Professional Documents
Culture Documents
Edited by
Bernd Kaspers
Department for Veterinary Sciences, Veterinary Immunology Study Group,
University of Munich, Munich, Germany
Karel A. Schat
Department of Microbiology and Immunology, College of Veterinary Medicine,
Cornell University, Ithaca, NY, United States
Thomas W. Göbel
Department for Veterinary Sciences, Veterinary Immunology Study Group,
University of Munich, Munich, Germany
Lonneke Vervelde
Division Infection and Immunity, The Roslin Institute and R(D)SVS,
The University of Edinburgh, Midlothian, United Kingdom
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vii
viii Contents
3.3.1 Cellular and molecular 4. B cells, the bursa of Fabricius, and the
identification of the clusters 46 generation of antibody repertoires 71
3.3.2 The paraaortic foci 46
3.3.3 Tracing the origins and fates Michael J.H. Ratcliffe and Sonja Härtle
of the aortic clusters 46 4.1 Introduction 71
3.4 Formation of the aorta: a dorsal 4.2 The generation of avian antibody
angioblastic lineage and a ventral repertoires 71
hemangioblasts lineage 48 4.2.1 Immunoglobulin light chains 71
3.4.1 Two endothelial lineages form the 4.2.2 Immunoglobulin heavy chains 72
vascular network of the embryo 48 4.2.3 Generation of Ig molecules by
3.4.2 Chimeric origin of the aortic V(D)J recombination 74
endothelial cells 48 4.2.4 Generation of Ig diversity by
3.5 Developing an in vitro model of somatic gene conversion 75
hemogenic endothelium commitment 4.2.5 Implications of gene conversion
and endothelial-to-hematopoietic for allelic exclusion 77
transition 50 4.3 The development of avian B cells 77
3.6 Spatiotemporal emergence and 4.3.1 Prebursal B cell development 77
organization of the chicken IAHCs 52 4.3.2 Colonization of the bursa by
3.7 Ecs of the late fetus/young adult bone B cell progenitors 78
marrow harbor hemogenic potential 4.3.3 Colonization of lymphoid
and generate multilineage follicles in the bursa 79
hematopoiesis 54 4.3.4 Growth of bursal B cells in
3.8 Spatial transcriptomics in the chicken bursal follicles 82
embryo reveals regulators of 4.3.5 Development of the bursa after
hematopoiesis 55 hatch 83
3.9 The avian thymus and T-cell 4.3.6 Role of cell adhesion molecules
development 57 and chemokines in bursal cell
3.9.1 Thymic development 57 development 85
3.9.2 Colonization of the thymus 57 4.3.7 Development of peripheral
3.9.3 T-cell differentiation 57 B cell populations 87
3.9.4 TCR rearrangement 58 4.3.8 Activation of peripheral B cells 89
3.10 The bursa of Fabricius, B-cell ontogeny, 4.3.9 Plasma cell development 90
and immunoglobulins 58 4.3.10 Cytokines in chicken B cell
3.10.1 Bursal development 58 development and activation 91
3.10.2 Formation of the bursal 4.3.11 Application of B cell cultures 92
epithelial anlage 58 References 93
3.10.3 Hematopoietic colonization
of the bursal rudiment and 5. Structure and evolution of avian
follicle bud formation 60
3.10.4 Development of the follicle-
immunoglobulins 101
associated epithelium and the Sonja Härtle, Katharine E. Magor,
follicular cortex 62 Thomas W. Göbel, Fred Davison and
3.10.5 Immunoglobulins 63 Bernd Kaspers
3.11 Lymphocyte-differentiating 5.1 The basic structure of immunoglobulins 101
hormones 63 5.2 Avian immunoglobulins 102
3.12 Development of the immune 5.2.1 Avian IgM 102
responses 64 5.2.2 Avian IgY (IgG) 103
3.12.1 Early immune responses 64 5.2.3 Avian IgA 104
3.12.2 Antibody isotype switching 5.2.4 Avian homologues of IgD
and hypersensitivity reaction 64 and IgE 105
3.12.3 Allograft rejection 64 5.2.5 L chains 105
3.13 Conclusion 64 5.2.6 Genomic organization of the
Acknowledgments 65 IgH and IgL locus 105
References 65 5.3 Ig half-life 107
Contents ix
8.6.3.7 TLR7 and TLR8 236 9.6.4 Cytokines and factors in other
8.6.3.8 The absence of TLR9 237 birds 261
8.6.3.9 Avian Toll-like receptor without 9.7 Chemokines 263
mammalian orthologues: 9.7.1 XC and CX3C chemokines 264
chTLR15 and chTLR21 237 9.7.2 CC Chemokines 264
8.6.3.10 Toll-like receptor signaling 9.7.3 CXC chemokines 265
pathways in chickens 238 9.8 Cytokine and chemokine receptors 265
8.6.3.11 Genetic diversity and 9.8.1 Type I receptors 266
evidence of selection in 9.8.2 Type II receptors 266
avian Toll-like receptors 238 9.8.3 Transforming growth factor-β
8.6.3.12 Other transmembrane family receptors 267
pattern recognition receptor 239 9.8.4 Tumor necrosis factor
8.6.4 Cytosolic pattern recognition superfamily receptors 267
receptor 240 9.8.5 Chemokine receptors 267
8.6.4.1 Nucleotide-binding 9.8.6 Interleukin-1 family receptors 267
oligomerization domain-like 9.9 The importance of regulation of
receptors 240 cytokine responses 267
8.6.4.2 Retinoic acid-inducible 9.10 Therapeutic potential of chicken
gene-like receptors 240 cytokines 268
8.6.5 Closing comments: general 9.10.1 Alternatives to antibiotic
considerations in pattern recognition 241 growth promoters 268
Acknowledgments 241 9.10.2 Potential use of cytokines as
References 242 vaccine adjuvants 269
9.11 Conclusion 270
9. Avian cytokines and their References 270
receptors 249
Andrew G.D. Bean and John W. Lowenthal
9.1 Introduction 249
10. Immunogenetics and the mapping
9.2 Avian cytokine and chemokine
families 250
of immunological functions 277
9.3 The interleukins 250 Susan J. Lamont, Jack C.M. Dekkers,
9.3.1 The interleukin-1 family 250 Anna Wolc and Huaijun Zhou
9.3.2 T-cell proliferative interleukins 251 10.1 Introduction 277
9.3.3 T-helper interleukins 252 10.2 Genetics and immunological traits
9.3.4 Th1 interleukins 254 in the chicken 277
9.3.5 Th2 interleukins 254 10.3 Key gene loci for immunological traits 279
9.3.6 Th1 Th2 paradigm 255 10.4 Detecting quantitative trait loci 280
9.3.7 Other Th subsets 255 10.4.1 Linkage disequilibrium 281
9.4 Other interleukins 257 10.4.2 Experimental designs to detect
9.4.1 The interleukin-10 family 257 quantitative trait loci 281
9.4.2 The interleukin-6 family 258 10.5 Statistical procedures for quantitative
9.4.3 Other interleukins 259 trait loci detection 283
9.5 The interferons 259 10.6 Strategies to use molecular data in
9.5.1 Type I interferon 260 genetic selection 285
9.5.2 Type II interferon 260 10.6.1 Marker-assisted selection 285
9.5.3 Type III interferon 260 10.6.2 Whole-genome prediction 285
9.6 Other factors 260 10.7 Systems biology 286
9.6.1 The transforming growth 10.8 Transgenic animals 288
factor-β family 260 10.9 Future directions for systems
9.6.2 The tumor necrosis factor biology in avian immunology 289
superfamily 261 Acknowledgments 290
9.6.3 Colony-stimulating factors 261 References 290
xii Contents
11. The mucosal immune system 299 11.2.7 The immune system in the
parabronchi 333
Bernd Kaspers and Karel A. Schat 11.2.8 The phagocytic system of the
References 300 respiratory tract 334
11.2.9 Handling of particles in the
11.1 The avian enteric immune respiratory tract 335
system in health and disease 303 11.2.10 The secretory IgA system in the
Adrian L. Smith, Claire Powers and Richard Beal respiratory tract 335
11.1.1 General considerations 303 11.2.11 Gene expression analysis as a tool
11.1.2 Gut structure and immune to investigate host pathogen
compartments 304 interaction 336
11.1.2.1 Chicken gut-associated References 337
lymphoid tissue structures 305
11.1.2.2 Cellular composition of the 11.3 The avian reproductive immune
avian gut-associated system 343
lymphoid tissues 306 Paul Wigley, Paul Barrow and Karel A. Schat
11.1.2.3 The enterocyte as part of 11.3.1 Introduction 343
an integrated gut immune 11.3.2 The structure and function of the
system 306 avian reproductive tract 343
11.1.3 Development of the enteric 11.3.3 Structure and development of the
immune system 307 reproductive tract-associated
11.1.3.1 Development of immune immune system in the chicken 344
responses to model antigens 309 11.3.3.1 Organization of
11.1.3.2 Immunity to enteric lymphocytes in the
pathogens 309 reproductive tract 344
11.1.3.3 Development of immunity 11.3.3.2 Distribution of macrophages
to enteric pathogens 309 and other cells 344
11.1.3.4 Maternal antibody and 11.3.4 Local and systemic changes to the
protection of the young immune system at the onset of
chick 310 sexual maturity in hens 344
11.1.4 Viral infections of the gut 310 11.3.5 The innate immune system and the
11.1.5 Bacterial infections of the gut 311 reproductive tract 346
11.1.5.1 Salmonella 311 11.3.6 The reproductive tract immune
11.1.5.2 Campylobacter 313 system in infection 346
11.1.5.3 Necrotic enteritis 314 11.3.6.1 Bacterial infections of the
11.1.6 Parasitic infections of the gut 314 reproductive tract 346
11.1.6.1 Eimeria spp 315 11.3.6.2 The immune response to
11.1.6.2 Other parasitic infections 316 Salmonella infection of the
11.1.7 Concluding remarks 317 reproductive tract 346
Acknowledgments 317 11.3.6.3 Responses to vaccination
References 317 in the reproductive tract 348
11.3.6.4 The chicken as a model-
11.2 The avian respiratory immune understanding immunity in
system 327 ovarian cancer 349
11.3.6.5 What do we need to
Sonja Härtle, Lonneke Vervelde and know—directions for
Bernd Kaspers future research? 349
11.2.1 Introduction 327 11.3.6.6 What are the functions
11.2.2 Anatomy of the respiratory tract 327 and phenotypes of the cells
11.2.3 The paraocular lymphoid tissue 329 in the reproductive tract? 349
11.2.4 Nasal-associated lymphoid tissue 330 11.3.6.7 How does the immune
11.2.5 The contribution of the trachea to tissue of the reproductive
respiratory tract immune responses 331 tract integrate with the rest
11.2.6 The bronchus-associated lymphoid of the immune system? 349
tissue 331 References 349
Contents xiii
12. Impact of the gut microbiota on 13.5.3 The allantoic sac 379
the immune system 353 13.6 Concluding remarks 380
References 380
Michael H. Kogut
12.1 Introduction to the microbiota and 14. Avian immunosuppressive
avian immune system 353
diseases and immune evasion 387
12.2 Microbiota, metagenome, and
microbiome 354 Karel A. Schat and Michael A. Skinner
12.3 GI tract and immune system of poultry 354 14.1 Introduction 387
12.3.1 Intestinal barrier system 354 14.2 Immunosuppression 387
12.4 Influence of the microbiota in 14.2.1 Introduction 387
immunity 355 14.2.2 Stress-induced
12.4.1 Germ-free chickens 355 immunosuppression 388
12.4.2 Antibiotic-treated chickens 356 14.2.3 Mycotoxin-induced
12.4.3 Fecal microbial transplants 356 immunosuppression 389
12.4.4 Layer-type chickens versus 14.2.4 Coccidia-induced
broiler chickens 356 immunosuppression 390
12.5 Gut microbiota immune system 14.2.5 Virus-induced
communication 356 immunosuppression 390
12.5.1 Components of the microbiota 357 14.3 Mechanisms of immunosuppression 399
12.5.2 Microbial metabolites 357 14.3.1 Corticosteroids and
12.5.3 Microbial epigenetic stress-induced
modifications 357 immunosuppression 399
12.6 Gut microbiota: immune homeostasis 358 14.3.2 Apoptosis, necroptosis, and
12.7 Gut microbiota: immune dysfunction: pyroptosis 399
dysbiosis and inflammation 358 14.3.3 Virus-induced changes in the
12.8 Managing the microbiome for regulation of immune responses 400
immune modulation 359 14.4 Immunoevasion 401
References 359 14.4.1 Introduction 401
14.4.2 Immunoevasion by viral
13. Innate defenses of the avian egg 365 proteases 401
14.4.3 Immunoevasion mechanisms of
Sophie Réhault-Godbert, Maxwell Hincke, avian coronaviruses 402
Rodrigo Guabiraba, Nicolas Guyot and
14.4.4 Immunoevasion mechanisms
Joel Gautron
of the avian herpesviruses 402
13.1 Introduction 365
14.4.5 Immunoevasion mechanism
13.2 Egg basic structures and their role
of the avian poxviruses 402
in innate defense 365
14.4.6 Immunoevasion mechanism
13.2.1 Physicochemical barriers 366
of the avian orthomyxoviruses 404
13.2.2 Antimicrobial molecules 371
14.4.7 Immunoevasion mechanism
13.3 Modification of egg structures during
of the avian paramyxoviruses 405
embryonic development 373
14.4.8 Immunoevasion mechanism
13.4 Embryonic immunity 374
of the avian reoviruses 405
13.4.1 Toll-like receptors 374
14.4.9 Immunoevasion mechanism
13.4.2 Macrophages 374
of the avian birnaviruses 406
13.4.3 Heterophils 374
14.5 Conclusions 406
13.4.4 Dendritic cells 375
References 406
13.4.5 T lymphocytes 375
13.4.6 Natural Killer cells 376
13.4.7 Cytokines and chemokines 376 15. Factors modulating the avian
13.5 Extraembryonic structures and innate immune system 419
immunity 376 Tina Sørensen Dalgaard, Johanna M.J. Rebel,
13.5.1 Amniotic sac 377 Cristiano Bortoluzzi and Michael H. Kogut
13.5.2 Yolk sac 378 15.1 Endocrine regulation of immunity 419
xiv Contents
21.1.5 Primordial germ cells 562 21.3 Genetically modified quails 568
21.2 Genetically modified chickens 563 References 569
21.2.1 Chicken models for
immunological research 564
Appendix 1: Genetic stocks for immunological
21.2.2 Disease-resistant chickens 567
research 573
21.2.3 Genetically engineered
Abbreviations 583
chickens for basic research
and agriculture 567 Index 589
List of contributors
James S. Adelman Department of Biological Sciences, Steven R. Fiddaman Department of Zoology, University
University of Memphis, Memphis, TN, United States of Oxford, Oxford, United Kingdom
Daniel R. Ardia Department of Biology, Franklin and Joel Gautron INRAE, University of Tours, BOA,
Marshall College, Lancaster, PA, United States Nouzilly, France
Jake Astill Department of Pathobiology, Ontario Thomas W. Göbel Department for Veterinary Sciences,
Veterinary College, University of Guelph, Guelph, Veterinary Immunology Study Group, University of
ON, Canada; Artemis Technologies Inc., Guelph, ON, Munich, Munich, Germany
Canada
Rodrigo Guabiraba INRAE, Université de Tours, ISP,
Adam Balic Division Infection and Immunity, The Nouzilly, France
Roslin Institute and R(D)SVS, University of
Nicolas Guyot INRAE, University of Tours, BOA,
Edinburgh, Midlothian, United Kingdom
Nouzilly, France
Paul Barrow The School of Veterinary Medicine and
Sonja Härtle Department for Veterinary Sciences,
Science, University of Nottingham, Sutton Bonington,
Veterinary Immunology Study Group, University of
Loughborough, United Kingdom
Munich, Munich, Germany
Richard Beal Ryedale School, Nawton, United Kingdom
Maxwell Hincke Department of Innovation in Medical
Andrew G.D. Bean CSIRO, Health & Biosecurity at the Education, University of Ottawa, Ottawa, ON,
Australian Centre for Disease Preparedness, Geelong, Canada; Department of Cellular and Molecular
VIC, Australia Medicine, University of Ottawa, Ottawa, ON, Canada;
Cristiano Bortoluzzi Southern Plains Agricultural Loire Valley Institute for Advanced Studies, Orléans
Research Center, USDA-ARS, College Station, TX, and Tours, France, with BOA–INRAE Centre Val de
United States Loire, Nouzilly, France
Tina Sørensen Dalgaard Department of Animal Science, Thierry Jaffredo Sorbonne Université, IBPS, CNRS
Aarhus University, Tjele, Denmark UMR7622, Inserm U 1156, Developmental Biology
Fred Davison Institute for Animal Health, Newbury, Laboratory, Paris, France
United Kingdom Bernd Kaspers Department for Veterinary Sciences,
J.J. (Sjaak) de Wit Royal GD, Deventer, The Veterinary Immunology Study Group, University of
Netherlands; Utrecht University, Utrecht, The Munich, Munich, Germany
Netherlands Jim Kaufman School of Biological Sciences, Institute for
Jack C.M. Dekkers Department of Animal Science, Iowa Immunology and Infection Research, Ashworth
State University, Ames, IA, United States Laboratories, University of Edinburgh, Edinburgh,
Timothy Doran Australian Animal Health Laboratory, United Kingdom; Department of Pathology, University
CSIRO Health and Biosecurity, Geelong, VIC, of Cambridge, Cambridge, United Kingdom
Australia Michael H. Kogut Southern Plains Agricultural Research
Dominique Dunon Sorbonne Université, IBPS, CNRS Center, USDA-ARS, College Station, TX, United
UMR7622, Inserm U 1156, Developmental Biology States
Laboratory, Paris, France Susan J. Lamont Department of Animal Science, Iowa
Gisela F. Erf University of Arkansas, Division of State University, Ames, IA, United States
Agriculture, Department of Poultry Science, John W. Lowenthal CSIRO, Infectious Diseases
Fayetteville, AR, United States Program, Geelong, VIC, Australia
xvii
xviii List of contributors
Katharine E. Magor Department of Biological Sciences, Adrian L. Smith Department of Zoology, Jenner
University of Alberta, Edmonton, AB, Canada Institute, Oxford, United Kingdom; University of
Enrique Montiel Anitox Corporation, Lawrenceville, GS, Oxford, Oxford, United Kingdom
United States Kate Sutton Division Infection and Immunity, The
Nándor Nagy Department of Anatomy, Histology and Roslin Institute and R(D)SVS, University of
Embryology, Faculty of Medicine, Semmelweis Edinburgh, Midlothian, United Kingdom
University, Budapest, Hungary Edwin J.A. Veldhuizen Department of Biomolecular
Venugopal Nair Viral Oncogenesis Group, Pirbright Health Sciences, Division Infectious Diseases &
Institute, Woking, United Kingdom Immunology, Faculty of Veterinary Medicine, Utrecht
University, Utrecht, The Netherlands
Imre Oláh Department of Anatomy, Histology and
Embryology, Faculty of Medicine, Semmelweis Lonneke Vervelde Division Infection and Immunity, The
University, Budapest, Hungary Roslin Institute and R(D)SVS, University of
Edinburgh, Midlothian, United Kingdom
Claire Powers University of Oxford, Oxford, United
Kingdom Michal Vinkler Department of Zoology, Faculty of
Science, Charles University, Prague, Czech
Michael J.H. Ratcliffe Department of Immunology,
Republic
University of Toronto, Toronto, ON, Canada
Paul Wigley Department of Infection and Microbiome,
Johanna M.J. Rebel Department of Animal Health and
Institute of Infection Veterinary and Ecological
Welfare, Wageningen University & Research,
Sciences, University of Liverpool, Leahurst, Neston,
Wageningen, The Netherlands
United Kingdom
Sophie Réhault-Godbert INRAE, University of Tours,
Anna Wolc Department of Animal Science, Iowa State
BOA, Nouzilly, France
University, Ames, IA, United States; Hy-Line
Catherine Robin Hubrecht Institute, Royal Netherlands International, Dallas Center, IA, United States
Academy of Arts and Sciences (KNAW), University
R. Darren Wood Department of Pathobiology, Ontario
Medical Center Utrecht, Utrecht, The Netherlands;
Veterinary College, University of Guelph, Guelph,
Regenerative Medicine Center, University Medical
ON, Canada
Center Utrecht, Utrecht, The Netherlands
Laurent Yvernogeau Hubrecht Institute, Royal
Karel A. Schat Department of Microbiology and
Netherlands Academy of Arts and Sciences (KNAW),
Immunology, College of Veterinary Medicine, Cornell
University Medical Center Utrecht, Utrecht, The
University, Ithaca, NY, United States
Netherlands; Sorbonne Université, IBPS, CNRS
Ursula Schultz CellGenix GmbH, Freiburg, Germany UMR7622, Inserm U 1156, Developmental Biology
Benjamin Schusser Reproductive Biotechnology, TUM Laboratory, Paris, France
School of Life Sciences, Freising, Germany Huaijun Zhou Department of Animal Science,
Shayan Sharif Department of Pathobiology, Ontario University of California-Davis, Davis, CA, United
Veterinary College, University of Guelph, Guelph, States
ON, Canada
Michael A. Skinner Section of Virology, Faculty of
Medicine, Imperial College London, London, United
Kingdom
Foreword
The fascinating world of avian immunology might be best lymph node development, which opens an array of
surmised by the quote from Dr. Jim Kauffman, who stated antigen-processing questions for immune recognition and
with obvious eloquence “chickens are not mice with stimulation. Birds also lack several Toll-like receptors
feathers.” The point being that while many immunologi- found in other vertebras, yet share many that recognize
cal attributes are shared between the species, many are similar molecular motifs. These are but two examples to
also different and that we as immunologists must work to highlight the differences, but there are many others.
discover them. Perhaps Winston Churchill, in 1939, was also speaking of
In the last two decades, there has been a great increase avian immunology when he said, “it is a riddle, wrapped
in our knowledge of avian immunology. Molecular immu- in a mystery, inside an enigma.”
nologists, utilizing new technologies, have expanded our It is impossible to discuss the context of this book
ability to determine and decipher what is encoded in the without acknowledging the current global COVID-19
avian genome for immunological defense. In addition, pandemic caused by the SARS-CoV-2 coronavirus. This
strategic funding capabilities provided primarily, but not relatively novel virus, without existing population immu-
exclusively, by the United States and EU have resulted in nity, has spread rapidly across transcontinental bound-
the development of a plethora of monoclonal antibodies aries. These events are reminiscent of reports in the early
against avian immunological proteins (e.g., cytokines and 1930s when chicks arrived at the veterinary laboratories
cell markers) through “toolbox” grants. These two driving of the agricultural college in Fargo, North Dakota. A new
forces have opened doors and windows in a field that has disease was raging across poultry farms in Minnesota and
historically struggled to provide reagents for intricate North Dakota resulting in sick birds that were undergoing
analysis. respiratory distress with high mortality. As researchers
This book arrives at a key time in our need for knowl- investigated the disease, it was eventually identified as a
edge to handle the ever-changing pressures for sustainable viral agent and termed infectious bronchitis virus (IBV)
solutions to poultry production issues on a global scale. based on disease characteristics. However, it was not until
The overall health of birds, in particular the immune sys- the advent of electron microscopy in the 1960s that
tem, is critical to feed an ever-growing human population. pathologists were able to get a visual look at the virus and
As an example, the loss of antibiotic treatment for poultry in 1964 identified “lollipop-shaped” protrusions at the end
flocks in most countries has created a need for immuno- of the spikes of the virus that resembled a halo of gas sur-
logical intervention strategies to enhance control against rounding the sun. The relevance of these observations is
bacterial pathogens. In addition to the applied aspect of notable in that approximately a year after these publica-
avian immunology, a comparative analysis, including tions, similar morphological characteristics were identi-
genomics, function, and evolution, is important to deter- fied in virus samples obtained from a boy undergoing
mine how and why the avian immune system works as it flu-like symptoms. Scientists utilized these morphological
does. similarities found in both avian and human samples to
The avian genome in general is regarded as condensed identify a new viral family, coronavirus.
in nature to mammalian species in terms of immune gene Parallels are also found in the immunological arena.
repertoire. Immunologically speaking, this translates in To combat the SAR-CoV-2 virus, in 2020, vaccines have
that birds have to do the same (protect the animal) with been developed and approved for use in humans. Of note
less. Despite this, the immune responses between birds is that two of the most used at this time in the United
and mammals display similar defining characteristics. States and EU are nucleic acid based on the messenger
Both species employ an innate and adaptive immune RNA sequence of the SARS spike (S) gene. The resulting
response to a variety of microbiological incursions from protein is the main coronavirus immunogen and contains
bacteria, viruses, protozoa, and parasites. However, differ- epitopes for inducing neutralizing antibodies. The rele-
ences also stand out. For example, chickens lack draining vance to avian immunology is that over 20 years ago we
xix
xx Foreword
and others reported on the development and use of DNA This book brings together highly distinguished and
vaccines expressing the S1 from IBV in chickens, which internationally representative authors who are specialists
provided protective efficacy and induced both antibodies in their respective fields of avian immunology. This book
and T-cell responses. will be valued by bench scientists, graduate students,
The future of avian immunology is a bright one. poultry veterinarians, commercial industry researchers,
Knowledge gleaned from the release of the avian genome, and avian ecologists as an “immunological-bible” for a
including conservation of synteny, has already resulted in better understanding of avian immunology.
increased scrutiny of what is and is not available for
Darrell R. Kapczynski
defense. Functional studies, such as epitope binding of
U.S. Department of Agriculture, Agricultural Research
avian MHCs, are shedding new light and possibilities for
Service, U.S. National Poultry Research Center, Athens,
enhanced immune responses. Research in disease resis-
GA, United States
tance breeding is being accelerated by the development of
transgenic technologies, including CRISPR, that allow for
manipulation and testing in a more timelier manner. As
these resources are brought to bear, past barriers in the
field of avian immunology will fall.
Acknowledgments
Gathering this wealth of information would not have been The editors also thank Dr. Sonja Härtle for her edito-
possible without the commitment, dedication, and gener- rial work on numerous chapters and Dr. Ton Schat for the
ous participation of the large number of contributors to pictures of birds on the back cover.
the third edition of this book. The editors are indebted to The editors would also like to thank Susan Ikeda for
them for the considerable amount of work, their enthusi- her efforts to get this project accomplished despite the
asm and willingness to set aside other priorities to con- problems caused by the ongoing pandemic.
tribute to this volume. The editors would like to thank Dr.
Fred Davison for his contribution as the lead editor for
the first edition of Avian Immunology, setting the stage
for the subsequent editions.
xxi
Chapter 1
1.1 Introduction the 1950s and 1960s, when lymphocyte function became
an active subject for research [1]. The immunological sig-
The avian immune system provides an invaluable model nificance of lymphocytes emanated from some seminal
for studies on basic immunology. Birds and mammals studies carried out by Gowans, Chase and Mitchison,
evolved from a common reptilian ancestor more than 250 Simonsen, and their contemporaries [1,2]. In elegant
million years ago and have inherited many common immu- experiments with laboratory mammals and using cell trans-
nological systems. They also have developed a number of fers, these workers demonstrated that lymphocytes are
very different, and in some cases remarkable, strategies. essential for generating immune responses and retaining
Due to their economic importance, and the ready availabil- memory of previous exposure to an antigen. However, evi-
ity of inbred lines, most avian immunology research has dence that lymphocytes play such a key role in protection
involved the domestic chicken, Gallus gallus domesticus. against infection and in tumor rejection had, in fact, been
A remarkable consequence of this research has been the discovered almost 40 years earlier [3 6], though little
seminal contributions it has made to understand the funda- attention was paid to it [7]. This was almost certainly
mental immunological concepts, especially the complete because at the time they were made the observations could
separation of developing bursa- (B-) and thymus- (T-) not properly be explained and, possibly, because the exper-
dependent lymphocyte lineages. Some of these observa- imental animal involved was the chicken.
tions have been made by chance, while others have Between 1912 and 1921, James Murphy, an experi-
resulted from painstaking work which took advantage of mental pathologist working at the Rockefeller Institute for
special avian features, such as ease of access to, and pre- Medical Research in New York, performed a series of
cise timing of, all the stages in embryonic development. remarkable experiments using chickens and their embryos
Some of the avian findings were described before they to study the growth and rejection of tumor grafts. His
were recognized as important and subsequently explained experiments appeared to prove beyond question that the
in mainstream immunology. The story of avian immunol- lymphocyte is the active component in tissue graft rejec-
ogy is a fascinating one and by no means complete, as tion, in protection against infection and, by implication, in
there is still the need for explanations of a number of innate and acquired immune responses [7]. Murphy [4]
unique features and different strategies adopted by birds. In observed that fragments of rat tumors would not grow in
this chapter, some of the “firsts,” rightly attributed to avian the adult chicken, just as they did not grow in other spe-
immunology, are described and the importance of further cies (xenogenic rejection). However, they could be grown
studies in avian immunology is highlighted. on the chorioallantoic membrane (CAM) of developing
chick embryos, although only up to about 18 days incuba-
1.2 The contribution from avian tion. In older embryos tumor grafts were rejected, just as
they were if grafted onto the newly hatched chick or an
lymphocytes adult bird. Interestingly, Murphy [3] observed that the
The advent of modern cellular immunology, and the funda- grafts which grew on embryos could be transferred to
mental role that lymphocytes play, is generally credited to fresh embryos without any evidence of them being
altered. They also retained their tumorigenic capacity if understood how such lymphocytes could be the same as
regrafted onto a rat. Murphy [3] commented that these those cells involved in cell-mediated functions. The bursa
cellular changes occurring when living tissue is grafted of Fabricius, an obscure sac-like structure attached to the
onto an unsuitable host are the same, regardless of the proctodeal region of the bird’s cloaca (Fig. 1.1), played a
type of host resistance, be it natural resistance because of crucial role in unraveling this problem.
species differences (allogeneic or xenogeneic rejection) or The cloacal bursa, takes its name from Hieronymus
acquired immunity due to recovery from a tumor Fabricius of Aquapendente (1537 1619), is also known
implanted earlier. The histological picture consisted of as Girolamo Fabrizi d’ Acquapendente (Fig. 1.2). He was
edema surrounded by fibroplasia in the host tissue, the a professor of surgery at the University of Padua, Italy,
budding out of blood vessels, and infiltration of surround- from 1565 to 1613 [9] and by all accounts a brilliant anat-
ing host tissues with small lymphocytic cells. The omist, embryologist, and teacher. For his pioneering
inevitable consequence was that cells in the graft died work, he was later credited in Italian medical science as
fairly quickly leaving only a scar. These were quite pro- the “Father of Embryology.” Fabricius not only carried
found though, at the time, unappreciated observations. out dissections on human cadavers but also extended his
Murphy [4] also performed a series of elegant experi- anatomical studies to other species, providing the most
ments using adult chicken tissues cografted onto the beautiful, detailed drawings of his work. From his obser-
CAM with fragments of rat tumors. He observed that vations of avian anatomy, he surmised that the cloacal
chicken tissues containing an abundant supply of lympho- bursa, a hollow structure connected by a duct to the proc-
cytes, such as the spleen or bone marrow, caused tumor todeal region of the cloaca, most likely acts as a recepta-
grafts to be rejected, whereas these were not rejected if cle for storing donated semen.
the cografted tissue lacked a rich supply of lymphocytes.
“Since the sac is pervious, so that there is an open passage
Later on, Murphy [5] showed that after grafting fragments
from the anus to the uterus itself and another from the
of adult chicken spleen onto the CAM of a 7-day embryo,
uterus to the sac, that is, above and below, and since it is
the embryo’s own spleen became grossly enlarged
closed at the other end, I think it is the place into which the
(splenomegaly). This is the first published record of a
cock introduces and delivers semen so that it may be stored
graft versus host response (GvHR). Much later, it was
there” [4].
explained by Simonsen [8] as the immunologically com-
petent lymphocytes of the adult responding to mismatched This is not the case, however, but the role of the
major histocompatibility complex (MHC) molecules cloacal bursa continued to puzzle researchers over the fol-
expressed by the embryonic cells. The embryonic cells lowing 350 years. Some surmised that since the bursa
were recognized as foreign causing the adult cells to repli- of Fabricius regresses with sexual maturity, its size
cate and destroy the embryonic host’s lymphocytes. Graft having an inverse relationship with the size testes and
versus host disease, in which allogeneic bone marrow the adrenals, it must be some sort of endocrine or lym-
transplants recognize the tissues of the graft recipient and phoid gland associated with growth and sexual develop-
cause severe inflammatory disease, is a serious problem ment [10].
for immunosuppressed recipients receiving bone marrow Over the years many investigated the function(s) of the
transplants. The phenomenon became a major concern bursa including one young researcher, Bruce Glick, work-
with the introduction of human bone marrow transplanta- ing at the Poultry Science Department at Ohio State
tion. Nonetheless, the phenomenon was first described University, United States. Glick surgically removed the
using chick embryos as long ago as 1916. bursa from young chicks to investigate the effect on
growth. By chance, after one experiment was concluded, a
colleague, Timothy Chang, asked if he could use some of
1.3 Contribution of the bursa of Fabricius the birds for a class demonstration on antibody production.
Without doubt, the most significant contribution that stud- A group of the chickens was injected with Salmonella
ies on the avian system have made to development of spp. “O” antigen but 1 week later, when the class carried
mainstream immunology was delineating the two major out tests with blood and antigen, there was no evidence of
arms of the adaptive immune system. As already pointed agglutination. Chang, somewhat perplexed, reported the
out, in the 1960 the significance of lymphocytes was just failure to Glick who was able to identify the nonresponder
becoming appreciated, and it was generally accepted that chickens as those that had been bursectomized. At the time
there are two types of adaptive immune responses: they did not seem to fully appreciate the singular impor-
humoral responses involving antibodies and cellular tance of this finding [10] but were able to confirm their ini-
responses mediated by macrophages and lymphocytes. tial observations in further experiments and wrote up an
Since antibodies are produced by plasma cells, which article entitled: “The role of the bursa of Fabricius in anti-
themselves are derived from lymphocytes, it was not body production.” This was submitted to Science but
The importance of the avian immune system and its unique features Chapter | 1 3
rejected on the grounds that further elucidation of the published their seminal paper on the delineation of the bur-
mechanisms was necessary before the article could be sal (B) and thymic (T) lymphoid systems in the chicken.
accepted for publication [10]. The article was subsequently These workers proposed that because of the similarities in
submitted to Poultry Science [11], where, for a time, it the lymphoid tissues and immune systems of birds and
failed to draw much attention. Several years passed before mammals, a mammalian equivalent for the bursa of
the significance of the work was properly appreciated and Fabricius must exist and provide a source of B-dependent
mainstream immunologists took an interest in the chicken’s lymphocytes to make antibodies. Later this bursa equiva-
immune system. It was eventually concluded that the avian lent was identified as bone marrow. The division of the
bursa must be essential for antibody-mediated immunity, adaptive immune system into B- and T-dependent compart-
whereas the thymus, which also undergoes involution dur- ments has remained a central tenet of immunological think-
ing sexual development, is necessary for cell-mediated ing ever since. The term B-lymphocyte is derived from
immunity [12 14]. Almost a decade after Glick and “bursa-derived lymphocyte” in honor of that peculiar avian
Chang’s initial observations [11], Cooper et al. [15] lymphoid structure which provided the original evidence.
4 Avian Immunology
express IgM on the surface (see Chapter 4). Within the evolved a number of methods for recognizing and
bursa, they undergo rapid rounds of cell division and only destroying cells with intracellular pathogens. These
within this unique environment gene conversion occurs. mechanisms allow the host’s effector lymphocytes to rec-
In the absence of the bursa, an antibody repertoire cannot ognize infected or neoplastic cells through the expression
be generated, and a major arm of the immune system of proteins that have been proteolyzed within the cell and
becomes nonfunctional. The chicken antibody repertoire are expressed on the cell surface as peptide fragments. In
is generated during the late embryonic stage and for a mammals, the molecules that present peptides on the sur-
short period after hatching. As the chick ages, its B cells face of cells are encoded by a highly polymorphic genetic
undergo additional rounds of somatic gene conversion region known as the MHC.
and the antibody repertoire becomes expanded until a The MHC region was originally recognized through
mature repertoire is achieved around 5 7 weeks when the its effects on tissue graft rejection and, of course, GvHR.
bursa is fully mature. Thereafter, the bursa begins to Two major types or classes of MHC molecules are
regress as sexual maturity approaches and the adult proba- encoded by genes in the mammalian MHC region and
bly relies on postbursal stem cells in the bone marrow as expressed on the surface of cells, class I and II MHC
the source of B cells. molecules (more fully described in Chapter 7). Although
Of course, generating the antibody repertoire in a burst both types of molecules are heterodimers: the class I
of activity in the young animal has its risks. Any pathogen MHC molecule consists of an alpha-chain encoded by the
that targets and destroys bursal cells will have a devastat- MHC together with an invariant β2-microglobulin mole-
ing effect on antibody-dependent immune responses. One cule from a gene outside of the MHC locus; the class II
such virus is the small RNA virus that causes infectious MHC molecule consists of two peptide chains (α and β)
bursal disease. Infection of the neonate chick with infec- whose genes are found in the MHC region. Unlike the
tious bursal disease virus (IBDV) may cause no clinical MHC class I heterodimer that is present on most types of
disease but destroys bursal B cells leaving the chick inca- cells, expression of class II MHC molecules is restricted
pable of mounting antibody responses to other pathogens, to antigen-presenting cells such as macrophages, den-
although paradoxically there is a good response to IBDV dritic, and B cells. During synthesis inside the cell, both
itself (see Chapter 14). The insidious nature of IBDV classes of MHC molecules trap peptide fragments in a
leaves the chick vulnerable to opportunistic infections and cleft on, what is to become, the outer surface of the extra-
unprotected by subsequent vaccinations. So, relying on cellular domain of the MHC heterodimer. On reaching the
the generation of the antibody repertoire in a single loca- cell surface, these peptides are displayed as peptide:MHC
tion and over a relatively short time span is not without complexes to signal T cells through their T-cell receptors.
danger and, perhaps, represents one of the more “risky” Since even the smallest virus produces a number of pro-
strategies birds have adopted. teins, and large pathogens can produce hundreds, there
needs to be a large repertoire of T-cell receptors
expressed by different T cells to recognize the multiplic-
1.4 The contribution of the chicken MHC ity of peptide fragments derived, not from the host’s own
Pathogens are diverse, cunning, and occupy different cells, but made by pathogens or neoplastic cells. This T-
niches within the body of the host. Apart from pathogens cell repertoire is developed within the thymus (described
that are found outside of cells, such as Clostridium, in Chapter 6), where developing T cells with receptors
Escherichia coli, and Bordatella, there are intracellular that recognize self-peptides associated with MHC mole-
pathogens that can be found in the cytoplasm or cellular cules are eliminated, or inactivated, before they mature to
vesicles. In addition, retroviruses and herpesviruses inte- prevent self-recognition and auto-immunity. Mature T
grate into the host’s own genome. To match this diversity, cells capable of recognizing “foreign” peptides are
and the different locations where pathogens are found, released from the thymus into the periphery and become
higher vertebrates have evolved a number of different activated if they recognize peptide fragments expressed
innate and adaptive immunological mechanisms to on MHC molecules. Interestingly, the T-cell repertoire in
improve the chances of survival. Antibodies recognize the chicken is developed in the thymus in a similar way
conformational epitopes on the pathogen’s molecules but to that of mammals. There is no evidence for a somatic
need to come into direct physical contact to neutralize a gene conversion mechanism such as occurs in the avian
pathogen, as well as recruit cells and other molecules that bursa.
bring about disposal. Ig molecules are large and cannot In mammals, the MHC is a large and complex region
easily enter a viable cell, so an intracellular pathogen can- that contains much redundancy [21]. In the human, it con-
not be recognized by an Ig molecule unless the patho- sists of about 4 million base pairs encoding at least 280
gen’s molecules are expressed on the surface of that genes. Separate regions contain several MHC class I and
infected cells. However, the cellular immune system has class II genes (there are a vast number of alleles) that are
6 Avian Immunology
highly expressed on cells. These regions are separated by heavily on vaccines to protect against a wide range of dif-
the third region that encodes immune response genes ferent infectious agents. Vaccinations are frequent and
(class III). Humans express two or three class I molecules begin from the day of hatching or even before. Chickens
and three or four class II molecules that are highly poly- are immunized with live-attenuated vaccines and killed
morphic. The high polymorphism is probably driven by vaccines delivered by various routes (injection, aerosol
the ever-changing variations in pathogens [22,23], spray, drinking water, etc.) in mass-vaccination programs
although different haplotypes appear to confer approxi- that dwarf such programs in human medicine.
mately the same degree of protection against most of the Every biology student knows that Edward Jenner is
infectious pathogens. In Chapter 7, Kaufman points out the founding father of vaccination. Jenner discovered that
that the associations between the human MHC and infec- cowpox pustules, obtained from an infected milkmaid,
tious disease are, actually, very slight. protected an 8-year-old boy, James Phipps, against the
The chicken MHC is known as the B locus since it related smallpox virus. However, further developments in
was first identified as a serological blood group locus [24] vaccination, and indeed the term vaccination, only came
encoding the polymorphic, and highly immunogenic BG into use about a century later, following studies by one of
antigen. This BG antigen is highly expressed on blood the greatest 19th-century scientists, Louis Pasteur. Here
cells and has no known mammalian equivalent. Later, it again the chicken had a privileged role and serendipity
was shown that that the B locus must constitute the avian played its part.
MHC, because of its strong association with cell- In 1878 Pasteur was investigating chicken cholera, a
mediated immune functions, such as graft rejection, disease with devastating effects, causing chickens to
mixed lymphocyte reactions, and GvHR. Remarkably, become anorexic, moribund, and usually leading to their
and in marked contrast to the large mammal MHC, the death. Pasteur investigated the causative agent, now
chicken B locus is minute, spanning only 92 kilobases known as the Pasteurella multocida, and succeeded in
and encoding 19 genes and making it approximately 20- growing the bacteria in culture. The story goes that
fold smaller than the human MHC [25]. Only two copies Pasteur’s research was interrupted by a holiday and a cul-
each of class I (BF) and class IIβ (B/Lβ) genes are found ture was left in a flask in the laboratory [29]. Upon
within the chicken B locus. The marked differences resuming his research, Pasteur inoculated chickens with
between the chicken MHC and its mammalian counterpart this stale culture. The chickens became sick but then
have led Jim Kaufman to argue that the chicken B locus recovered within a few days. We now know that the bac-
represents a minimal essential MHC that must have teria had become attenuated and no longer capable of
evolved after birds and mammals separated some 200 mil- causing mortality. Pasteur did not know this and decided
lion years ago. Another striking feature of the chicken to inject new chickens with a fresh bacterial culture but,
minimal MHC region is that not only does it affect a unfortunately (or fortunately!), his assistant found that
number of important cell-mediated immune functions but chickens at the local market were in short supply. A num-
also determines life or death in response to a number of ber of fresh birds were obtained but those that had recov-
pathogens [26 28]. In Chapter 7, Jim Kaufman develops ered from the inoculation with the stale culture needed to
the argument that chickens, with a minimal essential be reused. As expected, the new chickens all succumbed
MHC, appear to have adopted a completely different to the fresh pathogen and died but those that had recov-
strategy to that of mammals whose MHC is large and ered from the previous treatment with stale inoculum
complex. The close association between the chicken again recovered. Pasteur realized he had achieved with
MHC and disease resistance is fascinating and at first chicken cholera what Jenner had accomplished with
sight seems to be a suicidal strategy. However, many les- smallpox some 100 years earlier, only in this case he had
sons can be learned from studying avian immunology and attenuated (weakened) the pathogen by prolonged storage.
its parallel evolution. By investigating the minimal He called the attenuated culture a “vaccine” [30] in honor
chicken MHC, light should be thrown on the important of Edward Jenner and then began a, largely successful,
interactions between pathogens and the immune system search for similar vaccines against other infectious dis-
and the relevance of the different evolutionary strategies eases such as pig erysipelas, sheep anthrax, and rabies.
elucidated. The serendipitous discovery of attenuation was another
novel finding made using the chicken. Since then, the
development of vaccines has had far-reaching implica-
1.5 Contributions to vaccinology tions for the health and welfare of both humans and
In any review of the “firsts” credited to avian immunol- domesticated animals. The search for better, more effec-
ogy, tribute needs to be paid to pioneering developments tive vaccines still goes on apace.
in the practical uses of immunology, in other words the Another first in vaccine development was in the con-
use of vaccination. The modern poultry industry relies trol of Marek’s disease (MD) a naturally occurring
The importance of the avian immune system and its unique features Chapter | 1 7
neoplastic disease of chickens. MD became the scourge pressure of vaccine use. The risk, however, is that these
of the poultry industry in the 1950s and 1960s, causing hotter vaccines could themselves be capable of causing
major problems for animal health and welfare and becom- bursal damage and immunosuppression in chicks that are
ing a huge financial burden. Before the introduction of poorly protected by maternal antibodies or have a suscep-
MD vaccines, morbidity and mortality in laying flocks tible genotype. Here again, we have an example of a strat-
ranged from 0% 60% or greater, with losses of 30% egy that is holding at present but may not be sustainable
being common [31]. The development of an MD vaccine long term. More aggressive vaccines or vaccination
represents the first example of widespread use of vaccina- regimes cannot be introduced without the risk that the
tion to protect against a natural form of cancer [32]. Over vaccines themselves could be harmful.
the years it has been remarkably effective [33], although These issues have been addressed in epidemiological
not without problems. studies on implications of the use of vaccines on the evo-
MD was first described as a neurological disease lution of pathogen virulence. Researchers [38] were
(polyneuritis) by Josef Marek [34]. The condition caused chiefly concerned with the use of different vaccines
paralysis of the wings and legs and was associated with developed against the malaria parasite and its implications
mononuclear infiltrations and enlargement of the major for human populations. Mathematical modeling was car-
nerves. Later it was observed [35,36] that in addition to ried out based on the premise that most vaccines are
lesions in the nerves and central nervous system, chickens imperfect and rarely provide full protection from disease.
also developed lymphoid tumors in several visceral tis- Using various models, these authors predicted that vac-
sues (visceral lymphomatosis) such as the ovary, liver, cines designed to reduce the growth and/or toxicity of
kidneys, adrenal, and muscles. With intensification of pathogens actually diminished selection pressure against
poultry production, the acute form of MD became a domi- more virulent pathogens. Consequently, subsequent patho-
nant feature. Although the introduction of MD vaccines in gen evolution may lead to higher levels of intrinsic viru-
the 1970s controlled the disease, in some countries pro- lence and hence more severe disease in unvaccinated
blems with vaccine breaks have continued to occur with individuals. Such evolution would tend to erode any
regularity and there is now good evidence that the causa- population-wide benefits, such that overall mortality
tive herpesvirus (MDV) has been able to evade vaccine- could increase with the level of vaccination coverage.
induced immune responses by evolving to greater viru- Interestingly, the authors found evidence of this phenome-
lence. Since the 1980s the response of the industry in non in the practical problems arising from MD vaccina-
some countries has been to introduce more aggressive tion. Current MD vaccines target viral replication and do
vaccine strategies, using “hotter” vaccines, such as not prevent infection with MDV, consistent with mathe-
CVI988, either alone or in combination with other MD matical modeling that predicts the evolution of pathogen
vaccines (bivalent or trivalent combinations). The most virulence. Yet again, evidence from work on the chicken
efficacious current MD vaccine CVI988 is derived from a has proved to be the pathfinder and pointed to important
serotype 1 MDV that is weakly oncogenic in genetically problems to take account of in vaccine design.
susceptible chickens and this has led some to raise the
important question [37]: where do we go if hypervirulent
1.5.1 Embryonic (in ovo) vaccination
MDV pathotypes evolve that can break through the pro-
tection of CVI988? The problems associated with challenge from virulent
MDV is not the only example of a poultry virus that MDV when chicks are moved into rearing quarters, the
has changed in response to the introduction of widespread vast numbers of chicks requiring vaccinations at the
use of vaccines. More virulent isolates of another lympho- hatchery, as well as the occasional failures caused by
tropic virus, IBDV, were isolated in the late 1980s. IBDV manual vaccination, has led to a search for new ways for
is a small double-stranded RNA virus that encodes only mass vaccination that can be done at an even earlier stage
five viral proteins. As already pointed out, IBDV targets than the day-old chick. Sharma and colleagues at the East
B lymphocytes in the bursa of young chicks causing no Lansing Regional Poultry Laboratory, United States dem-
clinical signs in neonates but causing clinical disease and onstrated that chick embryos could be successfully vacci-
some mortality in older chicks (see earlier). Chicks are nated against MDV at 17 18 days incubation [39,40].
protected by maternal antibodies derived via the yolk, but The automated INOVOJECT system, which allows the
it became clear in the late 1980s that the very virulent automated mass application of vaccines to large numbers
IBDV being isolated from outbreaks was capable of caus- of eggs (up to 50,000 eggs per hour, [41]), was developed
ing disease in the presence of high levels of maternal anti- and has been widely applied in the poultry industries of
bodies. The response of the industry has been to introduce some countries (for a fuller description see Chapter 18).
more aggressive (hotter) vaccines to protect against the In the United States, almost all broilers (over 7 billion per
more virulent IBDV strains that have evolved under the year) are vaccinated by this method. In ovo vaccination is
8 Avian Immunology
achieved by puncturing a small hole through the blunt [7] Silverstein AM. The lymphocyte in immunology: from James B.
end of the egg with an oblique pointed needle then pass- Murphy to James L. Gowans. Nat Immunol 2001;2:569571.
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[9] Adelmann HB. The embryological treatises of Hieronymus
then taken up by the embryo. Interestingly, the reasons
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1.6 Conclusion immune response in the chicken. Nature 1962;196:784785.
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(neuro-lymphomatosis gallinarum). Storrs Agric Exp Stn Bull and comparative analysis of the chicken genome provide unique
1926;143:186290. perspectives on vertebrate evolution. Nature 2004;432:695716.
Chapter 2
interactions are prevented in vivo because of compart- corticomedullary border, leaving the medulla undivided
mentalization. Appreciation of the detailed structure of an (Fig. 2.1A and B and Fig. 1.1). Arteries travel in the sep-
organ is essential for complete understanding of its tum, entering the thymic parenchyma at the end. Besides
functioning. the arteries, capillaries, veins, and efferent lymphatics are
The development of secondary lymphoid organs
begins when T and B cells intermingle and then separate
into distinct T and B cell areas, followed by the induction
of GCs in T cell areas. With separation into T and B cell
areas, nonlymphoid cell populations develop at their char-
acteristic sites.
Nonlymphoid cells can be classified into two groups.
The first consists of epithelial cells, endothelial cells and
connective tissue cells, including reticular cells.
Endothelial cells form the inner lining of arteries, veins
and capillaries, and lymphocytes cross this endothelium
to enter lymphoid tissue. Specialized vessels with high
endothelial cells that bulge into the lumen are the so-
called HEV, which facilitate the entry of lymphocytes
using specific adhesion molecules that interact with
tissue-specific homing receptors on lymphocytes. These
endothelial cells have a high number of mitochondria and
ribosomes, suggesting an increased state of activity which
may be related to their function, transporting both cells
and antigens. Reticular cells and their extracellular matrix
form the reticulum, the basic framework of lymphoid tis-
sues. Little is known about the reticular cells, but they
could have direct involvement in the regulation of
immune functions by guiding the migration and anchor-
age of lymphocytes to their respective compartments.
Reticular cells contain glycoproteins for which many lym-
phocytes express adhesion molecules.
The second group consists of macrophages [8] and
antigen-presenting dendritic cells (DC), which belong to
the mononuclear phagocyte system. This system includes
all cells derived from monoblasts in the bone marrow.
This group of cells is described in detail in Chapter 8.1.
also found. Septae have a rich cellular composition con- which exhibit variations in shape and electron density
sisting of fibroblasts, plasma cells, lymphocytes, and, (Fig. 2.1D). The ends of the cytoplasmic processes con-
occasionally, a few granulocytes. The surface of the tain moderately developed smooth- and rough-surfaced
lobules is isolated from the capsule and septae by a basal endoplasmic reticulum. DEC205 (CD205) is expressed by
lamina (BL). The connective tissue of the thymic capsule the cytokeratin1 reticular epithelial cells of the cortex.
and septae develop from the cells of the cranial neural Their relationship to the cortical thymocytes is a remark-
crest. Lack, or impaired migration, of neural crest cells to able cytological phenomenon. Between each epithelial
the third and fourth brachial arches results in a thymic cell and thymocyte, several adhesion points occur, indi-
condition such as Di George syndrome in humans and cating that cell-to-cell contact may be significant in T-cell
nude mice. maturation (Fig. 2.1D), which suggests a nursing function
During embryonic development, the thymic mass for ERC [15]. Several granules contain very loose, low-
gradually increases with the colonization of hematopoietic density material, suggesting partial release of their granu-
stem cells, and this rapid increase, a few days before lar contents (Fig. 2.1D) and thus giving the appearance of
hatching, results in the appearance of the medulla. During vesicles. This type of cell occurs exclusively in the cortex.
the formation of the thymus, invasion of the connective Macrophages, identified by the CVI-ChNL-74.2 and
tissue septae results in cortical lobulation, which coin- KUL01 (MRC1L-B) monoclonal antibodies [16], are scat-
cides with the emigration of T cells from the thymus. tered over the cortex and medulla (Fig. 2.2F). The cortex
The thymic reticulum develops from the endoderm of is loaded with CD81 cells, while the medulla has far
the third and fourth branchial pouches [12]. In the epi- fewer CD8α1cells (Fig. 2.2D).
thelial anlage of the thymus, immature T lymphocytes
proliferate in the subcapsular zone, from where they
2.2.3 Thymic medulla
migrate toward the medulla during T-cell maturation.
Immunologically competent cells leave the thymus via The large number of epithelial and highly variable
medullary postcapillaries. epithelial-like cells and the smaller number of thymocytes
in the medulla result in less basophilic staining
(Fig. 2.1A). Epithelial cells can be classified as several
2.2.2 Thymic cortex
types, but it is not known whether they represent actual
Pale-stained, fine cytokeratin meshes of the epithelial cell types or stages of differentiation. The functional sig-
reticular cells (ERC) in the cortex are densely packed nificance of epithelial cells is not known, but an endo-
with thymocytes, contributing to cortical basophilia crine function has been suggested [17,18]. The two
(Fig. 2.1A and B). Moderate numbers of macrophages medullary regions can be distinguished in sections stained
also occur in the network of cortical epithelial cells. with anticytokeratin (Fig. 2.2A). Over the medulla, rough
Large- and medium-sized lymphocytes are situated under cytokeratin positive “cords” form a 3D network, which
the surface epithelial cells, which may proliferate, sug- initiates at the corticomedullary border joining to the fine
gesting that the subcapsular zone of the cortex is the cortical cytokeratin1 meshwork. The larger part of the
major site of proliferation. During T-cell maturation, the medulla among the cords is keratin-free and shows direct
cells migrate toward the corticomedullary border, where connection with the connective tissue of the interlobular
macrophages and thymic DC sentinel and select the thy- septae. This may indicate a common developmental origin
mocytes before these enter the medulla and circulation for the medulla and the connective tissue of the thymic
via the medullary postcapillaries. capsule and interlobular septae. The blood vessels of the
The fine cytokeratin network of the cortex is formed medulla locate in the keratin-free regions, and around the
by the cortical ERC (Fig. 2.1C). Two types of ERC can postcapillaries of the medulla, the CVI-ChNL-74.31 DC
be identified, though it is difficult to distinguish between are localized (Figs. 2.1B and 2.2C) [13].
them cytologically. One is located on the surface of the The classical histological structure in the thymic
lobe and contains few, or no, cytoplasmic granules, but medulla is Hassall’s corpuscle, an epithelial cell aggrega-
expresses more cytokeratin than cortical ERC. Possibly, tion (Fig. 2.2B). In chickens, Hassall’s corpuscles are
its major function is to isolate the cortical parenchyma small, poorly developed structures, unlike their human
from the mesenchyme [13,14], which surrounds the thy- counterparts. Their origin remains an enigma, although it
mic lobe (Fig. 2.1B) and participates in the cortical blood- has been proposed that they are the result of the turnover
thymus barrier. These cells are connected by of epithelial cells since the center frequently shows kerati-
desmosomes to one another and to the cortical ERC. The nization like that of the skin epidermis. Some regard
latter cells have large, irregular-shaped nuclei with evenly Hassall’s corpuscles as a place of thymocyte degenera-
dispersed chromatin substances (Fig. 2.1C). The cyto- tion, though thymocytes are rarely observed in them,
plasm is rich in mitochondria and loaded with granules, unlike neutrophils (mammals) or heterophils (birds).
14 Avian Immunology
These granulocytes are probably scavenger cells, elimi- pattern of the CD81 and CD41 cells of the thymus are
nating Hassall’s corpuscles. Over the past quarter-century, shown in Fig. 2.2D and E.
several researchers have reported the production of Skeletal muscle cells are a common feature in the thy-
biologically active substances, such as alpha- mic medulla (Fig. 2.3A). Some are round or ovoid in
naphthylesterase and leucinaminopeptidase [19], interleu- shape with striated myofibrils encircling the nucleus [27].
kin (IL)-7 [20], transforming growth factor-α [21], CD30 Others are elongated with their ends appear Y-shaped.
ligand [22] and an IL-7-like cytokine called thymic stro- There are no signs of innervation to these skeletal
mal lymphopoietin (TSLP) [23,24]. TSLP is produced by muscles.
Hassall’s corpuscles in the human thymus, and TSLP
receptors are preferentially expressed by immature medul- Thymic corticomedullary border
lary myeloid DC. TSLP activates myeloid DC, which The corticomedullary border contains a DC barrier which
generates regulatory T cells in the thymic medulla [24]. expresses the major histocompatibility class (MHC) II
The high-affinity receptor for TSLP and the IL-7 receptor antigen and may play a role in the negative selection of T
(IL-7R) share the IL-7Rα chain. The chicken IL-7Rα cells [28]. However, our observations, based on immuno-
chain is expressed on many cortical thymocytes (less so cytochemistry with anti-MHC II and CVI-ChNL-74.3,
toward the capsule) and some medullary cells, suggesting indicate that the thymic DC accumulate in the
a T cell maturation-dependent expression [25] similar to cytokeratin-free medullary region (Fig. 2.2C) [13]. The
that of the mammalian thymus [26]. The location and CVI-ChNL-74.31 thymic DC coexpress CD83 [14,29]. In
Structure of the avian lymphoid system Chapter | 2 15
addition to thymic DC, a large number of peroxidase- and the bursal lumen. As a diverticulum of the cloaca, the
positive cells occur at the corticomedullary border and bursa is lined with a cylindrical epithelium thought to be
may also contribute to the negative selection of T cells of endodermal origin; however, a recent experimental
[30]. These endogenous peroxidase-positive cells gener- study proposed an ectodermal origin [31]. The bursa
ally form groups, and single cells occur between them reaches its maximum size at 8 10 weeks of age; then,
(Fig. 2.3B). Their irregularly shaped granules show granu- like the thymus, it undergoes involution. By 6 7 months,
lolysis, and the cell surface is highly ruffled (Fig. 2.3C). most bursae are heavily involuted [11].
Peroxidase-positive cells do not have segmented nuclei, The bursa, like other hollow organs, is surrounded by
and the ultrastructure of their cytoplasmic granules is not a thick, smooth muscle layer. Studies generally neglect
comparable to that of eosinophilic granulocytes. this muscle coat, and its contractility is not considered in
Granulocyte-specific mAbs (Grl-1 and Grl-2) do not rec- bursal function. During muscle contraction, compression
ognize these thymic peroxidase-positive cells. of the follicles can promote the flow of cells within the
medulla and contribute to the emptying of lymphatics sit-
uated in the axis of the folds. In the bursal lumen, 15 20
2.3 The bursa of Fabricius longitudinal folds emerge, resulting in a slit-like space.
During muscle contraction, the surfaces of the folds come
2.3.1 Anatomy and histology into contact with one another, so the bursal lumen is
The chicken bursa of Fabricius has the shape and size of almost a virtual space. Inside each fold, follicles are orga-
a chestnut and is located between the cloaca and the nized into two layers separated by axial structures (arter-
sacrum (Fig. 1.1). A slot-like bursal duct provides a con- ies, veins, lymphatics, and connective tissues) (Fig. 2.4).
tinuous and free communication between the proctodeum Consequently, follicles are in contact with blood and
16 Avian Immunology
FIGURE 2.4 The structure of the bursa of Fabricius. This schematic of the histological organization of the bursa of Fabricius shows the corticome-
dullary epithelium (CME) (shaded) in the medulla; the follicle-associated epithelium supportive cell (FAE-SC); the epithelial reticular cell (ERC); the
bursal secretory dendritic cell (BSDC); the interfollicular epithelium (IFE). BL, basal lamina; Ly, lymphocyte; MØ, macrophage; M, medulla; C, cor-
tex; MRC, mesenchymal reticular cell in the cortex.
lymphatic vessels as well as the bursal lumen. In one of zone of the FAE cells may contribute to bidirectional
the ventral folds, peripheral (secondary) lymphoid tissue transport by these cells (as proposed by Bockman and
can form. Cooper [32]). The luminal or apical surface of the FAE,
unlike that of the IFE, is adhesive because Salmonella can
enter the bursal lumen [36] and attach exclusively to the
2.3.2 Bursal surface epithelium CSF1R2 FAE (Fig. 2.5D). Antigen or particles can gain
The surface epithelium of each fold consists of an inter- access to the medulla from the bursal lumen [33,37,38],
follicular epithelium (IFE) and a follicle-associated epi- and in the opposite direction products of bursal secretory
thelium (FAE) [32] that form about 90% and 10% of the DC (BSDC) are also taken up by the FAE [34,38]. During
surface, respectively (Fig. 2.5A) [33]. IFE is columnar late embryonic life, such products appear in large quanti-
and produces a mucin-like substance, which is released ties in the medulla and enter the FAE’s intercellular
into the bursal lumen and lubricates the surface of the space. FAE cells take up and possibly secrete these pro-
folds. The columnar epithelium is pseudostratified, since ducts into the bursal lumen [39]. Some FAE cells express
there is a layer of cuboidal basal cells with densely TIM4, CD44, and nonspecific esterase, like macrophages,
stained cytoplasm. Their function is not known. Perhaps which suggests their extrabursal origin. Interestingly,
these cells are predestined for the IFE, since the surface these cells do not stain with typical monocyte/macrophage
epithelium does not proliferate and no epithelial stem cell markers, like CVI-ChNL-68.1, CVI-ChNL-68.2, CVI-
has been identified. The IFE rests on a basement mem- ChNL-74.2) or KUL01 (mannose receptor MRC1L-B),
brane (BL), continuous with the corticomedullary border and may represent senescent BSDC that have migrated
separating the medulla from the cortex (Figs. 2.4 and from the medulla [40]. Anti-pan-cytokeratin staining indi-
2.5B). cates the reduced level or absence of keratin-intermediate
FAE covers the bursal fold above the follicles and pro- filaments in the FAE, although each epithelial medullary
vides a direct connection between the follicular medulla component expresses cytokeratin (Fig. 2.5E).
and the bursal lumen (Figs. 2.4 and 2.5B). Gamma-actin The FAE is an attachment point between the follicle
recognized by GIIF3 monoclonal antibody (Fig. 2.5C) is and the surface epithelium, so the number of FAE attach-
expressed by guinea fowl bursa of Fabricius FAE [34,35]. ments is identical to the number of follicles (Figs. 2.4 and
Using CSF1R-transgenic chickens, Balic et al. showed 2.5F). Under each FAE, the BL is absent and (unusual for
that FAE comprised a mixture of CSF1R1 epithelial cells an epithelium) replaced by 2 3 layers of squamous epi-
(Fig. 2.5C’) and CSF1R2 macrophage-like cells expres- thelial cells, known as FAE-supportive cells (Figs. 2.4
sing phosphatidylserine receptor T-cell immunoglobulin and 2.5D). The FAE cells are connected to the FAE-
and mucin domain-containing 4 antigen (TIM4) [36]. A supportive cells by desmosomes. The flat FAE-supportive
special smooth-surfaced vesicular system in the apical cells, together with the corticomedullary epithelial cells,
Structure of the avian lymphoid system Chapter | 2 17
envelop the medulla, separating it from the FAE and cor- providing a means to count the number of follicles, which
tex, respectively. The FAE is devoid of lymphocytes is estimated to be 20,000 per bursa. During folliculogen-
(Fig. 2.5D). However, because classical M cells can stim- esis, there is no cell migration between follicles.
ulate lymphocytes, the absence of any lymphocytes in the Therefore assuming a minimum of 1 B cell precursor per
FAE questions whether these FAE cells are in fact classi- follicle, this means a minimum of 20,000 pre-B cells are
cal M cells. required for bursal colonization [33].
Soluble substances dropped onto the cloacal lips are
rapidly sucked into the bursal lumen [33,36,38,41,42].
2.3.3 Bursal follicle
The mechanism of this suction is not known, although it
may be due to negative pressure within the bursal lumen. Each bursal follicle consists of a medulla and a cortex
The bursal duct is flat, not circular, and therefore gener- with a closely associated (both structurally and function-
ally closed. Absorption of the air from the bursal lumen ally) but not integral FAE [32]. During bursal ontogeny,
can result in negative pressure, with contraction of the the medullary anlage emerges on embryonic day 11 12
cloacal sphincter opening up the bursal duct. By this (E11 12) and is soon followed by FAE formation
mechanism, bacteria, viruses, and tracers can gain access (E14 15), with the first cortical cells appearing around
into the bursal lumen, to be absorbed by the FAE hatch [43 45]. The cortex is fully developed by 2 weeks
(Fig. 2.5D and F). The FAE takes up colloidal carbon, post-hatch (see Chapter 3).
18 Avian Immunology
Bursal folds are filled with follicles, which are flat- 2.3.4 Medulla
tened, oval-shaped and B0.2 0.4 mm in diameter
The medulla, like the thymus, is a classical lymphoepithe-
[33]. A collagen-rich capsule surrounds each follicle,
lial tissue. It consists of epithelial cells, which originate
which represents the structural, functional, and patho-
from the anal invaginations of ectoderm [31], and blood-
logical bursal unit. Each follicle has its own blood sup-
borne hematopoietic cells, including lymphoid cells, DC,
ply independent of neighboring follicles. Small
and perhaps macrophages; few plasma cells occur in the
precapillaries branch from the main artery in the fold
involuting bursa.
and cross the follicular cortex, creating a dense capil-
lary network at the corticomedullary border
(Fig. 2.6A). Blood vessels never enter the medulla. A
blood bursa barrier may exist in the medulla, though 2.3.5 Bursal medullary epithelial cells
not in the cortex. Early studies concerning phagocytic The ERC form a cellular network in the medulla, and
capability in the bursa support the existence of a there is no immunocytochemically identifiable extracellu-
blood bursa barrier, as the bursa contains no phagocy- lar matrix (ECM). This is unlike the cortex, where the
tosed substances [46]. desmin1 mesenchymal reticular cells (MRC) produce an
The corticomedullary BL provides a complete separa- ECM. Epithelial cells can be classified according to their
tion of the medulla from the cortex (Fig. 2.5B); these two location and cellular structure. The superficial epithelial
regions are different histologically, ontogenically, and cells form a layer on the surface of the medulla. At the
possibly functionally. Supporting cells in the medulla and corticomedullary border, they form arches on the medul-
the cortex are respectively of epithelial and mesenchymal lary side and are covered by a BL on the cortical side.
origin. Under the FAE, the arch-forming cells become flat and
serve as supportive cells for the FAE, replacing the BL. can be observed between BSDCs and lymphocytes [52].
Hence, the interior of the medulla is separated from the The senescent BSDC seems to be phagocytic; the cell
cortex and FAE. The corticomedullary epithelium enclose loses the vimentin-intermediate filament and surface
CD451 lymphoblast-like cells (Fig. 2.6B), which express receptor for IgY, but the membrane-bound electron-dense
neither B cell (e.g., chB6, CXCR4, and CD1) nor BSDC substance is preserved for a while (Fig. 2.6D and E).
(e.g., vimentin, IgY, CSF1R and 74.3) or macrophage The biochemical composition of the granules is not
(TIM4, 74.2, 68.2) antigens (Fig. 2.6C). Delta-Notch sig- known, but the mAb CVI ChNL-74.3 recognizes intracel-
naling in the corticomedullary border suggests that the lular BSDC antigens, possibly granules (Fig. 2.7B) [54].
function of the arch-forming cells differs from that of the IBDV infection eliminates the 74.31 staining, suggesting
medullary ERC [47]. These corticomedullary epithelia that BSDCs are an IBDV target. The antigen recognized
may have nursing or regulatory function for the hemato- by mAb 74.3 has not been characterized, but double-
poietic cells. Recently, Wu et al. showed that epithelial staining with vimentin clearly indicates colocalization
cells of the corticomedullary border produce macrophage [39]. Surface IgY (sIgY) appears on BSDC just before
colony-stimulating factor (CSF1) [48]. Ramm et al. [49] hatching and persists throughout life, so anti-IgY can also
suggested that the cells at the corticomedullary junction be used for their identification (Fig. 2.7C). IgY appears
are resistant to infectious bursal disease virus (IBDV) and on the cell membranes of BSDC before hatching and
represent a macrophage subpopulation. If the blast-like therefore should be of maternal origin. IBDV infection
cell population is exhausted after IBDV infection or tes- eliminates not only the B cells but also sIgY, confirming
tosterone treatment, regeneration of the follicle, but not the involvement of BSDC in IBDV pathogenesis. Besides
the entire bursa, is handicapped (N. Nagy, unpublished IgY, vimentin and 74.3 antigen avian BSDCs express
observation). putative CD11c, DEC205, and CSF1R (Fig. 2.7D) in
Medullary epithelial cells have stellate morphology chicken, NIC2, CSF1R in quail, gray partridge, and
and form a 3D network (Fig. 2.5E) for B cells guinea fowl, respectively [29,34,52,55,56] These cells
(Fig. 2.6C), bursal secretory dendritic cells (BSDCs) show no reaction with the classical avian monocyte/mac-
(Fig. 2.6D), and macrophages (Fig. 2.6E). Epithelial cells rophage-specific mAbs (Fig. 2.7D F), thus, these mAb
are connected by desmosomes to one another and to the are convenient tools for monitoring the BSDC’s condition
cells of the FAE-supportive and corticomedullary arch- [14]. Corticomedullary epithelium promote the prolifera-
forming cells. Their cytological structure is comparable to tion (Fig. 2.7G) of BSDC precursors and B cells
that of FAE-supportive cells; namely, they are rich in (Fig. 2.7H J), which are nearby, but inhibit their differ-
cytokeratin and have poorly developed cytoplasmic orga- entiation, suggesting a regulatory function; the ERC allow
nelles, suggesting a supportive rather than secretory them to differentiate [57]. At hatching, 5 8 vimentin-
function. positive BSDC per follicle are present, increasing to
65 70 by 4 6 weeks of age
2.3.6 Bursal secretory dendritic cells
2.3.7 Bursal macrophages
BSDCs, first identified by Oláh and Glick [50,51], are
found only in the medulla (Fig. 2.6D). Cyclophosphamide In histological sections, macrophages with phagocytic
eliminates B cells from both the medulla and cortex but substances are easily identified in both the cortex and
does not affect BSDCs. However, B cell depletion results medulla; however, no positive cells are recognized using
in aggregation of these cells in the center of follicles. mAb against monocyte/macrophage markers, such as
BSDCs have two cellular processes, which are different CVI-ChNL-74.2, 68.1, 68.2, and KUL01 (Fig. 2.7K). This
in size and cytological structure. The smaller one contains suggests either that the bursal follicle does not have
a few rough-surfaced endoplasmic reticulum cisternae, macrophages or that its macrophage population is unique.
free ribosomes, and a large number of vimentin- Recently, it has been shown that the bursal medullary
intermediate filaments (Fig. 2.7A). The larger one con- macrophages express lysosome-specific Lep100 antigen
tains many membrane-bound electron-dense granules (Lamp-1) and TIM4 [14,36,48]. After IBDV infection,
around a well-developed Golgi region, or it lines up along large cells loaded with cellular debris and virus particles
the cell membrane (Fig. 2.5D). The arrangement of the appear first in the medulla and later in the cortex, while
cytoplasmic organelles in the cell gives the mature BSDC BSDC cannot be identified using any cell marker.
a polarized appearance (Figs. 2.6D and 2.7A). The mature Electron micrographs show a surface substance on several
BSDC is highly elongated in shape and has a nuclear macrophages, comparable to that on BSDC (Fig. 2.6E and
chromatin structure similar to that of the lymphocyte. F). This finding, together with the MHC class II1
Granular contents are released and attach to the outer sur- macrophage-like cells in the FAE, indicates that the med-
face of the cell membrane [34,52,53]. Cell adhesion sites ullary macrophages are possibly senescent BSDC [40].
20 Avian Immunology
(Continued)
Structure of the avian lymphoid system Chapter | 2 21
2.3.8 Bursal lymphocytes dispersed uniformly throughout the cortex but only the
chB61 B cells located in the outer region of the follicular
At least 98% of bursal lymphocytes are B cells. Scattered
cortex coexpress CXCR4. It is suggested that B cells have
T cells occur in the cortex, but few of them enter the
to downregulate CXCR4, express Ov antigen before they
medulla. B cells proliferate in both cortex and medulla
can leave the CXCL12-rich environment of the follicle cor-
(Fig. 2.7G). After 8 10 weeks of age, the number of lym-
tex and migrate out to colonize the peripheral lymphoid
phocytes in the interior of the medulla decreases, indicating
organs [60]. The ECM is rich in collagen III and fibronectin,
the initiation of bursal involution, which is a process com-
indicating intensive cell migration through the cortex. The
pleted by 6 7 months of age. A few plasma cells may
complete absence of ECM in the medulla means that cell
develop in the cortex and medulla with age, but generally
migration is questionable, but the ERC surface serves as a
their number remains very low, suggesting some kind of
scaffold for it. The cortex has a rich capillary network adja-
inhibition of B-cell terminal maturation. However, in birds
cent to the corticomedullary BL (Fig. 2.6A) and is drained
surviving IBDV infection, the bursa has a remarkably large
by postcapillaries, which conjoin to form veins in the con-
number of plasma cells before fibrosis is complete.
nective tissue of the fold where the lymphatics emerge.
One perennial question remains: Are there any differ-
ences between cortical and medullary B cells? Trafficking
of bursal cells between the cortex and medulla occurs, and 2.3.10 Peripheral lymphoid tissue of the bursa of
there are some differences in phenotype; however, the Fabricius
functional differences are not fully understood (see
Chapter 4 for further discussion). Medullary chB6 1 B Close to the bursal duct, one of the ventral folds produces
cells express surface IgM [58] and CD15 (Lewis X) [59], peripheral (secondary) lymphoid tissue (Fig. 2.7F). This tis-
but the Ov antigen and CXCR4 appear only on cortical B sue is not likely to be a simple continuation of cloacal lym-
cells [60] (Fig. 2.7H J). This makes it difficult to deter- phoid infiltration because the fold is flat, it is wider than
mine the B-cell maturation process. During acute involu- others and its colonization by lymphoid precursors is
tion of the bursa, caused by IBDV treatment, T delayed about 1 day. The peripheral lymphoid tissue consists
lymphocytes accumulate in the follicles. The more severely of GC and an interfollicular dense lymphatic substance that
damaged follicles have more T cells, suggesting a delicate represents B- and T-dependent regions, respectively. This
balance between T and B cells. If B cells are destroyed or fold may provide communication between the cloacal con-
emigrate from the follicle, T cells can temporarily replace tents and the bursa of Fabricius [37,63,64].
them [61]. The accumulation of T cells in bursal follicles
seems to depend on B cell depletion and not just infection. 2.3.11 Germinal center of the peripheral
lymphoid organs
2.3.9 Cortex
Histologically, the GC is defined by aggregates of blast
The cortex begins to develop around hatching, and it is sepa- cells which, after antigenic stimulation, occur in defined
rated from the medulla by the BL of the corticomedullary areas within peripheral tissues. In chickens, two types of
border, its surface is covered by fine collagen-rich capsules. GC, fully and partially encapsulated, have been
The cortex, unlike the medulla, develops exclusively from described. Although their cell populations do not differ
the mesoderm. MRC form the supporting cells of the cortex cytologically, there is speculation that the two types rep-
and express vimentin- and desmin-intermediate filaments resent functionally different structures [65].
[62]. The 3D meshwork of the MRC is filled with chB61/ Alternatively, they may represent different stages of GC
CXCR41 B lymphocytes (Fig. 2.7J) and CSF1R1/Lep1001/ development [66]. Mature GC are surrounded by a cap-
TIM41 macrophages. BSDC are lacking in the cortex. sule of connective tissue and completely lack blood ves-
Cortical B cells are heterogeneous, chB61 cells are sels (Fig. 2.8A, G, and H).
L
FIGURE 2.7 The structure of the bursa of Fabricius. (A) A vimentin-intermediate filament is expressed by the bursal secretory dendritic cells
(BSDC). Inset: magnified from the outlined area. The BSDC generally have two well-developed cell processes, giving them a highly polarized appear-
ance. (B) CVI-ChNL-74.3 mAb recognizes cytoplasmic antigen in the BSDC. The 74.31 cells occur in the medulla, but occasionally they show up in
the FAE. (C) The anti-IgY-specific antibody identifies strong membrane-bound IgG immunoreaction on the cell surface of the BSDC. (D-F) Double
staining with CSF1R (green) and TIM4 (red) highlights the BSDC in bursal follicles. TIM4 is expressed by the macrophages-like cells of the follicular
medulla, but not the CSF1R1 BSDC. (G) BrdU incorporation shows that proliferating cells are closely associated with the marginal zone of the
medulla and cortex. (H, I) Medullary B cells express CD15 and IgM. (J) In adult bursa only cortical B cells express CXCR4 antigen. (K) The macro-
phage marker KUL01 does not stain cells in the follicles. MRC1L-B1 cells occur only in the interfollicular connective tissue. (L) Between two bursal
folds (B) the peripheral lymphoid tissue (outlined) consists of germinal centers and dense lymphoid tissue, as shown by chB6 (Bu-1) antigen
immunostaining.
22 Avian Immunology
In the spleen, the induction sites of GC seem to be [63]. Arakawa et al. [71] estimated the clonal complexity
directed by clusters of stromal cells, recognized by the in each GC of the spleen, suggesting that 6 12 B cell
mAb CVI-ChNL-74.3, which are located close to the T clones are expanding, with each producing a different
cell areas, adjacent arteries and arterioles. During a antibody.
humoral response, newly developing GC, consisting of Contradictory results concerning Ig expression in GC
bromodeoxyuridine-incorporating proliferating (BrdU1) B have been reported. Jeurissen et al. [70] described follicle
cells, are located adjacent to clusters of 74.31 cells [67]. B cells expressing sIgM but not sIgY. Yasuda et al. [72]
During development, the B cells grow around the clusters isolated individual GC from the spleen and analyzed GC
until they completely surround them. Cells in the middle cell populations at the individual cell level. They
of the GC do not incorporate BrdU; thus they are not described considerable differences in sIgM and sIgY
mitotically active and resemble centrocytes, while the expression between GC. Ig-expressing cells were found in
outer ring of proliferating cells represents centroblasts the center of the GC but not in the outer rim, where pro-
(Fig. 2.8B). Although a light zone of centrocytes and a liferating B cells were found. The relative proportions of
dark zone of centroblasts, as described in mammalian GC, sIgM1 and sIgY1 cells changed after intravenous inocula-
can be less easily discriminated morphologically in the tion of DNP-KLH, with the highest proportion of sIgM1
chicken, functional homology seems likely [65,66,68]. cells detected on day 7. The proportion of sIgY1 cells
Structurally similar to mammalian metallophilic macro- increased during the first 14 days, but, as observed for
phages, a mature GC is surrounded by TIM41 macro- sIgM1 cells, large individual variations were evident. In
phages [69]. In mature GC, 74.31 CSF1R1 cells, called contrast, the proportion of CD31 cells remained relatively
follicular dendritic cells (FDC), can trap immune com- constant at 10% 20% in each GC. Double-staining stud-
plexes on their surface. These trapped immune complexes ies of GC have elucidated the nature of the isolated sIg1
are important in memory formation. Specific antibodies cells, where the FDC appeared to possess IgG on their
or antibody-containing cells are detected in serum and surface [62].
spleen, respectively, before the formation of GC.
Therefore the site of GC formation is unrelated to that of
the simultaneously induced antigen-specific plasma cells, 2.4 The spleen
as reported for the mouse spleen [66,70].
2.4.1 Origin and anatomy
Transmission electron microscopy shows a dense
intercellular substance around the FDC. The structure of The splenic primordium first appears as a mass of mesen-
the FDC’s nuclear chromatin resembles that of small lym- chymal cells in the 48-hour embryo [73]. Sinusoids with
phocytes, and the cytoplasm contains a variable number erythrocytes appear in the mesenchyme at E5, granulopoi-
of electron-dense granules of various sizes (Fig. 2.8C). esis begins from E7 and erythropoiesis follows at E11. In
The surface membrane of 74.31/CSF1R1 FDCs contrast to that of mammals, the avian spleen is not con-
(Fig. 2.8D and E) binds (but does not express) IgY sidered a reservoir of erythrocytes for rapid release into
(Fig. 2.8F), and the FDC’s cytoplasm contains vimentin- the circulation [74]. Although not a primary site for lym-
intermediate filaments (Fig. 2.8G). The anti-vimentin- phocyte antigen-independent differentiation and prolifera-
specific antibody and anti-CSF1R are convenient markers tion, the spleen has an important role in embryonic
to identify avian FDC [14,55,56,62]. In addition to FDC, lymphopoiesis, for it is here, though not solely here, that
the GC contains other types of nonlymphoid cells such as B cell progenitors undergo rearrangement of their Ig
supporting reticular cells, which express desmin- genes before colonizing the bursa of Fabricius [75] (see
intermediate filaments (Fig. 2.8H). Tingible body macro- also Chapter 4). At the time of hatching, the spleen
phages are not stained by classical monocyte-macrophage becomes a secondary lymphoid organ which provides an
markers, such as 74.2 (Fig. 2.8I), 68.2, and KUL01 indispensable microenvironment for interaction between
(MRC1L-B), but they express TIM4 [69] and lysosomal lymphoid and nonlymphoid cells [14]. The contribution
membrane protein LAMP-1 (Lep100 antibody) of the avian spleen to the immune system as a whole may
(Fig. 2.8J). This may indicate a different developmental be more important than in mammals because of the poorly
stage or a different origin. The tingible body macrophages developed avian lymphatic vessels and lymph nodes.
engulf apoptotic B cells and cell debris. A mature GC The chicken spleen is a round or oval structure lying
consists of proliferating Bu-11 (chB6 antigen) B cells dorsal to and on the left side of the proventriculus
(Fig. 2.8K), a few T cells expressing CD3, CD4, and (Fig. 1.1C). One or more small accessory spleens some-
TCRαVβ1 (Fig. 2.8L), and 74.31 FDC. Arakawa et al. times occur nearby [76]. Major splenic development
[71] and Igyarto et al. [67] demonstrated that the antigen- occurs after hatching, following exposure to antigens
activated B cells of the spleen migrate into the GC, where [77]. The main blood supply to the spleen is provided by
they expand, as proposed for human GC development the Arteria lienalis cranialis and caudalis, with some small
Structure of the avian lymphoid system Chapter | 2 23
FIGURE 2.8 The structure of the germinal centers. (A) Schematic illustration of the germinal centers. Follicular dendritic cells (FDC) and macro-
phages are permanent constituents of the GC. Majority of the tingible-body macrophages occur in the outer layer of the GCs. (B) BrdU
(Continued)
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gray whiskers, while two of the others were Black Elrich and the man
who had first caught the big dog by the collar in the Hotel Metropole.
Elrich was at the head of the party, and he advanced straight toward
the diamond. As he drew near, he loudly said:
“So this is your team, Merriwell? I’m glad I found you here. I’ve
brought Dave Morley, manager of the Reds, along. This is Mr.
Morley.”
A short, stout, thick-set man came forward. He had a smooth-shaven
face of the bulldog cast, and he was smoking a black cigar. His first
words were:
“Mr. Merriwell, I see you are a squealer.”
CHAPTER XXVI.
A DESPERATE ATTEMPT.
The man’s manner was quite as offensive as his words, but Merry
looked at him calmly, betraying no emotion, as he asked:
“What do you mean, sir?”
“Your acceptance of my challenge was a squeal,” declared Morley.
“How?”
“My challenge was to play for a purse and the entire gate-money.”
“And I accepted, stating my reasons for declining to play for a purse.”
“Which was a squeal.”
“Which was nothing of the sort! I have not started out with the
intention of running this ball-team to make money. We are out for
sport, and nothing else. I am not a gambler, and I take no
satisfaction in playing ball for purses.”
“Oh, I don’t suppose you ever did such a thing in your life?” sneered
Morley.
Merry flushed.
“It makes no difference what I have done.”
“But you can’t deny that you have played for purses.”
“Never without protest—never unless practically forced to do so. In
this case, I refuse to be forced. The gate-money should be sufficient
to pay well the winning team.”
“My team is run under heavy expense, and there is no assurance
that your aggregation of amateurs will prove a drawing card.”
Hodge was at Frank’s elbow, scowling like a thundercloud, his heart
filled with hot anger over the insolent words of the man. Bart’s
fighting blood was being stirred, and he longed to give Mr. David
Morley just what he deserved.
“Then you have the privilege of declining to meet us,” said Frank.
“That will settle the whole matter in short order.”
“He knows we’ll draw!” exclaimed Hodge. “Your name alone,
Merriwell, will turn out a crowd.”
“I think you are mistaken,” said the manager of the Reds. “In the
East, Frank Merriwell may be regarded as something of a wonder,
but out here he does not count. We have plenty of better men.”
“I’ll bet you——” began Bart hotly.
“Ah!” grunted Morley; “at least this member of your team is not
adverse to making a little gamble, Mr. Merriwell.”
“That has nothing to do with him,” said Bart. “I’ll bet you ten dollars
we get more hits off your pitcher than you do off Merriwell.”
“Ten dollars!” came scornfully from the manager of the Denver team.
“Why don’t you make it ten cents? You’re putting the figures too high,
young man.”
His words and manner were calculated to enrage Bart still more.
Frank’s fingers fell with a firm grip on the arm of his friend, and he
quietly said:
“I do not think we’ll do any betting over the game. If you wish to play
us on the terms stated by me in my acceptance of your challenge,
well and good. If you do not, we’ll let the matter drop.”
“It’s plain enough, Morley,” put in Elrich, “that the young chap knows
which side his bread is buttered on.”
“He must think me a mark to put my salaried team against his
collection of non-salaried kids,” sneered the baseball man, “unless
there is something more than glory in it. It’s mighty little glory we’d
get defeating his team.”
“That’s right!” exclaimed Bart; “for you’d never defeat it.”
“Then we’ll have to call the game off,” said Frank, remaining
perfectly calm.
“That’s a shame!” muttered Berlin Carson, who had heard some of
the talk. “I’m a Colorado man, but I think I know what Merriwell’s
team can do, and——”
“We cuc-cuc-can do those fellows,” said Gamp, who also was
aroused.
“Why, it would be a snap!” chuckled Jack Ready. “All we wanted the
game for was to get a little practise.”
“You’re a lot of bluffers!” roughly declared Morley.
“I told you there was nothing in it, Dave,” said Elrich, with an air of
weariness. “The boys have not money enough to put up a purse.”
Then Frank felt some one tugging at his elbow, and he looked round
to see Dick there, his eyes gleaming and his face flushed with
indignation.
“Bet him, Frank!” palpitated the lad. “I wouldn’t stand it to have him
talk that way to me! You know father was dreadfully rich, and all his
money was left to us. I’ll bet every cent of my part that your team can
beat his!”
“Ho! ho!” laughed Morley. “And how much might your part be, kid?”
“Oh, a little trifle of eight or ten million dollars, that’s all,” said Frank,
who could not help being somewhat nettled by the insulting manner
of the man. “I think it would be quite enough to accommodate you, in
case it was staked against anything you could raise at twenty to
one.”
It was not often Merriwell said anything like this, but just now he had
been provoked to the limit, and he could not refrain.
“You don’t mean to say the kid is worth eight or ten million dollars, do
you?” asked Elrich, as if incredulous.
“He will inherit something like that amount when he comes of age,”
answered Merry, as he carelessly toyed with the ball he had held
throughout this conversation.
A swift look passed between Elrich and the man with the gray
whiskers, who stood slightly apart from the group.
“And you’re his brother?” Elrich questioned further.
“I am,” bowed Merry.
“Where do you come in?”
“What do you mean?”
“What part of this snug little fortune do you get?”
“Really, sir, I do not know as that is of any concern to you. Still, it is
no secret that I, also, will inherit a similar sum when he comes of
age.”
“When he does? That’s odd. You’re of age now. How does it happen
that——”
“I decline to speak of this matter further, sir, as I——”
“You’re a big bluffer, Merriwell. I do not take any stock in your
romance of millions.”
“And I care not a snap whether you do or not.”
“If you had so much money at your command, you’d not hesitate to
put up a few hundred to back your ball-team—that is, if you really
believe your team capable of playing ball.”
“I have reasons for not gambling in any way,” said Frank. “I do not
expect men like you to respect my scruples, so all this talk is
wasted.”
“Well, we can’t fool with you!” angrily sneered Morley. “I’ll bet you five
hundred dollars, even money, that the Denver Reds can defeat your
ball-team. If you will not cover the money, we’ll fool away no more
time.”
“If he will not cover your money, I reckon I will!” exclaimed a voice,
as a man, who had approached without attracting notice, pushed into
the excited group.
“Father!” exclaimed Berlin Carson.
“Mr. Carson?” came from Merry’s lips.
“That’s me!” nodded the rancher, extending his hand and giving
Merry a hearty grip. “Forgot to tell Berlin to attend to one little piece
of business while in the city, so I decided to follow him. Heard over at
the hotel that you were here, Merriwell, with a ball-team. They told
me where to find you, and I came right out. What sort of a game of
talk was this man giving you?”
“He was trying to force me into wagering money with him over a ball-
game to which he has challenged me. He is the manager of the
Denver Reds.”
“Well, I don’t often bet against a home team, but I know you, and I’ve
seen your men play ball, so, if he wants to plank down five hundred,
I judge I can accommodate the gentleman. I believe I have that
amount of money about my clothes.”
“Then you’re the man I’m looking for!” exclaimed Morley. “Mr. Elrich
is my backer, and he will put up the money.”
“Who’ll hold the stakes?”
“Why, Mr. Jordan here is a good man to——”
“I allow I don’t know anything about Mr. Jordan, but I do know
Charley Gans, down at the Metropole, and he’ll suit me to a T.”
“Gans is all right,” nodded Elrich, who seemed eager to get the bet.
“Then I’ll meet you there at six this evening, and we’ll put up the
dust,” said Mr. Carson, with a dismissing wave of his hand. “Good
day till later.”
“Hurrah!” cried Dick, flinging his hat into the air. “That’s the stuff!”
“Slang, my boy—slang!” said Ready, severely. “You’re catching on
altogether too quick. I’m afraid you have been associating with bad
company lately.”
“You’re a regular young sport!” said Elrich, with apparent admiration,
his words being intended to flatter the boy.
“Are you a sport?” asked Dick.
“Well, I allow I have some sporting blood in me.”
“Then I’m no sport!” the lad quickly asserted. “I don’t want to be like
you.”
Elrich’s smile turned to a frown, but he said:
“You’re pretty sharp with your tongue, but you may have some of
your flipness taken out of you some day. All the same, I like you, and
I’ll give you a drive back to the hotel in my private carriage, if you’ll
go.”
“Hardly,” said Merry. “He can have all the drives he likes at his own
expense.”
“Oh, very well!” said the gambler, turning away and starting to talk in
a low tone to Morley.
Mr. Carson was speaking with those of Frank’s friends whom he had
met before. Now he turned to Merry once more.
“I reckon I’ve got you to thank for getting my boy onto the Yale ball-
team,” he said. “Berlin said it was through you he got a chance to
show what he could do.”
“It was because I knew he had the right stuff in him,” asserted
Merriwell. “I presume you’ll let him play with us against the
Denvers?”
“Sure as you’re shouting! And I’ll disown him if he doesn’t put up a
good game.”
At this moment there came a sudden cry. They turned to see Dick
Merriwell, caught up by the man with the gray beard, being carried
swiftly toward the gate, which was standing open. The man was
running, holding the struggling lad under his arm.
For an instant every one seemed paralyzed with astonishment. Then
Frank Merriwell sprang out, his arm went back, and, with all his
strength, he threw the ball in his hand.
Straight as a bullet from a rifle flew the ball, and it struck the running
man fairly on the back of the head, knocking him forward on his face.
This caused him to drop the boy, and, quick as thought, Dick
scrambled up and leaped, like a young panther, on the back of the
man.
When Merriwell leaped forward, Black Elrich suddenly stepped into
his way, and there was a collision. Elrich staggered and caught hold
of Merriwell’s arm, to which he tried to cling.
Instantly Frank beat off the hand of the man, sprang round him, and
dashed to the aid of Dick. But the man had flung the boy off, and
now he rose to his feet, casting one quick look over his shoulder.
A surprising thing had happened, for the man was beardless now,
his gray whiskers being grasped in the fingers of the plucky lad.
Frank saw the face of the man.
“Mescal!” he cried.
It was Mescal and again he had made a desperate play to get
possession of Dick Merriwell, for Frank was confident it had been the
intention of the fellow to abduct the boy.
Mescal now fled like a deer out through the gate, sprang into the
carriage standing there, tore the reins from the hands of the driver,
snatched out the whip, cut the horses, and was carried away just as
Frank came up.
For a moment Merry contemplated pursuing the desperado, but he
quickly decided that it would be folly to make the attempt.
Black Elrich came rushing out through the gate, shouting:
“Stop! stop there! By heavens! he’s running off with my team!”
Frank faced the gambler, his eyes flashing.
“The job failed, Elrich,” he said cuttingly. “It was a daring attempt,
and rather foolish, I think.”
“The man is crazy!” said Black Ben.
“Crazy to make a play for ransom-money,” said Merry. “I know him,
and I’ll see that the police of Denver are put on his track. If he is
caught, he may squeal and expose his pals. In that case, Mr. Elrich,
you are liable to feel rather uncomfortable.”
“Do you mean to insinuate——”
“My words are plain. I saw the look that passed between you and
that man a short time ago. My eyes are pretty wide open.”
“Why, I don’t know the man. He came along to the gate as we were
entering, and walked in with us.”
“By appointment?”
“Nothing of the sort! I tell you I don’t know him. And anybody who
knows me will swear that my word is good.”
“In a matter of cards or other gambling it may be. But I wish you to
inform Anton Mescal that I shall be better prepared for him next time.
The ball that brings him down then will be of lead, and not a common
baseball.”
CHAPTER XXVII.
A DASTARDLY TRICK.
Frank Merriwell made a signal, and his men came trotting in from the
field.
But the eyes of the spectators were on the strangely handsome boy
and the wrinkled old Indian, the latter having his dirty red blanket
wrapped about his shoulders. At the home plate, to which the boy
seemed to lead the Indian, they stopped.
Some boys on the bleachers began to whoop like a whole pack of
redskins. Unheeding everything, Old Joe slowly walked round the
rubber plate, then stopped, extended his hands over it and made
some queer signs, as if he were weaving a spell. A hush had fallen
on the curious crowd.
Finally the aged Indian stooped and solemnly placed the flat of his
hand upon the plate, as if blessing it. This done, he turned, and,
accompanied by the boy, walked toward the bench. Again the
urchins began to whoop, and the crowd laughed.
The umpire appeared and advanced onto the field. The Reds, of
course, had their choice of innings, and they decided to go to bat
first.
Merriwell’s men were bunched about their leader, who was speaking
to them in low tones.
“All ready,” called the umpire.
Immediately, the Merries turned and trotted out onto the field once
more, while the first batter of the home team picked out his stick and
advanced toward the plate.
“Light on him right off the reel,” said Dave Morley, who was sitting on
the home bench. “Break his heart in the first inning.”