International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Non-proliferative and Proliferative Lesions of the Non-human Primate (M. fascicularis)

( Table 24 Table 24. Microscopic Findings of the Hepatobiliary System: Gall Bladder; Non-human primate Gallbladder and Biliary Tree Common Uncommon Not Observed but Potentially Relevant Not Applicable Congenital Ectopic tissue, pancreas x Ectopic tissue, hepatocytes x Non-proliferative Calculus x Cholecystitis 1 x Metaplasia, glandular x Hyalinosis x Infiltrate, [insert appropriate cell type] *^ x Inflammation ^ x Parasite ^ ( Enterocytozoon bieneusi *) x Proliferative (non-neoplastic) Hyperplasia x * Terminology with diagnostic criteria or comments described below. ^ Terminology addressed in the Systemic/General Pathology section. 1 The term cholecystitis is not preferred and the term inflammation, gall bladder should be used instead. ) * Terminology with diagnostic criteria or comments described below. ^ Terminology addressed in the Systemic/General Pathology section. 1 The term cholecystitis is not preferred and the term inflammation, gall bladder should be used instead. Spontaneous background (may represent MALT) Focal to diffuse, frequently lymphoplasmacytic, infiltrate in the gall bladder lamina propria 61 . May have germinal centers. Comments: Inflammatory cell infiltrates of the gall bladder have been reported in New World NHPs in association with Helicobacter sp. Infections, which have also been reported in macaques. ( Figure 105–106 Figure 105. Cynomolgus, Gall bladder, Parasite, Enterocytozoon bieneusi , H&E. Figure 106. Cynomolgus, Gall bladder, Parasite, Enterocytozoon bieneusi , IHC. ) Cynomolgus, Gall bladder, Parasite, Enterocytozoon bieneusi , H&E. Cynomolgus, Gall bladder, Parasite, Enterocytozoon bieneusi , IHC. Enterocytozoon bieneusi is a microsporidian parasite of primarily macaque species that causes proliferative cholecystits, choledochitis, and serositis Non-suppurative inflammation of the biliary tract. Single, sloughed epithelial cells that contain the organism are diagnostic for this infection. It is very difficult to see the organism without special stains. Marked proliferation of bile ducts in the portal tracts with concurrent fibrosis. Proliferative serositis associated with mesothelial hyperplasia and non-suppurative inflammation of the small intestine. Cryptosporidium parvum - Ascending biliary infections are associated with biliary hyperplasia and eosinophilic to neutrophilic inflammation admixed with mononuclear cells. - Organisms are seen attached to the apical surface of the biliary epithelial cells or free in the lumen. - Organisms can be very numerous. Immunohistochemistry for measles matrix protein will highlight the organism. A modified Weber’s trichrome is also diagnostic. Additional diagnostic testing can be performed using electron microscopy or PCR. Comments: This is an uncommon opportunistic infection in immunocompromised macaques 126 . Recently data suggest that M. fascicularis can also harbor the parasite although pathogenesis studies have not been performed to date 214 . The proliferative serositis lesion is an uncommon manifestation 215 . In severe cases of E. bieneusi , grossly the liver can have an accentuated lobular pattern due to the degree of biliary hyperplasia and accompanying fibrosis.

All procedures used to prepare macroscopic and microscopic images of animal specimens for this article were performed in accordance with regulations and established guidelines for humane treatment of research animals and were reviewed and approved in advance by an Institutional Animal Care and Use Committee.

( Figure 12–13 Figure 12. Cynomolgus, Tongue, Fungus, Candida spp., H&E, Low magnification; Courtesy of Dr. Cindy Farman. Figure 13. Cynomolgus, Tongue, Fungus, Candida spp., H&E, High magnification; Courtesy of Dr. Cindy Farman. ) Cynomolgus, Tongue, Fungus, Candida spp., H&E, Low magnification; Courtesy of Dr. Cindy Farman. Cynomolgus, Tongue, Fungus, Candida spp., H&E, High magnification; Courtesy of Dr. Cindy Farman. Host response to fungal infection can vary from neutrophilic to granulomatous inflammation in miliary to mass-like lesions. Multinucleate giant cells may also be present. Distinct fungal morphologic features are often visualized by light microscopy. Special histochemical stains (Grocott or Gomori methenamine silver [GMS] and periodic acid Schiff [PAS] being most commonly used), culture results, antigen detection and molecular diagnostics aid in diagnosis. Comments: Primary fungal infections are extremely rare in laboratory macaques. However, like many other mammalian species, NHP are susceptible to opportunistic infection. NHP are often temporarily housed or originate in geographic locations that are endemic for specific fungal pathogens, and thus can be exposed to a variety of fungal agents, most of which remain latent in immunocompetent animals. Reported fungal infections observed in immunosuppressed macaques include Pneumocystis carinii in the lung , Histoplasma capsulatum , Cryptococcus neoformans and non-neoformans spp ., Aspergillus spp., and Coccidioides spp . 16 , 17 , 18 Disseminated Talaromyces (Penicillium) marneffei infection was also recently reported in a laboratory cynomolgus macaque and directly linked to the immunosuppressive pharmacology of therapeutic agent given 19 . Candida albicans, while considered a normal saprophytic inhabitant of mucous membranes of the alimentary and genital tract in NHP, can also cause localized infection (thrush) or disseminated disease in neonates and immunocompromised animals 17 .

( Figure 7–9 Figure 7. Cynomolgus, Duodenum, Amyloid, H&E. Figure 8. Cynomolgus, Duodenum, Amyloid, congo red. Figure 9. Cynomolgus, Duodenum, Amyloid, crystal violet. ) Cynomolgus, Duodenum, Amyloid, H&E. Cynomolgus, Duodenum, Amyloid, congo red. Cynomolgus, Duodenum, Amyloid, crystal violet. Amyloid is a pathologic proteinaceous substance deposited between cells in various tissues and organs of the body in a wide variety of clinical settings. In AA-amyloidosis, there is an increased level of serum amyloid-A (SAA), which is common in inflammatory states. It is formed mainly in the liver upon stimulation by proinflammatory cytokines and normally plays a role in cholesterol transport and as a chemoattractant in the inflammatory processes 8 . When the concentration of this molecule is increased, typically as a result of chronic inflammation, certain isoforms of SAA are partially cleaved into fragments that have an increased propensity to form fibrillar aggregates of amyloid deposited systemically. Deposition of insoluble amyloid fibrils occurs either due to a defect in SAA-degrading enzyme in the system, or consequent to the synthesis of abnormal SAA protein resistant to the enzymatic degradation. In AL-amyloidosis, unstable monoclonal immunoglobulin light chains, produced by a plasma cell dyscrasia, lead to the formation and deposition of fibrils. Grossly, affected organs appear enlarged, moderately firm, and abnormally discolored (pale gray or yellowish orange). Microscopically, amyloid is deposited extracellularly in various affected tissues. Amyloid is a homogeneous, extracellular, hyaline to finely fibrillar waxy material. Can interfere with normal tissue function and eventually may produce pressure atrophy of adjacent cells. Grossly, affected organs appear enlarged, moderately firm, and abnormally discolored (pale gray or yellowish orange). Microscopically, amyloid is deposited extracellularly in various affected tissues. Amyloid is a homogeneous, extracellular, hyaline to finely fibrillar waxy material. Can interfere with normal tissue function and eventually may produce pressure atrophy of adjacent cells. Other similar-appearing extracellular deposits such as collagen and fibrin: Necrosis: - Congo red, Thioflavin T and immunohistochemical staining negative. - Disruption of cellular architecture - Nuclear pyknosis, karyolysis, or karyorrhexis. Fibrosis: - Fibrillary appearance with small numbers of fibroblasts - Congo red, Thioflavin T and immunohistochemical staining negative. - Positive collagen staining (van Gieson and Masson-trichrome). Necrosis: - Congo red, Thioflavin T and immunohistochemical staining negative. - Disruption of cellular architecture - Nuclear pyknosis, karyolysis, or karyorrhexis. Fibrosis: - Fibrillary appearance with small numbers of fibroblasts - Congo red, Thioflavin T and immunohistochemical staining negative. - Positive collagen staining (van Gieson and Masson-trichrome). Congo red staining orange to red with green birefringence under polarized light. Other special stains: crystal violet (amyloid stains purple-violet) and thioflavin T (brilliant orange fluorescence). Immunohistochemistry to identify amyloid deposits and the specific constituents composing the deposits such as the antiβ-light chain antibodies. TEM: non-branching fibrils of indefinite length and 0.7-1 mm diameter that form single to laterally aggregated bundles or interlocking mesh-like ribbons lacking periodicity. Comments: Different types and clinical forms of amyloidosis are known based on the deposition in tissues and organs of various domestic and wild animals, as well as humans. Systemic amyloid deposition can be due to AL-amyloidosis, AA-amyloidosis, or familial amyloidosis. Amyloid substance may be confined at a given area in the body in the form of localized amyloidosis. Amyloidosis involving several tissues and organs throughout the body is referred to as systemic amyloidosis 9 . AA-amyloidosis is the most common form of amyloidosis in NHP. Affected animals present weight loss, diarrhea and malabsorption. Common sites of amyloid deposition in NHP include spleen, lymph nodes, kidney, liver, pancreatic islets and lamina propria or submucosa of the small intestine; especially in aged macaques, often due to chronic enterocolitis or other inflammatory conditions 10 , 11 , 12 . Similar to humans, aging NHP can develop cerebrovascular amyloid angiopathy, within vessels of the cortex and leptomeninges or cerebral plaques composed of amyloid-β, which correlate with neurocognitive decline 13 .

( Figure 1–6 Figure 1. Cynomolgus, Thyroid, Ectopic tissue, thymus, H&E, Low magnification. Figure 2. Cynomolgus, Thyroid, Ectopic tissue, thymus, H&E, High magnification. Figure 3. Cynomolgus, Parathyroid gland, Ectopic tissue, thymus, H&E. Figure 4. Cynomolgus, Pancreas, Ectopic tissue spleen H&E. Figure 5. Cynomolgus, Kidney, Ectopic tissue, adrenal cortex, H&E. Figure 6. Cynomolgus, Kidney, Ectopic tissue, adrenal cortex, H&E. ) Cynomolgus, Thyroid, Ectopic tissue, thymus, H&E, Low magnification. Cynomolgus, Thyroid, Ectopic tissue, thymus, H&E, High magnification. Cynomolgus, Parathyroid gland, Ectopic tissue, thymus, H&E. Cynomolgus, Pancreas, Ectopic tissue spleen H&E. Cynomolgus, Kidney, Ectopic tissue, adrenal cortex, H&E. Cynomolgus, Kidney, Ectopic tissue, adrenal cortex, H&E. Histologically normal tissue in an aberrant location. Comments : Ectopic thymic tissue is commonly identified in or adjacent to the thyroid or parathyroid glands in cynomolgus monkeys. Adrenal cortical tissue can also be identified in or adjacent to the liver 6 . Accessory spleens, also referred to as ectopic spleens, spleniculi, or splenic nodules, are occasionally observed in cynomolgus monkeys. These are often embedded in or attached to the pancreas 7 . Several other ectopic locations for tissue are described under individual organs.

( Figure 28–34 Figure 28. Cynomolgus, Spleen, Pigment, malaria pigment, H&E. Figure 29. Cynomolgus, Lung, Pigment, dust-like with birefringent crystals, H&E. Figure 30. Cynomolgus, Lung, Pigment, peribronchiolar, H&E. Figure 31. Cynomolgus, Liver, Pigment, golden brown, H&E, Low magnification. Figure 32. Cynomolgus, Liver, Pigment, golden brown, H&E, High magnification. Figure 33. Cynomolgus, Skin, Skin/mammary gland, Pigment, tattoo ink, H&E, Low magnification. Figure 34. Cynomolgus, Skin, Skin/mammary gland, Pigment, tattoo ink, H&E, High magnification. ) Cynomolgus, Spleen, Pigment, malaria pigment, H&E. Cynomolgus, Lung, Pigment, dust-like with birefringent crystals, H&E. Cynomolgus, Lung, Pigment, peribronchiolar, H&E. Cynomolgus, Liver, Pigment, golden brown, H&E, Low magnification. Cynomolgus, Liver, Pigment, golden brown, H&E, High magnification. Cynomolgus, Skin, Skin/mammary gland, Pigment, tattoo ink, H&E, Low magnification. Cynomolgus, Skin, Skin/mammary gland, Pigment, tattoo ink, H&E, High magnification. May include the pigment type, if known Endogenous pigments Lipofuscin Melanin Hemosiderin Mite pigment Hematoidin Hemozoin (malaria pigment) Bilirubin Exogenous pigments Carbon (coal dust), anthracosis Tattoo ink The pathogenesis varies based on the type of pigment observed in tissue sections. Pigments are generally colored based substances that arise from either exogenous sources or endogenous processes. Endogenous pigments can be further classified as either hematogenous or nonhematogenous. In most cases, these products develop as a pathological consequence; however some may occur as normal physiological products. Lipofuscin or lipochrome is an insoluble pigmented byproduct of lipid peroxidation 30 . It appears as yellow-brown, finely granular cytoplasmic material in histological sections of tissue. Accumulates in macrophages in the heart, liver, spleen, gastrointestinal tract and brain. Ceroid is a golden acid-fast, sudanophilic pigment, probably a type of lipofuscin, although differing from true lipofuscins by failing to stain with Schmorl ferric-ferricyanide reduction stain 30 . Melanin is a naturally occurring substance formed the oxidation of tyrosine to dihydroxyphenylalanine in melanocytes 30 . The result is a brown-black pigment that is deposited mainly in the skin, meninges, uvea, iris, and reproductive tissues where it is sometimes known as melanosis 31 . Hemosiderin appears as golden brown granules within macrophages. Hemosiderin results when the intracellular protein ferritin binds with free iron 30 . Ferritin is highly abundant in the liver, spleen and bone marrow; therefore these tissues may contain numerous hemosiderin-laden macrophages. Hemosiderin may be observed in a variety of tissues subsequent to hemorrhage. Accumulation of perivascular hemosiderin-laden macrophages is a common finding in the lung 32 . Application of the Perls’ Prussian blue histochemical stain will detect this pigment in tissues. Mite pigment . This pigment results from Pneumonyssus simicola infections 33 . Although it is seen primarily in the lungs in proximity to the mite or bronchial lesions, it may also extend to the tracheobronchial lymph nodes. Histologically macrophages contain a golden brown to black pigment that is refractile. Acid hematin. This pigment develops in tissues fixed in unbuffered formalin 31 . It is most abundant in regions rich in erythrocytes or hemoglobin including regions of hemorrhage and congested blood vessels. Acid hematin presents as a granular to sometimes crystaline black to dark brown substance. The pigment can be bleached with the Kadasewitsch-method to avoid interference with endogenous pigments. Hematoidin is a golden-brown crystalline pigment. Morphologically the crystals may appear in a variety of forms including: 1) thread-like filaments that form star-shaped clusters (Medusa’s head); 2) small, irregular crystals; 3) spheroid crystals; and 4) rhomboid crystals 34 . Closed tissue compartments have low oxygen tension, which are suitable for the formation of hematoidin following degradation of hemoglobin from extravasated erythrocytes. Hemozoin also known as malaria pigment is the result of the Plasmodium parasite converting free heme to the insoluble b-hematin 35 . The pigment can be detected in blood films by light microscopy as intracytoplasmic yellow to green-brown pigment within granulocytes and/or monocytes. Hemozoin is recognized as granular birefringent material with polarization. Bilirubin is a breakdown product of erythrocytes and is also excreted by the hepatocytes as a bile pigment. Grossly it imparts a yellow discoloration to tissues also known as icterus. Bilirubin is a pigment found within bile and is therefore associated with cholestasis and observed histologically as yellow brown plugs or casts within the bile canaliculi. Carbon (coal dust). Anthracosis is the accumulation of inhaled carbon particles by alveolar and interstitial macrophages in the lungs. The carbon pigment may also extend to the tracheobronchial lymph nodes. Tattoo ink. This form of exogenous pigment is localized to the skin and draining lymph node. Dermal macrophages, as well as those located in nearby lymph nodes phagocytize this pigment where it remains indefinitely. Fine granular material: - May resemble bacteria, which can be differentiated by Gram stain. Calcium salts: - Impart a basophilic granular appearance, can be differentiated from pigmented substances using von Kossa histochemical staining. Copper: - May not be readily detected in hematoxylin and eosin stained tissues but can be differentiated from other granular material using rhodanine or rubeanic acid. Hematoidin: - Should be distinguished from other pigments based on its negative Perls’ Prussian blue staining. Light microscopy, special stains (Perls Prussian blue), electron microscopy, polarization

Intracellular or extracellular cocci, bacilli ,coccobacilli, rods or other less common forms visualized by light microscopy. Often associated with inflammation, tissue degeneration, and/or necrosis and can be obscured by cellular debris. Histochemical methods suchs as Gram, Acid Fast or Silver (Warthin-Starry) stains aid in visualization. Culture results and molecular diagnostics.aid in diagnosis. Necrosis, of other known or unknown causes. inflammation, of other known or unknown causes. Comments: Primary bacterial infections are rare in laboratory macaques. However, like many other mammalian species, NHP are susceptible to opportunistic infection. A complete review of bacterial lesions in macaques is beyond the scope of this manuscript. The reader is referred to the literature. Relevant emerging and reemerging bacterial pathogens of laboratory NHP are reviewed in Bailey and Mansfield 2010 14 .

Syndromes that may occur in the nonhuman primates include fatal fasting macaque syndrome, hemophagocytic syndrome and phospholipidosis, which are described below. The syndrome should not be recorded as a finding but should be mentioned as an observation comment in the data capture system and/or in the pathology narrative. The individual findings that together make the syndrome should be recorded seperately using INHAND terms where possible. ( Figure 35–37 Figure 35. Cynomolgus, Kidney, Tubular vacuolation, fatal fasting syndrome, H&E. Figure 36. Cynomolgus, Liver, Vacuolation, fatal fasting syndrome, H&E. Figure 37. Cynomolgus, Adipose tissue, Necrosis and inflammation, fatal fasting syndrome, H&E. ) Cynomolgus, Kidney, Tubular vacuolation, fatal fasting syndrome, H&E. Cynomolgus, Liver, Vacuolation, fatal fasting syndrome, H&E. Cynomolgus, Adipose tissue, Necrosis and inflammation, fatal fasting syndrome, H&E. Obese macaque syndrome; fatal fatty liver-kidney syndrome in obese monkeys; fatal fatty liver syndrome Although still poorly understood, the pathogenesis of this syndrome is considered multifactorial 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 . Stress-inducing conditions such as transportation, recaging, changes in husbandry and others, may cause decreased food consumption up to anorexia and subsequent rapid and/or repeated weight variation/losses in previously overfed/obese adult monkeys. This finally causes a negative energy/nutritional imbalance. The subsequent mobilization of lipid and fatty acids from adipose tissue throughout the body disrupts lipid metabolism and finally causes lipid accumulation in several organs, especially the liver and kidneys. Raised level of circulating triglycerides may be associated with morphological changes, but blood chemistry profile generally does not suggest liver failure. Macroscopically, the liver and kidneys may appear enlarged and pale, and multiple pale-tan foci can be observed in the fat tissue of the abdominal cavity at necropsy. Various degrees of cellular swelling due to cytoplasmic vacuolation are noted in several organs especially the liver (hepatocytes) and kidneys (epithelial lining of the proximal convoluted tubules). The vacuoles are of variable size but generally large and round-oval and contain lipids. They appear empty in HE-stained section and are positive to Oil Red-O stain and other lipidic special stains. Acute degenerative and inflammatory changes can be observed in the pancreas, such as decreased zymogen content in acinar cells, acinar cell necrosis, acute cell-type infiltration and steatonecrosis in the surrounding fat tissue. In some cases, adrenocortical hyperplasia and vacuolation, and thyroid follicular enlargement associated with flattening of the follicular epithelium have been reported in affected monkeys 37 . Xenobiotic-induced cellular degeneration in the liver and kidneys (with fatty change), pancreas and fat tissue. Traumatic events causing degenerative/inflammatory changes in fat tissue. Pancreatic cell degeneration associated with inflammation: - Adenoviral-related pancreatitis has been reported in M. mulatta 44 . Hypothyroidism and diabetes mellitus may be associated with fatty change in the liver. Serous atrophy of fat: - Generally not associated with steatonecrosis and lipidic accumulations in other organs. Special stains for lipid content such as Oil Red-O and Sudan Black. Comments: This syndrome has been reported in several species of non-human primates such as M. fascicularis, M. mulatta, M. arctoides, M. nemestrina and M. radiata 36 , 37 , 39 , 42 . It has been generally described in adult monkeys, and females seem to be predisposed. HPS HPS is a nonneoplastic condition characterized by proliferation of activated macrophages 45 . HPS is mediated by hyperinflammatory state comprising uncontrolled CD8+ T-cell activation leading to marked production by interferon-gamma (IFN-γ) 46 . This is followed by histiocytic proliferation and production of cytokines including tumour necrosis factor-alpha (TNF-α), IL-1, IL-6, IL-10, IL-18, and granulocyte colony-stimulating factor (G-CSF) 47 . Together this leads to multiorgan dysfunction. Bone marrow cytopenia Increased numbers of hemophagocytic macrophages in peripheral tissues. Epstein-Barr infections: - Often present with monocytosis, and Epstein-Barr Nuclear Antigen (EBNA) can be detected in sections of tissue by immunohistochemistry. Simian Parvovirus infections: - May present with a normocytic, normochromic, nonregenerative anemia, or bone marrow dysmaturation with the presence of large intranuclear inclusion bodies within bone marrow hematopoietic cells 14 . Infection with Simian Retrovirus: - A betaretrovirus, presents with cytopenic abnormalities that includes anemia, leukopenia and thrombocytopenia 48 . Earlier phases of infection may consist with bone marrow hyperplasia involving granulocytic and erythroid lineages 49 ; however late stage infection typically present as severe pancytopenia associated with bone marrow depletion. Light microscopy, complete blood counts, clinical history, viral titers Comments: The veterinary literature is lacking information regarding HPS in monkeys. A recent report described HPS in rhesus 50 associated with SRV infection. Much of what is known about hemophagocytic syndrome (HPS) comes from the human medical literature and scant reports in the veterinary medical literature including cats and dogs 51 , 52 . HPS is an often-fatal nonneoplastic proliferative disorder characterized by activated macrophages, multi lineage cytopenias in the bone marrow and profound phagocytosis occurring in the bone marrow and liver. Both genetic 53 and acquired 54 forms with Epstein-Barr infections have been reported. Comments: Similar to the other laboratory species, the macaque is susceptible to phospholipidosis 55 . Drug-induced phospholipidosis is characterized by cytoplasmic vacuolation seen in multiple tissues on light microscopic examination. The descriptive term vacuolation is the preferred terminology. A diagnosis of phospholipidosis is confirmed by demonstrating the accumulation of lamellar bodies in lysosomes on TEM.

( Figure 14–16 Figure 14. Cynomolgus, Skin, Intranuclear inclusions, simian varicella virus, H&E. Figure 15. Cynomolgus, Bone marrow, Intranuclear inclusions, simian parvovirus, H&E. Figure 16. Cynomolgus, Bone marrow, Erythroid blasts with viral inclusions, simian parvovirus, H&E. ) Cynomolgus, Skin, Intranuclear inclusions, simian varicella virus, H&E. Cynomolgus, Bone marrow, Intranuclear inclusions, simian parvovirus, H&E. Cynomolgus, Bone marrow, Erythroid blasts with viral inclusions, simian parvovirus, H&E. Viral infection Cytoplasm; intranuclear Intranuclear inclusions are typically round, may partially or almost completely fill the nucleus leading to margination of host nuclear chromatin. Contents of intranuclear inclusion bodies vary from eosinophilic, amphophilic to basophilic and may be granular or flocculent. Intracytoplasmic inclusions are typically eosinophilic, round to oval, homogenous, vary in size and occur as single or multiple structures in the cytoplasm. Comments: Laboratory cynomolgus monkeys are the natural hosts for several well-described retroviruses, herpesviruses (e.g. cytomegalovirus, lymphocryptovirus and simian varicella virus), small DNA viruses (parvovirus and SV40) and adenovirus. The majority of viral infections in laboratory cynomolgus monkey colonies are clinically silent, since host-virus coevolution has allowed for lifelong viral persistence, often with a limited detrimental effect on the immunocompetent natural host. While complete elimination of infection is not possible, present strategies for pathogen control in laboratory non-human primate colonies including pre-study viral screening for known NHP retroviruses, Herpes B and measles virus have effectively decreased the likelihood that viral infection will confound toxicology study results. However, these viruses do pose a risk of opportunistic infection under conditions of immunosuppression or immunomodulation 17 , 20 , 21 . Cytomegalovirus, lymphocryptovirus and adenovirus are among the most common latent viral infections in macaques 22 . Viral inclusions should be recorded as “inclusion, intranuclear and/or cytoplasmic” when observed, further specifications can be provided in observation comments or the pathology narrative. Inclusions may be recorded as “present” or graded, depending on pathologist discretion. Non-viral causes of inclusion bodies such as those due to lead exposure are less common, but have been reported in macaques 23 and should be considered.

Nonhuman primates used within routine toxicology studies may be subclinically infected with a variety of infectious organisms acquired from their breeding colony or holding facilites en-route to the testing facility. Evidence of these organisms or infections may be an incidental finding at necropsy, or they may become clinically activated or be transmitted while the animal is on a nonclinical study, particularly when one or more animals become immunologically compromised. Infectious agents seen within macaques include fungal, parasitic, bacterial and viral diseases, which should be recorded within the data capture system at a high level under the visible etiologic agent (fungus, parasite, bacteria) or inclusions (virus), and not under individual organisms or disease diagnosis (e.g. Plasmodium spp.). Any pathologic response(s) or finding(s) associated with the infectious agent should be recorded separately using appropriate INHAND terms where possible. Additional diagnostic tools such as in situ hybridization and immunohistochemistry can aid in identifying and investigating the impact of known or novel NHP pathogens on nonclinical studies 22 . A complete review of infectious agents in macaques is beyond the scope of this manuscript. The reader is referred to the extensive literature and to the individual INHAND terms (fungus, bacteria, parasite, inclusions).

Neutrophil, eosinophil, lymphocyte, plasma cell, mast cell, macrophage, mononuclear cell, lympho-histiocytic, lymphoplasmacytic, mixed cell Inflammatory cell infiltrate; aggregates, inflammatory cell The pathogenesis of inflammatory cell infiltrate is undetermined; however it is hypothesized that infiltrates may occur as a self-limiting immune response or as part of a tissue repair process. Usually manifests as variably sized focal to multifocal aggregates of inflammatory or immune cells in diverse tissues and body organs In general, lack of concomitant edema, congestion, hemorrhage, necrosis, and/or fibrosis, helps to differentiate from active or resolving inflammation Differential Diagnoses Inflammation: - In addition to inflammatory cell infiltrates, additional features include edema, tissue damage, hemorrhage, and/or fibrosis Mucosa-associated lymphoid tissue (MALT) - MALT structures are normal anatomical features of the mucosal immune system that are histologically identified as nonencapsulated subepithelial lymphoid follicles with parafollicular areas and overlying specialized follicle-associated epithelium (FAE). The reader is referred to the hematopoietic and lymphoid system section for a detailed discussion of MALT and MALT-like structures such as duct-associated lymphoid tissue (DALT). Infiltrates are easily identifiable in H&E stained tissue sections Immunohistochemistry may be performed to identify the lineage of leukocytes involved Comments: Mononuclear cell infiltrates in various tissues are among the most common spontaneous pathology findings in control cynomolgus monkeys 6 , but need to be differentiated from naturally occurring mucosa-associated lymphoid tissue (MALT) and duct-associated lymphoid tissue (DALT). Within the conjunctiva, mononuclear or lymphoid cell infiltrates may not be indicative of an inflammatory process but may instead represent conjunctiva-associated lymphoid tissue (CALT), a normal structure of the conjunctival mucosa. With chronic antigenic stimulation, lymphoid hyperplasia may occur in CALT structures.

Granulomatous, mixed cell, neutrophil, eosinophil, lymphocyte, mast cell, macrophage, mononuclear cell “-itis”, specific to the organ affected, e.g. encephalitis, gastritis, etc. Inflammation in a tissue may be evoked as a response of resident or systemic inflammatory cells in response to nocuous stimuli. Presence of a single cell lineage or multiple types of leukocytic infiltrates in tissues along with other features of tissue damage and/or repair such as edema, congestion, hemorrhage, necrosis, and fibrosis. Based on the cell type, inflammation can be classified as suppurative (mainly neutrophilic), mononuclear or mixed cell (consisting of neutrophils, eosinophils, lymphocytes, plasma cells, and macrophages), granulomatous (mainly consisting of macrophages with or without multinucleated giant cells), or pyogranulomatous (consisting of both macrophages and neutrophils). Infiltrate, [insert appropriate cell type] Abscess Leukocytic neoplasms Inflammation is easily identifiable in H&E stained tissue sections Immunohistochemistry may be performed to identify the lineage of leukocytes involved Comments: Inflammation can occur as a normal host defense or a repair process in response to tissue injury. It should be differentiated from an “inflammatory cell” infiltrate as the latter does not have other features of tissue damage and/or repair such as edema, congestion, hemorrhage, necrosis, and fibrosis. Inflammation is one of the most common spontaneous pathology findings reported in control cynomolgus monkeys 6 . Differentiation of systemic tumors e.g. by accumulation of “inflammatory cells” without a discernible stimulus, morphology of cells such as mitosis or pleomorphism. For organ specific, detailed characteristics refer to the description in the respective organ section. Various approaches to further characterize inflammation are used by pathologists, including chronicity (peracute, acute, subacute, etc.), location (perivascular, peribiliary, etc.), and others. To implement a descriptive terminology, the use of the term “inflammation” is preferred over the “-itis” terminology, and the indication of the predominant cell type(s) in the diagnosis is recommended over the conventional use of chronicity, e.g.: Inflammation, neutrophil Inflammation, lymphocyte Inflammation, plasma cell Inflammation, monocyte/macrophage When applicable the terms are combined, e.g.: Inflammation, lympho-plasmocytic Inflammation, lympho-histiocytic Further description by location and distribution is recommended.

( Figure 10–11 Figure 10. Cynomolgus, Lymph node, submandibular, Extramedullary hematopoiesis, H&E, Low magnification. Figure 11. Cynomolgus, Lymph node, submandibular, Extramedullary hematopoiesis, H&E, High magnification. ) Cynomolgus, Lymph node, submandibular, Extramedullary hematopoiesis, H&E, Low magnification. Cynomolgus, Lymph node, submandibular, Extramedullary hematopoiesis, H&E, High magnification. Increased Varying proportions of marture and immature myeloid, erythroid and megakaryocyte lineages, depending on the etiology. Common extramedullary hematopoiesis sites. - Medullary cords in lymph nodes - Sinusoids in the liver - Adrenal cortex - Renal interstitium Infiltrate [insert appropriate cell type]. - Infiltration of mature leukocytes into the tissue. - Presence of leukocytes but no other histologic criteria of inflammation. Hematopoietic neoplasms - Clonal disorders involving hematopoietic blast cells of myeloid lineage and their progenitors. - >20% blasts in bone marrow, neoplastic cells in circulation or infiltrating parenchyma tissue (refer to Hematolymphoid General Terms). Lymphoma - Distinguished by cell morphology, tissue distribution and clonality. Comments: Extrameduallary hematopoiesis (EMH) is rarely noted in the spleen in healthy control cynomolgus macaques 7 , yet is commonly seen in the mandibular lymph nodes, medullary cords as well as sporadically in other lymph nodes. When bone marrow is the target of toxicity, EMH can be seen in tissues such as the adrenal gland, liver (sinusoids) and kidneys (interstitium) 15 . Extramedullary hematopoiesis may be recorded when it is increased above background levels and the modifier “increased” may be used in such cases.

( Figure 17–27 Figure 17. Cynomolgus, Diaphram, Parasitic granuloma, cestode, H&E, Low magnification. Figure 18. Cynomolgus, Diaphram, Parasitic granuloma, cestode, H&E, High magnification. Figure 19. Cynomolgus, Cecum, Parasite, Balantidium coli , H&E. Figure 20. Cynomolgus, Lymph node, Parasitic granuloma, H&E. Figure 21. Cynomolgus, Lymph node, Parasite, remnants H&E. Figure 22. Cynomolgus, Lymph node, mesenteric, Parasite, H&E. Figure 23. Rhesus, Cecum, Parasite, nematode H&E. Figure 24. Rhesus, Brain, Parasitic granuloma, Baylisascaris spp. H&E. Figure 25. NHP, Rectum, Parasite, Balantidium coli and Trichuris spp. H&E. Figure 26. NHP, Skeletal muscle, Parasite, Sarcocystis H&E. Figure 27. Cynomolgus, Skin, Parasite, acarid H&E. ) Cynomolgus, Diaphram, Parasitic granuloma, cestode, H&E, Low magnification. Cynomolgus, Diaphram, Parasitic granuloma, cestode, H&E, High magnification. Cynomolgus, Cecum, Parasite, Balantidium coli , H&E. Cynomolgus, Lymph node, Parasitic granuloma, H&E. Cynomolgus, Lymph node, Parasite, remnants H&E. Cynomolgus, Lymph node, mesenteric, Parasite, H&E. Rhesus, Cecum, Parasite, nematode H&E. Rhesus, Brain, Parasitic granuloma, Baylisascaris spp. H&E. NHP, Rectum, Parasite, Balantidium coli and Trichuris spp. H&E. NHP, Skeletal muscle, Parasite, Sarcocystis H&E. Cynomolgus, Skin, Parasite, acarid H&E. Distinct helminth or protozoal morphologic features are often visualized by light microscopy. Host response to tissue invasion can vary from neutrophilic, eosinophilic to granulomatous inflammation in miliary to mass-like nodular lesions. Heminth tissue invasion commonly presents as organized, focal, well demarcated, nodular lesions. Parasite profiles are often encapsulated by fibroblasts, lymphocytes, and plasma cells, with limited to no active inflammation. Epithelioid macrophages and multinucleated giant cells (Langhans or foreign body types) are often present. Granulomas may distort normal tissue architecture. Comments: Many parasitic pathogens are endemic in NHP colonies and remain clinically inapparent in immunocompetent animals. Treatment with anti-helmintics prior to importation and during quarantine has significantly reduced or eliminated many parasitic infestations; however, a low incidence is still noted on post mortem examination. In healthy immunocompetent animals, most parasitic infections present as non-invasive disease with low numbers of organisms present. For example, Balantidium coli is commonly identified within the gastrointestinal tract lumen, specifically in the cecum and colon without mucosal invasion or alteration. Similarly, Sarcocystis spp. cysts are occasionally observed in skeletal muscle, and less frequently in the cardiac and smooth muscle 24 in the absence of active disease. Common helminths identified in cynomologus macaques include species of Strongyloides , Oesophagostomum , Anatrichosoma , Trichostrongylus , Trichuris, Gongylonema , larval stages of nematodes (ascarids, spirurids), cestodes ( Echinococcosis spp. ) and trematodes (the lung fluke – Paragonimus westermanii ) 25 . Cerebral Baylisascaris larva migrans has also recently been reported in cynomolgus macaques 26 . In most instances of infection, only a few organisms are observed and tissue damage is limited with parasitic granulomas being a common presentation. Protozoal infections encountered in macaques include Cryptosporidium parvum, Enterocytozoon bieneusi, Plasmodium spp., Trichomonas spp., Acanthamoeba spp., Toxoplasma gondii 27 and Trypanosoma cruzi 28 . Toxoplasma gondii systemic infection can be seen spontaneously in macaques 29 and is characterized by necrosis and inflammation in the brain, lungs, mesenteric lymph node, liver and/or heart. Trypanosoma cruzi infection can be the cause of inflammation in the heart and/or brain of cynomolgus monkeys. Lastly, pulmonary acariasis ( Pneumonyssus simicola ) was once prevalent, yet due to current pathogen control measures is now rarely identified in laboratory colonies. A complete review of parasitic lesions in macaques is beyond the scope of this manuscript. The reader is referred to the literature.