AVIAN HISTOPATHOLOGY | Third Edition Published by The American Association of Avian PathologistsLegends for Cover Images Lung, Granuloma. Cockatoo, 22 Years. A central core of caseous material surrounded by a ring of giant cells is typical of an avian response to necrosis. Staphylococcus sp. was isolated from this case. Bursa Fabricius, Atrophy. Broiler, 40 Days. Atrophy of bursal lymphoid follicles is diffuse, interfollicular tissue is expanded, and plical epithelium is thrown into folds, Intestine, Coccidiosis. Pheasant. Nearly all enterocytes contain stages of coccidia including zygotes with prominent peripheral wall-forming granules. Kidney, Renal Urate Deposit (Gout). Lori, 7 Years. A necrotic renal tubule is replaced by a urate deposit that shows a typical feathery pattern. Spinal Cord, Avian Encephalomyelitis. Chicken, 2.5 Weeks. Neuronal degeneration is indicated by central chromatolysis - a characteristic lesion of avian encephalomyelitis. Glial nodules also are shown. Keel Bone, Avian Leukosis Virus - J. Broiler Breeder. Marrow cavity is filled by neoplastic myeloid cells characteristic of myeloid tumor induced by avian leukosis virus - J. Kefo ye yy: MAN ; oe ae bye AVIAN HISTOPATHOLOGY Oscar J. Fletcher, Editor w Poultry Health Management, Department of Population Health and Pathobiology College of Veterinary Medicine NC State University Raleigh, NC 27606 Tahseen Abdul-Aziz, Associate Editor Rollins Animal Disease Diagnostic Laboratory North Carolina Department of Agriculture and Consumer Services Raleigh, North Carolina 27607 Third Edition Published by American Association of Avian PathologistsCopyright © 2008 by ‘American Association of Avian Pathologists, Inc. ‘All Rights Reserved Printed by Omni Press Madison, Wi 53704 Library of Congress Catalog Number 2008942157 International Standard Book Number 978-0-0789163-3-6 Copies available from ‘American Association of Avian Pathologists AAAP, Inc. 12627 San Jose Bivd., Suite 202 Jacksonville, Florida 32223-8638 AAAP@aaap infoPreface Third Edition Recognition of both basic responses of avian tissues to injury and the patterns of response are critical steps in collecting information on which to base differential and disease diagno- ses. This third edition of Avian Histopathology follows the foundation work of Craig Riddell in the First Edition. Material is organized by body system with some normal histology provided. Histologic changes and injury patterns are presented within the organizational framework of uses of injury. Different etiologies often cause similar histologic lesions so emphasis is placed on recognition of responses to injury and of those differential features or “things” that make specific disease diagnosis possible. Most of the material covered in this edition comes from traditional poultry, but some pet avian material is included. Reference material is provided as additional readings without providing specific citations within the text material. This is an effort to avoid citing nearly every statement in the text, thus the text represents our own experiences but acknowledged as being influenced and aided by the work of many others. Digital imaging technology and software for editing and management of images are essential tools that make the use of color images in this edition possible. Most of the images come from the collections of the authors. Material received from colleagues is acknowledged in the figure legends. The first edition of Avian Histopathology, written by Craig Riddell, was published by AAP in 1987. My copy of that first edition has on the inside of the cover page the following autograph: *O. J. Fletcher — With thanks for your support and a very enjoyable sabbatical leave at Athens when much of the work on this book was done. Craig Riddell”. Thanks are expressed to Craig for his inspiration and for the excellent contributions he made to avian histopathology. This is a full-cycle experience — from seeing Craig Riddell work on the First Edition to being able to make contributions to this Third Edition. | am grateful to AAAP for the opportunity. The coauthors were essential for any success of this edition and I thank them for their contributions, assistance, and encouragement. Special thanks is expressed to Dr. Tahseen Abdul-Aziz for his contributions as the associate editor. Oscar J. Fletcher iiAcknowledgements Fred Hoerr and Conrad Pope provided guidance in the initial planning for the Third Edition. We thank the following who reviewed chapters and made helpful suggestions: Richard (Mick) Fulton Fred Hoerr — Hepatobiliary Scott Hafner - Alimentary and Integumentary Richard Julian — Cardiovascular Craig Riddell — Skeletal Pat Wakenell — Lymphoid Susan Williams - Several Chapters Floyd Wilson — Hemic, Skeletal Rosemary Marusak reviewed all of the chapters for style and content. Valerie Hendrickson provided proof reading and standardized text and additional readings’ styles. Alice Harvey provided advice and technical assistance for the organization of the chapters into this book format. Sue Clanton of AAAP gave us support throughout.Dedication The Third Edition of Avian Histopathology is dedicated to Dr. Craig Riddell, who was the author of the First Edition and Editor of the Second Edition, and in recognition of his many contributions to the discipline of avian histopathology.Contributing Authors Tahseen Abdul-Aziz Rollins Animal Disease Diagnostic Laboratory North Carolina Department of Agriculture and Consumer Services Raleigh, NC 27607 H. John Barnes Poultry Health Management Population Health and Pathobiology College of Veterinary Medicine NC State University Raleigh, NC 27606 Oscar J. Fletcher Poultry Health Management Population Health and Pathobiology College of Veterinary Medicine NC State University Raleigh, NC 27606 H. L. Shivaprasad California Animal Health and Food Safety Laboratory System Fresno Branch School of Veterinary Medicine University of California, Davis Fresno, CA 93725 David E. Swayne USDA/ARS/SEPRL 934 College Station Road Athens, GA 30605 Susan Williams Poultry Diagnostic and Research Center Department of Population Health College of Veterinary Medicine University of Georgia Athens, GA 30602 viHemic System Hematopoiesis Hemic Cells Bone Marrow Anemia Bone marrow hyperplasia Bone marrow inflammation Pigments Inflammation Neoplasia Lymphoid System Bursa Fabricius Thymus Spleen General Responses to Injury Deficiencies and Toxicities Infectious Agents Viruses Bacteria Parasites Amyloid Pigments Neoplasia Skeletal System Deficiencies - Rickets Lesions of Cartilage General Responses of Bone to Injury Infectious Agents - Bacteria Fracture and Fracture Repair Medullary Bone Osteoporosis Neoplasia Joints and Tendons - General Responses Infectious Agents - Bacteria and Viruses Aticular Gout vii 24Muscular System General Responses to injury Hereditary Muscular Dystrophy Nutritional Myopathy Exertional/Capture Myopathy Deep Pectoral Myopathy Toxic Myopathies Myopathies of Uncertain Etiology Infectious Agents Parasites Injection Site Injury Neoplasia Cardiovascular System Ascites Noninfectious Conditions Mineral and Vitamin Deficiencies and Toxicities, Infectious Agents Viruses Bacteria Fungi Parasites Neoplasia Blood Vessels Vascul Fibromuscular Dysplasia Atherosclerosis, Neoplasia Respiratory System General Responses to Injury Noninfectious Agents. Infectious Agents. Viruses Bacteria, Including Mycoplasma Fungi Parasites Neoplasia Alimentary System Beak Upper Alimentary System General Responses to Injury Toxicities and Deficiencies Infectious Agents Viruses vill 130 131 131 131 132 133 133 134 165 165 165 165 165 165 128 164Bacteria Fungi Parasites Gizzard Toxicities and Deficiencies Infectious Agents Viruses Parasites Intestines General Responses and Histology Infectious Agents Viruses Bacteria Parasites Cloaca Neoplasia Hepatobiliary System General Responses to Injury Infectious Agents Viruses Bacteria Parasites Fungi Noninfectious Conditions Amyloid Urates - Visceral Gout Fatty Liver Toxicities Neoplasia Urinary System Glomerulopathies Pigments and Amyloid Nephrosis Noninfectious Agents and Toxicities, Urolithiasis Visceral Urate Deposition and Renal Gout Infectious Agents Viruses Bacteria Fungi Parasites Neoplasia Nervous System Degeneration and Necrosis Nutritional Deficiencies Toxicities Infectious Agents Viruses 166 168 166 167 167 167 167 167 167 167 168 168 169 170 171 71 203 204 204 205 206 207 207 207 207 207 208 208 240 240 240 241 241 241 242 242 242 242 242 242 281 262 262 263, 283 202 238 260Flaviviruses Herpesviruses Orthomyxoviruses Paramyxoviruses: Papovaviruses Picomaviruses Retroviruses Togaviruses Miscellaneous Viruses Bacterial Infections Mycotic Infections, Parasitic Infections Protozoa Metazoa Neoplasia Eye and Ear ‘Anatomy. Diseases Developmental and Genetic Nutritional Toxicities Infectious Diseases Viral Bacterial Fungal Parasites Neoplasia Miscellaneous Ocular Conditions Cataracts, Other Miscellaneous Conditions. Ear Introduction and Anatomy Diseases Diseases of the External Ear Diseases of the Middle Ear Diseases of the Inner Ear Endocrine Pancreas Introduction and Normal Histology General Responses to Injury Noninfectious Conditions Infectious Agents Viruses Bacteria Neoplasia Thyroid Glands Introduction and Normal Histology Noninfectious Conditions, Neoplasia Parathyroid Glands Adrenal Glands, Introduction and Normal Histology 292 324General Responses to injury Infectious Agents - Viruses. Neoplasia Pituitary Gland (Hypophysis) Reproductive System Male Reproductive System Degeneration Inflammation Neoplasia Female Reproductive System Ovary Oviduet Disturbances of Growth Right Ovary and Oviduet Degeneration Inflammation Cophoritis Salpingitis Vaginitis Neoplasia ‘Sex cord/gonadostromal tumors Germ cell tumors Epithelial tumors Leiomyoma Integumentary System ‘Anatomy Epidermis Feathers Color of Skin and Feathers Diseases ‘Avian Skin Reaction to Injury Genetic, Nutritional Toxicity Viral Bacterial Mycotic Parasitic Miscellaneous Diseases Neoplasia Index Index to Images 329 329 329 330 349 360 361 361 362 362 353 354 355 355 366 356 367 368 368 369 359 369 361 348 392 428 435Hemic System H. John Barnes, Oscar J. Fletcher, and Tahseen Abdul-AzizIntroduction Cells and tissues that arise from pluripotent hematopoietic stem cells including bone marrow, extramedullary hematopoietic foci, osteoclasts, osteoblasts, macrophages, lymphoreticular tissues, and blood comprise the hemic system. Lymphoreticular tissues are covered separately (See Chapter 2). Information on the pathology of hemic tissues is sparse compared to information on hematology. In general, hematology reflects the status of hemic tissues and only wil be discussed in the context of pathologic changes in the hemic system. Hematopoiesis Differentiation and maturation of blood cells is continuous throughout the life of the bird and follows precise, highly controlled pathways regulated primarily by cytokines that act to either inhibit or accelerate the process. Hematopoiesis is intimately associated with bone and supporting mesenchymal tissues, which differentiate from pluripotential mesenchymal stem cells and participate in the control of blood cell differentiation. Type of cell, percentage of the cell population, and point in the maturation pathway when ‘cells are affected, determine the impact of a disease process on blood and hemic tissues, @.g., chicken ‘anemia virus, which affects hematopoietic stern cells (hemocytoblasts), causes not only anemia, but also pancytopenia and lymphoid atrophy. Hematopoiesis normally occurs within the marrow cavities of skeletal bones and non-skeletal osseous tissues such as tracheal rings, ossified tendons, ‘ectopic bone that develops within the lungs, or areas of osseous metaplasia. Distribution of bone marrow in birds is restricted by air sacs that invade pneumatic bones. In laying birds, occurrence of bone marrow coincides with medullary bone. Medullary bone develops within the marrow cavity but does not eliminate the bone marrow. Bone marrow of young birds is typically more cellular than that of older birds. Age related conversion of red marrow to fatty ‘marrow is a normal process, but the precise time sequence in birds has not been determined. In the rmid-shaft of the femur of chickens, this conversion starts at around 40 days of age. Disease is another cause of red marrow depletion. In these birds small islands of active hematopoietic cells are usually found along cortical bone and metaphyseal areas while the center of the marrow cavity is filled with fat cells. In starvation, lipid is depleted in fat cells and the reticular stroma produces a mucoid substance ("serous ‘atrophy of fat’, “gelatinous transformation’). Marrow depletion resulting naturally because of aging or from disease varies among different hematopoietic sites. ‘When assessing bone marrow activity it is useful to ‘examine multiple sites. Red marrow sites need to be ‘examined for evidence of hypoplasia or aplasia, while hyperplasia is best determined by examination of fatty marrow sites (e.g., mid-shaft femur). Bone marrow in the ulna is considered the best site for determining starvation. In birds, hematopoiesis within the marrow is ‘compartmentalized; erythropoiesis occurs within ‘medullary sinuses whereas granulopoiesis occurs in the extravascular spaces between sinusoids. The amount of erythropoiesis and granulopoiesis are approximately equal in normal bone marrow. Medullary sinuses are composed of reticulin cells that lack a basement membrane. Hemocytoblasts in close contact or adhering to the internal surface of the sinus wall give rise to a gradient of maturing erythrocytes that can be followed to mature erythrocytes in the center of the sinuses. Enythropoiesis in birds is not ‘as easy to evaluate as itis in mammals because ‘of the normal nucleated red cells in birds. Sinuses ‘anastomose and eventually drain into the large ccentral vein allowing erythrocytes to enter the general circulation. In regenerative anemias, immature as well as mature cells leave the sinuses. Birds do not have megakaryooytes; thrombocytes arise from stem cells in the same way as erythrocytes. Granulocytes (heterophils, eosinophils, basophils) develop in the extravascular spaces from hemocytoblasts in ‘contact with the extemal sinusoidal membrane. Microscopically, hemocytoblasts that produce ‘granulocytes are indistinguishable from those that produce erythrocytes and thrombocytes. Granulocytes. eventually enter sinuses through gaps in the sinus wall where final maturation occurs. Variable amounts of fat and lymphoid aggregates or nodules are also located within the extrasinusoidal spaces. Lymphoid tissue within marrow generally increases with age due to repeated antigenic stimulation and may be focal or diffuse. Its often ‘admixed with myeloid cells. Hematopoiesis in soft tissues outside of the bone marrow is referred to as extramedullary (ectopic) hematopoiesis (hemopoiesis). Extramedullary hematopoiesis may occur in any tissue, but is most frequent in tissues where hematopoiesis occurred in the embryo including liver, intestine, spleen, kidney, thymus, gonads, heart, nerves, Mecke'’s diverticulum, and bursa of Fabricius. Extramedullary hematopoiesis frequently occurs in hepatic portal areas where it may be mixed with lymphocytes. Often hematopoietic foci are associated with vessels; tis normal for themto extend through the vessel wall. The amount of extramedullary hematopoiesis varies with the age and species of bird and is increased in diseases that affect bone marrow or blood. Enlarged, mottled livers of condemned chickens often have marked hematopoiesis in portal areas, which can be confused with cholangitis. Extramedullary hematopoiesis needs to be distinguished from inflammatory lesions and leukoses involving hematopoietic cells, e.g., myeloid leukosis. Hematopoietic foci consist of mixed cells in varying proportions representing different maturational stages of erythrocytic and granulocytic cells. ‘Occasionally only granulopoiesis is seen. Mitotic figures are usually present, but are not numerous, and surrounding tissues are normal, except for vacuolated hepatocytes that are occasionally seen when foci ‘occur in the liver. In some birds, hematopoietic and fat tissue form myelolipomatous foci, which may be ‘numerous in the liver. Granulocyte foc! in the intestinal wall of neonatal chicks may provide innate immunity during the first week of life Hemic Cells In contrast to mammals, all avian blood cells, including erythrocytes and thrombocytes, are nucleated. Presence of nucleated erythrocytes in an unknown tissue indicates the tissue is not mammalian, but the tissue could have originated from a bird, reptile, amphibian, or fish as they all have nucleated erythrocytes. Nucleated erythrocytes in avian tissues make them appear more cellular, which contrasts sharply with sections of similar tissues from mammals. ‘Avian erythrocytes are flattened, ellipsoidal cells with bright, red-orange, eosinophilic cytoplasm and deeply stained basophilic oval nuclei that are located centrally inthe cell. They normally are intravascular and are readily identified in tissue sections. Autolysis or exposure to hemolytic toxins such those produced by Clostridium sp. causes the cytoplasm of erythrocytes to lyse leaving “ghost cells” or only the intensely stained nuclei. The latter can be confused with small lymphocytes or thrombocytes. The relatively large, uniform, dark oval nuclei without cytoplasmic staining that are usually within vessels helps to identity the cells as lysed erythrocytes. Only very immature erythroid cells are identifiable in tissue sections. They are larger and more round than mature erythrocytes, and have basophilic cytoplasm that lacks granules, The nucleus of immature erythroid cells remains centrally located but is rounder, becomes less condensed with an identifiable ‘chromatin pattem, and is typically surrounded by a narrow pale zone in the cytoplasm. Nucieoli may be seen in erythroblasts, Immature lymphocytes have less cytoplasm that is pale staining, which helps to distinguish the two cell types. There are no granules in the cytoplasm of erythroblasts, which is useful in differentiating them from myeloblasts. However, in ‘some cases, cytoplasmic granules may be difficult to identify in myeloblasts making it necessary to rely on ‘ther morphologic features to distinguish the two cell types. Immature erythroid cells are intravascular and are uncommon in tissues other than bone marrow, although they may rarely be found in the sinusoids of the liver and spleen when erythropoiesis is intense. Avian thrombocytes function similarly to mammalian Platelets. Their aggregation provides an important Physical occlusion to reduce hemorrhage from ‘damaged vessels, however, they produce litle thromboplastin and play oniy a minor role in initiating blood clotting. Granules contain 5-hydroxytryptamine (serotonin), which is an early inflammatory mediator. ‘Thrombocytes rapidly clear particulates in the blood ‘and are considered the primary phagocyte in the circulation. Morphologically, avian thrombocytes are similar to erythrocytes but are smaller, have clear cytoplasm, are less elongated, and have rounded nucleus. Their cytoplasm stains positively with periodic acid-Schiff because of its high carbohydrate content. They rapidly degenerate and lose their oval shape in blood smears and tissue sections. Thrombocytes are dificult to recognize in tissues unless there is intravascular aggregation from vascular damage. Intravascular clumping of thrombooytes is increased by particulates in the ‘circulation. Aggregated thrombocytes do not fuse to form giant cells. Thrombocyte aggregates are most often observed in the small and intermediate vessels of the lungs. In tissue sections, their location within intravascular aggregates and small size help distinguish thrombocytes from nuclei of lysed erythrocytes or small lymphocytes. Birds have three granular leukocytes — heterophils, eosinophils, and basophils. Each avian granulocyte contains more than one type of granule and there are differences among avian species. Both heterophils and eosinophils have eosinophilic cytoplasmic granules that are often dificult to distinguish in tissues by conventional stains. Following prolonged storage in aqueous fixatives, heterophil granules can degrade and lose their characteristic spindle shape. ‘After heterophils die, which can occur both ante- or Post-mortem, heterophil granules become spherical ‘as the degenerate. Also, immature heterophils have spherical granules and in some avian species (e.g., waterfow)), heterophil granules are normally round. In‘most tissue sections heterophil granules generally are spherical making it difficult to distinguish heterophils from eosinophils. Special staining for phospholipids with Sudan black B or peroxidase can be used to differentiate the two cell types — heterophils are negative for both stains while eosinophils are positive. In descriptions of tissue changes, heterophils and eosinophils are often grouped together and collectively referred to as granulocytes, unless the defining granules of each cell type can be definitively identified Heterophils are the most abundant avian granular leukocytes. Those of the chicken and turkey are round with a lobulated nucleus, have numerous, spindle- or rod-shaped, eosinophilic, cytoplasmic granules, and pale staining cytoplasm. In well-preserved tissues, ‘mature heterophils in areas of acute inflammation ccan usually be identified. Granules often are seen extracellularly where heterophils are numerous. It is ‘common to find heterophils marginated in pulmonary vessels, especially venules, of normal lungs. Perhaps: this represents a pool of cells that can be rapidly mobilized. In addition to being increased in response to acute inflammation, heterophils also are increased during mild to moderate stress, but decreased if stress extreme. The ratio of heterophils to lymphocytes (HIL ratio) in blood of stressed birds increases (heterophils increase, lymphocytes decrease) making ita good method for assessing the onset, duration, and intensity of stress responses in birds. Heterophils are functionally equivalent to the mammalian neutrophil and are the primary cell involved in early acute inflammation. Although they lack myeloperoxidase, catalase, and alkaline phosphatase found in mammalian neutrophils (see Harmon, 1998), avian heterophils are highly efficient at phagocytosis and bacterial Kiling by non-oxidative processes. Oxidative burst, degranulation, and up regulation of pro-inflammatory cytokine production by heterophils results from stimulation of Toll-like receptors on the cell surface. Heterophils contain several cationic B-defensins that are capable of killing ‘a variety of bacteria both intra- and extracellularly Differences in enzyme content also are believed to account for the caseation of heterophilic exudate that occurs in birds compared to liquefaction and formation of purulent exudate in mammals. Activated heterophils have increased functional capabilities (adherence, chemotaxis, phagocytosis, and bacterial kiling) compared to non-stimulated heterophils. Heterophils show intense chemoattraction to endotoxin and are the principal cell that responds to gram-negative bacterial infections. Evidence indicates that heterophils also ‘may be the main effector cells in both antibody- dependent and natural cytotoxicity. Morphologic. changes in heterophils in response to severe stress include increased lobulation and fragmentation. Toxic heterophils characterized by swelling, condensed nucleus, loss of eosinophilic staining and granules, and vacuolation are seen in blood films from birds with inflammatory lesions. In tissues, these changes appear as degeneration and necrosis of the cells. Eventually heterophils undergo caseous necrosis. that results in a mass of inspissated exudate. ‘Macrophages progressively increase on the surface of the exudate and fuse to form multinucleated giant cells that form a complete layer covering the central caseated mass. Microscopically this appears as a palisade of giant cells and macrophages surrounding a mass of caseated exudate, Eosinophils of chickens and turkeys appear similar to heterophils, but have densely packed, round, eosinophilic, cytoplasmic granules, and pale basophilic cytoplasm. The latter may not be seen because of numerous granules filing the cytoplasm. As noted above, its often difficult to distinguish heterophils and eosinophils from each other in routine stained histologic sections. Experimental eosinophilia in birds is difficult to produce and there are few reports of naturally occurring eosinophilia. Unless confirmed by special stains, most reports of significant numbers Cf eosinophils in avian lesions are debatable. Eosinophils are suspected of being involved in the response of the bird to parasitism and allergic. response, but confirmation that the cells are actually eosinophils has been done in only a limited number of studies. Eosinophils have been shown to be involved in the early stages of acute inflammation induced by certain experimental substances, suggesting they play a modulating role in delayed-type hypersensitivity reactions. Cells morphologically consistent with eosinophils frequently are found in the deep lamina propria of the intestine, but their identity has not been confirmed. ‘Avian basophils are round cells with an oval nucleus that have many intensely basophilic, spherical, cytoplasmic granules that contain histamine. They appear early in the inflammatory response, and, like eosinophils, are thought to play a role in modulating the early events in the inflammatory process. Endotoxin is strongly chemotactic for basophils. Basophilia with degranulation of cells often occurs: in response to stressful situations. While basophils are readily identified in blood films, they are not ‘easily recognized in conventionally stained histologic sections. Mast cells contain several inflammatory mediators, of which histamine has been best studied, that are released from their cytoplasmic granules following stimulation. They are important effectorcells in inflammation, immediate but not delayed hypersensitivity, anaphylaxis, and play a significant role in intestinal immunity, especially in response to coccidiosis. The relationship between basophils in the Circulation and mast cells in tissues remains uncertain, but current evidence suggests they arise from different cell ineages. Staining methods can differentiate basophils and mast cells, as well as two types of mast cells - mucosal and connective ti Mast cells are widely distributed in birds. They can be found in tissues of the alimentary tract, lung, ovary, oviduct, lymphoid organs, thyroid glands, peritoneum, brain, nerves, eye, and skin. Distribution and number of mast cells varies within and among avian species, ‘Typically they are located in perivascular connective tissue and are especially numerous in the lamina propria of the digestive and reproductive tracts. Mast cells respond to parasitic infections in the intestinal tract and are significantly increased in the duodenum of turkeys that develop intestinal lesions following infection with hemorrhagic enteritis virus. Mast cells in the brain produce gonadotropic hormones and function in reproduction. Birds with muscular dystrophy have decreased mast cells in the thymus. Special stains (toluidine blue, Alcian blue/safranine, acid-fast) are Necessary to identify mast cells in tissues; fixation in alcoholic formalin or Camoy's fixative improves preservation of the granules. Nongranular avian leukocytes in birds — lymphocytes and monocytes ~ are similar to those in mammals. Lymphooytes are classified morphologically as small, medium, or large. Small and medium lymphocytes are considered most important; large lymphocytes are considered immature cells. Functionally, lymphocytes are divided into those cells that differentiate in the thymus (T-cells) and those that differentiate in the bursa of Fabricius (B-cells). T-cells posses. specific surface antigens (CD3, CD4, CD8) and proliferate following exposure to certain lectins (phytohemagglutinin and concanavalin A), which is referred to as a mitogenic response. Natural killer cells are small lymphocytes (5-6 ym) with a low cytoplasm:nucleus ratio and many surface projections. They are CD8+ but lack both B- and T-cell markers. B-cells can be identified by the presence of markers including Fc receptors and surface immunoglobulins. Lymphocytes within inflammatory lesions are considered to be responding to antigens or abnormal cells. Responses of these cells are closely regulated and interwoven with those of the monocyte-derived macrophages through chemicals (cytokines) ‘elaborated by the cell types; lymphokines produced by lymphooytes and monokines produced by macrophages. Organized lymphoid nodules mature into germinal centers that contain large B-lymphocytes centrally. Smaller T-cells form a more diffuse infiltrate around the periphery of the B-cells and within interstitial tissues. Natural Killer cells are important in intestinal mucosal immunity and destruction of tumor cells. Under appropriate stimulation, B-lymphocytes differentiate into plasma cells, which can be identified in tissue sections by their eccentricaly located hucleus, often with a roughly radially arranged chromatin pattem; thin, pale perinuclear area of cytoplasm, and rich, basophilic staining of the remaining cytoplasm. Single or multiple accumulations of globulin may occur in the cytoplasm of plasma cells that are actively producing antibody. They appear as hyaline, eosinophilic globules and are termed Russell bodies. Cells containing Russell bodies are referred to as Mott cells. Lymphocytic or lymphoplasmacytic inflammation occurs most frequently in viral infections and tumor diseases. Monocytes are larger than lymphocytes with more cytoplasm that is less basophilic and often contains vacuoles. They have an elongated, indented nucleus surrounding a pale area of cytoplasm known as the "Hof, which contains well-developed Golgi complexes. Monocytes and large lymphocytes appear similar, especially in tissues. Extravascular ‘monocytes become activated and morph into tissue macrophages, which exhibit chemotaxis, are phagocytic, and participate in antigen processing and immune regulation. Macrophages can develop an epithelioid appearance and fuse to form multinucleated giant cells. When differentiating into epithelioid cells they lose IgG receptors, which may be related to an inability to remove necrotic material resulting in the persistent caseous exudate that characterizes chicken heterophilic granulomas. Initial macrophages recruited to an area of inflammation are less phagocytic ‘compared to later macrophages. Bactericidal activity ‘of macrophages is less than that of heterophils. ‘Concanavalin A binding can be used to specifically identify macrophages in tissues. Staining for lysozyme also is useful for identifying macrophages, but is. less specific. In turkeys, intravascular macrophages are primarily found in liver sinusoids along with lower numbers in the spleen and bone marrow, but are not present in the lung. Viruses can adversely affect macrophage function and may replicate in the cells causing necrosis. Similarly, endotoxin impairs intracellular bacterial kiling by pulmonary macrophages, but does not affect oxidative burst. Bone Marrow Changes in bone marrow include disturbances of ‘growth (atrophy, hyperplasia), pigment deposition,inflammation, and neoplasia. They result from direct involvement of the marrow, or as reflections of pathologic processes in other organ systems. Bone ‘marrow responses are limited to either a decrease or increase in cells or inflammation. Cellular depletion results in atrophy and is usually caused by direct damage to marrow cells. Bone marrow atrophy is manifested in the bird by anemia, leukopeni thrombocytopenia, or pancytopenia depending on Which cell type is affected. Survival and recovery from the cause of bone marrow atrophy results in regeneration and eventual return to a normal cell population. Bone marrow hyperplasia occurs during recovery from bone marrow atrophy or in response to an extemal stimulus to the hemic system. For example, hypoxia stimulates erythroid hyperplasia, which results in polycythemia, while acute inflammation stimulates granulocytic hyperplasia resulting in leukocytosis. Compared to mammals, bone marrow of birds is substantially more reactive, and hyperplasia resulting from sepsis, bacterial infections, or exposure to endotoxin can be so pronounced that it is difficult to distinguish it from myeloid neoplasia. Lack of tissue effacement or invasion, presence of cells representing all stages of maturation, and absence of tumors in other tissues are Useful features for identifying hyperplasia. In contrast, expansive nodules composed of a monomorphic population of myeloid cells, and destruction or spread into adjacent tissues characterizes neoplastic myeloid lesions. Presence of numerous mitotic figures is not helpful in differentiating myeloid hyperplasia and neoplasia as cell proliferation is a feature of both. Manifestations of bone marrow changes have been associated with a variety of diseases, but their pathogenesis and the histopathologic appearance of the bone marrow have been studied infrequentty This is particularly true of anemia, which can easily be determined by procedures such as hematocrits. AS identification of decreased numbers of thrombooytes. and leukooytes is more difficult, involvement of other blood cells besides erythrocytes can go unrecognized if bone marrow is not evaluated. Many ‘anemic especially those that result from either toxic or infectious agents, are actually pancytopenias. In birds with diseases that manifest a hematologic disorder, histopathological evaluation of the bone marrow should be done. Anemia ‘Anemia is characterized by an abnormally low number of erythrocytes or hemoglobin concentration within the erythrocytes, which results in impaired delivery of oxygen to tissues. Hypoxia signals release of erythropoietin, which initiates erythropoiesis. Anemia results from either loss of erythrocytes in excess of the abilty to produce them or a failure to produce new cells to replace those lost through attrition. If there is a bone marrow response, the anemia is classified as regenerative, and there is increased erythropoiesis in the marrow and immature red cells inthe circulation. Ifthe bone marrow is unresponsive to an increased demand for oxygen and erythrocytes, the anemia is classified as non-regenerative. In non-regenerative anemia, bone marrow is depleted and immature red cells are not present in the blood. If sufficient residual hematopoietic stem cells survive in cases of direct destruction by toxic substances, infectious agents, or radiation, what begins as a non-regenerative anemia will transition to a regenerative anemia, as the erythron is re-established. ‘Anemia also can be classified according to the basic, causative mechanism as hemorrhagic, hemolytic, aplastic, or myelopathic (myelophthisic). Hemorrhagic and hemolytic anemias are usually regenerative while aplastic and myelopathic anemias are usually non-regenerative. Aplastic anemias are generally only part of a pancytopenia in which leukocytes and thrombocytes also are decreased in the circulation, Many causes of anemia have been described in birds (Table 1). Hemorrhagic Anemia. Hemorrhagic anemia results when blood loss exceeds replacement. It ‘occurs in conditions characterized by acute or chronic blood loss. Birds are capable of rapid Fecovery through intense erythropoiesis following ‘an episode of hemorrhage or chronic, sub-lethal blood loss. Identifying the cause of hemorrhagic ‘anemia should present litle difficulty unless blood loss has resulted from a seemingly minor lesion. Other than hemorrhage at the site of blood loss and possible identification of a cause, there are no specific microscopic lesions. Normal variation in the number of red blood cells within vessels obscures any ‘overall deficit of erythrocytes. If blood loss is chronic, erythroid hyperplasia in the marrow, and possibly in extramedullary sites (hepatic sinusoids, spleen), can be seen. Hemolytic Anemia. Hemolytic anemia results from erythrocyte destruction exceeding the ability of the bird to replace the destroyed cells. Red cell destruction may occur by cell lysis within the vascular compartment (intravascular hemolytic anemia) or extravascularly following phagocytosis by macrophages (extravascular hemolytic‘anemia). Hemoglobinuric nephrosis can result if sufficient intravascular hemolysis occurs, but this has rarely been recognized in birds and has not been reported from poultry. In hemoglobinuric nephrosis, bright orange hemoglobin fils Bowman's space of glomeruli and renal tubules and some tubules show acute epithelial necrosis. Splenic hyperplasia, erythrophagocytosis, and excess hemosiderin (hemosiderosis) in hepatic and splenic macrophages, hepatocytes, and, less frequently, renal tubular epithelium, are characteristic microscopic changes seen in extravascular hemolytic anemias. Degenerative changes resulting from hypoxia may be seen in the liver (periacinar degeneration and necrosis) and myocardium if the anemia is severe. Hemoprotozoan infections, bacterial infections, and toxins cause hemolytic anemias in birds. Hemoprotozoa can be seen in heavy infections or when parasites are large, 0.9. Leucocytozoon, but are best diagnosed from blood smears or tissue impressions rather than histologic sections. If present, exoerythrocytic stages of some parasites are usually easier to identify than erythrocytic stages. Destruction of parasitized red cells can be mediated through antibodies or by anti-erythrocytic factors. Autoimmune hemolytic anemia has rarely been described in birds and has not been reported in poultry. When bacteria cause hemolytic anemia, they can generally be identified in lesions in affected tissues although special stains may be needed. Finding bacteria in the bone marrow is useful for identifying septicemia. Toxic causes of hemolytic anemia are more dificult to identify, as tissue changes are often not specific. However, Heinz body anemias have been produced experimentally in birds with dimethy! disulfide and phenylhydrazine, acid-fast intranuclear inclusion bodies in liver and kidney can occasionally be found in cases of lead poisoning, and Mallory's stain may reveal blue granules in sinusoidal lining cells (Kupffer cells) indicative of copper accumulation in copper toxicty Myelopathic anemia. Myelopathic (myelophthisic) anemia results from replacement of marrow by another tissue. The cause is usually neoplasia that proliferates within the bone effacing the marrow. An exception is mycobacteriosis in which epithelioid macrophages and giant cells packed with acid-fast bacilli replace normal bone marrow. Other Types of Anemia. Inherited and nutritional anemias are of litle significance in poultry. The former is apparently rare while nutritional anemia is Lnikely to occur in poultry fed commercially formulated rations. However, iron-deficiency anemia can result from interference with iron uptake and utilization by ochratoxin, and feeding 10% rapeseed meal to adult chickens caused a regenerative macrocytic a but the mechanism responsible for the anemia was not determined. Significant anemia without tumor formation in young chickens and turkeys has been associated with infection by certain strains of leukosis/sarcoma viruses. In most instances, the pathogenesis is. unknown. Avian leukosis viruses in subgroups B and D caused the most severe anemia in experimentally inoculated chickens. Some chickens that survived the initial anemia, later died from a nonregenerative anemia following reactivation of virus infection. Pancytopenia. Pancytopenia (aplastic anemia) ‘occurs when pluripotent hematopoietic stem cells are impaired or lost, which also results in leukopenia and thrombocytopenia in addition to anemia. Pure red cell hypoplasia refers to conditions in which there is a nonregenerative anemia because of a lack of erythroid stem cells, but normal numbers of other blood cells. Until the diseases in birds characterized by extensive loss of bone marrow cells are better defined, it ‘seems best that they should be treated together and referred to as pancytopenia. Diseases that cause pancytopenia are characterized by anemia, which is initially nonregenerative; thrombocytopenia associated with bleeding disorders; increased susceptibility to ‘bacterial infections related to leukopenia, decreased macrophages, and immunosuppression; pale, atrophied hypocellular bone marrow with increased fat content; and lymphoid organ atrophy. Viral infections, toxicities, and starvation are the main causes of pancytopenia in birds (Table 1). Irradiation also causes pancytopenia, but its of little practical significance to the diagnostician. Pancytopenia is recognized microscopically by marked depletion of the bone marrow leaving only the reticular framework of the sinusoids and fat cells. In the early stages, necrosis of marrow cells characterized by pyknosis (karyopyknosis) and karyorthexis is seen. Inclusions typical of adenoviruses and chicken infectious anemia virus may be seen in poultry while those of polyomavirus, herpesvirus, and psittacine circovirus have been reported from other avian species. Death due to sepsis is a common cause of mortality in birds with pancytopenia. in such cases, intravascular bacteria usually are seen in the marrow. ‘When bone marrow depletion is severe, it often is still possible to find single or small clusters of very large hemocytoblasts. These will serve as the source {or repletion of the marrow and undergo hyperplasia to regenerate the bone marrow if the bird survives. and the initial cause of the pancytopenia has beeneliminated. Depletion of lymphocytes from lymphoid organs typically parallels bone marrow changes in Pancytopenia. Bone marrow hyperplasia In contrast to anemia, polycythemia is an abnormal inorease in the number of red cells in the circulation that is manifested by elevated packed cell volumes and increased hemoglobin concentrations. Hemoconcentration caused by dehydration results in a relative increase in red cells that must be differentiated from polycythemia. Absolute increases of red cells are uncommon, but occur in meat-type chickens. in response to hypoxia from altitude, insufficient cardiopulmonary capacity, pulmonary or skeletal disease, or low ambient temperatures. Polycythemia increases blood viscosity, which predisposes to pulmonary hypertension, right heart failure, and ascites. Decreased deformability of red cells also contributes to pulmonary hypertension. Bone marrow changes in polycythemia have not been reported but erythroid hyperplasia would be expected. Increased white blood cells in the circulation (leukocytosis), which occurs frequently in many avian infectious diseases and as a response to stress, would be expected to be associated with myeloid hyperplasia. However, there appears to be only limited information correlating the histopathology of the bone ‘marrow with episodes of leukocytosis. Acute viral, bacterial, and fungal infections can cause myeloid hyperplasia. Hematopoietic stem cell proliferation and hyperplasia accompanied leukocytosis and selective thrombocytopenia in chickens experimentally infected with virulent influenza (H5N2) virus. Non-specific myeloid hyperplasia is frequently seen in diagnostic case material. Myeloid hyperplasia can be marked and needs to be distinguished from myeloid leukosis in which clonal populations of granulocytes at a single maturation stage predominate, there is tissue destruction or invasion, and tumors are present in other tissues. Bone marrow inflammation ‘Atthough bone marrow actively participates in inflammatory processes in the body, there is litle information on the local response of bone marrow to injury. Necrosis of bone marrow occurs in some ‘acute viral diseases and toxicities, but the response to the injury is not inflammation, but regenerative hyperplasia. Particulate uptake by bone marrow ‘approximates that of the spleen, and bone marrow is recognized as an excellent site for isolating systemic infectious agents. In avian mycobacteriosis, ‘granulomas frequently form in bone marrow and can, become so extensive that a myelopathic anemia results. Increasing diffuse and nodular lymphoid tissue ‘seen in commercial birds as they age suggests bone marrow participates in systemic responses to antigens. Pigments Hemosiderin can be found in bone marrow, especially in cases of extravascular hemolytic anemia. Dark brown to black protoporphyrin pigment accumulates in bone marrow in addition to the liver and spleen in birds with protoporphyria, an inherited defect in heme synthesis. The pigment has a Maltese cross appearance when viewed with polarized light. Inflammation Inflammation occurs in response to an initant. The degree and type of inflammatory response depends cn the tissue that is damaged, degree to which the irritant can damage tissue, ability of the bird to respond, and duration and severity ofthe injury. Necrotic tissue increases the degree of inflammation and persistence of a lesion; the dead tissue serves as an additional irtant unti itis removed or can be sequestered by a granulomatous response. In ‘general, the inflammatory process in birds is similar to that of mammals, except 1) basophils and eosinophils. ‘are more involved in modulating the early phases of inflammation in birds, 2) fibrinous and heterophilic ‘exudates undergo caseation instead of liquefaction and are slowly removed by macrophages and giant cells, 3) the role of eosinophils in allergic reactions and response to metazoan parasites is less well known in birds, and 4) the stages of inflammation proceed at a rapid rate. Stages of inflammation requiring days to weeks in mammals occur within hours to days in birds. Similarities between inflammation in mammals and birds have resulted in terminology used for ‘mammals being applied to birds. In some instances, (eg, catarthal, serous, fibrinous, etc.) the application is appropriate, while with other types of inflammation, Use of terms more descriptive for birds are preferred (Table 2). Liquid to semisolid purulent exudate that is common in acute mammalian inflammation occurs rarely in birds making use of the term purulent to describe what is actually solid caseous exudate in birds inacourate. The term fbriscess is preferred for focal inflammatory lesions in birds and reptiles inwhich multiple layers of fibrin entrap inflammatory cells and causative agents, instead of the mammalian term abscess, a lesion which forms by a wholly different process, Mid irritation of secretory membranes stimulates: hypersecretion resulting in catarthal inflammation of mucous membranes, serositis of serosal membranes, ‘or synovitis of synovial membranes. In catarrhal inflammation, goblet cells and mucous gland activity are increased and excess mucus is present on the mucosal surface and in the lumen of the affected ‘organ. Increased eosinophilic staining reflects ‘changes in mucus composition. Often mucociliary clearance in airways is compromised in catarthal inflammation of the respiratory tract, which can lead to accumulation of mucus on the mucosal epithelium. Microscopic changes in mild inflammation of serosal and synovial membranes are more difficult to detect. ‘Membranes may be thickened and edematous because of the excess fluid, and epithelial cells are swollen and rounded. Epithelial hyperplasia that progresses to polypoid proliferations is a feature of chronic lesions of secretory membranes. Lymphoid inftrates often accompany chronic proliferative lesions. Fibrin exudation and cellular infitration are not features of this stage of inflammation. With intense irritants, the inflammatory process proceeds so rapidly that these early changes are often not seen. Continued or more severe irritation leads to vascular changes and leakage of proteins causing edema with or without accompanying fibrin (serous, serofibrinous, or fibrinous inflammation). Early vascular changes, most likely in response to the release of vasoactive ‘amines from mast cells and basophils, affect the post-capillary venules. Serous fluids containing protein are homogenous and stain pink with H&E stains, the intensity of color being proportional to the protein content. Proteinaceous fluid, often seen ‘around sheathed capillaries in the spleen of birds with bacterial sepsis, needs to be distinguished from amyloid because of its similar appearance and ‘occurrence in that organ. Fibrin forms eosinophilic strands or networks that appear more solid as it condenses. Interconnections of fibrin are marked by a characteristic, small, bead-like consolidation, which aids in its histologic identification. At this stage, there may be small, intravascular aggregates of thrombocytes or thrombooytes adhering to the vasculature intima. Intravascular fibrin thrombi in the liver, spleen, kidneys, and, less commonly, the lungs also may be present and indicate a systemic process. Disseminated intravascular coagulation can occur, usually because of bacterial septicemia. ‘Accompanying leakage of proteins, margination ‘and emigration of cells accelerates to become the predominant feature of the inflammatory process. ‘Within 30-60 minutes of exposure of a tissue to an intant, there is dilation of venules, margination of heterophils and monocytes, and emigration of these cells into surrounding tissues in response to locally generated chemoattractants. Heterophils may predominate, be less than, or approximately equal to the number of monocytoid cells depending on the nature of the irritant. Endotoxin is strongly chemotactic for heterophils. While heterophils and monocytoid cells continue to increase, variable numbers of basophils also migrate to the damaged site. This ‘constellation of rapid mobilization and emigration of heterophils and monocytes with basophil participation is considered characteristic of early inflammation in the chicken and an essential survival mechanism. Marginated intravascular reserves and circulating pool of leukocytes provide the cells for the initial response. Transient heteropenia occurs simultaneously with, the rapid increase in tissue heterophils. Subsequent heterophilia likely results from bone marrow activation and hyperplasia. As inflammation develops, monocytes become activated into macrophages and. heterophils undergo degeneration and degranulation releasing toxic substances into surrounding tissues. By approximately 12 hours basophils are no longer a feature of the process and heterophils, many of which are degenerating, reach maximal numbers. and begin to decrease. Over the next 12-24 hours, lymphoid cells and macrophages continue to increase and become the prominent feature of the lesior macrophages fuse to form multinucleated giant cells. Presence of giant cells in avian inflammation should Not be interpreted as an indication of chronicity Macrophages play a central role in avian inflammation. Controversy exists concerning the relative significance of macrophages and heterophils as primary phagocytic cells in vivo. Both are capable of phagocytosis and probably perform this function although variations likely exist depending on the specific circumstances. involved in the inflammatory process. Cytokines, produced primarily by macrophages, with activities similar to mammalian IL-1, IL-2, IL-6, TNF, and granulocyte colony-stimulating factor, orchestrate local and systemic responses. Toll-like receptors on the ccell membrane recognize basic molecular pattems. of infectious agents, which stimulate production of reactive oxygen products, nitric oxide, and pro- inflammatory cytokines. Macrophages also are responsible for recruiting lymphocytes to the area and stimulating their multiplication and differentiation intoplasma cells. They also process antigens and deliver ‘them to lymphocytes initiating an acquired immune response. Exudate that results from tissue necrosis and massive fibrin and heterophil accumulation caseates to form a heterophilicfbriscess. This may not be a static. process as repeated episodes of inflammation can ‘occur resulting in enlargement of the original mass and a laminated “onion-skin” appearance grossly and microscopically. Often intralesional bacterial colonies are located within the caseous exudate. Although the bacteria are sequestered within the exudate, they remain viable and can be isolated using appropriate cultural methods. Colonial morphology may provide ‘a presumptive identification, but isolation and identification should be relied on to confirm the identity of the organism. Macrophages accumulate around the core of necrotic fibrinoheterophilic debris; many fuse to become giant cells. After a few days, a layer ‘of macrophages and giant cells forms on the surface of the necrotic core isolating it from adjacent viable tissue, which results in the formation of a heterophilic, granuloma. Microscopically, a palisade of radially arranged multinucleated giant cells rims the caseous core. An incomplete layer of macrophages and giant cells or focal necrosis of the cells with extension of ‘exudation through this cell layer indicates the process is stil active and has not been completely controlled. ‘A broader zone of predominantly macrophages, ‘often mixed with a few heterophils and giant cells, surrounds the core rimmed by giant cells. The heterophilic granuloma later becomes surrounded by fibrous tissue that condenses with age and may contain variable numbers of lymphocytes and plasma cells. Phagocytic cells slowly erode the caseous exudate over an extended period. However, variable ‘numbers of heterophils continue to be present in these lesions suggesting the exudate itself serves as an irritant provoking a continued mild inflammatory reaction. Ifthe caseated central mass is eventually removed, the damaged site fills wth granulation tissue, which matures to fibrous connective tissue. With the possible exception of parasitic granulomas, Calcification of exudate is not a feature of either heterophilc or histiocytic granulomas in birds. Histiocytic granulomas develop by an entirely different process. The initial lesion is a collection of foamy macrophages, which contain the causative organism (e.g., Mycobacteria). As the lesion expands, centrally located cells undergo degeneration and necrosis, often provoking a mild heterophilic response. This is ‘accompanied by marked proliferation of giant cells. The causative agent may be demonstrable in these cells or free in the necrotic central area. At certain stages, particularly terminally histiocytic granulomas can appear similar to heterophilic granulomas. The primary difference between heterophilic and histiocytic - granulomas is the type of cell forming the necrotic center of the lesion. Inflammation in chiokens also has been studied 4 by examining wound repair. In wound healing, inflammatory and reparative processes occur - together. Fibroplasia begins at around 18 hours following the injury. By day three angiogenesis and re-epithelialization are occurring, healing is essentially ‘complete by day 7, and tissue is nearly normal at day 10. Beginning on day 5, there is a period when rmult-nucieated cells are present in granulation = tissue. Newly formed vessels within granulation tissue persist and remain patent. This same process ‘occurs in response to ulceration of the skin or mucous membranes. Neoplasia AA variety of mesenchymal and some epithelial neoplasms result from infection with avian retroviruses (leukosis/sarcoma viruses) including ones involving Cells of the hematopoietic system — lymphoid leukosis (See Chap. 2), myeloid leukosis (myeloblastosis, e myelocytomatosis), and erythroblastosis (erythroid leukosis). The type of neoplasia that develops depends on the strain and dose of virus; genotype, age, and sex of the bird; route of inoculation; and presence or absence of “helper” viruses. As noted above hematopoietic neoplasia needs to be differentiated from bone marrow hyperplasia, ‘extra-medullary hematopoiesis, and inflammation. Hematopoietic neoplasia may be aleukemic or leukemic. Circulating tumor cells are often abundant and readily identified in hepatic sinusoids when leukemia is present, which aids in diagnosis. Myeloid tumors may be comprised of cells in any stage of differentiation from blast cells (myeloblastosis, myeloblastomas) to nearly mature granulocytes (myelocytomatosis, myelocytomas). When myeloid leukosis is caused by serogroup J avian leukosis Virus (ALV-J), tumors composed of cell populations in different stages of maturation can be found in the same bird. Collectively granulocytic tumors are referred to as myeloid leukosis regardless of the maturational stage of the cell type, which is useful since tumors represent the full gradient of cell maturation and they have the same etiology. However, myeloblastomas and myelocytomas differ in both gross ‘and microscopic pathology. 10Myelocytomas are cream-colored, soft, nodular, multiple, tumors that are commonly associated with the periosteum and cartilaginous junctions of bones, especially ribs, stemum, vertebrae, synsacrum, and fiat bones of the skull. Bone marrow is pale. Visceral ‘organs (ver, spleen, kidney, gonads, lungs, etc.) also are affected. Microscopically, tumors consist ‘of sheets of uniform myelocytes characterized by medium size; large, round to elongated nuciel; and ‘cytoplasm containing abundant, intensely eosinophilic round granules. Mitotic figures are common. Marrow is effaced and tumors extend through bone along Haversian and Volkmann’s canals into adjacent soft tissues. Initial lesions begin in the bone marrow of the epiphysis. Atrophy of bone and periosteal proliferation of bone accompany myeloid lesions. Lesions also may occur in the dura mater and ossified respiratory tissues. Myeloblastomas are characterized by enlargement of liver, spleen, and kidneys, which are pale, mottled or, on rare occasions, contain gray nodules. Bone marrow is pale gray to white. Organ enlargement is due to massive intra- and extravascular accumulations of immature myeloid cells (myeloblasts, promyelocytes). Myeloblasts tend to be larger than rmyelocytes but smaller than erythroblasts; have a ‘more acidophilic cytoplasm, often with faintly visible, large, pale granules; and are polygonal or angular shaped compared to the round to oval shape of erythroblasts. Their nucteus is denser and nucleoli may be visible, but they are not as prominent as those seen in erythroblasts. Affected birds are usually leukemic and large numbers of these cells can be seen in blood smears. Extramedullary hematopoiesis is often prominent, and should not be interpreted as representing a different type of neoplasm. Erythroblastosis (erythroid leukosis) is much less ‘common compared to lymphoid or myeloid leukosis. ‘Two distinct diseases result from infection with erythrobiastosis viruses — a proliferative form and ‘an anemic form. In the proliferative form, there is diffuse hepatic and splenic enlargement. The ‘organs and bone marrow are bright red. Sinusoids in these organs and the bone marrow are packed with erythroblasts. Tumors per se do not develop in erythroblastosis; lesions remain intravascular. In the anemic form, visceral organs are normal or atrophic, birds are severely anemic, and bone marrow is pale, almost acellular, and partly replaced by massive proliferation of endosteal bone. Erythroblasts have a large round nucleus with fine chromatin, one or two nucleoli, and abundant basophilic cytoplasm that is often pale around the nucleus. They are generally larger than myeloblasts. Presence of cytoplasmic granules in the cells distinguishes myelobiastosis " from erythroblastosis, but occasionally the granules, in myeloblasts can be difficult to identify. Both myeloblasts and erythroblasts may be intravasculat but erythroblasts are only intravascular. If necessary, cell markers specific for each cell type can be identified by immunohistochemistry. In blood smears, large numbers of erythroblasts and other immature erythrocytes can be seen in both forms. As with myeloblastosis, extramedullary hematopoiesis can be extensive in birds with erythroblastosis and should not be confused with neoplasia. Additionally, mixed erythroblastosis and myeloblastosis cases, and rare tumors comprised of hemopoietic stem cells, which must be differentiated from myelobiasts and erythroblasts occur. Cells in hemopoietic stem cell tumors are much larger and more basophilic than either erythroblasts or myeloblasts. They have a large ‘nucleus with prominent nucleoli and abundant intense staining cytoplasm that lacks visible granules. ‘Acknowledgement. Critical review of this chapter and glass slides of normal and hyperplastic bone marrow provided by Dr. Floyd Wilson are gratefully acknowledged. Additional Readings ‘Andreasen, C. B., and K. S. Latimer. 1990. Cytochemical staining characteristics of chicken heterophils and eosinophils. Vet Clin Pathol 19:51-54. ‘Awadhiya, RP. J.L. Vegad, and G.N. Kolte. 1980. Demonstration of the phagocytic activity of chicken thrombocytes using colloidal carbon. Res Vet Sci 29:120-122. ‘Awadhiya, R. P., J. L. Vegad, and G.N. Kolte. 1980. Studies on acute inflammation in the chicken using mesentery as a test system. Res Vet Sci 29:172-180. Bames, H. J. 1996. Hemic System. In, Avian Histopathology, C. Riddell (ed.), Am Assoc Avian Pathol, Kennett Square, PA. pp. 1-16. Bar-Shira, E., and A. Friedman, 2006. Development and adaptations of innate immunity in the gastrointestinal tract of the newly hatched chick. Dev ‘Comp Immunol 30:930-941. Bermudez, A. J., and B. A. Hopkins. 1995. Hemoglobinuric nephrosis in a rhea (Rhea americana). Avian Dis 39:661-665.Bounous, D.l., and N.L. Stedman. 2000. Normal avian hematology: chicken and turkey. In Schalm’'s Veterinary Hematology, 5° ed., B.F. Feldman, J.G. Zinki, and N.C. Jain, Eds. Lippincott Willams & Wikkins, Baltimore, MD. pp. 1147-1154. Brooks, R. L. Jr, D. |. Bounous, and C. B. Andreasen. 1996. Functional comparison of avian heterophils with human and canine neutrophils. Comp. Haematol. Int. 6:153-159, Calderon, N. L., F. Galindo-Muniz, M. Ortiz, B. Lomniczi, T. Fehervari, and L. H. Paasch, 2005. Thrombocytopenia in Newcastle disease: haematological evaluation and study of bone marrow. Acta Vet Hung 53:507-513. Caldwell, D. J., H. D. Danforth, B. C. Mortis, K.A. ‘Ameiss, and A. P. McElroy. 2004. Participation of the intestinal epithelium and mast cells in local mucosal immune responses in commercial poultry. Poult Sci 83:591-599, Campbell, T. W,, and C. K. Els. 2007. Avian and Exotic Animal Hematology and Cytology. Blackwell, Publishing Professional, Ames, 1A. pp. 3-50. Chiu, H., and D. Lagunoff. (1972). Histochemical ‘comparison of vertebrate mast cells. Histochem J 4: 135-144, Clark, M. W., R. P. Gildersleeve, J. P. Thaxton, C.R. Parkhurst, C. R., and D. |. McRee. 1988. Hematological effects of ethyl methanesulfonate, paraquat and phenylhydrazine in Japanese quall ‘Comp Biochem Physiol C 89:15-30. Crespo, R., and R. P. Chin. 2004. Effect of feeding green onions (Allium ascalonicum) to White Chinese geese (Threskiomis spinicollis). J Vet Diagn Invest 16:321-325, Gobel, T. W., B. Kaspers, and M. Stangassinger. 2001. NK and T cells constitute two major, functionally distinct intestinal epithelial lymphocyte subsets in the chicken. Int Immunol 13:757-762. Harmon, B. G. 1998. Avian heterophils in inflammation and disease resistance. Poult Sci 77:972-977. He, H., K. J. Genovese, D. J. Nisbet, and M. H. Kogut. 2006. Profile of Toll-like receptor expressions and induction of nitric oxide synthesis by Tol-like receptor agonists in chicken monocytes. Mol Immunol 43:783- 788, Huchzermeyer, F. W., and J. E. Cooper. 2000. Fibriscess, not abscess, resulting from a localised inflammatory response to infection in reptiles and. birds. Vet Rec 147:515-517. Hutchinson, A. E., and R. B. Owen. 1984. Bone marrow fat in waterfowl. J Wild! Manage 48:585-591. Inoue, M.,A. Fujita, and K. Maeda. 1999. Lysis of myelocytes in chickens infected with infectious bursal disease virus. Vet Patho! 36:146-151. Johnston, M. S., T. T. Son, and K. L. Rosenthal. 2007. Immune-mediated hemolytic anemia in an eclectus parrot. J Am Vet Med Assoc 230:1028-1031. Kaiser, P. 2007. The avian immune genome - a glass half-full or haif-empty? Cytogenet Genome Res 117:221-230. Klasing, K. C. 1998. Avian macrophages: regulators of local and systemic immune responses. Poult. Sci 77: 1983-989, Kogut, M. H., I. Muhammad, H. He, V. Philbin, P. Kaiser, and A. Smith. 2005. Expression and function of Toll-like receptors in chicken heterophils. Develop ‘Comp immuno! 29:791-807. Kunkle, R.A., and R. B. Rimler. 1996. Pathology of acute aspergillosis in turkeys. Avian Dis 40: 875-886. Latimer, K. S., K.N. Tang, MA. Goodwin, WL. Steffens, and J. Brown. 1988. Leukocyte changes associated with acute inflammation in chickens. Avian Dis 32:760-72. 2 Lowry, V. K., K. J. Genovese, LL. Bowen, and M. = H. Kogut. 1997. Ontogeny of the phagocytic and bactericidal activities of turkey heterophils and - their potentiation by Salmonella enteriids-immune lymphokines. FEMS Immunol Med Microbiol 19:95- 100. Maxwell, M. H. 1987. The avian eosinophil ~ a review. World's Poult Sci J 43:190-207. Maxwell, M. H. 1995. The avian basophilic leukocyte: review. World's Poult Sci J 51:307-325 Mayne, R. K.,F. Powell, R. W. Else, P. Kaiser and P.M. Hocking. 2007. Foot pad dermatitis in growing turkeys is associated with cytokine and cellular changes indicative of an inflammatory immune response. Avian Patho! 36:453-459. 12Montali, RJ. 1988. Comparative pathology of inflammation in the higher vertebrates (reptiles, birds and mammals). J Comp Pathol 99:1-26. Nair, M. K. 1973. The early inflammatory reaction in the fowl. A light microscopical, ultrastructural and ‘autoradiographic study. Acta Vet Scand (Suppl) 42:1- 103. Nakamura, K., M. Ogiso, K. Tsukamoto, N. Hamazaki, H. Hihara, and N. Yuasa. 2000. Lesions of bone and bone marrow in myeloid leukosis occurring naturally in adult broiler breeders. Avian Dis 44:215-221 National Research Council. 1994. Nutrient Requirements of Poultry, 9th ed. National Academy Press, Washington D.C. pp. 46-57. hitov/www.nap, ‘edulopenbook php?isbn=0308048023 Pantin-Jackwood, M. J., T. P. Brown, and G. R. Huff. 2004. Proventricultis in broiler chickens: immunohistochemical characterization of the lymphocytes infiltrating the proventricular glands. Vet Pathol 41:641-648. Petrone, V. M., C. F. Constantino, and P. Pradal-Roa 2002. Identification and quantification of granulocytes in caecal mucosa and submucosa of chickens experimentally infected with Eimeria tenella and ‘Salmonella enteritidis. Br Poult Sci 43:653-661. ‘Qureshi, M. A. 2003. Avian macrophage and immune response: an overview. Poult Sci 82:691-698. Schepelmann, K. 1990. Erythropoietic bone marrow in the pigeon: development of its distribution and volume during growth and pneumatization of bones. J ‘Morphol 203:21-34. Siatskas, C., and R. Boyd. 2000. Regulation of chicken haemopoiesis by cytokines. Develop Comp Immunol 2437-68, Silverman, A-J., L. Asarian, M. Khali, and R. Silver. 2002. GnRH, brain mast cells and behavior. In, Progress in Brain Research, Vol. 141, L. S. Prahar, Ed. Elsevier Science B.V. pp.317-327. ‘Smyth, J.A.,D.A. Moffett, TJ. Connor, and M. S. McNulty, 2006. Chicken anaemia virus inoculated by the oral route causes lymphocyte depletion in the thymus in 3-week-old and 6-week-old chickens. Avian Pathol 36:254-259, ‘Takami, S.,M. Goryo, T. Masegi, and K. Okada. 2005, Systemic spindle-cell proliferative disease in broiler chickens, J Vet Med Sci. 67:13-18, Ueda, ¥., ¥. Aohagi, and S. Nakahara. 2005. Distribution of protoporphyrin in female broiler chickens affected with protoporphyria. J Vet Med Sci 67:1289-1291. Vegad, J. L., and A. K. Katiyar. 1995, The acute inflammatory response in the chicken. The acute inflammatory response in the chicken. Vet Bull 65:399-409. Venugopal, K. 1999. Avian leukosis virus subgroup J: a rapidly evolving group of oncogenic retroviruses. Res Vet Soi67:113-119. Wade, L. L., and S. J. Newman. 2004. Hemogiobinuric nephrosis and hepatosplenic erythrophagocytosis iin a dusky-headed conure (Aratinga weddell) after ingestion of garlic (Allium sativum). J Avian Med Surg 18:155-161. ‘Weber, M.A. S. P. Terrell, D. L. Neiffer, M.A. Miller, ‘and B. J. Mangold. 2002. Bone marrow hypoplasia and intestinal crypt cell necrosis associated with fenbendazole administration in five painted storks. J ‘Am Vet Med Assoc 221:417-419. Zhang, Z., F. Wilson, R. Read, L. Pace, and S. Zhang. 2006. Detection and characterization of naturally ‘acquired West Nile virus infection in a female wild turkey. J Vet Diagn Invest 18:204-208. 13Table 1. Causes of Anemi A. Hemorrhagic Anemia 1. Blood Loss. a. Trauma b. Persecution (cannibalism) c. Servicing (beak trimming, te trimming) 2. Disease ‘a. Aortic rupture b. Fatty liver syndrome . Neoplasia — hemangioma, hemangiosarcoma 3. Parasites ‘a. Black fies (Simullum spp.) b. Ticks (e.g. Argas spp., hard ticks) . Mites (e.g. Dermanyssus, Omithonyssus) 4. Internal helminths (e.g. Amidostomum, Omithostrongylus, Trichostrongylus, Capillaria, etc.) €. Intestinal and cecal coccidiosis 4. Rodenticides — warfarin, brodifacoum, diphacinone B. Hemolytic Anemi 1. Viral infections a, Marek’s disease virus? b. Myeloblastosis virus 2. Bacterial Infections a. Fow typhoid (Salmonella gallinarum) b. Salmonellosis ©. Erysipelas d. Streptococcosis @. Spirochetosis (Borrelia anserina) - f. Aegyptianellosis (Aegyptianella pullorum) 3. Parasitic a. Hemoprotozoa (Plasmodium, Haemoproteus, Leucocytozoon) 4, Toxic 2 a. Aflatoxin b. Lead — acute ©. Copper d. Dimethyl disulfide — plants in the Brassica family e. Phenylhydrazine f. Methylsulfonate A 9. Paraquat hh. Fenbendazole i. Crude oi! |. Green onions es k. Garlic C. Nutritional Anemia 1. Minerals a. Iron deficiency i, Ochratoxin-induced li, Myeloblastosis virus b. Copper deficiency 2. Vitamins ‘a. Pyridoxine - ducklings 14». Folic acid ~ megaloblastic anemia x cc. Vitamin K — bone marrow atrophy — D. Inherited Anemia E. Undetermined m a. Rapeseed meal b. Duck anemia (reticuloendotheliosis) virus cc. Avian leukosis viruses ~ certain types d. Beak necrosis F, Myelopathic (myelophthisic) Anemia 2, Myeloid leukosis = b. Erythroid leukosis ©. Osteopetrosis a 4, Spindle-Cell Proliferative Disease fe, Mycobacteriosis G. Pancytopenia/Aplastic Anemia a 4. Viral Infections bes ‘a. Chicken infectious anemia virus (vertical or neonatal infection) Infectious bursal disease virus - very virulent forms = ©. Velogenic Neweastie disease virus 4. Virulentreoviruses pe e. Adenoviruses (usually serogroup 8) f. Avian leukosis viruses ~ myeloblastosis virus be @. Marek's disease virus — absence of maternal immunity 2. Toxie bs a, Sulfonamides ». Lead — chronic be ©. Methyl mercury ~ high exposure 4. Mycotoxins Pe i, Trichothecene mycotoxins i, Ochratoxin Be i. Citrinin . Cyclophosphamide fe {Irradiation 3. Nutrition pg a. Starvation 16Table 2. Comparative Terminology for Inflammation in Birds. Mammalian Term(s) Avian Term(s) Justification Suppurative, Heterophilic, ‘Acute cellular exudate in birds is composed primarily of Purulent Granulocytic, heterophils and undergoes caseation, not liquefaction Nonsuppurative, Nongranulocytic, Same as above; cellular composition of exudate Nonpurulent Lymphocytic, provides best descriptor i a cell type is predominant Lymphopiasmacytic, Histiocytic, etc. (ther compound words containing suppurative or purulent should be similarly modified to describe types of inflammation in avian species, e.g. fibrinopurulent in mammals would become fibrinoheterophilc or fibrinogranulocytic in birds. Pyogranulomatous —_Heterophilic. Virtually all inflammation of any duration in birds has a granuloma granulomatous component; itis useful to distinguish those resulting from caseated heterophilic exudate from those caused by intracellular microorganisms Granulomatous Histiocytic, ‘Term for granulomas resulting from intracellular bacteria ‘granuloma eg. Mycoplasma or fungin which macrophages rather than heterophils are the predominant cell type ‘Abscess Fibriscess. Localized inflammatory lesions in birds consist of multiple layers of fibrin that entrap causative agents and inflammatory cells, not by necrosis and neutrophilic exudation as in mammals: 16or a @ a & r =) r ) a @ rer @ @ re) O G a @ @e aa r ) @ @ ie) @ @ e C) eo () nid to worse od gutt 4 lebioeuintestixe ed! ai sonkg athib 18 eedyoolunene to 9 asia 1Hot ai 9s m nig #i auogt obiek ssroni HM noienater oli. 6d teuen: HM: retobn 2H001. Bone Marrow, Femur, Mid-Shaft - Normal. Chicken, 41 Days. A. Hematopoiesis is compartmentalized in the bone marrow of birds. Erythropoiesis occurs within sinusoids while myelopoiesis takes place in the extrasinusoidal spaces. B. Focus of myelopoiesis composed of granulocytes in different stages of maturation. C. Lymphoid foci occur frequently in bone marrow and increase in both size and number with age. D. Myeloid and lymphoid cells often ocour together. (Case courtesy of Dr. F. Wilson) H002. Liver — Extramedullary Hematopoiesis [EMH], Chronic Right Heart Failure. Broiler Breeder, 43 Weeks. Hematopoiesis in organs outside of the bone marrow (extramedullary hematopoiesis [EMH)) is common in both normal and diseased birds. This hematopoietic focus is primarily myelopoietic because it is composed of myelocytes in different stages of maturation. EMH increases in birds when there is a greater demand for blood cells. EMH must be differentiated from inflammatory and neoplastic lesions HO003. Liver — Extramedullary Erythropoiesis, Normal. Violet Euphonia (Tanagra violacea ), Adult. Similar to its occurrence in bone marrow, extramedullary erythropoiesis occurs within vessels. Large round to oval, intensely basophilic cells, with a central nucleus line the sinusoids in which erythropoiesis is occurring. Extramedullary erythropoiesis without concurrent myelopoiesis is uncommon. H004, Heart - Extramedullary Hematopoiesis [EMH] — Normal. Broiler Chicken. Extramedullary hematopoiesis (EMH) occurs in the heart of young birds. Both myelo- and erythropoiesis are evident here. EMH can be differentiated from inflammatory and neoplastic lesions by the mix of cells and lack of invasion or adverse affects on adjacent tissues. HO005. Air Sac — Experimental Fowl Cholera, 6 Hours. Turkey, 8 Weeks. In well preserved acute inflammatory lesions, heterophils of chickens and turkeys can be recognized by their spiculate, eosinophilic, cytoplasmic granules. More commonly, the granules are not as distinct and differentiation of heterophils from eosinophils requires special staining methods. Heterophils are aggregating where bacteria have breached the air sac epithelium, as endotoxin is strongly chemotactic. Protein-rich fluid also is accumulating in the air sac interstitium H006. Lung — Intravascular Hemolysis, Undetermined Etiology. Broiler Breeder, Female, 27 Weeks. Pulmonary capillaries and small vessels contain lysed erythrocytes. Phagocytic cells within the vessels are capturing hemoglobin, which is in the process of being converted into hemosiderin. Insert. Detail showing early formation of hemosiderin (*). The cause of the hemolysis was not determined. 8Ho04 HO06 9 HO003 HOO5 BGeeecoconetceceoeoaotg@dtscaeecee eCeeeeecee se Gi007. Kidney — Hemoglobinuria, Undetermined Etiology. Guinea Fowl, 2 Years. A. as Excess hemoglobin resulting from intravascular hemolysis is filtered by the glomeruli and may accumulate in renal tubules as hemoglobinuric casts. Casts are acellular, hyaline, and stain bright orange with H&E stain in contrast to the more common pink-staining protein casts. Degeneration and necrosis of tubular epithelium (arrowheads) can occur in severe cases. B. Detail showing hemoglobinuric casts. Note lysis of erythrocytes in vessels. H008. Bone Marrow — Pancytopenia, Chicken Infectious Anemia. SPF Chickens, 7 & 14 Ee Days. A. There is a mild loss of hemopoietic cells in bone marrow at 7 days after infection with chicken infectious anemia virus on day 1. B. By 14 days post-infection, the marrow is virtually devoid of hemopoietic cells. (Case courtesy of Dr. M. Goryo) H009. Bone Marrow ~ Pancytopenia, Chicken Infectious Anemia. SPF Chicken, 7 Days. At7 days post infection, hemocytoblasts (center) frequently contain multiple small eosinophilic inclusion bodies (arrows) within their nuclei. Inclusions are rarely seen after day 12 and are no - longer present by day 16. (Case courtesy of Dr. M. Gory) H010. Bone Marrow, Femur, Mid-Shaft — Hyperplasia, Type 1 Avian Adenovirus Infection [Inclusion Body Hepatitis]. Chicken, 3 Weeks. Viruses are not usually considered as causes of bone marrow hyperplasia, but in this natural infection with Type 1 avian pi adenovirus, there is marked bone marrow hyperplasia. A. Basophilic cells along sinusoids, and extrasinusoidal spaces expanded by confluent granulocytes characterize bone marrow hyperplasia. B,C. Detail showing hyperplastic changes. D. Several blast cells. Whether these are myeloblasts, erythroblasts, or even more primitive hemocytoblasts cannot be determined with certainty. (Case courtesy of Dr. F. Wilson) H011. Bone Marrow, Femur, Mid-Shaft - Hyperplasia, Aspergillus sp. Infection pa [Asper . Victoria Crowned-Pigeon (Goura victoria), Adult. In addition to viral and bacterial diseases, fungal infections can also stimulate bone marrow hyperplasia, especially of myeloid cells. Intrasinusoidal spaces are markedly expanded by immature myeloid cells in various stages of development. Note the mitotic figures and compression of adjacent sinusoids. Presence of immature myeloid cells in different stages of development distinguishes hyperplasia from neoplasia in which cell populations are usually monoclonal. H012. Air Sac - Experimental Airsacculitis, 3 Hours. Turkey, 8 Weeks. The post- capillary venule is the site of inflammatory changes. This air sac was exposed to phorbol myristate acetate, a potent initiator of inflammation. A normal unaffected arteriole (far right) and a severely inflamed venule (center to left) can be seen. Within the venule is a thrombus composed of degenerating thrombocytes that is surrounded by heterophils. Emigrating heterophils are remaining close to the venule, possibly because of a lack of chemotactic factors to draw them away. Dense proteinaceous fluid is leaking from the venule indicating severe damage to the vessel wall (lower left). 20J, 5 ay EEy FY J J O10 =. 2HO013. Liver - Leukocytosis, Heterophilia, Liver Necrosis, Suspected Clostridium sp. Infection. Broiler Chicken, 6 Days. Dilated sinusoids containing numerous granulocytes provide histopathologic evidence of leukocytosis and heterophilia. Compare the number of leukocytes with the number of erythrocytes in this section. Normally sinusoids contain mostly erythrocytes with fewer leukocytes. H014. Liver - Granulomatous Hepatitis, Mycobacteriosis. Duck, 9 Years. Multiple nodules of pale cells replace much of the liver parenchyma. Small nodules associated with larger nodules indicate this is an active process. Alarge nodule contains a central mass of caseous exudate that is ringed with multinucleated giant cells. Granulomas are common in birds and come in two types — heterophilic and histiocytic depending on the celll type making up the caseous center of the lesion. This is an example of a histiocytic granuloma caused by Mycobacterium sp. Insert. Detail of granuloma wall. Caseous center is in the upper left. HO015. Liver - Myeloid Leukosis. Broiler Breeder, Adult, Avian Leukosis Virus ~ J Serotype Positive Flock. A focus of tumor cells has replaced liver tissue to form a tumor nodule. Cells comprising the tumor are a uniform population with sparse, but well defined, eosinophilic granules in the cytoplasm, a round, open nucleus with distinct nucleololus, and often an angular shape. These features define the cells as myeloid cells. Occasional mitotic figures are present, Observe the expansion of tumor cells along sinusoids, which has resulted in isolation, compression, and loss of liver cells. Compare with extramedullary hematopoiesis (H002). Insert. Detail of neoplastic myeloid cells. HO016. Liver - Myeloid Leukosis, Myeloblastosis. Broiler Breeder, Adult, Avian Leukosis Virus (ALV) - J Serotype Positive Flock. A portal area is infiltrated by neoplastic myeloid cells, which form a layer 2 to 3 cells thick around a vessel. On the opposite side (upper right) is an infiltrate of myeloblasts. Myeloblasts approximate the size of liver cells. It is common in ALV-J virus infections to have multiple neoplastic cell types. Insert. Neoplastic myeloid cells (left) compared to myeloblasts (right). H017. Ovary — Myeloblastosis. Broiler Breeder, Adult, Avian Leukosis Virus — J Serotype Positive Flock. An expanding mass of tumor cells fills a space within the ovary. Cells are large, basophilic, have an eccentric round nucleus with prominent nucleolus, and the cytoplasm of most cells contains pale pink, hyaline granules. These features define the cells as myeloblastic. Compare the size of neoplastic myeloblasts with the normal granulocytes that are in the ovarian stroma. Insert. Detail of myeloblasts showing their characteristic features. H018. Spleen - Erythroid Leukosis. Broiler Breeder, Adult, Avian Leukosis Virus - J Serotype Positive Flock. Neoplastic erythroid cells can be recognized by their location within vascular channels, large size, basophilic staining, and round to oval shape with a similarly shaped centrally located nucleus. Occasionally cells are binucleate. Cells lack cytoplasmic granules. Insert. Detail of neoplastic erythroid cells. Note the binucleate cells. Compare with two myeloblasts (*) located within the tumor. 22HO14 O13 HO16 HO15 HO18 ‘O17 23Lymphoid System Oscar J. Fletcher and Tahseen Abdul-Aziz ra 24,Introduction and Normal Histology This system is composed of two primary (the only sites for programmed education) lymphoid organs, the bursa of Fabricius (BF) (cloacal bursa) and the thymus, and the frequently well-organized, nodular or diffuse aggregates, including mural nodules and ectopic foci, of bursa-dependent (B) lymphocytes and thymus-dependent (T) lymphocytes in the spleen and, other organs and tissues. The term lymphoid refers to the round, basophilic cells seen in sections stained with H&E. The lymphoid system includes, in addition to the B-lymphocytes responsible for immunoglobulin production and the T-lymphocytes responsible for cell- ‘mediated immune functions, the supporting framework of reticular cells, the histiocytes or macrophages, and dendritic cells. B- and T-lymphocytes can be tentatively identified based on location and histologic patter in the spleen and other organs or tissues, but definitive identification requires special immunohistochemistry staining. B-lymphocytes have ‘surface immunoglobulin, and T-lymphocytes have antigenic markers including CD4 and CD8. In general, the nodular aggregates are of BF origin, and the diffuse collections are of thymus origin. Significant populations of lymphoid cells are commonly seen throughout the digestive tract (gut-associated lymphoid tissue or GALT), being especially prominent at the junction of the esophagus and proventriculus, and in the lamina propria of the proventriculus, duodenum, ileum (the distinct Peyer's patch in the distal leur), and cecum (the cecal tonsils). Mecke'’s diverticulum contains a large population of B and T-lymphocytes. Ducks have distinct annular bands of lymphoid tissue in the proximal and distal jejunum and ileum. The wall of the gall bladder also contains lymphoid cells, and scattered nodules are present in the pancreas, Lymphoid cells accumulate in the respiratory system, being especially prominent beneath the mucosa in the upper respiratory tract, in the tracheal mucosa, and in primary and secondary bronchi (bronchial- associated lymphoid tissue or BALT). Lymphoid tissue is prominent in the occulonasal region, with collections in the conjunctiva (conjunctiva-associated lymphoid tissue or CALT), beneath the mucosa of the turbinates in the nasal cavity, and in the lachrymal gland and its duct, lateral nasal glands and their ducts, larynx, and dorsal nasopharynx. The Harderian gland (gland of the nicttating membrane), an organ located in the orbit behind the eye, has a large population of plasma cells in the interstitial tissue, and itis a major component of lymphoid cells making up the head-associated lymphoid tissue (HALT). Accumulations of B and ‘Tlymphocytes can be found in the walls of lymphatics (the mural lymphoid nodules). Paired lumbar iymph odes are present in some species of water, marsh, 25 and shore birds. Stem cells of yolk sac origin are critical for populating BF and thymus during embryonic development. Bone marrow is not a primary lymphoid ‘organ in birds but also contains scattered collections of lymphoid cells and thus is also qualified as a secondary lymphoid organ. Lymphatic vessels are less numerous in birds than in mammals, and they are closely associated with blood vessels, surrounded by a sheath of collagen. fibers, and relatively dificult to visualize in H&E stained sections. The absence of well-defined lymph nodes in most avian species does not mean less need for a highly functional lymphoreticular system, but rather, that birds have dispersed their lymphoid cells throughout many tissues. The avian lymphoid system has the capacity when stimulated to respond with the formation of new, widely distributed lymphoid foci, so their presence in many tissues should not be interpreted as evidence of lymphoid neoplasia Proper fixation of tissues of the lymphoid system is critical for accurate diagnosis. Impression smears or imprints of the spleen are useful in identification of cell types in neoplasia and for diagnosis of parasitic infections. Autolysis of the spleen is likely unless the capsule is opened before being placed in fixative. Identification of specific cells is dificult in H&E stained sections, and impossible in poorly fixed material. Bursa Fabricius The bursa of Fabricius (cloacal bursa) has a thin serosal layer, a smooth muscle layer, and a mucosal, layer having many folds or plicae (about 12 major ‘ones) that project into the bursal lumen. A layer of epithelial cells covers the plicae and there is often a visible tuft of specialized epithelial cells capable of pinocytosis located at the points of contact of the follicles with the mucosal epithelium. Bursal follicles are populated with large numbers of Iymphoid cells. Each follicle has a distinct outer cortex and inner medulla, except in ratites where this pattern is reversed. The medulla and cortex are separated by a layer of undifferentiated epithelial cells that are not easily seen in the normal follcie, but are more visible as follicles become depleted of lymphoid cells. Thin strands of interfolicular connective tissue separate the lymphoid folicies from one another. The increase in the space between follicles is termed expansion of the interfolicular connective tissue, and itis an indication of atrophy of the follicles or age-related regression. ‘The BF in the chicken grows rapidly after hatching, ‘teaches maximum size between 4 and 12 weeks, andbegins regression after 20-24 weeks. Regression is characterized by reduction in size and number of follicles, formation of cystic folicies, marked increase in the interfolicular fibrous connective tissue, and in- folding of the epithelium, Thymus ‘The lobes of the avian thymus are composed of lobules having an outer cortex containing many densely packed, relatively small lymphoid cells. ‘The medulla is less densely populated, contains a mixed population of cells, and is more palely stained than the cortex with H&E stain. Cells in the medulla include macrophages, plasma cells, myeloid cells, ‘erythrocytes, and occasional granulocytes. The medulla contains diffuse, pale stained areas that are ‘composed of reticular cells within which are scattered vesicles containing eosinophilic material likely representing debris of degenerating cells. Hassall's ‘corpuscles are nodules of squamous epithelial cells and are less numerous in avian than in mammalian, thymuses. The thymus reaches a maximum size in the chicken around 16 weeks and regresses by sexual maturity. Spleen ‘capsule of connective tissue and smooth muscle surrounds the spleen, but true trabeculae do not ‘occur. The so-called trabecular arteries give rise to numerous branches of central arteries; each is. surrounded primarily by a diffuse, dense population of ‘T-lymphocytes, which form the so-called periarterial lymphoid sheath (PALS). Nodular aggregates with germinal centers of B-iymphocytes occasionally occur within the PALS and adjacent to central arteries. The ‘number of lymphoid folicles with germinal centers increases in response to antigenic stimulation. ‘The central arteries give rise to the penicilform capillaries that are recognized by having high or plump endothelial cells and being surrounded by a sheath of reticular cells and dendritic cells (thus the name sheathed capillaries). The sheath is also known as the ellipsoid or Schweigger-Seidel (SS) sheath. These SS sheaths are very prominent in owls and, to a lesser degree, prominent in psittacines. Prominent SS sheaths should not be confused with tumors. Lymphoid cells that are considered to be B- lymphocytes Surround the ellipsoid sheath.. Depletion of these B-lymphocytes causes the reticular cells of the ellipsoids to become more prominent. The PALS and the lymphocytes around the ellipsoid form the white pulp region. The red pulp is the region containing erythrocytes and fewer lymphoid cells that is located between the peniciliform capillaries and the venous sinusoids. The distinction between the white and red pulp regions is much less evident in the avian spleen as compared to mammals as these regions intermingle in the avian spleen. Unlike the spleen of mammals, the avian spleen does not have a major blood storage or reservoir function. Recognition of the pattern of splenic architecture is critical for appreciation of the changes associated with diseases. Interpretation of changes in the spleen is aided by experience in examining the organ in a variety of normal conditions and diseases. For example, the number and size of lymphoid follcies, with germinal centers can vary from few to multiple per 10X microscope field. Hyperplasia of lymphoid cells is often difficult to interpret as it could be due to antigenic stimulation following vaccination, or to disease. General Responses to Injury Major responses to injury of the lymphoid system include atrophy, necrosis, hyperplasia of either lymphoid cells or reticular cells or both, inflammation, intranuclear (UN) viral inclusions, and neoplasia. Hyperplasia of the epithelium lining the plicae of the bursa of Fabricius (BF) frequently accompanies injury to the BF, with cryptosporidial infection causing a ‘model for epithelial hyperplasia. Eimeria sp. can infect epithelial cells of the BF. Type | avian adenovirus can infect the epithelial cells of the BF plicae and cause IN inclusions. These inclusions frequently are associated with atrophy of bursal folicies, although it is not known whether the atrophy is caused by the adenovirus. Deficiencies and Toxicities Most, if not all diseases, including nutritional deficiencies and toxicities, can result in bursal atrophy, but generally the atrophy is not as diffuse ‘as that associated with IBD or MD. Nutritional and toxic causes of bursal atrophy include: Vitamin A deficiency, selenium deficiency, vitamin E deficiency, cy, aflatoxins, citrinin, cyclopiazonic acid, in B1, ochratoxin, rubratoxin,trichothecenes, ‘and zearalenone. Vitamin A deficiency can cause ‘squamous metaplasia of the epithelium of the plicae of the BF, and this may be accompanied by an ‘accumulation of caseous material in the bursal lumen,Infectious Agents Viruses ‘The major causes of atrophy of the BF and/or thymus _ in chickens is infection with chicken anemia virus (CAV), infectious bursal disease virus (IBDV), or Mare results in very ‘severe atrophy of the thymus, in addition to atrophy of hematopoietic tissue in the bone marrow. Chickens frequently die from secondary infections, especially adenoviral hepatitis, following CAV infection. Classical IBDV causes necrosis of bursal lymphoid cells with {frequent formation of cystic cavities in the medullae of follicles. Inflammation is a prominent feature and includes intrafolicular and interfollicular edema, infiltration of variable numbers of heterophils, and congestion and/or hemorrhage. As the disease progresses, fibroplasia of the interfolicular connective tissue occurs and the plical epithelium becomes irregular due to hyperplasia of epithelial cells and infolding of the epithelial layer. The result is atrophy of the lymphoid follicles and marked increase in the interfolicular fibrous connective tissue. Variant strains of IBDV cause bursal atrophy without inflammation and without the more diffuse and acute necrosis seen in the classical form of IBD. Virulent strains of IBDV also cause hemorrhage in the bone marrow and thymus, in addition to the necrosis of lymphoid cells. MDV infection of the BF can cause lesions of atrophy resembling that seen in IBD, but the presence Of diffuse inflammatory lesions supports a diagnosis of IBDV. CAV causes a major loss of lymphoid cells in the thymus, beginning with the outer cortical thymocytes. and proceeding, in the absence of inflammation, to deplete the cortex and then the medulla. CAV produces a protein (VP3 or apoptin) the luces apoptosis, and it is this apoptotic mechanism of destruction of thymocytes that likely explains the atrophy in the absence of significant inflammation. ‘CAV in experimental infections rarely damages bursal lymphocytes, so bursal atrophy in field cases of CAVis likely due to secondary rather than to direct CAV effects. CAV produces small eosinophilic /N inclusion bodies, first in infected cells of the outer thymic cortex. Small eosinophilic I/N inclusions may also be found in the bone marrow, the lamina propria of the proventriculus, intestine, and in other organs including kidney, lung, and heart. These IN inclusions are transitory, being found during the first 10 days following experimental infection with CAV, thus have not been relied upon in the diagnosis of naturally occurring CAV. These inclusions are found in cells with enlarged nuclei, and careful examination Of the lymphoid cell populations in the lamina propria of the intestine and in the kidneys and lung, in addition to the thymus and spleen, is required to find the inclusion bodies. These small eosinophilic UN inclusions are in contrast to the large, morulla- like, basophilic INN inclusions found in circovirus infections of pigeons and other avian species. CAV infects and kills hemocytoblasts in the bone marrow resulting in a severe depletion of both erythroid and myeloid cell populations, Anemia is severe and loss of thrombocytes leads to frequent bleeding (blue wing syndrome). (Other viral infections that cause necrosis of bursal lymphoid cells and depletion of lymphocytes in the spleen and other organs include reovit Teovirus infection, viscerotropic Newcastle disease (V/ND), Seo rnees a auae viae ios Tt omacs UNTinclusvons in bursal macrophages. Vaccination of chickens with a virulent laryngotracheitis (LT) virus, applied to the vent, an older method of immunization, ‘can cause necrosis in the BF with the formation of syncytial cells containing IN inclusions in the plical epithelium and in medulla (likely in the epithelium separating medulla from cortex) of follicles. Regeneration frequently occurs in the BF following bursal injury and is characterized by irregularity of follicle size with many folicies being depleted of lymphoid cells and having a prominent epithelial layer between medulla and cortex, and an irregular plical epithelium. Regenerative changes are similar to the changes that occur during natural (age-related) atrophy of the BF. Regeneration of the BF does not occur after involution. \Viral infections may cause fibrinoid necrosis in the ‘spleen, but more typically cause depletion of lymphoid ccells with real or apparent hyperplasia of reticular ccalls. The loss of lymphoid cells is more generalized rather than focal. Reticular cell hyperplasia could be real but it may reflect the loss of populations of B and T-lymphocytes. Variable numbers of lymphoid cells with small dark stained nuclei are expected in the BF, thymus, spleen and dispersed lymphoid cell collections, and they result from the process of programmed cell death or apoptosis. Apoptosis may be accelerated or stimulated by some viral infections, CAV being a classic example, so that loss of lymphoid cells is caused by an indirect mechanism rather than direct viral injury leading to cell death. Viral infections should be considered in the. “Gifferentials when the pattern of reticular jonis observed. Inclusions (VN) may be present and are diagnostic — a7 in cases of hemorrhagic enteritis (HE) of turkeys.Marble spleen disease of pheasants presents a similar histologic pattern with similar UN inclusions in reticular cells. Awidespread necrosis of lymphoid cells is a feature of avian influenza and VVND. Hemorrhages: in the lymphoid aggregates of the intestinal tract can be a prominent feature of these diseases, but bacterial infections, Pasteurellosis for example, also can cause hemorrhage and necrosis in lymphoid tissues. Bacteria Bacterial infections in the BF can result in focal areas of caseous necrosis. The bursal lumen can. become filed with a core of caseous necrotic material Clostridia can colonize plical epithelium and cause epithelial necrosis. Bacteria typically cause accumulations of proteinaceous exudate, seen as irregular, multifocal eosinophilic deposits in the spleen. These areas are called foci of fbrinoid necrosis or fibrinold changes. Fibrinoid necrosis is a common change found in the ellipsoids of the spleen, and it suggests damage to blood vessels with subsequent leakage of protein- rich exudate into the surrounding tissues. Bacterial colonies may or may not be seen, but the presence of fibrinoid necrosis indicates that bacterial infection is a primary rule-out. Bacterial infections can progress. to cause large zones of necrosis in the spleen that may coalesce to involve the entire organ. Fibrin may accumulate on the capsule of the spleen and is a. common feature in colibacillosis. Congestion, hemorrhage, and fibrin thrombi usually accompany necrosis of Iymphoid cells and thus are features of bacterial infections in the spleen. Bacterial toxins produced by Clostridia, gangrenous dermatitis being an example, cause lymphoid cell depletion and fibrinoid necrosis in the perielipsoidal sheaths. The presence of aggregates of macrophages or histiocytes isa feature of mycobacterial infections. These macrophage-histiooyte nodules frequently resemble neoplasia. The macrophages are large with granular cytoplasm. Acid-fast staining reveals many acid-fast bacteria in the cytoplasm. Parasites In Leucocytozoon infections, numerous parasites (megaloschizonts) may be found in the spleen, ‘and their presence is accompanied by reticular cell hyperplasia. Intracellular accumulation of hemosiderin pigment is prominent due to destruction of erythrocytes. Amyloid ‘Accumulation of amorphous, eosinophilic material that distorts the splenic architecture is characteristic ‘of amyloidosis. Amyloid is more common in waterfow! but can occur in chronic infectious diseases in any avian species and is seen in brown breed laying chickens, Pigments ‘Accumulation of hemosiderin pigment in the cytoplasm ‘of macrophages can result from excessive destruction of erythrocytes or from unknown causes as in hemosiderosis. Erythrophagocytosis is characterized by the presence of erythrocytes or, more commonly, red or red-brown globules, representing partially digested red blood cells, in the cytoplasm of numerous ‘macrophages in the sinusoids of the spleen Neoplasia Neoplasia involving the various cell populations of the Iymphoid system is a major diagnostic challenge. The avian retroviruses cause a variety of morphologic types of tumors including lymphoid leukosis, erythroblastosis, myeloblastosis/myelocytomatosis. (myeloid cell tumors or J virus tumors), fibrosarcomas, ‘myxomatous sarcomas, and hemangiomas. Lymphoi leukosis (LL) originates in the BF and is a tumor of B-lymphocytes. Within involved organs, including the spleen, LL tumors have a nodular pattern and they ‘grow by expansion. The tumor nodules are composed of a relatively uniform population of lymphoid cells. LL tumors in the BF are located within the folictes (intrafolicular) although, if arge, the folcles and interfolicular tissue are obliterated by tumor cells, thus making the bursal origin of cells difficult or impossible to discern. Differentiation of lymphoid leukosis from the lymphoid cell tumors of Marek’s disease (MD) is a Continuing challenge at the level of light microscopy. Findings of lymphoid cell infitrations in peripheral and/or brain support a diagnosis of MD, but such infitrates occur also in peripheral nerve neuropathy, reticuloendotheliosis and some other lymphoid tumors (see below). MD is the primary rule- out when massive enlargement of the chicken spleen 28is found on histopathologic examination caused by lymphoid cell proliferation that effaces normal splenic structure. Cells in MD tumors are small to medium lymphocytes, lymphoblasts, and reticular cells and these neoplastic cells infitrate tissues rather than ‘grow by expansion. Neoplastic involvement of the BF in MD is not always present, but when found has a characteristic interfolicular pattern. As mentioned earlier, bursal atrophy is a common lesion caused by MoV. Myeloid cell tumors (myelocytomatosis) are diagnosed by the presence of eosinophilic granules in the cytoplasm of the tumor cells. Reticuloendotheliosis virus (REV) causes lymphoid neoplasia in chickens, turkeys, and other avian species including ducks, quail, pheasants, geese, peafowl, and prairie chickens, REV can cause runting and stunting when vaccines contaminated with REV are given to young chickens. REV also can cause bureal lymphomas composed of a uniform population of lymphoblasts that are indistinguishable from LL tumors in chickens. REV also may cause in chickens nonbursal lymphomas located in the thymus and spleen as well as liver. These nonbursal lymphomas are composed of a population of immature iymphoreticular cells. They are usually not uniform 50 can be confused with MDV. REV lymphomas in turkeys are characterized by a relatively uniform population of lymphoreticular cells in the neoplastic nodules found in the spleen, liver, intestine, and other ‘organs. In the runting and stunting syndrome as well ‘as some of the lymphomas, peripheral nerve lesions ‘characterized by infitration of lymphooytes and plasma cells may be present. Lymphoproliferative disease of turkeys occurred in Europe and Israel but has not been reported in the U. S. Lymphoproliferative disease is characterized by marked enlargement of the spleen due to proliferation of pleomorphic mononuclear cells. The tumors are composed of lymphocytes, lymphoblasts, plasma cells, ‘and macrophages. Nerve lesions resembling those seen in MD may be found. Mutticentric histiocytosis is characterized by proliferation of spindle-shaped cells having abundant eosinophilic cytoplasm and located in the periarteriolar lymphoid sheaths of the spleen. There is marked ‘expansion of the periarteriolar lymphoid sheaths that results in a nodular pattern seen both grossly and microscopically. Similar nodules composed of histiocytic cells can be found also in the liver and kidneys. Multicentric histiocytosis is likely the same 29 condition as spindle-cell proliferative disease. Metastatic tumors may be found in the spleen but they may also infiltrate the splenic capsule. In poultry, ‘adenocarcinomas of reproductive tract origin are probably the most common tumor that metastasizes to the spleen by intracoelomic implantation Additional Readings ‘Adair, B. M. 2000. Immunopathogenesis of chicken ‘anemia virus infection. Dev Comp Immun 24:247-255, Bacha, W. J. J., and L. M. Wood. 1990. Color Atlas. of Veterinary Histology. 65-79. Wiliams and Wikins, Baltimore. Bang, B. G., and F. B. Bang. 1968. Localized lymphoid tissues and plasma cells in paraocular and paranasal organ systems in chickens. Am J Pathol 53:735-751. Bang, B. G., F. B. Bang, and M.A. Foard, 1972. Lymphocyte depression induced in chickens on diets deficient in vitamin A and other components. Am J Pathol 68:147-162. Bang, 8. G., M.A. Foard, and F. B. Bang, 1973. The effect of vitamin A deficiency and Newcastle disease on lymphoid cell systems in chickens, Proc Soc Exp Biol Mod 143:1140-1146. Biggs, P. M. 1991. Lymphoproliferative disease of turkeys. In: B. W. Calnek, H. J. Bames, C. W. Beard, W. M. Reid, and H. W. Yoder Jr. (eds.) Diseases of Pouitry, 9" ed. lowa State University Press: Ames, IA. 456-459, Cheville, N. F, 1967. Studies on the pathogenesis of ‘Gumboro disease in the bursa of Fabricius, spleen, and thymus of the chicken. Am J Path 51:527-551. Cheville, N. F. 1970. The influence of thymic and bursal lymphoid systems in the pathogenesis of avian encephalomyelitis. Am J Pathol 58:105-125. Cheville, N. F., and W. D. Richards. 1971. The influence of thymic and bursal lymphoid systems in avian tuberculosis. Am J Pathol 64:97-122. Cheville, N. F., and C. W. Beard. 1972. Cytopathology of Newcastle disease. The influence of bursal and thymic lymphoid systems in the chicken. Lab Invest 2129-143.Cheuille, N., and S. Sato. 1977. Pathology of adenoviral infection in turkeys (Meleagris gallopavo) with respiratory disease and colisepticemia. Vet Patho! 14:567-581, Cheville, N. F., W. Okazaki, P. D. Lukert, and H. G. Purchase. 1978. Prevention of avian lymphoid leukosis by induction of bursal atrophy with infectious bursal disease viruses. Vet Pathol 15: 376-382. Cheville, N. F. 1979. Environmental factors affecting the immune response of birds: A review. Avian Dis 23:308-314. Fadly, A. M., and L. N. Payne. 2003. Leukosis/sarcoma group. In: Y. M. Saif, H. J. Barnes, J.R. Glisson, A. M. Fadley, L. R. McDougald, and D. E. Swayne (eds.) Diseases of Poultry, 11" ed. lowa State University Press: Ames, 1A. 465-516. Hafner, W. S., and M. A. Goodwin. 2003. Multicentric histiocytosis. In: Y. M. Saif, H. J. Bames, J.R. Glisson, ‘A.M. Fadley, L. R. McDougaid, and D. E. Swayne (eds.) Diseases of Poultry, 11” ed. lowa State University Press: Ames, IA. 539-540, Hodges, R. D. 1974. The Histology of the Fow. ‘Academic Press, New York. Jeurissen, S. H. M., E. M. Janse, G. Koch, and G. F. DeBoer. 1989. Postnatal development of mucosa- ‘associated lymphoid tissues in chickens. Cel! Tissue Res 258:119-124. Jeurissen, S. H. M. 1991. Structure and function in the chicken spleen. Res Immunol 142:352-355. Jeurissen, S. H. M., E. Claassen, and E. M. Janse. 1992. Histological and functional differentiation of ‘non-lymphoid cells in the chicken spleen. Immunol 7775-80. Jeurissen, S. H. M., F. Wagenaar, J. M.A. Pol, A. J. van der Eb, and M.H. M. Noteborn. 1982. Chicken anemia virus causes apoptosis of thymocytes after in Vivo infection and of cell ines after in vitro infection. J Virol 66:7383-7388, John, J. L. 1994. The avian spleen: A neglected organ. Quart Rev Bio! 69:327-351 Jones, Y. L., and D. E. Swayne. 2004. Comparative pathobiology of low and high pathogenicity H7N3 Chilean avian influenza viruses in chickens. Avian Dis 48:119-128. Nagy, N. E., Biro, A. Takacs, M. Polos, A. Magyar, and |. Olah. 2005. Peripheral blood fibrocytes contribute to the formation of the avian spleen. Devel Dynamics 232:55-66. Nagy, NB. layarto, A. Magyar, E. Gazdag, V. Palya, and I. Olah. 2005. Oesophageal tonsil of the chicken. Acta Vet Hung 53:173-188. Nakamura, K., K. Waseda, Y. Yamamoto, M. Yamada, M. Nakazawa, E. Hata, T. Terazaki, A. Enya, T: Imada, and K. Imai. 2006. Pathology of cutaneous fowipox with amyloidosis in layer hens inoculated with fowipox vaccine. Avian Dis 50:152-156. Olah, 1, N. Nagy, A. Magyar, and V. Palya. 2003. Esophageal tonsil: A novel gut-associated lymphoid organ. Poult Sci 82:767-770. Payne, L. N., and P. C. Powell. 1984. The lymphoid system. In: B. M. Freeman (ed.) Physiology and Biochemistry of the Domestic Fowl. Academic Press, London, 277-321. Pope, C. R. 1991. Pathology of lymphoid organs with emphasis on immunosuppression. Vet Immunol & Immunopathol 30:31-44. Pope, C. R. 1996. Lymphoid system. In: C. Riddell (ed.) Avian Histopathology. AAAP, New Bolton. Center. 17-44. Press, C. M., and T. Landsvak. 2006. The lymphoid system. In: J. A. Eurell and L. Brain (eds.) Dellmann’s ‘Textbook of Veterinary Histology. Blackwell Publishing: Ames, A. 134-152 Riddell, C. 1987. Avian Histopathology. 1st ed. AAAP, New Bolton Center. 7-17. Rose, M. E. 1981. Lymphatic system. In: A. S. King land J. McLelland (eds.) Form and Function in Birds. ‘Academic Press: New York, NY. 341-384. Sarr, C. F.,T. ¥. Morishita, and B. Mersessian. 2005. The effect of route of inoculation and challenge dosage on Riemereila anatipestifer infection in pekin ducks (Anas platyrhyrehos). Avian Dis 49:104-107. ‘Smyth, J., D. Moffett, R. T. Conno, and M. McNulty. 2006. Chicken anaemia virus inoculated by the oral route causes lymphocyte depletion in the thymus in 3-week-old and 6-week-old chickens. Avian Pathol 35:254-259,‘Smyth, J., D. A. Moffett, M. S. McNulty, D. Todd, and D. P. Mackle. 1993. A sequential histopathologic and immunocytocherical study of chicken anemia virus infection at one day of age. Avian Dis 37:324-338. ‘Smyth, J., D. Soike, D. Moffett, J. H. Weston, and D. ‘Todd. 2005. Circovirus-infected geese studied by in situ hybridization. Avian Pathol 34:227-232. ‘Swayne, D. E. 1997. Pathobiology of HSN2 Mexican avian influenza virus infection of chickens. Vet Pathol 34:557-567. Talami, S., M. Goryo, T. Masegi, and K. Okada. 2004. Histopathological characteristics of spindle- cell proliferative disease in broiler chickens and its experimental reproduction in specific pathogen-free chickens. J Vet Med Sci 66:231-235. Van Santen, V. L., K. S. Joiner, C. Murray, N. Petrenko, F. J. Hoerr, and H, Toro. 2004. Pathogenesis of chicken anemia virus: Comparison of the oral and the intramuscular routes of infection. Avian Dis 48:494- 504, Williams, A. E., and T. F. Davison. 2005. Enhanced immunopathology induced by very virulent infectious bursal disease virus. Avian Pathol 34:4-14. Witter, R. L., and A. M. Fadly. 2003. Reticuloendotheliosis. In: YM. Saif, H. J. Bames, J.R. Glisson, A. M. Fadiey, L. R. McDougald, and D. E. ‘Swayne (eds.) Diseases of Poultry, 11" ed. lowa State University Press: Ames, IA. 517-536. Witter, R. L., and K.A. Schat. 2003. Marek’s disease, In: ¥. M. Saif, H. J. Bames, J.R. Glisson, A. M. Fadley, LR McDougald, and D. E. Swayne (eds.) Diseases of Poultry, 11" ed. lowa State University Press: Ames, 1A. 407-465. 3L001. Bursa of Fabricius - Normal. Chicken, 3 Weeks. A typical fold or plica is covered by columnar epithelium and contains numerous lymphoid follicles separated by thin bands of fibrous connective tissue. L002. Bursa of Fabricius - Normal. Chicken, 3 Weeks. Note the relative proportions of cortex (the more darkly stained outer zone) and medulla in the follicles. The thin bands of connective tissue between the follicles are the interfollicular connective tissue. L003. Bursa of Fabricius - Normal. Turkey, 21 Days. The arrows identify the focal and highly specialized epithelium (insert for higher magnification) that caps follicles and has pinocytotic activity important in the presentation of antigenic material to lymphoid cells. L004. Thymus - Normal. SPF Chicken, 23 Days. A wide, darkly stained cortex C contains numerous lymphoid cells, and a smaller medulla M is composed of a more diverse population of cells. L005. Spleen - Normal. Chicken, Layer, 21 Days. Central arteries (arrows) are surrounded by a diffuse population of T-lymphocytes that form the periarterial lymphoid sheath. There is also a nodule or follicle composed of B-lymphocytes. L006. Spleen - Normal. SPF Chicken, 23 Days. A central artery is surrounded by the periarterial lymphoid sheath (PALS) and has an adjacent bursa-dependent lymphoid follicle. 32Loo4 Looz 33 Loo1 Loo3 Loos eeooce@e@eaaeeCgceCc@ea« ogee eoee eae eoaeCee ee CeeL007. Spleen - Normal. SPF Chicken, 23 Days. A bursa-dependent lymphoid follicie is located within a periarterial lymphoid sheath. L008. Spleen - Normal. SPF Chicken, 23 Days. Two sheathed capillaries are surrounded by pale-staining reticular cells. These are ellipsoid sheaths. The lymphoid cells within and around these capillaries are primarily B-cells and constitute the periellipsoidal lymphoid sheath (PELS). L009. Conjunctiva - Normal. Collections of nodular and diffuse lymphoid tissue beneath the conjunctival epithelium constitute the conjunctival-associated-lymphoid-tissue (CALT). L010. Trachea - Normal. Turkey, 16 Days. This nodular lymphoid tissue is a normal component of the mucosal-associated lymphoid tissue found in many organs in the avian ‘species. L011. Lung - Normal. Turkey, Five Weeks. Multiple nodular and diffuse collections of lymphoid cells are in the mucosa of this secondary bronchus. This is an example of bronchial- associated lymphoid tissue (BALT). See figure L012 for a higher magnification. L012. Lung - Normal. Turkey, Five Weeks. Nodular and diffuse bronchial-associated lymphoid tissue (BALT) is prominent in the mucosa of this secondary bronchus.L007 35L013. Esophageal - Proventricular Junction — Normal. Broiler Beeder. Nodular and diffuse collections of lymphoid cells are prominent in this location. L014, Duodenum - Normal. Turkey. Nodular and diffuse collections of lymphoid cells are in the lamina propria of several villi. This is an example of gut-associated lymphoid tissue (GALT). L015. Meckel’s Diverticulum - Normal. Turkey, 18 Weeks. Nodular and diffuse collections of lymphoid cells are prominent in this location. The small cystic structure is the remnant of the yolk sac. The amount of lymphoid tissue easily could be interpreted ad lymphoid hyperplasia, but probably is within normal limits. L016. Pancreas - Normal. Turkey. Two lymphoid nodules are in this field. Scattered nodular deposits of lymphoid cells are examples of the normal lymphoid cell populations dispersed through many organs. L017. Proventriculus - Normal. Chicken. Lymphoid nodules scattered in the proventricular glands are normal findings. L018, Mural Lymphoid Nodule - Normal. Broiler, 5 Weeks. This lymphoid cell nodule is located in the wall of a lymphatic vessel (or a vein) adjacent to the pancreas. Mural nodules may be found as part of the normal lymphoid cell population in many locations and are often overlooked. 36L013 L014 L015 L016 L017L019. Bone Marrow - Normal. Broiler Breeder, 3 Weeks. Two lymphoid nodules surrounded by hematopoietic cells illustrate a normal population of lymphoid cells in bone marrow. L020. Bursa of Fabricius - Infectious Bursal Disease. Chicken, 23 Days. Extensive necrosis of bursal follicular lymphoid cells, inflammation in follicles, and edema and heterophils in the interfollicular connective tissue are lesions typical of the classical form of infectious bursal disease. Initially, some follicles may be unaffected or only partially involved, but the disease progresses to invoke all, or most, follicles. L021. Bursa of Fabricius - Infectious Bursal Disease. Chicken, 23 Days. This higher magnification of figure L020 illustrates the depletion of lymphoid cells in both cortex and medulla and the expansion of interfollicular connective tissue by edema and inflammatory cells. L022. Bursa of Fabricius - Infectious Bursal Disease. Chicken, 23 Days. Cyst formation within follicles is common following massive destruction of lymphoid cells in the bursal follicles. 023. Bursa of Fabricius - Infectious Bursal Disease. Chicken, 23 Days. Edema and cellular infiltration by heterophils and macrophages are responsible for expansion of the interfollicular connective tissue. L024. Bursa of Fabricius - Atrophy. Chicken, 40 Days. Variation in the size of bursal follicles and irregular folding of the epithelium surface of the plicae are features of atrophy. These changes also are characteristic of bursal regeneration following injury and of age- associated regression.Lo19 L020 39L025. Bursa of Fabricius - Atrophy. Chicken, 40 Days. Atrophy of follicles is diffuse, and the epithelium is irregular due to in-folding. These changes are characteristic of bursal atrophy regardless of the etiology, and are also features found in age-related regression of the bursa of Fabricius. The insert illustrates the inclusions at higher magnification. L026. Bursa of Fabricius - Adenoviral Infection. Turkey, 3 Weeks. Type | adenovirus inclusions are in the plical epithelium (circles). The insert illustrates the inclusions at higher magnification. 027. Bursa of Fabricius - Adenoviral Infection and Cryptosporidiosis. Turkey, 5 Weeks. Cryptosporidia are the small spherical structures on the epithelial surface. A large basophilic inclusion typical of a Type | avian adenovirus is in an epithelial cell. L028. Bursa of Fabricius - Circovirus Infection. Pigeon. Necrosis of bursal follicles, presence of large inclusion bodies, formation of numerous cysts, fluid in the lumen of the bursa, and marked atrophy of follicles are features of pigeon circovirus infection. L029. Thymus - Chicken Anemia Virus. Broiler Chicken, 5 Weeks. Necrosis is extensive and accompanied by hemorrhage. Some thymic lobules are relatively normal. Diffuse atrophy of the bone marrow was another lesion found in chickens from this flock. L030. Thymus - Chicken Anemia Virus. Broiler Chicken, 5 Weeks. Some lobes are diffusely affected and hemorrhagic while others are relatively unaffected. See figure L035 for a higher magnification. 40atL031. Thymus - Chicken Anemia Virus. Broiler Chicken, 5 Weeks. Loss of thymic lymphoid cells is diffuse and accompanied by hemorrhage and fibrosis within the affected lobule as well as on the capsule of the thymus. There is no heterophilic inflammation. L032. Thymus - Chicken Anemia Virus. Broiler Chicken, 4 Weeks. There is diffuse loss of cells in the bone marrow. Several cells have enlarged nuclei with a small intranuclear inclusion (insert) ms L033. Small Intestine - Chicken Anemia Virus. Chicken. Hyperplasia of lymphoid tissue is, associated with expansion of the lamina propria. Arrows identify cells having enlarged nuclei = some containing small eosinophilic inclusions characteristic of infection with chicken anemia virus. See figure L038 for a higher magnification, L034. Small Intestine - Chicken Anemia Virus. Chicken. Arrows identify cells with enlarged nuclei and small eosinophilic intranuclear inclusions within the expanded lamina propria of the small intestine. L035. Thymus - Severe Atrophy. Chicken. Severe atrophy of the thymus with no associated inflammatory response is highly suggestive of CAV infection. L036. Thymus - Atrophy. Turkey. Cortical width is decreased giving the appearance of an - increase in the medulla. These features indicate thymic atrophy of moderate severity. The changes are not disease specific and may represent thymic atrophy due to age. Note the scattered clear areas or holes that contain small dark nuclei. This is characteristic of apoptosis = in lymphoid organs. 42Lo32 L031 L034 L036 L035 43L037. Thymus - Atrophy. Turkey. Very few lymphoid cells remain in this remnant of thymus indicating diffuse, severe (marked) thymic atrophy that could be age related. Thymic (Hassall's) corpuscles are prominent in the medulla. L038. Spleen - Hemorrhagic Enteritis. Turkey, 2 Weeks. Lymphoid cell depletion and reticular cell hyperplasia around sheathed capillaries are patterns indicating higher magnification examination to determine if there are intranuclear inclusions typical of avian adenovirus Type I L039. Spleen - Hemorrhagic Enteritis. Turkey, 2 Weeks. Many macrophages or reticular cells have enlarged nuclei (insert) containing intranuclear inclusion bodies. L040. Spleen, Bursa of Fabricius, Liver, and Kidney. Polyoma. A. Spleen. Lovebird, 6 Months. Lymphoid depletion is accompanied by many large, pale intranuclear inclusions in reticular cells. B, Bursa of Fabricius. Lovebird, 5 Weeks. Loss of cortical and medullary lymphocytes is complete and remaining reticular cells contain intranuclear inclusions typical of polyoma virus. C. Liver. Lovebird, 6 Months. Necrosis and hemorrhage are extensive. Inclusions can be infrequent and difficult to find. D. Kidney. Ring-Necked Parrot, Fledging. Many mesangial cells in glomeruli contain intranuclear inclusions typical of polyoma virus. L041. Bursa of Fabricius - Atrophy and Necrosis - Bacterial Etiology. Chicken, 35 Days. Alarge focal area of necrosis located at the base of a plica is typical of a bacterial infection. This lesion commonly is called a bursal abscess and there may be multiple such foci. Note the generalized atrophy of bursal follicles and the unevenness of the epithelial surface. The necrotic area is not likely the cause of the generalized bursal atrophy, but may be an indication of impaired disease resistance due to a compromised immune system. L042. Spleen - Perisplenitis. Broiler, 5 Days. The surface exudate consists of fibrin, inflammatory cells and bacterial colonies. The lesion is not specific, but common in E. coli infections.L037 L038 45L043. Spleen - Colibacillosis. Chicken. Lesions are congestion and areas of fibrinoid necrosis (within the box). Note the depletion of lymphoid cells. L044. Spleen - Colibacillosis. Turkey. The arrow identifies a fibrin thrombus in a splenic artery. Congestion and depletion of lymphoid cells are present. L045. Spleen - Colibacillosis. Turkey. Fibrin deposition (arrow) and necrosis in a bursa- dependent follicle (*) are prominent lesions. Bacteria (insert) are identified by the arrow. L046. Spleen - Fowl Cholera. Turkey, 4 Weeks. Large numbers of bacteria are in areas of fibrinoid necrosis. L047. Spleen - Riemerella anatipestifer. Turkey, 8 Weeks. A fibrin thrombus in an artery (arrow) in the splenic capsule is identified by an arrow. See insert for higher magnification. L048. Spleen - Erysipelas. Turkey. Large zones of necrosis are characteristic.L043 L045 47 Lo44 Lo46L049. Spleen - Erysipelas. Turkey. A large necrotic zone, probably an infarct, has congestion and hemorrhage at the margins. L050. Spleen - Erysipelas. Turkey, 8 Weeks. Eosinophilic hyaline deposits of protein including fibrin form distinct bands around many of the sheathed capillaries. Few lymphoid cells remain in the periellipsoidal sheath. L051. Spleen - Erysipelas. Turkey. Bacteria (arrow) are in the periphery of an area of fibrinoid necrosis. L052. Spleen- Mycobacteriosis. Duck, 2 Years. Multiple, variable-sized nodules of macrophages (histiocytes) are prominent. See figure L053 for higher magnification. 053. Spleen- Mycobacteriosis. Duck, 2 Years. Splenic nodules are composed of a uniform population of large macrophages having abundant granular cytoplasm. L054. Spleen- Mycobacteriosis. Duck, 2 Years. Acid-fast stain reveals numerous positive (red) bacteria in the cytoplasm of macrophages and is diagnostic for avian mycobacteriosis. 48Lo49 LOso L051 Los2 49L055. Bursa of Fabricius - Cryptosporidiosis. Chicken, 6 Weeks. Cryptosporidia are the small spherical structures closely associated with the bursal epithelial cells (insert). Epithelial hyperplasia is characteristic of cryptosporidiosis. Atrophy of lymphoid follicles is diffuse. L056. Bursa of Fal Coccidiosis. Pheasant. Macrogametocytes (arrow and insert) are in plical epithelial cells. All stages of coccidia, including oocysts, may be found in bursal epithelial cells. L057. Spleen - Amyloidosis. Duck, 7 Weeks. Large amounts of eosinophilic hyaline material fil the sinusoids and replaces normal parenchyma resulting in loss of the normal lymphoid cell populations. See figure LO76 for a higher magnification. L058. Spleen - Amyloidosis. Duck, 7 Weeks. A central artery is surrounded by eosinophilic. deposits of amyloid. There is no cellular reaction or other indication of an inflammatory response. L059. Spleen - Hemosiderosis. Attwater Prairie Chicken. A large amount of brown pigment is evident in the cytoplasm of macrophages (insert). L060. Spleen - Erythrophagocytosis. Turkey, 5 Weeks. Macrophages with distended cytoplasm that contains a red-brown pigment are numerous (illustrated by arrows) due to increased phagocytosis and destruction of erythrocytes. The cause was not determined. Two sheathed capillaries are present (within the box).51L061. Bursa of Fabricius - Marek’s Disease. Chicken, 18 Weeks. Proliferation of neoplastic lymphoid cells that expand the interfollicular tissue and atrophy of bursal follicles are characteristic lesions. Few recognizable follicles (box) remain. See figure L063 for a higher magnification. L062. Bursa of Fabricius - Marek's disease. Chicken, 18 Weeks. Expansion of the interfollicular connective tissue is due to infiltration by neoplastic lymphoid cells. Few remaining identifiable follicles are atrophic. L063. Bursa of Fabricius - Marek’s disease. Chicken, 18 Weeks. The cortex of the bursal follicle in the left corner of this figure has only a thin layer of cells. The remainder of the section is filled by neoplastic lymphoid cells within the interfollicular connective tissue. L064. Spleen - Marek’s Disease. Chicken, 7 Weeks. Multiple, variably sized foci of neoplastic cells are coalescing and replacing splenic parenchyma. L065. Spleen - Marek’s Disease. Chicken. Neoplastic lymphoid cells are expanding and obliterating sinusoids. See figure L066 for a higher magnification. L066. Spleen - Marek’s Disease. Chicken. A pleomorphic population of lymphocytes constitutes the lymphoid tumor. Note numerous small darkly stained nuclei scattered through the tumor. These represent apoptotic or necrotic cells. Normal splenic architecture is replaced by tumor cells.i) J Bo) J a CP - =L067. Spleen - Lymphoid Leukosis. Broiler Breeder, 25 Weeks. Multiple, variably sized nodules of neoplastic lymphoid cells creates a nodular pattern suggestive of the bursal (B-cell) origin of this type of lymphoid tumor. L068. Spleen - Lymphoid Leukosis. Broiler Breeder, 25 Weeks. Large masses of neoplastic lymphoid cells are replacing splenic parenchyma. Growth of the tumor appears to be from expansion of smaller nodular tumors rather than infiltration. See figures L069 and “ L070 for higher magnifications. L069. Spleen - Lymphoid Leukosis. Broiler Breeder, 25 Weeks. A nodule of neoplastic lymphoid cells is separated from adjacent splenic parenchyma by a thin band of connective tissue. See LO70 for a higher magnification. = L070. Spleen - Lymphoid Leukosis. Broiler Breeder, 25 Weeks. Tumor nodules are composed of a relatively uniform population of lymphoid cells (lymphoblasts). Autolytic changes are common. The nodule is expanding and compressing the adjacent tissue. L071. Bursa of Fabricius - Lymphoid Leukosis. Chicken. Proliferation of lymphoid cells within bursal follicles (intrafollicluar lymphoid tumors) results in a nodular pattern. Expansive growth of intrafollicular tumors leads to replacement of normal bursal follicles. See figure LO72 for a higher magnification. 1072. Bursa of Fabricius - Lymphoid Leukosis. Chicken. A relatively uniform population of neoplastic lymphoid cells is characteristic.L067 55L073. Spleen - Reticuloendotheliosis. Turkey. Proliferating neoplastic lymphoid cells are prominent around blood vessels. Areas of fibrinoid necrosis are prominent (arrows). L074. Spleen - Reticuloendotheliosis. Turkey. A nodular and diffuse pattern results from proliferation of pleomorphic tumor cells (insert). Numerous apoptotic cells are surrounded by a clear space and fibrinoid necrosis is around the sheathed capillaries. L075. Spleen - Reticuloendotheliosis. Turkey. Tumor growth by expansion results in destruction of splenic architecture. Some areas with relatively normal architecture (arrow) are at the periphery of the proliferating tumor cells. The pleomorphic character of the neoplastic cells is illustrated in the insert. L076. Spleen - Metastatic Adenocarcinoma. Backyard Chicken, 3 Years. Adenocarcinoma of reproductive tract origin is implanted on the splenic capsule. 1077. Spleen - Metastatic Adenocarcinoma. Backyard Chicken, 3 Years. An adenocarcinoma of reproductive tract origin is expanding and replacing splenic parenchyma. See figure LO78 for a higher magnification. L078. Spleen - Metastatic Adenocarcinoma. Backyard Chicken, 3 Years. Expanding adenocarcinoma results in compression and distortion of normal splenic tissue.L073 Lo74 L075 L076 L077 7Skeletal System Oscar J. Fletcher, Tahseen Abdul-Aziz, and H. John BarnesIntroduction and Normal Histology Bone is the core component of the skeletal system and serves as the major reservoir for calcium and phosphorus. It can be easy when examining histologic ‘sections of bones to forget that bone is a highly active and complex metabolic system. The skeletal system is divided into the appendicular skeleton and the axial skeleton. The appendicular skeleton, comprised ‘of the long bones of the body, consists primarily ‘of compact (cortical or lamellar) bone arranged in ‘concentric layers around Haversian canals. The lat bones of the skull are composed of cancellous bone arranged in a framework of bony struts. These flat bones and the vertebrae (cortical bone) of the spinal column constitute the axial skeleton. Birds also have pneumatic bones that contain extensions of the air sacs. Pneumatic bones develop by replacement of bone marrow by air sacs. During this process proliferating fibroblasts and osteoclasts erode bone. The proliferating fibroblast and osteoclast population resembles the proliferative response seen in some forms of rickets and must be interpreted as a normal developmental event. Egg-aying females have medullary bone in the marrow cavities of nonpneumatic bones in both the appendicular and axial skeleton. Bone is composed of mineral, collagen, cellular components, and blood vessels. The three major cell types of bone are osteoblasts that secrete matrix (osteoid), osteocytes that are formed when osteoblasts become entrapped in matrix, and osteoclasts, the multinucleated cells responsible for bone resorption. Cartilage is another major component of the skeletal system. Long bones develop through the process of endochondral ossification in which cartilage tissue that serves as a template is replaced by bone. Longitudinal growth of bones is accomplished at the proximal and distal ends of long bones and the ends of vertebrae at the region of the growth plate or physis. ‘Young birds have extensive amounts of cartilage filling the core (medullary cavity) of the long bones. This core of embryonic cartilage is rapidly replaced during the first 7-14 days of life. The fiat bones of the axial skeleton develop through the process of intramembranous ossification with usually one or two centers of ossification in each flat bone. Appositional ‘growth is the increase in bone diameter that is accomplished by the activity of periosteal osteoblasts creating matrix followed by deposition of mineral and balanced by endosteal bone resorption by osteoclasts. The articular surfaces of the long bones are covered with cartilage. Joints are surrounded by a capsule of fibrous connective tissue having a lining of secretory 59 cells forming the synovial membrane. Synovial cell secretions plus the uitrafitrate derived from blood. plasma creates the joint fluid for lubrication. Tendons are composed of dense collagenous connective tissue and connect bones to skeletal muscles. Tendons are enclosed in a sheath lined by secretory synovial cells. Some tendons including the extensor and flexor tendons of the wings and legs develop regions of osseous metaplasia with aging. Terminology used to describe the region of endochondral ossification can be confusing thus adding to the difficulties of recognizing, describing, and interpreting lesions. In this manual the following terms are used to describe the regions that can be identified by examination of the ends of decalcified long bones, cut in longitudinal section and stained with H&E. Epiphysis the cartilaginous region that caps the tends of long bones. Some authors use the term epiphyseal growth plate to include all ofthe regions of endochondral ossification. We are restricting the region by using the above definition. Physis — the growth plate; lies immediately beneath the epiphysis and is partially penetrated by blood vessels from the epiphysis. The physis has several distinct zones or regions: Resting chondrocytes — germinal or basal chondrocytes that give rise to the cells of the layer of proliferating chondrocytes. These cells are often not seen or appreciated in HBE sections. Proliferating chondrocytes or the zone of proliferation - the zone of proliferation is characterized by columns of chondrocytes stacked Closely together thus resembling a stack of coins viewed on edge. Each column of chondrocytes is separated by extracellular cartilage matrix. Chondrocytes become larger, more spherical, and separated by more cartilage matrix with distance from the epiphysis thus forming the zone of prehypertrophy (maturingjtransitional chondrocytes), The blood vessels from the epiphysis descend to this, transitional zone. The zone of hypertrophy consists of enlarged rounded chondrocytes separated from each other by extracellular cartilage matrix. Mineralization ocours in the extracellular matrix around the hypertrophic chondrocytes. The term zone of mineralization is sometimes used for this region between the zone of hypertrophy and the metaphysis. Because ‘mineralization cannot be assessed acourately by ‘examination of decalcified bone, the usual way that bone is prepared for diagnostic histopathology, we do not use the term, Metaphysis-the region located beneath the physi. Mineralization has occurred and bone is being formed in this region. Blood vessels from the metaphysis, penetrate into the zone of hypertrophy of the physi.Metaphyseal blood vessels do not communicate with epiphyseal blood vessels. An avascular zone of approximately 15 cells thick separates the vessels from the epiphysis from the vessels from the metaphysis. This dual blood supply to the physis is important in that infections in joints can extend through epiphyseal vessels into the physis. The bind or closed ends of the epiphyseal and metaphyseal blood vessels provide an ideal site for bacteria to localize. Diaphysis (Shaf)-contains blood vessels and bone ‘marrow or air sacs, and trabecular bone surrounded by cortical bone. Most long bones do not have centers of ossification in the epiphysis. The only such ossification center in the epiphysis of chickens is in the proximal epiphysis of the tibiotarsus. Osteoblasts form the ‘matrix on which inorganic salts of calcium phosphate are deposited. Osteocytes are cells that are surrounded by and trapped in the calcium phosphate (hydroxyapatite) matrix. Osteoclasts are large, most are multinucleated, cell that resorb bone. The process of bone resorption results in the creation of lacunae or pits in the bone. Fibroblast proliferation is a feature indicative of excessive bone resorption, and also is common in fracture repair. Bone is highly active ‘metabolically and responds or remodels through a series of complex processes in response to the loads and stresses experienced. The endocrine system, especially the parathyroid glands, and the kidney play important roles in these complex metabolic processes that take place in the skeletal system. Rapid growth is a contributing factor in development of lesions of the skeletal system. Deficiencies - Rickets Increases in the width of the physis due to increase in the depth with disorganization of the zone of proliferation along with a decrease in vascular invasion by metaphyseal vessels are features of vitamin (D3) deficiency. Similar lesions are caused by deficiencies of calcium in rapialy growing poults and chicks. Bone formation is reduced resulting in bones that are easily bent or twisted. Osteoid is prominent around trabecular bone in the metaphysis and fibrous connective tissue often replaces bone marrow in cases of Vitamin D3 or calcium deficiency. These are the classical lesions of rickets. Deficiencies in phosphorus lead to similar decreased bone strength. Physeal lesions of phosphorus deficiency include an increase in the depth of the zone of hypertrophy with metaphyseal vascular invasion of the physis. In many ‘cases of rickets from field case material (field rickets), this distinction between vitamin Dicalcium deficiency and phosphorus deficiency can not be made because a combination of physeal lesions is found suggesting the involvement of multiple factors in the etiology. Rickets also can be caused by an imbalance of calcium and phosphorus. Interpretation of physeal lesions depends upon recognition of the normal differences in physeal structure between different types of birds. The physis of broilers is thicker, more irregular, and has more irregular vascular penetration than that of Leghom chickens. Age is an important consideration in the interpretation of physeal lesions as long bone development occurs in a cartilage model. ‘Young chicks and poults have large amounts of normal cartilage in the long bones forthe first 7-14 days of life. Rickets in birds with diets adequate in Vitamin D3, calcium, and phosphorus can be caused by enteric lesions such as found in malabsorption syndrome! runting-stunting, magnesium toxicity, excess vitamin A, ‘excess fat, or mycotoxicosis. Increased width of the zone of hypertrophy with limited or little vascular invasion and decreased numbers of bone trabeculae in the metaphysis may be caused by deficiency in copper. These changes must be differentiated from dyschondroplasia. Increased width in the zone of proliferation and the zone of hypertrophy with irregular vascular invasion or tunneling and thick irregular bone trabeculae are lesions that may be found in rapidly growing broilers with excess vitamin A (hypervitaminosis A). Slower growing Leghoms with ‘excess vitamin A may have a decrease in the width of the zone of proliferation, increased width of the zone of hypertrophy and a thin periosteum with osteoporosis. Broilers also have this thinning of the periosteum with ‘osteoporosis as a result of hypervitaminosis A ‘Table 1 summarizes the physeal changes in rickets associated with Vitamin D3/calcium deficiency and. those associated with phosphorus deficiency. Lesions of Cai Definitions of terms used in this chapter to describe lesions of cartilage other than rickets described above are summarized below: Dyschondroplasia is the persistence of cartilage that contains apoptotic or necrotic chondrocytes in the physis associated with failure of differentiation to hypertrophic chondrocytes and failure of vascularization resulting in a plug of cartilage in the physeal region. 60Chondrosis or Osteochondrosis are degenerative lesions that'include eosinophilic streaks, changes in cartilage matrix, and tears or separation of cartilage found in the physeal cartilage. Vasculature may be disrupted by thrombi Chondrodystrophy Failure of physeal chondrocytes to proliferate when mineralization and appositional growth of bone is normal resulting in a shortening of the long bones. Shortening of vertebral bodies may also occur. The zone of proliferation is narrow with disorganization of chondrocytes in regions distal to epiphyseal blood vessels. Disorganization results when groups of chondrocytes within the zone of proliferation become hypertrophic. Chondrodystrophy replaces the older term of perosis. The gastrocnemius tendon may be displaced. Dyschondroplasia and chondrosis are discussed below in more detail, Dyschondroplasia Persistence of cartilage, frequently as a large mass of cartilage located in the distal physis, results from failure of differentiation of transitional chondrocytes, to become hypertrophic chondrocytes leading to failure of vascular invasion: This is the lesion of dyschondroplasia and, while common in the proximal tibia (tibial dyschondroplasia), can be found in any bones that have a physis, including vertebrae. ‘The cartilage plug contains necrotic or apoptotic chondrocytes that stain eosinophically with H&E. Causes of dyschondroplasia include feeding low calcium or low calciur-high phosphorus diets to rapidly growing broilers. Several toxic compounds including the fusarium mold toxin fusarochromanone also cause this lesion. Other causes include toxicity from thiram, a fungicide used to treat seed, excess dietary levels of cysteine and homocysteine, diets deficient in copper, and metabolic acidosis. Genetics and the rearing environment are factors that influence the incidence of dyschondroplasia. Chondrosis (Osteochondrosis) Degenerative lesions that may be found in physeal cartilage include the presence of eosinophilic streaks across or parallel to the zone of proliferation, matrix necrosis, lakes of amorphous material, and tears or ‘separation of cartilage. Thrombosis of blood vessels ‘occurs. Endochondral ossification is abnormal. This ‘combination of lesions is characteristic of chondrosis ‘or osteochondrosis. Osteochondrosis is the term 61 most commonly used. The synovial joints on either side of T6 (T5:T6 and T6:T7) and the proximal femur are the most common sites in broilers. Deformation of the articulating vertebra results in displacement with impingement on the spinal cord and paralysis. The condition is called spondylothesis. Gener | Responses of Bone to Injury Histopathology often is not done as a part of the investigation of leg problems having certain clinical ‘signs including valgus and varus deformities, long bone distortion, rotated tibia, bowing of the tibia, and shortened long bones. Many of these clinical conditions are associated with defects in physeal cartilage that include dyschondroplasia, ‘osteochondrosis, or chondrodystrophy. Lesions of bacterial osteomyelitis are under diagnosed without the use of histopathology. Also many clinical leg ‘weakness problems are not accurately diagnosed ‘when examination of the vertebral column and spinal Cord is not done. Multiple lesions, sometimes in the same bone or in different sites within the skeletal system like long bones, vertebrae, or ribs, are found often in diagnostic ‘material from field cases. Examples include fractures associated with rickets or with dyschondroplasia, fractures in bones with osteomyelitis, and fractures with lesions of osteochondrosis in vertebrae. Lesions of rickets and dyschondroplasia are often seen together as are lesions of osteochondrosis with lesions of dyschondroplasia. Infectious Agents - Bacteria Inflammation and necrosis in bone is osteomyelitis and is commonly caused by bacterial infection. Heterophils and fibrin are major components of ‘osteomyelitis. Intralesional bacteria are seen in blood vessels and in necrotic tissue. Lesions may be found within the physis, metaphysis, bone marrow, or cortex of long bones. Osteomyelitis in vertebrae is common. As previously mentioned, the terminal ‘ends of epiphyseal and metaphyseal blood vessels are excellent locations for bacterial emboli to lodge and large numbers of bacteria may be found at these locations. Staphylococcus aureus (S. aureus) is the most common bacterial cause of osteomyelitis in commercial poultry. Other bacteria commonly associated with osteomyelitis include Escherichia coll, Salmonella sp., Enterococcus faecalis, Yersinia pseudotuberculosis, and Mycobacterium avium. Fungi‘can cause osteomyelitis and intralesional mycelia can bbe demonstrated in the lesions. Air sac infections frequently extend into pneumatic bones resulting in osteomyelitis. Enterococcus faecalis osteomyelitis may be associated with the deposition of amyloid in periartcular tissue, synovium, and articular cartilage. Lesions ranging from the presence of small clusters of macrophages located anywhere within the bone ‘marrow to large areas of necrosis surrounded by giant cells and having an outer fibrous capsule are characteristic of infection by Mycobacterium avium. ‘Acid-fast stains will reveal numerous red staining organisms within the cytoplasm of the macrophages. Mycobacterial osteomyelitis is more common in backyard chickens than in commercial flocks. ‘The femoral head is a common site for bacterial osteomyelitis caused by S. aureus. This lesion can result in necrosis of the femoral head. The term femoral head necrosis should not be used for this condition due to confusion with the artifactual separation of the proximal femoral epiphysis from the femur on disarticulation at necropsy. The lesion of dyschondroplasia followed by cartilage degeneration is properly termed epiphysiolysis of the femoral head. Epiphysiolysis may be a traumatic lesion and not associated with dyschondroplasia. Fracture and Fracture Repair Distortion of the shaft of long bones and changes in shape and location of vertebrae can often best be appreciated by low power microscopic examination. ‘Several patterns of response may be found in bone repair following fractures. Acute hemorrhage and thrombosis form the initial callus and are replaced by fibrosis and remodeled bone. Osteoclasts and osteoblasts proliferate extensively during the process of new bone formation and remodeling. Rapid removal of bone (osteociasis) with increased amounts of fibrous connective tissue replacing bone occurs in primary and secondary hyperparathyroidism. These endocrine related lesions are termed osteodystrophia fibrosa. Differentiation between these changes and those found both in vitamin D3/caleium deficiency rickets and the proliferative reaction associated with fracture repair is aided by examination of multiple ones. Deficiency and endocrine induced lesions should be generalized in the skeleton. Medullary Bone Medullary bone, a type of nonwoven bone with different staining characteristics, blue to gray with H&E stain rather than cortical (lamellar) bone that is eosinophilic with H&E, develops when ovarian follicles begin maturation. Medullary bone is found in, the marrow cavity of bones having a vascular bone marrow and grows from endosteal surfaces. Medullary bone disappears rapidly as calcium is required and removed for egashell formation and then reforms as the next follcie develops. Laying hens need to provide about 10% of their total body calcium for each eggshell produced. Additional calcium is deposited in the yolk 0 that an effective mechanism involving medullary bone for mobilization of calcium evolved. Osteoporosis Osteoporosis is the loss of bone resulting in a thinner cortex due to increased porosity of the cortical bone caused by failure to repiace lost calcium. Trabeculae in the diaphysis are reduced in size. ‘These changes cause decreased bone volume, ‘The number of resorption cavities in cortical bone is increased. Changes are not found in growth Plates because longitudinal growth has ceased. ‘Osteoporosis is a major problem in caged layers. ‘Osteomalacia is a softening of bones in adult birds because of a decrease in bone density resulting from failure of osteoid to mineralize. Osteomalacia ‘often accompanies osteoporosis in caged layers, ‘Osteopenia occurs as part of the aging process and is characterized by increased porosity of cortical bone. Osteoporosis can occur in young birds. Cage layer osteoporosis is a specific syndrome in which ‘osteoporosis affects cortical bone and osteomalacia affects medullary bone. Pericortical proliferation of osteoblasts in the region of the mid-diaphysis of long bones results in the formation of immature bone that is hypercellular. This lesion is caused by avian retroviruses and is termed osteopetrosis. Osteopetrosis occurs infrequently in chickens and can be experimentally produced in guinea fowi. An osteopetrosis-lke lesion may be found in turkeys. Neoplasia ‘Tumors of bone and cartilage are not common, but include osteomoas, osteosarcomas, chondromas, and chondrosarcomas. Neoplasia of respiratory tract origin 62‘may extend into pneumonic bones from tumor cells originating in the air sacs or by extension of primary Jung tumors through the air sacs. Tumors may arise from the synovial cells causing synoviomas or synovial cell carcinomas. Joints and Tendons - General Responses: ‘Thinning of the articular cartilage with erosions, fissures, and areas of necrosis are features of degenerative joint disease. Inflammation usually is not present in these true degenerative diseases. However, inflammatory exudates within the joint space often cause degenerative and necrotic lesions in articular cartilage. Joint capsules are lined by a synovial membrane. Synovial cells form the epithelial lining of the synovial membrane and are normally no ‘more than 2 to 4 cell layers in thickness. Injury to synovial tissue results in inflammation with hypertrophy and hyperplasia of the synovial lining cells. Infectious Agents - Bacteria and Viruses Hyperplasia associated with mycoplasmal infections, ‘Mycoplasma synoviae being the classic agent causing infectious tenosynovitis, can result in synovial epithelium that is up to 40 cells thick leading to formation of villous projections of the synovial membrane. Necrotic and cellular debris is common Con the synovial surface and on the surface of the articular cartilage in cases of synovitis from any ‘cause. Fibrinoheterophilic exudates with bacterial colonies within the lesions are characteristic of bacterial infections. Exudate accumulates in the joint cavity where itis often associated with erosion of articular cartilage and accumulates in tendon sheaths. ‘Osteomyelitis is frequent. Staphylococcus aureus is most frequently bacterium isolated, but E. cof, Pasteurella multocida, Salmonella sp., Erysipelas, Enterococcus, and Pseudomonas may be involved Heterophils are the primary inflammatory cell in bacterial infections of joints and tendons. Fibrinoheterophilic inflammation is also an early event (frst 48-72 hours) in viral arthritis caused by reoviruses and in mycoplasmal infections of the joints and tendons. This acute reaction to reoviruses. and mycoplasma is followed quickly by infiltration and proliferation of lymphocytes and by synovial hyperplasia so that by 5-7 days post-infection the lymphoid proliferation predominates and few if any heterophils are seen. The presence of significant ‘numbers of heterophils with no or few lymphocytes helps differentiate bacterial infections from reoviral and mycoplasmal infections in birds that at least 5-7 days post-infection. In both reovirus and mycoplasmal 63 infections lesions are characterized by increasing numbers of lymphocytes and plasma cells and by the development of lymphoid nodules (germinal centers). The presence of lymphoid nodules, also termed the Iymphofollicular reaction, is characteristic ‘of Mycoplasma synoviae (MS) infections. Histologic hallmark lesions of MS are synovial cell hyperplasia, synovial villus formation, and dense infitration of lymphocytes and plasma cells. Differentiation on the basis of histopathology between reovirus induced viral arthritis and mycoplasmal arthritis can be diffcut. The lymphocytic and plasma cell response with Iymphoid nodule formation and the degree of synovial hyperplasia usually is more extensive and dense in mycopiasmal infections. Tenosynovitis caused by either bacteria or reovirus leads to fibrosis of the flexor tendons thus these causes cannot be differentiated in this chronic stage (10 weeks post-infection). Articular Gout ‘Swelling around the joints with accumulation of eosinophilic to basophilic granular to amorphous. ‘material surrounded by giant cells, macrophage: and fibrous connective tissue are features of articular gout. This combination of features defines a tophus (tophi for multiple lesions). Sometimes the feathery basophilic deposits of urates can be found within the lesions, Tendons have synovial sheaths with similar histologic structure and function as joints so inflammatory processes in tendon sheaths are similar to those described above. Some tendons contain cartilage as a normal component and cartilaginous metaplasia due to aging or injury leads to increased levels of replacement of collagenous tendon with cartilage. Focal areas of ossification develop normally in the gastrocnemius tendon and are located in the tendon after it branches as it passes over the hock joint. Rupture of tendons results in hemorrhage, and inflammation followed by proliferation of fibrous connective tissue. Fibrosis with collagen deposition can be extensive. The gastrocnemius and flexor muscle tendons are prone to rupture. Tendon rupture can occur in association with tenosynovitis, but some cases have no evidence of prior inflammation.Additional Readings Duff, S. R. |. 1988. Abnormalities in the axial skeleton Of broiler breeding fowl. Avian Pathol 17:239-258. Duff, S. R. l. 1989. Disturbed endochondral ossification in the axial skeleton of young broiler fowls. J Comp Pathol 101:399-409. Duff, S. R. |. 1989. Physeal clefts and disturbed ‘endochondral ossification in broiler fowls. J Comp Pathol 101:75-86. Duff, S. R. I. 1990. Do different forms of spondylolisthesis occur in broiler fowis? Avian Patho! 19:279-294 Duff, S. R. I, and |. A Anderson. 1986. The gastrocnemius tendon of domestic fowl: Histological findings in different strains. Res Vet Sci 41:402-409, Duff, S. R. I, and C. J. Randall. 1986. Tendon lesions in broiler fowls. Res Vet Sci 40: 333-338. Edwards, H. M., Jr. 1992. Nutritional factors and leg disorders. In: C. C. Whitehead (ed.) Bone Biology and Skeletal Disorders in Poultry. 167-193. Carfax Publishing Co, Abingdon. Haynes, J. S. 1990. Immunohistochemical localization of type X collagen in the proximal tibiotarsi of broiler chickens and turkeys. Anat Rec 227:307-313. Hil, J. E., G. N. Rowland, J. R. Glisson, and P. Villegas. 1989. Comparative microscopic lesions in reoviral and staphylococcal tenosynovitis. Avian Dis 33: 01-410. Hodges, R. D. 1974. The Histology of the Fowl. ‘Academic Press, New York. Hurwitz, S. 1982. The role of vitamin D in poultry bone biology. In: C. C. Whitehead (ed.) Bone Biology and Skeletal Disorders in Poultry. 87-102. Carfax Publishing Co, Abingdon. Julian, R. J. 1985. Osteochondrosis, dyschondroplasia, and osteomyelitis causing femoral- head necrosis in turkeys. Avian Dis 29:854-866. Lulian, RJ. 1998. Rapid growth problems: Ascites and skeletal deformities in broilers. Poult Sci 77:1773- 1780 Landis, W. J., and F. H. Silver. 2002. The structure and function of normally mineralizing avian tendons. Comp Biochem and Physiol A, 133:1135-1157. Landman, W. J. M., K.T. Veldman, D. J. Mevius, and J.H.H. van Eck. 2003, Investigations of Enterococcus faecalis-induced bacteraemia in brown layer pullets through different inoculation routes in relation to the production of arthritis. Avian Pathol 32:463-471. Leach, R. M., Jr., and C. V. Gay. 1987. Role of epiphyseal cartilage in endochondral bone formation. J ‘Nutrition 117:784-790. Leach, R. M., Jr, and M. S. Lilburn. 1992. Current knowledge on the etiology of tibial dyschondroplasia in the avian species. Poult Soi Rev 4:57-65. Long, P.H., S. R. Lee, G. N. Rowland, and W. M. Britton. 1984. Experimental rickets in broilers: Gross microscopic, and radiographic lesions. Il. Calcium deficiency. Avian Dis 28: 921-932. Long, P.H., S. R. Lee, G. N. Rowiand, and W. M. Britton, 1984, Experimental rickets in broilers: Gross, microscopic, and radiographic lesions. |. Phosphorus deficiency and calcium excess. Avian Dis 28:460-474, Long, P. H., S. R. Lee, G. N. Rowland, and W. M. Britton. 1984c. Experimental rickets in broilers: Gross, microscopic, and radiographic lesions. Ill. Vitamin D deficiency. Avian Dis 28: 933-943. Loveridge, N., B. M. Thomson, and C. Farquharson. 1992. Bone growth and tumover. In: C. C. Whitehead (ed.) Bone Biology and Skeletal Disorders in Poultry. 3-17. Carfax Publishing Company, Abingdon, McCaskey, P. C., G. N. Rowland, R. K. Page, and LR. Minear. 1982. Focal failures of endochondral, ossification in the broiler. Avian Dis 26:701-717. McNamee, P.T, J. J. McCullagh, J. D. Rogers, B. H. ‘Thorp, H. J. Ball, TJ. Connor, D. McConaghy, and 4J-A. Smyth. 1999. Development of an experimental ‘model of bacterial chondronecrosis with osteomyelitis in broilers following exposure to Staphylococcus ‘aureus by aerosol, and inoculation with chicken anaemia and infectious bursal disease viruses. Avian Pathol 28:26-35.McNamee, P. T., and J. A. Smyth. 2000. Bacterial chondronecrosis with osteomyelitis (femoral head necrosis’) of broiler chickens: A review. Avian Pathol 29:253-270. Miller, S. C. 1992. Calcium homeostasis and mineral tumover in the laying hen. In: C. C. Whitehead (ed.) Bone Biology and Skeletal Disorders in Poultry. 103- 416. Carfax Publishing Co., Abingdon, Miller, S. C., B.M. Bowman, and R. L. Myers. 1984. Morphological and ultrastructural aspects of the activation of avian medullary bone osteoclasts by parathyroid hormone. Anat Rec 208: 223-231 Morrow, C. J., J. M. Bradbury, M. J. Gentle, and B. H. Thorp. 1997. The development of lameness and bone deformity in the broiler following experimental infection ‘with Mycoplasma gallisepticum or Mycoplasma synoviae. Avian Pathol 26:169-187. Orth, M. W,, and M. E. Cook. 1994. Avian tibial dyschondroplasia: A morphological and biochemical review of the growth plate lesion and its causes. Vet Pathol 31:403-414, Peperkamp, N. H. M., W. J. Landman, P. C. Tooten, A. Ultee, W. F. Voorhout, and E. Gruys. 1997. Light microscopic, immunohistochemical, and electron microscopic features of amyloid arthropathy in chickens. Vet Pathol 34:271-278. Perry, R. W., G. N. Rowland, and W. M. Britton, 1991. Pathology of experimental vitamin d deficiency in turkeys and the effects of various vitamin d ‘supplements. Avian Dis 35:542-553. Perry, R. W., G. N. Rowland, and J. R. Glisson. 1991. Poult malabsorption syndrome. |. Malabsorption in poult enteritis. Avian Dis 35:685-693, Perry, R. W., G. N. Rowland, and J. R. Glisson. 1991 Poult malabsorption syndrome. Il. Pathogenesis of skeletal lesions. Avian Dis 35:694-706. Perry, R. W, G. N. Rowland, T. L. Foutz, and J. R. Glisson. 1991. Poult malabsorption syndrome. It ‘Skeletal lesions in market-age turkeys. Avian Dis 36:707-713. Perry, R. W,, G. N. Rowiand, J. R. Glisson, W.L. Steffens, and J. A. Quinn. 1891. Skeletal lesions associated with a naturally occurring poult enteritis. Avian Dis 35:158-164. Peters, T. L., R. M. Fulton, K. D. Roberson, and M. W. Orth. 2002. Effect of antibiotics on in vitro and in vivo avian cartilage degradation. Avian Dis 46:75-86. Pines, M., A. Hasdai, and E. Monsonego-Oman. 2005. Tibial dyschondroplasia — tools, new insights and future prospects. World Poult Sci J 61:285-297, 321, 326-327, 331-332, 338, 334. Pines, M., and S. Hurtwitz. 1991. The role of the growth plate in longitudinal bone growth. Poult Sci 70:1806-1814, Praul, C.A, B.C. Ford, C. V. Gay, M. Pines, and R. M. Leach. 2000. Gene expression and tibial dyschondroplasia. Poult Sci 79:1009-1013. Price, J. S., and R. G. G, Russell, 1992, Bone remodelling: Regulation by systemic and local factors. In: C. C. Whitehead (ed.) Bone Biology and Skeletal Disorders in Poultry, 39-60. Carfax Publishing Co., Abingdon. Rath, N. C., W. E. Huff, G. R. Bayyari, and J. M. Balog 1998, Cell death in avian tibial dyschondroplasia. Avian Dis 42:72-79. Rath, N. C.,.M. P. Richards, W. E. Huff, G. R. Huff, and J. M. Balog. 2005. Changes in the tibial growth plates Of chickens with thiram-induced dyschondroplasia. J Comp Pathol 133:41-52. Reece, R. L. 1992. The role of infectious agents in leg abnormalities on growing birds. In: C. C. Whitehead (€4.) Bone Biology and Skeletal Disorders in Poultry. 231-263. Carfax Publishing Co, Abingdon Riddell, C. 1983. Pathology of the skeleton and tendons of broiler chickens reared to roaster weights. |. Crippled chickens. Avian Dis 27:950-962 Riddell, C. 1983. Pathology of the skeleton and tendons of broiler chickens reared to roaster weights. |. Normal chickens. Avian Dis 27:980-991. Riddell, C. 1992. Non-infectious skeletal disorders of poultry: An overview. In: C. C. Whitehead (ed.) Bone Biology and Skeletal Disorders in Poultry. 119-145. Carfax Publishing Co., Abingdon. Riddell, C. 1996. Skeletal system. In: C. Riddell (ed.) Avian Histopathology (2~ ed). 45-60. AAAP, New Bolton Center.Senties-Cue, G., H. L. Shivaprasad, and R. P. Chin. 2005. Systemic Mycoplasma synoviae infection in broiler chickens. Avian Pathol 34:137-142. Srinivasan, S., S.A. Kellin, S. Judex, R. C. Bray, R. F. Zemicke, and T. S. Gross. 2000. Aging-induced ‘osteopenia in avian cortical bone. Bone 26:361-365. ‘Thorp, B. H., C. C. Whitehead, and J. S. Rennie. 1991. Avian tibial dyschondroplasia: A comparison of the incidence and severity as assessed by gross ‘examination and histopathology. Res Vet Sci 51:48-54. ‘Trampel, D. W., and F. Goll, Jt. 1994. Outbreak of Mycoplasma iowae infection in commercial turkey pouits. Avian Dis 38:905-909. ‘Waldenstedt, L. 2006. Nutritional factors of importance {or optimal leg health in broilers: A review. Animal Feed Sci Technol 126:281-307. ‘Webster, S. V., C. Farquharson, D. Jefferies, and A. P. L. Kwan. 2003. Expression of type X collagen, Indian hedgehog and parathyroid hormone related-protein in normal and tibial dyschondroplastic chick growth plates. Avian Pathol 32:69-80. Wilson, S., S. R. |. Duff, and C. C. Whitehead. 1992. Effects of age, sex and housing on the trabecular bone of laying strain domestic fowl. Res Vot Sci 53:52-58, Wu, W.D., M. E. Cook, Q. L. Chu, and E. B. Smalley. 41993. Tibial dyschondroplasia of chickens induced by fusarochromanone, a mycotoxin. Avian Dis 37:302- 308.Table 1. Comparison of Lesions in the Growth Plate (Physis) Caused by Vitamin D3/Calcium and Phosphorus Deficiency Marin DSTCaTctumm DenCTency + Tncreased wid Of Zone oF proMeration (@) + Irregular ZP — loss of organized col- umns of proliferating chondrocytes. ‘Reduced width of zone of hypertrophy (z) + Limited vascular invasion ‘+ lregular base of physeal cartilage ‘+ Few calcified trabeculae in metaphysis + Abundant osteociasts ‘+ Fibrous tissue may replace bone mar- row | Phosphorus Detictency = Tncreased Wath Of Zone OF hypertrophy (2H) ‘= Normal vascular invasion ‘= Decrease in number of osteoclasts Increase in number of osteoblasts 67$001. Proximal Tibiotarsus, Normal. Turkey, 2 Weeks. Illustrated are the epiphysis E, physis P, metaphysis M, diaphysis D, and cortex (box and see Figure S005 for higher magnifications). BM, bone marrow and attachment of a tendon (arrow) are shown. $002. Proximal Tibiotarsus, Normal. Turkey, 2 Weeks. Higher magnification of figure S001 illustrates the tendon T, epiphysis E, and the regions of the physis including the zone of proliferation ZP, zone of pre-hypertrophy ZPH, and the zone of hypertrophy ZH. $003. Proximal Tibiotarsus, Normal. Turkey, 2 Weeks. Regions of the physis illustrated are: A. The junction of the epiphysis and the physis shows resting chondrocytes and proximal zone of proliferation (ZP). B. The zone of proliferation illustrates flat chondrocytes arranged in columns creating a stacked coin appearance. C. The zone of pre-hypertrophy is characterized by rounded chondrocytes separated by more matrix than those in the ZP. D. The zone of hypertrophy illustrates the round chondrocytes surrounded by relatively large amounts of matrix and the vascular invasion by vessels from the metaphysis. $004. Proximal Tibiotarsus, Normal. Turkey, 1 Day. A large plug of cartilage that fils the metaphyseal region and extends deeply into the diaphysis is the cartilaginous template for bone development and is normally present for the first 7 - 14 days. The epiphysis, E and physis, P are shown, $005. Proximal Tibiotarsus, Normal. Turkey, 2 Weeks. Higher magnification of the region bounded by the box in Figure S001 illustrates: A. Periosteum and cortical bone with a prominent row of osteoblasts. B. Higher magnification of cortical bone illustrates osteocytes surrounded by mineralized bone matrix. The arrow identifies the row of osteoblasts on the growing bone surface. C. Trabecular bone and bone marrow. D. A higher magnification of C, illustrates a multinucleated osteoclast and bone marrow. S006. Medullary Bone, Normal. Broiler Breeder Hen, 29 Weeks. Medullary bone is replacing bone marrow in these vertebrae. A, B, C, and D represent progressively higher magnifications to illustrate the location and staining characteristics of medullary bone. Note the contrast in staining quality between medullary bone and regular bone.$002 04 Sol $00" a a a Il C C T Cee € eee $003 69$007. Proximal Tibiotarsus - Rickets - Vitamin D3/Calcium Deficiency. Turkey, 2 Weeks. Illustrated are: widening of the physis below the epiphysis E; with disorganization of the zone of proliferation P; and failure of blood vessels from the metaphysis M, to invade the physis. $008. Proximal Tibiotarsus - Rickets - Vitamin D3/Calcium Deficiency. Turkey, 2 Weeks. This higher magnification of Figure $007 illustrates the increased width of the zone of proliferation (ZP) A; disorganization of the ZP B; failure of vascular invasion C; and proliferation of fibrous tissue in metaphysis D $009. Proximal Tibiotarsus - Rickets - Vitamin D3/Calcium Deficiency. Turkey, 2 Weeks. Aseries of progressively higher magnifications of the cortical region of the metaphysis shown at lower magnification in Figure S007 illustrates: a relatively thin cortex A; proliferation of fibrous tissue and osteoblasts B, and large numbers of osteoblasts C and D. Trabecular bone is relatively thin. The osteoblast activity may be in response to an early fracture in this region S010. Proximal Tibiotarsus - Rickets - Phosphorus Deficiency. Broiler Chicken, 18 Days. Illustrated are a relatively normal zone of proliferation, ZP beneath the epiphysis E, increased width of the zone of hypertrophy, ZH, and vascular invasion of the physis by blood vessels from the metaphysis. $011. Proximal Tibiotarsus - Rickets - Phosphorus Deficiency. Broiler Chicken, 18 Days. This higher magnification of the physis from Figure $010 illustrates a relatively normal orientation of the ZP (A and C), and increased width of the ZH (B and D). A blood vessel from the epiphysis is penetrating the ZP (C). Metaphyseal vessel invasion of the ZH is shown in D. $012, Proximal Tibiotarsus - Rickets. Broiler, 2 Weeks. Illustrated are: increase in width of the physis beneath the epiphysis E, a distinct zone of proliferation (ZP), a persistent zone of hypertrophy (ZH) with invasion by blood vessels from the metaphysis. Cortical bone is thin. See Figure S013 for higher magnifications of the physis. 70$007 S010 nm$013. Proximal Tibiotarsus - Rickets. Broiler, 2 Weeks. Illustrated are: A. Increase in width of the zone of proliferation (2P), but relatively orderly arrangement of chondrocytes. Epiphysis is shown, E. The zone of hypertrophy (ZH) is increased in width. B. The zone of proliferation (ZP) is relatively wide. An epiphyseal blood vessel (EBV) is invading the ZP and this is normal. C. The zone of pre-hypertrophy (ZPH) is very thin and almost indistinct as it merges with the zone of hypertrophy (ZH). D. Distal zone of hypertrophy illustrates deposition of some weakly stained matrix. Osteoblasts are numerous and there is proliferation of fibrous tissue. This case has some features of phosphorus deficiency rickets and some features of vitamin D3/calcium deficiency. $014, Proximal Tibiotarsus - Fracture and Rickets. Turkey, 4 Weeks. This is a saggital section of the tibiotarsus with the epiphysis E, physis P, metaphysis M, and diaphysis D identified. A folding fracture (arrow) is present at the junction of the physis P, and metaphysis M. The zone of proliferation has increased width as does the zone of hypertrophy thus rickets is present. Fractures are common in birds with rickets. S015. Proximal Tibiotarsus — Fracture and Rickets. Turkey, 4 Weeks. A series of progressively higher magnifications of the region of fracture in the tibiotarsus illustrated in Figure S014 illustrate the distortion of hypertrophic chondrocytes in the distal physis (A and B). Osteoblast and fibroblast proliferation is extensive and focal areas of necrosis are present, D. $016. Proximal Tibiotarsus - Fracture and Rickets. Turkey, 4 Weeks. This higher magnification of the region of fracture shown in Figure S014 illustrates the presence of multiple cell types including osteoblasts and osteoclasts. $017. Rib - Fracture with Callus. Turkey. A large callus (arrow) developed in response to fracture of this rib $018. Rib - Fracture with Callus. Turkey. A series of progressively higher magnifications illustrate the cartilaginous and fibrous connective tissue proliferation in response to a fracture. 72$013 $014 S015 ‘S016 73$019. Tibiotarsus - Tibial Dyschondroplasia. Chicken. This saggital section shows the epiphysis E, physis P, and a large plug of cartilage GP, that has no vascular invasion. This is the characteristic lesion of tibial dyschondroplasia. $020. Tibiotarsus- Tibial Dyschondroplasia. Chicken. A series of progressively higher magnifications of the TD lesion shown in Figure S019 shows the tip of the nonvascularized cartilage plug A, the avascular zone of hypertrophy B, and the apoptotic/necrotic chondrocytes (C and D) within the persisting hypertrophic zone. $021. Vertebra - Tibial Dyschondroplasia. Turkey. This cross section illustrates persistence of cartilage typical of dyschondroplasia $022. Vertebra - Tibial Dyschondroplasia. Turkey. A series of higher magnifications of the lesion shown in Figure S021 demonstrates persistence of cartilage with no vascular invasion, A, and the disorganized pattern of hypertrophic chondrocytes with small lakes of matrix material (B, C and D). The chondrocytes in this lesion are viable thus the combination of lesions supports a diagnosis of osteochondrosis. The failure of vascular invasion supports a diagnosis of dyschondroplasia. $023. Vertebrae - Osteochondrosis. Turkey. This longitudinal section illustrates distortion of a vertebral body resulting in compression of the spinal cord (box — A). Necrosis of articular cartilage with underlying osteomyelitis (box — B), and persistence of cartilage (box — C) are illustrated. Mycoplasma iowae was isolated and confirmed by PCR. $024. Vertebrae - Osteochondrosis. Turkey. Higher magnification of the areas illustrated in Figure $023 demonstrate distortion and necrosis of cartilage with osteomyelitis A, necrosis of cartilage with necrotic debris in the expanded intervertebral joint B, necrosis of cartilage in a higher magnification of the area within the box - B and osteomyelitis C, as well as persistent cartilage and expansion of the ventral surface of the vertebra. These lesions were associated with Mycoplasma iowae and include osteochondrosis, dyschondroplasia, and osteomyelitis. 74$020 $022 $019 $021 18$025. Tibiotarsus - Osteomyelitis. Broiler breeder, 9 Weeks. This saggital contains multiple variable-sized areas of necrosis in the epiphysis E, physis P, and metaphyseal M regions. The cause was Staphylococcus aureus. $026. Tibiotarsus - Osteomyelitis. Broiler breeder, 9 Weeks. This series of higher magnifications of the osteomyelitis shown in Figure $025 illustrates necrosis in the epiphysis A, a large area of necrosis in the physis B, and multiple bacterial colonies within the necrotic foci (C and D), $027. Distal Tibiotarsus and Proximal Tarsometatarsus - Relatively Normal. Turkey, 1 Week. This saggital section illustrates the relationships between the distal tibiotarsus and proximal tarsometatarsus and shows the joint space JS, a portion of the synovium §, and the flexor tendons. Cartilage (*) is the normal early age cartilage template for bone formation. $028. Flexor Tendons - Tenosynovitis. Turkey, 1 Week. The flexor tendons shown in figure $027 and illustrated at higher magnification have mild heterophil accumulation in the peritendonal space (* in A and at higher magnification in B). Normal tendon structure is illustrated in © and D. The tenosynovitis was caused by Staphylococcus aureus. $029. Distal Tibiotarsus and Proximal Tarsometatarsus - Tenosynovitis and Osteomyelitis. Turkey, 1 Week. This saggital section of the distal tibiotarsus and tibiotarsus illustrates lesions of tenosynovitis and osteomyelitis caused by Staphylococcus aureus. The joint space (*) contains a cellular exudate. Multiple areas of necrosis are in epiphysis and physis of the distal tibiotarsus. These lesions are illustrated at higher magnifications in Figures $030 and $031. $030. Distal Tibiotarsus and Proximal Tarsometatarsus -Tenosynovitis and Osteomyelitis. Turkey, 1 Week. This series of higher magnifications illustrate lesions of Staphylococcus aureus induced osteomyelitis with necrosis extending through the epiphysis A, necrotic cartilage in the physis B, multiple foci of necrosis in the physis C, and multiple colonies of bacteria (intralesional) in the necrotic foci D. 76$025 $026 $027 Désta Tinotarsu Proximal $030$031. Distal Tibiotarsus and Proximal Tarsometatarsus - Tenosynovitis and Osteomyelitis. Turkey, 1 Week. A series of higher magnifications of the lesions shown in Figure S029 illustrate fibrinoheterophilic synovitis (A and B), and tendonitis (C and D) in a case of Staphylococcus aureus tenosynovitis and osteomyelitis. $032. Tendon - Reoviral Tendosynovitis. Chicken. Lesions of tenosynovitis caused by Reovirus include acute hemorrhage and peritendonal fibrosis. Figures A-D show focal areas of acute hemorrhage and extensive proliferation of fibroblasts. $033. Tendon - Reoviral Tendosynovitis. Chicken. A series of higher magnifications of the lesions illustrated in Figure S032 show focal to multifocal accumulations of lymphocytes that characterize reoviral tenosynovitis. Acute hemorrhage and peritendonal fibrosis with lymphoid follicle development is shown in A. A higher magnification of the lymphoid nodules is in B and shows involvement of the tendon as well as peritendonal tissue. Accumulations of lymphocytes are shown in C and D. Note the absence of fibrin and heterophils. $034. Joint - Mycoplasma Synovitis. Chicken. Synovitis caused by Mycoplasma synoviae is characterized by lesions that include pronounced hyperplasia of the synovium (A and B) and accumulation of multiple nodular deposits of lymphocytes in the synovium (A, B, and C). Synovial epithelial hyperplasia and accumulation of fibrin in the joint space are illustrated in $035. Joint - Articular Gout. Chicken. Articular gout is characterized by deposition of urates in periarticular tissue. Distortion and expansion of tissue with urate deposition and granulomatous response are shown. The feathery appearance of the urates is shown in the insert. 78$035Muscular System Figures and Figure Legends ‘Oscar J. Fletcher and Tahseen Abdul-Aziz 80Introduction and Normal Histology The basic unit of the muscle system is the muscle fiber, an elongated multinucleated cell. It has an outer membrane called the sarcolemma. Within the fiber is the sarcoplasma that is composed of specialized cell organelles and cytoplasm. The nuclel are ovoid, elongated in shape and lie near the sarcolemma. ‘Some nuclei in avian muscle are also located within the central region of the fiber, between the myofiri. Satelite cells are thin, nucleated cells with a small ‘amount of sarcoplasm that are located between the ‘sarcolemma and the extemal lamina. Satellite cells are stimulated to proliferate when muscle is damaged and proliferating nuclei of satelite cells contribute to the increased cellularity that is a common response in muscle injury. In skeletal muscle, the myofibers are striated and composed of numerous myofilaments. ‘The contractile units or sarcomeres extend from one Z band to another and consist of bands created by the overlapping of thick and thin filaments (myosin and ac- tin). The zone with no overlapping next to the Z band is called the | band that is pale or lightly stained with H&E. The central zone between the ends of the thin filaments where only the thick filaments are seen is, called the H band. A thick dense line of M substance (on each filament, which is collectively called the M line, divides the H band. The band, darkly stained with H&E, is the wide central zone which extends from one end of the thick filaments to the other and alter- nates with the | band. When the sarcomere contracts, the Z bands move closer together, reducing the | and. H band widths. Myofibris are surrounded by the sarcoplasma. Two important components include the T system and the sarcoplasmic reticulum. The T system consists of invaginations of the sarcolemma, which allows action potentials to be quickly transmitted throughout the whole fiber. The sarcoplasmic reticulum is a complex network of tubules that act as a transporter and ‘supply system by carrying energy and metabolites to and from the fibrils. Individual muscle fibers and the satelite cells that accompany them are grouped together in fascicles. Each fascicle is surrounded by ‘a perimysium of loose connective tissue. Nerves and larger vessels are also located in the perimysium. Groups of fascicles are bound together by a sheath of dense connective tissue, the epimysium. ‘The variation in color of normal avian muscle is well documented and is related to the prevalence of various types of fibers. Most muscles are composed of mixture of fiber types, which explain the variation in fiber size seen in microscopic sections of avian muscle. There are two main types of fibers and one at lesser type. The red fiber is smaller, contains high levels of myoglobin and numerous mitochondria. It contains small amounts of glycogen but abundant fat. Ithas a good blood supply and is capable of sustained activity. The white fiber is larger and contains ‘more myoglobin and few mitochondria. It contains substantially less glycogen and lite fat. It has a poor blood supply. It is capable of sudden rapid movement. The lesser type of fiber is the intermediate fiber, which is between the red and white fibers. General Responses to Injury There are several different syndromes that are categorized by muscle lesions in birds. Five important syndromes are well defined and will be discussed in this section. Muscular dystrophy should be used only with inherited progressive muscular disease of chickens, turkeys, and Japanese quail. Other syndromes are myopathies caused by deficiency, toxins, excessive activity, or compartmental disease. Hereditary Muscular Dystrophy Hereditary muscular dystrophy of chickens is characterized by intial hypertrophy of muscles. followed by irregular atrophy. Pectoral muscle lesions are most striking but leg muscles are also involved. Initia microscopic changes include increased numbers of nuclei, variable fiber size, and ringed fibers. The ‘muscle fibers then disappear and are replaced by adipose tissue. Turkeys also have a hereditary ‘muscular dystrophy characterized by muscles of the pectoral and wing being affected with only atrophy. Numerous fibers become necrotic, vanish, and are replaced by adipose tissue. Japanese quail of the mutant LWC strain also have an autosomal dominant trait for hereditary muscular dystrophy that is similar to myotonic dystrophy in humans. Changes are similar to those described above with replacement by adipose tissue of the type II muscle fibers of the pectoral region, Nutritional Myopathy Nutritional myopathy is due to deficiency of vitamin E or selenium. Histological changes include loss of ‘cross striations, hyalinization, necrosis, and segmental {fragmentation of fibers. There is a proliferation Of satellite nuclei and invasion by macrophages.alization may also occur in affected fibers. When combined with the exudative diathesis form of the deficiency, the lesions are more severe and vascular changes are apparent. Thrombosis can occur in capillaries and veins such that the endothelium is. disrupted and fibrinoid degeneration of arterioles can ‘occur. Immunohistochemical staining for copper zinc superoxide dismutase and manganese superoxide dismutase results in granular aggregates or homogenous precipitates in the cytoplasm of affected muscle indicating up-regulation of the superoxide dismutase is occurring in order to scavenge the oxidative radicals. Exertional/Capture Myopathy Exertional myopathy/capture myopathy was first Gescribed in flamingos and now has been described in many other species of birds. Grossly, there are large areas of pale muscle. Lactic acid accumulations ‘Tap be Ws Gauss the unc necroia, RN chronic lesions are similar to nutritional myopathy. Changes include loss of cross striations, necrosis of fibers, fragmentation, and hyalinization along with increased numbers of satelite nuciel and macrophages phagocytosing the necrotic fibers. Without a good history, itis very difficult to determine the cause Deep Pectoral Myopathy Deep pectoral myopathy has been described in meat type chickens and turkeys. It follows exertion but has a different mechanism or pathogenesis than exertional myopathy. Only one muscle is affected and hangar are dbo to aSvonia PaaS lactic acid accumulations. In meat type poultry, the deep pector ‘muscle is encased by the sternum and tight fascia. When there is excessive exercise of the deep pectoral ‘muscle, it swells and the fascia does not expand, resulting in compression ischemia. The center of the muscle becomes necrotic and green (thus the lay term “Green muscle disease’). Histologically the fibers in the necrotic muscle are intact but just ghosts of the nuclei or no nuclei are apparent. The fibers have homogenous transverse fissuring due to discoid necrosis. Blood vessels contain lysed erythrocytes in the affected muscle. The necrotic. muscle is surrounded by fibrous granulation tissue and giant cells and macrophages infiltrate the necrotic and surrounding normal muscle fibers. There may be attempts of regeneration in the reactive zone. ‘Vascular thrombi can also occur in the zone and are considered secondary to the ischemia and necrosis. Toxic Myopathies Two types of toxic myopathies occur in poultry: lonophore and poisonous plants (Senna occidentalis). lonophore antibiotics are widely used in commercial poultry for control of coccidia. These compounds can be toxic if used improperty and can cause significant mortality in chickens, turkeys, guinea fowl and ostriches. Affected birds may be paralyzed before death, and this has been associated with degeneration Of skeletal muscles. lonophores interfere with ion transfer at the muscle cell membrane. Sodium ions from the sodium ionophore monensin can increase uptake of Ca++ at the muscle cell membrane by ‘changing the integrity of the membrane resulting in muscle fiber damage. Ca++ dependent phospholipases A2 may help with the movement ‘of Ca++ ions across the sarcolemma membrane to Keep balance. Compounds like ionophores disrupt this balance and can result in skeletal muscle damage. The amount of histological change seen in the muscle fibers can vary greatly. In some field ‘outbreaks lesions are absent or minimal while other ‘cases have hyalinized fibers, loss of cross striations, ‘and even fragmentation and mineralization. There can be infitration by macrophages and proliferation of satellite cell and fibroblast cell nuclei. Experimental trials have reported similar results. Tiamutin, an antimicrobial used to treat mycoplasmal infections, has been reported to interact with ionophores resulting in toxicosis because it prevents the degradation of the ionophore. An experimental trail with Tiamulin and. ionophores resulted in striking microscopic changes within the muscle fibers. There was hyalinization, fragmentation, and macrophage infitration in some fibers with severely enlarged nuclei associated with pale to basophilic sarcoplasma in other fibers. The affected nucle have prominent nucleoli. These changes were restricted to leg muscles only. Poisonous plants like Senna occidentalis (formerly Cassia sp.) are recognized to cause muscle degeneration in cattle and poultry. All parts of the plant are poisonous but the seeds are most toxic. The toxin in the seed remains unidentified. Some studies demonstrate that the external tegument of the seed is most toxic. ‘Grossi the sfected mates are pele and edematouis. Histological changes include swolfen fibers and fragmentation and proliferating nuclei. Less ‘severe changes like type 1 and type 2 fiber atrophy have been reported along with changes that can been seen by electron microscopy. These include lipid storage in fibers and enlarged mitochondria with isrupted or excessively branched cristae.Myopathies of Uncertain Etiology Other reported myopathies involving poultry have been inadequately explained as to the pathogenesis of the lesions. Many have limited histological descriptions. ‘An example of acute pectoral myopathy was described in broiler breeders that die suddenly with good flesh ‘The description ofthe lesions included hyalinization and fragmentation of muscle fibers, which suggested an exertional myopathy, taking into account the clinical aspects of the report. Tom turkeys with degenerative polymyopathy occurring in the first few days of the photoperiod being increased are reported. Affected turkeys have paralysis of the neck and may be unable to stand. The lesions reported include widespread degeneration of the muscle with cervical muscles most affected. Because many of the cervical muscles are surrounded by tight fascia, this may represent an example of compartment disease like deep pectoral myopathy. ‘Turkey leg edema syndrome is characterized by subcutaneous edema in the inguinal space and around the leg. It has been associated with disseminated focal myopathy with muscle fibers hyalinized or fragmented. There are some macrophages and heterophils infitrating fibers and some fibers are mineralized. Since the syndrome is primarily found at the processing plant, itis thought that overexertion during transport may play a role. A similar ddisserninated focal myopathy has been reported previously in normal and lame or crippled chickens, turkeys, and ducks. The lesions are more severe in lame birds but it has not been proven that the lesions cause the lameness. Myopathies that are localized have been reported ‘ducks affected with idiopathic torticolis and in broiler chickens at processing. The ducks have the epaxial cervical muscles affected with microscopic lesions of muscle fiber necrosis, mononuclear cell infiltrates, fatty infiltrates, and a thickened perimysium. A significant percentage of chickens with degenerated pubio-ischio- femnoralis muscle have been identified at processing. Most of the muscle was necrotic and contained large fibers with intemalized and clustered nuclei Infectious Agents ‘Muscovy ducks with parvovirus infections (Muscovy duck origin) have been reported to have degeneration of skeletal muscle along with numerous other lesions in other organ systems. Experimentally infected at day 1, by 3 weeks of age, the ducks have swelling and loss of cross striation of the muscle fiber and a reparative fibroblast proliferation. ‘Avian encephalomyeitis (AE) infection of chickens ‘can cause lymphocytic infiltration usually as multiple variable-sized collections within skeletal muscle. ‘These muscle lesions often are overlooked because tissue selection in suspected cases of AE commonly is limited to brain, gizzard, and proventriculus. Bacterial and mycotic infections involving muscle usually occur as extensions of processes in other organs. Parasites ‘Schizonts of protozoan parasites are common in the muscles of some avian species and have a characteristic appearance. There is little inflammation associated with the parasite except when the parasite is aberrant, and then a marked inflammatory reaction occurs. Hyaline degeneration of fibers, edema, fibrosis, calcification, and infiltration of lymphoid cells, macrophages, and heterophils have been reported. Injection Site Injury Injection of antibiotics or oil emulsion vaccines (bacterins) can result in necrosis and inflammation, Infitrates include macrophages, multinucleated giant cells, lymphoid cells, and heterophils, Neoplasia Marek’s disease induced lymphosarcoma is the ‘most common neoplasm involving the muscle of chickens. Although the neoplasm is characterized by pleomorphic populations of lymphoid cells, several sections may have to be examined histologically to see the variation of lymphoid cell sizes. Rhabdomyosarcoma and fibrosarcoma involving the muscle of chickens and other types of birds are rare. Fibrosarcomas and fibromas can be induced by avian leukosis virus infection besides spontaneous development. Ad jonal Readings Baird, G. J., G. L. Caldow, |. 8. Peeks, and D. A. Grant. 1997. Monensin toxicity in a flock of ostriches. Vet Rec 140:624-626.Bergeland, M., and D. Bares. 1965. Another problem appears degenerative polymyopathy of breeder tom turkeys. Gobbles 19:33. Blom, L. and F. Rasmussen. 1976. Tissue damage at the injection site after intramuscular injection of drugs: in hens. Br Poult Sci 17:1-4. Braga, |. S. 3°, K. Oda, T. Kikuchi, S. Tanaka, ¥. Shin, M. Sento, C. Itakura, and M. Mizutani. 1995. A new inherited muscular disorder in Japanese quails, (Cotumix cotumix japonica). Vet Patho! 32:351-360. Brooks, W. S. and S. E. Garrett. 1970. The mechanism Of pipping in birds. Auk 87:458-466. Cavaliere, M. J., E. E. Calore, M. Haraguchi, S. L. Gérniak, M. L. Dagli, PC. Raspantini, N.M. Galore, and R. Weg. 1997. Mitochondrial myopathy in Senna occidentalis-seed-fed chicken. Exotoxicol Envrion Saf 37:181-185. Chiasson, R. B. and E. W. Goulet. 1984. Muscle fiber types in the dystrophic pubischiofemoralis of ‘commercial broilers. Avian Dis 28:489-496. Dowling, L. 1992. lonophore toxicity in chickens: Areview of pathology and diagnosis. Avian Pathol 21:355-368, Droual, R., A. A. Bickford, B. R. Charlton and D. R. Kuney. 1990. Investigation of problems associated with intramuscular breast injection of oil-adjuvanted killed vaccines in chickens. Avian Dis 34:473-478. Droual, R., A.A. Bickford, and G. J. Cutter. 1993. Local reaction and serological response in commercial layer Chickens injected intramuscularly in the leg with oil- adjuvanted Mycoplasma gallisepticum bacterin. Avian Dis 37:1001-1008. Fadly, A.M. and L. N. Payne. 2003.Leukosis/ Sarcoma Group. In: M. Y. Saif, H. J. Barnes, J. R. Glisson, A. M. Fadly, R. L. McDougald, and D. E. ‘Swayne (eds.). Diseases of Poultry, 11th ed. lowa State University Press: Ames, lowa. 465-516. Fisher, H. . 1958. The ‘hatching muscle in the chick ‘Auk 75:391-399. George, J. C., and A. J. Berger. 1966. Avian Myology. ‘Academic Press: New York, NY. Gir, D. K., D. L. Miller, L. J. Thompson, L. Mailer, E. ‘Styer, and C. Balwin. 2007. Superoxide dismutase expression and oxidative damage in a case of myopathy in brown pelicans (Pelecanus occidentalis). J Vet Diagn Invest 19:301-304 Glavits, R., A. Zolnai, E. Szabo, E. Ivanics, P. Zarka, TT. Mato, and V. Palya. 2005. Comparative pathological studies on domestic geese (Anser anser domestica) and Muscovy ducks (Cairina moschata) experimentally infected with parvovirus stains of goose and Muscovy duck origin. Acta Vet Hung 53:73-89, Gopalakrishnakone, P.1984, Pathological features of idiopathic torticollis in the duck—a model for human disease. J Comp Pathol 94:453-462, Hanley, C. S., N. J. Thomas, J. Paul-Murphy, and B. K. Hartup. 2005. Exertional myopathy in whooping cranes (Grus americana) with prognostic guidelines. J Zoo Wild! Med 36:489-497. Haraguchi, M., E. E. Calore, M. L. Dat Cavaliere, N. M. Calore, R. Weg, P. C. Ra: 'S. L. Gomiak. 1998. Muscle atrophy induced in broiler chicks by parts of Senna occidentalis seeds. Vet Res Com 22:265-271 Haraguchi, M., M. L. Dagli,P. C. Paspantini, and S. L, Gémiak. 2003, The effect of low doses of Senna ‘occidentalis seeds on broiler chickens. Vet Res Com 27:321-328, Hassan, S., J. Hakkarianen, L. Jonsson, and J. ‘Tyopponen. 1990. Histopathological and biochemical changes associated with selenium and vitamin E deficiency in chicks. J Vet Med 37A:708-720. Hodges, R. D. 1978. The Histology of the Fowl. Academic Press: New York, NY. Kemp, J. Monensin poisoning in turkeys. Vet Rec 102:467. Marco, |, G. Mentaberre, A. Ponjoan, G. Bota, Mafiosa, and S. Lavin. 2008. Capture myopathy in little bustards after trapping and marking. J Wald! Dis 42:889-891. Montgomery, C.A. 1978. Muscle diseases. In: K. Benirschke, F. M. Gamer, and T. C. Jones, (eds). Pathology of Laboratory Animals. Vol. 1. Springer- Verlag: New York, NY. 821-887. Munday, B. L., J. D. Humphrey, and V. Kila. 1977. Pathology produced by, prevalence of, and probable life-cycie of a species of Sarcocystis in the domestic fowl. Avian Dis 21:697-703.Opitz, H. M., H. J. Jakob, E. Weinsenhuetter, and V. \V. Devi. 1982. A myopathy associated with protozoan schizonts in chickens in commercial farms in peninsular Malaysia. Avian Pathol 11:527-534. Randall, C. J. 1882. Acute pectoral myopathy in broiler breeders. Avian Pathol 11:245-252. Reece, R. L. 2003. Other tumors of unknown etiology. In: M. ¥. Saif, H. J. Bames, J. R. Glisson, A. M. Fadly, R. L. McDougald, and D. E. Swayne (eds.). Diseases of Poultry, 11th ed. lowa State University Press: Ames, lowa, 541-564. Rigdon, R. H. 1961. Spontaneous muscular dystrophy in the white Pekin duck. Am J Pathol 39:27-40, Rigdon, R. H., T. M. Ferguson, and J. R. Couch. 1962. Spontaneous muscular necrosis in the chicken. Poult ‘Soi 41:398-409. Rigdon, R.H., T. M. Ferguson, J. L. Trammel, J. R. Couch, and H. L. German. 1968. Necrosis in the “pipping” muscle of the chick. Poult Sci 47:873-877. ‘Sandercock, D. A. and M. A. Mitchell. 2003. Myopathy in broiler chickens: A role for Ca (2+)-activated phospholipases A2? Poult Sci 82:1307-12. Sandercock, D. A. and M. A. Mitchell. 2004. The role Of sodium ions in the pathogenesis of skeletal muscle damage in broiler chickens. Poult Sci 83:701-706. Shivaprasad, H. L., R. Crespo, B. Puschner, S. Lynch, and L. Wright. 2002. Myopathy in brown pelicans (Pelicanus occidentalis) associated with rancid feed Vot Rec 150:307-311 Siller, W. G. and P. A. L. Wight. 1978. The pathology of deep pectoral myopathy of turkeys. Avian Pathol 7:583-617. ‘Simpson, C. F., B. L. Damron, and RH. Harms. 1971. ‘Toxic myopathy of chicks fed Cassia occidentalis seeds. Avian Dis 15:284-290. ‘Skarda, R., V. Konrad, and J. Marcanik. 1978. Pathological changes in the skeletal musculature Of chicks after experimental hypokinesis. Folia Veterinaria 22:25-35. Sosnicki, A., R. G. Cassens, D. R. Mcintyre, and R. J \Vimini. 1988. Structural alterations in oedematous and apparently normal skeletal muscle of domestic turkey. Avian Pathol 17:775-791. Sosnicki, A. A., R. G. Cassens, R. J. Vimini, and M. L. Greaser. 1991. Histopathological and ultrastructural alterations of turkey skeletal muscle. Poult Sci 70:349- 357, Stuart , J. C. 1978. An outbreak of monensin poisoning in adult turkeys. Vet Rec 102:303-304. Umemura, T., H. Nakamura, M. Goryo, and C. Itakura 1984. Histopathology of monensin-tiamulin myopathy in broiler chicks. Avian Pathol 13:459-467. Wight, P.A.L., and W. G. Siller. 1980. The pathology of deep pectoral myopathy of broilers. Vet Patho! 17:29-38. Witter, R. L., and K.A. Schat. 2003. Marek’s Disease. In: M.Y. Saif, H. J. Bames, J. R. Glisson, A. M. Fadly, R.L. McDougald, and D. E. Swayne (eds.). Diseases of Poultry, 11th ed. lowa State University Press: Ames, Iowa. 407-465. Wobeser, G. A. 1981. Capture myopathy. In: Diseases of Wild Waterfowl. Plenum Press: New York, NY. 208- 210. ‘Young, E. 1967. Leg paralysis in the greater flamingo and lesser flamingo following capture and transportation. Int Zoo Yearb 7:226-227.M001. Skeletal Muscle - Normal. Broiler Breeder, 27 Weeks. This section of normal pectoral muscle shows striations (insert) and the central location of some myofiber nuclei. M002. Skeletal Muscle - Atrophy. Broiler, 15 Days. Muscle fibers are very small and nuclei are numerous. Broiler was from a flock experiencing runting-stunting syndrome. M003. Skeletal Muscle - Myopathy. Broiler Breeder, 27 Weeks. Myopathy is characterized by swelling and fragmentation of multiple muscle fibers with a minimal reactive response. This female was mobility — impaired, but the cause of this muscle lesion is not known. The lesions are similar to exertional myopathy and nutritional myopathy. M004. Skeletal Muscle - Myopathy. Broiler Breeder, 27 Weeks. Illustrated in this higher magnification of figure M003 are the swollen and fragmented muscle fibers. There is minimal proliferation of muscle nuclei at the periphery of the fiber. MOOS. Skeletal Muscle - Lymphocytic Myositis with Muscle Atrophy and Lipidosis. Backyard Chicken. Muscle fibers are variable in size. Some fibers are surrounded by lymphocytes and others are missing and replaced by fat. Replacement of muscle fibers by fat is a characteristic of muscular dystrophy. A hereditary basis for this lesion cannot be established and the lymphocytic response is not a usual feature of muscular dystrophy. This may be an immune mediated myopathy, but the cause is unknown. M006. Skeletal Muscle - Lymphocytic Myositis with Muscle Atrophy and Lipidosis. Backyard Chicken. This higher magnification of figure M005 shows the lymphocytic. infiltration and variations in muscle fiber size. Also note the replacement of muscle by adipose tissue.ye ) J ‘moot moo2 7M007. Skeletal Muscle - Lymphocytic Myositis with Muscle Atrophy and Lipidosis. Backyard Chicken. This higher magnification of figure M006 shows variations in fiber size and accumulation of adipose tissue. M008. Skeletal Muscle - Lymphocytic Myositis with Muscle Atrophy and Lipidosis. Backyard Chicken. This higher magnification of figure M007 shows atrophy of muscle fibers and accumulation of adipose tissue. This lesion has similarities to muscular dystrophy, but there was no data to support this conclusion in this case. M009. Skeletal Muscle - Chronic Myositis with Mineralization. Broiler Breeder, 28 Weeks. Necrosis and mineralization of large regions within pectoral muscle are accompanied by lymphocytic and histiocytic inflammation. A mineralized fiber is illustrated (box — lower right). The cause of this lesion is not known, but considered to be a chronic exertional myopathy lesion M010. Skeletal Muscle - Deep Pectoral Myopathy (Green Muscle Disease). Broiler Breeder. Pectoral muscle fibers have lost nuclei and are necrotic. Note the absence of any reactive response with progression from the margin toward the central part of the tissue. Proliferation of nuclei is confined to the periphery. M011. Skeletal Muscle - Deep Pectoral Myopathy (Green Muscle Disease). Broiler Breeder. The margin of the affected deep pectoral muscle has loss of muscle fibers and an inflammatory response in the adjacent connective tissue. M012. Skeletal Muscle - Deep Pectoral Myopathy (Green Muscle Disease). Chicken. An artery with a thrombus is shown. Necrotic muscle adjacent to this artery has no accompanying reactive or inflammatory response. 88Moo7 ‘Moos ‘moo9 MotoM013. Skeletal Muscle - Deep Pectoral Myopathy (Green Muscle Disease). Chicken. This higher magnification of figure M012 shows the thrombus in an affected artery. Fibrosis is confined to the tissue immediately adjacent to the blood vessel. M014, Skeletal Muscle - lonophore Toxicity. Turkey, 35 Days. This section of thigh muscle (red or “slow-twitch” fibers) has swelling and fragmentation of multiple myofibers with proliferation of nuclei M015. Skeletal Muscle - lonophore Toxicity. Turkey, 35 Days. A higher magnification of figure M014 illustrates the swelling and fragmentation with loss of cross-striations of myofibers and the proliferation of nuclei. M016. Skeletal Muscle - lonophore Toxicity - Trachea. Turkey. There is diffuse involvement of skeletal muscle of the trachea characterized by loss of myofibers, proliferation of nuclei, infiltration by histiocytes and fibroplasia. This lesion may result in a change in vocalization of affected turkeys. M017. Skeletal Muscle - lonophore Toxicity - Trachea. Turkey. Degeneration and loss of myofibers with proliferation of muscle nuclei is accompanied by increased numbers of histiocytes and fibroblast proliferation. Higher magnification of figure M016. M018. Skeletal Muscle - Avian Encephalomyelitis. Broiler, 18 Days. Focal collections of lymphocytes are scattered through this section of lumbar muscle. Lymphocytes are shown at higher magnification in the insert. 90Mots Mota M015 Mo16 a”M019. Skeletal Muscle - Necrosis - Clostridial Infection. Broiler, 38 Days. This lesion is characterized by diffuse necrosis of myofibers, erythrocytes, and blood vessels. This was an extension of gangrenous dermatitis caused by Clostridium septicum. The condition may be called Clostridial myositis because of the edema. Insert shows necrotic myofibers and bacteria M020. Skeletal Muscle - Mycotic Myositis. Broiler Breeder, 5 Weeks. A large zone of necrosis has a prominent peripheral infiltration of lymphocytes. This lesion was one of multiple mycotic granulomas that were in various organs including kidney and air sac. This lesion likely was an extension of a severe mycotic airsacculitis. M021. Skeletal Muscle - Mycotic Myositis. Broiler Breeder, 5 Weeks. This higher magnification of figure M020 shows the margin between necrotic muscle and adjacent viable fibers with histiocytes and lymphocytes infiltrating between muscle fibers. M022. Skeletal Muscle - Mycotic Myositis. Broiler Breeder, 5 Weeks. This higher magnification of figure M021 shows the swelling, fragmentation and loss of striations of muscle fibers. Fragments of fungi (arrow) are visible. Occasional longer segments of mycelia (box — lower right) can be seen within the necrotic zone M023. Skeletal Muscle - Sarcocystis. Turkey. A sarcocyst is within a skeletal muscle fiber eliciting no host response. Bradyzoites are numerous within the sarcocyst. M024. Skeletal Muscle - Sarcocystis. Turkey. This higher magnification of figure M023 illustrates the wall of the sarcocyst and the internal bradyzoites. Note striations in adjacent muscle fibers and the absence of host response. 92mo21 Mo22 93M025. Skeletal Muscle - Myositis (Injection Site Injury). Broiler Breeder, 28 Weeks. Vaccines containing oil-based adjuvants may cause intense granulomatous inflammation. Large clear spaces represent accumulation of oillipid material. Adjacent muscle fibers are variable in size with many being necrotic. M026. Skeletal Muscle - Myositis (Injection Site Injury). Broiler Breeder, 28 Weeks. Multiple granulomas have a central core of caseous necrosis and are surrounded by giant cells. Lymphocytes and histiocytes (Iymphoid cells) extend into skeletal muscle. M027. Skeletal Muscle - Myositis (Injection Site Injury). Broiler Breeder, 28 Weeks. This is a higher magnification of figure M026 that illustrates the granulomatous myositis with loss of muscle fibers. M028. Skeletal Muscle - Marek’s Disease. Chicken. Muscle fibers are infiltrated by neoplastic lymphoid cells. This is the most common tumor of skeletal muscle in chickens. M029. Skeletal Muscle - Marek’s Disease. Chicken. This higher magnification of figure M028 shows the pleomorphic lymphoid tumor cells replacing muscle fibers. Several atrophic fibers are in this field. M030. Skeletal Muscle - Rhabdomyosarcoma. Conure, 9 Years. The tumor is composed of elongated to fusiform cells that are arranged haphazardly. The cells have abundant eosinophilic to lightly basophilic cytoplasm that lack cross striation. Multinucleated cells are common and their presence is a characteristic feature of rhabdomyosarcoma,Mo27 95Cardiovascular System Oscar J. Fletcher and Tahseen Abdul-Aziz 96Introduction and Normal Histology ‘The cardiovascular system includes the heart and the arteries, veins, capillaries, and lymphatics that are distributed throughout the body. The avian heart is Conical, lies in the midline within the thoracic cavi just behind the thoracic inlet, and is enclosed withi the pericardial sac that contains serous fluid. It has four chambers, two ventricles and two atria. The left ventricle is somewhat larger than the right. The right ventricle extends only about two thirds of the way to the apex of the heart, and thus the apex is formed solely by the left ventricle. The wall ofthe left ventricle is three to four times thicker than that of the right ventricle. In relation to mammalian valves, the left atrioventricular valve in birds has poorly defined cusps that are attached to chordae tendinae. The right atrioventricular valve is a triangular muscle flap or plate that is devoid of chordae tendinae, arises from the muscle wall of the right ventricle, and closes against the wall ofthe left ventricle. The anatomy of this valve makes birds very susceptible to valvular insufficiency that often leads to right heart failure. ‘The major blood vessels that enter or leave the heart include the aorta, the pulmonary artery, the common pulmonary vein, the anterior vena cava, and the posterior vena cava. The exit from the right ventricle to the pulmonary artery is controlled by the pulmonary valve, which consists of three semilunar cups. A similar valve controls the exit from the left ventricle to the aorta. The cardiac valves are leaflets arising from the endocardium. They consist of a central core of dense irregular connective tissue (lamina fibrosa) covered by a single layer of flat endothelial cells. In the atrioventricular valve, the central connective issue, which is predominantly collagen, connects to the papillary muscles by collagenous strands, called chordae tendinae. A cartilaginous plaque is normal in the wall where major blood vessels leave the heart. The heart muscle (myocardium) consists of bundl of muscle fibers (myofibers) bound together by small ‘amounts of connective tissue. The myofibers are long, cylindrical, branching cells with usually a single, ovoid to elongated nucleus centrally located within each cell. The long axis of the nucleus is parallel to that of the fibers. Each muscle fiber is made of numerous myofibrils, which lie parallel to each another within the sarcoplasm (cytoplasm) and are enclosed by sarcolemma (plasma membrane). A delicate network of supporting tissue that contains an extensive network of capillaries fils the spaces between musole fibers. ‘The inner layer of the heart is the endocardium, the surface of which consists of a single-cell layer of flattened endothelial cells that continue with the endothelium of the vessels entering and leaving the heart. Beneath the endothelium is a delicate supporting layer of collagenous and elastic tissue. ‘The external surface of the heart is the epicardium, covered by a single-cell layer of flattened epithelial cells, the mesothelium, that also line the parietal surface of the pericardial sac. These mesothelial cells, secrete the serous fluid in the pericardial sac. A thin layer of fibroelastic tissue lies beneath and supports the mesothelial cells. Variable amounts of adipose tissue may be found between the epicardium and the myocardium. Large modified and specialized cardiac muscle fibers called Purkinje cells that comprise the conducting system of the heart are found beneath the endocardium either as single fibers or well-developed tracts. They are also found within the myocardium, usually in association with blood vessels, and ‘occasionally in the epicardium. Purkinje cells are relatively easy to recognize Because of their large size ‘and extensive pale cytoplasm. Focal accumulations of hematopoietic and myeloid cells are scattered through the myocardium. Occasional focal nodules of lymphoid cells are found in the myocardium of clinically normal birds. Both hematopoietic cell foci and lymphoid cell foci may be mistakenly interpreted as lesions. In many cases only cross sections of the myocardium are submitted for histologic evaluation. Such samples may provide useful information, but saggital sections that include the base of the major arteries and the valves must be included ifthe submitter wants to improve the opportunities for accurate diagnosis. ‘Some diseases have striking gross lesions that often. include ascites, hydropericardium, and enlargement of the heart, but microscopic changes in the myocardium are likely the end-stage effects of impaired cardiac function rather than being the primary causes of the {gr085 lesions. Lesions of visceral gout often include deposition of urates on the pericardium resulting in increased pericardial thickness. Urate deposition ‘on the pericardium appears as a band of basophilic material. Occasionally urate deposits can be found in the myocardium. Myofibers cannot regenerate following necrosis, but they do undergo hypertrophy in response to increased workload demands. Increased cardiac ‘workload results from increased demands for oxygen, or increased resistance to blood flow following constriction, stenosis, and/or obstruction of arteries, arterioles, and capillaries. Physiologic hypertrophy is not associated with damage to myofibers. The process of cardiac enlargment with damage to myofibers is called cardiomyopathy, which can lead to heart failure. 7‘Sudden death syndrome in broilers (acute death, heart attack, flip-over) has no significant histologic changes inthe heart. Sudden death syndrome in turkeys is characterized by perirenal hemorrhage, hypertrophic cardiomyopathy, and fibromuscular dystrophy in arteries and arterioles, Ascites Ascites is not a specific disease, but isa clinical ‘syndrome having characteristic gross lesions. Ascites has muttiple causes that include damage to vascular endothelium, right atrioventricular valvular insufficiency, increased venous pressure, primary right- sided valvular endocarditis, and right-sided ventricular cardiomyopathy with cardiac failure as an end-stage of increased pulmonary arterial pressure (pulmonary hypertension syndrome). Factors causing or involved in pulmonary hypertension include polycythemia, high altitude, cold exposure, and increased rigiity of erythrocytes, severe pulmonary lesions, and the inoreased oxygen demands of rapidly growing birds. ‘Sodium toxicity can cause increased erythrocyte rigidity, increased blood volume, glomerulosclerosis, and lung edema. Pulmonary hypertension syndrome (PHS, ascites) in fast-growing broilers is the result of elevated biood pressure (pulmonary hypertension) in the circulatory system of the lung and is not associated with lesions in the myocardium unti the terminal stages of right ventricular hypertrophy are reached. Histologic changes in the heart in these terminal stages include myofiber degeneration with swelling and fragmentation, occasional hyaline deposits within myofibers, focal necrosis, edema, and fibrosis. Noninfectious Conditions Endocardiosis Endocardiosis, which is a focal thickening of a valve oF of the subendothelium due to the accumulation of collagenous and often myxomatous tissue, is frequent in cardiomyopathies. In more chronic cases of cardiomyopathies (for example dilated cardiomyopathy in turkeys and in the right AV valve in chronic right heart failure), the accumulation of collagenous and ‘myxomatous tissue is more diffuse and this process is called fibroelastosis, Fibroelastosis is more prominent in the subendothelium of the left ventric ‘A focal area of epicardial fibrosis may be found at, the apex of the heart where it touches the sternum. This accumulation of fibrous tissue is referred to as a “friction rub” lesion as it likely results from the contact, (trauma) of the tip of the heart with the sternum, Cardiomyopathy - Round Heart Cardiomyopathy is a general term meaning disease of heart muscle. This term is more useful in gross pathology as there are no defining histologic changes, Cardiomyopathy indicates gross enlargement of the heart that usually reflects impaired cardiac performance or cardiac failure usually with gross lesions of ascites and/or hydropericardium, Dilated cardiomyopathy is characterized by one or more dilated (enlarged) chambers with thin walls. This is a degenerative condition in which myofibers are lost. Myofibers are long, thin, and disorganized in focal to muttfocal areas. Connective tissue and collagen are increased. Restrictive cardiomyopathy is characterized by fibrosis of the pericardium, epicardium, and/or endocardium, ‘This type often follows pericarditis caused by bacterial infections. Cardiomyopathy is a significant gross lesion in spon- taneous cardiomyopathy of turkeys (round heart) and round heart disease (not to be confused with pulmo- nary hypertension syndrome) of chickens. ‘Spontaneous cardiomyopathy of turkeys, also called round heart disease, is an example of dilated cardiomyopathy characterized by enlargement of both ventricles. The primary lesion is focal myofiber degeneration with vacuolation. Enlargement ‘of muscle nuciel and dilation of capillaries may be present as secondary lesions. Endocardial fibroelastosis that can include fibrocartlaginous tissue is in the subendothelium of the left ventricle in chronic, ‘cases. Furazolidone-induced cardiomyopathy is another example of dilated cardiomyopathy and the histologic lesions are similar to those of spontaneous cardiomyopathy of turkeys. Round heart disease of chickens, a disease of historical interest and probably not currently seen, is characterized by hypertrophy of the left ventricle. Histologic changes may include myofiber degeneration ‘with swelling, increased granularity, and fragmentation, deposits within myofibers, focal xdema, and fibrosis. Coalescence of small vesicles creates larger spaces seen around nuclei. Endocardiosis is offen present. Fat accumulation or lipidosis is characterized by the presence of multiplevacuoles within myofibers, Multiple vacuoles may be Present in round heart disease of chickens and is a feature in fatty liver-kidney syndrome. A large amount of fat is normal in the heart muscle of broiler Mi and Vitamin Deficiencies and Toxicities lonophore toxicity produces the range of degenerative changes in myofibers described above but usually without the mineralization that is frequent in degeneration and necrosis resulting from vitamin E /selenium deficiency. Other causes of myofiber degeneration and necrosis include furazolidone toxicity, toxic fat syndrome (dioxin toxicity), silver and cobalt toxicity, selenium toxicity, deficiency of ‘copper, and poisonous plant (Cassia and Crotalaria as examples) toxicities. Features of potassium deficiency include hypertrophy of myofibers, degenerative changes in myofibers as described above and endocardial fibroelastosis. Epicardial fibrosis and subendocardial fibrosis are chronic lesions that follow edema and may be changes caused by toxicity from monensin, rapeseed oil or rapeseed meal if used as a protein source, soybean oil, or polychlorinated biphenyls. Fat can accumulate in myofibers in fatty liver and hemorrhagic syndrome and in rapeseed oil (erucic acid) toxicity. Vitamin D3 toxicity causes deposition of mineral, most likely secondary to kidney damage, in the wall of arteries in the myocardium. Infectious Agents Viruses Viral infections can result in myocardial hemorrhage, ‘edema, necrosis and, if the bird survives the acute phase, Significant damage with limited or very subtle microscopic changes can be caused by viral infections of the heart. Avian influenza (Al) can cause edema and muitifocal degenerative and necrotic changes in myofibers. Immunohistochemistry reveals much more AA\ viral antigen in both nuclei and cytoplasm than one might expect from the changes seen in H&E stained sections alone. Some viruses produce inclusions in the nuclei of myofibers — examples include parvovirus infections of geese and polyomavirus infections in pet birds. Avian encephalomyelitis (AE) virus causes increased numbers of lymphoid cell foci, but these are more characteristic (less chance of misinterpretation ‘of “normal” lymphoid foci in the muscle wall of the ventriculus and intestine than in the myocardium. in ‘some cases of AE, difuse lymphocytic myocarditis ‘can be extensive and may involve walls of the auricles. Interstitial, lymphocytic myocarditis and lymphocytic, sometimes lymphofollicular, pericarditis are lesions that may be found in young chickens and turkeys affected with viral arthrtisenosynovitis caused by reovirus. Mild to severe myofiber necrosis, infiltration by lymphocytes mixed with few heterophils, macrophages, plasma cells and multinucleated giant cells can be caused by reovirus infection in young, turkey poults. Eastern equine encephalitis virus, Highlands J virus, and West Nile virus cause necrosis and lymphocytic inflammation of the myocardium. Bacteria Epicardits is inflammation of the epicardium. tt is characterized by an accumulation of inflammatory ‘exudate on the mesothelial layer of the epicardium. Epicarditis is common in systemic bacterial infections, ‘especially with Escherichia coli(E. coll) and Salmonelfa. In early lesions the inflammatory exudate is composed of mainly heterophils and fibrin; later lymphoid cells and macrophages predominate. If the bird survives, fibrin-tich exudate usually undergoes fibrovasoular organization in which endothelial ‘cells and fibroblasts grow from the surface of the ‘epicardium and replace fibrin with granulation tissue, characterized by ingrowth of new capillary branches with proliferation of fibroblasts. Lymphoid inflammatory cells, including macrophages and giant cells are common. The granulation tissue matures to {orm fibrous tissue that results in adhesions between the parietal and visceral surface of the epicardial sac — a condition defined as restrictive cardiomyopathy. In acute bacterial septicemias, clusters of bacteria. ‘and fibrin thrombi may be seen in the capillaries of the myocardium, and there may be varying degrees of vasculitis, Bacterial infections are common causes of pericardial and myocardial lesions with . coli being frequently involved. Lesions of colibacilosis are usually more extensive in the pericardium and epicardium. The microscopic changes include edema, fibrin exudation, heterophil infiltration and perivascular lymphoid cell accumulations. More chronic lesions of colibacillosis have more pronounced accumulations of lymphocytes ‘and macrophages that may be admixed with fibrin. Focal lymphocytic myocarditis may occur in the subepicardial muscle. Chronic colibacillosis also is characterized by necrotic debris and fibrin surrounded by multinucleated giant cells, macrophages, andlymphocytes. Bacterial colonies may be numerous in the necrotic debris. Riemerella anatipestifer infection of turkeys and ducks is an example of the fibrinoheterophilic response. Fibrin thrombi are common. Chlamydophilosis (chiamydiosis) should be considered when the pericardial lesions are fibrinous with large numbers of lymphoid cells and relatively few heterophils. Staphylococcus aureus can cause septicemia resulting in large areas of necrosis in the myocardium. Vasculitis is prominent and bacteria are observed in multiple blood vessels in the myocardium. ‘Salmonella pullorum can cause multiple variably sized areas of myocardial necrosis and inflammation. The inflammatory response is lymphoid and includes many large cells with foamy cytoplasm (macrophages) and some multinucleated giant cells sufficient to justify a diagnosis of granulomatous myocarditis. Endocardiosis and/or endocardial fibroelastosis are possible secondary lesions, Valvular endocarditis or vegetat ‘endocarditis usually is a large lesion and is found more commonly in the left atrioventricular and aortic valves. The necrotic debris on the surface usually contains large numbers of bacteria. A granulomatous inflammatory reaction is typical at the base of the endocardial lesion. Bacteria associated with ‘endocarditis include Enterococcus, Staphylococcus, Pasteurella, and Erysipelothrix. Focal to multifocal necrotizing myocarditis is frequent with these bacterial infections. Infections with Mycobacteria can involve the he but lesions are usually found in the digestive tract with multiple nodular lesions in the liver, spleen, and possible skin involvement. Accumulations of large macrophages having foamy or granular cytoplasm. should cause consideration of mycobacteriosis as the disease. Central cores of caseous necrosis are common, but not always present, in mycobacteriosis in chickens with acid-fast organisms found in the center of the granulomas. The finding of numerous acid-fast bacill in the cytoplasm of the macrophages is diagnostic. Fungi Mycotic infections may be localized but replace large areas of myocardium. Multinucleated giant cells are ‘numerous among the lymphocytic and histiocytic cell population, and remnants of fungal elements can be found in giant cells and macrophages. Mycotic. infections of the heart are relatively uncommon. Adjacent myocardium may have focal areas of lymphocytic myocarditis. Parasites ‘Sarcocystis is one of the more common protozoans found in the myocardium. The cysts in myofibers usually are not associated with any inflammatory reaction. Toxoplasmosis causes extensive necrosis and granulomatous inflammation with organisms visible within the necrotic lesions. Caseous necrosis with large numbers of trichomads within the myocardi lesions may be found in young pigeons with systemic trichomoniasis. Neoplas Lymphoid tumors of Marek’s disease are the most ‘common tumors found in the hearts of chickens. Marek’s disease lymphomas are characterized by a pleomorphic lymphoid cell infiltration of the pericardium and, often, the myocardium. Lymphoid leukosis tumors in chickens and lymphoid tumors. induced by reticuloendotheliosis virus in turkeys may involve the heart. Primary tumors of the heart include rhabdomyosarcomas, myxomas, fibromas, and fibrosarcomas. Blood Vessels Blood vessels can be divided into elastic arteries, ‘muscular arteries, veins, capillaries, and venules. The wall of large arteries is composed from inside to outside of three concentric layers: Tunica intima, tunica media, and tunica adventitia. The tunica intima is composed of a single-cell layer of endothelial cells, and various amounts of subendothelial connective tissue. The tunica media, usually the thickest of the three layers, is composed of circularly arranged smooth muscle cells and fibroelastic connective tissue. The tunica adventitia is the outer layer of the vessel wall and composed of loose connective tissue of predominantly collagen fibers with fewer elastic bers; the cellular component of this layer consists mainly of fibroblasts. The tunica media of the aorta consists of concentrically fenestrated sheaths of elastic fibers separated by collagenous tissue and relatively few muscle fibers. The tunica media of large muscular arteries is composed of concentric layers of smooth muscle fibers between which pass few elastic fibers. Condensed elastic fibers form a well-defined lamina (internal elastic lamina) that separates the tunica intima from the tunica media and the less-defined external elastic lamina that separates the tunica media from the tunica adventitia. In small muscular arteries 100and large arterioles, the elastic fibers are fewer, ‘and only a thin elastic lamina may be recognized; there is no external elastic lamina, and the tunica adventitia may be as thick as the tunica media and merge with the surrounding collagenous tissues. In ‘small arterioles, the tunica intima is only distinguished by the nuclei of endothelial cells, the tunica media ‘consists of a layer of smooth muscle cells two- to three-cells thick, and the adventitia merges imperceptibly with the surrounding supporting tissue. In the lung, and perhaps elsewhere, small muscular arterioles respond to pressure by hypertrophy of muscle cells (as in myofiber hypertrophy in the heart) thus reducing luminal size. In capillaries, a one-cell layer of endothelial ces is the structure that is discemible, muscular and adventitial layers are absent. Occasionally embracing the endothelial cells are flattened cells called perioytes that have contractile functions. The diameter of capillaries is usually that of red blood cells except in the lung where capillaries have a smaller diameter than erythrocytes. In comparably sized vessels, veins have thinner walls, relatively less muscle tissue in the tunica media, and, a tunica adventitia thicker than the tunica media. Because of these features, veins do not retain their shape. They often appear irregular in stained tissue sections, and the lumen may not be patent. Lymphatic vessels have similar features. Small and medium- sized veins have a thin tunica media of circularly arranged smooth muscle fibers, and a thick tuni adventitia of longitudinally arranged collagen fibers. ‘The medium-sized veins have a relatively thick tunica media consisting of several layers of smooth muscle separated by layers of collagen fibers; the tunica adventitia is a tick layer of collagen and elastic fbers. In large veins, the tunica media is reduced to a very thin muscular layer, but the adventitia is a very thick layer of collagen fibers between which thick elastic, fibers are present. The tunica adventitia of some large blood vessels contains numerous small arteries (vasa vasorum), which provide nutrients to the walls of the vessels. Vasculitis Vasculitis is characterized by inflammation in and damage to the walls of blood vessels. The most ‘common lesion of vasculitis is segmental or diffuse infiltration of the vessel wall with inflammatory cells. Necrotizing vasculitis is characterized by necrosis of the vessel wall that is usually accompanied by intact ‘and necrotic inflammatory cells, either heterophils ‘or lymphoid cells. Vascults is a lesion that may be caused by bacterial, viral, fungal, protozoal, or parasitic agents (example is schistosomiasis in Brant 101 geese) infections. Aspergillus has the tendency to invade the wall of blood vessels and cause extensive vasculitis and, occasionally thrombosis. Fibrinoid necrosis is defined as the accumulation of a homogenous, eosinophilic, and glassy material in blood vessel walls. This accumulated material usually results from leaked proteinaceous fluid, including fibrin, and/or deposition of immune complexes in the vessel wall, Areas of fibrinoid necrosis contain various amounts of immunoglobulin and complement, albumin, breakdown products of cells and of the vessel wall components, and fibrin. ‘Athrombus is a mass that is formed by the clotting process and located within the vascular lumen and attached to the wall of blood vessels. The ‘most common cause of thrombosis is injury to the endothelial lining of blood vessels that triggers a series of cascading reactions resulting in the formation of ‘a coagulum composed of fibrin and platelets. Fibrin microthrombi may be found in the capillaries of birds dying from systemic bacterial infection such as E. coli ‘septicemia and in the capillaries in the brain in free radical damage associated with vitamin E deficiency (encephalomalacia). An embolus is an abnormal mass (Solid, liquid, or gas) that is free in the lumen. ‘of blood vessels and is transported through the vascular system to a different site than where it was formed. Emboli usually cause obstruction or occlusion of a blood vessel. Most emboli are fragments of thrombi that break free from the original mass and get cartied in the blood stream to sites of lodgment. In septicemia, bacterial emboli (septic emboli) may be seen in the lumen of capillaries in diferent organs and tissues, including the heart, Bacterial toxins or endotoxins are potential causes of endothelial injury. Fibromuscular Dysplasia Fibromuscular dysplasia (FMD) is a non-inflammatory, non-atherosclerotic lesion in the wall of arteries that ultimately lead to narrowing of the vessel lumen. The lesions of FMD vary, depending on the predominant arterial wall layer involved. Three primary types are identified, and these include: intimal fibroplasia, medial dysplasia, and adventitial (periarterial) fibroplasia, Fibromuscular dysplasia can be found as incidental observation at almost any age and is more commonly found in turkeys than in chickens.Atherosclerosis Atheroscelrosis is the accumulation of lipids and. ‘macrophages (foam cells = macrophages containing cytoplasmic lipid droplets) with or without collagenous fibrous tissue or myxoid tissue in the walls of larger arteries. The microscopic appearance of atheromatous lesions depends on the stage of the development. Atheromatous changes are more frequently found in older birds. The lesions can be produced by diets high in cholesterol and genetic factors. Marek’s disease herpes viral infection is ‘associated with a higher incidence of atherosclerosis, in chickens. Neoplasia ‘Tumors of blood vessels include angiomas and hemangiosarcomas. Vascular tumors are included ‘among the tumors caused by avian retroviruses. Additional Readings Ayala, |., B. Garcia Perez, G. Domenech, M. T. Castells, and M. Valdes. 2005. Use of the chicken as an experimental animal model in atherosclerosis. Avian & Poul Biol Rev 16:151-159. Balamurugan, V,, and J. M, Kataria. 2004. The hydropericardium syndrome in poultry ~ a current scenario. Vet Res Com 28:127-148, Balog, J. M. 2003. Ascites syndrome (pulmonary hypertension syndrome) in broiler chickens: Are we seeing the light at the end of the tunnel? Avian & Poul Biol Rev 14:99-126. Bezuidenhout, A. J. 1983. The valva atrioventricularis dextra of the avian heart. Anat Histol Embryol 12:104- 108, Bickford, A. A., R. E. 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