor cell control? Considerable circum­ stantial evidence has suggested a role for viruses (Levy, 1974). If viruses are the cause of the decline in suppressor cell function, one must predict a highly selective tropism by viruses for such cells. Al­ though examples of selective destruction by viruses of T cells or of B cells has been described, as have differences in virus susceptibility of lymphocytes in varying stages of development (Schneider and ZurHausen, 1975; Cerny and Waner, 1975; Pelton et al., 1977), the authors are not aware of examples of viruses being able to discriminate between closely similar T-cell subsets. However, suppressor cells and other T-cell sub­ sets do manifest alloantigenic differences in their cell membranes (Cantor and Boyse, 1975), so differential susceptibility to viruses is possible. In support of the causative role of viruses in AID of NZB mice and their hybrids are the observations that these mice express high levels

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of type C xenotropic viruses (Levy, 1975). One of the coat antigens of these viruses (gp 70) circulates in high concentration and is found in the immune complex lesions (Yoshiki et al., 1974). Recently, how­ ever, the virus hypothesis took a setback when genetic studies using backcrosses showed a disassociation between viral expression and autoimmunity (Datta et al, 1978). In contrast, in the canine model viruses are strongly suspected to be etioiogic agents (Lewis, 1974), but whether or not they function in this role by destruction of suppressor cells is open for conjecture. The viral hypothesis of canine SLE was supported by the work of Lewis and Schwartz (1971). In an attempt to prove the genetic basis of canine SLE, these workers produced a large colony of dogs using as a nucleus natural clinical cases of SLE. Although they did not succeed in producing clinical cases of SLE, the dogs had disease as judged by the criterion of antibody development to native double-stranded DNA. Since almost all the dogs developed SLE, including backcrosses that on genetic predictions should not have done so, the workers strongly suspected an infectious agent trans­ mitted either vertically or horizontally. Evidence was produced for this by showing that SLE could be transmitted not only to puppies, but also to mice, by spleen filtrates from affected dogs. Furthermore, mice receiving the spleen filtrates frequently developed neoplasms attributable to the activation of endogenous murine leukemia viruses. Extracts of these mouse tumors not only were shown to induce tumors in susceptible mice, but also the recipient mice produced antibodies to native double-stranded DNA, and, if injected back into dogs, antiDNA antibodies developed but not any other signs of SLE. Studies by Lewis and Schwartz suggest an as yet unidentified infectious agent, possibly a viral agent, however, these studies await verification by other investigators. The hypothesis favored by Lewis and Schwartz to explain their data is that susceptible individuals contain an inte­ grated quiescent genetic sequence called a "lupogene" that may be activated by a virus and indeed may be virus coded. Datta et al. (1978) have termed this putative sequence the autoimmunity response locus but have not suggested that it is virus coded. Both hypotheses sug­ gested that a variety of cofactors may activate the lupogene and SLE develops; all cofactors could function by their effects on suppressor cells. It should be noted that antibody to double-stranded DNA cannot be produced by any immunization schedule attempted. Although removal of suppressor cell control is currently the most popular explanation for AID, aberrant immunoregulation could equally well occur as the result of viruses stimulating helper cell function (Allison, 1973) or perhaps by acting as nonspecific adjuvants (Patter-

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son, 1973). That viruses may act as adjuvants for responses to ex­ traneous antigens has been reported (Patterson, 1973). The idea of virus-reinforced helper-cell function becomes theoretically attractive when considered in light of the fact that, in normal individuals, B .cells that react with self antigens can readily be demonstrated (Allison, 1976). The responsiveness is assumed to occur because of a lack of antigen-reactive helper T cells. Allison and colleagues (1971) have lucidly argued that viruses could activate and trigger helper cells and lead to autoimmunity, but direct evidence for such a hypothesis needs to be obtained.

VII. Initiation of Lymphoid Neoplasms It would be unrealistic to attempt to review the topic of viruses as causes of neoplasms, and it would be wrong to interpret all neoplasms as examples of immunopathologic reactions. However, persuasive argu­ ments have been presented by others to suggest that many tumors may occur as a result of the loss of suppressor cell control, and other reviews should be consulted for a full discussion of this topic (Gershwin and Steinberg, 1973; Essex, 1975). Certain lymphoid tumors appear to start out as immunopathologic reactions. The herpesvirus-induced lymphoproliferations and neoplasms are good examples of this phenomenon (Payne et al, 1977; Epstein and Achong, 1977). Infectious mononucleosis of man for example, is considered to represent a T-cell proliferative response against B cells that are infected with the Epstein-Barr virus (Epstein and Achong, 1977). Ultimately, the T-cell immunopatho­ logic reaction subsides and recovery occurs, but the virus persists in a latent form. It could be that, in the presence of unknown cofactors, a lymphoma may occur. This idea is supported by the fact that in Marek's disease of chickens, caused by herpesvirus similar to EBV, neoplastic lymphoid transformation commonly follows the immunopathologic re­ action and recovery is uncommon (reviewed by Payne et al., 1977). Two major hypotheses have been presented to interpret the etiology and pathogenesis of Marek's disease (MD), namely, the intrinsic and extrinsic theories. The former hypothesis, which attracts the most adherents, states that the lymphoid proliferations result from each cell being infected by the MD virus genome. According to this hypothesis, the MD lesions should not be considered as immunopathologic. The extrinsic hypothesis interprets the lymphoid infiltrâtes, which have been shown to be mainly T cells (Rouse et al.y 1973), to be inflammatory cells reacting against, and destroying, target cells, which in turn are

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infected by the MD virus. The target cells may carry integrated genomes, and the antigens against which the T cells are directed may be either virus-coded antigens, unmasked host antigens, or perhaps derepressed fetal antigens. Seen in this light, the lesions are immunopathologic and benign, and in fact the reactions may regress, especially in genetically resistant birds (Sharma et al., 1973). Supporting the immunopathologic interpretation of the MD lesions are the findings that thymectomy and immunosuppressive drugs may reduce the inci­ dence and severity of disease (Payne et al., 1976). However, in MD, the lymphoid proliferation ultimately takes on the appearance of a neoplasm and is considered as a lymphoma. The cofactors responsible for this change have not been identified, but a loss of suppressor cells, perhaps as a result of secondary infection, or activation of endogenous C-type viruses that selectively affect the suppressor cell function, seems to us to be the most likely explanation. Alternatively, the proliferating T cells themselves may eventually become transformed upon ultimate infection by the MD oncogene (Rouse and Warner, 1974). VIII. Direct Effects of Viruses on the Immune System Viruses can cause immunopathologic effects by directly affecting the function of cells, such as lymphocytes and macrophages, that are in­ volved in immunity. Several viruses are known that depress the activity of the immune response, and the topic of viruses and immunosuppression has received several reviews (Notkins et al., 1970; AVoodruff and Wood­ ruff, 1975a,b; Dent, 1972). A few viruses are known to enhance im­ munologie activity (Oldstone, 1975), but this topic will receive no further discussion in this chapter. Virus-induced immunosuppressive effects can vary from barely apparent to very severe, and many aspects of the immune response may be affected. Thus, following the initial report of virus-induced immunosuppression in 1908 by von Pirquet with measles virus infections, reports of effects of many different viruses on delayed hypersensitivity, allograft response, in vitro responses to mitogens and antigens, levels of immunoglobulins, antibody formation, and the function of the reticuloendothelial system have all appeared. The topic was succinctly reviewed by Woodruff and Woodruff (1975a,b). Some of the virus infections of veterinary interest that suppress various aspects of immunity are listed in Table IV. In many cases, the virus-induced immunodepression is considered to have clinical significance. Some have preferred to cite such conse­ quences as examples of microbial synergism (Machowiak, 1978). The

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shipping fever complex and neonatal diarrhea of calves, both of which are more severe in combined viral-bacterial infections (Jericho et al., 1976), may be examples of such synergism. Perhaps the best-documented example in the veterinary literature of virus-induced immunosuppression is feline leukemia-affected cats, which may show profound depression of immunity and develop a wide spectrum of secondary infections (re­ viewed by Mackey, 1965; Essex, 1975). Canine distemper virus also has been shown to be immunosuppressive both by in vitro and in vivo assays (McCullough et al, 1974a; Krakowka and Koestner, 1977). The clinical result of this immunosuppression is viral persistence and possibly late sequelae, such as subacute distemper-associated encephalitis (Appel and Gillespie, 1972; Appel, 1969; Koestner, 1975), or secondary bacterial infection, such as Bordetella pneumonitis (Schultz, 1978). Although the concept of viruses as causes of immunosuppression is well accepted, more work is required, especially with domestic animal disease models, to determine the mechanism and to document fully the clinical relevance of the immunosuppression. Some of the possible mechanisms by which viruses could induce immunosuppression are listed in Table V. They are discussed below. 1. DAMAGE TO RETICULOENDOTHELIAL FUNCTION

Considerable speculation has appeared suggesting that viruses may damage the function of the reticuloendothelial system so that bacterial clearance is impaired and secondary infection occurs. However, there are few hard data to attest to this hypothesis and some evidence to

TABLE V POSSIBLE M E C H A N I S M S OF VIRUS-INDUCED IMMUNODEPRESSION

1. Damage to reticuloendothelial function 2. Direct destruction of lymphocytes a. Resulting from virus replication b. Because of generation of lymphocytotoxins 3. Suppression of lymphocyte function a. By altering surface receptor function b. Because of stimulation of interferon(s) 4. Alteration of lymphocyte traffic 5. By means of lymphocytotoxic antibody production 6. Impairment of suppressor-cell control 7. Neoplastic transformation of antibody-forming cell precursors

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refute it. Thus some viruses known to replicate in macrophages and to produce different effects on in vivo responses to antigen may react with antigens identically in vitro. This has been shown with two viral in­ fections of mice: LCM, which suppresses immune responses, and lactic dehydrogenase virus, which enhances responses (Oldstone, 1975; Oldstone et al, 1974). Theoretically, viruses could suppress macrophage and neutrophil function by impairing their response to chemotactic stimuli. Some evi­ dence for this has been reported for cytomegalovirus infection in macrophages (Notkins et al., 1970) and for influenza infections in neutrophils (Ruutu et al., 1977). Similarly, phagocytosis itself may be suppressed by some viral infections (Merchant and Morgan, 1950; Larson and Blandes, 1976). Both phagocytosis and chemotaxis are measured by relatively imprecise in vitro assays, and it is by no means clear whether the effect is of sufficient magnitude to be of significance in vivo. It would be of interest if viruses were found to affect the microbicidal activity of phagocytes, but the authors know of no such reports. 2. VIRUS-INDUCED DESTRUCTION OF LYMPHOCYTES

Direct destruction of lymphocytes is one obvious way by which virus infection can lead to immunosuppression. Indeed, one of the hallmarks of several virus infections is lymphopenia—this being especially pro­ nounced in feline panleukopenia (Povey, 1976), canine distemper (Ap­ pel and Gillespie, 1972), and several pestivirus infections (Brunner and Gillespie, 1973). Although the mechanisms of lymphopenia could be multiple, it may result in some cases from virus replication in lympho­ cytes or their precursor cells. Canine distemper (Appel and Gillespie, 1972) and feline panleukopenia (Povey, 1976) provide respective ex­ amples of the two mechanisms. Other examples of lymphopenia may be explained by differences in lymphocyte traffic (discussed below), by cytotoxic immune responses against virus-infected lymphocytes, or by the indirect effect of the generation of lympholytic substances, such as corticosteroids. It seems probable that the thymic atrophy that may occur in cats with feline leukemia may be an example of indirect cell destruction possibly mediated by corticosteroids. No direct evidence for the notion is available, but at least in the mouse certain thymocytes are extremely sensitive to corticosteroids (Claman, 1972). Although some viruses, such as measles, CDV, and herpes simplex, can replicate in, and destroy, resting lymphocytes, stimulated cells support replication more readily (Woodruff and Woodruff, 1975b).

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In fact, many viruses can replicate only if lymphocytes are previously stimulated. Examples of this phenomenon were reported with vesicular stomatitits virus (VSV) (Edelman and Wheelock, 1966), polio (Willems et al., 1969), and mumps (DucNguyen and Henle, 1966). Selective suppressive effects on humoral or cell-mediated immunity (CMI) by viral infection are expected to occur, since some viruses appear to be selective for T or B lymphocytes. Thus herpes simplex and VSV replicate preferentially in T cells whereas Epstein-Barr virus selectively affects B cells (Bloom et al., 1970; Pelton et al., 1977; Epstein and Achong, 1977). It is of interest that measles infection suppresses CMI and helper T-cell function but does not affect B-cell activity, but whether this suppression of CMI by measles virus is due to a selective destruction of T cells has not been reported (McFarland, 1974). Explanations for the selective effects on T cells or B cells probably lie with the differential expression of viral receptors. Evidence for this was provided with EBV, where only B-cells express receptors for this virus (Jondal and Klein, 1973). 3. SUPPRESSION OF LYMPHOCYTE FUNCTION

Overlapping the explanation of attributing immunosuppression to lymphocyte function is the possibility that suppression results from virus-induced depression of lymphocyte function. That such suppression occurs can be observed by measuring the blastogenic responses of lym­ phocytes from infected individuals to a variety of stimuli. An alternative approach, which yields more information about mechanisms, is to compare the responsiveness of normal lymphocytes to mitogens or antigens in the presence and in the absence of virus. By this means, several viruses have been shown to be immunosuppressive, and the explanation is usually not one of simple virus-induced cytotoxicity to lymphocytes (reviewed by Woodruff and Woodruff, 1975a). Probably a variety of mechanisms can explain the suppression, since in some cases suppression is observed only if viruses are added shortly after culture initiation (e.g., measles—Olson, 1973), whereas suppression with other viruses may be observed if virus is added up to 3 days after culture initiation (e.g., poliovirus—AVillens and Rawls, 1969). Explana­ tions for the functional impairment of lymphocytes may include a competition for membrane receptors between the stimulant and the viral antigen, a central depression of DNA synthesis by virus infection or possibly because of the binding of antigen-antibody complexes. Additional explanations include the stimulation of suppressor cells (discussed below), and suppression mediated by interferon. Evidence for suppression by immune complexes comes from clinical data documenting

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the correlation between the presence of immune complexes and immunosuppression and in vitro evidence where nonsuppressive levels of some viruses can be made suppressive by adding antiviral antibody (Lee and Sigel, 1974). The possible role of interferon as the mediator of the suppression has recently attracted much attention (reviewed by Gresser, 1977). Thus, interferon was shown to inhibit cell growth and to suppress the development of cell-mediated and humoral immune responses both in vitro and in vivo. Recently, cell growth inhibition was shown to be mediated by purified interferon, substantiating the idea that the inter­ feron molecule itself has immune regulatory effects (DeMaeyer-Guignard et al., 1978). It will be of interest to see data on possible correlations be­ tween levels of interferon induced by different viruses and the degree of immunosuppression observed. Interferon also has other effects on cells that may have immunopathologic significance. Examples include the increased expression of certain membrane antigens (Lindahl et al., 1974) and the enhancement of effector cell function (Lindahl et al., 1972; Babiuk and Rouse, 1978; Wardley et al., 1976a). 4. ALTERATION OF LYMPHOCYTE TRAFFIC

For the immune system to function normally, many cell types must interact. This normally occurs in well defined areas of the lymphoid system. Impairment of immunity is to be expected if recirculating lymphocytes, T or B, have an altered traffic pattern and home to organs, such as the liver, where they will be less likely to respond optimally to antigen since vital accessory cells will be absent. In vitro studies have shown that only slight membrane changes are sufficient to cause differences in lymphocytoxic homing patterns, and virus in­ fection or attachment of viruses or immune complexes to cells, might be sufficient to cause the change. Indeed, Woodruff and Woodruff (1975a,b), who have examined the lymphocyte traffic question in many communications and who have lucidly made the case for it being an explanation for immunosuppression, have shown that treatment of cells for as little as 15 minutes with Newcastle disease virus or influ­ enza virus is sufficient to cause lymphocytes to home to the liver rather than the lymph nodes or spleen. Clearly, virus replication was not necessary in this case to cause the redistribution. The neuraminidase enzyme on the virus envelope was reported to be responsible for the changes in membrane characteristics. Influenza viruses can also cause changes in membrane behavior of lymphocytes as evidenced by the temporary reduction in T-cell rosettes during acute influenza (Schei­ berg et al., 1976).

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5. SUPPRESSION BY LYMPHOCYTOTOXIC ANTIBODY

A further way of suppressing immunity could be by the development of lymphocytotoxic antibody. This type of antibody is produced in old NZB mice and is assumed to be responsible, at least in part, for the immunosuppression that these mice develop with age (Talal, 1977). Although, as discussed previously, much evidence favors a role for C-type viruses in the etiology of the immunopathologic problems of NZB mice, how such viruses actually trigger the production of the lymphocytotoxic antibody needs to be elucidated. 6. IMPAIRMENT OF SUPPRESSOR-CELL CONTROL

A currently popular explanation of immune imbalance is that of an impairment in suppressor cell control (Gershon, 1974). Thus immuno­ suppression may result from an overactivity of suppressor cells. This explanation has been used to explain the immunosuppression of several murine leukemias (Gorczynski, 1974; AVeislow and Wheelock, 1975; Essex, 1975; Kirchner et al., 1975) as well as of feline leukemia. How­ ever, the mechanism of increased suppressor cell activity is not known. Of the many theoretical possibilities (e.g., direct infection and stimula­ tion of suppressor cells or stimulation by immune complexes), the one most appealing to us entails interferon as a mediator. Thus, as suggested by Gresser (1977), interferon, although it may suppress cell division, in doing so may permit or expand expression of the other functions of the treated cell. Thus the activity of individual suppressor cells, which may act by the elaboration of prostaglandins (Goodwin et al., 1977), may be enhanced, and immunosuppression results. Finally, neoplastic transformation of immune cell precursors and immunosuppression may occur as a result of viruses infecting and transforming lymphocyte precursors (Dent, 1972). As a result, trans­ formed cells ultimately crowd out and replace normal immunocompetent cells. This explanation for immunosuppression may be the most likely one for advanced leukemia.

IX. Speculations Immunologie poets have painted a picture of the body as floating on a life raft of immunity in a sea of bacteria (Friedman, 1978). The immunity vessel succeeds in repelling all but 200 or so of the thousands of species of bacteria. With respect to viruses, the relation­ ship is more complex, since the continued existence of all species of

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viruses requires that they at least occasionally be given a berth on the raft. Although some viruses reward this generosity with overindul­ gence, the function of the immune system is such that most viruses are ultimately rejected or accepted. Under rather uncommon circumstances, however, the fine tuning of the immune network is disturbed and re­ sults in unacceptable damage to the body—a situation usually referred to as immunopathology. This state of affairs is most common if viruses affect the actual cells of the immune system and either destroy them or subtly change their luxury functions. In this review, we have described many circumstances under which antiviral immune responses can be considered as immunopathologic. Most of the mechanisms de­ scribed were recognized from in vitro models so that their actual in vivo importance is speculative at best. However, if one wished to unite all immunopathologic mechanisms under a common pattern, we suggest that there is a perturbation of the delicate balance between cells that make up the immune reticulum. Thus it would not be surprising if all ex­ amples of virus-induced autoimmune disease and lymphoid neoplasia are shown to result from viral damage to the function of some regulator cell whose normal target in the body is another cell of the immune system. Similarly, it will not come as a surprise if immune complex disease is ultimately shown to result from aberrant regulation of phagocytic cells that normally serve to rapidly remove potentially toxic complexes from the circulation. Finally, we expect that, as knowledge expands concerning the actual mechanism of the immune imbalance that constitutes immunopathology, the interferon family of molecules may be intricately involved. ACKNOWLEDGMENTS We thank Dr. Arthur Brown, Dr. Carl Wust, and Dr. Bob Michel for helpful criticisms and Darlene DeLoskey for typing the manuscript. We are grateful for financial support from N I H Grant RR09012-01. REFERENCES Abdou, N . I., Segawa, A., Pascual, E., Herbert, J., and Sadeghee, S. (1976). Clin. Immunol. Immunopath. 6, 192-199. Allison, A. C. (1973). Ann. Rheum. Dis. 2>2, 283-293. Allison, A. C. (1976). N. Engl. J. Med. 295, 821-827. Allison, A. C , Denman. A. M., and Barnes, C. D. (1971). Lancet 2, 135-140. Amako, K., and Dales. S. (1967). Virology 32, 201-215. Appel, M. J. G. (1969). Am. Med. Vet. Res. 30, 1167-1182. Appel, M. J. G , and Gillespie, J. H. (1972). "Virology Monographs," Vol. 11. Springer-Verlag, Berlin and New York. Armstrong, R. M. (1972). Med. Clin. North Am. 51, 515-520.

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ADVANCES IN VETERINARY SCIENCE AND COMPARATIVE MEDICINE, VOL. 2 3

Immunology of a Persistent Retrovirus Infection— Equine Infectious Anemia TRAVIS C. McGUIRE AND TIMOTHY B. CRAWFORD Department

I. II.

III.

IV.

V.

VI.

VII. VIII. IX.

of Microbiology and Pathology, College of Veterinary Washington State University, Pullman, Washington

Introduction Virus Characteristics 1. Physicochemical Properties 2. Morphology 3. RNA-Directed D N A Polymerase ( R D D P ) 4. Structural Proteins and Glycoproteins 5. Virus Classification Virion Surface Glycoproteins 1. Neutralization 2. Antigenic Variation 3. T Lymphocytes Sensitive to Viral Antigens 4. Glomerulitis Major Internal Virion Polypeptide (p29) 1. Demonstration 2. Immunoglobulin Response 3. Antigenic Relationship to Other Viruses 4. Immune Response to Other Virion Polypeptides Viral-Infected Cell Membrane Antigen 1. Detection 2. Complement Lysis 3. Antibody-Dependent Cell-Mediated Cytotoxicity 4. Lymphocyte Cytotoxicity to Viral Infected Cells Anemia 1. Viral Hemagglutination 2. Mechanisms of Anemia Immunosuppression Viral Persistence Discussion References

Medicine,

.

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137 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-039223-2

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I. Introduction Equine infectious anemia virus (EIAV) is noted for its persistence in infected horses causing a recurring disease with respect to clinical signs and lesions. After infection most horses experience multiple episodes of pyrexia lasting a few days, accompanied by weight loss and anemia, interspersed with clinically normal periods of a few days to a few weeks. Horses that survive the early phases of the disease experience a gradual decline in the number of clinically apparent episodes until they are asymptomatic. Even though infected horses do not eliminate the virus, levels in the blood and tissues vary markedly (Kono, 1969), being generally proportional to the severity of the clinical disease. Since natural transmission is predominantly by biting insects, transmission is most likely when viremia is high and least likely during asymptomatic stages. Reviews of EIA published within the last 10 years cover general aspects (Ishitani, 1970; Henson and McGuire, 1974; Ishii and Ishitani, 1975), pathology (Kono and Yamamoto, 1970), immunology and pathogenesis (Squire, 1968; Henson and McGuire, 1971; McGuire and Henson, 1973), virology and persistence (Kono, 1973; Coggins, 1975; Crawford et al, 1978), and transmission (Shen et al, 1972). The recent classification of EIAV as a nononcogenic retrovirus (Charman et al, 1976; Archer et al, 1977; Cheevers et al, 1977, 1978; see also earlier evidence reviewed in McGuire and Henson, 1973) allows comparison with the RNA tumor viruses and other nononcogenic retroviruses (such as visna, maedi, and progressive pneumonia viruses of sheep) providing a tremendous background of information relevant to EIA research. With this background in mind, immunologie aspects of EIA can be set in a clearer prospective. This is the purpose of this article. II. Virus Characteristics 1. PHYSICOCHEMICAL PROPERTIES

The virion of EIAV has a lipid-containing envelope evidenced by its buoyant density of about 1.16 gm/cm 3 (Matheka et al, 1976; Cheevers et al., 1977) and its sensitivity to lipid solvents. It was resistant to trypsin, RNAase, and DNAase. Uridine was incorporated but not thymidine, indicating the genome was RNA, but replication was sensitive to DNA inhibitors in the early phases (for references, see Henson and McGuire, 1974; Ishii and Ishitani, 1975). Workers examining the EIAV virion for reverse transcriptase origi-

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nally noted that the RNA genome had a high sedimentation coefficient (Charman et al., 1976; Archer et al., 1977). A definitive physical char­ acterization of the genome was subsequently reported (Cheevers et al., 1977). The RNA genome was predominantly single stranded, had a molecular weight of about 5.5 X 10G and sedimented at 62 S. The virion also contained considerable 4 S RNA which was probably host-derived tRNA, and a small amount of 5 S RNA of uncertain origin. The 62 S genome could be dissociated into two 34 S subunits with molecular weights of about 2.8 X 106. 2. MORPHOLOGY

The size and shape of the mature particle varied, but generally was spherical, averaging about 110-120 nm in diameter. It was bounded by a double-layered envelope with surface projections (Nakajima et al., 1969; Tajima et al., 1969; Weiland et al, 1977) derived from the cell plasma membrane during the budding process. The composition of the surface projections is still a matter of conjecture, but, based on analogy with other viruses of the same class (Shafer and Bolognesi, 1977), they seem likely to be the site of hemagglutinin as shown by Sentsui and Kono (1976) and of the major surface glycoprotein (gp77/79) of the virion (Crawford et al., 1978; Cheevers et al., 1978). The core of the particle was remarkably pleomorphic, ranging from cone shaped to tubular (Weiland et al., 1977), a feature with only rare parallels among similar viruses—one being a syncytia-producing virus isolated from cattle (Boothe and Van Der Maaten, 1974). Infected cells also had intracytoplasmic tubular structures arranged in circles and matrices. Though Ito (1974) reported that the mature extracellular particle was derived from these intracytoplasmic structures, this point needs further verification. In the authors' laboratory no direct relationship between the two entities has been seen. It is our experience that the crescentshaped precursors of the virion core consistently form directly beneath the plasma membrane rather than being formed in the cytoplasmic matrices (W. C. Davis, and T. B. Crawford, unpublished observations). These cytoplasmic structures are significantly different from classical A-type intracytoplasmic particles that are precursors to the mature virion of B-type retroviruses (Sarkar and Whittington, 1977). 3. RNA-DIRECTED DNA

POLYMERASE (RDDP)

The presence within the EIA virion of an R D D P enzyme that will catalyze synthesis of DNA from synthetic template (Charman et al., 1976) or from the EIAV genome (Archer et al., 1977) was recently re-

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ported. The enzyme was similar to those from a number of RNA tumor viruses. It catalyzed the production of a DNA provirus that integrated into the genome of, at least, the persistently infected cell. The DNA product synthesized in the in vitro reaction in the absence of actinomycin D was double stranded and represented 30 to 50% of the RNA genome sequences; it was used as a probe to locate EIA sequences integrated in the DNA genome of infected flbroblasts (Crawford et al., 1978). 4. STRUCTURAL PROTEINS AND GLYCOPROTEINS

Until lately, reports relating to proteins of EIAV were only semiquantitative in nature, and concerned those proteins that reacted with infected horse serum in immunodiffusion and complement-fixation tests (Section I V ) . Descriptions of the complete structural polypeptide com­ plement of the EIA virus using more precise biochemical characteriza­ tion techniques have recently begun to appear (Ishizaki et al., 1978; Crawford et al., 1978; Cheevers et al., 1978). Five polypeptides com­ prised 80% of the virion protein. The major structural protein was nonglycosylated, making up about 27%; of the virion protein and representing the analog of the gag gene product of other mammalian retroviruses (Oroszlan et al., 1978) which is the major group-specific antigen of these viruses. It was not labeled when the intact virion was subjected to surface-labeling reagents (Crawford et al., 1978; Cheevers et al., 1978) confirming earlier suggestions (Kono, 1968) that it was internal, not a component of the envelope. It had an isoelectric point of 5.8 and reports of its molecular weight ranged from 25,000 to 28,000 (Norcross and Coggins, 1971; Charman et al., 1976; Ishizaki et al., 1978). Based on repetitive determinations in this laboratory we will henceforth refer to it as p29 (Cheevers et al., 1978). Ishizaki et al. (1978) reported one other nonglycosylated peptide in the EIA virion, whereas Cheevers et al. (1978) reported two more that were probably virus coded and six minor peptides that were presumed to be products of the host cell. These virus-coded peptides had a molecular weight between 12,000 and 14,000. The heavier of the two apparently represented the low-molecular weight precipitating antigen previously referred to by others (Toma and Goret, 1974; Malmquist and Becvar, 1975). Our best estimate for the molecular weight was 13,000; hence it will be referred to as pl3. As noted by Cheevers et al. (1978), inherent technical variability of SDS-polyacrylamide gel molecular weight estimation in the 10,000-15,000 range rendered it impossible to be certain that this protein was not in fact glycosylated,

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as there was a small component that migrated in this region that in­ corporated glucosamine. Ishizaki et al. (1978) failed to find evidence that this was a protein and considered it a glycolipid. Cheevers et al. (1978) reported incorporation of labeled amino acids and therefore designated it as a small glycoprotein. Though it seemed to be smaller than the pl3, the possibility thus remains that pl3, which comprises about 2 1 % of the virion protein, is a component of the virion membrane similar to the pl5 of Rauscher murine leukemia virus (Barbacid and Aaronson, 1978). A major virion glycoprotein of EIAV migrated as a duplex peak with molecular weights of about 77,000 and 79,000 and was designated p77/79 (Cheevers et al, 1978). Similar results were reported by Ishizaki et al. (1978) except that only a single migrating species of MW 80,000 was reported. Surface iodination confirmed that this glycoprotein was in the envelope, as was the major glycoprotein of type-C virions (Stephenson et al., 1978). A glycopeptide of MW 40,000 was consistently found in PAGE separations. It was very poorly labeled by lactoperoxidase, and may simply represent a breakdown product of the major envelope glyco­ protein, the gp77/79 (Stephenson et al., 1978). There was one other glycopeptide consistently found in gel separations with a molecular weight of 64,000 which represented about 10-15% of the virion protein. Its location within the virion, its origin, and its significance are still uncertain. It does not appear to have an analog among mammalian type-C viruses (Cheevers et al., 1978). Thus the EIA virion appears to contain two virus-coded glycoproteins, gp77/79 and gplO, and two other glycoproteins, gp40 and gp64, that may represent degradation products of the major envelope glycoprotein, gp77/79. There are also two major'nonglycosylated viral coded peptides (p29 and pl3), and several minor ones of uncertain origin. The other major virion protein (nonstructural) is the RDDP. The proteins de­ tailed account for practically all the coding capacity of the EIAV genome (Cheevers et al., 1978). 5. VIRUS CLASSIFICATION

In conformity with the system of virus classification approved by the International Committee on Virus Nomenclature (Fenner, 1976), EIAV must be classified in the family Retroviridae. These viruses contain a high-molecular weight RNA genome, an RDDP, from five to eight structural proteins, and they mature by budding from the cell surface. Retroviridae has been divided into three subfamilies; however, the sub-

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classification of EIAV is not complete. Certain of its characteristics exclude it from the foamy viruses (subfamily Spumavirinae) which have submembrane crescents during core formation, shorter surface pro­ jections, no clear intracytoplasmic A particles, and a Mg 2+ rather than Mn 2+ -dependent RDDP. EIAV, in contrast to the subfamily Oncovirinae (C-type RNA tumor \ r iruses), has intracytoplasmic structures and an irregular shaped nucleoid, and its RDDP is Mg- + dependent. EIAV bears considerable resemblance to visna virus (Ito, 1976) and the bovine syncytia-producing virus (Boothe and Van Der Maaten, 1974) in their morphological features (AVeiland et al., 1977). Further­ more, the R D D P of both visna and EIA prefers Mg 2+ . This parameter has not been described for the bovine virus. EIAV does not form syncytia in either persistently infected fibroblasts or macrophages, although it produces cytopathology and cell death when it replicates in macro­ phages. Visna is also cytopathic in vitro, a relatively uncommon feature among the retroviruses. Visna and related viruses (maedi and progressive pneumonia) have been tentatively placed under the subfamily Lentivirinae. The morphologic and biochemical similarities between EIA and visna virus suggest a fundamental relationship which needs further examination, including nucleic acid hybridization. III. Virion Surface Glycoproteins 1. NEUTRALIZATION

Neutralizing antibodies in EIAV-infected horse sera were first dem­ onstrated by titrating surviving virus in susceptible horses (Stein and Gates, 1950; Tanaka and Sakaki, 1962). Later, titration in horse leukocyte cultures was used for virus neutralization (Kono, 1969; Henson et al., 1969) and it became apparent that there were antigenic differences between viral isolates (Kono, 1969). Indeed, there was no cross-neutralization between eight different isolates from several dif­ ferent countries when studied by virus neutralization (Kono et al., 1971). This observation was supported by cross-protection studies in horses (Kono et al., 1970). The ability of antibody to neutralize circulating virus during acute disease is hampered by antigenic variation (Section III, 2) and perhaps infectious virus-antibody complexes (McGuire et al., 1972). Antiimmunoglobulin treatment caused a 99% reduction in infectious virus in horse sera when compared with control treatment. This showed that a large percentage of infectious virus in serum from acutely

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infected horses was bound to antibody. This observation does not clarify the quantitative importance of infectious virus-antibody com­ plexes in infection of susceptible cells. However, it is our opinion that whatever importance they might have is limited to the pathogenesis of acute disease (see Section VIII). The surface of type-C retroviruses contains a glycoprotein designated gp69/71 (Strand and August, 1973). Antibody to purified gp69/71 of a murine leukemia virus neutralized virus infectivity (Steeves et al., 1974). Immunochemical analysis of gp69/71 from feline and murine leukemia viruses revealed several kinds of determinants (Strand and August, 1974). The type-specific determinants were unique to the virus isolate while group-specific determinants were shared by similar viruses iso­ lated from the same species. The gp69/71 molecules had small amounts of a third kind of determinant, designated interspecies, which was shared by similar viruses isolated from different species. The gp77/79 of EIAV (Section 11,4) is located in the envelope, and is analogous to the major glycoprotein of other retroviruses. Antibody to purified gp77/79 should neutralize EIAV showing that this antigen is involved in viral neutralization and therefore antigenic variation (Section 111,2). 2. ANTIGENIC VARIATION

The virus isolated from horses during febrile episodes was antigenicly distinct from both the inoculating virus and virus isolated from subsequent febrile episodes (Kono et al., 1973a). Antibodies to isolated viruses were prepared in rabbits and used to confirm the antigenic drift of EIAV in infected horses (Kono et al., 1973a,b). This observation provided an explanation for the episodes of clinical disease associated with bursts of virus in the blood (Kono, 1969). However, antigenic variation is not the only and possibly not the primary mechanism of viral persistence (Section VIII). Another nononcogenic retrovirus, visna virus of sheep, had antigenic differences between isolates (Thormar and Helgadottir, 1965) and underwent antigenic variation in individual animals (Gudnadottir, 1974; Pétursson et al., 1976; Narayan et al., 1977a,b). Antigenic varia­ tion of visna virus isolated from peripheral blood leukocytes did not occur prior to development of neutralizing antibody in the animal (Narayan et al., 1977b). In addition, the selection of antigenic variants under antibody pressure occurred in vitro when sheep cell cultures were inoculated with plaque-purified visna virus and maintained with antibody in the medium (Narayan et al., 1977b).

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Little is known about the mechanism of antigenic variation of visna virus and x even less about EIAV. The gp77/79 is likely the protein which undergoes antigenic variation. Furthermore, the obser­ vation of type-specific determinants on the gp69/71 associated with more constant determinants (group and interspecies) seemingly con­ served (Strand and August, 1974), provides a framework for antigenic variation. The area containing type-specific determinants could vary with the remainder of the molecule being constant. An interesting parallel of antigenic variation is found in African trypanosomes. Episodes of disease are associated with the appearance of new antigenic variants (reviewed in Vickerman, 1974 ). These organisms have a surface "coat" on the cell membrane composed of a 64,000-MW glycoprotein. Structural changes in this variant-specific surface glycoprotein cause antigenic variation (Cross, 1975). Even though only the variant-specific determinants of the molecules are accessible on live trypanosomes, recent studies have shown that isolated variant-specific surface glycoproteins do indeed have cross-reacting determinants (Barbet and McGuire, 1978). In trypanosomes either several genes could be present for surface antigens, or recombinations and mutations could provide the \^ariation or a combination of the possibilities. In EIAV the restricted size of the RNA genome (Cheevers et al., 1977) narrows the genetic mechanisms to recombination and mutation. The number of known EIAV variants makes the possibility of recombination seem plausible. A recent paper has suggested that recombination provides a mechanism for variability of the immunoglobulin system (Kabat et al., 1978). The antigenic shifts in visna virus apparently reflect changes in viral RNA: Restriction maps of proviral DNA from variants are different (Clements et al., 1978). 3. T LYMPHOCYTES SENSITIVE TO VIRAL ANTIGENS

T lymphocytes sensitive to viral antigen have been demonstrated using in vitro lymphocyte proliferation studies (Kono et al., 1976; Banks et al., 1978). In one case (Banks et al., 1978) the antigen was gradient purified virus; the stimulating antigen was therefore likely the gp77/79. The responding cells (T lymphocytes) were defined by their failure to bind to nylon wool columns and lack of surface immunoglobulin and complement receptors (Banks et al., 1978). Sensitive lym­ phocytes appeared shortly after infection, declined during asymptomatic stages of the disease only to increase with the recurrence of clinical disease (Kono et al., 1976). The lymphocyte stimulation did not appear

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to be strain specific, indicating the recognition of common antigenic determinants between strains. 4. GLOMERULITIS

Proliferative glomerulitis was found in most EIAV infected horses examined, with immunoglobulin and complement deposits in the glomerular mesangium (Banks et al., 1972). Immunoglobulin eluted from glo­ meruli of infected horses reacted with viral antigen in indirect immunofluorescence (Crawford et al., 1971). The specific antigens involved in the glomerulitis are still unidentified, but certain possibilities are sug­ gested by other data. For instance, studies with New Zealand mice have shown glomerular deposition of viral envelope glycoprotein (gp69/71) of murine leukemia virus (Yoshiki et al., 1974). The large ratio gy69/71 to p30 complexed with antibody in the glomeruli demonstrated its importance in the induction of glomerulonephritis. Immunoglobulin eluted from EIAV infected horse glomeruli should be tested for gp77/79, p29, and other proteins of EIAV to determine which antigens are involved in the kidney lesions. IV. Major Internal Virion Polypeptide (p29) 1. DEMONSTRATION

Following the demonstration of neutralizing antibody (Section 111,1) in infected horse serum, antibodies were found which fixed complement with antigens from infected horse leukocyte cultures (Kono and Kobayashi, 1966a). The antigen was not detectible on isolated virus, but could be detected after disruption (Kono, 1968) and appeared to be com­ mon to all viral isolates tested (Hensen et al., 1970; Kono and Kobayashi, 1967; Kono et al., 1971; Henson et al., 1973). Infected horse spleen con­ tained a group-specific antigen which reacted in immunodiffusion with sera of infected horses (Coggins and Norcross, 1970). This spleen antigen was also detectible by complement-fixation (Henson et al., 1971 ; Norcross and Coggins, 1971). Purified disrupted EIA virus from infected horse leukocyte cultures reacted with infected horse serum in immunodiffusion (Nakajima and Ushimi, 1971, 1972; Nakajima et al, 1973). The anti­ gens from infected spleen and purified virus were subsequently shown to be identical (Nakajima et al., 1972). The group-specific determinants are carried on an internal structural protein herein designated p29 (Section 11,4).

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2. IMMUNOGLOBTJLIN RESPONSE

The immunoglobulin classes of the horse are both taxonomically and functionally complex. Several classes and subclasses have been de­ scribed and include IgM, IgA, aggregating immunoglobulin (also referred to as gamma-1 component and IgB ), and IgG subclasses (IgGa, IgGb, IgGc, and IgG(T) (nomenclature and characterization reviewed in McGuire et al, 1973; McGuire and Crawford, 1973). Of interest are the contrasting properties of IgGab (IgGa and IgGb are very difficult to separate) and IgG(T) (summarized in Table I ) . Notable among these is the failure of IgG(T) anti-hapten antibody to precipitate with haptencarrier (Klinman et al., 1964; Klinman and Karush, 1967) and flocula­ tion of IgG(T) antibody with protein antigens (Nakamura and Katsura. 1964; Johnson and Allen, 1968a). IgGab yields typical precipitation reactions with the same antigens. These differences are a reflection of the more limited flexibility of the hinge region of IgG(T): Data show equine IgG has an average distance between binding sites greater than that of IgG(T) (Archer and Krakauer, 1977a,b). Apparently differences in the Fc portion of IgG and IgG(T) influence their functional ac­ tivities as IgG(T) will not fix complement by the classical pathway (Nakamura and Katsura, 1964; Johnson and Allen, 1968a,b) and will not bind to neutrophils and macrophages when attached to erythrocytes (Banks and McGuire, 1975) as will IgGab antibodies. Both IgG and IgG(T) will agglutinate antigen-coated erythrocytes (Crawford etal, 1978). The complement-fixation test proved unreliable for the diagnosis of EIA because most horse sera were nonreactive after an initial period of reactivity 14-40 days after infection (Kono and Kobayashi, 1966b; Henson et al, 1969, 1970; Kono, 1969). The defective complementfixing ability of IgG(T) led to the suggestion that the failure of in­ fected horse sera to fix complement with EIA viral antigen was due TABLE I COMPARISON OF FUNCTIONAL PROPERTIES OF EQUINE IgG AND I g G ( T )

Property

IgGab

IgG(T)

Hemagglutination Complement-fixation

Yes Yes

Yes No

Binding to monocytes and neutrophils Precipitation (hapten-repeating determinants) Precipitation (protein-nonrepeating determinants)

Yes Yes Yes

No No Yes (floculation)

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to its presence. Purified IgGab fixes complement with EIAV antigen in a normal manner. With the same antigen, IgG(T) not only does not fix complement but competitively inhibits complement-fixation by IgGab. The activity of a given serum from infected horses was shown to be a reflection of the relative levels of IgG(T) and IgGab antibodies it contained (McGuire et al., 1971b). Fluctuating levels of IgG(T) in most horse sera render the complement-fixation reaction unreliable as a test for anti-EIAV antibody. The observation of unusual precipitating properties of IgG(T) prompted a study of how IgG(T) reacted with EIAV p29. Precipitation analysis of IgG(T) and isolated p29 showed that floculation occurred (McGuire, 1977) with soluble complexes in both antigen and antibody excess. In immunodiffusion tests, no precipitation line occurred in IgG(T) antibody excess. In the diagnostic test for EIA, suspect sera are placed next to known positive serum (Coggins et al., 1973) so that IgG(T)-rich sera are recognized by inhibition of a portion of the positive control line (McGuire, 1977). Antibodies to p29 antigen were present as early as 14 days after infection and were continually present (Coggins and Norcross, 1970; Henson et al., 1971; Nakajima, 1973); indeed, horses having antibody by the immunodiffusion test were shown to have virus by horse inocula­ tion (Coggins et al., 1972, 1973). This test is now extensively used for EIA diagnosis incorporating viral antigen prepared from persistently EIAV infected dermal cells (Malmquist et al., 1973). 3. ANTIGENIC RELATIONSHIP TO OTHER VIRUSES

Similar to the gp69/71 surface glycoprotein, the p30 has group-specific and type-specific determinants as well as interspecies determinants (Geering et al., 1970; Strand and August, 1974). Unpublished experiments by the authors have been unable to show cross-reactions between the major structural proteins of EIA and Rauscher murine leukemia and the Rickard strain of feline leukemia virus. Radioimmunoassays using both precipitation and inhibition were negative using four antisera to EIA p29, four anti-feline leukemia virus sera, and two anti-murine leukemia virus sera. Other workers reported finding no antigenic similarities between EIA and Mason-Pfizer monkey virus, feline leukemia virus, murine leukemia virus, mouse mammary tumor virus, bovine leukemia virus, and squirrel monkey virus using immunodiffusion (Schochetman et al., 1977), complement-fixation, and radioimmunoassay (Charman et al., 1976). Antigenic comparisons to the visna group (Weiss et al., 1977) have not been reported.

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4. IMMUNE RESPONSE TO OTHER VIRION POLYPEPTIDES

Antibodies to EIAV pl5 (pl3 in Section 11,4) were found in infected horse sera by immunodiffusion (Toma and Goret, 1974; Malmquist and Becvar, 1975). Ishizaki et al. (1978) using radioimmunoprecipitation showed antibody in horse antiserum to gp77/79, the major structural protein, and two other peptides, one 14,000 MW (our pl3) and another of 11,000 MW. The significance of these viral antigen-antibody systems in the disease process is not clear. Antigens detected by the precipitin test have not been defined (Moore et al., 1966). V. Viral-Infected Cell Membrane Antigen 1. DETECTION

An EIAV-specific antigen was demonstrated on the surface of per­ sistently infected fibroblasts using both a radioimmune binding test (McGuire and Crawford, 1978) and lymphocyte cytotoxicity (Section V,4) · Antibody against this antigen appeared within 1 month after infec­ tion and persisted at least 4 years. Cells infected with retroviruses ex­ press a number of antigens on the cell surface including gp69/71 (Cloyd et al., 1977), p30 (Shellam et al, 1976), p. 15 (Lejneva et al, 1976), and apparently nonvirion antigens (Rogers et al., 1977; Siegert et al., 1977). The antigen type expressed on EIAV infected cells is not known; how­ ever, the presence of a new antigen provides several potential mechanisms for destruction of infected cells in vivo as well as rationale for im­ munization. 2. COMPLEMENT LYSIS

Viral-induced antigen on the surface of EIAV infected cells and antibody in infected horse sera suggest that complement lysis might be a method of removing infected cells in vivo. The efficiency of this mechanism in other systems is decreased by non-complement-fixing antibodies and by lowered complement levels. Lysis of EIAV infected cells has not been demonstrated either in vitro or in vivo making further speculation ^premature. 3. ANTIBODY-DEPENDENT CELL-MEDIATED CYTOTOXICITY

Retrovirus infected cells expressing recognizable surface antigen can be killed by antibody-dependent cell-mediated cytotoxicity (ADCC) (Lamon et al., 1977). The ADCC mechanism functions in horses as

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well: Equine monocyte-lymphocyte mixtures, neutrophils, and macro­ phages lysed DNP-coated erythrocytes after incubation with equine IgG anti-DNP antibodies. However, in vitro killing of cells infected with EIAV by ADCC has been difficult to demonstrate (Fujimiya et al., 1979). The difficulty in killing EIAV infected cells by ADCC may be due to low antigen density on the cell surface. 4. LYMPHOCYTE CYTOTOXICITY OF VIRAL INFECTED CELLS

Low level in vitro killing of EIAV infected cells by lymphocytes from infected horses was demonstrated (Fujimiya et al., 1979). This system was allogeneic with respect to the effector and target cells and perhaps better killing would occur in a syngeneic system. Other data suggest an important role for cell-mediated immunity in the eventual cessation of clinical disease cycles in EIAV infected horses. Asymp­ tomatic infected horses had viremia and clinical disease shortly after treatment with either dexamethosone or cyclophosphamide (Kono et al., 1975). The time interval between treatment and disease ex­ pression was too short to be explained by an effect on antibody levels. In another experiment, horses with chronic EIA resisted infection by another serotype to which they had no demonstrable neutralizing anti­ body (Kono et al., 1973b). Taken together the experiments suggest that sensitive lymphocytes destroy infected cells at a sufficient rate to keep virus amounts at a low level in the face of reinfection. Immunosuppressive drugs may diminish this response and allow virus production of a new variant resulting in clinical disease (see Section VIII for more details).

VI. Anemia 1. VIRAL HEMAGGLTJTINATION

Purified EIAV hemagglutinated guinea pig erythrocytes (Sentsui and Kono, 1976). The activity was destroyed by lipid solvents and proteolytic enzymes, but not by neuraminidase and phospholipase C. Receptors on erythrocytes were sensitive to proteolytic enzymes, but not neuraminidase. Though its location in the envelope has not been directly shown, it is probably a constituent of membrane spikes (Shafer and Bolognesi, 1977; Weiland et al., 1977; see also Section 11,2). Hemagglutination-inhibiting antibodies appeared in the sera of horses 60 to 150 days after infection and persisted. Interaction of intact virus

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or hemagglutinin subunits with erythrocytes and antibody of infected horses might lead to erythrocyte destruction (Section VI,2). 2. MECHANISMS OF ANEMIA

Anemia is a consistent feature of active disease and has received considerable attention. Two mechanisms are involved: bone marrow suppression and hemolysis. Evidence for bone marrow suppression was from ferrokinetic experi­ ments and morphologic evaluation and quantitation of the bone mar­ row cells. Ferrokinetic studies showed a decrease in both plasma iron turnover and percentage utilization of radioactive iron in active disease, evidence of decreased production of erythrocytes (Obara and Nakajima, 1961b; McGuire et al., 1969b). Normal or increased myeloid-erythroid ratios in the presence of anemia was further evidence of hyporesponsiveness (Pehl, 1953; McGuire et al., 1969). Alterations of iron metabolism included marked decreases in serum iron associated with low saturation of transferrin with iron (Obara et al., 1957; McGuire et al., 1969b), decreased bone marrow sideroblasts, and increased stainable reticuloendothelial iron (reviewed in McGuire et al., 1969b) combine to suggest that part of the bone marrow depression is caused by iron deficiency. This iron deficiency was temporary and reflected a failure of the reticuloendothelial cells to release iron properly. Other functional alterations of circulating monocytes in infected horses have been shown (Banks, 1975) as has the presence of virus in acute disease (McGuire et al., 1971a). Increased destruction of erythrocytes was documented by decreased erythrocyte life spans (Obara and Nakajima, 1961a; Obara et al., 1962; McGuire et al., 1969a). Plasma hemoglobin and decreased haptoglobin during severe clinical EIA showed that part of the hemolysis was intravascular (McGuire et al., 1969a). Some of the proposals for erythrocyte destruction follow. (1) Intraerythrocytic inclusions called Heinz bodies were described in EIA and a casual relationship was proposed (Matthias and Schmidt, 1956; Zakopal, 1958). Studies with splenectomized horses and in vitro tests for sus­ ceptibility to Heinz body formation in erythrocytes from infected horses indicated that Heinz bodies were secondary to other erythrocyte damage and were not the primary cause of anemia (McGuire et al., 1970). (2) Monocyte activation in EIA infected horses was demonstrated and these monocytes had increased in vitro binding of autologous erythro­ cytes and erythrocytes from other horses, sheep, and rabbits fBanks, 1975,). Activated monocytes were present in infected horses when anemia was

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severe and erythrophagocytosis was noted. A causal relationship between altered macrophages and anemia was suggested (Banks, 1975). (3) Com­ plement (C3) coated erythrocytes were found in infected horses and associated with increased osmotic fragility and decreased life-span of erythrocytes (McGuire et al., 1969c). Even though no immunöglobulin could be detected on the C3-coated erythrocytes by Coombs' tests it was felt that it resulted from antigen-antibody activation. Cold hemagglutinins were found in infected horse sera but were diffi­ cult to incriminate in the induction of C3-coated erythrocytes because of low amounts and inability to bind at 37°C (McGuire and Gorham, 1969). (4) Both warm and cold hemagglutinins have been noted in infected horse serum (Oki and Inoue, 1966, 1967). Direct positive Coombs' tests were noted using anti-human IgM serum in horses with the warm IgM hemagglutinin (Oki and Miura, 1970). The role of this autoantibody reaction in the cause of anemia is uncertain. We were unable to confirm this later reaction although other verification should be sought. Information to date implicates immunologie damage and an activated macrophage system in red cell destruction. The description of a viral hemagglutinin (Sentsui and Kono, 1976) offers new information for designing experiments about immunologie mechanisms resulting in erythrocyte destruction. VII. Immunosuppression Initial studies on lymphocyte function in EIAV infected horses showed that the number of lymphocytes with surface immunoglobulin was normal (Banks and Henson, 1973). Lymphocyte responses to phytolectins were the same in infected and control lymphocytes (Banks and Henson, 1973). Immunoglobulin quantitation revealed a mild hypergammaglobulinemia (Henson et al., 1967). Thus, it was concluded that no generalized immunosuppression resulted from E l A infection. While quantitating the serum immunoglobulin types to determine the composition of the hypergammaglobulinemia it was noted that serum IgG(T) concentrations gradually decreased with time following infection. Further studies showed significant depression of IgG(T) at 2 months and at 1 year after infection while IgG was significantly elevated at both time intervals (McGuire, 1976). In comparison, infected horses produced less IgG(T) antibody to DNP immunization than control horses. Metabolism studies with 125I-labeled immunoglobulins showed that the synthesis of IgG(T) was suppressed in infected horses. Neither

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the mechanism nor significance of suppression of synthesis of an IgG subclass, IgG(T), in persistent EIAV infection is presently known.

VIII. Viral Persistence The two biggest factors retarding research on EIA are the facts that the horse is the only experimental animal available, and that the exceedingly cumbersome leukocyte culture system is the only available in vitro assay for field strains of virus. Development of a better in vitro titration system is the greatest single need for EIA investigation. Because of these impediments, progress toward a mechanistic under­ standing of EIA has been exasperatingly slow, allowing plenty of time for speculation about potential mechanisms of persistence (Kono, 1973; McGuire and Henson, 1973; Henson and McGuire, 1974; Coggins, 1975; Crawford et al., 1978). Mechanisms posed most frequently as potential contributors have included infectious virus-antibody com­ plexes, malfunction of the reticuloendothelial system, a defective immune response, antigenic drift of the virus, and integration of DNA provirus into host cell DNA. The problem now is not showing which phenomena exists in EIA—it is now plain that they all do—but to decide which are important ones and how they interact, The vast majority of EIAV circulating in the plasma of acutely ill horses has antibody bound to its surface, but remains infectious for macrophages (McGuire et al, 1972). These complexes are not likely important in maintaining persistence, for two reasons. First, they have not been shown to be infectious in any cell other than the macrophage, and it is our opinion that the macrophage is probably not the site of persistence in vitro (Crawford et al., 1978). Second, data of Coggins and Kernen (1976) suggests that in asymptomatic horses, most of the circulating infectivity is associated with leukocytes, not with the plasma. Viral infection of macrophages resulting in depression of virus clear­ ance and antigen processing necessary for the immune response has been proposed as contributing to persistence in EIA (Henson and McGuire, 1974; Coggins, 1975) as well as in other persistent virus infections (Mims, 1975) and indeed there is depression of reticuloendothelial (RE) func­ tion during acute EIA infection (McGuire and Henson, 1973). This is a result of widespread replication in macrophages during cycles; however, this is not attractive as an important contributor to persis­ tence. First, there is no evidence that this temporary depression of RE function compromises antigen processing sufficiently to significantly depress the immune response. Second, any effects of RE depression

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would be seen only during acute disease; none is present during clinical quiescence. Yet the virus quite successfully weathers the quiescent periods. Thus, any contribution this mechanism might have to per­ sistence is probably negligible. Though demonstration of antigenic drift of EIAV stimulated initial excitement that the explanation for persistence might be at hand, this phenomena's importance probably lies in allowing "cycles" or periodic bouts of rapid virus replication more than in persistence per se. Even if it does not play the primary role in persistence, it is probably useful in periodically increasing the population of persistently infected cells, whatever their identity. This leaves two candidates for the leading role in persistence: some defect (s) in the immune response to the infection, and the capability of the virus to exploit the phenomenon of integration of the provirus with incomplete expression of the viral information. The first of these may be important to persistence; the latter almost certainly is. The profound inhibitory effects of IgG(T) on the ADCC reaction against certain antigens (Section V,3), if operable against EIA antigens as well, might significantly impair the ability of the host to eliminate pro­ ductively infected cells. Also, appearance of suppressor T cells that effectively inhibit lymphocyte cytotoxic killing of infected cells could be a very important modulating influence. More work is needed to assess these factors. Finally, the mechanism that has been obvious since the classification of the agent became certain: proviral integration with latency. Recent studies from this laboratory have been directed toward this area. Using the method of acceleration of reassociation of a double stranded DNA "probe" synthesized in vitro by the endogenous EIAV R D D P reaction, we have found that the EIAV genome is indeed integrated into persistently infected fibroblasts. The reassociation kinetics are con­ sistent with 40-50 copies of the "probe" sequences per cell (Crawford et al., 1978). We have not yet examined macrophages or infected horse tissues. We believe that EIA probably persists in vivo in non-RE cells, the analog of the persistently infected fibroblast system in vitro. If this is true, and if the EIA provirus is capable of remaining latent within the ceil genome without production of viral protein or mature virus, then it can easily persist even in the face of an aggressive immune response. In visna, the vast majority of cells are latently infected, producing neither viral protein nor RNA (Haase et oX., 1978). Only a small pro­ portion of these cells at a given time are actively synthesizing viral

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protein and thus immunologically recognizable. Upon appearance of an antigenic variant (Narayan et al, 1977b), a temporary burst of viral replication and infection of new cells occurs until immunologie control is reestablished. This is proposed as the means whereby the lesions progress and the pool of infected cells is replenished. The evidence suggests that a similar situation may exist in EIA. Still needed is demonstration of nonproductive or latent infection by EIAV. An attractive hypothesis is that a large pool of infected non-RE cells exists, most of which are not synthesizing viral protein, in which a few are constantly undergoing spontaneous activation. The cellmediated system promptly eliminates recognizable cells as they appear, preventing prolonged virus synthesis. Any virus that reaches the extracellular compartment is neutralized by antibodies, blocking entry into the macrophage population. But, when an antigenic variant virus appears in a newly activated cell, that cell is not recognized by the CMI system, allowing it to shed virus that also eludes antibody and starts a surge of replication in macrophages. During this phase, the supply of persistently infected cells is replenished and immunological mediation systems produce the signs and lesions of EIA. As recognition of cross-strain antigens improves with repetitive exposure to new variants, the efficiency of the immune system improves until eventually no new variants are allowed to "slip through." With no new cycles to replenish the pool of infected cells, it would gradually diminish as infected cells undergo activation and elimination. Thus the probability of an active cycle occurring would decrease in proportion to the length of time the animal had been asymptomatic.

IX. Discussion Presently available information permits construction of a plausible scheme of pathogenetic events for EIA. Upon entry virus infects macro­ phages and possibly other, as yet unidentified, cell types. Macrophages apparently succumb to the infection and release large amounts of virus and constituent antigens. An immune response occurs to a variety of the antigens including gp77/79, p29, pl3, and others. Antigen-antibody complexes form in the serum inducing glomerulitis, complement de­ pletion (Perryman et al., 1971), and fever. Viral antigen, possibly hemagglutinin subunits, binds to erythrocytes followed by interaction with antibody and complement leading to either hemolysis or phagocytosis by an activated reticuloendothelial system. Iron release by macrophages is

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delayed causing temporary iron-deficient erythropoiesis. Lesions in the liver and other organs result from lymphocyte response to viral-infected cells since lesions are directly correlated with virus amounts. Pathologic processes subside as neutralizing antibody appears and exerts control over this macrophage replication phase. When a new antigenic variant of the virus appears replication and consequent phlogogenic events recur. These episodes of disease occur with decreasing frequency until most infected horses eventually become asymptomatic. The reasons for failure of horses to eliminate the virus during the initial immune response are complex. Since EIAV is capable of intergration into the host genome, the most prominent possibility for persis­ tence is the existence of latently infected cells unrecognized by the immune system. A small percentage of these cells are being continually activated to produce virus and only those cells producing antigenic types of virus unrecognized by the immune system would survive to start a new wave of viral replication. This process would eventually yield enough virus to produce overt disease. The eventual suppression of disease recurrences may be due to progressive recognition of common surface antigens on infected cells. This response is probably predominantly cell mediated. Whether only T lymphocytes or ADCC mechanisms are involved is not known. Among the immunologie problems remaining in EIAV research, defini­ tion of antigenic determinants interacting with the immune system appears foremost. Since the number of these is unmanageably large (Strand et al., 1976), reduction of the problem to consider those viral antigens most important to diagnosis and immunization seems useful. The problem of diagnosis of infection has been effectively solved by exploiting the group-specific determinants residing on the p29.- Still needed is an easy quantitative assay for circulating virus and viral antigens. At present it is very difficult to determine the transmission risk from infected horses identified by the presence of serum anti-viral antibody. Immunization against EIA is indeed a difficult problem especially in view of the variation that the neutralizing determinants readily undergo. However, the suggestion that common determinants exist between serotypes offers some hope for eventual immunoprophylaxis in EIA. It seems possible that appropriately designed immunologie regimens to exploit common determinants associated with the gp77/79 on either the virion or infected cell membranes might be effective, as they may be in certain oncogenic retrovirus infections (Shafer et al., 1976; De Noronha et al, 1977).

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REFERENCES

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ADVANCES IN VETERINARY SCIENCE AND COMPARATIVE MEDICINE, VOL. 2 3

Immunology and Pathogenesis of African Animal Trypanosomiasis J. B. H E N S O N * and JAN C. NOEL f International

Laboratory

for Research

on Animal

I. II.

Introduction Trypanosomes 1. Structure 2. Antigens 3. Toxins and Products 4. In Vitro Propagation I I I . Immunology 1. Immune Response 2. Survival of Animals in Endemic Areas 3. Experimental Vaccination IV. Pathogenesis V. Conclusions References

Diseases, Nairobi,

Kenya

.

161 164 164 164 166 166 167 167 171 174 175 179 180

I. Introduction Hemoparasite infections represent some of the most important uncon­ trolled diseases of man and animals in the tropics and other parts of the world. These conditions are especially prevalent in many developing countries and are significant constraints to livestock production and improved human health. Examples are malaria and trypanosomiasis (African and American) of man and babesiosis, anaplasmosis, trypano­ somiasis, and theileriosis of animals. The maladies are complex in terms of their pathogenesis and immunology. They are characterized by chronic or persistent infections in some or all infected species, frequently for the life of the infected host. * Present address : Department of Microbiology and Pathology, College of Veteri­ nary Medicine, and f Graduate School, Washington State University, Pullman, Washington 99164. 161 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-039223-2

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The persistence of the organisms and chronic nature of the disease often occurs in association with a vigorous host immune response. Such a phenomenon is not unique, however, to the hemoprotozoa, but also occurs in certain viral and bacterial infections. The mechanisms by which the parasites evade the host defense mechanisms are not completely clear, but the pathogenesis in some diseases is associated with complex alterations of the immune response and the lymphoreticular system. Methods for vaccination against these diseases for prevention under field conditions are not readily available. Details of the biology of the organisms, the host-parasite relation­ ships, pathogenesis, immunology, epidemiology, and other aspects of the hemoparasites and the disease they induce are not completely known and are the subject of investigations ongoing in national and interna­ tional organizations in various parts of the world. The data accruing from these investigations, frequently resulting from renewed interest in the diseases and their impact on man and animals in the developing countries, are becoming available and are allowing insight into many heretofore poorly understood aspects of the hemoprotozoal infections. This report will summarize recent research on African trypanosomiasis of animals to indicate findings making possible a better under­ standing of this important disease and to indicate the role(s) played by the host immune response and the various factors influencing it in the pathogenesis of the disease. Also, the disease may serve as a model system whose mechanisms might have broader implications in other hemoprotozoal and persistent infections. Since there are fairly recent reviews on certain aspects of the disease, in some instances the reader will be referred to these reviews with little details on the specific subjects given in this paper. African trypanosomiasis has been recognized for many years (Harris, 1839; Cummings, 1850; Bruce, 1895; Preller, 1917), yet the disease re­ mains endemic in the animal and human populations across vast ex­ panses of Africa. Bruce in 1895 established that trypanosomes caused disease in livestock and that the disease could be transmitted by tsetse flies. As early as 1839 (Harris), disease in domestic livestock was at­ tributed to infection carried by tsetse flies. In addition to Africa, African trypanosomiasis caused by Trypanosoma vivax occurs in South America (E. A. Wells, personal communication, 1977). As is the cause with many tropical diseases, there is a paucity of concise data relating to the occurrence of trypanosomiasis in both hu­ man and animal populations. It has been estimated that 10,000 new cases of human sleeping sickness are diagnosed annually (de Roadt and

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Seed, 1977) with millions at risk (World Health Organization, 1979). Undoubtedly this is an underestimate of unknown magnitude, since many of the countries where the disease is endemic do not have available ac­ curate data necessary for determining the concise occurrence and distri­ bution of the diease. In animal trypanosomiasis, concrete data are also lacking, and data frequently are based upon the number of doses of trypanocidal drugs used in the country in question. It appears that many thousands of animals are affected by the acute disease each year. How­ ever, chronic trypanosomiasis in cattle is probably more important than the acute condition, affecting hundreds of thousands of animals. Trypanosomiasis in Africa occurs in man, cattle, sheep, goats, camels, horses, pigs, and wildlife species. The important species causing disease in cattle are T. congolense, T. vivax, and T. brucei. Other important species of trypanosomes are T. gambiense (man), T. rhodesiense (man), T. evansi (camels, horses, dogs, and others), T. suis (pigs), T. equinum (horses), and others. Tsetse flies are the primary transmitters, with wild­ life serving as one of the reservoirs. T. vivax is also transmitted by biting flies, which are the principal transmitters in South America, where tsetse flies do not occur. The primary means of controlling trypanosomiasis have been at­ tempts to control the tsetse populations. This has been based upon such activities as bush clearing, wildlife destruction, human settlement, and the widespread application of insecticides to the tsetse habitat. The insecticide method has been used in reclaiming large areas in Nigeria and to a lesser degree in other parts of Africa. Recently, research has been and is still being carried out on the possible utilization of the release of sterilized male tsetse flies as a means of decreasing the fly populations. The difficulties encountered with the control pro­ cedures to date have been the necessity of application of insecticide in the proper concentration and amounts over vast areas of Africa, the continual efforts required to prevent reintroduction of tsetse flies into the reclaimed areas, regrowth of bush, and others. Investigations on the immunologie control of trypanosomiasis have been described (see Murray and Urguhart, 1977), but successful field immunization against heterologous challenge has not been reported. An immunization approach, however, would be one of the contrai methods of choice were it possible to do so effectively under prevailing con­ ditions in Africa. Along with the lack of immunologie control procedures, there is a lack of information about the immunology of the disease, its pathogenesis, and the makeup of the causative organisms, including its antigens.

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II. Trypanosomes 1. STRUCTURE

The morphology of African trypanosomes has been described in detail and will not be elaborated again here (see Mulligan and Potts, 1970; Vickerman, 1969; Vickerman and Luckins, 1969; Hoare, 1972). A simplistic approach will be used here, with the term "bloodstream form" to mean the infectious form(s) that occur in the blood of the mammalian host after needle- or fly-induced infection and the term "metacyclic" to mean the form of the parasite extruded in the saliva of the infected tsetse fly during the feeding process. This is the form of the parasite introduced into the host at the time of the tsetse bite. Other developmental forms occur, but will not be referred to here. Emphasis will be placed on the antigens of the parasite, especially on the surface of the bloodstream forms. The structural components observed in trypanosomes include a nucleus, kinetoplast, mitochondria, granules, filaments, and tubules. A limiting membrane encloses these structures and associated cytoplasm. External to the limiting membrane is a protein coat. 2. ANTIGENS

The trypanosome is a complicated parasite with the above struc­ tures, metabolic products, and enzymes, etc., representing a complex of antigens to which the host is exposed during the life and death of the parasite in the blood and tissues. An immune response against "somatic" antigens (nonsurface) can be demonstrated by a number of sérologie techniques. Vickerman (1969) and Vickerman and Luckins (1969) demonstrated the presence of a proteinaceous layer on the outer surface of the mem­ brane of bloodstream forms. This "coat" is absent during the develop­ ment of the parasite in Glossina spp., but reappears on the metacyclic forms. The presence of the coat on the bloodstream forms, its loss during development stages in the tsetse fly, and subsequent reappearance on metacyclic forms, are important in considering the immunology of trypanosomiasis. This proteinaceous coat is the point of contact between the host and the parasite, and its antigenic composition determines, as will be discussed later, resistance following artificial immunization and other aspects of the disease. The chemical composition of the coat has been further defined, and it is now known as the "variant specific surface antigen" (VSSA) described by a number of authors (Vickerman and Luckins, 1969; Mulligan and Potts, 1970; Njogu, 1974; Cross,

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1975; Cross and Johnson, 1976; Barbet and McGuire, 1977; Rovis, 1977; Rovis et al, 1978; see also Doyle, 1977). The characterization of the VSSA of T. brucei has been extensively studied by Cross (1975) and Cross and Johnson (1976). It is a glycoprotein with a molecular weight of approximately 65,000. Cross studied the biochemical composition of the VSSA of variant populations of trypanosomes (see below) in infected animals. The work from this group indicated that each variant antigen had considerably different amino acid sequences, but it must be realized that the total area of the antigen studied was limited. Recent work based upon immunologie analysis of the VSSA has indicated, however, that common antigenic site(s) occur in different VSSAs (Barbet and McGuire, 1977). These common sites result in cross-reactivity between different variants of the same trypanosome species and between different trypanosome species. Investigations currently under way indicate that some infected animals mount an immune response against both the common and variable de­ terminants. The role these determinants play in the disease and its potential prevention remains to be elucidated. Much less work has been done on the VSSA of T. congolense and T. vivax. Preliminary results have suggested, however, that the molecular weight of the VSSAs from these species are slightly smaller than that of T. brucei (Rovis, 1977). A given trypanosome or trypanosome population with a single VSSA on their surfaces demonstrable within the limitations of present tech­ nology, will, upon introduction into an animal either by needle or by tsetse, result in the occurrence over time of populations of the organism with different antigenic determinants (antigenic variants) in the blood­ stream of an individual infected animal. This phenomenon is antigenic variation and has been recognized for years (Doyle, 1977; Gray, 1965, 1970; Leach, 1973; Nantulya et al, 1978; Njogu, 1974; Seed, 1974; Van Meirvenne et al., 1975). Gray (1975) has studied the sequential occurrence of different antigens during the course of T. gambiense in animals. Frequently occurring, or "basic," antigens were noted. Other researchers have observed similar occurrences and have also suggested a reversion to a predominant or frequently occurring antigen upon passage of trypano­ somes from infected animals through the tsetse (Cunningham, 1966; Gray, 1965, 1970; Seed, 1974; Van Meirvenne, 1975; see also Doyle, 1977). Therefore, animals in a given geographic area could be infected with parasites that undergo antigenic variation with ultimate exposure to these "basic" or frequently occurring antigens. It appears that al­ though different populations of trypanosomes with different surface antigens can be detected in the blood of infected animals, the appearance

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of the antigens does not follow a specific sequence (see Doyle, 1977). Also, Gray (1970) has shown the presence of similar antigens in trypanosome populations isolated from the same geographic area during a 5-year study. It should be kept in mind that present studies on antigenic variation generally utilize relatively insensitive techniques. Therefore, subtle differences in antigenic makeup of trypanosomes or small numbers of trypanosomes with different surface antigens may be difficult to detect. The potential use of metacyclic antigens for immunization is worthy of consideration, since the destruction or prevention of replica­ tion of trypanosomes at the site and time of the fly bite would seem a logical place to prevent the disease. The situation is undoubtedly com­ plicated by multiple infections in tsetse and by other considerations. Since the number of metacyclic trypanosomes obtained from the saliva of infected flies limit this research, efforts are being carried out to grow metacyclics in culture (Hirumi et al., 1978). The results have shown that the bloodstream forms of T. brucei in culture (elaborated further below) can be made to undergo morphologic changes similar to those occurring in the fly, including metacyclic forms, by appropriate manipu­ lation of the culture system. The number of metacyclics (by mor­ phology) at this stage of development of this system, however, is small, but efforts are being made to develop techniques to increase the number of metacyclics obtainable in vitro. 3. TOXINS AND PRODUCTS

Tizard et ai. (1978) have recently reviewed the biologically active products and components that have been demonstrated in African try­ panosomes. These authors have postulated mechanisms by which these biologically active substances may play a role in the pathogenesis of the disease. The reader is referred to this review which describes a number of substances including enzymes, lipids, hemolysins, mitogens, inflammatory factors, and others. Although the exact role these sub­ stances play in the disease is not clear, some of them undoubtedly contribute to the pathogenesis. 4. In Vitro PROPAGATION

One of the constraints to research on trypanosomiasis has been the inability to propagate the infectious bloodstream forms of the parasite in vitro. Previous efforts have enabled researchers to maintain the para­ site in culture for a short time with loss of the VSSA, loss of infectivity for mammals, and other changes. It has only been recently reported by

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Hirumi et al. (1977) that the infective bloodstream forms of T. brucei could be replicated continuously in culture. These authors have now shown that T. brucei can be propagated for approximately 2 years in culture (Hirumi et al, 1978), that infectivity for mice can be maintained, and that the cultured organisms have VSSA and other characteristics of the bloodstream forms. In addition, these researchers have been. able to manipulate the culture system to enable the parasite to undergo morpho­ logic alterations that appear analogous to the developmental states in both the mammalian and insect hosts. The same investigators have also been able to clone individual trypanosomes in culture. This significant accomplishment is making it possible to begin to investigate many aspects of the parasite and disease that were previously impossible. Such questions as the mechanism of antigenic variation, attenuation by in vitro propagation, relationship between replication rates and pathogenecity can now be investigated. Additional recent advances in the study of the parasite relate to the mechanism of antigenic variation. The phenomenon is well described in infected animals, but the mechanisms are unclear. A dual approach using the in vitro propagation system (Hirumi et al, 1978) and bio­ chemical investigations (Williams, 1978; Dube et al., 1979) are beginning to yield important results. Variation occurs in vitro in the absence of anti­ body and other host influences. Additionally, research has demonstrated that it is possible to isolate mRNA from T. brucei and that this mRNA can direct the synthesis of VSSA in a cell-free system. These findings are being pursued and should provide data on the mechanisms of antigenic variation and could theoretically provide a means for producing large amounts of pure antigens for vaccine and other uses. III. Immunology 1. IMMUNE RESPONSE

The antibody response in African trypanosomiasis has been investi­ gated to some degree in several species of animals. Mattern in 1961 described an increase in ß2 macroglobulins (IgM) in sleeping sickness patients. The high IgM levels have been found in humans, mice, cattle, rabbits, dogs, monkeys, sheep, and others (Mattern et al., 1961; Cun­ ningham et al, 1967; Rees, 1969; Seed et al, 1969; Frommel et al, 1970; Luckins, 1972, 1976; Clarkson, 1975; Clarkson et al, 1975; Kobayashi and Tizard, 1976; Murray and Urquhart, 1977). The elevated IgM levels in human sleeping sickness have been used as a nonspecific diag­ nostic test (Cunningham et al, 1967; Bing et W.,il968). The IgM increase observed in experimentally infected calves occurs

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within 1-3 weeks after inoculation, and IgM generally remains high for long periods. The levels of IgM reported by Kobayashi and Tizard for Holstein and Holstein-Hereford cross calves infected with T. congolense were 6 to 24 times higher than the preinfection levels by 2 to 3 weeks after infection. Similar increases have been reported by others for T. vivax and T. brucei infected cattle. The IgM levels in the animals reported by Kobayashi and Tizard gradually declined, so that some calves have levels comparable to the control after several months. Simi­ larly, Clarkson et al. (1975) found that the IgM levels in cattle infected with trypanosome strains of low pathogenecity declined to normal or nearnormal levels after being initially elevated. Luckins (1972) reported that domestic animals reared in tsetse areas had elevated IgM, probably due to repeated challenge and/or infection. Kobayashi and Tizard (1976) measured IgGi and IgG 2 in the experi­ mentally infected calves described above. The mean IgGi level increased approximately 2- to 3-fold by 7 weeks postinfection followed by decreases to slightly above preinfection levels by 7 weeks. IgG 2 levels increased to a lesser degree than did IgGi and did not reach levels twice that detected in the control animals. Other workers have reported similar results indicating less increase in IgG than in IgM. The functions of the elevated immunoglobulins and immunoglobulin classes and subclasses have not been investigated in depth. Both IgM and IgG have been shown to be functional in various sérologie pro­ cedures and in trypanosome neutralization. In addition, increased production of antibodies directed against nontrypanosome (heterophile) antigens, such as sheep erythrocytes, has been reported (Clarkson et al., 1975; Houba et al., 1975). It appears, therefore, that African trypanosomiasis reuslts in a pronounced immuno­ globulin response, especially of IgM but also of IgG. The immuno­ globulins produced have specificity for somatic antigens as well as VSSA of the trypanosome. Stimulation of antibodies directed against nontrypanosome antigens occurs at the same time, but the quantitative amounts of the total IgM and IgG directed against trypanosome and nontrypanosome antigens is unclear. The antibodies are detectable by the common sérologie techniques, such as fluorescent antibody (FA), precipitation, agglutinations, complement fixation, radioimmunoassay, etc. By the use of some of these techniques, especially FA, it is possible to differentiate trypanosomes with different VSSA specificity. Using somatic antigens it is possible to detect antibodies with widespread cross-reactivity due to the occurrence of common somatic antigens in practically all trypanosome populations. Until recently, techniques like FA have detected only the surface antigen common to that particular

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variant population and has been the basis of studies on antigenic varia­ tion. Barbet and McGuire (1977), however, have shown common reacting sites within the VSSA, using radioimmunoassay as the detection system. Preliminary results suggest that some infected animals mount an immune response against the common site(s) as well as the noncommon (variant) site(s). This work is being pursued further at the present time. The antibodies produced are effective in the killing of trypanosomes (Leach, 1973; Lowrie and O'Connor, 1936). This has been shown by neutralization tests with and without complement, but killing is more effective with complement. Anti-trypanosome antibodies can be detected within 1 to 2 weeks after infection and persist for months in infected animals. Exactly how long these antibodies remain detectable in the circulation without restimulation by the same or cross-reacting antigens is not known. It has been shown, however, that animals that have been vaccinated or infected with a trypanosome population with a given VSSA specificity and do not die or are treated with a trypanocidal drug after having time to mount an immune response against a given popula­ tion, cannot be reinfected by trypanosomes with the same VSSA specifi­ city for months after the initial infection or immunization. Longevity studies in this regard, however, are limited. In addition to the above-mentioned antibodies, low molecular weight IgM has been detected in trypanosomiasis as have anti-tissue anti­ bodies (Frommel et al., 1970; MacKenzie and Borham, 1974). Whether the anti-tissue antibodies are significant in the pathogenesis of the disease or are a reflection of nonspecific antibody production is not clear. No evidence is available that suggests that cellular immune responses per se kill trypanosomes, so that a direct role for this arm of the immune response in the elimination of the parasite from the infected host is not evident. Phagocytized parasites can be observed in macrophages in the spleen and other tissues. Current information on the immunology of African trypanosomiasis indicates that the initial proliferation of trypanosomes in the infected host results in an antibody response directed against the VSSA and other antigens. As the antibody response occurs, the parasites in the circulation with the VSSA to which the antibodies are directed are killed. Death and lysis of the parasites in the circulation and/or their removal by the phagocytic system releases a number of antigens and products (Tizard et al., 1978) into the circulation, against which the host also responds and which may directly or indirectly influence the immune and other sys­ tems. Subsequently, or concurrent with the decline in parasite numbers, an­ other population of parasites with a different VSSA specificity replicates and can be recognized by FA staining using specific antisera. The host

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subsequently responds against the VSSA on these proliferating para­ sites with destruction of part or all of them. This sequence of events in many instances continues to occur for months. Thus, the infected host is responding to parasite antigens, but the parasite is able to elude the response and/or stay sufficiently in advance of the immune response by antigenic variation and other possible mechanisms to be described below. Work by Hirumi and his colleagues (1978) has shown, however, that antibody is not required for variation to occur in vitro. The host immune response and the various factors that influence it in determining the ability of an animal to "tolerate" infection or eliminate parasites have only superficially been investigated, the results primarily having been developed from the mouse system. Whether a specific component(s) of the host immune system is more effective in killing the trypanosomes in the host blood and tissues remains to be proved. The role of immunoglobulin classes and subclasses and their interrelationships needs further clarification. Investigations carried out to date are rather superficial and probably would not detect subtle differences in the neutralizing ability of various immunoglobulin classes. If differences become evident, investigations on how the most effective mechanism (s) could be stimulated need to move forward. Current research suggests that the trypanosome, or trypanosome com­ ponents, products, or perhaps the VSSA have a significant influence on the immune system of infected mice. Immunosuppression occurs (Goodwin et al, 1972; Longstaff, 1974; P. K. Murray et al, 1974a,b; Hodson et al, 1975; Roelants et al, 1977), and the results indicate that there is a nonspecific, polyclonal activation of the immune system that may limit the ability of the system to "see" and respond to certain antigens. The lymphoid system appears to be "turned on" and stimulated to produce antibodies against a variety of antigens that the animal has previously encountered, including trypanosome antigens. A mitogenic effect by the parasite or its products has been suggested (Green­ wood, 1977; Esuruoso, 1976; Assoku et al, 1977). Other investigations indicate that there may be influences on the number and potential inter­ actions between various cells comprising the immune system and that the interactions of the cells necessary for an effective immune response may be altered in such a way as to prevent the system functioning optimally (Morrison et al, 1978). This suppression appears to be the result of the activity of suppressor and/or cytotoxic cells demonstrable in the spleen (Pearson et al, 1978). Thus, the host defense mechanisms may be "de­ flected" sufficiently by the parasite to ensure its continued persistence. Such studies have not been carried out in cattle, and the role of im­ munosuppression in this species is still to be determined.

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The mechanism (s) of the predominant IgM increase in trypanosomiasis is unknown. As stated above, a mitogenic effect by a trypanosome component or product could occur and cause the elevation (Greenwood, 1974; Esuruoso, 1976; Assoku et al, 1977). The composition of the causative components or products could play a role in stimulating primarily IgM production and/or impede the transition from IgM to IgG. The cellular interactions in the immune response could be important as well with data currently available suggesting that the cellular com­ position and possible cell-cell interactions are not normal in infected mice (Roelants et al, 1977; Pearson et al, 1978). Continued stimulation of the immune system occurs with the release of large amounts of anti­ gen, formation and deposition of immune complexes, decrease of circu­ lating C3 levels (Kobayashi and Tizard, 1976; Musoki and Barbet, 1977), activation of the kinin system (Boreham, 1968), etc. All of these could interplay in influencing the immune system. To learn the importance of antigenic variation and/or deflection or dysfunction of the immune sys­ tem individually or collectively for parasite persistence requires additional research and could be important in gaining the necessary information for controlling the disease. The role of nonspecific stimulation of the immune system on trypanosomiasis has not been investigated in depth, but there have been indications that the nonspecificaily stimulated immune system can significantly alter the clinical course of trypanosome infections in mice (Murray, 1978). These studies demonstrated that mice given C. parvum before infection had a significantly prolonged clinical course when com­ pared to nonadjuvant stimulated controls. Whether such experimental, nonspecific stimulation of the immune system in cattle can be of benefit remains to be shown. Likewise, whether "priming" of the immune system occurs in the field and is important in the survival of cattle needs study. The success of these and other more specific immunologie ap­ proaches, however, could depend upon the elucidation of the optimal effector mechanism (s) in the host and the selection and use of an immunostimulant best suited to obtain the most appropriate response (s). 2. SURVIVAL OF ANIMALS IN ENDEMIC AREAS

The ability of the host immune response to prevent infection or allow an infected animal to live with the infection while remaining clinically asymptomatic are important considerations in appreciating the role(s) of immunopathology in the pathogenesis of the disease and the potential for immunoprophylaxis preventing the disease. For many years it has been known that there are small humpless, shorthorned

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cattle in Central and West Africa (Fiennes, 1950; Chandler, 1952, 1958; Desowitz, 1959; Stephen, 1966, Roberts and Gray, 1973) able to survive in tsetse areas. These include the N'Dama, Muturu, and other breeds. The ability of cattle to survive in trypanosomiasis areas has been called trypanotolerance and is of considerable interest to national govern­ ments and international organizations concerned with exploiting this tolerance for cattle production in endemic areas. Previous reports indi­ cated that trypanotolerant cattle could survive in tsetse-infested areas, but some animals developed infection and some died of the disease. Therefore, trypanotolerance means the ability of an animal/breed to live with the disease (tolerance) after becoming infected. Also, reports have indicated that N'Dama cattle surviving in one geographic area would develop disease and some would die when moved to another geographic area (Gates, 1952). This suggests an "acclimatization" to a new spectrum of parasites in the new area. Other workers, however, have stressed the "inherent" resistance more than exposure to the trypanosome population in the new area (Chandler, 1958; Desowitz, 1959; Roberts and Gray, 1973). Recent research that has been carried out in West Africa on trypano­ tolerance has been reported by Murray (1978). The studies compared trypanosomiasis in a trypanotolerant breed (N'Dama) with a nontrypanotolerant breed (Zebu) in the Gambia (Murray and Morrison, 1977; Murray, 1978). The animals were experimentally infected by injection of T. congolense, T. brucei, or both, and a large number of parameters were evaluated. Both N'Dama and Zebu became infected with T. brucei, T. congolense or both, depending upon the experimental group in ques­ tion. The clinical course of the disease was shorter, the anemia was less severe and the parasitemias lower in the N'Dama than in the Zebu. Of the animals at risk for the total period of the observation, approxi­ mately 75% of the Zebu died whereas none of the N'Dama died. The N'Dama, however, were clinically ill and showed the signs and tissue and body fluid alterations characteristic of the disease. Thus, the N'Dama cattle were able to live with the infection although they did develop, in many cases, severe disease. In a continuation of this investigation, the survivors along with additional noninfected controls were subjected to field exposure by tsetse challenge along the Gambia river. The results of this exposure indicated that the Zebu that had not died from the experimental in­ fection became ill, most dying as did the previously nonexposed con­ trols. The N'Dama which had recovered from the experimental disease, however, regardless of whether they had previously been infected with T. brucei, T. congolense, or both, did not develop significant anemia or

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parasitemia and were generally clinically normal. N'Dama controls that had not previously been experimentally infected became infected, de­ veloped clinical disease, and, at this writing, none died. These results suggest that the N'Dama animals were better able to cope with trypano­ somiasis than the Zebu and that previous experimental infection rendered the N'Dama more resistant to tsetse-transmitted infections than either the N'Dama controls or Zebu, whether the latter were previous infected or not. The trypanosomes used for the experimental infections had been isolated from infected cattle from the area into which the animals were introduced for field exposure. It appears from this in­ formation that some breeds of cattle can be made more resistant or tolerant to trypanosomiasis. The genetic implications in regard to the host appear to be significant, but have not been examined in cattle. In other experiments carried out in Senegal (S. Toure, personal communication, 1978), N'Dama and Zebu cattle were placed in the field under tsetse challenge. Both breeds developed severe disease and would have died had not chemotherapy been initiated. This would suggest that tolerance is relative and can be overcome by severe chal­ lenge. It has also been observed in Africa that cattle other than the small, humpless, shorthorns can survive in areas where trypanosomiasis is endemic. This has been suggested by Cunningham (1966) and others who have observed the rearing of cattle without chemoprophylactic cover in such areas. All of the above suggests that cattle can be maintained in endemic areas. The observations that N'Dama that are able to survive in one area develop disease when moved to another area which the animals had not previously occupied further suggests that an immune response against appropriate antigens may play a role (Gates, 1972; Chandler, 1958). Certain strains of sheep and goats also appear to be more tolerant to trypanosomiasis than are others. This is based upon observation of sheep and goats maintained in trypanosome areas where cattle cannot be successfully raised and in limited work directed to comparing the course of the disease in different sheep and goat breeds. In addition to the domestic species, there have been limited investi­ gations on the ability of wildlife species to tolerate trypanosomiasis infection. The suggestion has been fairly widely accepted that wildlife are the primary reservoir of trypanosomiasis in the field and that wildlife species are tolerant or resistant to the disease. Cunningham (1966), however, suggested that wild animals do get the disease, but that this goe& unrecognized for a variety of reasons including the lack of close scrutiny by diagnosticians. Limited work carried out in Africa in which

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different species of wild animals were inoculated with trypanosomes indicated that there is a variation in the ability of different species to "tolerate" infection. This work (Ashcroft et al, 1959) primarily involved T. rhodensiense and T. brucei and that some animals, such as Thompson's gazelle, dikdik, Blue forest duiker, and hyrax were usually continuously parasitemic and were killed by the infection. Other species, such as common duiker, eland, bohor reedbuck, oribi, bushbuck, and impala became infected, but usually did not die from the infection. The blood of these species contained detectable parasites for a considerable period of time. Warthog, bushpig, and porcupine were found to be capable of infection, but trypanosomes were very scanty in the blood. These differences should be exploited for evaluating the mechanisms of host resistance to trypanosomiasis. Host factors and mechanisms in trypanotolerance have been studied using the mouse system and have shown that different inbred strains of mice vary in a number of parameters following experimental infection (Morrison et al, 1977; Morrison et al., 1978). A / J mice infected with a standard challenge dose of T. congolense died within approximately 10 days whereas C57/B1 mice lived for over 90 days. Strains of mice with survival times between these two extremes were also demonstrated. Various hématologie and other parameters differed as exemplified by levels of parasitemia and decrease in PCV. The more tolerant mice had lower initial and subsequent parasitemias than did the strains of mice that died more quickly (Morrison et al., 1977). In addition, these animals became less anemic than did the more susceptible counterparts. Cellular events in terms of numbers of lymphoid cells, type of cells, immune responsiveness, etc., have been examined by Roelants et al. (1977) and indicate that the less tolerant mice were more subject to polyclonal activation. Studies on specific functions of various lymphoid and other cell populations are currently underway. These investigations have further suggested that the different strains of mice vary in their ability to cope with the initial parasitemia and that the ability to keep the initial parasitemia low significantly influences the clinical course, although all mice eventually die. 3. EXPERIMENTAL VACCINATION

It has been recognized for many years that resistance to experimental trypanosome infection can be produced in various animal species when antigenically homologous trypanosomes are used for vaccination and challenge. Various antigens have been effective, including purified VSSA, irradiated whole parasite, killed parasites with adjuvant (Bevan, 1936; Lapierre and Rousset, 1961; Johnson et al., 1963; Herbert and Lumsden,

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1968; Duxbury and Sadun, 1969; Wilson, 1971; Duxbury et al, 1972; Lanham and Taylor, 1972; Wilson et al, 1976; Murray and Urquhart, 1977). Thus, good immunogens are present on and in the trypanosome, and these have been shown to elicit a protective host response which is probably mediated through antibody. As indicated above, successful vaccination has utilized homologous systems in which the parasites with the same VSSA antigenic specificity immunization as are used for challenge. Challenge with heterologous parasite, those with the different VSSA, results in infection and no protection. One reason cited for lack of protection to heterologous challenge has been antigenic variation. Although present evidence sug­ gests that the spectrum of these antigens is large, studies have shown a frequency of occurrence of certain antigens (basic or predominant antigenic types, etc.) in the bloodstream during the course of the ex­ perimental and natural disease as discussed above. Also, limited passage of trypanosomes through the tsetse fly may result in a return to either a common antigenic type or to a number of types. This would imply that a population of animals from a given geographic area might ultimately encounter a similar antigen or group of antigens. Since animals are able to survive in endemic areas, such may occur under field conditions. A few authors have described protection against heterologous chal­ lenge by experimental immunization, field exposure or repeated in­ fected tsetse bites (Bevan, 1936; Whiteside, 1962; Stephen, 1966; Fiennes, 1970; Wilson et al, 1976). These studies need further elabora­ tion and only suggest that protection can be engendered.

IV. Pathogenesis Much of the information on the pathogenesis of African trypano­ somiasis (Fiennes, 1970; Losos and Ikede, 1972; Murray, 1974; M. Mur­ ray et al, 1974) has resulted from studies in the mouse. Considerably less information is available concerning bovine trypanosomiasis, and even less is known about the disease in other species. In contrast to the field disease in cattle, experimental infection of mice almost always kills the animals. Experimental infection of cattle frequently kills, whereas the disease in the field may produce a chronic syndrome. The disease in mice is characterized by rapid parasitemia (1 to 3 days), anemia, hypergammaglobulinemia, severe changes in the lymphoreticular system, myocarditis and other tissue alterations, and death. In cattle the initial parasitemia occurs after approximately 6 to 14 days, frequently followed by cycles of parasite replication. Some ex-

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perimentally infected animals recover from the initial stages of the disease after several months, become clinically normal, and are in­ frequently parasitemic. This is exemplified by five boran steers that were experimentally infected with T. congolense. These animals became severely ill, were very anemic, and were repeatedly parasitemic. After approximately 6 months, however, the animals began to improve clinically, the anemia became less severe, and they began to gain weight. They have now been clinically asymptomatic for 1.5 years, although it is still possible to detect low numbers of parasites in the blood on rare occasions. Similar recoveries undoubtedly occur in the field, but rein­ fection, stresses of trekking for food and water, plane of nutrition, and other factors would influence such "recoveries." The lesions in infected cattle are loss of body condition, anemia, hypergammaglobulinemia, reactive bone marrow (in some cattle), lymphadenopathy, splenomegaly, and, at terminus, an enlarged flabby heart. The microscopic changes are dominated early by a lymphoproliferative response with active germinal centers and lymphoid hyperplasia. Later, the cell populations in lymph nodes and spleen are largely plasma cells and macrophages. Lymphoid infiltrates may be observed perivascularily in a number of tissues, and necrosis of the myocardial muscle fibers may be observed. The host-parasite relationships and the mechanisms of disease in Africa trypanosomiasis are extremely complex. The balance between disease and no disease after the bite of an infected fly and the intro­ duction of trypanosomes is due to a number of possible factors, some of which are listed below. A.

B.

Parasite factors 1. Number and species of introduced trypanosome(s) 2. Pathogenicity (virulence) 3. Ability of trypanosomes to replicate to high levels before sup­ pression by host factors 4. Introduction into the host of additional parasites by continuing exposure to infected flies 5. Antigenic variation 6. Biological activity of the parasite and its products and com­ ponents to abrogate or deflect functional host defense mechan­ isms and to influence other systems and functions 7. Release of antigens and other structural and metabolic products into the circulation of the host Host factors 1. Status, timing, and effectiveness of the immune system and its response (s)

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Previous exposure to similar or cross-reacting antigens Genetics Age Colostral transfer of antibody (?) Antigen-antibody complexes Nutritional status and stress

Introduction of trypanosome by an infected tsetse fly results in replication (in a fully susceptible host) with parasitemias evident from one to several days after inoculation. The methods for detecting parasitemias are not very sensitive, so that parasite replication prob­ ably reaches significant levels before it is detectable microscopically. The level of the initial parasitemia appears to indicate to a degree the course of the disease that will result, based upon data from mice and cattle experiments. A low parasitemia may indicate a more rapid and/or efficient response by the host or may be the result of less-virulent trypanosomes. The virulence and numbers of organisms inoculated influences the disease produced. This has been well documented by Murray and Morrison (1977) in cattle. Antigenic variation occurs with parasites with varying VSSA antigenicity detectable at time intervals from approximately 3 to 12 days in many instances. The host mounts an immune response directed against the various trypanosome antigens including the VSSA. Trypanosome components and/or products directly affect the host lymphoid system, directly decrease complement levels, and have a direct toxic effect on host erythrocytes. The parasite (in the mouse) has a possible mitogenic effect on the lymphoid system, and immunosuppression, hypergammaglobulinemia, and anemia also occur. Antigen-antibody complexes are formed and are deposited in kidneys and heart (Murray et al, 1975). Polyclonal activation of the lymphocytes occurs and elevated heterophile as well as antitrypanosome antibodies are detectable. The lymphoid system appears to be stimulated and very active, as evidenced by morphology and immunoglobulin pro­ duction, whereas some aspects of the immune response are defective or suppressed (in the mouse). If the host has previously been exposed to trypanosomes with an analogous VSSA to that of the inoculated population, the parasites are destroyed. If the host immune system has been nonspecifically stimu­ lated before inoculation, the host may be better able to cope with the infection. Genetic factors play a role in determining the clinical disease course, level of parasitemia, degree of anemia and trypanotolerance in general. The mechanisms are not clear, but in the mouse may indicate the

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degree of susceptibility to immune system alteration by the trypanosome and/or its products. The mechanism of tolerance in cattle may reflect the responsiveness and/or effectiveness of the immune response or could relate to physiologic and other factors that allow the N'Dama and similar breeds to better cope with infection. It seems likely that a number of the lesions and functional alterations result directly from the presence of the parasites and their products. The N'Dama cow becomes infected, but in some cases restricts the para­ site and the resultant deleterious effects. After recovering from infection, the N'Dama appears able to cope with mild and/or moderate tsetse challenge and may become clinically asymptomatic. That this partially results from exposure to the trypanosome population in the area in which the animal is resident is suggested by the reappearance of clinical disease and sometimes death when N'Dama previously asymptomatic in one location are moved to another. It is assumed that the animals have been exposed to a different population of trypanosomes in the new location. It must be pointed out again, however, that N'Dama become clinically ill and die from trypanosomiasis. The mouse data from re­ search examining trypanosomiasis in different inbred strains of mice indicate that the genetics of the host influence the experimental disease. It appears that in mice the genetic factors may be attributable to immune mechanisms, but this remains to be proved. Similar data are not available for cattle. Other factors, including age, colostral transfer of antibody, nutritional status, and stress, are indicated as playing a role in the disease in the field. Calves from recovered N'Dama cows frequently do not get disease or get mild disease. Some have indicated that calves generally get milder disease, but this needs further study. Cattle in a poor state of nutrition and having to walk considerable distances for food and water may become parasitemic and die after having been clinically asymptomatic for some time. Cattle whose immune system is capable of rapid responses that de­ press or abrogate parasite proliferation appear more likely to be able to cope with the disease. Previous exposure to similar antigens, the rapidtiy of the immune response, and the degree of immunodepression would seem important, as are the factors mentioned previously. Treat­ ment by trypanocidal drugs will frequently result in rapid clinical improvement including the anemia. That infected animals are con­ tinually exposed to antigens and antigen—antibody complexes seems evident, but the role of the complexes in disease is not clear. Some workers have suggested that antigen-antibody complexes are important in the genesis of the anemia (Kobayashi et al., 1976). It must also be kept in mind that infections by more than one

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species of trypanosome are frequent and that continual inoculation by additional fly bites occur in the field. Within a given animal, the above-mentioned processes are going on, with the possible introduction at any time of other parasites to which the host has not previously been exposed. The interrelationships and dynamics of the factors discussed above and others are important in trypanosomiasis. Surprisingly little is known about the pathogenesis of the disease in cattle and man,, which points out the importance of additional in-depth studies being carried out. Parasite influences on various aspects of the host immune capabili­ ties seem paramount. V. Conclusions African animal trypanosomiasis is characterized by a complex host-parasite relationship in which the organism has evolved rather sophisticated mechanisms for evading the host defense mechanisms. The parasite, after introduction into the host, changes its own surface antigen (s) and also appears to directly and/or indirectly influence various host factors, such as directly activating complement; directly, and possibly indirectly through antigen-antibody complexes, destroy­ ing or causing premature removal of erythrocytes, activating the kinin system causing immunodepression and perhaps otherwise deflect­ ing the immune responses ; producing hypergammaglobulinemia primarily composed of IgM ; and having a mitogenic effect that may account for some of the above. That parasite components and/or products can cause host cell and tissue alterations directly seems evident. Likewise, host responses and parasite components and products may cause dysfunction and lesions. It appears that the host may be more capable of either destroying or living with the parasite if the animal has previously encountered similar antigens ; if its immune system or some component of the system has been specifically or nonspecifically stimulated and is capable of "rapid" responses to prevent the parasite from replicating to sufficient numbers to cause severe disease; if the animal is able to survive in a given geographic area long enough to have been exposed to the necessary spectrum of antigens to keep parasitemias low or eliminated; or if the animal genetically may be more resistant to the influences of the apparent effects. The genetic background of the host appears to play an undetermined but influential role in enabling the host to tolerate infections. The mechanisms for such tolerance in cattle and in wild ruminants would seem to be important areas for further research. The

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mouse systems may also be able to contribute to an understanding of such mechanisms, but such studies should be closely correlated with the disease in the field in domestic livestock. The rapid recovery of animals with clinical trypanosomiasis following chemotherapy with trypanocidal drugs further suggests that the direct influence of the parasite in causing many of the lesions and alterations. Thus, domestic animals that can naturally or artifically by immunoprophylaxis keep the numbers of parasites low and/or respond more quickly to the antigens of each variant population have an advantage, although the advantage appears relative and can be overcome by severe challenge. The information necessary to completely understand African animal trypanosomiasis is lacking. Research on the parasite, the host, and the host-parasite relationship as well as the epidemiology are necessary if sufficient data are to become available to enable an appreciation of the disease mechanisms and the development of successful immunoprophylactic approaches that can be applied to the field in Africa. REFERENCES Ashcroft, M. T., Burtt, E., and Fairbairn, H. (1959). Ann. Trop. Med. Parasitol. 53, 147-141. Assoku, R. K. G , Tizard, I. R., and Nielsen, K. H. (1977). Lancet ii, 956-959. Barbet, A. F., and McGuire, T. (1977). 1976 Research Report. International Labora­ tory for Research on Animal Diseases, Nairobi, Kenya. Barbet, A, F., and McGuire, T. C. (1978). Proc. Nat. Acad. Sei. USA 75, 1989-1993. Bevan, L. E. W. (1936). Trans. R. Soc. Trop. Med. Hyg. 30, 9-206. Bing, G., Timperman, G., and Hutchinson, M. P. (1968). Bull. W. H. 0. 38, 523-545. Boreham, P. F . L. (1968). Br. J. Pharmacol. Chemother. 32, 493-504. Bruce, D. (1895). Burnat and Davis, Durban. Chandler, R. L. (1952). Ann. Trop. Med. Parasitol. 46, 127-134. Chandler, R. L. (1958). J. Comp. Pathol. 68, 253-260. Clarkson, M. J. (1975). Trans. R. Soc. Trop. Med. Hyg. 69, 272. Clarkson, M. J., Penkale, W. J., and McKenna, R. B. (1975). J. Comp. Pathol. 85, 97-410. Cross, G. A. M. (1975). Parasitology 7 1 , 393-417. Cross, G. A. M., and Johnson, J. G. (1976). In "Proceedings of Host-Parasite Rela­ tions" (H. Van den Bosseche, ed.), pp. 413-420. Elsevier, Amsterdam. Cummings, R. G. (1850). "Five Years of a Hunter's Life in the "Far Interior of Southern Africa," Vol. I I . Murray, London. Cunningham, M. P. (1966a). East Afr. Med. J. 43, 394-397. Cunningham, M. P. (1966b). Trans. R. Soc. Trop. Med. Hyg. 60, 126. Cunningham, M. P., Bailey, N . M., and Kimber. C. D. (1967). Trans. R. Soc. Trop. Med. Hyg. 6 1 , 688-695. de Roadt, P., and Seed, J. R. (1977). "Parasitic Protozoa" (J. P. Kreier, ed.), Vol. 1, p. 176. Academic Press, New York. Desowitz, R. S. (1959). Ann. Trop. Med. Parasitol. 53, 293-313.

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Doyle, J. J. (1977). Adv. Exp. Med. Biol. 93, 31-36. Dube, D. K , Williams, R. O., Seal, G., and Williams, C. (1979). Biochem. Biophys. Acta, in press. Duxbury, R. E., and Sadun, E. H. (1969). J. Parasitol. 55, 859-865. Duxbury, R. E., Sadun, E. H., and Anderson, J. S. (1972). Am. J. Trop. Med. Hyg. 21, 885-888. Esuruoso, G. 0 . (1976). Clin. Exp. Immunol. 23, 314-317. Fiennes, R. N . T W (1950). Ann. Trop. Med. Parasitol. 44, 42-54. Fiennes, R. N . T W (1970). In "The African Trypanosomiases" H. W. Mulligan, ed.), pp. 729-750. Allen & Unwin, London. Frommel, D., Perey, D. Y. E., Masseyeff, R., and Good, R. (1970). Nature (London) 228, 1208-1210. Galvao-Castre, B., Hockmann, A., and Lambert, P. H. (1978). Clin. Exp. Immunol. 33, 12-24. Gates, G. M. (1952). Farm For. 11, 19-43. Goodwin, L. G., Green, D. G., Guy, M. W., and Voller, A. (1972). Br. J. Exp. PathoL 53, 40-43. Gray, A. R. (1965). J. Gen. Microbiol. 4 1 , 195-214. Gray, A. R. (1970). J. Gen. Microbiol. 62, 301-313. Gray, A. R. (1975). Trans. R. Soc. Trop. Med. Hyg. 69, 131-138. Harris, W. C. (1839). "The Wild Sports of Southern Africa." H. Bohn, London. Herbert, W. J., and Lumsden, W. H. (1968). J. Med. Microbiol. 1, 23-32. Hirumi, H., Doyle, J., and Hirumi, K. (1977). Science 196, 992-994. Hirumi, H., Doyle, J., and Hirumi, K. (1978). 1977 Research Report. International Laboratory for Research on Animal Diseases, Nairobi, Kenya. Hoare, C. A. (1972). "The Trypanosomes of Animals." Blackwell, Oxford. Hodson, K. M., Frieman, J. C , Fyner, C , and Terry, R. J. (1975). Trans. R. Soc. Trop. Med. Hyg. 69, 273. Houba, V., Brown, K. N., and Allison, A. C. (1969). Clin. Exp. Immunol. 4, 113-123. Johnson, P., Neal, R. A., and Gall, D. (1963). Nature (London) 200, 83. Kobayashi, A., and Tizard, I. R. (1976). Tropenmed. Parasitol. 27, 441-417 . Kobayashi, A., Tizard, I. R., and Woo, P. T. K. (1976). Am. J. Trop. Med. Hyg. 25, 401-406. Lanham, S. M., and Taylor, A. E. R. (1972). J. Gen. Microbiol. 72, 101-116. Lapierre, J., and Rousset, J. J. (1961). Bull. Soc. Pathol. Exot. 54, 332-336. Leach, T. M. (1973). Adv. Vet. Sei. Comp. Med. 17, 119-162. Longstaff, J. A. (1974). Trans. R. Soc. Trop. Med. Hyg. 68, 150. Losos, G. J., and Ikede, B. O. (1972). Vet. Pathol. 9, Suppl. Lowrie, E. M., and O'Connor, R. J. (1936). Ann. Trop. Med. Parasitol. 30, 365-388. Luckins, A. G. (1972). Br. Vet. J. 128, 523-528. Luckins, A. G. (1976). Ann. Trop. Med. Parasitol. 70, 133-145. MacKenzie, A. R., and Borham, P. F . L. (1974). Immunology 26, 1225-1238. Mattern, P., Massepff, R., Michel, R., and Peretti, P. (1961). Ann. Inst. Pasteur, Paris 101, 382-383. Morrison, I., Murray, M., and Roelants, G. (1977). 1976 Research Report. Interna­ tional Laboratory for Research on Animal Diseases, Nairobi, Kenya. Morrison, W. I., Roelants, G. E., Mayor-Witkney, K. S., and Murray, M. (1978). Clin. Exp. Immunol. 32, 25. Morrison, W. I., Roelants, G. E., Pearson, T. W., and Murray, M. (1979). Adv. Exp. Med. Biol. 69, in press.

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Mulligan, H. W., and Potts, W. H., eds. (1970). "The African Trypanosomiases." Allen & Unwin, London. Murray, M. (1974). Prog. Immunol., Int. Congr. Immunol., 2nd, 1974 Vol. 4, p. 181. Murray, M. (1978). 1977 Research Report. International Laboratory for Research on Animal Diseases, Nairobi, Kenya. Murray, M., Lambert, P. H., and Morrison, W. I. (1975). Med. Malad. Inf. 12, 638-641. Murray, M., and Morrison, I. (1977). 1976 Research Report. International Labora­ tory for Research in Animal Diseases, Nairobi, Kenya. Murray, M., and Urquhart, G. M. (1977). Adv. Exp. Med. Biol. 93, 209-241. Murray, M., Murray, P. K., Jennings, F. W., Fisher, E. W., and Urquhart, G. M. (1974). Res. Vet. Sei. 16, 77-84. Murray, P. K., Jennings, F . W., Murray, M., and Urquhart, G. M. (1974a). Immunology 27, 815-824. Murray, P. K., Jennings, F . W., Murray, M., and Urquhart, G. M. (1974b). Immunology 27, 825-840. Musoke, A. J., and Barbet, A. F. (1977). Nature 270, 438-440. Nantulya, V., Doyle, J., and Jenni, L. (1978). 1977 Research Report. International Laboratory for Research on Animal Diseases, Nairobi, Kenya. Njogu, A. R. (1974). Proc. Int. Congr. Parasitol. 3rd, 1974 Vol. 2, p. 1094. Pearson, T. W., Lunden, L. B., Roelants, G. E., and Mayor-Witkney, K. S. (1978). Research Report. International Laboratory for Research on Animal Diseases, Nairobi, Kenya. Pearson, T. W., Roelants, G. E., Lunden, L. B., and Mayor-Witkney, K. S. (1978). Eur. J. Immunol., in press. Preller, G. S. (1917). Trans. R. Soc. Trop. Med. Hyg. 63, 124-125. Rees, J. M. (1969). Trans. R. Soc. Trop. Med. Hyg. 63, 124-125. Roberts, C. J., and Gray, A. R. (1973). Trop. Anim. Health Prod. 5, 220-233. Roelants, G., Morrison, I., and Mayer-Witkney, K. (1977). 1976 Research Report. International Laboratory for Research on Animal Diseases, Nairobi, Kenya. Rovis, L. (1977). 1976 Research Report. International Laboratory for Research on Animal Diseases, Nairobi, Kenya. Rovis, L., Barbet, A. F., and Williams, R. O. (1978). Nature 241, 641-649. Seed, J. R. (1974). J. Parasitol. 52, 1134-1140. Seed, J. R., Cornille, R., Ruby, E. L., and Gram, A. A. (1969). Parasitology 59, 283-292. Stephen, L. E. (1966). Ann. Trop. Med. Parasitol. 60, 230-246. Van Meirvene, N., Jannsens, P. G., Magnus, E., Lumsden, W. H. R., and Herbert, W. S. (1975). Ann. Soc. Belge Med. Trop. 55, 25-38. Vickerman, K. (1969). J. Cell Sei. 5, 163-193. Vickerman, K., and Luckins, A. G. (1969). Nature (London) 224, 1125-1126. Whiteside, E. F . (1962). In "Drugs, Parasites and Hosts" (L. G. Goodwin and R. H. Nimmo-Smith, eds.), pp. 116-141. Churchill, London. Williams, R. (1978). 1977 Research Report. International Laboratory for Research on Animal Diseases, Nairobi, Kenya. Wilson, A. J. (1971). Trop. Anim. Health Prod. 3, 14-22. Wilson, A. J., Paris, J., and Dar, F. K. (1975). Trop. Anim. Health Prod. 7, 63-71. Wilson, A. J., Paris, J., Luckins, A. G., Dar, F . K., and Gray, A. R. (1976). Trop. Anim. Health Prod. 8, 1-11. World Health Organization (1978). W H O Special Programs for Research and Train­ ing in Tropical Diseases, 2nd Annual Report, p. 15.

ADVANCES I N VETERINARY SCIENCE AND COMPARATIVE MEDICINE, VOL. 2 3

Tumor Immunology in Domestic Animals M. ESSEX AND C. K. GRANT Department

I. II.

III.

IV.

V.

VI. VII. VIII. IX.

of Microbiology, Boston,

Harvard School of Public Massachusetts

Health,

Immune Surveillance Demonstration of Spécifie Tumor Immunity Tumor Antigens . 1. Tissue-Specific and Histocompatibility Antigens of Tumors . . . 2. Antigens of Carcinogen-Induced Tumors 3. Antigens of Virus-Associated Tumors 4. Oncofetal or Tumor-Associated Embryonic Antigens 5. Soluble Oncofetal Antigens Antitumor Immune Effector Mechanisms 1. Immune Function of Cancer Patients 2. Mechanisms of Escape from Surveillance Immunotherapy of Cancer 1. Nonspecific Immunotherapy 2. Specific Immunotherapy Virus-Associated Tumors of Cats 1. Pathobiology and Epidemiology . 2. Immune Response to the Virus 3. Feline Oncornavirus-Associated Cell Membrane Antigen (FOCMA) 4. Immune Response to FOCMA .· Avian Tumors Caused by Retroviruses Marek's Disease Bovine Leukosis Transmissible Venereal Sarcoma of Dogs References

184 185 186 186 186 187 188 189 190 193 193 197 197 198 200 200 205 208 210 214 216 217 219 220

In most cases, malignant change appears to be an irreversible event and the characteristic, when once acquired, is passed on to progeny. The basis for such a permanent change must lie in the DNA, and can result only from alterations in the expression or repression of existing DNA sequences or in the introduction new DNA sequences (such as the src gene) following oncogenic virus infection. Whatever changes occur in DNA will be reflected in the molecular chemistry of the cell, and at 183 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-039223-2

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least some of the changes will involve the metabolism of proteins and glycoproteins which are, by nature, antigenic. As a result, malignant cells will sometimes bear new or altered protein antigens, and just as the proteins of an invading pathogen are recognized by an immunologically competent host as foreign, so the new or altered proteins of freshly arising tumors should also stimulate an immune response. The idea that a tumor might be recognized by its host as foreign is not particularly new, and the first recorded attempts to take therapeutic advantage of the putative human tumor antigens were made around the turn of the century. Hericourt and Richet (1895) treated patients with "tumor antisera" raised in heterospecies, and von Leyden and Blumenthal (1902) attempted "vaccination" using autologous tumor. The pioneering experimental studies of Ehrlich unavoidably involved tumor transplantation between outbred animals. Nevertheless, he first suggested a major tenet of current theory, namely, that the frequency at which tumors arise is far greater than their clinical incidence, and that it is the immune response which eliminates all but the clinically detected minority in their early and asymptomatic stages (Ehrlich, 1909). Development of inbred strains of mice, within which all individuals of a strain are syngeneic, subsequently facilitated experiments which proved the existence of tumor-specific antigens of laboratory-induced tumors (Gross, 1943; Foley, 1953). Prehn and Main (1957) provided convincing evidence for the individual nature of tumor antigens when they showed that within syngeneic tumor-immunized mice, rejection of a second transplant of tumor occurred while normal skin grafts from the tumor donors persisted indefinitely.

I. Immune Surveillance Burnet (1969, 1970) crystallized most current thinking in tumor immunology when he originally advanced the theory of immune surveil­ lance. Malignant change can be viewed as a predictable, relatively frequent, and unavoidable by-product of cell division rather than as a one-time event resulting in a serious or fatal lesion. The causes of transformation may be unrepaired DNA damage which results from both environmental insult and mistakes that occur during DNA replication and cell division. Differences between normal and malignant cells result in the appearance of tumor-specific proteins, and as the host has had no prior tolerizing experience of such proteins in early

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life, these have the capacity to stimulate an immune response, i.e., tumor-specific antigens are non-self. As the disparate antigenicity of cancer cells is usually recognized, the immune response destroys the majority of tumors in their early stages. The aberrant event in tumor progression can therefore be viewed as a failure of the immune response to recognize and destroy every microscopic tumor cell focus. Thus, Burnet suggests that a major function of the immune response is to survey all dividing cells regularly, and to eliminate those which have naturally become malignant. The immune mechanisms which destroy tumors may be the same as those which destroy surgically transplanted healthy foreign grafts. Indeed, while it seems unlikely that such mechanisms evolved to prevent organ transplantation, it does seem possible that they evolved to prevent accidental cell transfer. If it were not for the unique histocompatibility differences of individuals, the horrific possibility would exist for contagious tumors of the skin, nasopharyngeal, and genital areas (see Section I X ) . DEMONSTRATION OF SPECIFIC TUMOR IMMUNITY

Experimentally, specific tumor immunity is demonstrated when im­ munized animals survive a challenge dose of live tumor cells sufficient to kill syngeneic controls (i.e., nonimmunized mice, or mice immunized with an unrelated tumor). To effect immunization with tumor, the cells used are attenuated by irradiation (γ or X rays), formalin, or glutaraldehyde fixation, or by treatment with metabolic inhibitors (e.g., mitomycin C) ; alternatively, tumor membrane extracts are used (Johnson et al., 1975; Reisfeld et al, 1977; Sanderson and Frost, 1974). When tumors grow in strictly localized fashion, ligation or amputation of the tumor mass is sometimes sufficient to induce resistance to a second tumor challenge (Alexander, 1968; Prehn and Main, 1957). Protection resulting from immunization with non-virus-associated tumors is relatively weak, and although immunized animals resist higher doses of live tumor cells than are necessary to kill controls, immunity can usually be overcome by inoculation of extremely large numbers of challenge cells. The evidence for antitumor immunity in inbred animals is now irrefutable, but the experimental design has been, for the most part, prophylactic. As the progress of established primary tumors—or tumor métastases—has only rarely been shown to be affected by subse­ quent induction of immunity (Mathe et al, 1977), the clinical relevance of some experiments is not clear.

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II. Tumor Antigens Localization of tumor antigens may be internal or on the cell mem­ brane: The former will be antigenic only when the cells are destroyed or if the antigen is secreted as a soluble product; the latter are antigenic on intact cells unless they are masked in some way. Cell surface anti­ gens are relatively stable but they are not fixed structures, and they may migrate or be endocytosed or shed in certain circumstances. Efforts to analyze tumor antigens have been directed mainly at the cell surface, but a means toward monitoring the progress of cancer therapy may be provided by measuring changing levels of soluble tumor cell products. The ability of a tumor to elicit a specific immune response in the host of origin or in syngeneic normal individuals is its immunogenic capacity. Two classes of tumor antigens may give rise to immunogenicity, one being tumor-specific antigens (TSA) and the other being tumor-associated antigens (TAA or TATA). The latter may be detected on certain nonmalignant cells, e.g., fetal cells or foreign normal tissue, but they are not found on the normal tissue from which the tumor arose. 1. TISSUE-SPECIFIC AND HISTOCOMPATIBILITY ANTIGENS OF TUMORS

Many antigens expressed by tumor cells are also expressed by the normal cells from which they arise. Tumor cells transplanted into allogeneic mice are usually rejected in the same manner as allografts of normal tissue. Occasionally, malignant change results in the loss of expression of tissue-specific antigens (Baldwin and Glaves, 1972) or of certain histocompatibility antigens. In fact, it has been suggested that certain tumor antigens result from expression of alien histocompati­ bility antigens, because an inverse relationship sometimes exists between detection of TAA and of normal histocompatibility antigens, and be­ cause immunization with normal allogeneic tissue may produce im­ munity to transplanted syngeneic tumor (Parmiani and Invernizzi, 1975; Wrathmell et al, 1976). 2. ANTIGENS OF CARCINOGEN-INDUCED TUMORS

Two explanations for the appearance of specific antigens on chemi­ cally induced tumors exist. Spécifie antigenicity may arise because the carcinogen causes alterations of gene expression in the target cell, and consequently changes occur in biochemistry and in the manufacture of new cell proteins (Baldwin, 1973). Alternatively, some oncogeneic agents (e.g., methylcholanthrene) have immunosuppressive properties,

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and therefore they may facilitate the growth of a greater number of naturally occurring tumors, including those bearing strong tumor anti­ gens which would be resolutely attacked if local surveillance was not suppressed (Prehn, 1976). The latter explanation may account for the variation in immunogenicity of chemically induced tumors, but does not explain (a) the appearance of tumor antigens on normal cells trans­ formed in vitro by exposure to carcinogens (Baldwin, 1977) or (b) the specific and variable tumor antigenicity of cancers induced by physical agents such as ultraviolet irradiation or celluloid implants (Kripke, 1977). The properties of chemically induced tumors have been recently reviewed by Baldwin (1977). Separate tumors induced by the same carcinogen vary in their ability to immunize syngeneic animals against a live tumor cell challenge. Relatively few tumors are strongly immunogenic, and these arise rapidly following carcinogen application. Tumors which arise after a long latent period are weakly immunogenic or stimulate no detectable immune response. Tumor-specific antigenicity also correlates somewhat with the dose of carcinogen employed, the higher the dose the more immunogenic the tumor. Chemically induced antigens seem to be relatively stable cell mem­ brane-associated proteins or glycoproteins. Their major characteristic is one of unique specificity, for the immunogenic properties of chemi­ cally induced tumors rarely cross-react. Even in the extreme case of two discrete tumors induced in the same animals by the same carcinogen, the likelihood of cross-reactive immunogenicity is small. Nevertheless, the absolute specificity of chemically induced tumor antigens has been questioned by the finding that cross-reactions are sometimes detected in microcytotoxicity assays in vitro (Steele and Sjogren, 1974). These cross-reactions do not appear to have detectable counterparts in vivo, and may be explained by the presence of fetal antigens (Baldwin, 1977). 3. ANTIGENS OF VIRUS-ASSOCIATED TUMORS

Tumor viruses occur in many species (Friend, 1977), and they may be DNA containing (e.g., polyoma virus of mice, SV40 of primate) or RNA containing (e.g., sarcoma and leukemia viruses of birds and mammals). The oncogenic potential of tumor viruses is sometimes masked, and not all cause tumors in the host species from which they were isolated (e.g., SV40). Polyoma virus is ubiquitous in mice, but normally complete resistance to tumor occurs. When injected into immunosuppressed adults or immunoincompetent neonates, however, they induce progressive tumors. Subsequent transplantation of virus-

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induced tumors into normal syngeneic recipients leads to continued tumor growth in the new environment. If the adult recipients were first immunized with virus then growth of the transplanted tumor is inhibited. Immunity to virus-induced tumors does not result directly from im­ munity to the tumor virus, and there is strong evidence that viruses induce membrane-associated transformation specific cellular antigens which are unrelated to the virus antigens per se (Klein and Klein, 1977). In some experimental systems it is possible to separate virus and tumor immunity and thereby prove the independence of these twro immunological mechanisms. Virus-induced tumor immunity originates indirectly, but functions effectively because all tumors induced by the same virus share cross-reactive TA A. When virus is injected into normal adults it is thought to induce microscopic tumor cell foci which are highly immunogenic. These foci are destroyed by an immune response which is then primed to recognize any tumor cells induced by that virus. As a result, mice immunized with virus will subsequently reject transplanted tumor due to the cross-reacting virus-induced TAA. The immune response to virus-induced tumor antigens may be mediated by thymus-dependent lymphocytes (T cells) in the case of polyoma virus, or by antibody (feline oncornavirus-associated cell membrane antigen of feline leukemia). Viremic animals manifest tumor-immune responses, and immunity is expressed against tumor cells in which no virus replication occurs. Both these findings strongly support the argument that immune responses to virus and tumor are independent (see Section V). Immunity to virus antigens may take the form of neutralizing anti­ body to virus envelope antigens (e.g., gp70, pl5E). Complement-fixing antibodies may be produced against soluble viral antigens, which are thought to be the unique enzymes necessary in virus nucleic acid interaction (e.g., reverse transcriptase). 4. ONCOFETAL OR TUMOR-ASSOCIATED EMBRYONIC ANTIGENS

Schone (1906) drew attention to the analogy between fetal and tumor tissue: While normal tissue is derived from fetal tissue by differentiation, tumor tissue frequently seems to be derived from normal tissue via a process of dedifferentiation. Hence, it is possible that tumor antigens have their counterparts in fetal antigens. Sera removed from multiparous females, or from adults immunized with attentuated fetal tissue, do frequently react in vitro with a variety of tumor types including spontaneous, chemical, and viral-induced tumors, suggesting that some tumors express cross-reactive fetal-like

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antigens. Nevertheless, it is possible to elicit an immune response to oncofetal antigens in tumor-bearing animals without causing tumor rejection or influencing tumor progression (Baldwin, 1977). This finding may reflect the nature of fetal antigens which do not appear to be integral cell membrane components, but rather soluble products secreted by tumor cells.

5. SOLUBLE ONCOFETAL ANTIGENS

a. Carcinoembryonic Antigens

(CEA)

CEA were first detected associated with colonie adenocarcinomas (Gold and Freedman, 1965), and later in sera from patients with colonie and pancreatic cancers (Thompson et ai, 1969). As the sensitivity of CEA assays increased, it was found associated with other forms of cancer (e.g., breast and lung) and also in some nonmalignant diseases (e.g., alcoholic cirrhosis, pancreatitis). Several CEA-like molecules with distinct characteristics have now been detected, and their properties vary according to the tissue from which they are derived ; they are highly antigenic glycoproteins (MW approximately 180,000) and they are located in part in the tumor cell glycocalyx from where they may be released into the circulation (von Kleist, 1976). b. a-Fetoprotein

(AFP)

AFP is an oncofetal glycoprotein of MW 70,000 which is often found in high serum concentration of patients with hepatomas. Hepatic syn­ thesis seems to relate to the degree of dedifferentiation accompanying malignancy, the more dedifferentiated the tumor, the higher the serum concentration is likely to be. AFP is found in high concentration in fetal sera, but low concentration in sera from newborns and pregnant women. Particularly in children, a simple immunodiffusion test for AFP seems a relatively specific indicator of hepatic carcinoma (von Kleist, 1976). c. Human Chorionic Gonadotrophin

(HCG)

HCG is a fetal hormone secreted by choriocarcinoma cells. The tumor is really a hemiallograft, as it arises from the placental trophoblast and contains paternal antigens. Invasive growth of these tumors is usually accompanied by moderate lymphocytic infiltration. Patients who respond to chemotherapy frequently do so with celerity, and this might indicate an immune response that was active—but unable to cope with the growing tumor burden until it was reduced in size. On

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the other hand, patients who do not respond to chemotherapy appear to retain their husband's skin grafts for a protracted period, though they reject unrelated skin grafts in the normal time. It is tempting to postulate that the specific immune response to paternal antigens, which would result in rejection of both skin grafts and tumor, is being sup­ pressed or blocked in some way. Bagshawe (1969) has achieved dramatic therapeutic success follow­ ing application of a radioimmune assay for HCG. Susceptible women— those with a history of hydatidiform mole—have been: screened, and the HCG assay has permitted early detection of both disease and re­ currence. As the synthetic rate of HCG per tumor cell is known, determination of serum concentration of fetal hormone facilitates an estimate of tumor burden at a given time. Other oncofetal substances have been detected, e.g., a-2H-ferroglobulin, fetosulfoglycoprotein, and there is little doubt more will be detected in the future. While oncofetal products are rarely specific indicators of malignancy, elevated concentrations often indicate some form of tissue abnormality. As titers of CEA, AFP, and HCG vary with tumor burden, increasing during progression and decreasing during remission, it is likely that radioimmune assays for oncofetal products will become increasingly important to monitor the efficacy of cancer therapy, and to provide warning of residual or recurrent disease. III. Antitumor Immune Effector Mechanisms Effector mechanisms have been defined through two basic approaches using syngeneic animals: 1. Adoptive transfer: Separated components of the immune system removed from tumor-immunized mice are transferred to syngeneic normal recipients, and these mice are then challenged to determine if tumor immunity was imparted. Controls receive similar treatment using tissues from normal mice or mice immunized with non-cross-reacting tumor. Passive transfer of immune serum occasionally confers immunity (Gorer and Amos, 1956). More commonly, adoptive transfer of syn­ geneic immune lymphocytes is required to confer tumor resistance (Klein et al., 1960), and specifically the thymus-derived T cells have been shown to carry antitumor memory (Rouse et al., 1973; Ting et al, 1976). 2. Cytotoxicity assays: Tumor cell killing is determined in vitro after incubation with serum or cells from immunized animals. Cytotox-

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icity may be determined microscopically or by use of radioisotope release assays (Fossati et al., 1975). Immunization with tumor, like transplantation of foreign tissue, pro­ duces a multitude of immune mechanisms as detected in vitro. For detailed study, it is necessary to isolate each mechanism, but in vivo it seems unlikely that they each operate in isolation, so it may be rather naive to attribute the rejection process to any one immune mechanism. The major effector mechanisms are summarized in Table I, and the overall complexity is further increased by modulatory mechanisms with helper or suppressor function (Benacerraf, 1978). In animal models, a good correlation between tumor immunity in vitro and in vivo is frequently obtained. In clinical situations, how­ ever, correlation is more elusive (Baldwin, 1975; Heppner et al., 1975; Herberman and Oldham, 1975). One major problem involves the preparation of appropriate target tumor cells. Fresh tumor cells may TABLE I ANTITUMOR I M M U N E M E C H A N I S M S

Specific immunity 1. Complement-dependent antibody (CDA) Specific antibody -f- complement = tumor cell lysis (e.g., Grant et ah, 1978) 2. Antibody-dependent cellular cytotoxicity (ADCC) Specific antibody + Fc receptor lymphoid cells = tumor cell lysis (e.g., Lamon et al, 1976) 3. T-cell-mediated cytotoxicity Immune thymus-derived lymphocytes = tumor cell lysis (no requirement for for serum components (e.g., Rouse et al, 1973; Ting et al., 1976) 4. Macrophage-mediated cytotoxicity Immune T cells + tumor cells —* macrophage activating factor —» cytotoxic macrophages = tumor cell death (Alexander et al., 1972) Nonspecific immune mechanisms 1. Macrophages. Activated by nontumor antigens, macrophages appear to differen­ tiate neoplastic from normal cells. This mechanism may explain the antitumor effect of certain bacterial infections (Alexander and Evans, 1971; Hibbs, 1976) 2. Natural killer cells ( N K ) . Non-T lymphoid cells in man may be similar to ADCC effector cells. Cells occur naturally, tumor cells killed without par­ ticipation of serum components. N K activity is depleted in tumor-bearing subjects (Klein, 1977; Bonnard et al., 1977) 3. Complement. Normal serum from some species destroys R N A tumor viruses without participation of specific antibody (Cooper et al.} 1976) ; complement receptor of virus may be pl5E (Bartholomew et al., 1978)

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be used, but commonly tumor cell lines are employed which have been prepared from solid tissue and manipulated in culture following enzymatic treatment. Trypsin digestion temporarily removes some sur­ face antigens though they may be replaced with a few hours (Thomas et al., 1976), and long-term tissue culture may result in loss or even expression of new membrane antigens. The cytotoxic effect of immune T lymphocytes on virus infected cells has been" shown to be restricted by the H-2 gene complex (Zinkernagel and Doherty, 1974). Virus-infected target cells are killed by immune T cells only when the sensitizing cells, the effector cells, and the target cells share at least some histocompatibility antigens. If such restriction operates in noninbred systems in tumor cell killing, then tumor cells from one patient will probably not be killed by immune T cells removed from an unrelated individual. In Rous sarcomas of chicken, immune spleen cells were more strongly cytotoxic to autochtonous than allogeneic tumor cells (Wainberg et al., 1974), and in other studies, killing of allogeneic tumor has been shown to occur more slowly and to a lesser extent than killing of syngeneic tumor (Pfizenmaier et al., 1976; Ting and Law, 1977). Nevertheless, the tumor cell killing detected in microcytotoxicity assays is frequently cross-reactive, and occurs whether the target is autochtonous or derived from the tumor of another patient with the same disease (Hellström and Hellström, 1969; Currie and Basham, 1972). It has recently been suggested that such cross-reactiona are caused predominantly by the presence of fetal antigens, and due to the relative instability of these tumor cell products in the cell mem­ brane, immune responses to them are unlikely to have much influence on the course of tumor progression (Baldwin, 1977). Nonspecific mechanisms [natural killer (NK) cells, activated macro­ phages, complement] may represent a strong, naturally occurring barrier to tumor induction or progression, but at present the real value of these mechanisms in vivo is not known. Destruction of injected tumor cells—and possibly of naturally occurring métastases—occurs in certain organs by what may prove to be a nonspecific mechanism. When tumor cells are radiolabeled in vitro and then injected intravenously into syngeneic recipients, the injected cells are trapped by various organs (e.g., lung, liver), and the distribution of trapped cells is determined by the route of injection (Proctor, 1976). Radiolabeled leukemia or sarcoma cells injected intravenously are trapped predominantly by the lung, and destruction occurs most rapidly in lungs of immunized recipients, or in animals whose primary tumor was previously excised (Proctor et al., 1976). In leukemic animals, the lung still destroys

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injected tumor cells though the efficiency is decreased, presumably because of the preexisting tumor burden (Sadler and Alexander, 1976). 1. IMMUNE FUNCTION OF CANCER PATIENTS

Autoimmunization of patients with their own live or irradiated tumor cells results in an immune response when the tumor is injected into sites distant from the natural regions of growth (Southam et al., 1966; Currie et al., 1971). Using live tumor cells, implantation of small num­ bers of cells was resisted, though resistance was overcome by increasing the cell dose (Southam et al., 1966). Observations from microcytotoxicity assays (Hellström and Hellström, 1969; Currie and Basham, 1972) suggest that during tumor growth the immune system becomes sensitized to tumor antigens, but in vivo the reaction is not sufficient to contain tumor growth completely or it is actively suppressed. Skin tests to a panel of antigens have been used to determine the cell-mediated immune status of cancer patients, in case those with progressing tumors are subject to suppression of their immune respon­ siveness. Both the primary delayed hypersensitivity reaction to dinitrochlorobenzene (DNCB) and also recall reactions to mumps, Candida, tuberculin, and streptokinase-streptodornase have been used. In some studies, normal immune reactivity has been correlated with good prognosis (Eilber and Morton, 1970; Fahey et al., 1977; Hersh et al., 1976) ; however, other workers report a poor correlation between results and prognosis (Cochran et al., 1976). In cases of bladder carcinoma use of DNCB appears to provide a more accurate assessment of patient condition than assays using recall antigens (Fahey et al., 1977; Golub et al., 1974). If skin reactions are reduced as a result of massive tumor burden, reactivity often returns to normal following tumor excision; skin reactivity may also be artificially suppressed as a result of treatment with certain drugs or radiotherapy. It appears that nonspecific skin tests often provide information of clinical value, but that immunological evaluation should be based on use of more than one test antigen. 2. MECHANISMS OF ESCAPE FROM SURVEILLANCE

If it is correct that all tumors that arise bear specific antigens denoting malignancy, then those which progress to cause disease have circumvented immune surveillance. In the case of virus-induced tumors which bear strong tumor-associated antigens, a process of escape from

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immune destruction may be the most common mechanism that facili­ tates tumor development. The fact that such tumors frequently arise only in immunosuppressed or immunoincompetent animals supports that contention. The situation may be different, however, in circumstances where tumors are induced by carcinogens or where they arise spon­ taneously. In the former case, carcinogens are known to induce tumors each with a specific immunogenicity which falls within a range from strong to undetectable (Prehn, 1976; Baldwin, 1977). Possibly, this immunogenic range is normally distributed; then strongly immunogenic tumors escape surveillance, but the tumors detected at the other end of the distribution are simply nonimmunogenic. Many progres­ sive, spontaneously arising tumors apparently express no immunogenic properties (Baldwin, 1977; Hewitt et al, 1976; Prehn, 1976). These observations do not imply that all spontaneously arising tumors are nonimmunogenic, or that immune surveillance plays no role in con­ trolling appearance of naturally occurring tumors. Until the technology is developed to quantitate malignant change at a much earlier stage than is presently possible by clinical diagnosis, we can only suspect that the majority of tumors that arise spontaneously are indeed eliminated by surveillance in their early development, and that the tumors which progress are the minority lacking overt immunogenicity. Phenotypic variation may account for lack of immunogenicity in certain cases. Whether tumors arise monoclonally (Möller and Möller, 1975; Prehn, 1976), or because areas of tissue progress through multiple independent stepwise changes (Foulds, 1958), subpopulations of cells with different characteristics exist within the tumor. If growing tumor stimulates a destructive immune response, this will select for variant cells expressing least specific antigenic determinants, and consequently tumor growth may be accompanied by subtle changes in tumor antigenicity. In some tumors, cell morphology differs detectably according to whether the area is invasive or noninvasive, and antigenic expression has been reported to vary in different tumor areas (Byers and Johnson, 1977). Changes in the characteristics of cellular infiltration have been observed accompanying tumor progression (Betz and Simar, 1976). The theoretical point of critical tumor mass is the point at which progressive tumor growth is ensured because too many tumor cells exist for the host immune response to kill. If spontaneous regression (Everson, 1964) is immunologically mediated, then it follows that tumors must continue to evade attack by immune mechanisms throughout the life of the host. Nevertheless, critical tumor mass has been evoked to explain the exquisite sensitivity of certain tumors (Burkitt's lymphoma, neuroblastoma, choriocarcinoma) to short-term

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chemotherapy (Burkitt, 1967; Hertz et al., 1964). Other fairly common observations are consistent with an immune mechanism that can control small tumor deposits, e.g., spontaneous regressions of lung métastases following excision of primary hypernephroma, prolonged survival of some breast carcinoma patients tumor free after surgery for years before métastases becomes evident, individual behavior of discrete de­ posits of malignant melanoma which regress and progress independently. In some cases it is possible that the tumors arose in relatively nonimmunogenic form, but that selective advantages endowed certain immunogenic cell variants was sufficient to facilitate their eventual succession when sheer tumor mass provided protection from immune destruction. Blocking is a mechanism that may explain escape of immunogenic tumors in some circumstances. Originally, Hellström and Hellström (1969) demonstrated that lymphocytes from tumor-bearing subjects were cytotoxic to syngeneic or autochthonous tumor cells, and that cytotoxicity effects cross-reacted between target tumors of the same tissue origin. Invariably, the cytotoxic effect of lymphocytes was reduced or abated by the addition of serum from the same tumor-bearing indi­ viduals. The blocking activity of tumor-bearing serum was attributed to blocking antibody which bound to tumor cells and thereby in­ hibited the action of cytotoxic lymphocytes. The blocking agent is now generally believed to be either antigen (shed from tumor cells) or a soluble complex of antigen and antibody. Free antigen will bind to specifically reactive lymphocytes, and im­ mune complex may also bind to cells via the activated antibody Fc receptor. Thorough washing of lymphocytes from cancer patients prior to cytotoxicity assay has been shown to produce maximal tumor cell killing, and the blocking factor can be recovered from washing media (Currie and Basham, 1972). The blocking activity of tumor bearer sera is consistent with antigen as the source of inhibitory activity, for maximum blocking is usually associated with sera removed when the tumor burden is large, and minimal activity is found in sera taken during remission. The ability of certain tumors to metastasize has been correlated with the rate at which the tumors spontaneously shed antigen from their cell surfaces (Alexander, 1974). While most tumors may seed métastases into the circulation to lodge and grow at distant sites, those métastases which shed low levels of antigen would be less capable of blocking local immune attack, therefore less likely to survive and establish growing secondaries. Antigenic modulation (enhancement) may explain tumor escape in

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some circumstances. Antibody specifically bound to cell membranes may cause lysis via complement activation, or alternatively capping can occur whereby antigen-antibody complexes migrate around the cell surface, become polarized, and subsequently internalized or shed (Taylor et al., 1971). In the absence of lysis, continual bombardment of cells with antibody produces a continuous capping process until the cells no longer express surface antigen. Mice immunized with T L + leukemias develop antibodies to TL antigen, and these mice support growth of a second T L + tumor innoculum. When these growing tumor cells are removed TL antigen is found to have disappeared from the cell surface, yet if these cells are reinjected into normal recipients the TL antigen reappears (Boyse et al., 1976). Some doubt exists about the subsequent sensitivity of antibody-modulated cells to immune attack. The cells may remain sensitive to lysis by immune T lymphocytes after they have lost the ability to fix antibody (Edidin and Henney, 1973), or they may be refractory to lysis by both antibody and T cells. As cells which undergo antigenic modulation can be lysed by complement in some circumstances (Stackpole et al., 1978), one suggestion is that modulation occurs when a deficit in lytic complement exists. Capping and disappearance of antigen from human cancer cells, induced by antibody in cancer patients sera, has been demonstrated with Burkitt's lymphoma, melanoma, and breast carcinoma cells (Nordquist et al., 1977). Masking of tumor antigens by increased concentrations of sialic acids on tumor cells surfaces may act to prevent immune recognition. Neuraminidase appears to unmask tumor antigens by enzymatically digesting sialic acids, with the result that some treated tumor cells become more immunogenic. Nevertheless, treatment does not reveal strong tumor antigens where none were detected previously. Rios and Simmons (1974) reported that some established tumors underwent regression following injection of the tumor-bearing hosts with neuraminidase-treated tumor cells. Lymph node paralysis may occur in nodes draining the site of pro­ gressing tumor while distant nodes respond normally to unrelated antigens (Alexander et al., 1969). Immunogenic tumor stimulates an apparently normal response within local nodes, but the subsequent release via the efferent lymphatics of antigen-sensitized effector cells is inhibited. Imbalance of suppressor cells has been advanced to explain the welldocumented occurrence of sneaking through (Mengersen et al., 1975). When very low doses of tumor cells are injected, they may progress to form lethal tumors., whereas larger doses of the same tumor stimulate

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the response resulting in tumor regression. It appears that specific immunological unresponsiveness on the part of a host can be attributed to abnormal suppressor T-cell function, and lack of response can be rectified by eliminating this cell population (Chiorazzi et al., 1977). In normal circumstances, the generation of immune effector cells is regulated by generation of suppressor cells, but it is possible that some tumors progress because they disturb this balance in favor of specific immunosuppression. It seems unlikely that a unifying explanation for tumor escape from surveillance will emerge, for the immunologie properties: of each tumor probably vary according to both the tissue of origin and the cause of the malignancy. Excluding virus-associated tumors, one view is that a minority of naturally occurring tumors escape immune surveillance because they do not express tumor-specific antigens. Nevertheless, it seems doubtful that any morphological class of neoplasm will be found where every example characteristically lacks tumor antigens, for evidence accumulates for an ever-widening array of tumors exhibiting specific antigens. The strongest clinical evidence that immunity plays a losing battle with neoplasia is found with Burkitt's lymphoma, choriocarcinoma, hypernephroma, neuroblastoma, and malignant mela­ noma; but there is a second group of tumors for which evidence of immune involvement is now apparent; conservatively these are breast, cervical, gastric, testicular, and bladder carcinomas, and also some forms of leukemia.

IV. Immunotherapy of Cancer Historically, there have been many varied approaches toward manipu­ lating the immune response of cancer patients, but little of practical benefit has so far emerged (Currie, 1972). Nevertheless, the inherent specificity of immune responses remains the most promising key to final eradication of tumor cells remaining after more conventional forms of treatment, and indeed it may be the only approach available to reach those malignant cells which presently survive chemotherapy (Mathe et al, 1977). 1. NONSPECIFIC IMMUNOTHERAPY

This approach encompasses attempts to increase the immune reac­ tivity of cancer subjects nonspecifically by using non-tumor-related vaccines, e.g., BCG or Corynebacterium parvum. BCG has been

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studied in detail in both animals and man and some generalized com­ ments are possible. Beneficial effects against leukemia and other cancers in man or animals have been obtained (Gutterman et al., 1976; Mathe et al., 1977). One major factor which appears to determine therapeutic effect is the size of the preexisting tumor burden which should be small, < 105 cells in one mouse leukemia model system (Mathe et al., 1977). In clinical application it has been sometimes overlooked that the small improvement in immune function obtained by BCG immuniza­ tion can hardly be expected to have much impact on patients with excessive tumor burden, or that overenthusiastic use of BCG may result in a state of immunological anergy. There appears to be a risk that BCG immunotherapy will sometimes enhance tumor growth which involves suppression of the host immune response. On the other hand, BCG may stimulate surviving bone marrow stem cells and there­ fore aid blood cell restoration following chemotherapy. The route of application of BCG appears critical (Mathe et al., 1977). In some circumstances, the optimal antitumor effect of BCG is achieved by combination immunotherapy also incorporating a specific tumor vaccine (Parr, 1972; Mathe et al., 1977). Induction of delayed hypersensitivity at the site of basal cell car­ cinomas was sometimes successful in controlling tumor growth (Klein, 1968). Patients were sensitized to triethyleneiminobenzoquinone solu­ tion, and then the tumor areas painted a second time. A marked in­ flammatory response occurred at the site of second application, and although the local secondary responses involved both surrounding normal tissue and tumor, the tumors were apparently more sensitive to the cell-mediated hypersensitivity damage and frequently regressed. 2. SPECIFIC IMMUNOTHERAPY

Active immunization employs attenuated tumor cells, or tumor cell extracts, as a vaccine. Using this approach animals have been rendered immune to a subsequent challenge with live tumor cells (Alexander, 1968; Prehn, 1976). Generally, animal experiments have proven im­ munization against tumors possible, but rarely has immunization caused rejection of an already established and progressing tumor. In the future, it may be possible to immunize subjects with heterologous soluble tumor antigens, e.g., CEA, AFP, and thereby provoke immune responses specifically against the similar but nonimmunogenic autologous tumor products, with the result that an effective antitumor immune response ensues (Binz and Wigzell, 1978). Immunization of cats with feline leukemia virus-infected tumor cells

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has been shown to prevent the appearance of virus-associated tumors in laboratory trials (Jarrett et al, 1975; Olsen et al., 1976; Yohn et al., 1976). Adoptive immunization has obvious application in inbred animal models when large numbers of genetically identical animals are avail­ able; in these circumstances, the transfer of tumor-immune lymphoid cells to normal syngeneic recipients confers resistance to subsequent tumor challenge (Klein et al., 1960; Rouse et al., 1973; Ting et al., 1976). Clinically, adoptive transfer is beset with problems, for the trans­ ferred lymphoid cells both bear and respond to histocompatibility anti­ gens. On one hand, injected foreign lymphocytes will be destroyed rapidly by a host immune response directed to foreign histocompati­ bility antigens. Conceivably, if the tumor-immune lymphoid cells were injected at the site of tumor, they might effect some tumor destruction before they were themselves destroyed; some tumor regressions were documented following injection of tumor-immune sheep lymphocytes into tumor-bearing rats (Alexander, 1968). On the other hand, injection of large numbers of foreign lymphocytes may result in development of graft-versus-host (GVH) disease, when the injected cells recognize the host histocompatibility antigens as foreign and respond by mounting a destructive attack. When immunocompetence is reduced, the host is even less likely to be able to contain GVH, however, when controlled a GVH reaction appears to have an antitumor effect that is most pro­ nounced on leukemic cells (Bortin et al., 1974). One alternative, perhaps with an exciting future, is the possibility that the tumor host's own lymphoid cells can be adequately sensitized in vitro to cultured tumor cells, in conditions whereby the culture is controlled to ensure maximal reactivity and minimal suppression. Subsequently, the highly reactive sensitized cells would be returned to the patient to effect tumor cell destruction (Bernstein, 1977; Trêves et al, 1975). Apart from the use of intact lymphoid cells, possibilities exist to use irradiated immune ceils when the ability to mount a GVH reaction is inhibited but the antitumor effect remains unaltered. Extracts of im­ mune lymphocytes, such as RNA or transfer factor, also confer tumor immunity to an otherwise unreactive host (Alexander, 1968; Fudenberg, 1976). Passive immunization or transfer of antitumor antisera sometimes produces tumor immunity. The technique has, on occasion, been markedly successful in protecting inbred mice from syngeneic tumor (Gorer and Amos, 1956; Motta, 1971; Zigheiboim et al, 1974). Al­ though the concept of blocking antibody may have, until recently,

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reduced interest in passive immunotherapy, potential applications are considerable. Possibly, specificity can be imparted on otherwise broadacting anticancer drugs by coupling them to tumor antibodies (Rubens, 1974). Human tumors have been shown to concentrate antibody pre­ pared to CEA (Primus et al., 1977), and tumor-specific antibody to neuroblastoma has been localized in vivo (Terman et al., 1975). Daunomycin and adriamycin can both be covalently bound to antibody with retention of drug and antibody activity (Hurwitz et al., 1975). If antisera can be used to confer specificity to some cytotoxic drugs, the necessary drug dose would probably be much reduced and an additive effect may also occur as antibody increases the antitumor effect of the drug (Madewell and Grant, 1977). Other possibilities include the coupling of very high specific activity radioisotopes with antibody, thereby inducing concentrated radiation damage at the site of tumor. Boron also might be incorporated into antibody and consequently localized at the tumor site; when the boron nucleus absorbs thermal neutrons it releases high-energy fission products, so neutron bombard­ ment of tumor would result in selective local tissue damage. V. Virus-Associated Tumors of Cats 1. PATHOBIOLOGY AND EPIDEMIOLOGY

The leukemias, lymphomas, and multicentric fibrosarcomas of cats represent the most thoroughly understood spontaneous tumors that occur in domestic animals, largely because of an etiologic association between C-type retroviruses and these mesodermal tumors which was firmly established by Jarrett and colleagues (1964a,b). Lymphoid tumors were originally categorized on the basis of gross pathology as thymic, alimentary, multicentric, and unclassified forms (Jarrett, 1971). The thymic or anterior mediastinal form originates in the thoracic cavity, appearing after involution and disappearance of the normal thymic lymphoid tissue; thymic lymphoma cells regularly have T-cell characteristics (Cockerell et al., 1976; Hardy et al., 1977). The thymic form regularly occurs when young kittens are inoculated with the Rickard strain of feline leukemia virus (FeLV), the strain most commonly used in recent laboratory pathogenesis studies (Hoover et al, 1976, 1977a,b; de Noronha et al., 1978). Multicentric tumors appear to originate in a generalized manner in lymphoid tissues, and often include bilateral involvement of nodes. Most frequently this tumor appears to be of T cell origin (Hardy et al., 1977). The alimentary form originates in gut wall lymphoid

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tissue and it is apparently a B cell tumor (Mackey and Jarrett, 1972; Hardy et al, 1977). Included in the original "unclassified" category were tumors that originate as localized lymphomas though in unusual "nonlymphoid" sites, such as the kidney, skin, or spinal cord. Also included in this category were the lymphoid leukemias, which originate in blood and/or bone marrow, and present in a manner comparable to acute lymphoblastic leukemia (ALL) of children (Cotter and Essex, 1977). The ALL form is ordinarily a T cell disease. The relative incidences of each form of lymphoid neoplasia vary with geographical location, and therefore presumably with the strain of virus and/or the genetics of the cat population (Essex et al, 1975b). In Glasgow, for example, the alimentary form of lymphoma is the most frequent type of lymphoid malignancy, while in Boston ALL and thymic lymphoma are the most frequent forms (Francis et al, 1979a). Feline leukemia virus (FeLV) is also associated with myelogenous leukemias and myeloproliferative diseases as well as aplastic and hemolytic anemias (Herz et al, 1970; Mackay et al, 1972, 1975; Hoover et al, 1973). The anemias can occur as either preleukemic syndromes or terminal diseases, and the relative frequency of FeLVassociated anemias is similar to the incidence of lymphoid malignancies in this species (Cotter et al, 1975). Infection with FeLV also causes a profound immunosuppression in cats (Anderson et al, 1971; Perryman et al, 1972; Hoover et al, 1973; Essex et al, 1975c). Immunosuppression has been associated with atrophy of the thymus (Anderson et al, 1971; Hoover et al, 1973), decreased numbers of peripheral blood lymphoid cells (Essex et al, 1975c), prolonged graft rejection times (Perryman et al, 1972), increase in suppressor cell activity (Cerny and Essex, unpublished observations), decreased lymphocyte blastogenesis following concanavalin A (con A) stimulation (Mathes et al, 1978), and diminished lymphocyte mem­ brane capping activity (Dunlap et al, 1979). One of the FeLV proteins (pl5E) has been shown to cause both depressed con A blastogenesis and depressed membrane capping when added to lymphoid cells in vitro (Mathes et al, 1978; Dunlap et al, 1979). The ability of cats naturally infected with FeLV to resist other viral and bacterial infections such as infectious peritonitis and septicemia is substantially reduced (Cotter et al, 1975; Essex et al, 1975c). Glomerulonephritis also occurs in FeLV-infected cats at frequencies much higher than would be ex­ pected due to chance alone (Anderson and Jarrett, 1971; Francis et al, 1979b). Feline sarcoma virus (FeSV) is found in association with most

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multicentric fibrosarcomas that occur in young cats (Snyder and Theilen, 1969; Gardner et al., 1970); these tumors grow rapidly with frequent métastases (Snyder and Dungworth, 1973). Under laboratory conditions, FeSV induces tumors with very short latent periods when inoculated into young kittens (Essex et al., 1971 a,b; Essex and Snyder, 1973; Snyder and Dungworth, 1973). When inoculated intracutaneously rather than subcutaneously, it frequently produces melanomas (McCullough et al., 1972). FeSV is defective for replication and it is found in association with helper FeLV (Sarma et al., 1971a,b) ; FeSV transforms flbroblasts in vitro whereas FeLV does not. Three genes and/or gene products have thus far been identified for FeLV (Khan and Stephenson, 1977; Kurth et al., 1979). Starting from the 5' end of the mRNA, the first gene, gag (for group-specific anti­ gen), codes for a polyprotein of approximately 75,000 MW which undergoes posttranslational cleavage. The products are peptides desig­ nated pl5, pl2, p30, and plO due to their molecular weight (X 1000). All four gag cleavage products become localized in the core of the virion with p30 as the major capsid subunit. The second gene, designated pol (for polymerase) makes an enzyme, RNA-dependent DNA polymerase or reverse transcriptase, that is carried in virus particles as a necessary component for virus replication. The third gene, env (for virus envelope) makes a protein which becomes glycosylated at about 90,000 MW and then undergoes posttranslational cleavage to form both the virion envelope spike protein, a glycoprotein of about 70,000 MW (gp70), and also a 15,000-MAV protein which becomes the backbone of the virion envelope (pl5E). All of the virion components are immunogenic (Essex et al., 1979), and all but pl5E have a specific FeLV antigenic component (pl5E is broadly cross-reactive among mammalian retroviruses). Several of the FeLV proteins also have subgroup-specific and/or interspecies-specific antigenicity (Essex et al., 1979). The p30 and pl5 molecules, for example, have at least one antigenic determinant that is spécifie for all FeLVs, and another that will cross-react with murine and primate retroviruses. The gp70 also has antigenic determinants that are both group and interspecies specific, but the determinant that is most im­ munogenic in cats has three subgroups specificities, and as a result FeLV can be categorized into subgroups A, B, and C (Sarma and Log, 1973). This subgroup specificity correlates with the spectrum of host-cell infectivity and also with host cell surface attachment interference (Sarma and Log, 1971; Sarma et al., 1975). Subgroup A viruses are defined as ecotropic because they grow well only in cat cells. Sub­ group B viruses, designated amphotropic, grow well in both feline

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and nonfeline cells. Subgroup C viruses show xenotropic tendencies, replicating poorly in cat cells but more efficiently in nonfeline cells. Cats infected with FeLV under natural conditions always yield sub­ group A virus which frequently occurs alone, or in association with subgroups B and C. Subgroups B or C have so far not been isolated alone, and isolations of subgroup C are rare (Jarrett and Russell, 1978). No clear association has been made between any particular disease form and a given subgroup, though a more frequent association between subgroups A and B has been described in animals with lymphoid malignancies than in healthy viremic animals (Jarrett and Russell, 1978). Additionally, in multicat households, the proportion of cats that are persistently infected with FeLV is higher if the cats are infected with AB as opposed to A alone. Feline retroviruses are transmitted horizontally as contagious agents (See Essex, 1975, for review). This event was initially suspected when several cases of leukemias of lymphomas were observed within the same multiple cat households (Schneider et al, 1967; Cotter et al, 1973), but the significance of the clusters in confirming horizontal transmission was established when (a) additional cases of leukemia or lymphoma continued to occur (in a prospective sense) in the same multiple cat households (Cotter et al., 1974; Essex et al., 1975e), indi­ cating that the cases were not due to chance alone; and (b) healthy cats in cluster households were found to have sérologie evidence of FeLV infection much more frequently than cats in multiple cat households where cases of lymphoid malignancy had not been de­ tected (Hardy et al., 1973, 1976a; Cotter et al., 1974; Essex et al, 1975d, 1976, 1977a; Charman et al, 1976; Grant et al, 1977, 1978; Stephenson et al, 1977a). The primary route of excretion for FeLV appears to be saliva (Francis et al, 1977). Persistently viremic cats, whether healthy or suffering from lymphoid malignancies, regularly shed 104 to 105 infectious FeLV per milliliter of saliva. In contrast, urine, feces, and feeding fleas appear relatively free of FeLV. FeLV survives for several days at room temperature when kept moist but it is rapidly inactivated at 56°C (Francis et al, 1979c). The mean induction period for development of leukemia and/or lymphoma following the initial infection with FeLV under natural conditions is at least 17.6 months with a range of 3 to 41 months for 18 animals that were followed (Francis et al, 1979c). The induction period appears longer under natural conditions than would be predicted from laboratory inoculation studies (Hoover et al, 1976). Additionally, ,cats appear more likely to develop persistent viremia when first ex-

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posed to the virus as adults under natural conditions and/or by natural routes than if they are inoculated as adults in the laboratory (Hardy et al, 1973, 1976b). Healthy cats which are known to be horizontally exposed to FeLV under natural conditions have greatly increased rates of persistent viremia (Hardy et al, 1973; Cotter et'al, 1974; Essex et al, 1975d,e), and greatly increased rates of detectable antibodies to the feline oncornavirus-associated cell membrane antigen (FOCMA) (Essex et al, 1975d,e; Rogerson et al, 1975)," to FeLV gp70 (Essex et al, 1977a,b; Stephenson et al, 1977a), and to FeLV reverse transcriptase (Jacquemin et al, 1978). Additionally the mean titers of antibodies found in healthy cats which reside in leukemia cluster households, where they receive continuous exposure to the virus, are generally much higher than the mean titers in stray cats which have no evidence of prior exposure to FeLV (Essex, 1974; Essex et al, 1975d). Additional evidence that FeLV was transmitted in a horizontal manner was gained by prospective seroepidemiological studies. First, 10 specific pathogen-free "tracer" cats, which were known to be free of prior FeLV exposure, were placed in well-characterized FeLV-positive leukemia cluster households (Cotter et al, 1973, 1974). All of the tracers developed evidence of FeLV infection by numerous sérologie criteria within a few months (Essex et al, 1977a). Second, a controlled study was undertaken to remove the FeLV excretor cats from some index FeLV-positive multiple cat households but not from others (Hardy et al, 1976b). The removal of FeLV excretor cats in this manner prevented subsequent FeLV infections from occurring in these houses. Not all lymphoid malignancies occur in cats that have detectable 'infectious FeLV. The overall proportion of cats with leukemia or lymphoma that are overtly infected with FeLV varies from 70 to 90% in the case of thymic lymphoma down to 50% or less for cats with alimentary lymphoma (Jarrett et al, 1973a; Essex et al, 1975b; Francis et al, 1979a). Cats with B-cell tumors are more frequently "leukemia virus negative" (LVN) than cats with T-cell tumors, and older cats that develop lymphoma are much more likely to develop LVN disease than young cats (Essex et al, 1975b, 1978a; Gardner et al, 1977; Francis et al, 1979a). LVN lymphomas were first designated so on the basis of lacking the major gag antigen by immunodiffusion (Hardy et al, 1969) or fixed cell immunofluorescence (Hardy et al, 1973; Essex et al, 1975b), and by electron microscopic examination (Jarrett et al, 1973b). The absence of replicating virus in such cases was subsequently confirmed by virus isolation procedures (Hardy et al, 1976a; Jarrett and Russell, 1978).

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Additionally, using the sensitive technique of radioimmunoprecipitation, LVN lymphomas were found to lack detectable levels of all the major virus structural proteins pl5, pl2, p30, plO, and gp70 (Stephenson et al, 1977b; Essex et al, 1979). The role of FeLV in inducing LVN tumors is thus unclear. Two observations suggest an association between the development of the LVN tumors and FeLV. First, LVN lymphomä cells express FOCMA, which is known to be FeSV coded (see Section V,3). Second, the epidemiologic studies suggest that LVN tumors occur in FeLV-infected multiple-cat households more often than in the population-at-large (Essex et al., 1978a). Counteracting the association observations are nucleic acid hybridization studies, which do not reveal increased levels of FeLV mRNA (Levin et al, 1976; Frankel and Gilbert, per­ sonal communication) or FeLV-specific provirus DNA (Koshy et al., 1979) in tumors over the levels found in normal tissues. The sensitivity levels of the hybridization assays did not rule out the possibility that transformation-related partial provirus sequences might be present in the tumor cells. Similarly, the possibility that FeLV could have caused transformation in stemline cells and be subsequently lost by a "hit-and-run" mechanism cannot be eliminated. Nevertheless, current evidence is insufficient to establish that the LVN lymphoid tumors of cats are caused by FeLV. 2. IMMUNE RESPONSE TO THE VIRUS

As far as has been determined, most or all of the structural proteins of FeLV are immunogenic in cats. Moreover, most of the proteins have more than one antigenic determinant. The antigenic determinants have differing degrees of cross-reactivity, and they are generally categorized as (a) interspecies specific, (b) group or species specific, (c) subgroup specific, and (d) type or strain specific. The interspeciesspecific determinants are normally shared by all type-C retroviruses of mammals. The group- or species-specific determinants are usually shared by all C-type retroviruses of a single species. Exceptions do occur in some species however, and the cat is one in having two distinct categories of retroviruses. One category, FeLV-FeSV, is the subject of this section; the other, designated the CCC or RD-114 viruses, bears group antigens which are distinct from those of the FeLV group (for review, see Essex, 1975). As opposed to FeLV, the RDr-114 viral agents have never been associated with disease, and their entire genome is regularly transmitted in a vertical (genetic) fashion in all domestic cats. The third class of antigenic determinants is subgroup specific, and

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their individual differences facilitate the categorization of spontaneous isolates of FeLV into three subclasses. Within each subgroup type or strain-specific antigens might be present though none have as yet been clearly defined. The possibility exists, however, because such differences have been detected with the murine retroviruses (Eckner and Steeves, 1972). The gp70 molecules represent the spikes or knobs which protrude from the virion surface and they are the major target for virusneutralizing antibody. Most cat antisera directed to gp70 show primarily a subgroup specificity (Sarma et al., 1974; Hardy et al., 1976a; Jarrett and Russell, 1978) ; they also show some group-specific reactivity but apparently no interspecies activity (Stephenson et al., 1977a). Goats inoculated with disrupted FeLV, on the other hand, do develop some interspecies-specific antibodies which cross-react with nonfeline gp70s (de Noronha et al, 1977, 1978). Cats that become persistently viremic with one subgroup of FeLV do not have free antibodies to the same subgroup of gp70. In theory at least, cats viremic with one subgroup might contain free gp70 anti­ bodies to a second or third subgroup which was previously present, but in practice naturally infected cats that were viremic with any of the subgroups regularly lacked antibodies to all determinants of gp70. These studies were conducted by radioimmunoprecipitation, using purified FeLV gp70s which were combinations of the various subgroups (Stephenson et al., 1977a). Conversely, nonviremic cats which have been naturally exposed to FeLV usually do have antibodies to the FeLV gp70 (Essex et al., 1977a,b; Stephenson et al., 1977a). Significant titers of antibody indicate resistance to subsequent infections with at least one subgroup (Hardy et al., 1976a). The proportion of cats in a given population that have virus-neutralizing antibodies and/or radioimmunoprecipitating antibodies to the gp70 is roughly proportional to the number that have antibodies to FOCMA or p30 (Essex et al., 1977a,b; Stephenson et al., 1977a). Cats also develop antibodies to the other protein components of the env gene, pl5e (Worley, personal communication). The major and possibly the only immunogènic component of this molecule is interspecies specific (Ikeda et al., 1975). The solubility characteristics of this pro­ tein have made purification and production of it very difficult, and little detailed information on antibody response to this molecule is available. Due, however, to the close correlation between pl5E and immunosuppression of various lymphoid functions, we might hypothesize that a good immune response to this component would be correlated

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with a good prognosis, especially within the class of healthy exposed cats that have persistent viremia. The gag protein which has been studied most extensively from the standpoint of immunity is p80 (Charman et al., 1976; Essex et al., 1977a,b; Stephenson et al., 1977a). Nonviremic cats exposed to FeLV produce antibodies to group- and/or interspecies-specific determinants on the p30 molecule (Charman et al., 1976; Essex et al., 1977a; Stephenson et al., 1977a), but apparently antibodies to p30 or any of the other gag proteins play no role in neutralization of virus. Goat antiserum to FeLV p30 reacts with the membranes of FeLV-infected cells (Essex et al., 1978a), yet cat antiserum produced by inoculation with purified FeLV p30 did not react with infected cell surfaces, even though the antiserum gave high titers of anti-p30 by radioimmunoprecipitation with the purified molecule (Charman et al.} 1976). All cats that develop antibodies to p30 appear to develop them to the group-specific component. Many cats also respond to the interspecies component of the p30, and this type of response appears to be cor­ related with the evolutionary background of the cat (Essex and Stephenson, unpublished observations). Using radioimmunoprecipitation, antibodies to all of the smaller peptides of the gag gene, pl5, pl2, and plO, have been found in cats that were naturally exposed to FeLV (Stephenson and Essex, unpublished observations). The pl5 also appears on the surface of infected cells, but apparently in concentrations that are lower than for the gp70 or p30 (Essex et al., 1978a). The product of the remaining viral gene is reverse transcriptase (RT). Antibodies to RT can be assayed by checking for neutralization of enzyme activity. Using this technique, antibodies to FeLV RT were often found in the same cats that had significant levels of antibodies to the gag and env proteins (Jacquemin et al., 1978). The titers of anti­ body to RT did not necessarily parallel the titers to any of the other virion antigens. Immune responses to FeLV are complicated because the antigenic determinants of each type (interspecies, group, and subgroup specific) may occur on most of the virion proteins. Further complications occur because each FeLV protein and each class of antigenic determinant on that protein may show degrees of immunogenicity which vary ac­ cording to the species and/or the individual which is exposed. Rabbits or goats, for*example, show a better response to an interspecies or group determinant of FeLV gp70, while most cats respond predominately to the subgroup-specific determinant on the same molecule. Individual cats show varying responses to group vs interspecies determinants on

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the FeLV p30 molecule. Possibly, these differences can be rationalized on the basis of immune response genetics, rather than just the age, dose, and rate of exposure to a given immunogen. Many of the virus structural proteins are found in or on the surface of cells that are replicating virus. Virion surface proteins, gp70 and pl5E, obviously present immune targets at the sites of virus budding from cell membranes. Additionally, however, some peptides that are located internally in virus particles are also found on cell surfaces, and in at least some instances they occur at locations distinct from budding sites. Antigenic determinants of disrupted and purified virus components which react with antibody in a radioimmunoprecipitation assay may, nevertheless, not be assessible to react with the same anti­ body when the antigen is naturally located at either the cell surface or the virus surface. Similarly, some antibodies may react with an antigenic determinant of a component (eg., gp70) which is presented at the cell surface but not at the virus surface, or vice versa. 3. FELINE ONCORNAVIRUS-ASSOCIATED CELL MEMBRANE ANTIGEN (FOCMA)

The feline oncornavirus-associated cell membrane antigen (FOCMA) was initially defined on the basis of a reaction seen between certain cat antisera and cultured FeLV-producer feline lymphoma cells (Essex et al., 1971a,b). The reaction was observed at the tumor cell surface. Serum samples which reacted at the highest dilutions usually origi­ nated from cats that resisted the development of lethal tumors after either natural or laboratory exposure to FeSV or FeLV (Essex et al., 1971a,b, 1975d,e; Essex and Snyder, 1973). At the time FOCMA was described on FeLV producer cells, it was not possible to determine whether the antibodies were directed to a tumor-specific antigen or to the virus itself. The highest titers of FOCMA antibody were initially found in cats that had undergone regression of FeSV-induced tumor (Essex et al., 1971a,b; Essex and Snyder, 1973). Since in such regressor cats viremia continued to persist (Aldrich and Pedersen, 1974), it seemed likely that the detectable free antibody was not reacting with a major virion component (Essex, 1974). Similarly, analyses revealed that healthy cats with persistent FeLV infections also frequently had significant FOCMA antibody titers (Essex et al., 1975d). Subsequently many other observations have confirmed that FOCMA antibody is not directed at FeLV, but is directed at a FeSV transformation specific cell surface antigen. These observations include the total lack of correlation found between

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titers of virus-neutralizing antibody and anti-FOCMA (Schaller et al, 1975), between radioimmunoprecipitating antibodies to the gag or env proteins and anti-FOCMA (Essex et al, 1977a,b; Stephenson et al., 1977a,b), and between antibody titers to FeLV RT and anti-FOCMA (Jacquemin et al., 1978). Examination of FeSV transformed cat fibroblasts revealed that such cells expressed FOCMA (Sliski et al., 1977). The same cells, when transformed within murine RNA tumor viruses, did not express FOCMA, nor did cat fibroblasts that were infected with FeLV but not transformed. Cat cells that were infected with FeLV but not trans­ formed did express the virus structural proteins gp70 and p30. FeSV has also been used to transform cells from other species, and such cells regularly express FOCMA (Sliski et al., 1977; Essex et al., 1978b, 1979; Sliski and Essex, 1979). Mink, dog, mouse, or primate cells, for example, express FOCMA if transformed by FeSV, but they are not FOCMA positive if infected with FeLV while remaining untransformed. Such cells are also FOCMA negative if transformed with nonfeline RNA sarcoma viruses, irregardless of whether or not they are superinfected with FeLV or another RNA helper virus. Nonproducer clones of FeSV-transformed cells have been selected (Sliski et al, 1977; Essex et al, 1978b, 1979; Sliski and Essex, 1979). These cells express FOCMA even though virus is not released. Analysis of the cells using ultrasensitive techniques revealed that they lack de­ tectable gp70, p30, and plO, but they do contain pl5 and pl2 (Stephenson et al, 1977b; Essex et al, 1979). While providing additional con­ firmation that FOCMA is unrelated to p30, plO, and gp70, these studies revealed a 65,000-MW molecule that represents FOCMA activity, as well as an 85,000-MW molecule which includes FOCMA activity (65,000 MW) covalently linked to pl5 and pl2 (Stephenson et al, 1977b). The studies also revealed that FOCMA must be FeSV coded, since it could be induced across species barriers as well as being distinguished from the other virus proteins. Although not present in either FeLV or mixtures of defective FeSV pseudotyped with FeLV, the 85,000-MW form of FOCMA was found in FeSV particles rescued with helper viruses of heterologous origin, and a radioimmunoassay was developed for detection of the molecule (Sherr et al, 1978a,b). Studies have also been conducted to evaluate the expression of FOCMA on malignant lymphoid cells derived from cases of leukemia and lymphoma (Hardy et al, 1977; Essex et al, 1978a,b, 1979). Both freshly biopsied and cultured lymphoid cells are regularly positive for FOCMA, irregardless of whether the tumor cells are from cases of

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thymic, alimentary, or multicentric lymphoma, or from cases of lymphoid leukemia. T-cell tumors as well as B-cell tumors express the antigen, as do both virus-negative and virus-positive lymphoid tumor cells. Unlike the FeSV-transformed fibroblasts, FOCMA-positive, virusnegative, lymphoid tumor cells lack all detectable env and gag gene proteins, including pl5 and pl2 (Stephenson et al., 1977b). Normal lymphoid cells are negative for FOCMA, whether infected with FeLV or not. 4.

IMMUNE RESPONSE TO

FOCMA

Following infection with virus, the latent period prior to tumor ap­ pearance is usually shorter for fibrosarcomas than for lymphomas or leukemias. Additionally, since the fibrosarcomas usually originate at the site of infection, which is commonly subcutaneous, tumor appear­ ance and progression can be monitored with ease. For these and other reasons, more studies on tumor immunity have been conducted with retrovirus-induced fibrosarcomas than with virus-induced leukemias, even though virus-associated leukemias and lymphomas occur more frequently than sarcomas in most animal populations. When cats of different ages are given a constant dose of FeSV, rapidly progressing tumors develop in most animals that are inoculated at a young age (Essex et al., 1971b; Essex and Snyder, 1973; Snyder, 1971; Snyder and Dungworth, 1973). Nevertheless, a few young ani­ mals resist tumor development particularly after inoculation with low virus doses, and some older cats succumb to progressing tumors when other cats of the same age are refractory (Essex et al., 1971a,b). Progressing tumors grow rapidly at the injection site, but also metastasize in the majority of cases to such organs as brain, lungs, and liver (Snyder, 1971; Snyder and Dungworth, 1973). Animals that resist the development of lethal tumors often only develop localized tumors which subsequently regress. Cats with progressing tumors usually lack significant levels of anti­ body to FOCMA (Essex et al, 1971a,b; Essex and Snyder, 1973; Schaller et al., 1975). In a study of FeSV-induced tumors in cats, which involved older as as well as newborn animals and inoculation with various strains of FeSV, only 1 progressor animal in 57 had a FOCMA antibody titer of 8 or higher when tested at various intervals up to the time of death (Essex et al., 1976). In direct contrast to the cats that develop progressor fibrosarcomas, those that develop no tumors following inoculation with FeSV have high anti-FOCMA titers (Essex et al., 1971a,b). Of 8 animals inocu-

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lated with FeSV that never developed palpable tumors, all had anti­ body titers of 8 or higher, and the geometric mean titer was about 30. Similarly, cats that developed tumors that transiently progressed and then regressed had strong anti-FOCMA humoral responses; of 25 such animals checked, all had antibody titers of 4 or higher and the geometric mean antibody titer was about 20. There are two possible explanations for reduced FOCMA antibody titers in cats with progressively growing tumors. The first is that some cats respond to retrovirus exposure by producing FOCMA antibody (at a titer sufficient to prevent tumor growth) while nonresponder cats succumb to tumor because they do not develop protective FOCMA antibody levels. The second is that all cats produce FOCMA antibody, but that in cats with progressive tumors the antibody is absorbed by the malignant growth and therefore it is not detected. We have no evidence to support the second explanation, and the data obtained are consistent with the first hypothesis of FOCMA responder and nonresponder animals for the following reasons. FOCMA antibody titers were determined in inoculated cats before palpable tumors were de­ tected at the injection site, and low titers or lack of detectable antibody was clearly associated with subsequent tumor progression (Schaller et al., 1975). In cats inoculated at older ages (e.g., 3 months), large tumors sometimes developed which subsequently regressed. High levels of free FOCMA antibody were detected in such individuals at around the peak of tumor growth and subsequently during regression (Essex et al., 1971b). Finally, attempts to detect FOCMA antibody bound to, or coating, progressor tumor cells were unsuccessful (Essex et al., 1976). Passively administered FOCMA antibody has been shown to be effective in causing regression of fibrosarcomas (Rolosin et al., 1979). Antisera containing FOCMA antibody has also been shown to prevent tumor development following virus challenge when suckling kittens have obtained the antibody from immune dams (Essex et al., 1971a; Hoover et al., 1977b). In the latter case, however, the possibility that the protective effect is at least partly due to virus-neutralizing anti­ body has not been ruled out. Since FOCMA antigen was first demonstrated on cultured lymphoma cells (Essex et al., 1971a,b), it was logical to ask if FOCMA antibody activity had a significant role in lymphoma and leukemia as well as in fibrosarcoma development. Studies with leukemias and lymphomas in­ duced in the laboratory with FeLV revealed an analogous relationship. Cats that developed lymphomas failed to demonstrate significant levels of anti-FOCMA while those that resisted lymphoma development had

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high anti-FOCMA titers (Jarrett et al, 1973a,b; Essex, 1974; Hoover et al, 1976). Although it was clear that tumor development was correlated with anti-FOCMA titers under laboratory conditions, where the animals were inoculated with FeLV or FeSV, it was important to determine if the same correlation was present under natural conditions. As the initial approach to this question, cats with naturally occurring lymphoma and leukemia were checked for humoral anti-FOCMA titers (Essex et al, 1975b, 1976). More than 100 cats with either lymphoid leukemia or lymphoma were checked, and almost all had no detectable antibody or only very low titers; less than 5% of the cats tested had titers higher than 4. The lack*of detectable FOCMA antibody or the presence of very low levels was therefore readily apparent for cats with both leukemia and lymphoma. As controls, analogous to FeLV-inoculated cats that did not develop tumors, we selected healthy virus-exposed cats residing in two "leukemiacluster" households (Cotter et al, 1974; Essex et al, 1975d,e). About 140 healthy cats known to be naturally exposed to FeLV were available for study. As a group, they had a geometric mean FOCMA antibody titer that was 10-fold higher than the mean for cats with leukemia and lymphoma (Essex et al, 1975d, 1976). Approximately two-thirds of the healthy but virus-exposed cats had FOCMA antibody detectable at titers of 4 or higher. Half of all the healthy cats in both cluster households were actually viremic with FeLV, and as a group these viremic cats had antibody titers that were slightly lower than the titers for the FeLV-exposed healthy nonviremic cats. Nevertheless, both groups had mean titers that were significantly higher than the titers for neoplastic. cats. Healthy cats from controlled environments where exposure to FeLV is absent regularly lack FOCMA antibodies (Essex et al, 1975a). By the time pet cats are diagnosed with leukemia or lymphoma, the disease has usually progressed to a stage where the animal is debilitated and immunosuppressed. Since serum samples from tumorous cats were generally collected at this stage, it was possible that the poor FOCMA antibody responses were a result, rather than a cause of the malignancies. To address this question, a large population of FeLV-exposed cats were screened for FOCMA antibodies, in a prospective sense, until a statistically significant number developed leukemia or lymphoma (Essex et al, 1975e). This study clearly established that cats which developed tumors could regularly be identified as "high-risk" candidates for lymphoma development on the grounds of FOCMA antibody titers many months before any clinical signs of the disease were present.

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The close correlation between humoral antibody response and tumor resistance we observed would not have been predicted from the majority of studies based on laboratory models (Herberman, 1973), which generally support the concept that T-cell immune effector mecha­ nisms are more relevant for tumor immunity (Burnet, 1970). In a generalized way, the latter assumption may be correct, but in immune surveillance and control of this particular series of tumors, we would project that B-cell-dependent, immunoglobulin-mediated pathways would be important. As mentioned above, the lymphoid cat tumors are predominately T-cell tumors, a situation which might logically lead to the evolution of T-cell-independent immune control mechanisms. Al­ though tumors other than T-cell tumors also occur in lower frequency (e.g., fibrosarcomas and B-cells lymphomas), FeLV has a predilection for infection of T cells (Azocar, Mandel, and Essex, unpublished obser­ vations). The B-cell tumors, which are caused by a defective variant of the virus, may represent a more recent evolutionary development between the agent and its host. The mechanism by which FOCMA antibody induces protection against tumors in cats appears tq be complement dependent. A very close cor­ relation was observed between the presence of FOCMA antibody and the presence of complement-dependent lytic antibody (CDA) in cat sera (Grant et al., 1978). Of particular significance, the correlation between antibody activities was greater when CDA was assayed in vitro using cat complement, when other more commonly employed heterologous complement sources were less effective (Grant et al.y 1977, 1978). Like FOCMA antibody, CDA is detected in sera only from virus exposed cats, and it appears in serum between 8 and 32 weeks after exposure through natural contact with viremic cats commences. In a randomized survey of cats in Davis (California), 25-30% of the privately owned cats tested had detectable levels of CDA in their sera. CDA is not directed at either of the major antigenic components of FeLV (p30 or gp70) (Grant et al., 1978). High titers of CDA are found in the majority of healthy cats in leukemia cluster multicat households, and approxi­ mately 50% of healthy but persistently viremic cats in these environ­ ments have strong CDA activity. Only 12% of 208 cats with diagnosed leukemia/lymphoma had serum CDA compared with 45% of com­ parable FeLV-exposed controls (Grant et al., 1979b). As FOCMA antibody appears to be complement dependent in order to effect tumor protection, complement levels were examined in a large group of healthy and sick animals. The levels were found to vary dramatically from week to week in healthy viremic cats but not in normal controls, and reduced complement levels were detected in association with FeLV-

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related anemia. Some cats with leukemia or lymphoma had reduced complement activity but others had normal levels in the terminal stages of disease. At present, it seems that some virus-associated tumors progress in the face of an anti-FOCMA humoral response at times of transient reduction in complement levels, but this event occurs in only some (30-50%) of the cats that develop progressive tumors in the presence of anti-FOCMA or CDA immune activity.

VI. Avian Tumors Caused by Retroviruses C-type retroviruses cause a range of tumors in chickens. The most important, from the incidence standpoint, is visceral lymphomatosis, which is a B-cell lymphoma resulting after a long incubation period. Also caused by the same family of viruses are the acute erythroid and myeloid leukemias, and the fibrosarcomas, all of which have a shorter latent period. The virus that causes lymphomatosis is very similar to FeLV, having analogous gag, pol, and env genes which code for virus structural polypeptides (Kurth et al., 1979). Although similar in structure and function, none of the proteins are evolutionarily close enough to show antigenic cross-reactivities with FeLV or the other mammalian retro­ viruses. Like the FeLVs, the avian lymphoma viruses (ALV) are divided into alphabetical subgroups on the basis of the type-specific antigen of the major virion env protein gp85. Along with the distinct subgroup patterns of virus-neutralizing activity, the subgroups demon­ strate host cell-surface attachment interference. Chicken cells can be categorized on a genetic basis because of their viral receptors, and effective resistance against disease development can be bred into chicken strains based on this characteristic (Crittenden, 1976). Subgroup A ALV is the most common cause of lymphoma in chicken flocks (Calnek, 1968). The virus is transmitted both horizontally and congenitally from the dam (Rubin et al., 1962). Chicks that become congenitally infected at birth are usually immunologically tolerant to the virus. As a result they remain persistently viremic throughout life, and have a high risk for disease development. Conversely, chickens that become horizontally infected at older ages develop effective titers of virus-neutralizing antibodies which protect against persistent infections. Although chickens also maintain complete retrovirus genomes that are genetically inherited (the subgroup E viruses), these agents have not been linked to the development of neoplastic diseases (Crit­ tenden, 1976).

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The viruses that cause the acute leukemias and fibrosarcomas are defective for replication, as is FeSV (Graf and Beug, 1978; Kurth et al., 1979). This group of viruses, particularly the Rous sarcoma viruses (RSV), have served as prototype models for studying malignant transformation of fibroblasts in vitro. A distinct transformation-specific protein of about 60,000 MW has been identified as encoded by RSV (Brugge and Erikson, 1977; Purchio et al., 1977). In the case of the acute avian leukemia viruses, transformation-specific proteins of a slightly higher molecular weight have been identified (Bistner et al., 1977). Both of the above proteins are partially or completely distinct from the viral structural proteins. The protein associated with the acute leukemia viruses, like the higher molecular weight form of FOCMA, contains a transformation-specific portion linked to the protein product of the 5' end of the gag mRNA. Although the 60,000-MW transformation-specific protein described above has not been identified on the cell membrane of RSV-transformed cells, several other virus-related cell surface proteins have been identi­ fied as potential targets for tumor immunity. Among these are (a) type- and group-specific determinants of the major virion env glycoprotein (gp85), (b) an embryonic antigen, which is induced by chemical transformation or by RSV, and (c) an RSV tumor-specific surface antigen (TSSA) which is not a virion structural component (Ignjatovic et al, 1978). The type-specific component of gp85 is expressed on virus-infected cells, irregardless of whether or not they are transformed, but the group-specific determinant of gp85 appears to be expressed only on transformed cells. Although TSSA is expressed only following RSV transformation in cultured fibroblasts, it seems likely that the same antigen may be expressed on B lymphoid cells transformed in vivo by ALV, because antibodies to TSSA are induced in chickens exposed to infectious ALV (Kurth et al, 1979). Like FOCMA, TSSA appears to be virus coded, because it can be induced by transformation across species barriers in vitro (Kurth and MacPherson, 1976). The antigen is also expressed on transformed nonproducer cells, which do not make the replication-competent helper virus. TSSA is group specific, expressed independently of the subgroup of the associated helper virus (Kurth and Bauer, 1972). TSSA is believed to be a major target for tumor immunity in vivo (Kurth et al., 1979), but the majority of studies have been conducted with animals or chickens inoculated with RSV-transformed cells rather than chickens with naturally occurring tumors. Many studies to define the antigens have involved lymph node or spleen immune effector cells

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in cell-mediated cytotoxicity tests (Hayami et al., 1979), and ap­ parently the cytotoxic effect of lymphocytes is partially restricted by the histocompatibility antigen complex (Wainberg et al., 1974). Anti­ bodies from RSV regressor chickens also react with the TSSA (Kurth and Bauer, 1972). The bursa of Fabricius is required as an initial transformation site in the development of ALV-induced lymphoma (Crittenden, 1976), but little is known about the relative importance of humoral and cell-mediated immune effector mechanisms in lymphoma development.

VII. Marek's Disease Marek's disease (MD) of chickens is a lymphoid cell tumor which occurs at a high incidence in young chickens, accounting for major financial losses in the poultry industry. An effective vaccine against MD has now been available for 6 to 8 years (Purchase, 1976; Nazerian, 1979). Along with the symbolic importance of this as the first effective vaccine against a neoplastic disease in any species, the availability of the vaccine has been of major economic importance for the industry. The major pathologic manifestation of MD is the infiltration of major nerve tracks with malignant T lymphoid cells (Payne and Biggs, 1967). MD is caused by a herpesvirus (MDV) which is efficiently transmitted in a highly contagious manner. Replication of MDV occurs primarily in feather follicle epithelium, and sloughed feather follicle debris appears to aid both airborne transmission and the preservation of infectivity (Calnek et al., 1970; Nazerian and AVitter, 1970). The malignant T cells themselves do not normally produce virus, but maintain multiple complete copies of the viral genome in the host cell DNA (Nazerian et al., 1973; Nazerian and Lee, 1974). Several intracellular viral antigens may be seen in MDV-infected cells, but these are probably not targets for immunity in MD. Two antigens appear on the surface of MD tumor cells which may be important in immunity. One is the viral membrane antigen (MA) which is probably also the target for neutralization of viral infectivity (Chen and Purchase, 1970). The second is the Marek's associated tumor-specific antigen (MATSA), which is not a virus structural pro­ tein (Powell et al., 1974; Witter et al., 1975; Nazerian, 1979). MATSA is distinct from histocompatibility antigens and embryonic antigens. Retrovirus-transformed tumor cells are negative for MATSA. Not all lymphoid cells in MD tumor cell preparations are positive for MATSA, but this is presumably because immune effector cells infiltrate the tumor.

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Chickens demonstrate a genetic resistance to MD which is closely linked to a few genes related to the major histocompatibility locus (Longenecker et al., 1973). Since both tumor-resistant and sensitive strains of chickens seem equally to support virus replication, it appears that the genetic effect must be mediated either at the level of sensitivity of target cells for transformation, or at the level of the antitumor immune response. An age-related resistance is also operative in MD (Nazerian, 1979). Chickens develop both humoral and cell-mediated immune responses against development of MD tumors. As in the case of genetic resistance, the immunity produced by the viral vaccines does not appear to be directed at viral replication. Chickens that were successfully vaccinated against tumor development, with either attenuated strains of MDV or the related herpesvirus of turkeys (HVT), do not resist virus in­ fection upon subsequent challenge (Purchase, 1976). Humoral antibodies may play some role in resistance. Antibodies against both MA and MATSA can be detected in some chickens. Chickens that develop high antibody titers survive longer than those that do not (Witter et al., 1970; Nazerian, 1979), and chicks that pas­ sively acquire maternal antibodies appear to have a longer incubation period before developing MD (Calnek, 1972). However, MDV is an immunosuppressive agent, and one major effect appears to involve a decreased ability to produce IgG (Jakowski et al., 1973). Despite this, many researchers feel that the major antitumor effect in MD is mediated by T cells, and T-cell functions are also suppressed by MDV infection (Purchase et al., 1968; Theis et al, 1975). T-killer cell activity has been demonstrated against MATSA containing lymphoblastoid cell lines (Sharma and Coulson, 1977).

VIII. Bovine Leukosis Four morphologic forms of bovine leukosis have been recognized. The most common form is enzootic bovine leukosis (EBL) which occurs primarily as a multicentric lymphoma of adult animals. The other three forms are designated skin leukosis, the juvenile thymic form, and the generalized sporadic form which frequently occurs in young animals (Straub, 1978). Little or nothing is known about the etiology of the latter three forms which also may occur much less frequently than EBL. Recent studies have conclusively demonstrated that EBL is caused by a C-type oncornavirus (see Burny et al., 1978, for review). The evidence that EBL was caused by a virus had long been suspected,

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since virus-like particles were often seen in tissue by electron micro­ scopy (Kawakami et al, 1970), and virus could be induced from lymphoid cells (Miller et ai, 1969; Ferrer et al, 1971; Stock and Ferrer, 1972). As soon as sufficient amounts of virus became available it was shown that the agent could induce lymphoma in sheep (Olson et al., 1972) and cattle (Burny et al., 1978). Elegant seroepidemiologic studies have confirmed that the agent was localized in animals and herds that were infected with EBL (Piper et al., 1975; Devare et al., 1976; Kaaden et al., 1977; Levy et al., 1977). Of equal importance, nucleic acid hy­ bridization studies indicated that EBL tumor cells regularly contain DNA proviral sequences related to BLV, whereas the same sequences are absent in normal bovine cells and tumor cells from the other three forms of bovine leukosis (Callahan et al., 1976; Kettman et al., 1976). Several structural proteins associated with BLV are known to be immunogenic for cattle under natural conditions. These include the major core antigen p24, which is comparable to the p30 of FeLV (Devare et al, 1976; McDonald et al, 1976; Levy et al, 1977), the virion core pl5 (Kaaden et al., 1977), and the virion envelope glycoprotein (Driscoll et al, 1977). The latter protein appears to be the target for virus-neutralizing antibody, and also a target on genome-carrying cells for prevention of virus release (Ferrer and Diglio, 1976; Driscoll et al, 1977). Surprisingly, none of the major virion structural proteins appear to cross-react with analogous proteins from other mammalian C-type viruses (Ferrer, 1972; McDonald et al., 1976; Van der Maaten and Miller, 1977). Antibodies to the virus structural proteins are regularly present in most cattle residing in infected herds, but are also present in cattle from EBL-free herds. Both the sérologie studies and the hybridization studies strongly support the concept that EBL is horizontally rather than vertically transmitted, which is in turn compatible with the high degree of immunogenicity associated with the agent. Since cattle with clinical EBL appear to have various antibodies to virus proteins just as frequently as disease-free contact animals, it appears that these antibodies do not prevent disease development. BLV-infected animals frequently experience very long incubation periods before development of EBL, and during this phase persistent lymphocytosis is sometimes present. One might speculate that an immune surveillance mechanism is usually operative to prevent de­ velopment of EBL during the long phase of persistent lymphocytosis, but no experimental evidence is yet available to support this concept. Relatively little has been done to identify tumor-specific cell surface antigens. Hollinshead and Valli (1976) found soluble cell membrane

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antigens in tumor tissue that were not present in normal tissue, and the fractions induced a delayed-type hypersensitivity reaction when inoculated into exposed cattle. Onuma and Olson (1977) also reported the existence of cytoplasmic and membrane antigens that were as­ sociated with EBL. In both studies, however, the possibility that the agents are virus structural proteins and/or equally expressed on BLVinfected normal lymphoid cells has not been ruled out. Additionally, little or nothing is known about what immune effector mechanisms may be mounted to these or other cell surface antigens on EBL tumor cells.

IX. Transmissible Venereal Sarcoma of Dogs The transmissible venereal sarcoma (TVS) of dogs is a very unusual tumor for at least two reasons. First, it is transmitted in a horizontal manner as a transplant of tumor cells rather than by an infectious virus which subsequently induces the disease. Second, after an initial phase of rapid growth with highly malignant characteristics, the tumors often regress, but may also metastisize. The tumor cells are transmitted by sexual intercourse and develop on both male and female genitalia, but only after abrasions have occurred in mucosal membranes. The TVS has a worldwide distribu­ tion, but a higher incidence in warm climates (Cohen, 1978). Evidence that the tumor is due to horizontally transmitted cells is based partly on the ease of laboratory induction by transplantation, but more importantly on the characteristics of the tumor cells, which are marked by highly specific chromosomal aberrations (Barski and CornefertJensen, 1966) and also lack the histocompatibility antigens of the host (Cohen, 1978). Although TVS sometimes metastasizes to kill the host, the tumors more frequently regress, and various lines of evidence suggest that the immune response plays an important role in the regression process. Using the mixed lymphocyte stimulation procedure, the degree of reac­ tivity was found to be partially correlated with tumor growth (Hess et al., 1975). Dogs in which tumors eventually regressed had more active blastogenic responses during the progressive phase of develop­ ment as compared to dogs that did not experience such marked tumor regression, and animals that developed lethal métastases had lympho­ cytes which were nonreactive. Antibodies to TVS cell surface antigens may also play an important role in the regression response (Cohen, 1972, 1973). Most exposed dogs

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had detectable antibodies, irregardless of whether they were in pro­ gressive stages with métastases or in regressive stages of tumor de­ velopment. However, regressor sera were most effective in inhibiting tumor cell growth in vitro (Bennett et al., 1975), and also in preventing tumor growth when passively transferred in a therapeutic manner (Powers, 1968). Other assorted observations are compatible with the hypothesis that the immune response plays a regulatory role in the development of TVS. For example, the tumor shows a higher degree of malignancy when cells are transplanted to irradiated dogs (Cohen, 1973). BCG, a non­ specific immune stimulant, causes earlier and more frequent regressions when administered to tumor-bearing dogs (Hess et al., 1979). Addi­ tionally, puppies from immune dams show greater resistance to the development of lethal tumors upon challenge than do puppies from unexposed dams (Yang and Jones, 1973). While evidence exists to suggest that the immune response plays an important role in surveillance against TVS transmission, much of interest remains to be learned about both the nature of the immune response and the target tumor antigens. ACKOWLEDGMENTS

Research conducted in the laboratories of the authors was supported by National Cancer Institute grants CA-13885 and CA-18216, National Cancer Institute contract CB-64001, grant DT-32 from the American Cancer Society, and grant 1483-C-l from the Massachusetts Branch of the American Cancer Society. C. K. G. is a Scholar of the Leukemia Society of America. We are grateful to several colleagues, Drs. H. Bauer, A. Burny, R. Kurth, K. Nazerian, and R. Olsen for providing prepublication information.

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ADVANCES IN VETERINARY SCIENCE AND COMPARATIVE MEDICINE, VOL. 2 3

Applications of Transplantation Immunology in the Dog H. M. VRIESENDORP Radiohiological Institute GO-TNO, Rijswijk, The Netherlands, and Laboratory for Experimental Surgery, Erasmus University, Rotterdam, The Netherlands

I. Introduction I I . Present Status of Histocompatibility Testing in Dogs . . . . . . 1. Regions of the Major Histocompatibility Complex 2. Genetic Organization of the DLA Complex 3. Minor Histocompatibility Systems III. Transplantation Immunology in Dogs 1. Donor Selection 2. Immunosuppression IV. New Applications of Transplantation Immunology in Dogs . . . . 1. Extrapolation of H u m a n and Experimental Animal Transplantation Immunology 2. Exploration of Remaining Problems in Transplantation Research . V. Conclusions References

229 233 234 238 238 239 239 247 255 255 258 259 260

I. Introduction Over the past two decades the field of transplantation has reached the stage of clinical application. Numerous kidney, bone marrow, heart, and liver transplants have been performed in human patients. One of the so-called preclinical experimental animal models used in the de­ velopment of this field has been the dog. In this chapter a review will be given of the results obtained in transplantation research in dogs and the possibilities for further applications of this knowledge in dog biology and dog medicine as well as in further laboratory studies of transplantation. The dog is chosen as an example above other domesticated species because it has been the most actively studied species in the field of transplantation. However, the conclusions reached might be applicable to the other domesticated species as well. In this introduction, a short survey of the historical development of concepts 229 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-039223-2

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in transplantation will be given as an orientation to the reader for subsequent sections in this chapter. In 1937 Gorer successfully predicted the "take" of a mouse tumor after transplantation. To achieve this he tested red cells of recipient mouse strains with rabbit anti-mouse sera. Only strains with certain red-cell types were susceptible to the injection of tumor-cell suspen­ sions. Since this discovery many more attempts were made to identify cellular membrane antigens that control tissue rejection and its re­ verse: histocompatibility. For historical as well as economical reasons the mouse has been the most frequently used experimental animal in histocompatibility research. However, important results have been ob­ tained in other experimental animal species since the advantages that these models offer over mice have been exploited. Examples of such data obtained in dogs are discussed in Sections II and III. Since the work of Counce et al. (1956), histocompatibility (H) loci have been subdivided into one strong or major locus and many weak or minor loci (i.e., all other H loci). The differences between these loci is that incompatibility for the major locus between donor and recipient leads to early rejection which is difficult to influence by tolerance in­ duction (Uphoff, 1961) or nonspecific immunosuppressive treatment (MacQuarrie et al., 1965). In the presence of a minor locus difference, donor tissues survive for a prolonged time after transplantation. Toler­ ance induction and further prolongation of graft survival by immunosuppression is more easily achieved over such a difference. In other mammalian species such as man (Kissmeyer-Nielsen and Thorsby, 1970), rhesus monkey (Balner et al., 1971), dog (Vriesendorp et al., 1971), pig (Vaiman et al., 1970), and rat (Stark et al., 1967), a similar subdivision between major and minor histocompatibility loci can be made. Examples in chickens (Pazderka et al., 1975) and frogs (DuPasquier et al., 1975) indicate that this principle is operative in nonmammalian species as well. In a recent review of the current state of histocompatibility typing in the mouse, Klein (1975) estimated that several hundred, different, H loci exist in this species in addition to the 38 that have been identified so far. If similar numbers of H systems are present in other species it is evidently impossible to match donor recipient pairs for all of them, unless they belong to the same inbred strain (rodents) or are monozygotic twins (man). Other donor recipient pairs can be matched only to a certain degree by introducing identity for major H genes first and subsequently for as many minor H genes as possible. Immunosuppression will be required to suppress the effects of the inevitable

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remaining minor H differences. Problems in the application of this approach arose when it became clear that the major H locus was not one locus but consisted of a cluster of numerous closely linked loci each of which was highly polymorphic. Therefore, instead of one major histocompatibility locus, a major histocompatibility complex (MHC) was postulated. The total number of loci within the MHC is presently unknown because the majority of loci within the MHC region have not yet been identified. Through an analysis of MHC recombinants in mice it became clear that not all parts (or regions) of the MHC are of equal importance in the determination of graft survival (Klein, 1975). Apparently at least three regions within the murine MHC exist that have a histocompatibility effect whereas others have no histocom­ patibility effect. Given the lack of a precise identification of all the loci in the MHC, it is presently preferable to indicate regions in the MHC by the loci occurring in them and to ascribe functions of the MHC to regions rather than isolated loci. This approach evades the danger of ascribing a function to a locus while in fact an unidentified locus or loci nearby in the same region control that function. Also, in experimental animals other than mice, i.e., rhesus monkeys (Balner and van Vreeswijk, 1975; van Es et al., 1977a) and dogs (Westbroek et al., 1975; Vriesendorp et al., 1975a), indications have been obtained for different effects of the various currently known regions of the MHC on graft survival. One of the remaining challenges to donor selection studies is the identi­ fication of the regions in the human MHC that are most relevant to donor selection. In addition to the search for the most effective and practical way to identify a histocompatible organ donor, investigators have turned their attention to the possible biological significance of H genes. In the not too distant past every genetic variation within a species was thought to be of biological significance (Falconer, 1964; Ford, 1965). However, more recently it was shown that the majority of genetic polymorphisms have probably evolved by chance, not by pressures of natural selection (Harris, 1971). Therefore the extreme polymorphism of the MHC is not acceptable as a priori evidence of the biological importance of the MHC outside the artificial situation of tissue transplantation. How­ ever, suggestive evidence has been obtained in man as well as in ex­ perimental animals for a biological significance of the MHC. This evidence is of necessity indirect as long as the true biological functions of the MHC have not been identified. It includes associations between MHC antigens and diseases (Dausset, 1977) as well as between MHC antigens and parameters of biological "fitness" as immune response

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genes (Benacerraf and McDevitt, 1972). In addition, population studies of MHC genes have indicated that present-day MHC phenotype fre­ quencies, in particular the occurrence of so-called linkage disequilibrium (see Section III,l,b), are best explained by assuming natural selection to act on MHC-coded structures (Bodmer et al., 1973; Grosse Wilde et al., 1975). Several hypotheses on the exact nature of the biological significance of MHC genes have been put forward (Jerne, 1971 ; Bodmer, 1972; Benacerraf and McDevitt, 1972; Zinkernagel and Doherty, 1974). In one form or another they all suggest a regulatory role for the MHC in the development or control of tissue differentiation or immune respon­ siveness. Studies testing these hypotheses are ongoing in many labora­ tories at the time of the writing of this chapter. In view of the complexity of the models proposed for the biological significance of H genes, it is not surprising that a definite answer to this question is not available yet. The definition of the biological significance of H genes would help in the development of further applications of histo­ compatibility studies outside the field of transplantation.

II. Current Status of Histocompatibility Testing in Dogs Dogs have been used as experimental animals since the early days of organ transplantation (Ullmann, 1902). Histocompatibility was not studied in this species until Rubinstein and Ferrebee (1964), Cleton (1965), and Epstein et al. (1968) started to look at various sérologie methods of determining polymorphic dog cellular antigens. Later it was shown that skin transplants between dogs were a reliable way of in­ ducing dog anti-dog antibodies that were reactive with antigens on dog leukocyte membranes (Vriesendorp et al., 1971). In the same study it was demonstrated that the genetic control of these antigens was located in one chromosomal area that was also of great importance for graft survival. This genetic area was called the DLA complex, in which D stands for dog and L for leukocytes; A indicates that it was the first and most important H system to be identified. Also, mixed lymphocyte cultures (see Section 11,1) appeared to be under the control of one genetic area (Templeton and Thomas, 1971), which later was found to be part of the same histocompatibility complex DLA (van der Does et al., 1973; Templeton et al., 1973). Subsequently other regions in the DLA complex were recognized. Table I is a summary of the currently known regions of the DLA complex and the methodology for the recognition of the gene products of loci within them.

DLA-A DLA-B DLA-C DLA-D DLA-E IrGA IrGL IrGT R

PGM 3

Lymphocyte-defined (LD) antigens Immune-response genes (Ir)

Resistance against bone marrow transplants (R)

Phosphoglucomutase-3 ( P G M 3)

Nomenclature of recognized loci within region

Serologically denned (SD) antigens

Type of region

TABLE I REGIONS I N THE DLA

Electrophoresis (Meera Khan et al., 1973)

Hemopoietic recovery of supralethally irradiated animal after infusion of 4 X 10 8 allogeneic bone marrow cells per kilogram body weight (Vriesendorp et al., 1976)

Unilateral mixed-fymphocyte cultures (Grosse Wilde et al., 1973) with homozygous typing cells as stimulators Immunization with low dose of antigen and Farr assay to quantify antibodies produced (Vriesendorp et al., 1977a)

Antisera in a microlymphocytotoxicity assay in a one-stage (Vriesendorp, 1973) or a two-stage (Albert et al.} 1973) modification

Technique and references for recognition of gene products

COMPLEX

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H . M. VRIESENDORP

1.. REGIONS OF THE MAJOR HISTOCOMPATIBILITY COMPLEX

a. SD With large antisera panels (50-100 different sera per panel) leuko­ cytes of unrelated dogs and dog families were investigated. As serological techniques, dog modifictions of microlymphocytotoxicity tests were used. With the appropriate immunogenetic analyses, antigens and their genetic control can be identified in such studies. Initially two (Vriesendorp et al, 1972), later three (Vriesendorp et al., 1977a), series of serologically defined (SD) multiple alleles have been found. The loci for these series were labeled DLA-A, DLA-B, and DLA-C. They are distinct loci and different from each other as well as from the other loci in Table I. This has been established in population studies of the alleles of the different loci in which nonoverlapping dis­ tribution patterns of the various alleles were found as well as in DLA recombinant families (see Fig. 1) in which alleles of different DLA loci showed different inheritance patterns. b. LD When the leukocytes of two dogs are mixed and cultured for several days under appropriate conditions, an increase in DNA synthesis can be demonstrated. In several studies it was shown that at least two loci within the DLA complex controlled this increased DNA synthesis (= reactivity) in mixed lymphocyte cultures (MLC) in the dog (Grosse Wilde et al, 1975; Bynen et al., 1977; Bynen, 1978). A separate con­ trol of MLC reactivity and serologically defined antigens was demon­ strated in DLA recombinant families (van der Tweel et al., 1974). Provisional labels for the MLC or LD (lymphocyte defined) loci are DLA-D and DLA-E. Alleles of these loci can be recognized through the use of so-called homozygous typing cells. The principle of this approach is shown in Fig. 2, where a pedigree is given of a mating between an MLC negative brother and sister leading to a third, inbred, generation, part of which is homozygous for the DLA complex. Homo­ zygous typing cells, indicated in Fig. 2, can be irradiated so that they will not "respond" (i.e., not contribute to the increased DNA synthesis in the mixture). Unirradiated cells will fail to respond to such irradiated typing cells if they carry the LD genes of the typing cell. Homozygous typing cells can be identified by the peculiar one-way nonreactivity in a checker board MLC like the one shown in Fig. 2. The encircled and squared MLC reactions in Fig. 2 illustrate this phenomenon. Presently available homozygous typing cells might be homozygous for more than one LD specificity. Therefore a clear-cut allocation of MLC alleles to one

235

TRANSPLANTATION IMMUNOLOGY I N T H E DOG

IrGT

DLA-D DLA-A DLA-C 9

Rd

DLA-B DLA-E ?

9

Chromosome

Recombination

2IU days

200

Days after transplantation

FIG. 3. Survival times of organs from DLA identical donor-recipient pairs. a One standard deviation of mean is given. Each mean is computed from at least six different animals with the exception of liver grafts, where only two animals per group have been grafted so far. l) Recipients received 750 rads of total body irradia­ tion (TBI) and 100 mg of silica per kilogram of body weight, given intravenously to suppress host versus graft reactions. Bone marrow cells were given intravenously 24 hours after T B I in a dose of 4 χ 108 cells per kilogram of body weight of recipient. [Reproduced with permission from Genetics and Immunology of the DLA Complex. Vriesendorp et al. (1977b).]

unrelated donor-recipient pairs are given in Section III,l,b. SD typing and/or MLC tests were used to identify the donors in Fig. 3 with a favorable organ graft survival time. It is a summary of results ob­ tained in or in cooperation with the Rotterdam Laboratory of Experi­ mental Surgery; similar results have been reported from the Cooperstown Laboratory (Dausset et al., 1971). The dog is the only experimental animal in which the effect of MHC typing on organ allograft survival can be compared for so many different organs. The data in Fig. 3 show that skin grafts benefit the least and liver and frozen nerve grafts benefit the most from donor selection. Possible explanations for the differences in efficacy of MHC matching for different organs are as follows. 1. Different end points. Obviously the (subjective) interpretation of the 100% rejection point of a skin graft differs from the (objective) death of an animal from, e.g., uremia or heart failure. 2. Difference in susceptibility to rejection. Some organs might be more susceptible to the same immunologie attack of the host than others. This could be due to differences in susceptibility of cells of different organs to antibodies or aggressive lymphocytes or to the

TRANSPLANTATION IMMUNOLOGY IN THE DOG

241

presence of a "bottleneck" structure in a given organ (e.g., the rejection of the conduction system of the heart makes the rest of the organ useless), or to the presence or the absence of reserve or regeneration capacity in a transplanted organ. 3. Blood transfusions. For the more complicated surgical procedures, such as heart and liver transplants, intraoperative blood transfusions are required that might cause some immunosuppression in the host. Liver and heart are among the longer surviving organs. However, with­ out additional postoperative immunosuppressive therapy, third-party blood transfusions are probably not an effective or predictable immuno­ suppressive regimen (Bull et al., 1978; Obertop, 1978) [see also Section ΙΙΙ,2,β,(1)]. 4. Minor histocompatibility differences. The survival of each organ might be influenced by a different set of minor histocompatibility sys­ tems differing in number and strength for each organ (see Sections 11,3 and III,l,b). 5. Differences in immunization procedure. Avascular transplants as a skin graft might be more immunogenic and therefore more rapidly rejected because they are presented to the host intradermally. For many antigens that are less complicated as allografts, the intradermal route was found to be more immunogenic than the intravenous route. In all probability several of these above-listed possible explanations for variation of MHC typing efficacy are operating in vivo. Further studies are required to identify the ones of the greatest clinical signifi­ cance, i.e., the ones that could be used for further improvement on the survival of MHC identical grafts in the unimmunosuppressed host. The efficacy of immunosuppression in DLA identical donors recipient pairs is currently unknown. In view of the results in rodents, where immunosuppressive treatment was much more effective in suppressing minor histocompatibility differences than major histocompatibility differences (MacQuarrie et al., 1965; Silvers et al., 1967), one would anticipate that a prolonged and possibly indefinite graft survival could be obtained in DLA identical littermate combinations with the currently available modes of immunosuppression. In addition to the influence of DLA typing on graft survival effects on histology and function of rejecting organs were found (Westbroek et al., 1972; Penn et al., 1976). The histology of rejection can be sub­ divided in two components: (1) a mononuclear cellular infiltrate of the donor tissue, and (2) a proliferative endarteritis in the graft. DLA identity between donor and recipient effectively decreases the severity of the cellular infiltrate, but has a less pronounced effect on the graft arteritis, which is the ultimate cause of failure in DLA identical grafts.

242

H. M. VRIESENDORP

In DLA mismatched grafts early occurrence of a massive cellular in­ filtrate is the cause of organ rejection. A study of blood and urine chemistry in renal allografts demonstrated that in the case of DLA identity tubular lesions gradually develop in the graft initially accompanied by polyuria, whereas in DLA mis­ matched transplants an almost immediate whole kidney ischemia occurs after 7 days, with an equally sudden onset of anuria. b. Unrelated Donor-Recipient

Pairs

The success of donor selection in unrelated donor recipient pairs is much more limited in comparison to results in littermate donor-recipient pairs. In the dog data are available for kidney allografts and hostversus-graft and graft-versus-host reactions in bone marrow trans­ plantation (Bynen, 1978; Bvnen et al., 1979; Westbroek et al, 1975; Vriesendorp et al., 1975a). Kidney data are given in Fig. 4 and are to be compared to the littermate data in Fig. 3. The four most-evident explanations that can be offered for this difference in efficacy of DLA matching are as follows: (1) imperfect SD and LD matching techniques; (2) an important role for minor histocompatibility systems; (3) SD and LD regions are not histocompatibility regions; (4) ex­ istence in addition to SD and LD regions, of other histocompatibility regions within the MHC. These possibilities will be discussed in greater detail below. 1. Presently SD and MLC matching techniques in the dog are im­ perfect. As shown in Table II some DLA SD antigens are not recognized yet. Therefore animals might be erroneously assigned as identical, but in fact be different for unidentified antigens. The recogniIUU

75

50

:! : L, :.j | i-i

I i T

i I

i__ ~~Li

I

'l·-! ""I j '".. \ I

25

_l 10

I 20

····

L* 30

identical nonsiblings identical siblings one haplotype different siblings two haplotypes different siblings

~"\ " I

."_

'

DLA DLA DLA DLA

I 40

S

II 50

I 60

I 70

//

I 150

Days after transplantation

FIG. 4. Kidney allograft survival in immunogenetically different groups of boagle donor-recipient pairs. [Figure kindly supplied by Bynen et al. (1979a).]

TRANSPLANTATION

IMMUNOLOGY I N T H E DOG

243

tion of known DLA SD antigens might be imperfect through crossreactivity phenomena (Vriesendorp et al., 1973; Saison and Doble, 1975; Joint Report of the Second International Workshop on Canine Immunogenetics, 1976). Thus, animals might be selected as identical for DLA SD antigens while an analysis with antisera without cross reactivity problems would have indicated SD antigen differences. Also the determination of LD phenotypes is presently provisional, as in­ dicated in Section II,l,b. Imperfect DLA typing in littermate donorrecipient pairs will not lead to accidental mismatching as long as the inheritance patterns of the parental DLA information are cor­ rectly interpreted. In unrelated donor recipient pairs, imperfect DLA typing cannot be rescued through a segregation analysis and more erroneously assigned good matches will occur, which might explain the found difference in efficacy in donor selection in the two groups. 2. Minor histocompatibility differences do play a role. A pronounced influence of minor histocompatibility differences was found in studies of kidney allograft survival in beagles (Bynen, 1978; Bynen et al., 1979b) (Fig. 4). Through a comparison of groups of appropriately se­ lected experimental animals, it could be shown that cumulative minor histocompatibility differences had about the same effect on graft survival as a one DLA haplotype mismatch.* The laws of inheritance dictate that a random littermate donor-recipient pair will on average be identical for 50% of their genes. In pairs of more distantly related or unrelated individuals, this percentage of shared genes will be much lower. Therefore, the difference in survival times of kidneys of DLA identical littermate and DLA identical nonlittermate donors in Fig. 4 can be explained by more minor histoincompatibilities in the latter group. A comparison with predictions based on simplified immunogenetic models (Simonsen, 1966; Vriesendorp et al., 1978) showed that a minimum of two or three minor H loci with a low number of alleles per locus could explain the obtained results. Therefore, the lower efficacy of DLA matching in unrelated donor-recipient pairs might be due to the presence of more minor histocompatibility differences. Typing and matching for these loci is presently impossible, because the ap­ propriate antisera or other means of prospectively identifying minor H differences are not available. However, even if this would be possible, the availability of donors would limit the application of minor as well as major histocompatibility matching. As mentioned earlier, at* A parental into offspring graft or an interlittermate graft where the littermates received identical DLA information ( = haplotype) from one parent and a different haplotype from the other parent.

244

H . M . VRIESENDORP

tempts to suppress minor histocompatibility by immunosuppressive treatment might be more realistic than a search for the low frequency ideal donor matched for major as well as minor histocompatibility systems. 3. LD and SD regions could not be major histocompatibility regions themselves but neighbors of true, as yet unidentified, histocompatibility regions. In littermate donor-recipient pairs, matching for one region of the MHC will be accompanied by matching for the other regions of the MHC because of the close linkage of these regions in their inheritance. In unrelated donor-recipient pairs, matching for one region will generally not lead to matching for a closely linked genetic region (Li, 1961). Therefore, the high efficacy of MHC matching in DLA identical littermates could be caused by accidental matching for true histocompatibility determinants through the selection of SD and/or LD identity. In SD, LD identical nonlittermate pairs, this matching for the unidentified locus or loci will not occur. The slight beneficial influence sometimes found in these donor-recipient pairs could be ex­ plained by the presence of so-called linkage disequilibrium between LD/SD loci and the true—unidentified—histocompatibility loci. In the normal situation, linkage equilibrium is present; it describes the inde­ pendence of the occurrence of alleles of linked loci after they have reached an equilibrium state in the random population. This can be illustrated by the example that the presence of a given allele a of locus A in a given individual does not influence the chance that another allele b of linked locus B is present in this individual. Linkage dis­ equilibrium (Cavalli Sforza and Bodmer, 1971) is the reverse situation, in which, for example, a and b are often found in the same individual and in which the frequency of the phenotype a,b is much higher than the product of the frequency of a and b. Linkage disequilibrium can be found under a number of conditions: (a) during a period in which a new allele originating from a recent mutation has not yet reached equilibrium in the population; (b) after a so-called founder, or bottle­ neck, effect; i.e., populations in which a low number of ancestors con­ tributed most of the genes to much more numerous next generations; (c) in the case of natural selection, favoring certain combinations of linked genes over others. Suggestive evidence for the last cause for linkage disequilibrium has been found for the MHC determinants of man and dog (Bodmer et al., 1973; Grosse Wilde et al., 1975). In the dog a high degree of linkage disequilibrium has been found between SD alleles of different loci (Vriesendorp, 1973) as well as between SD and LD alleles (van der Tweel et al, 1974; Grosse Wilde et al., 1974). The degree of linkage disequilibrium is higher in the DLA

TRANSPLANTATION IMMUNOLOGY IN THE DOG

245

complex than in HLA in man (Mattiuz et al., 1970) or RhLA in rhesus monkeys (Balner and van Vreeswijk, 1975) and can be ex­ plained by a different population structure in dogs causing a more pronounced "founder" effect (Vriesendorp, 1973). Linkage disequilibrium within the MHC makes donor selection in unrelated donor-recipient pairs for a system without a histocompatibility effect nevertheless pro­ duce a significant graft prolongation, through accidental matching for alleles of the linked histocompatibility system. This matching will less often be complete, since linkage disequilibrium is never 100%. Therefore this indirect matching will never be as good as matching through a direct identification of the true histocompatibility de­ terminants. 4. In addition to LD and/or SD regions, other still unidentified regions might exist within the MHC with a histocompatibility effect. In this situation again selection of LD, SD identical littermate donorrecipient pairs will also introduce identity for all other histocompati­ bility loci within the MHC, which do not have to be identified in the donor selection procedure. Selection of unrelated donors will only guarantee identity for the positively identified loci. Thus, LD and SD matching alone will lead to less complete MHC matching in this group in comparison to the littermate group and could therefore explain a lower efficacy of donor selection in the former group. A further analysis of tha problem by Bynen and co-workers (1979a,b) in the dog has shown that of the above-mentioned possible explanations for the lower efficacy of current techniques of host-donor matching in unrelated donor-recipient pairs, possibility 3 is probably not operative, as LD and SD regions do have some histocompatibility effect. The relative importance of possibilities 1, 2, and 4 remain to be determined. Some results of these studies are given in Figs. 5 and 6. In Figure 5 the data are shown for the efficacy of LD and SD match­ ing in prolonging kidney allograft survival in mongrel dogs. No immunosuppressive treatment was given to the recipients. Analysis of the data by a Wilcoxon test indicates that the difference in survival times between LD matched and mismatched combinations is significant {p = 0.001 for groups 1 and 3, p = 0.04 for groups 2 and 4, and p < 0.001 for groups 1 and 2 compared to groups 3 and 4). SD matching alone does not lead to a prolonged graft survival (p = 0.39, groups 3 and 4), but a slight and possibly significant prolongation is seen in the LD matched combination (p = 0.07, groups 1 and 2). Keeping in mind the limitations outlined in possibilities 1 to 4 above, this does seem to indicate that for kidney graft survival LD matching is more im­ portant than SD matching. The longest mean survival time in Fig. 5

246

H . M. VRIESENDORP 100

ig

50 · · · SD = LD = ( I ) ooo SD^

LD=(2)

AAA SD= ΔΔΔ S D /

LD/(3) LD/(4) 10

15

20

25

Days after transplantation

FIG. 5. Kidney allograft survival in immunogenetically different groups of mongrel dogs. [Figure kindly supplied by Bynen et al. (1979b).]

for matched unrelated donor recipient pairs is 18 days, which is disap­ pointingly short in comparison to the 43 days found in littermate donor-recipient pairs (see Fig. 3). Another approach, illustrated in Fig. 6, consists of the identification of the region or regions within the MHC with a histocompatibility effect through transplantation studies in DLA recombinants. By transplanting organs from such a recombinant to a littermate and vice versa, smaller regions of the DLA complex can be investigated for their histocompatiIrGT

DLA-D

DLA-A DLA-C

DLA-B

DLA-E

-O

Graft survival (days) I O - 4 4 ( n = IO, medians-. 17, 20) 10, I I , >ll >I50 19, 31 19->150(η = 21, median: 42)

Identity and

Difference for corresponding chrosomal region

LD locus

Çj

SD locus

?

\r gene Exact relative position not known

FIG. 6. Genetic organization of DLA complex and kidney allograft survival in DLA recombinant littermate donor-recipient pairs. [Figure kindly supplied by Bynen et al. (1979b).]

TRANSPLANTATION

IMMUNOLOGY I N T H E DOG

247

bility effects in analogy to the work done in H-2 recombinants in mice (Klein, 1975). A drawback to this approach is the low frequency of recombinants. Therefore many families need to be screened before a single DLA recombinant can be identified. The advantages of this ap­ proach are that further studies can be concentrated on the most im­ portant genetic areas (as attempts to uncover new polymorphic sys­ tems or a better gene definition for already known loci), and that less of the tissue typer's efforts are wasted on studies of areas in which the phenomenon of his interest, i.e., histocompatibility, is not controlled. The preliminary conclusion on kidney graft survival in DLA recom­ binant combinations is that the most relevant histocompatibility in­ formation is located in the region around or to the left of the DLA-D locus. This is in accordance with the observation of the significant graft prolongation in unrelated mongrel dogs by LD matching (Westbroek et al, 1975; Bynen, 1978; Bynen et al, 1979b). The data underline the need for a more intensive immunogenetic as well as immunobiologic analysis of that MHC region. Presently the conclusion for dogs (and this in the author's opinion holds for other mammals as well) is that the small effects of donor selection in unrelated donor recipient pairs do not justify the appli­ cation of logistically difficult and expensive procedures in that setting. A further analysis of the problems listed under 1, 2, and 4 above is required to increase the efficacy and applicability of donor selection in unrelated donor-recipient combinations. 2.

IMMUNOSUPPRESSION

The need for immunosuppression in addition to donor selection is illustrated in Fig. 3, where most of the organs are rejected notwith­ standing DLA identity after a sometimes prolonged time of initial acceptance by the host. Suppressing the immune system carries the well-known risk of exposing the recipient to an increased susceptibility to so-called "opportunistic" infections. In the immunosuppressed host, numbers of microorganisms that under normal circumstances are nonpathogenic will lead to extremely dangerous, and not infrequently lethal, infections. The immune suppression required to prevent allograft rejection is quite severe and often leads to this complication. For a successful transplant only the immune response against the donor tissues need be suppressed. Various approaches have been tried in the laboratory to achieve limited—donor specific—immunosuppression and thus to circumvent infectious complications of nonspecific immuno-

248

H . M . VRIESENDORP

suppression. Nonspecific as well as specific immunosuppression has also been investigated in the dog, and a short review of results is given in two separate sections below. a. Nonspecific

Immunosuppression

(1) Immunosuppressive agents. The difficulties in suppressing the allograft rejection can be illustrated by the observation that many drugs are labeled immunosuppressive because they are able to suppress some part of the chain of events that leads to an immune response to a simple protein antigen. However, they often fail to prolong allo­ graft survival (Bach, 1975; Hersh, 1974). Presently three drugs are commonly used in man for the suppression of the kidney allograft rejection: azathioprine, prednisone, and antilymphocyte serum. Other immunosuppressive drugs effective in prolonging dog allograft survival, and used not at all or less frequently for that purpose in man, are cyclophosphamide, procarbazine, L-aspariginase, methotrexate, and the recently reported cyclosporine A. A summary of results with these drugs in dogs is given in Table III. The drugs have mainly been used as single agents with the exception of the combination of azathioprine and prednisone. Following the example set by multiple drug programs in human oncology (DeVita et al., 1970), new studies of combinations of drugs with different mechanisms and different dose-limiting toxicities might lead to the definition of more effective, less toxic immunosuppressive protocols. In addition to the mentioned increased risk of opportunistic infections with chronic immunosuppression, an increased incidence of malignancies has been reported in immunosuppressed human patients (MacKhann, 1969). These complications and, last but not least, the high failure rate of current immunosuppressive regimens in man [approximately 25% of MHC identical kidneys and 50% of MHC mismatched kidneys are rejected within 2 years (The 13th Report of the Human Renal Transplant Registry, 1977) ] underline the need for the identification of new immunosuppressive regimens. The interactions between immunosuppressive treatment and donor selection still need to be further defined in a preclinical model, such as the dog. In this species the influence of new and old immunosuppressive agents can be investigated in the same donor-recipient combinations as those available in man. Results of such studies could lead to a definition of the most effective and least toxic mode of immunosup­ pression required for optimally matched, partially matched, or totally mismatched donors. (2) Blood transfusions. In man, retrospective studies have shown that

P.O.: 5, daily

Biological

Alkylating agent Alkylating agent Biological

Antimetabolite

Antifungicide

Antilymphocyte serum

Cyclophosphamide

Methotrexate

Cyclosporine

I.m.: 50 in alcohol days 1-7; p.o.: 50 in olive oil daily from day 7

Unknown

Sensitization to bac­ terial protein; weight loss I.v. : 0.5, every other Bone marrow, G I tract day

I.V.: 100-1000 U / k g daily

Bone marrow, behavior

Corticosteroid side effects Sensitization to foreign animal protein, thrombopenia, viral infections Bone marrow, bladder

Bone marrow, liver

Toxicity

++

N o t done, effective in skin grafts and G v H prophylaxis

+

+

+

++ +++

++

Prolongation kidney allograft survival a

Zukoski et al, 1963; Reams, 1963 MacDonald et al, 1971 St. Pierre et al, 1970; Rapaport et al, 1971 Hechtman et al, 1972; Storb et al, 1970 Calne and Dunn, 1977

Abaza et al, 1966; Monaco et al, 1966

Zukoski et al, 1965

Calne et al, 1962; Zukoski et al, 1963

References

b

Controls survive 10 days; survival in experimental animals: + , 10-20 days; ++, 20-30 days; + + + > 30 days. Procarbizine was found to be useful in combination with anti lymphocyte serum in the prevention of the rejection of a bone marrow allograft in dogs sensitized by prior blood transfusions (Storb et al, 1974).

a

L-Asparaginase

P.O. : 5, daily

S.c: 1 ml/kg, daily; i.V.: 3 ml/kg, daily

Hormone

Prednisone

Procarbazine &

P.O.: 10,2 days; 5, 4 days; 2.5, daily P.O.: 3, daily

Antimetabolite

Class

Common drug schedule (mg/kg)

Azathioprine

Agent

TABLE III

SUMMARY OF EXPERIENCE W I T H IMMUNOSUPPRESSIVE AGENTS AND K I D N E Y ALLOGRAFT REJECTION I N DOGS

250

H. M. VRIESENDORP

blood transfusions prior to kidney transplantation sometimes have a beneficial influence on the survival of an allogeneic kidney (Opelz et al., 1972). Although the exact mechanism of this graft prolongation effect of prior blood transfusions is unknown, this phenomenon is in­ cluded here under the heading of nonspecific immunosuppression. The induction of kidney donor, specific, nonreactivity in the host by blood is unlikely. This would require that at least some of the important kidney donor histocompatibility antigens also occur on the cells of the blood donors. The probability of that event is very low, owing to the high degree of polymorphism in the MHC. Therefore the blood transfusion effect is probably caused by the induction of a—presently ill understood—state of nonspecific immunosuppression. The magnitude of the effect is quite important: e.g., in man approximately 60% versus 30% 2-year graft survival in transfused versus nontransfused individuals. However, the effect is also quite unpredictable. In some human studies a correlation was found between prolonged graft survival and patients in which no antibodies were demonstrated after blood transfusions, while a shortened graft survival was seen in patients with antibodies (van Hooff et al, 1972; Oliver et al., 1973; Patel et al, 1971). However, this observation was not confirmed by other investigators (Beizer et al., 1974; Callender et al., 1974; Dausset et al., 1974). This irreproducibility is probably caused by the complicated, multivariable situation of human kidney transplantation. A further prospective analysis in man of mechanisms and manipulation of the effects of blood trans­ fusions is severely limited by ethical constraints on experiments in human patients. However, recently a rhesus monkey as well as a dog model has been reported in which a further analysis of this interestingphenomenon can proceed without these constraints (van Es et al., 1977b; Abouna et al., 1977; Obertop, 1978; Obertop et al., 1978a). One of the dog models will be described here. Obertop et al. (1975) reported that blood transfusions of the future kidney donor do not influence graft survival in the dog if the crossmatch between donor lymphocytes and recipient serum is negative. In the case of a positive crossmatch, an ultrashort survival time is seen. This holds also true for the situation in which a positive crossmatch with kidney donor lymphocytes is induced by third party dog blood (Bull et al., 1978). If repeated (3 times at weekly intervals) blood transfusions were given, some effect was demonstrated on DLA mismatched kidney graft survival if no anti­ bodies, or antibodies with a low frequency of reactivity, were made by the recipient prior to the transplant. This effect became much more pronounced if low-dose immunosuppression (prednisone 1 mg per kilo­ gram of body weight and azathioprine 2 mg per kilogram of body

251

TRANSPLANTATION IMMUNOLOGY I N T H E DOG

% Graft survival 100 I

I·

75

50

25 ·-

NTR

! c 30

20

40

50

60

Days after transplantation

FIG. 7. Kidney allograft survival in beagles and mongrels with or without prior blood transfusions, with or without immunosuppression. T R , Transfused beagles receiving mongrel dog kidney with postoperative immunosuppression; N T R , nontransfused beagles (littermates from T R ) receiving other mongrel dog kidney with post operative immunosuppression; M, nontransfused mongrel dogs receiving beagle kidney with postoperative immunosuppression; C, mongrel dogs receiving mongrel dog kidney without immunosuppression. Immunosuppression: 1 mg of prednisone and 2 mg of azathiopine per kilogram of body weight intravenously daily, starting the day of operation. [Reproduced with permission from Obertop et al. (1978a). © 1978 The Williams & Wilkins Co., Baltimore.]

weight i.V., daily) was given (see Fig. 7) (Obertop et al., 1978a). A clear-cut negative correlation between immune reactivity in recipient's sera against a panel of lymphocytes before kidney transplantation and subsequent graft survival was found. Low reactivity in a one-stage microcytotoxicity test or a more sensitive two-stage microcytotoxicity test was found only in the long-surviving animals. This promising model is currently being further explored to obtain a better insight in the mechanism and optimal control of the immunosuppressive effect of blood transfusions. b. Specific

Immunosuppression

For historical reasons, specific immunosuppression is subdivided into (1) enhancement and (2) tolerance. A working definition of enhance­ ment is that prolongation of graft survival is achieved by antidonor

252

H . M . VRIESENDORP

immunity, which is "protective" instead of "destructive." Kaliss (1958) has limited enhancement by definition to protective antibodies. How­ ever, in a wider definition of the phenomenon, graft prolongation by protective ("suppressor") cells could also be included. Transplantation tolerance can be defined as the absence of an immune response to donor antigens while other immune responses are normally present (Medawar, 1973). It has been argued that the separation of specific immunosuppression into tolerance and enhancement is a laboratory artifact (Hellström et al, 1971), because if sufficiently sensitive techniques are used antidonor immunity can be demonstrated under all circumstances. The discussion about this possibility is still open and will not be pursued here. Only brief reviews of results in both areas in the dog will be given. (1) Enhancement. Before the label enhancement can be applied, two conditions need to be fulfilled. First, antidonor immunity has to be documented and, second, specificity needs to be present: i.e., prolonged survival should be found only for tissue of the donor against which immunity is present; tissue of third parties against which no immunity can be demonstrated should be rejected at the regular speed. Prolonga­ tion of graft survival in the absence of the first prerequisite is due to tolerance; in the absence of the second or both prerequisites, it is due to aspecific immunosuppression. In rodents enhancement has been de­ scribed in two different experimental protocols called passive and active enhancement, respectively. In the first modification, antidonor antisera are used to prolong graft survival; in the second, the recipient is actively immunized before transplantation with donor tissues. A summary of enhancement results in dogs is given in Table IV. In the majority of studies the requirements for the definition of enhance­ ment were not met, but they are nonetheless included in Table IV because they are generally alluded to in the transplantation literature as showing evidence for the possibility of inducing enhancement in other animal species than rodents. The following conclusions can be drawn from the dog enhancement experience, (a) The injection of so-called subcellular spleen donor antigen is the most effective method of prolonging graft survival, es­ pecially when this method is combined with low-dose, nonspecific im­ munosuppression. Important variables in the success rate of this protocol appear to be route of injection and antigen dose. The intravenous route and a low starting dose of antigen correlated with longer survival (Halasz et al, 1966; Zimmerman et al, 1968; Wilson et al, 1969). Spleen appeared to be a better source of antigen than lymph nodes (Holt-Allen et al, 1970). (b) Some of the successful results were not

a

N D , not done.

Allogeneic Antidonor Antiserum

PASSIVE

Subcellular antigen Subcellular antigen Subcellular antigen Subcellular antigen

Spleen, bone marrow lymph node Bone marrow Bone marrow Blood, s.c. Blood, s.c, i.v.

ACTIVE

Donor antigen cells

TABLE IV

None

Azathioprine and prednisone Azathioprine and prednisone

2 out of 6 long survivors

Yes

Possibly

ND

Possibly

ND

ND

44/10

Yes

Yes

ND ND ND ND

ND ND ND In some animals ND

Possibly

ND

Specificity

ND