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Avian Immunology
Avian Immunology
Third Edition

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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Dedication

Dedicated to

Professor Pete Kaiser 1964 2016

Coeditor Second Edition Avian Immunology
Valued Avian Immunologist and Sorely Missed by the Editors of the
Third Edition of Avian Immunology.
Contents

List of contributors xvii 2.3.11 Germinal center of the

Foreword xix peripheral lymphoid organs 21
Acknowledgments xxi 2.4 The spleen 22
2.4.1 Origin and anatomy 22
1. The importance of the avian immune 2.4.2 Periarteriolar lymphoid sheath 25
system and its unique features 1 2.4.3 Ellipsoids and periellipsoid
white pulp 25
Fred Davison 2.4.4 The marginal-zone equivalent
1.1 Introduction 1 and antigen handling 27
1.2 The contribution from avian 2.5 Gut-associated lymphoid tissue 28
lymphocytes 1 2.5.1 Follicle-associated epithelium or
1.3 Contribution of the bursa of Fabricius 2 lymphoepithelium 30
1.3.1 Gene conversion and the bursa 4 2.5.2 Esophageal and pyloric tonsils 30
1.4 The contribution of the chicken MHC 5 2.5.3 Peyer’s patches 31
1.5 Contributions to vaccinology 6 2.5.4 Meckel’s diverticulum 31
1.5.1 Embryonic (in ovo) vaccination 7 2.5.5 Cecal tonsils 32
1.6 Conclusion 8 2.6 Harderian gland 33
References 8 2.7 Mural lymph node 34
2.8 Ectopic lymphatic tissue and pineal
2. Structure of the avian lymphoid gland 36
system 11 2.9 Bone marrow 37
2.10 Blood 38
Nándor Nagy, Imre Oláh and
References 39
Lonneke Vervelde
2.1 Introduction 11
2.2 The thymus 12 3. Development of the avian
2.2.1 Anatomy and histological hematopoietic and immune
organization 12 systems 45
2.2.2 Thymic cortex 13
Laurent Yvernogeau, Nándor Nagy,
2.2.3 Thymic medulla 13
Dominique Dunon, Catherine Robin and
2.3 The bursa of Fabricius 15 Thierry Jaffredo
2.3.1 Anatomy and histology 15 3.1 Introduction 45
2.3.2 Bursal surface epithelium 16 3.2 Origins and migration routes of
2.3.3 Bursal follicle 17 hematopoietic cells using quail/chicken
2.3.4 Medulla 18 complementary chimeras 45
2.3.5 Bursal medullary epithelial 3.2.1 Looking for the source of
cells 18 hematopoeietic cells during
2.3.6 Bursal secretory dendritic cells 19 development 45
2.3.7 Bursal macrophages 19 3.2.2 Macrophage production by the
2.3.8 Bursal lymphocytes 21 yolk sac 46
2.3.9 Cortex 21 3.2.3 The aortic region produces HSCs 46
2.3.10 Peripheral lymphoid tissue of 3.3 Aortic clusters as the intraembryonic
the bursa of Fabricius 21 source of definitive hematopoiesis 46

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

5.4 Natural antibodies 107 7.7 Gene coevolution in the chicken

5.5 Maternal antibodies 108 major histocompatibility complex 142
5.6 Fc receptors 109 7.8 Other chicken genes important for
5.6.1 Chicken polymeric Ig receptor 109 the major histocompatibility
5.6.2 Chicken FcRn homologue 110 complex 144
5.6.3 Chicken Fc receptor cluster 110 7.9 Polymorphism and typing chicken
5.6.4 ggFcR 110 major histocompatibility complex
5.6.5 CHIR-AB1 110 genes 145
5.7 Avian antibody responses 110 7.10 Avian major histocompatibility
5.8 The chicken egg as a source of complexes 146
antibodies 112 7.11 Immunity, disease resistance, and the
5.8.1 Avian antibodies as tools for major histocompatibility complex in
research 112 wild birds 148
References 113 7.12 Sexual selection and the major
histocompatibility complex in wild
6. Avian T cells: Antigen Recognition birds 149
and Lineages 121 7.13 Origin and evolution of the immune
system 150
Adrian L. Smith and Thomas W. Göbel Acknowledgments 151
6.1 Introduction 121 References 151
6.2 T cell receptor structure and lineages 121
6.2.1 Somatic DNA recombination 121 8. Introduction to the avian innate
6.2.2 Organization of the T cell
immune system; properties,
receptor clusters 123
6.3 CD3 signaling complex 125 effects, and integration with other
6.3.1 Mammalian CD3 125 parts of the immune system 163
6.3.2 Chicken CD3γ/δ and CD3ε 126 Thomas W. Göbel and Adrian L. Smith
6.3.3 ζζ homodimer 126
6.3.4 T cell receptor complex—structural
8.1 Macrophages and dendritic cells 167
models 126
6.3.5 T cell receptor signal transduction 127 Kate Sutton, Adam Balic, Bernd Kaspers and
6.4 CD4 and CD8 127 Lonneke Vervelde
6.5 Costimulatory molecules 128 8.1.1 Introduction 167
6.6 T cell lineages 129 8.1.1.1 Antigen presentation 167
6.7 Methods to study T cell function 130 8.1.1.2 Dendritic cells 168
6.8 Perspectives 130 8.1.1.3 Macrophages 169
References 131 8.1.1.4 Development of myeloid
cells 171
7. The avian major histocompatibility 8.1.1.5 Sources of avian macrophages
and dendritic cells 172
complex 135
8.1.1.6 Avian myeloid cell lines 175
Jim Kaufman 8.1.1.7 Cell surface markers for
7.1 Introduction 135 avian myeloid cells 175
7.2 The biology of the major 8.1.1.8 Characterization of
histocompatibility complex 135 macrophages and DC in
7.3 The major histocompatibility complex: tissue sections 178
a genomic region or a biological unit? 136 8.1.1.9 Functional properties of
7.4 The chicken major histocompatibility chicken macrophages 178
complex and the major histocompatibility 8.1.1.10 Macrophage migration 178
complex syntenic region 137 8.1.1.11 Phagocytosis 179
7.5 Classical and nonclassical major 8.1.1.12 Respiratory burst activity 180
histocompatibility complex molecules 139 8.1.1.13 Nitric oxide production: a
7.6 Chicken classical major readout system for avian
histocompatibility complex molecules 140 macrophage activation 180
x Contents

8.1.1.14 Cytokine response of avian 8.5.1 Soluble components 217

macrophages 182 8.5.1.1 Host defense peptides 217
8.1.2 Functional properties of chicken 8.5.1.2 Collagenous lectins 219
antigen-presenting cells 183 8.5.1.3 Surfactant protein A and cLL 219
8.1.2.1 Maturation from antigen 8.5.1.4 Mannose-binding lectin 220
sampling to antigen 8.5.1.5 Collectin 10, -11, and -12 220
presenting 183 8.5.1.6 Complement 221
8.1.2.2 Migration 184 8.5.1.7 Components of the classical
8.1.2.3 Other nonmyeloid pathway 222
antigen-presenting cells 185 8.5.1.8 Components of the lectin
8.1.3 Concluding remarks 185 pathway 222
References 186 8.5.1.9 Components of the
alternative pathway 222
8.2 Avian granulocytes 197 8.5.1.10 Downstream components of
complement 223
Michael H. Kogut
8.5.2 The acute-phase response 223
8.2.1 Functional activities of heterophils 197
8.5.2.1 C-reactive protein 223
8.2.2 Receptors 198
8.5.2.2 Serum amyloid A 224
8.2.3 Other innate immune receptors 199
8.5.2.3 α1-acid glycoprotein 224
8.2.4 Genetic effects on heterophil
8.5.2.4 (Ovo)transferrin 224
genotype and phenotype 200
8.5.2.5 PIT54 225
8.2.5 Heterophil isolation 201
8.5.2.6 Hemopexin 225
References 201
8.5.2.7 Ceruloplasmin 225
Further reading 203
8.5.2.8 Fibrinogen 225
8.5.2.9 Other potential chicken APPs 225
8.3 Thrombocyte functions in the References 226
avian immune system 205
Jake Astill, R. Darren Wood and Shayan Sharif 8.6 Pattern recognition receptors 231
8.3.1 Introduction 205 Adrian L. Smith and Steven R. Fiddaman
8.3.2 Avian thrombocyte structure 205 8.6.1 Introduction 231
8.3.2.1 Physical characteristics 205 8.6.2 Tissue fluid and secreted pattern
8.3.2.2 Surface protein expression 206 recognition receptors 232
8.3.3 Avian thrombocytes and immune 8.6.2.1 C-reactive protein 232
responses 208 8.6.2.2 Collectins 232
8.3.3.1 Innate responses 208 8.6.2.3 Mannose-binding lectin 232
8.3.3.2 Adaptive immune responses 209 8.6.2.4 Ficolins 233
8.3.4 Infection of thrombocytes 210 8.6.2.5 Surfactants: surfactant
8.3.5 Conclusion 210 protein A and surfactant
References 210 protein D 233
8.6.2.6 Other collectins 233
8.4 Natural killer cells 213 8.6.2.7 Chicken mannose (or mannan)-
Thomas W. Göbel binding lectin-associated serine
8.4.1 Potential natural killer cell receptor protease proteins, linking soluble
families 213 pattern recognition receptor to
8.4.2 Phenotype of chicken natural killer complement activation 234
cells 214 8.6.3 Cell-associated pattern recognition
8.4.3 Natural killer cell function 214 receptors 234
References 215 8.6.3.1 Avian Toll-like receptors 234
8.6.3.2 TLR1/6/10-related molecules 235
8.6.3.3 TLR2 235
8.5 Soluble components and
8.6.3.4 TLR3 235
acute-phase proteins 217
8.6.3.5 TLR4 236
Edwin J.A. Veldhuizen and Tina Sørensen Dalgaard 8.6.3.6 TLR5 236
 Contents xi

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

15.1.1 Stress hormones: epinephrine, 16.4.3 Immunological mechanisms 450

norepinephrine, dopamine, 16.4.4 Summary 452
and glucosteroids 419 Acknowledgments 452
15.1.2 Sex hormones 420 References 452
15.1.3 Metabolic hormones: thyroid
hormone, growth hormone, 17. Tumors of the avian immune
and leptin 422 system 457
15.1.4 Environmentally responsive
hormones: melatonin 422 Venugopal Nair
15.2 Physiological states 423 17.1 Introduction 457
15.2.1 Temperature and housing as 17.2 Tumors of the immune system 457
immune modulators 423 17.2.1 Marek’s disease 457
15.3 Dietary effects on immunity 424 17.2.2 Avian leukosis 459
15.3.1 Contribution of the 17.2.3 Reticuloendotheliosis 460
microbiome 425 17.3 Oncogenic mechanisms of tumor
15.3.2 Immunomodulatory nutrients viruses 460
and feed additives 425 17.3.1 Oncogenic mechanisms of
15.3.3 Immunometabolism 427 retroviruses 461
15.4 Assessment of immunocompetence 428 17.3.2 Oncogenic mechanisms of
15.4.1 Functional activity of the DNA tumor viruses 461
immune response 428 17.4 Immune responses to oncogenic
References 429 viruses 461
17.4.1 Immune responses to
16. Autoimmune diseases of poultry 437 leukosis/sarcoma viruses 462
17.4.2 Immune responses to
Gisela F. Erf reticuloendotheliosis virus 462
16.1 General characteristics of autoimmune 17.4.3 Immune responses to Marek’s
diseases 437 disease virus 462
16.2 Autoimmune vitiligo in Smyth-line 17.5 Antitumor responses 463
chickens 440 17.6 Conclusion 464
16.2.1 Introduction 440
References 464
16.2.2 The Smyth line chicken model
for autoimmune vitiligo 440
16.2.3 Characteristics of the
18. Practical aspects of poultry
Smyth-line chicken 441 vaccination 469
16.2.4 Pigmentation and normal J.J. (Sjaak) de Wit and Enrique Montiel
melanocyte function 442 18.1 Introduction 469
16.2.5 Target cell defects 442 18.2 Vaccine types 469
16.2.6 Immunological mechanisms 443 18.2.1 Live vaccines 469
16.2.7 Environmental factors 445 18.2.2 Inactivated vaccines 470
16.2.8 Summary 446 18.2.3 Poultry vaccine adjuvants 471
16.3 Spontaneous autoimmune 18.3 Vaccine application 471
(Hashimoto’s) thyroiditis in 18.3.1 Mass application 471
obese-strain chickens 446 18.3.2 Individual applications 472
16.3.1 Introduction 446 18.4 Factors influencing vaccine
16.3.2 Development and responses 474
characteristics of OS chickens 447 18.4.1 Status of the immune system
16.3.3 Immunological mechanisms 447 at the time of vaccination 474
16.3.4 Target cell/organ defects 448 18.4.2 Maternally derived antibodies 474
16.3.5 Summary 449 18.4.3 Vaccine storage, preparation,
16.4 Scleroderma in UCD 200/206 chickens 449 and administration 475
16.4.1 Introduction 449 18.4.4 Age at vaccination 476
16.4.2 Development and characteristics 18.4.5 Duration of immunity 476
of the UCD 200/206 lines 450 18.4.6 Interference between vaccines 476
 Contents xv

18.4.7 Time intervals between 20. Evolutionary and ecological

vaccinations 477 immunology 519
18.5 Immunosuppression 477
18.5.1 Stress and immunosuppression 477 Michal Vinkler, James S. Adelman and
18.5.2 Mycotoxins 478 Daniel R. Ardia
18.5.3 Immunosuppression by 20.1 Introduction 519
vaccines 478 20.2 Assessing immune function in
18.5.4 Influence of immunosuppression free-living birds 520
on vaccination 478 20.2.1 Single-time-point assays 520
18.6 Quality control of response to 20.2.2 Multiple-time-point assays 522
vaccination 479 20.3 Development of the immune
18.6.1 Serology 479 system in free-living birds 524
18.6.2 PCR/culture 480 20.3.1 Ontogeny 524
Acknowledgments 480 20.3.2 Parental transmission of
References 480 antibodies 525
20.4 Factors causing variation in immune
19. Comparative immunology of responses 526
20.4.1 Age-related variation 526
agricultural birds 489
20.4.2 Social environment 527
Ursula Schultz and Katharine E. Magor 20.4.3 Condition, nutrition and
19.1 Introduction 489 individual quality 528
19.2 Innate immunity 490 20.4.4 Seasonality/annual cycles 530
19.2.1 Toll-like receptors 490 20.4.5 Parasite exposure 531
19.2.2 Retinoic acid induced gene-I 20.4.6 Other factors with
(R)-like receptors 491 immunomodulating effects 531
19.2.3 Antimicrobial peptides 492 20.5 Molecular variation and evolution in
19.3 Cytokines 492 immune genes 532
19.3.1 Interferons 492 20.5.1 The major histocompatibility
19.3.2 Interleukins 493 complex 532
19.3.3 Tumor necrosis factor family 498 20.5.2 Innate immune genes 535
19.3.4 Th2 cytokines 499 20.6 Immune function as an evolving life
19.4 Chemokines 499 history trait 536
19.4.1 CXC chemokines 499 20.6.1 Costs of mounting immune
19.4.2 CC chemokines 500 responses 536
19.5 CCR7 500 20.6.2 Parasite-mediated natural
selection and immune
19.6 Cell surface antigens 500 function 539
19.6.1 Anti-chicken monoclonal 20.6.3 Links with male secondary
antibodies cross-reacting with characters 542
turkey, quail, and duck 20.7 Priorities for future research 544
leukocytes 500 Acknowledgment 545
19.6.2 Evidence for T and B cell References 545
populations in ducks 503
19.6.3 Antigens expressed on duck 21. Advances in genetic engineering
lymphocyte subsets 503
of the avian genome 559
19.6.4 C-type lectin immune
receptors 506 Benjamin Schusser and Timothy Doran
19.6.5 Surface immunoglobulin 506 21.1 Methods to manipulate the avian
19.6.6 Major histocompatibility genome 559
complex 506 21.1.1 Viral vectors 559
19.7 Secreted antibodies 507 21.1.2 Transposons 560
19.8 Cell lines 508 21.1.3 Direct in vivo transfection 561
Acknowledgments 509 21.1.4 Sperm transfection-assisted
References 509 gene editing 561
xvi 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,
University of Memphis, Memphis, TN, United States
Daniel R. Ardia Department of Biology, Franklin and
Marshall College, Lancaster, PA, United States
Jake Astill Department of Pathobiology, Ontario
Veterinary College, University of Guelph, Guelph,
ON, Canada; Artemis Technologies Inc., Guelph, ON,
Canada
Adam Balic Division Infection and Immunity, The
Roslin Institute and R(D)SVS, University of
Edinburgh, Midlothian, United Kingdom
Paul Barrow The School of Veterinary Medicine and
Science, University of Nottingham, Sutton Bonington,
Loughborough, United Kingdom
Richard Beal Ryedale School, Nawton, United Kingdom
Andrew G.D. Bean CSIRO, Health & Biosecurity at the
Australian Centre for Disease Preparedness, Geelong,
VIC, Australia
Cristiano Bortoluzzi Southern Plains Agricultural
Research Center, USDA-ARS, College Station, TX,
United States
Tina Sørensen Dalgaard Department of Animal Science,
Aarhus University, Tjele, Denmark
Fred Davison Institute for Animal Health, Newbury,
United Kingdom
J.J. (Sjaak) de Wit Royal GD, Deventer, The
Netherlands; Utrecht University, Utrecht, The
Netherlands
Jack C.M. Dekkers Department of Animal Science, Iowa
State University, Ames, IA, United States
Timothy Doran Australian Animal Health Laboratory,
CSIRO Health and Biosecurity, Geelong, VIC,
Australia
Dominique Dunon Sorbonne Université, IBPS, CNRS
UMR7622, Inserm U 1156, Developmental Biology
Laboratory, Paris, France
Gisela F. Erf University of Arkansas, Division of
Agriculture, Department of Poultry Science,
Fayetteville, AR, United States
Steven R. Fiddaman Department of Zoology, University
of Oxford, Oxford, United Kingdom
Joel Gautron INRAE, University of Tours, BOA,
Nouzilly, France
Thomas W. Göbel Department for Veterinary Sciences,
Veterinary Immunology Study Group, University of
Munich, Munich, Germany
Rodrigo Guabiraba INRAE, Université de Tours, ISP,
Nouzilly, France
Nicolas Guyot INRAE, University of Tours, BOA,
Nouzilly, France
Sonja Härtle Department for Veterinary Sciences,
Veterinary Immunology Study Group, University of
Munich, Munich, Germany
Maxwell Hincke Department of Innovation in Medical
Education, University of Ottawa, Ottawa, ON,
Canada; Department of Cellular and Molecular
Medicine, University of Ottawa, Ottawa, ON, Canada;
Loire Valley Institute for Advanced Studies, Orléans
and Tours, France, with BOA–INRAE Centre Val de
Loire, Nouzilly, France
Thierry Jaffredo Sorbonne Université, IBPS, CNRS
UMR7622, Inserm U 1156, Developmental Biology
Laboratory, Paris, France
Bernd Kaspers Department for Veterinary Sciences,
Veterinary Immunology Study Group, University of
Munich, Munich, Germany
Jim Kaufman School of Biological Sciences, Institute for
Immunology and Infection Research, Ashworth
Laboratories, University of Edinburgh, Edinburgh,
United Kingdom; Department of Pathology, University
of Cambridge, Cambridge, United Kingdom
Michael H. Kogut Southern Plains Agricultural Research
Center, USDA-ARS, College Station, TX, United
States
Susan J. Lamont Department of Animal Science, Iowa
State University, Ames, IA, United States
John W. Lowenthal CSIRO, Infectious Diseases
Program, Geelong, VIC, Australia
xvii
xviii List of contributors
Katharine E. Magor Department of Biological Sciences,
University of Alberta, Edmonton, AB, Canada
Enrique Montiel Anitox Corporation, Lawrenceville, GS,
United States
Nándor Nagy Department of Anatomy, Histology and
Embryology, Faculty of Medicine, Semmelweis
University, Budapest, Hungary
Venugopal Nair Viral Oncogenesis Group, Pirbright
Institute, Woking, United Kingdom
Imre Oláh Department of Anatomy, Histology and
Embryology, Faculty of Medicine, Semmelweis
University, Budapest, Hungary
Claire Powers University of Oxford, Oxford, United
Kingdom
Michael J.H. Ratcliffe Department of Immunology,
University of Toronto, Toronto, ON, Canada
Johanna M.J. Rebel Department of Animal Health and
Welfare, Wageningen University & Research,
Wageningen, The Netherlands
Sophie Réhault-Godbert INRAE, University of Tours,
BOA, Nouzilly, France
Catherine Robin Hubrecht Institute, Royal Netherlands
Academy of Arts and Sciences (KNAW), University
Medical Center Utrecht, Utrecht, The Netherlands;
Regenerative Medicine Center, University Medical
Center Utrecht, Utrecht, The Netherlands
Karel A. Schat Department of Microbiology and
Immunology, College of Veterinary Medicine, Cornell
University, Ithaca, NY, United States
Ursula Schultz CellGenix GmbH, Freiburg, Germany
Benjamin Schusser Reproductive Biotechnology, TUM
School of Life Sciences, Freising, Germany
Shayan Sharif Department of Pathobiology, Ontario
Veterinary College, University of Guelph, Guelph,
ON, Canada
Michael A. Skinner Section of Virology, Faculty of
Medicine, Imperial College London, London, United
Kingdom
Adrian L. Smith Department of Zoology, Jenner
Institute, Oxford, United Kingdom; University of
Oxford, Oxford, United Kingdom
Kate Sutton Division Infection and Immunity, The
Roslin Institute and R(D)SVS, University of
Edinburgh, Midlothian, United Kingdom
Edwin J.A. Veldhuizen Department of Biomolecular
Health Sciences, Division Infectious Diseases &
Immunology, Faculty of Veterinary Medicine, Utrecht
University, Utrecht, The Netherlands
Lonneke Vervelde Division Infection and Immunity, The
Roslin Institute and R(D)SVS, University of
Edinburgh, Midlothian, United Kingdom
Michal Vinkler Department of Zoology, Faculty of
Science, Charles University, Prague, Czech
Republic
Paul Wigley Department of Infection and Microbiome,
Institute of Infection Veterinary and Ecological
Sciences, University of Liverpool, Leahurst, Neston,
United Kingdom
Anna Wolc Department of Animal Science, Iowa State
University, Ames, IA, United States; Hy-Line
International, Dallas Center, IA, United States
R. Darren Wood Department of Pathobiology, Ontario
Veterinary College, University of Guelph, Guelph,
ON, Canada
Laurent Yvernogeau Hubrecht Institute, Royal
Netherlands Academy of Arts and Sciences (KNAW),
University Medical Center Utrecht, Utrecht, The
Netherlands; Sorbonne Université, IBPS, CNRS
UMR7622, Inserm U 1156, Developmental Biology
Laboratory, Paris, France
Huaijun Zhou Department of Animal Science,
University of California-Davis, Davis, CA, United
States
Foreword
The fascinating world of avian immunology might be best
surmised by the quote from Dr. Jim Kauffman, who stated
with obvious eloquence “chickens are not mice with
feathers.” The point being that while many immunologi-
cal attributes are shared between the species, many are
also different and that we as immunologists must work to
discover them.
In the last two decades, there has been a great increase
in our knowledge of avian immunology. Molecular immu-
nologists, utilizing new technologies, have expanded our
ability to determine and decipher what is encoded in the
avian genome for immunological defense. In addition,
strategic funding capabilities provided primarily, but not
exclusively, by the United States and EU have resulted in
the development of a plethora of monoclonal antibodies
against avian immunological proteins (e.g., cytokines and
cell markers) through “toolbox” grants. These two driving
forces have opened doors and windows in a field that has
historically struggled to provide reagents for intricate
analysis.
This book arrives at a key time in our need for knowl-
edge to handle the ever-changing pressures for sustainable
solutions to poultry production issues on a global scale.
The overall health of birds, in particular the immune sys-
tem, is critical to feed an ever-growing human population.
As an example, the loss of antibiotic treatment for poultry
flocks in most countries has created a need for immuno-
logical intervention strategies to enhance control against
bacterial pathogens. In addition to the applied aspect of
avian immunology, a comparative analysis, including
genomics, function, and evolution, is important to deter-
mine how and why the avian immune system works as it
does.
The avian genome in general is regarded as condensed
in nature to mammalian species in terms of immune gene
repertoire. Immunologically speaking, this translates in
that birds have to do the same (protect the animal) with
less. Despite this, the immune responses between birds
and mammals display similar defining characteristics.
Both species employ an innate and adaptive immune
response to a variety of microbiological incursions from
bacteria, viruses, protozoa, and parasites. However, differ-
ences also stand out. For example, chickens lack draining
lymph node development, which opens an array of
antigen-processing questions for immune recognition and
stimulation. Birds also lack several Toll-like receptors
found in other vertebras, yet share many that recognize
similar molecular motifs. These are but two examples to
highlight the differences, but there are many others.
Perhaps Winston Churchill, in 1939, was also speaking of
avian immunology when he said, “it is a riddle, wrapped
in a mystery, inside an enigma.”
It is impossible to discuss the context of this book
without acknowledging the current global COVID-19
pandemic caused by the SARS-CoV-2 coronavirus. This
relatively novel virus, without existing population immu-
nity, has spread rapidly across transcontinental bound-
aries. These events are reminiscent of reports in the early
1930s when chicks arrived at the veterinary laboratories
of the agricultural college in Fargo, North Dakota. A new
disease was raging across poultry farms in Minnesota and
North Dakota resulting in sick birds that were undergoing
respiratory distress with high mortality. As researchers
investigated the disease, it was eventually identified as a
viral agent and termed infectious bronchitis virus (IBV)
based on disease characteristics. However, it was not until
the advent of electron microscopy in the 1960s that
pathologists were able to get a visual look at the virus and
in 1964 identified “lollipop-shaped” protrusions at the end
of the spikes of the virus that resembled a halo of gas sur-
rounding the sun. The relevance of these observations is
notable in that approximately a year after these publica-
tions, similar morphological characteristics were identi-
fied in virus samples obtained from a boy undergoing
flu-like symptoms. Scientists utilized these morphological
similarities found in both avian and human samples to
identify a new viral family, coronavirus.
Parallels are also found in the immunological arena.
To combat the SAR-CoV-2 virus, in 2020, vaccines have
been developed and approved for use in humans. Of note
is that two of the most used at this time in the United
States and EU are nucleic acid based on the messenger
RNA sequence of the SARS spike (S) gene. The resulting
protein is the main coronavirus immunogen and contains
epitopes for inducing neutralizing antibodies. The rele-
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
possible without the commitment, dedication, and gener-
ous participation of the large number of contributors to
the third edition of this book. The editors are indebted to
them for the considerable amount of work, their enthusi-
asm and willingness to set aside other priorities to con-
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.
The editors also thank Dr. Sonja Härtle for her edito-
rial work on numerous chapters and Dr. Ton Schat for the
pictures of birds on the back cover.
The editors would also like to thank Susan Ikeda for
her efforts to get this project accomplished despite the
problems caused by the ongoing pandemic.

xxi
Chapter 1
The importance of the avian immune
system and its unique features
Fred Davison
Institute for Animal Health, Newbury, United Kingdom
1.1 Introduction
The avian immune system provides an invaluable model
for studies on basic immunology. Birds and mammals
evolved from a common reptilian ancestor more than 250
million years ago and have inherited many common immu-
nological systems. They also have developed a number of
very different, and in some cases remarkable, strategies.
Due to their economic importance, and the ready availabil-
ity of inbred lines, most avian immunology research has
involved the domestic chicken, Gallus gallus domesticus.
A remarkable consequence of this research has been the
seminal contributions it has made to understand the funda-
mental immunological concepts, especially the complete
separation of developing bursa- (B-) and thymus- (T-)
dependent lymphocyte lineages. Some of these observa-
tions have been made by chance, while others have
resulted from painstaking work which took advantage of
special avian features, such as ease of access to, and pre-
cise timing of, all the stages in embryonic development.
Some of the avian findings were described before they
were recognized as important and subsequently explained
in mainstream immunology. The story of avian immunol-
ogy is a fascinating one and by no means complete, as
there is still the need for explanations of a number of
unique features and different strategies adopted by birds. In
this chapter, some of the “firsts,” rightly attributed to avian
immunology, are described and the importance of further
studies in avian immunology is highlighted.
1.2 The contribution from avian
lymphocytes
The advent of modern cellular immunology, and the funda-
mental role that lymphocytes play, is generally credited to
Avian Immunology. DOI: https://doi.org/10.1016/B978-0-12-818708-1.00010-5
© 2022 Elsevier Ltd. All rights reserved.
the 1950s and 1960s, when lymphocyte function became
an active subject for research [1]. The immunological sig-
nificance of lymphocytes emanated from some seminal
studies carried out by Gowans, Chase and Mitchison,
Simonsen, and their contemporaries [1,2]. In elegant
experiments with laboratory mammals and using cell trans-
fers, these workers demonstrated that lymphocytes are
essential for generating immune responses and retaining
memory of previous exposure to an antigen. However, evi-
dence that lymphocytes play such a key role in protection
against infection and in tumor rejection had, in fact, been
discovered almost 40 years earlier [3 6], though little
attention was paid to it [7]. This was almost certainly
because at the time they were made the observations could
not properly be explained and, possibly, because the exper-
imental animal involved was the chicken.
Between 1912 and 1921, James Murphy, an experi-
mental pathologist working at the Rockefeller Institute for
Medical Research in New York, performed a series of
remarkable experiments using chickens and their embryos
to study the growth and rejection of tumor grafts. His
experiments appeared to prove beyond question that the
lymphocyte is the active component in tissue graft rejec-
tion, in protection against infection and, by implication, in
innate and acquired immune responses [7]. Murphy [4]
observed that fragments of rat tumors would not grow in
the adult chicken, just as they did not grow in other spe-
cies (xenogenic rejection). However, they could be grown
on the chorioallantoic membrane (CAM) of developing
chick embryos, although only up to about 18 days incuba-
tion. In older embryos tumor grafts were rejected, just as
they were if grafted onto the newly hatched chick or an
adult bird. Interestingly, Murphy [3] observed that the
grafts which grew on embryos could be transferred to
fresh embryos without any evidence of them being
1
2 Avian Immunology
altered. They also retained their tumorigenic capacity if
regrafted onto a rat. Murphy [3] commented that these
cellular changes occurring when living tissue is grafted
onto an unsuitable host are the same, regardless of the
type of host resistance, be it natural resistance because of
species differences (allogeneic or xenogeneic rejection) or
acquired immunity due to recovery from a tumor
implanted earlier. The histological picture consisted of
edema surrounded by fibroplasia in the host tissue, the
budding out of blood vessels, and infiltration of surround-
ing host tissues with small lymphocytic cells. The
inevitable consequence was that cells in the graft died
fairly quickly leaving only a scar. These were quite pro-
found though, at the time, unappreciated observations.
Murphy [4] also performed a series of elegant experi-
ments using adult chicken tissues cografted onto the
CAM with fragments of rat tumors. He observed that
chicken tissues containing an abundant supply of lympho-
cytes, such as the spleen or bone marrow, caused tumor
grafts to be rejected, whereas these were not rejected if
the cografted tissue lacked a rich supply of lymphocytes.
Later on, Murphy [5] showed that after grafting fragments
of adult chicken spleen onto the CAM of a 7-day embryo,
the embryo’s own spleen became grossly enlarged
(splenomegaly). This is the first published record of a
graft versus host response (GvHR). Much later, it was
explained by Simonsen [8] as the immunologically com-
petent lymphocytes of the adult responding to mismatched
major histocompatibility complex (MHC) molecules
expressed by the embryonic cells. The embryonic cells
were recognized as foreign causing the adult cells to repli-
cate and destroy the embryonic host’s lymphocytes. Graft
versus host disease, in which allogeneic bone marrow
transplants recognize the tissues of the graft recipient and
cause severe inflammatory disease, is a serious problem
for immunosuppressed recipients receiving bone marrow
transplants. The phenomenon became a major concern
with the introduction of human bone marrow transplanta-
tion. Nonetheless, the phenomenon was first described
using chick embryos as long ago as 1916.
1.3 Contribution of the bursa of Fabricius
Without doubt, the most significant contribution that stud-
ies on the avian system have made to development of
mainstream immunology was delineating the two major
arms of the adaptive immune system. As already pointed
out, in the 1960 the significance of lymphocytes was just
becoming appreciated, and it was generally accepted that
there are two types of adaptive immune responses:
humoral responses involving antibodies and cellular
responses mediated by macrophages and lymphocytes.
Since antibodies are produced by plasma cells, which
themselves are derived from lymphocytes, it was not
understood how such lymphocytes could be the same as
those cells involved in cell-mediated functions. The bursa
of Fabricius, an obscure sac-like structure attached to the
proctodeal region of the bird’s cloaca (Fig. 1.1), played a
crucial role in unraveling this problem.
The cloacal bursa, takes its name from Hieronymus
Fabricius of Aquapendente (1537 1619), is also known
as Girolamo Fabrizi d’ Acquapendente (Fig. 1.2). He was
a professor of surgery at the University of Padua, Italy,
from 1565 to 1613 [9] and by all accounts a brilliant anat-
omist, embryologist, and teacher. For his pioneering
work, he was later credited in Italian medical science as
the “Father of Embryology.” Fabricius not only carried
out dissections on human cadavers but also extended his
anatomical studies to other species, providing the most
beautiful, detailed drawings of his work. From his obser-
vations of avian anatomy, he surmised that the cloacal
bursa, a hollow structure connected by a duct to the proc-
todeal region of the cloaca, most likely acts as a recepta-
cle for storing donated semen.
“Since the sac is pervious, so that there is an open passage
from the anus to the uterus itself and another from the
uterus to the sac, that is, above and below, and since it is
closed at the other end, I think it is the place into which the
cock introduces and delivers semen so that it may be stored
there” [4].
This is not the case, however, but the role of the
cloacal bursa continued to puzzle researchers over the fol-
lowing 350 years. Some surmised that since the bursa
of Fabricius regresses with sexual maturity, its size
having an inverse relationship with the size testes and
the adrenals, it must be some sort of endocrine or lym-
phoid gland associated with growth and sexual develop-
ment [10].
Over the years many investigated the function(s) of the
bursa including one young researcher, Bruce Glick, work-
ing at the Poultry Science Department at Ohio State
University, United States. Glick surgically removed the
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
the birds for a class demonstration on antibody production.
A group of the chickens was injected with Salmonella
spp. “O” antigen but 1 week later, when the class carried
out tests with blood and antigen, there was no evidence of
agglutination. Chang, somewhat perplexed, reported the
failure to Glick who was able to identify the nonresponder
chickens as those that had been bursectomized. At the time
they did not seem to fully appreciate the singular impor-
tance of this finding [10] but were able to confirm their ini-
tial observations in further experiments and wrote up an
article entitled: “The role of the bursa of Fabricius in anti-
body production.” This was submitted to Science but
 The importance of the avian immune system and its unique features Chapter | 1
rejected on the grounds that further elucidation of the
mechanisms was necessary before the article could be
accepted for publication [10]. The article was subsequently
submitted to Poultry Science [11], where, for a time, it
failed to draw much attention. Several years passed before
the significance of the work was properly appreciated and
mainstream immunologists took an interest in the chicken’s
immune system. It was eventually concluded that the avian
bursa must be essential for antibody-mediated immunity,
whereas the thymus, which also undergoes involution dur-
ing sexual development, is necessary for cell-mediated
immunity [12 14]. Almost a decade after Glick and
Chang’s initial observations [11], Cooper et al. [15]
3
FIGURE 1.1 A dissection showing the
positions of the major lymphoid tissues in
the chick. Insets provide details of: (A) the
seven thymic lobes lying alongside the left
jugular vein with the crop lying beneath;
(B) the spleen can be seen below the pro-
ventriculus, the dark-colored gall bladder
lies alongside; (C) cecal tonsils are found
at the proximal ends of the two blind cae-
ca, close to the ileo-cecal junction; (D) the
ovoid bursa of Fabricius is situated within
the pelvic girdle immediately dorsal to the
cloaca. From: Dr. Sven Reese, Institute for
Anatomy, Histology and Embryology, Faculty
of Veterinary Medicine, LMU Munich.
published their seminal paper on the delineation of the bur-
sal (B) and thymic (T) lymphoid systems in the chicken.
These workers proposed that because of the similarities in
the lymphoid tissues and immune systems of birds and
mammals, a mammalian equivalent for the bursa of
Fabricius must exist and provide a source of B-dependent
lymphocytes to make antibodies. Later this bursa equiva-
lent was identified as bone marrow. The division of the
adaptive immune system into B- and T-dependent compart-
ments has remained a central tenet of immunological think-
ing ever since. The term B-lymphocyte is derived from
“bursa-derived lymphocyte” in honor of that peculiar avian
lymphoid structure which provided the original evidence.
4 Avian Immunology
FIGURE 1.2 Hieronymous Fabricius of Aquapendente, Professor of
Surgery at the University of Padua from 1565 1613. Fabricius was the
first to describe the bursa, which is now known to be a primary lym-
phoid organ, attached by a duct to the proctodeum in birds and essential
for the development of bursal-derived (B) lymphocytes and the avian
antibody repertoire.
1.3.1 Gene conversion and the bursa
Antibodies recognize specific conformational molecular
shapes on their targets through the immunoglobulin (Ig)
molecule variable region. Antigenic shapes are legion so
an immunocompetent individual must be capable of gen-
erating an antibody repertoire with a huge number of Ig
specificities [16]. Different B cells produce Ig molecules
of different specificities, but each B cell is capable of pro-
ducing only one Ig specificity. In man and mouse, the
antibody repertoire of B cells is generated by a process
known as Ig gene rearrangement, which is ongoing
throughout life. In the case of the Ig light chain, genes
that encode the variable region (VL gene segment), a join-
ing region (JL gene segment), and the constant region (CL
gene segment) are spatially separated by noncoding seg-
ments in germline DNA. Noncoding segments are spliced
to allow the joining of VLJL regions in genomic DNA,
while excision of the noncoding section in the RNA tran-
script allows the VLJLCL regions to combine permitting
translation of a functional Ig light chain molecule [17]. In
the case of the Ig heavy chain, a further diversity (D)
gene is involved, making a VHDJH rearrangement, other-
wise the process is similar. Since multiple copies of V, J,
and D genes exist in the mammalian Ig locus (1) random
recombination of the different gene segments, (2) differ-
ent combinations of heavy and light chains paired
together; diversity introduced at the joints between the
different gene segments by the variable addition and sub-
traction of nucleotides and, finally, (3) point mutations
introduced into the rearranged sequence diversifying these
region further (somatic hypermutation), allow for a vast
(B1011) antibody repertoire to be generated [16].
In contrast, the cluster of genes encoding the chicken
Ig light chain has only a single copy of the functional VL
and JL genes [18]. Hence, diversity due to VLJL joining is
very limited and the effects of VJ rearrangement are mini-
mal (a detailed description can be found in Chapter 4).
Likewise, with the Ig heavy chain locus, the presence of
single functional VH and JH genes means that little diver-
sity can be generated through VHDJH rearrangement.
However, clusters of pseudogenes, upstream of the heavy
and light chain Ig loci have a critical role in the genera-
tion of chicken antibody diversity. By a process known as
somatic gene conversion, VL and VH sequences are
replaced with pseudogene sequences. An enormous
amount of diversity is generated by substantial diversity
in the hypervariable regions of the donor V pseudogenes
and somatic gene conversion events accumulate within
single functional VL or VH genes. Perhaps, chickens rep-
resent the extreme situation with only one functional VL
gene, while other species such as the duck have up to four
functional VL genes, although they still use gene conver-
sion to introduce variability. It seems that, uniquely, birds
rely on somatic gene conversion for generating their anti-
body repertoire, which is the equal of that in immuno-
competent mammals. Interestingly, it has recently been
shown that if gene conversion is blocked in chicken
B cells then somatic hypermutation occurs instead [19].
It has also been observed that gene conversion is not just
limited to birds, it also occurs in rabbits [20] pigs, and
other mammalian species, though none appear to rely on
it as the exclusive means for generating the antibody
repertoire.
The striking fact about avian somatic gene conversion
is that it only occurs in the bursa of Fabricius (see
Chapter 4). For instance, if the bursa is destroyed early in
development (60 hours), then those chicks that hatch pro-
duce only nonspecific IgM and are unable to mount spe-
cific antibody responses. In other words, they do not have
an antibody repertoire and are incapable of eliciting typi-
cal responses or isotype switching to produce IgG or IgA.
However, if the bursa is removed much later during
embryonic development, but before 18 days when the B
lymphocytes have begun to migrate from the bursa into
the peripheral lymphoid tissues, then the hatched chicks
lack circulating Ig and are incapable of eliciting specific
antibody responses. We know that prebursal stem cells
enter the bursa between 8 and 14 days incubation and
have already undergone gene Ig rearrangement, probably
in the embryonic spleen and bone marrow, for they
 The importance of the avian immune system and its unique features Chapter | 1
express IgM on the surface (see Chapter 4). Within the
bursa, they undergo rapid rounds of cell division and only
within this unique environment gene conversion occurs.
In the absence of the bursa, an antibody repertoire cannot
be generated, and a major arm of the immune system
becomes nonfunctional. The chicken antibody repertoire
is generated during the late embryonic stage and for a
short period after hatching. As the chick ages, its B cells
undergo additional rounds of somatic gene conversion
and the antibody repertoire becomes expanded until a
mature repertoire is achieved around 5 7 weeks when the
bursa is fully mature. Thereafter, the bursa begins to
regress as sexual maturity approaches and the adult proba-
bly relies on postbursal stem cells in the bone marrow as
the source of B cells.
Of course, generating the antibody repertoire in a burst
of activity in the young animal has its risks. Any pathogen
that targets and destroys bursal cells will have a devastat-
ing effect on antibody-dependent immune responses. One
such virus is the small RNA virus that causes infectious
bursal disease. Infection of the neonate chick with infec-
tious bursal disease virus (IBDV) may cause no clinical
disease but destroys bursal B cells leaving the chick inca-
pable of mounting antibody responses to other pathogens,
although paradoxically there is a good response to IBDV
itself (see Chapter 14). The insidious nature of IBDV
leaves the chick vulnerable to opportunistic infections and
unprotected by subsequent vaccinations. So, relying on
the generation of the antibody repertoire in a single loca-
tion and over a relatively short time span is not without
danger and, perhaps, represents one of the more “risky”
strategies birds have adopted.
1.4 The contribution of the chicken MHC
Pathogens are diverse, cunning, and occupy different
niches within the body of the host. Apart from pathogens
that are found outside of cells, such as Clostridium,
Escherichia coli, and Bordatella, there are intracellular
pathogens that can be found in the cytoplasm or cellular
vesicles. In addition, retroviruses and herpesviruses inte-
grate into the host’s own genome. To match this diversity,
and the different locations where pathogens are found,
higher vertebrates have evolved a number of different
innate and adaptive immunological mechanisms to
improve the chances of survival. Antibodies recognize
conformational epitopes on the pathogen’s molecules but
need to come into direct physical contact to neutralize a
pathogen, as well as recruit cells and other molecules that
bring about disposal. Ig molecules are large and cannot
easily enter a viable cell, so an intracellular pathogen can-
not be recognized by an Ig molecule unless the patho-
gen’s molecules are expressed on the surface of that
infected cells. However, the cellular immune system has
5
evolved a number of methods for recognizing and
destroying cells with intracellular pathogens. These
mechanisms allow the host’s effector lymphocytes to rec-
ognize infected or neoplastic cells through the expression
of proteins that have been proteolyzed within the cell and
are expressed on the cell surface as peptide fragments. In
mammals, the molecules that present peptides on the sur-
face of cells are encoded by a highly polymorphic genetic
region known as the MHC.
The MHC region was originally recognized through
its effects on tissue graft rejection and, of course, GvHR.
Two major types or classes of MHC molecules are
encoded by genes in the mammalian MHC region and
expressed on the surface of cells, class I and II MHC
molecules (more fully described in Chapter 7). Although
both types of molecules are heterodimers: the class I
MHC molecule consists of an alpha-chain encoded by the
MHC together with an invariant β2-microglobulin mole-
cule from a gene outside of the MHC locus; the class II
MHC molecule consists of two peptide chains (α and β)
whose genes are found in the MHC region. Unlike the
MHC class I heterodimer that is present on most types of
cells, expression of class II MHC molecules is restricted
to antigen-presenting cells such as macrophages, den-
dritic, and B cells. During synthesis inside the cell, both
classes of MHC molecules trap peptide fragments in a
cleft on, what is to become, the outer surface of the extra-
cellular domain of the MHC heterodimer. On reaching the
cell surface, these peptides are displayed as peptide:MHC
complexes to signal T cells through their T-cell receptors.
Since even the smallest virus produces a number of pro-
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-
ity of peptide fragments derived, not from the host’s own
cells, but made by pathogens or neoplastic cells. This T-
cell repertoire is developed within the thymus (described
in Chapter 6), where developing T cells with receptors
that recognize self-peptides associated with MHC mole-
cules are eliminated, or inactivated, before they mature to
prevent self-recognition and auto-immunity. Mature T
cells capable of recognizing “foreign” peptides are
released from the thymus into the periphery and become
activated if they recognize peptide fragments expressed
on MHC molecules. Interestingly, the T-cell repertoire in
the chicken is developed in the thymus in a similar way
to that of mammals. There is no evidence for a somatic
gene conversion mechanism such as occurs in the avian
bursa.
In mammals, the MHC is a large and complex region
that contains much redundancy [21]. In the human, it con-
sists of about 4 million base pairs encoding at least 280
genes. Separate regions contain several MHC class I and
class II genes (there are a vast number of alleles) that are
6 Avian Immunology
highly expressed on cells. These regions are separated by
the third region that encodes immune response genes
(class III). Humans express two or three class I molecules
and three or four class II molecules that are highly poly-
morphic. The high polymorphism is probably driven by
the ever-changing variations in pathogens [22,23],
although different haplotypes appear to confer approxi-
mately the same degree of protection against most of the
infectious pathogens. In Chapter 7, Kaufman points out
that the associations between the human MHC and infec-
tious disease are, actually, very slight.
The chicken MHC is known as the B locus since it
was first identified as a serological blood group locus [24]
encoding the polymorphic, and highly immunogenic BG
antigen. This BG antigen is highly expressed on blood
cells and has no known mammalian equivalent. Later, it
was shown that that the B locus must constitute the avian
MHC, because of its strong association with cell-
mediated immune functions, such as graft rejection,
mixed lymphocyte reactions, and GvHR. Remarkably,
and in marked contrast to the large mammal MHC, the
chicken B locus is minute, spanning only 92 kilobases
and encoding 19 genes and making it approximately 20-
fold smaller than the human MHC [25]. Only two copies
each of class I (BF) and class IIβ (B/Lβ) genes are found
within the chicken B locus. The marked differences
between the chicken MHC and its mammalian counterpart
have led Jim Kaufman to argue that the chicken B locus
represents a minimal essential MHC that must have
evolved after birds and mammals separated some 200 mil-
lion years ago. Another striking feature of the chicken
minimal MHC region is that not only does it affect a
number of important cell-mediated immune functions but
also determines life or death in response to a number of
pathogens [26 28]. In Chapter 7, Jim Kaufman develops
the argument that chickens, with a minimal essential
MHC, appear to have adopted a completely different
strategy to that of mammals whose MHC is large and
complex. The close association between the chicken
MHC and disease resistance is fascinating and at first
sight seems to be a suicidal strategy. However, many les-
sons can be learned from studying avian immunology and
its parallel evolution. By investigating the minimal
chicken MHC, light should be thrown on the important
interactions between pathogens and the immune system
and the relevance of the different evolutionary strategies
elucidated.
1.5 Contributions to vaccinology
In any review of the “firsts” credited to avian immunol-
ogy, tribute needs to be paid to pioneering developments
in the practical uses of immunology, in other words the
use of vaccination. The modern poultry industry relies
heavily on vaccines to protect against a wide range of dif-
ferent infectious agents. Vaccinations are frequent and
begin from the day of hatching or even before. Chickens
are immunized with live-attenuated vaccines and killed
vaccines delivered by various routes (injection, aerosol
spray, drinking water, etc.) in mass-vaccination programs
that dwarf such programs in human medicine.
Every biology student knows that Edward Jenner is
the founding father of vaccination. Jenner discovered that
cowpox pustules, obtained from an infected milkmaid,
protected an 8-year-old boy, James Phipps, against the
related smallpox virus. However, further developments in
vaccination, and indeed the term vaccination, only came
into use about a century later, following studies by one of
the greatest 19th-century scientists, Louis Pasteur. Here
again the chicken had a privileged role and serendipity
played its part.
In 1878 Pasteur was investigating chicken cholera, a
disease with devastating effects, causing chickens to
become anorexic, moribund, and usually leading to their
death. Pasteur investigated the causative agent, now
known as the Pasteurella multocida, and succeeded in
growing the bacteria in culture. The story goes that
Pasteur’s research was interrupted by a holiday and a cul-
ture was left in a flask in the laboratory [29]. Upon
resuming his research, Pasteur inoculated chickens with
this stale culture. The chickens became sick but then
recovered within a few days. We now know that the bac-
teria had become attenuated and no longer capable of
causing mortality. Pasteur did not know this and decided
to inject new chickens with a fresh bacterial culture but,
unfortunately (or fortunately!), his assistant found that
chickens at the local market were in short supply. A num-
ber of fresh birds were obtained but those that had recov-
ered from the inoculation with the stale culture needed to
be reused. As expected, the new chickens all succumbed
to the fresh pathogen and died but those that had recov-
ered from the previous treatment with stale inoculum
again recovered. Pasteur realized he had achieved with
chicken cholera what Jenner had accomplished with
smallpox some 100 years earlier, only in this case he had
attenuated (weakened) the pathogen by prolonged storage.
He called the attenuated culture a “vaccine” [30] in honor
of Edward Jenner and then began a, largely successful,
search for similar vaccines against other infectious dis-
eases such as pig erysipelas, sheep anthrax, and rabies.
The serendipitous discovery of attenuation was another
novel finding made using the chicken. Since then, the
development of vaccines has had far-reaching implica-
tions for the health and welfare of both humans and
domesticated animals. The search for better, more effec-
tive vaccines still goes on apace.
Another first in vaccine development was in the con-
trol of Marek’s disease (MD) a naturally occurring
 The importance of the avian immune system and its unique features Chapter | 1
neoplastic disease of chickens. MD became the scourge
of the poultry industry in the 1950s and 1960s, causing
major problems for animal health and welfare and becom-
ing a huge financial burden. Before the introduction of
MD vaccines, morbidity and mortality in laying flocks
ranged from 0% 60% or greater, with losses of 30%
being common [31]. The development of an MD vaccine
represents the first example of widespread use of vaccina-
tion to protect against a natural form of cancer [32]. Over
the years it has been remarkably effective [33], although
not without problems.
MD was first described as a neurological disease
(polyneuritis) by Josef Marek [34]. The condition caused
paralysis of the wings and legs and was associated with
mononuclear infiltrations and enlargement of the major
nerves. Later it was observed [35,36] that in addition to
lesions in the nerves and central nervous system, chickens
also developed lymphoid tumors in several visceral tis-
sues (visceral lymphomatosis) such as the ovary, liver,
kidneys, adrenal, and muscles. With intensification of
poultry production, the acute form of MD became a domi-
nant feature. Although the introduction of MD vaccines in
the 1970s controlled the disease, in some countries pro-
blems with vaccine breaks have continued to occur with
regularity and there is now good evidence that the causa-
tive herpesvirus (MDV) has been able to evade vaccine-
induced immune responses by evolving to greater viru-
lence. Since the 1980s the response of the industry in
some countries has been to introduce more aggressive
vaccine strategies, using “hotter” vaccines, such as
CVI988, either alone or in combination with other MD
vaccines (bivalent or trivalent combinations). The most
efficacious current MD vaccine CVI988 is derived from a
serotype 1 MDV that is weakly oncogenic in genetically
susceptible chickens and this has led some to raise the
important question [37]: where do we go if hypervirulent
MDV pathotypes evolve that can break through the pro-
tection of CVI988?
MDV is not the only example of a poultry virus that
has changed in response to the introduction of widespread
use of vaccines. More virulent isolates of another lympho-
tropic virus, IBDV, were isolated in the late 1980s. IBDV
is a small double-stranded RNA virus that encodes only
five viral proteins. As already pointed out, IBDV targets
B lymphocytes in the bursa of young chicks causing no
clinical signs in neonates but causing clinical disease and
some mortality in older chicks (see earlier). Chicks are
protected by maternal antibodies derived via the yolk, but
it became clear in the late 1980s that the very virulent
IBDV being isolated from outbreaks was capable of caus-
ing disease in the presence of high levels of maternal anti-
bodies. The response of the industry has been to introduce
more aggressive (hotter) vaccines to protect against the
more virulent IBDV strains that have evolved under the
7
pressure of vaccine use. The risk, however, is that these
hotter vaccines could themselves be capable of causing
bursal damage and immunosuppression in chicks that are
poorly protected by maternal antibodies or have a suscep-
tible genotype. Here again, we have an example of a strat-
egy that is holding at present but may not be sustainable
long term. More aggressive vaccines or vaccination
regimes cannot be introduced without the risk that the
vaccines themselves could be harmful.
These issues have been addressed in epidemiological
studies on implications of the use of vaccines on the evo-
lution of pathogen virulence. Researchers [38] were
chiefly concerned with the use of different vaccines
developed against the malaria parasite and its implications
for human populations. Mathematical modeling was car-
ried out based on the premise that most vaccines are
imperfect and rarely provide full protection from disease.
Using various models, these authors predicted that vac-
cines designed to reduce the growth and/or toxicity of
pathogens actually diminished selection pressure against
more virulent pathogens. Consequently, subsequent patho-
gen evolution may lead to higher levels of intrinsic viru-
lence and hence more severe disease in unvaccinated
individuals. Such evolution would tend to erode any
population-wide benefits, such that overall mortality
could increase with the level of vaccination coverage.
Interestingly, the authors found evidence of this phenome-
non in the practical problems arising from MD vaccina-
tion. Current MD vaccines target viral replication and do
not prevent infection with MDV, consistent with mathe-
matical modeling that predicts the evolution of pathogen
virulence. Yet again, evidence from work on the chicken
has proved to be the pathfinder and pointed to important
problems to take account of in vaccine design.
1.5.1 Embryonic (in ovo) vaccination
The problems associated with challenge from virulent
MDV when chicks are moved into rearing quarters, the
vast numbers of chicks requiring vaccinations at the
hatchery, as well as the occasional failures caused by
manual vaccination, has led to a search for new ways for
mass vaccination that can be done at an even earlier stage
than the day-old chick. Sharma and colleagues at the East
Lansing Regional Poultry Laboratory, United States dem-
onstrated that chick embryos could be successfully vacci-
nated against MDV at 17 18 days incubation [39,40].
The automated INOVOJECT system, which allows the
automated mass application of vaccines to large numbers
of eggs (up to 50,000 eggs per hour, [41]), was developed
and has been widely applied in the poultry industries of
some countries (for a fuller description see Chapter 18).
In the United States, almost all broilers (over 7 billion per
year) are vaccinated by this method. In ovo vaccination is
8 Avian Immunology
achieved by puncturing a small hole through the blunt
end of the egg with an oblique pointed needle then pass-
ing down a smaller needle to deliver a small amount (usu-
ally 50 μL) vaccine into the amniotic cavity. Since the
amniotic fluid is imbibed prior to hatching the vaccine is
then taken up by the embryo. Interestingly, the reasons
why in ovo vaccination, applied to such an immunologi-
cally immature individual, is so effective remains to be
fully explained. Nevertheless, the finding that a higher
vertebrate can be protected against challenge early after
hatching (birth) by vaccinating the embryo is quite revo-
lutionary and its practical application in mass vaccination
a great achievement. The immunological explanations
will surely come in due course
1.6 Conclusion
Compared to those of mouse and man the avian immune
system may seem like a poor relation and yet it has pro-
vided important insights into fundamental immunological
mechanisms and can claim a number of important “firsts”
especially with regard to vaccinology. From the immuno-
logical viewpoint, the chicken is, perhaps, the best-
studied nonmammalian species. The publication of the
chicken genome [42] and recent developments in reverse
genetic technology (see Chapter 21) provide the opportu-
nity for a “quantum shift” in the search for new chicken
genes and exciting possibilities for developing new tools
and reagents to study immune responses and immunoge-
netics. Interest in studying immune responses of other
avian species is increasing, including species of wild
birds. Ecologists are now taking an interest in measuring
immunocompetence and determining its importance as a
heritable trait for the survival, both of the individual and
the population. Avian immunology is a fascinating and
growing field and will surely provide new and exciting
insights for mainstream immunology in the future.
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Chapter 2
Structure of the avian lymphoid system
Nándor Nagy1, Imre Oláh1 and Lonneke Vervelde2
1
Department of Anatomy, Histology and Embryology, Faculty of Medicine, Semmelweis University, Budapest, Hungary, 2Division Infection and
Immunity, The Roslin Institute and R(D)SVS, University of Edinburgh, Midlothian, United Kingdom
2.1 Introduction
Understanding the physiology and immunology of the
lymphoid system is handicapped without knowledge of its
basic structure. Lymphomyeloid tissues develop from epi-
thelial (bursa of Fabricius and thymus) or mesenchymal
(spleen, lymph nodes, bone marrow) anlages, which are
colonized by blood-borne hemopoietic cells. In the case
of central lymphoid organs, hematopoietic stem cells
enter the thymic or bursal anlages and develop to become
immunologically competent T and B cells, respectively.
Hence, T and B cells are of extrinsic origin, as proposed
by Moore and Owen [1,2].
Immunologically mature cells enter the circulation and
colonize the peripheral lymphoid organs: spleen; Harderian
gland; and gut-, bronchus-, and skin-associated lymphoid tis-
sues. This peripheralization process can be experimentally
manipulated by surgical or chemical interventions. Homing
of lymphocytes may take place through the high endothelial
venules (HEV), which are located in the T-dependent zones
of the peripheral lymphoid organs. In these organs, the T
and B cells occupy different compartments, called T- and B-
dependent zones, respectively. In the avian spleen, the T-
dependent zone—called the periarterial lymphatic sheath
(PALS)—surrounds the splenic central artery. In other lym-
phoid organs, this is not well defined, and the interfollicular
region is regarded as a T-dependent area. B-cell compart-
ments are the germinal centers (GCs). In addition to the GC,
the periellipsoidal white pulp (PWP) of the spleen is also a
B-dependent region, like the marginal zone of the mamma-
lian spleen. Possibly, this B-cell population of the PWP is
responsible for antibody production against bacterial capsu-
lar antigens (e.g., Pneumococcus). Splenectomy of the
pigeon abrogates the antibody response, and the birds die
soon after Pneumococcus infection [3].
The splenic anlage develops from the dorsal meso-
gastrium, ventral to the notochord, and its ablation at
Avian Immunology. DOI: https://doi.org/10.1016/B978-0-12-818708-1.00027-0
© 2022 Elsevier Ltd. All rights reserved.
the lower thoracic level abrogates splenic development,
suggesting a notochordal inductive effect.
Hematopoietic colonization of the splenic anlage starts
at around 6.5 days of incubation, identical to that of the
thymus. It is worth mentioning that, in the spleen, not
only the cells of the hematopoietic compartment but
also other resident cells, such as the ellipsoid-
associated cells (phagocytic) and supporting cells of the
ellipsoid (capable of collagen production), are of blood-
borne origin [4,5].
Several peripheral or secondary lymphoid tissues can
be distinguished in the chicken, most notably the spleen
and all mucosa-associated tissues, such as the eye-
associated lymphoid tissues (the Harderian gland (HG)
and the conjunctiva-associated lymphoid tissue) of the
lower eyelid; the nasal-, bronchus-, genital- and gut-
associated lymphoid tissues (esophageal and pyloric ton-
sils, Peyer’s patches (PP), cecal tonsils, and Meckel’s
diverticulum); and the skin- and pineal-associated lym-
phoid tissues.
Secondary lymphoid organs begin to develop in predi-
lected sites before hatching. Their formation requires the
so-called lymphoid tissue inducer cells (Lti), which have
been identified in the mouse and the human [6] but not
yet in the chicken. Further maturation of the lymphoid tis-
sues is antigen driven, as can be demonstrated in germ-
free chickens [7].
Lymphoid organs are highly compartmentalized. Each
anatomical compartment has its own arrangement of Lti
cells in B cell- and T cell-dependent areas. The different
types of nonlymphoid cells create a specific microenvi-
ronment in each of the compartments. Separate areas exist
where antigen presentation to T cells by nonlymphoid
cells occurs, T cells interact with B cells, and immuno-
globulin production takes place. It is therefore important
to realize that in vitro studies generate information on the
possible interactions between cells, though some of these
11
12 Avian Immunology

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.

2.2 The thymus

2.2.1 Anatomy and histological organization
The avian thymus lies parallel to the vagus nerve and
internal jugular veins [9]. On each side of the neck are
7 8 separate lobes, extending from the third cervical ver-
tebra to the upper thoracal segments [10]. Each lobe is
encapsulated with a fine fibrous connective tissue capsule
and embedded in adipose tissue (refer to Chapter 1,
Fig. 1.1). From the capsule, septae invade the thymic
parenchyma and incompletely divide the lobe into
lobules. The thymic lobes reach a maximum size of
6 12 mm in diameter by 3 4 months of age, before
physiological involution begins [11]. The parenchyma of
each lobe consists of a centrally located uniform medulla
surrounded by a lobulated cortex. Septae end at the
FIGURE 2.1 The detailed structure of the thymus. (A) Hematoxylin-
eosin-stained thymic lobe. The undivided pale-stained medulla is sur-
rounded by lobulated heavily stained cortical lobules. (B) Schematic
illustration of the avian thymus. Thymic dendritic cells are located in the
epithelial-free zone of the thymic medulla, concentrated around the
blood vessels. (C) The densely packed cortical thymocytes result in
basophilic staining. The large, pale nuclei belong to the epithelial reticu-
lar cell (ERC) (outlined). Granules show different electron densities, and
the membrane-bound vesicles contain a fine flocculated substance. (D)
Attachment spots between the cortical thymocytes and the ERC. epith,
Epithelial reticular cells, M, medulla, C, cortex. (B) Adapted from Nagy
et al., Dev Comp Immunol; 2016. 58: 47 59

also found. Septae have a rich cellular composition con-
sisting of fibroblasts, plasma cells, lymphocytes, and,
occasionally, a few granulocytes. The surface of the
lobules is isolated from the capsule and septae by a basal
lamina (BL). The connective tissue of the thymic capsule
and septae develop from the cells of the cranial neural
crest. Lack, or impaired migration, of neural crest cells to
the third and fourth brachial arches results in a thymic
condition such as Di George syndrome in humans and
nude mice.
During embryonic development, the thymic mass
gradually increases with the colonization of hematopoietic
stem cells, and this rapid increase, a few days before
hatching, results in the appearance of the medulla. During
the formation of the thymus, invasion of the connective
tissue septae results in cortical lobulation, which coin-
cides with the emigration of T cells from the thymus.
The thymic reticulum develops from the endoderm of
the third and fourth branchial pouches [12]. In the epi-
thelial anlage of the thymus, immature T lymphocytes
proliferate in the subcapsular zone, from where they
migrate toward the medulla during T-cell maturation.
Immunologically competent cells leave the thymus via
medullary postcapillaries.
2.2.2 Thymic cortex
Pale-stained, fine cytokeratin meshes of the epithelial
reticular cells (ERC) in the cortex are densely packed
with thymocytes, contributing to cortical basophilia
(Fig. 2.1A and B). Moderate numbers of macrophages
also occur in the network of cortical epithelial cells.
Large- and medium-sized lymphocytes are situated under
the surface epithelial cells, which may proliferate, sug-
gesting that the subcapsular zone of the cortex is the
major site of proliferation. During T-cell maturation, the
cells migrate toward the corticomedullary border, where
macrophages and thymic DC sentinel and select the thy-
mocytes before these enter the medulla and circulation
via the medullary postcapillaries.
The fine cytokeratin network of the cortex is formed
by the cortical ERC (Fig. 2.1C). Two types of ERC can
be identified, though it is difficult to distinguish between
them cytologically. One is located on the surface of the
lobe and contains few, or no, cytoplasmic granules, but
expresses more cytokeratin than cortical ERC. Possibly,
its major function is to isolate the cortical parenchyma
from the mesenchyme [13,14], which surrounds the thy-
mic lobe (Fig. 2.1B) and participates in the cortical blood-
thymus barrier. These cells are connected by
desmosomes to one another and to the cortical ERC. The
latter cells have large, irregular-shaped nuclei with evenly
dispersed chromatin substances (Fig. 2.1C). The cyto-
plasm is rich in mitochondria and loaded with granules,
Structure of the avian lymphoid system Chapter | 2 13
which exhibit variations in shape and electron density
(Fig. 2.1D). The ends of the cytoplasmic processes con-
tain moderately developed smooth- and rough-surfaced
endoplasmic reticulum. DEC205 (CD205) is expressed by
the cytokeratin1 reticular epithelial cells of the cortex.
Their relationship to the cortical thymocytes is a remark-
able cytological phenomenon. Between each epithelial
cell and thymocyte, several adhesion points occur, indi-
cating that cell-to-cell contact may be significant in T-cell
maturation (Fig. 2.1D), which suggests a nursing function
for ERC [15]. Several granules contain very loose, low-
density material, suggesting partial release of their granu-
lar contents (Fig. 2.1D) and thus giving the appearance of
vesicles. This type of cell occurs exclusively in the cortex.
Macrophages, identified by the CVI-ChNL-74.2 and
KUL01 (MRC1L-B) monoclonal antibodies [16], are scat-
tered over the cortex and medulla (Fig. 2.2F). The cortex
is loaded with CD81 cells, while the medulla has far
fewer CD8α1cells (Fig. 2.2D).
2.2.3 Thymic medulla
The large number of epithelial and highly variable
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
types, but it is not known whether they represent actual
cell types or stages of differentiation. The functional sig-
nificance of epithelial cells is not known, but an endo-
crine function has been suggested [17,18]. The two
medullary regions can be distinguished in sections stained
with anticytokeratin (Fig. 2.2A). Over the medulla, rough
cytokeratin positive “cords” form a 3D network, which
initiates at the corticomedullary border joining to the fine
cortical cytokeratin1 meshwork. The larger part of the
medulla among the cords is keratin-free and shows direct
connection with the connective tissue of the interlobular
septae. This may indicate a common developmental origin
for the medulla and the connective tissue of the thymic
capsule and interlobular septae. The blood vessels of the
medulla locate in the keratin-free regions, and around the
postcapillaries of the medulla, the CVI-ChNL-74.31 DC
are localized (Figs. 2.1B and 2.2C) [13].
The classical histological structure in the thymic
medulla is Hassall’s corpuscle, an epithelial cell aggrega-
tion (Fig. 2.2B). In chickens, Hassall’s corpuscles are
small, poorly developed structures, unlike their human
counterparts. Their origin remains an enigma, although it
has been proposed that they are the result of the turnover
of epithelial cells since the center frequently shows kerati-
nization like that of the skin epidermis. Some regard
Hassall’s corpuscles as a place of thymocyte degenera-
tion, though thymocytes are rarely observed in them,
unlike neutrophils (mammals) or heterophils (birds).
14 Avian Immunology
These granulocytes are probably scavenger cells, elimi-
nating Hassall’s corpuscles. Over the past quarter-century,
several researchers have reported the production of
biologically active substances, such as alpha-
naphthylesterase and leucinaminopeptidase [19], interleu-
kin (IL)-7 [20], transforming growth factor-α [21], CD30
ligand [22] and an IL-7-like cytokine called thymic stro-
mal lymphopoietin (TSLP) [23,24]. TSLP is produced by
Hassall’s corpuscles in the human thymus, and TSLP
receptors are preferentially expressed by immature medul-
lary myeloid DC. TSLP activates myeloid DC, which
generates regulatory T cells in the thymic medulla [24].
The high-affinity receptor for TSLP and the IL-7 receptor
(IL-7R) share the IL-7Rα chain. The chicken IL-7Rα
chain is expressed on many cortical thymocytes (less so
toward the capsule) and some medullary cells, suggesting
a T cell maturation-dependent expression [25] similar to
that of the mammalian thymus [26]. The location and
FIGURE 2.2 The detailed structure of the
thymus. (A) Pan-cytokeratin immunostaining
shows a very fine network in the cortex, while
the space of the septae (arrow) is continuous
with the epithelium-free medullary (efm)
region. The medullary epithelium has a rough
cytokeratin network. (B) Hassall’s body (H)
shows a concentric appearance. (C) 74.3 anti-
gen is expressed by thymic dendritic cells
(red) located in the reticular epithelial-free
zone (green) of the medulla. (D) The pattern
of the CD8α1 cytotoxic T cells in the thymus.
(E) CD41 helper T cells in the thymus. (F)
The CVI-ChNL-74.2 monoclonal antibody
shows uniformly scattered macrophages over
the cortex and medulla.
pattern of the CD81 and CD41 cells of the thymus are
shown in Fig. 2.2D and E.
Skeletal muscle cells are a common feature in the thy-
mic medulla (Fig. 2.3A). Some are round or ovoid in
shape with striated myofibrils encircling the nucleus [27].
Others are elongated with their ends appear Y-shaped.
There are no signs of innervation to these skeletal
muscles.
Thymic corticomedullary border
The corticomedullary border contains a DC barrier which
expresses the major histocompatibility class (MHC) II
antigen and may play a role in the negative selection of T
cells [28]. However, our observations, based on immuno-
cytochemistry with anti-MHC II and CVI-ChNL-74.3,
indicate that the thymic DC accumulate in the
cytokeratin-free medullary region (Fig. 2.2C) [13]. The
CVI-ChNL-74.31 thymic DC coexpress CD83 [14,29]. In

addition to thymic DC, a large number of peroxidase-
positive cells occur at the corticomedullary border and
may also contribute to the negative selection of T cells
[30]. These endogenous peroxidase-positive cells gener-
ally form groups, and single cells occur between them
(Fig. 2.3B). Their irregularly shaped granules show granu-
lolysis, and the cell surface is highly ruffled (Fig. 2.3C).
Peroxidase-positive cells do not have segmented nuclei,
and the ultrastructure of their cytoplasmic granules is not
comparable to that of eosinophilic granulocytes.
Granulocyte-specific mAbs (Grl-1 and Grl-2) do not rec-
ognize these thymic peroxidase-positive cells.
2.3 The bursa of Fabricius
2.3.1 Anatomy and histology
The chicken bursa of Fabricius has the shape and size of
a chestnut and is located between the cloaca and the
sacrum (Fig. 1.1). A slot-like bursal duct provides a con-
tinuous and free communication between the proctodeum
Structure of the avian lymphoid system Chapter | 2 15
FIGURE 2.3 The detailed structure of the
thymus. (A) A skeletal muscle cell (myoid) in
the medulla shows regular striations (I and A
bands of the sarcomere). Peroxidase-positive
cells (arrow). (B) At the corticomedullary bor-
der, there is a peroxidase1 cell barrier. The
lobe and the corticomedullary border are out-
lined. The peroxidase1 cells form small
groups, suggesting their clonal origin (inset).
(C) An electron micrograph of the peroxidase-
positive cell. The irregularly shaped nucleus is
surrounded by granules. Many of them show
granulolysis.
and the bursal lumen. As a diverticulum of the cloaca, the
bursa is lined with a cylindrical epithelium thought to be
of endodermal origin; however, a recent experimental
study proposed an ectodermal origin [31]. The bursa
reaches its maximum size at 8 10 weeks of age; then,
like the thymus, it undergoes involution. By 6 7 months,
most bursae are heavily involuted [11].
The bursa, like other hollow organs, is surrounded by
a thick, smooth muscle layer. Studies generally neglect
this muscle coat, and its contractility is not considered in
bursal function. During muscle contraction, compression
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
longitudinal folds emerge, resulting in a slit-like space.
During muscle contraction, the surfaces of the folds come
into contact with one another, so the bursal lumen is
almost a virtual space. Inside each fold, follicles are orga-
nized into two layers separated by axial structures (arter-
ies, veins, lymphatics, and connective tissues) (Fig. 2.4).
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
the ventral folds, peripheral (secondary) lymphoid tissue
can form.
2.3.2 Bursal surface epithelium
The surface epithelium of each fold consists of an inter-
follicular epithelium (IFE) and a follicle-associated epi-
thelium (FAE) [32] that form about 90% and 10% of the
surface, respectively (Fig. 2.5A) [33]. IFE is columnar
and produces a mucin-like substance, which is released
into the bursal lumen and lubricates the surface of the
folds. The columnar epithelium is pseudostratified, since
there is a layer of cuboidal basal cells with densely
stained cytoplasm. Their function is not known. Perhaps
these cells are predestined for the IFE, since the surface
epithelium does not proliferate and no epithelial stem cell
has been identified. The IFE rests on a basement mem-
brane (BL), continuous with the corticomedullary border
separating the medulla from the cortex (Figs. 2.4 and
2.5B).
FAE covers the bursal fold above the follicles and pro-
vides a direct connection between the follicular medulla
and the bursal lumen (Figs. 2.4 and 2.5B). Gamma-actin
recognized by GIIF3 monoclonal antibody (Fig. 2.5C) is
expressed by guinea fowl bursa of Fabricius FAE [34,35].
Using CSF1R-transgenic chickens, Balic et al. showed
that FAE comprised a mixture of CSF1R1 epithelial cells
(Fig. 2.5C’) and CSF1R2 macrophage-like cells expres-
sing phosphatidylserine receptor T-cell immunoglobulin
and mucin domain-containing 4 antigen (TIM4) [36]. A
special smooth-surfaced vesicular system in the apical
zone of the FAE cells may contribute to bidirectional
transport by these cells (as proposed by Bockman and
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
CSF1R2 FAE (Fig. 2.5D). Antigen or particles can gain
access to the medulla from the bursal lumen [33,37,38],
and in the opposite direction products of bursal secretory
DC (BSDC) are also taken up by the FAE [34,38]. During
late embryonic life, such products appear in large quanti-
ties in the medulla and enter the FAE’s intercellular
space. FAE cells take up and possibly secrete these pro-
ducts into the bursal lumen [39]. Some FAE cells express
TIM4, CD44, and nonspecific esterase, like macrophages,
which suggests their extrabursal origin. Interestingly,
these cells do not stain with typical monocyte/macrophage
markers, like CVI-ChNL-68.1, CVI-ChNL-68.2, CVI-
ChNL-74.2) or KUL01 (mannose receptor MRC1L-B),
and may represent senescent BSDC that have migrated
from the medulla [40]. Anti-pan-cytokeratin staining indi-
cates the reduced level or absence of keratin-intermediate
filaments in the FAE, although each epithelial medullary
component expresses cytokeratin (Fig. 2.5E).
The FAE is an attachment point between the follicle
and the surface epithelium, so the number of FAE attach-
ments is identical to the number of follicles (Figs. 2.4 and
2.5F). Under each FAE, the BL is absent and (unusual for
an epithelium) replaced by 2 3 layers of squamous epi-
thelial cells, known as FAE-supportive cells (Figs. 2.4
and 2.5D). The FAE cells are connected to the FAE-
supportive cells by desmosomes. The flat FAE-supportive
cells, together with the corticomedullary epithelial cells,

envelop the medulla, separating it from the FAE and cor-
tex, respectively. The FAE is devoid of lymphocytes
(Fig. 2.5D). However, because classical M cells can stim-
ulate lymphocytes, the absence of any lymphocytes in the
FAE questions whether these FAE cells are in fact classi-
cal M cells.
Soluble substances dropped onto the cloacal lips are
rapidly sucked into the bursal lumen [33,36,38,41,42].
The mechanism of this suction is not known, although it
may be due to negative pressure within the bursal lumen.
The bursal duct is flat, not circular, and therefore gener-
ally closed. Absorption of the air from the bursal lumen
can result in negative pressure, with contraction of the
cloacal sphincter opening up the bursal duct. By this
mechanism, bacteria, viruses, and tracers can gain access
into the bursal lumen, to be absorbed by the FAE
(Fig. 2.5D and F). The FAE takes up colloidal carbon,
Structure of the avian lymphoid system Chapter | 2 17
FIGURE 2.5 The structure of the
bursa of Fabricius. (A)
Hematoxylin-eosin staining indi-
cates the follicle-associated epithe-
lium (FAE) and the interfollicular
epithelium (IFE). The FAE is
above the medulla. (B) Antilaminin
immunostaining indicates that the
basal lamina (BL) under the inter-
follicular epithelium (IFE) con-
tinues at the corticomedullary
border (arrow). The FAE, the
medulla (M), and the cortex (C).
(C, C’) Gamma-actin1 and
CSF1R1 FAE cells. (D) The FAE
sits on top of the follicle and is
separated from the IFE by a shal-
low epithelial trough (bracket). In
the FAE, three macrophage-like
cells can be seen. Under the FAE,
the basal lamina is absent. A large
number of bacteria are attached to
the FAE. (E) Anticytokeratin stain-
ing identifies IFE, FAE-supportive
cells (epithelial trough), and ERC.
(F) A carbon particle suspension
dropped onto the anal lips is
sucked up into the bursal lumen.
The carbon is absorbed by the
FAE, allowing counting of the
number of follicles. Each black dot
covers one FAE.
providing a means to count the number of follicles, which
is estimated to be 20,000 per bursa. During folliculogen-
esis, there is no cell migration between follicles.
Therefore assuming a minimum of 1 B cell precursor per
follicle, this means a minimum of 20,000 pre-B cells are
required for bursal colonization [33].
2.3.3 Bursal follicle
Each bursal follicle consists of a medulla and a cortex
with a closely associated (both structurally and function-
ally) but not integral FAE [32]. During bursal ontogeny,
the medullary anlage emerges on embryonic day 11 12
(E11 12) and is soon followed by FAE formation
(E14 15), with the first cortical cells appearing around
hatch [43 45]. The cortex is fully developed by 2 weeks
post-hatch (see Chapter 3).
18 Avian Immunology
Bursal folds are filled with follicles, which are flat-
tened, oval-shaped and B0.2 0.4 mm in diameter
[33]. A collagen-rich capsule surrounds each follicle,
which represents the structural, functional, and patho-
logical bursal unit. Each follicle has its own blood sup-
ply independent of neighboring follicles. Small
precapillaries branch from the main artery in the fold
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
not in the cortex. Early studies concerning phagocytic
capability in the bursa support the existence of a
blood bursa barrier, as the bursa contains no phagocy-
tosed substances [46].
The corticomedullary BL provides a complete separa-
tion of the medulla from the cortex (Fig. 2.5B); these two
regions are different histologically, ontogenically, and
possibly functionally. Supporting cells in the medulla and
the cortex are respectively of epithelial and mesenchymal
origin.
2.3.4 Medulla
The medulla, like the thymus, is a classical lymphoepithe-
lial tissue. It consists of epithelial cells, which originate
from the anal invaginations of ectoderm [31], and blood-
borne hematopoietic cells, including lymphoid cells, DC,
and perhaps macrophages; few plasma cells occur in the
involuting bursa.
2.3.5 Bursal medullary epithelial cells
The ERC form a cellular network in the medulla, and
there is no immunocytochemically identifiable extracellu-
lar matrix (ECM). This is unlike the cortex, where the
desmin1 mesenchymal reticular cells (MRC) produce an
ECM. Epithelial cells can be classified according to their
location and cellular structure. The superficial epithelial
cells form a layer on the surface of the medulla. At the
corticomedullary border, they form arches on the medul-
lary side and are covered by a BL on the cortical side.
Under the FAE, the arch-forming cells become flat and
FIGURE 2.6 The structure of the
bursa of Fabricius. (A) The MEP-
21 (antithrombomucin) monoclonal
antibody recognizes thrombocytes
and endothelial cells. There is a
dense capillary network along the
corticomedullary basal lamina in
the cortex; there are no blood ves-
sels inside the medulla. (B)
Epithelial cells of the corticome-
dullary border form arches, which
are filled with blast-like cells
(bracket). The arch opening toward
the medulla and blast-like cells join
to the medulla. BL, basal lamina;
(C) Both medulla and cortex are
densely packed with chB61 cells,
but the corticomedullary border
appears as an unstained, light line.
(D) An electron micrograph of the
medulla showing that the mature
dendritic cell (DC) has an eccentri-
cally located nucleus with a large
cell process that is loaded with
electron-dense granules. The mod-
erately ruffled cell surface is cov-
ered by a fine filamentous
substance. (E) A macrophage-like
cell in the medulla, loaded with
apoptotic lymphocytes in different
stages of digestion. (F) A detail of
a macrophage-like cell. The surface
membrane is covered by a fine
flocculated substance like the DC.

serve as supportive cells for the FAE, replacing the BL.
Hence, the interior of the medulla is separated from the
cortex and FAE. The corticomedullary epithelium enclose
CD451 lymphoblast-like cells (Fig. 2.6B), which express
neither B cell (e.g., chB6, CXCR4, and CD1) nor BSDC
(e.g., vimentin, IgY, CSF1R and 74.3) or macrophage
(TIM4, 74.2, 68.2) antigens (Fig. 2.6C). Delta-Notch sig-
naling in the corticomedullary border suggests that the
function of the arch-forming cells differs from that of the
medullary ERC [47]. These corticomedullary epithelia
may have nursing or regulatory function for the hemato-
poietic cells. Recently, Wu et al. showed that epithelial
cells of the corticomedullary border produce macrophage
colony-stimulating factor (CSF1) [48]. Ramm et al. [49]
suggested that the cells at the corticomedullary junction
are resistant to infectious bursal disease virus (IBDV) and
represent a macrophage subpopulation. If the blast-like
cell population is exhausted after IBDV infection or tes-
tosterone treatment, regeneration of the follicle, but not
the entire bursa, is handicapped (N. Nagy, unpublished
observation).
Medullary epithelial cells have stellate morphology
and form a 3D network (Fig. 2.5E) for B cells
(Fig. 2.6C), bursal secretory dendritic cells (BSDCs)
(Fig. 2.6D), and macrophages (Fig. 2.6E). Epithelial cells
are connected by desmosomes to one another and to the
cells of the FAE-supportive and corticomedullary arch-
forming cells. Their cytological structure is comparable to
that of FAE-supportive cells; namely, they are rich in
cytokeratin and have poorly developed cytoplasmic orga-
nelles, suggesting a supportive rather than secretory
function.
2.3.6 Bursal secretory dendritic cells
BSDCs, first identified by Oláh and Glick [50,51], are
found only in the medulla (Fig. 2.6D). Cyclophosphamide
eliminates B cells from both the medulla and cortex but
does not affect BSDCs. However, B cell depletion results
in aggregation of these cells in the center of follicles.
BSDCs have two cellular processes, which are different
in size and cytological structure. The smaller one contains
a few rough-surfaced endoplasmic reticulum cisternae,
free ribosomes, and a large number of vimentin-
intermediate filaments (Fig. 2.7A). The larger one con-
tains many membrane-bound electron-dense granules
around a well-developed Golgi region, or it lines up along
the cell membrane (Fig. 2.5D). The arrangement of the
cytoplasmic organelles in the cell gives the mature BSDC
a polarized appearance (Figs. 2.6D and 2.7A). The mature
BSDC is highly elongated in shape and has a nuclear
chromatin structure similar to that of the lymphocyte.
Granular contents are released and attach to the outer sur-
face of the cell membrane [34,52,53]. Cell adhesion sites
Structure of the avian lymphoid system Chapter | 2 19
can be observed between BSDCs and lymphocytes [52].
The senescent BSDC seems to be phagocytic; the cell
loses the vimentin-intermediate filament and surface
receptor for IgY, but the membrane-bound electron-dense
substance is preserved for a while (Fig. 2.6D and E).
The biochemical composition of the granules is not
known, but the mAb CVI ChNL-74.3 recognizes intracel-
lular BSDC antigens, possibly granules (Fig. 2.7B) [54].
IBDV infection eliminates the 74.31 staining, suggesting
that BSDCs are an IBDV target. The antigen recognized
by mAb 74.3 has not been characterized, but double-
staining with vimentin clearly indicates colocalization
[39]. Surface IgY (sIgY) appears on BSDC just before
hatching and persists throughout life, so anti-IgY can also
be used for their identification (Fig. 2.7C). IgY appears
on the cell membranes of BSDC before hatching and
therefore should be of maternal origin. IBDV infection
eliminates not only the B cells but also sIgY, confirming
the involvement of BSDC in IBDV pathogenesis. Besides
IgY, vimentin and 74.3 antigen avian BSDCs express
putative CD11c, DEC205, and CSF1R (Fig. 2.7D) in
chicken, NIC2, CSF1R in quail, gray partridge, and
guinea fowl, respectively [29,34,52,55,56] These cells
show no reaction with the classical avian monocyte/mac-
rophage-specific mAbs (Fig. 2.7D F), thus, these mAb
are convenient tools for monitoring the BSDC’s condition
[14]. Corticomedullary epithelium promote the prolifera-
tion (Fig. 2.7G) of BSDC precursors and B cells
(Fig. 2.7H J), which are nearby, but inhibit their differ-
entiation, suggesting a regulatory function; the ERC allow
them to differentiate [57]. At hatching, 5 8 vimentin-
positive BSDC per follicle are present, increasing to
65 70 by 4 6 weeks of age
2.3.7 Bursal macrophages
In histological sections, macrophages with phagocytic
substances are easily identified in both the cortex and
medulla; however, no positive cells are recognized using
mAb against monocyte/macrophage markers, such as
CVI-ChNL-74.2, 68.1, 68.2, and KUL01 (Fig. 2.7K). This
suggests either that the bursal follicle does not have
macrophages or that its macrophage population is unique.
Recently, it has been shown that the bursal medullary
macrophages express lysosome-specific Lep100 antigen
(Lamp-1) and TIM4 [14,36,48]. After IBDV infection,
large cells loaded with cellular debris and virus particles
appear first in the medulla and later in the cortex, while
BSDC cannot be identified using any cell marker.
Electron micrographs show a surface substance on several
macrophages, comparable to that on BSDC (Fig. 2.6E and
F). This finding, together with the MHC class II1
macrophage-like cells in the FAE, indicates that the med-
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
At least 98% of bursal lymphocytes are B cells. Scattered
T cells occur in the cortex, but few of them enter the
medulla. B cells proliferate in both cortex and medulla
(Fig. 2.7G). After 8 10 weeks of age, the number of lym-
phocytes in the interior of the medulla decreases, indicating
the initiation of bursal involution, which is a process com-
pleted by 6 7 months of age. A few plasma cells may
develop in the cortex and medulla with age, but generally
their number remains very low, suggesting some kind of
inhibition of B-cell terminal maturation. However, in birds
surviving IBDV infection, the bursa has a remarkably large
number of plasma cells before fibrosis is complete.
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
there are some differences in phenotype; however, the
functional differences are not fully understood (see
Chapter 4 for further discussion). Medullary chB6 1 B
cells express surface IgM [58] and CD15 (Lewis X) [59],
but the Ov antigen and CXCR4 appear only on cortical B
cells [60] (Fig. 2.7H J). This makes it difficult to deter-
mine the B-cell maturation process. During acute involu-
tion of the bursa, caused by IBDV treatment, T
lymphocytes accumulate in the follicles. The more severely
damaged follicles have more T cells, suggesting a delicate
balance between T and B cells. If B cells are destroyed or
emigrate from the follicle, T cells can temporarily replace
them [61]. The accumulation of T cells in bursal follicles
seems to depend on B cell depletion and not just infection.
2.3.9 Cortex
The cortex begins to develop around hatching, and it is sepa-
rated from the medulla by the BL of the corticomedullary
border, its surface is covered by fine collagen-rich capsules.
The cortex, unlike the medulla, develops exclusively from
the mesoderm. MRC form the supporting cells of the cortex
and express vimentin- and desmin-intermediate filaments
[62]. The 3D meshwork of the MRC is filled with chB61/
CXCR41 B lymphocytes (Fig. 2.7J) and CSF1R1/Lep1001/
TIM41 macrophages. BSDC are lacking in the cortex.
Cortical B cells are heterogeneous, chB61 cells are
L
dispersed uniformly throughout the cortex but only the
chB61 B cells located in the outer region of the follicular
cortex coexpress CXCR4. It is suggested that B cells have
to downregulate CXCR4, express Ov antigen before they
can leave the CXCL12-rich environment of the follicle cor-
tex and migrate out to colonize the peripheral lymphoid
organs [60]. The ECM is rich in collagen III and fibronectin,
indicating intensive cell migration through the cortex. The
complete absence of ECM in the medulla means that cell
migration is questionable, but the ERC surface serves as a
scaffold for it. The cortex has a rich capillary network adja-
cent to the corticomedullary BL (Fig. 2.6A) and is drained
by postcapillaries, which conjoin to form veins in the con-
nective tissue of the fold where the lymphatics emerge.
2.3.10 Peripheral lymphoid tissue of the bursa of
Fabricius
Close to the bursal duct, one of the ventral folds produces
peripheral (secondary) lymphoid tissue (Fig. 2.7F). This tis-
sue is not likely to be a simple continuation of cloacal lym-
phoid infiltration because the fold is flat, it is wider than
others and its colonization by lymphoid precursors is
delayed about 1 day. The peripheral lymphoid tissue consists
of GC and an interfollicular dense lymphatic substance that
represents B- and T-dependent regions, respectively. This
fold may provide communication between the cloacal con-
tents and the bursa of Fabricius [37,63,64].
2.3.11 Germinal center of the peripheral
lymphoid organs
Histologically, the GC is defined by aggregates of blast
cells which, after antigenic stimulation, occur in defined
areas within peripheral tissues. In chickens, two types of
GC, fully and partially encapsulated, have been
described. Although their cell populations do not differ
cytologically, there is speculation that the two types rep-
resent functionally different structures [65].
Alternatively, they may represent different stages of GC
development [66]. Mature GC are surrounded by a cap-
sule of connective tissue and completely lack blood ves-
sels (Fig. 2.8A, G, and H).

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
directed by clusters of stromal cells, recognized by the
mAb CVI-ChNL-74.3, which are located close to the T
cell areas, adjacent arteries and arterioles. During a
humoral response, newly developing GC, consisting of
bromodeoxyuridine-incorporating proliferating (BrdU1) B
cells, are located adjacent to clusters of 74.31 cells [67].
During development, the B cells grow around the clusters
until they completely surround them. Cells in the middle
of the GC do not incorporate BrdU; thus they are not
mitotically active and resemble centrocytes, while the
outer ring of proliferating cells represents centroblasts
(Fig. 2.8B). Although a light zone of centrocytes and a
dark zone of centroblasts, as described in mammalian GC,
can be less easily discriminated morphologically in the
chicken, functional homology seems likely [65,66,68].
Structurally similar to mammalian metallophilic macro-
phages, a mature GC is surrounded by TIM41 macro-
phages [69]. In mature GC, 74.31 CSF1R1 cells, called
follicular dendritic cells (FDC), can trap immune com-
plexes on their surface. These trapped immune complexes
are important in memory formation. Specific antibodies
or antibody-containing cells are detected in serum and
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,
as reported for the mouse spleen [66,70].
Transmission electron microscopy shows a dense
intercellular substance around the FDC. The structure of
the FDC’s nuclear chromatin resembles that of small lym-
phocytes, and the cytoplasm contains a variable number
of electron-dense granules of various sizes (Fig. 2.8C).
The surface membrane of 74.31/CSF1R1 FDCs
(Fig. 2.8D and E) binds (but does not express) IgY
(Fig. 2.8F), and the FDC’s cytoplasm contains vimentin-
intermediate filaments (Fig. 2.8G). The anti-vimentin-
specific antibody and anti-CSF1R are convenient markers
to identify avian FDC [14,55,56,62]. In addition to FDC,
the GC contains other types of nonlymphoid cells such as
supporting reticular cells, which express desmin-
intermediate filaments (Fig. 2.8H). Tingible body macro-
phages are not stained by classical monocyte-macrophage
markers, such as 74.2 (Fig. 2.8I), 68.2, and KUL01
(MRC1L-B), but they express TIM4 [69] and lysosomal
membrane protein LAMP-1 (Lep100 antibody)
(Fig. 2.8J). This may indicate a different developmental
stage or a different origin. The tingible body macrophages
engulf apoptotic B cells and cell debris. A mature GC
consists of proliferating Bu-11 (chB6 antigen) B cells
(Fig. 2.8K), a few T cells expressing CD3, CD4, and
TCRαVβ1 (Fig. 2.8L), and 74.31 FDC. Arakawa et al.
[71] and Igyarto et al. [67] demonstrated that the antigen-
activated B cells of the spleen migrate into the GC, where
they expand, as proposed for human GC development
[63]. Arakawa et al. [71] estimated the clonal complexity
in each GC of the spleen, suggesting that 6 12 B cell
clones are expanding, with each producing a different
antibody.
Contradictory results concerning Ig expression in GC
have been reported. Jeurissen et al. [70] described follicle
B cells expressing sIgM but not sIgY. Yasuda et al. [72]
isolated individual GC from the spleen and analyzed GC
cell populations at the individual cell level. They
described considerable differences in sIgM and sIgY
expression between GC. Ig-expressing cells were found in
the center of the GC but not in the outer rim, where pro-
liferating B cells were found. The relative proportions of
sIgM1 and sIgY1 cells changed after intravenous inocula-
tion of DNP-KLH, with the highest proportion of sIgM1
cells detected on day 7. The proportion of sIgY1 cells
increased during the first 14 days, but, as observed for
sIgM1 cells, large individual variations were evident. In
contrast, the proportion of CD31 cells remained relatively
constant at 10% 20% in each GC. Double-staining stud-
ies of GC have elucidated the nature of the isolated sIg1
cells, where the FDC appeared to possess IgG on their
surface [62].
2.4 The spleen
2.4.1 Origin and anatomy
The splenic primordium first appears as a mass of mesen-
chymal cells in the 48-hour embryo [73]. Sinusoids with
erythrocytes appear in the mesenchyme at E5, granulopoi-
esis begins from E7 and erythropoiesis follows at E11. In
contrast to that of mammals, the avian spleen is not con-
sidered a reservoir of erythrocytes for rapid release into
the circulation [74]. Although not a primary site for lym-
phocyte antigen-independent differentiation and prolifera-
tion, the spleen has an important role in embryonic
lymphopoiesis, for it is here, though not solely here, that
B cell progenitors undergo rearrangement of their Ig
genes before colonizing the bursa of Fabricius [75] (see
also Chapter 4). At the time of hatching, the spleen
becomes a secondary lymphoid organ which provides an
indispensable microenvironment for interaction between
lymphoid and nonlymphoid cells [14]. The contribution
of the avian spleen to the immune system as a whole may
be more important than in mammals because of the poorly
developed avian lymphatic vessels and lymph nodes.
The chicken spleen is a round or oval structure lying
dorsal to and on the left side of the proventriculus
(Fig. 1.1C). One or more small accessory spleens some-
times occur nearby [76]. Major splenic development
occurs after hatching, following exposure to antigens
[77]. The main blood supply to the spleen is provided by
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)
Another random document with
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 On they ran wildly.
But the Steam Horse had gone from sight. All effort to catch up
with it was futile.
Thoroughly terrified the two servitors were obliged finally to halt.
They were completely exhausted.
They paused by a narrow stream or brook of saline water to rest.
Suddenly Pomp sprung up with a hoarse cry of terror.
“Golly, what am dat?” he gasped. “Jes so suah as yo’ am bo’n,
I’ish, dar am dem Injuns a’comin’ agin.”
The clatter of ponies’ hoofs was plainly heard, and they saw the
forms of a band of savages outlined against the sky as they rode
along an eminence near.
The effect of this upon Barney and Pomp was thrilling.
They stood for a moment so overcome with horror that they could
not act.
The savages seemed coming directly toward them.
To fall into the hands of the Comanches would be the worst kind of
a fate, and our two friends had no desire in that direction.
Barney was the first to recover.
“Bejabers, we niver must shtay here!” he cried. “Shure it’s out av
this we must get.”
“Yo’ bet!” cried Pomp.
“Shure, phwere will we go?”
“Fo’sho’ I donno nuffin’ ’tall.”
But Barney, in despair, started down the course of the stream.
 Pomp followed him.
It proved the very luckiest sort of a move for them.
The Steam Horse had taken this very course.
Just below it had run into a patch of chaparral, which was
surrounded with a dense growth of grass and trailing vines.
For fifty yards the Horse had cleaved its way through this growth.
Then it had come to a stop.
The clinging vines had clogged the axles and a swaying branch
had caught the throttle rein and shut off power, bringing the monster
to a dead stop.
Barney saw the lights of the Horse first and a yell escaped his lips.
“Whurroo! we’ve found him at lasht,” he roared. “Cum on, naygur.
Run fer yer loife.”
The savages were coming in hot pursuit.
The way the two jokers fled down that slope was a caution to race
horses.
Barney reached the wagon first.
He sprung in, seized an ax, and then springing out again began
hacking away the obstructions which held the Horse immovable.
The clinging vines yielded of course beneath such treatment.
Pomp cleared the axles and then both dodged into the wagon.
They were not a moment too soon.
Down came the savages howling like dervishes. In a twinkling they
had surrounded the Horse.
Arrows and bullets rattled against the lattice work of the wagon.
 But Barney seized the throttle rein and turned the Horse about.
Pomp busied himself with his rifle at the loopholes and every shot
he fired was sure to count.
The Comanches before such a deadly fire fell back for a moment.
This gave Barney time to turn the Horse about.
The wagon was now free from the incumbrance of the trailing
vines and the Horse quickly galloped out of the chaparral.
The Comanches swarmed about now as thick as bees.
But Barney did not deviate in his course.
He pulled the throttle wide open and sent the Steam Horse ahead
at a powerful rate of speed.
Into the crowd of savages the machine plunged.
The shock drove them back. The light ponies were thrown aside
and their riders unceremoniously tossed.
There was not weight enough to resist the progress of the Steam
Horse.
With baffled yells the savages broke ranks and the Horse went
through like a thunderbolt.
It was so dark that Barney was at a loss to know just what course
to pursue.
But he went straight ahead as it looked to be the safest course.
The savages, like a pack of wolves, came yelling on behind.
It was easy for the Steam Horse, however, to keep ahead of them.
If it had been daylight Barney could have amused himself giving
the savages a wild chase.
But it was so exceedingly dark that he dared not go too fast.
 Pomp kept up his destructive fire just the same, and the savages
dropped at every shot.
On and on in the night ran the Steam Horse.
Fortunately thus far they had encountered no obstacle.
The course had been perfectly smooth, with scarcely a pebble to
jar them.
But Barney could not hope for this to last.
It seemed as if they had covered several miles when a startling
thing happened.
Suddenly lights gleamed just ahead. As well as Barney could
determine they were torches.
A chill struck the brave Celt.
What did it mean?
Were there more of the foe in front? If so, they were literally
hemmed in.
This would mean a serious matter, and the Celt’s grip tightened on
the handle of the brake.
“Bejabers, I wondher phwativer it is?” he muttered.
Certainly there was a band of men in front of them.
The glare of torches could be seen, and the dim outlines of horses
and men in the gloom.
Barney saw no way to turn out.
To come to a stop would have been to allow the Comanches to
descend upon the Horse.
What was to be done?
 There seemed no other way but to forge straight ahead, and take
chances of breaking through the line of the new foe.
But suddenly a great light flashed up, which made for some
seconds the whole vicinity as plain as day.
It was a signal light, burned by those in front, and Barney saw to
his amazement and delight the outlines of a large body of soldiers.
A great cry of joy and triumph went up from the Celt’s lips.
“Och hone, an’ if it ain’t sogers they be!” he cried. “Shure, the luck
is wid us an’ we’re safe the while!”
“Golly! yo’ don’ mean it,” cried Pomp, “den dot am a berry lucky
fing. Dem sogers can jes’ help Marse Frank fo’ to cotch dem
countingfitters.”
“Yez are right!” cried Barney.
Then he closed the throttle and applied the brake.
The Steam Horse quickly came to a halt. The result was thrilling.
The Comanches had not yet seen the soldiers and came about the
Steam Horse in a legion.
But now up the plain came the stentorian order:
“Attention battalion! Draw sabre! double-quick, forward! Charge!”
The clatter of hoofs, the jingling of accouterments and the wild
cheer of the soldiers rose upon the air.
Forward rode the troop in headlong fashion and led by a man with
as commanding a figure as Murat.
The flash of their sabres could be seen in the torches’ light.
The savages saw them and a panic was created.
At first they seemed inclined to stand their ground.
 But their experience with Uncle Sam’s troops had heretofore been
of the most expensive sort.
So they did not deem it best to risk an encounter.
There was a series of baffled yells, a hustling rush, and away they
went into the darkness and out of sight.
But the cavalry came on and in a moment surrounded the Steam
Horse.
The tall Murat like captain rode forward, and touching the flat of his
sword to the visor of his cap, asked:
“Who are you? Friend or foe?”
“Well, sor,” replied Barney, readily, “shure, we’re not an inemy.”
“No? Well, what in thunder kind of a rig do you call that?”
“Can’t ye see fer yersilf?” cried Barney, a little out of patience.
“It looks like a horse made of iron.”
“Shure, an’ that’s phwat it is, but I’d advise ye not to do it any
harm.”
“Fear not,” cried the tall officer. “I am Captain Elmo, of Fort Forty-
Five. You don’t mean to say that that iron horse can travel of itself?”
“Shure it’s a steam horse!” replied Barney. “Did yez niver hear of it
afore?”
“No, never!” was the reply. “But who are you?”
“I’m Barney O’Shea!”
“Do you own that Steam Horse?”
“No, sor. It’s the property av Misther Frank Reade, Jr.”
“Ah, I see it now!” cried Captain Elmo. “I have heard of Frank
Reade, Jr. Did he not travel out here with a Steam Man once?”
 “That he did, sor!”
“Then I understand it all. Then you two chaps are Barney and
Pomp, eh? Well, I’m glad to see you.”
“The same, sor!”
“But where is your master, Mr. Frank Reade. Jr.?”
“Shure, sor, him an’ a thrapper chap named Beaver Bill, they have
gone up into ther Death Valley Pass, sor, lookin’ fer Bert Mason, sor.
Yez know him?”
“Bert Mason!” cried Captain Elmo. “Why he is the chap I have
been sent up here to find.”
“Mither Mary presarve us, an’ phat for, sor, do yez want him?”
“He is said to be the leader of a band of counterfeiters who are
supposed to have headquarters somewheres about here.”
“Shure, sor, it’s glad I am to hear av that,” cried Barney. “An’ it’s
mesilf as kin tell yez, just where to foind yure man?”
“Where?”
“Shure, sor, up in the Death Pass.”
“Are you sure of that?”
“I was well informed, sor!”
“Enough!” cried the captain, “that is then our objective point.”
“All right, sor! Are yez goin’ there right away?” asked Barney.
“At once!”
“Shure, sor, don’t yez want the sarvices av ther Stheam Horse?”
“Why certainly!”
“Ye’re welcome quite!”
 “Very well. Fall into line with us.”
Barney was not loath to do this. The cavalcade set out at once for
Death Valley, and the Steam Horse and Barney and Pomp followed.
The gray light of dawn was just appearing in the east. The events
of the night were rapidly drawing to a close. But fresh scenes were at
hand.
 CHAPTER XI.
TO THE RESCUE.

The sensations of Frank Reade, Jr., and the trapper, Beaver Bill,
was of a fearful sort when they found themselves surrounded by the
outlaws.
It was the Mexican, Castrello, who stood before them.
There was no use in offering resistance.
They would have only been shot down in cold blood for it.
Frank Reade, Jr., realized this, and he was wise enough to keep
perfectly quiet.
“We’re in for it, Bill,” he whispered. “But never mind. Keep up a
good heart.”
“Wall, you bet.”
In the hands of the counterfeiters the position of the two captured
spies was not of the most reassuring.
Death, of course, was the fate of spies, and Frank reflected that
the counterfeiters had no reason for sparing them.
“Hard luck!” groaned Frank. “We are done for, Bill!”
“Wall, I reckon,” agreed the big trapper, “we kain’t die but once.”
“There is consolation in that thought,” declared Frank.
Castrello seemed beside himself with joy at the capture. He had
the two captives securely bound hand and foot.
Then he said:
 “Perdito! take them into the strong house. See to it that they do not
escape!”
“Si, senor!” replied one of the men, who appeared to be a
Spaniard.
The two captives were led to a door in the stone house.
It was evident that the upper end of the house was used for such
purposes as this.
There was not a sign of a window anywhere. The door was
opened and the prisoners were thrust into a small ten by twelve
chamber.
This was as dark as Erebus when the door was closed.
The door formed the only means of ingress and egress. Windows
there were none, and no lamp or torch was left to cheer their
drooping spirits.
Frank was very much disgusted.
“I don’t understand it,” he muttered, dismally.
“What mought that be, pard?” asked the big trapper.
“How we came to fall into the hands of these miscreants.”
“I kin see it easily enuff.”
“How?”
“Why, we jest made cussed fools of ourselves. We hadn’t orter
risked lookin’ in that window. They spotted us then.”
“Well, I’m inclined to believe you,” muttered Frank, “but the fates
have seemed against us. Is there no way that we can escape from
this place?”
“Don’t see any way.”
 “We can’t saw a window bar out, for windows there are none.
Really, Bill, I’m at my wits’ end.”
“I’m sorry fer ye, pard. I don’t see as we kin do anything better
than to wait fer mornin’.”
“You are right!” cried Frank. “That is the best we can do.”
Both were much tired and gradually dropped into a light sleep.
This lasted for several hours and was of great benefit to them.
Sleep is truly said to be a great restorer.
Frank felt better when he awoke.
It was lighter in the prison cell now and Frank knew that daylight
had come.
He was secretly wondering what was to come next. Suddenly the
door opened and a rough looking man came in.
“So ye’ve raised, eh?” he said with a gruff manner. “Wall, it’s about
time.”
“Who are you?” asked Frank, curtly.
“Eh? Wall I’m the chap what watched at this door last night to see
that ye didn’t get out.”
“You are, eh?”
“Yas!”
“Well, what do you want?”
“The cap’en, Bert Mason, sent me in to fetch ye two chaps out.
He’s got a bit of an inquest to hold over ye.”
The fellow laughed coarsely at what he considered his witty
remark.
Frank experienced a queer thrill.
 He felt that he was going forth to face death. But it occurred that at
last he would be face to face with Bert Mason, the murderer and
fugitive from justice.
He offered no resistance, nor did Beaver Bill, and the two men
were led out into the open air.
It was now after sunrise some while and the counterfeiters’ camp
in every detail was revealed.
Frank looked about with curiosity.
He saw a squad of fully a score of armed men congregated near
the western verge of the plateau.
The two prisoners were led thither and into the center of a circle of
the foe.
Sullen glances were bestowed upon them which Frank
disregarded wholly. The young inventor knew that his life was sought
by these men, and that he was never nearer death in his life.
One of the gang, a short, thickset fellow with lowering eyes and a
brutish cast of countenance, advanced.
“Wall, what have you two pilgrims got to say for yourselves?”
Frank Reade, Jr., knew instinctively that this was the ruffian, Bert
Mason, and he eyed him coolly.
“I have nothing to say for myself,” he replied, coldly.
“Ye haven’t, eh?”
“I said so.”
Mason glared savagely at the young inventor and hissed:
“So ye’re the chap whom they call Frank Reade, Jr., the inventor,
eh?”
“That is my name!”
 “Ye’re the man what owns that wonderful Steam Hoss?”
“I am the man.”
“Where’s yer hoss now?”
“I do not know.”
Mason laughed coarsely.
“Had lots of sand to cum into this region with the idee of bringing
me to my milk, didn’t ye?”
“Perhaps so.”
Frank was cool and perfectly fearless. This maddened the wretch.
“Cuss ye, I kin tell ye that ye’ll never succeed. Ye’re in my grip
now, an’ yer fate will be the same as has overtaken every man
heretofore who has tried the same game. That’s death.”
Frank gazed unflinchingly at Mason.
“Very well,” he said, coldly. “I am not afraid to die.”
“Look yonder!”
Mason pointed to the Death Valley.
“No man who ever goes in there comes out alive,” he declared.
“Well, what of it?”
“That’s where I’m going to send you.”
The villain laughed in a fiendish way. He motioned to his men.
“Bring ’em along to the edge, boys,” he cried. “We’ll soon end the
argument.”
Beaver Bill was just as cool and nervy as Frank was.
Both men had the pluck to face death coolly and without fear.
 They were led to the verge of the plateau by the counterfeiters.
Below down a sheer descent of fully a thousand feet was Death
Valley.
The hot sands lay below, baking in the blistering sun. Frank saw
with an awful chill the purpose of the gang.
This was to throw them over the edge of the plateau.
It was an awful thought.
If even one should survive the fall, the deadly noxious gases
would be sure to terminate life.
Frank realized this with a terrible appalling sense, such as comes
to one upon the verge of an awful death.
“God help us!” he reflected. “I think this is the end.”
They were led forward to the verge of the precipice.
“Now I’m going to give ye a chance,” cried Mason. “If ye think ye
can make the jump ye have a chance for your life. Cut their bonds,
men. Ha, ha, ha! I wouldn’t give much for your chance.”
Two men sprung forward, and with sharp knives cut their bonds.
“Now say yer prayers!” cried Mason. “Ye’ll soon be in Hades.”
The villain laughed again uproariously at his ghastly wit.
But Frank Reade, Jr., conceived of a daring plan at that moment.
He whispered to the trapper:
“Bill, we have the use of our hands and feet. It is death anyway.
Why not make a fight for it?”
“Kerect! I’m with ye!” replied the trapper. “What’s the move?”
“You dash to the right, and I’ll go to the left.”
“Ready!” yelled Mason. “One——”
 He did not finish counting. With the rapidity of lightning the two
prisoners leaped in opposite directions.
There was the report of a pistol held in Mason’s hand.
The bullet whistled by Frank Reade, Jr.’s, ear.
But the young inventor did not pause. He rushed madly into the
arms of one of the guards.
Before the fellow could make resistance Frank had struck him a
terrific blow in the stomach which stretched him out upon the ground.
Then he sped away across the plateau like a deer.
Rifle bullets followed him, and though his clothes were shot full of
holes, miraculously he was not injured.
But Beaver Bill was having a decidedly harder time.
He had been clutched by several of the foe, and a lively struggle
followed.
The trapper was the center of a struggling mass of the
counterfeiters, but was making a valiant struggle, when a loud yell of
alarm went up on the air.
Frank, in his flight across the plateau, suddenly heard a thrilling
sound.
He turned his head and clear to his hearing came the shrill whistle
of the Steam Horse.
“Barney and Pomp!” he gasped. “What has brought them here?”
Sure enough, across the plateau, at whirlwind speed, came the
Steam Horse.
But directly behind the horse was a body of United States troops,
with Captain Elmo at their head.
 They came down at a swinging gallop upon the counterfeiter’s
den.
Bert Mason with dismay had seen the whole affair. The villains
gave a yell of terror and running to the edge of the plateau
disappeared.
In a twinkling every one of the counterfeiters had disappeared in
the same manner.
The trapper Beaver Bill was left standing alone in the middle of the
plateau not in the least harmed.
“Wall, by thunder!” he cried. “What skeered them off that way?”
Then he chanced to look up and see the soldiers and the Steam
Horse.
A yell of triumph went up from the excited trapper’s lips.
“Hooray!” he shouted, “that’s the stuff. We’re all hunky now, I
swan!”
The next moment the two erstwhile prisoners were standing
beside the Steam Horse, and talking with Barney and Pomp and
Captain Elmo.
The rescuers had come just in the nick of time.
 CHAPTER XII.
FATE OF THE COUNTERFEITERS.

There was no question about this.

A little delay and the two prisoners would no doubt have passed in
their final accounts.
As it was hardly likely that Frank would have been able to have
escaped from the plateau.
It was, however, they were safe and there was good reason for
self-congratulations. It had been a narrow escape.
The soldiers were looting the stone house of the counterfeiters.
Bushels of counterfeit coin was seized and all the tools and dies in
use.
It was a big haul, and not for years had so extensive a den as this
been discovered.
But Captain Elmo was not satisfied.
The ringleader and indeed nearly all of the gang were at large.
Frank Reade, Jr., was desirous of effecting the capture of Bert
Mason, as the villain’s identification was necessary to save the life of
Benjamin Astley.
Captain Elmo was presented to Frank Reade, Jr., by Barney and
the two men had a lively conversation.
“I have been looking for Mason for a long while!” declared Elmo. “It
is lucky we happened to meet, for if I had caught him I should have
hung him to the nearest tree, and then your man, Astley, would have
remained unidentified.”
 “Yet if we do not capture Mason, I fear my man is lost!” said Frank.
“You’re right; he is at large now at least.”
“Yes.”
“But we must capture him. Where do you think he has gone?”
“Come with me and I will answer that question!” said Frank.
The young inventor led the way to the verge of the plateau.
“He went out of sight in this direction,” he said.
“What!” exclaimed the captain, aghast. “You don’t mean to say that
he actually leaped over into the valley?”
“I don’t know. I only saw him disappear about here.”
“But I can see no way of climbing down here.”
“Nor I. It may be possible that he committed suicide here, but I
doubt it.”
“What then is your opinion?”
“There must be some secret way of getting down the face of the
cliff here.”
“Let us examine.”
For some while the two men made a close examination of the
vicinity. But this resulted in nothing.
“The mystery is yet unexplained,” declared Captain Elmo.
“So it is,” agreed Frank. “Now I will advance a theory.”
“Pray, do so.”
“Of course you understand the nature of that valley down there
and its sandy trail of death?”
 “I understand that the bodies of many victims to the peculiar
atmosphere of the valley yet lie down there.”
“Yes.”
“And that it is not safe for any person to venture into the place.”
“Quite wrong, sir!”
“What?”
“It is quite safe for anybody to cross that valley.”
The captain looked astonished.
“I fail to comprehend,” he said.
“I will modify my statement,” said Frank, “and affirm that there are
times when it is safe to cross the valley.”
“Ah, that is quite as surprising. What time or when is it safe?”
“At night.”
The captain cleared his throat and looked quizzically at Frank.
“Have you tried it?”
“No.”
“Ah! Then do not.”
“To the contrary, I will try it, and prove to you that I mean what I
say this very night.”
“Do not do anything rash.”
“What is more, I will capture the counterfeiters for you at the same
time if you will follow my directions.”
“What are they?”
“Post soldiers at near intervals all around the valley. The rascals
certainly are hiding in some place in the cliffs.”
 “Ah! what is their purpose?”
“Escape! They will in the night cross the death valley and get away
from us.”
The captain was much impressed with Frank’s statements.
“I have no intention of disputing you, Mr. Reade,” he said, quietly,
“or of seeking to know how you get your information.”
“Simply by observing a few natural phenomena in connection with
the valley.”
“Ah!”
“I will show you more clearly to-night.”
“Then I will follow your directions to a dot. I suppose that by to-
morrow we shall have the counterfeiters all in our power.”
“I think so.”
The captain went back to his men.
He proceeded to follow Frank’s instructions carefully.
Men were placed at intervals along the plateau and with rifle shot
completely around the valley.
Unless there was some unknown exit by means of a secret
cavern, Frank could not see how the counterfeiters could escape.
The day was spent in overhauling and exploring Mason’s den.
Towards night Frank, with Barney and the trapper, Beaver Bill,
went to the verge of the plateau.
Pomp was left with the Steam Horse by the stone house.
Frank’s theory was that the counterfeiters were secreted beneath
the cliffs and were waiting simply for night to enable them to find
their way across the valley.