Sampled: 06/05/2017 Paramus, NJ Received: 06/13/2017 (201) Finalized: 07/27/2017. Anatomic Pathology

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1 Owner: 240 Farrier Road,, Ithaca, NY Ph: Fax: Page 1 of 7 Oradell Animal Hospital Inc - (3382) Dr John Lucy 580 Winters Ave Sampled: 06/05/2017 Paramus, NJ Received: 06/13/2017 (201) Finalized: 07/27/2017 Anatomic Pathology Department of Biomedical Sciences Phone: Fax: Body, Whole Addendum (Pathology) Addendum Consultation Results: Dr. Andrew D. Miller, DVM, DACVP, a veterinary pathologist, was consulted for his opinion on this case. Dr. Miller agrees that the most significant changes are the granulomatous lymphadenitis noted in the mesenteric lymph node, and the pleuritis in the lung sections. The lymphadenitis is consistent with feline infectious peritonitis (FIP), based on the positive immunoreactivity of macrophages to feline coronavirus antigen. The rare positive immunoreactivity noted in the pleura appears to be equivocal, and is not consistent or striking enough to warrant a firm diagnosis of coronavirus within the pleural tissue. However, this does not exclude an FIP-related pleuritis, as coronavirus may be present in other areas not examined on histology. A differential diagnosis of the pleuritis, given its marked fibrinosuppurative component, would be a bacterial pleuritis. Due to the severe tissue artifact related to autolysis and freeze-thaw, determining the source of bacterial infection is not possible. It is possible that systemic immune suppression related to FIP predisposed this animal to secondary bacterial infection. Culture of frozen lung tissue is available if requested. However, culture may not yield a positive result, and if it does, it will only indicate the underlying bacteria involved; it cannot elucidate the source of the infection. The remaining changes are expected findings and are largely incidental. In addition to the lesions described in the original report, an adrenal cortical adenoma, composed of proliferative, well-differentiated adrenal cortical cells surrounded by a thin capsule, was noted. This lesion is incidental, and often seen in aged animals. The renal adenocarcinoma is a potentially significant lesion; however, there is enough remaining renal parenchyma unaffected by the tumor that it likely did not cause renal insufficiency, and there is no observed metastasis in any of the other examined tissues. The chronic interstitial nephritis may have caused clinical renal signs, but this is an expected lesion in aged cats, the etiology of which is uncertain. Finally, rare, well demarcated, lightly basophilic, homogenous, round deposits (Lafora bodies) are noted in multiple sections of brain. These are age-related changes with no pathologic significance. Dr. Teresa Southard, DVM, PhD, DACVP, a veterinary pathologist, was also consulted and concurs with these findings.

2 Page 2 of 7 Addendum Comments: A Masson's trichrome histochemical stain for collagen confirms mild pancreatic fibrosis. A panel of histochemical stains does not reveal bacterial or fungal organisms in areas of pleuritis. Histochemical Stain Results:Histochemical staining of sections of lung (slide 4) reveals the following: Gram for detection of bacteria: No bacterial organisms are observed in the examined sections. Gomori's methenamine silver (GMS) for detection of fungi: No fungal organisms are observed in the examined sections. Modified Steiner silver for detection of argyrophilic bacteria: No argyrophilic organisms are observed in the examined sections. A Masson's trichrome histochemical stain of a section of pancreas (slide 6) reveals focally coalescing tracts of mature deeply basophilic collagen coursing between multiple lobules. Necropsy, Final Final Report Final Diagnosis: Feline infectious peritonitis Thyroid adenomatous hyperplasia Hepatic biliary cystadenoma Hepatic lipidosis Renal adenocarcinoma Final Comment: Histologic changes and immunohistochemical staining confirm the clinical diagnosis of feline infectious peritonitis (FIP) in this cat and likely account for the demise of this cat. Feline infectious peritonitis (FIP) is a fatal, progressive disease resulting from mutations of feline enteric coronavirus leading to systemic viral infection of macrophages. In this case FIP viral antigen is demonstrated within macrophages primarily in the mesenteric lymph node, but also in the pleural exudate, and likely account for the pleural effusion seen on gross examination. Histochemical stains to rule out the presence of bacterial and fungal agents in areas of pleuritis are pending and results will follow in an addendum to this report. Histological evidence of thyroid gland adenomatous hyperplasia, one of the most common proliferative diseases resulting in

3 Page 3 of 7 hyperthyroidism in cats, confirms the clinical diagnosis of hyperthyroidism leading to thyrotoxic hypertrophic cardiomyopathy seen on gross examination. The large liver mass seen on gross examination has histologic features consistent with a biliary cystadenoma, a common, benign neoplasm of cats. Additionally, hepatic lipidosis, a common sequela to prolonged anorexia is confirmed. The renal mass is a primary renal adenocarcinoma rather than a lymphoma as previously suggested from the gross examination. This renal adenocarcinoma is relatively small and unlikely to have clinical significance. The chronic lymphoplasmacytic interstitial nephritis with glomerulosclerosis is considered an incidental age-related finding, while the multifocal renal cortical infacts might be secondary to thromboembolism. A mild focus of fibrosis within the pancreas likely represents scarring from a previous pancreatitis; however, there is no evidence of acute pancreatitis in the sections of tissue examined. A histochemical stain to assess the degree of collagen deposition within the pancreas is pending and results will follow in an addendum to this report. Histologic Findings: Lungs: Moderate, generalized, acute fibrinous pleuritis Mesenteric lymph node: Severe, generalized granulomatous lymphadenitis with lymphoid depletion and fibrosis Right thyroid gland: Generalized adenomatous hyperplasia Left thyroid gland: Focal adenomatous hyperplasia Pancreas: Mild, focal pancreatic fibrosis Kidney: 1. Moderate, generalized, chronic lymphoplasmacytic interstitial nephritis with glomerulosclerosis 2. Renal adenocarcinoma 3. Multifocal, chronic renal cortical infarcts Liver: 1. Biliary cystadenoma 2. Moderate, multifocal, centrilobular hepatic lipidosis Histologic Description: The following tissues are examined: Slide 1: Liver, gallbladder

4 Page 4 of 7 Slide 2: Kidney, urinary bladder Slide 3: Spleen, adrenal gland, right thyroid gland, bone marrow Slide 4: Lung, left thyroid gland Slide 5: Heart Slide 6: Stomach, duodenum, pancreas, jejunum Slide 7: Cecum, colon, mesenteric lymph node Slide 8: Brain Slide 9: Brain Slide 10: Brain Slide 11: Eye Histopathologic changes are detected in these tissues: Liver (Slide 1, 3 sections): In two sections, the parenchyma is expanded and replaced by a well-demarcated, unencapsulated, non-infiltrating mass composed of low cuboidal and flattened polygonal cells arranged in tubules, acini, and cystic spaces on a robust, fibrous stroma. These cells have moderate to scant eosinophilic cytoplasm and central, round, basophilic nuclei with finely stippled chromatin. Anisokaryosis and anisocytosis are mild. Hepatocytes of the remaining hepatic parenchyma are expanded by large, round, well-demarcated, cytoplasmic clear vacuoles (lipidosis). Centrilobular hepatocytes are most frequently affected by lipidosis. Kidney (Slide 2, 3 section): In one section, the cortex is expanded by a large, well-demarcated, unencapsulated, non-infiltrating, moderately cellular nodule that comprises 50% of the section. This mass is composed of low cuboidal, polygonal cells arranged in loose trabeculae and rudimentary tubules on a robust fibrous matrix. Occasional tubules, mostly at the periphery, are dilated and filled with necrotic debris. The polygonal have central, dark nuclei with coarse chromatin and moderate, eosinophilic cytoplasm. Anisokaryosis and anisocytosis are mild. The adjacent cortical tissue is severely displaced and compressed by this mass. In other sections, scattered glomeruli are shrunken and irregular, with distinct loss of capillary loops (sclerosis). The Bowman s capsule of many glomeruli is mildly thickened. Throughout all sections, low to moderate numbers of lymphocytes and plasma cells infiltrate and expand the interstitium, forming loose cords and tight clusters. The interstitial fibrous connective tissue is generally thickened and expanded. In multiple sections, small, sharply demarcated regions of the cortex are shrunken, with tubular loss and close apposition of glomeruli and increased fibrous connective tissue (infarct). Right thyroid gland (Slide 3, 1 section): The entire section is composed of irregular tubules and acini that are variably dilated and filled with colloid, forming variably sized, dilated and folded follicles. In some areas, these tubules are markedly dilated, forming cystic follicles. These tubules and acini are lined by low cuboidal polygonal cells with moderate amounts of eosinophilic cytoplasm and central, dark, round nuclei with coarse chromatin. Adrenal gland (Slide 3, 1 section): The medulla is obscured by an expansile, well-demarcated, densely cellular nodule composed of large polygonal cells with abundant, variably sized, clear cytoplasmic vacuoles arranged in irregular islands and tight packets. These cells have small, dark nuclei with coarse chromatin. Anisokaryosis and anisocytosis are minimal.

5 Page 5 of 7 Bone marrow (Slide 3, 1 section): All lineages are represented. The myeloid:erythroid ratio is approximately 3:1 (myeloid hyperplasia). There are adequate megakaryocytes. Lungs (Slide 4, 4 sections): The pleural surfaces of all sections are generally expanded by a thick layer composed of fibrin and degenerate neutrophils. In some areas, this infiltrate extends into the underlying parenchyma. An immunohistochemical stain for feline enteric coronavirus antigen (feline infectious peritonitis virus) reveals cytoplasmic immunostainning of rare, small aggregates of macrophages within the pleural exudate. Left thyroid gland (Slide 4, 1 section): Approximately 70% of the examined section is slightly shrunken, with small follicles and mild depletion of colloid. In one area, comprising the remaining 30% of the parenchyma, the follicular cells are expanded and numeorus, forming loose tubules and variably sized, often folded follicles similar to those in the right thyroid. Pancreas (Slide 6, 1 section): A poorly demarcated region of the pancreas is expanded by large, thick tracts of coarse collagen, admixed with shrunken, irregular ducts. The exocrine acini are effaced and replaced in this area by the fibrosis. Mesenteric lymph node (Slide 8, 2 sections): The cortex is hypocellular with depleted follicles and replaced by abundant, hyalinized, fibrillar, eosinophilic material. The cortex and medulla are generally dissected by large, anastomosing tracts of fibrosis, obscuring the cortico-medullary junction and effacing normal sinus architecture. Immunohistochemical stain for feline enteric coronavirus antigen (feline infectious peritonitis virus) reveals cytoplasmic immunoreactivity of frequent, scattered, large aggregates of macrophages. Necropsy, Gross Gross Report Preliminary Diagnosis: Thyroid gland adenoma and contralateral atrophy Thyrotoxic cardiomyopathy Biliary cystadenoma Renal lymphoma Pleural effusion Preliminary Comment: The most significant gross finding is a severely enlarged left thyroid gland with concurrent atrophy of the right thyroid. Given the history of hyperthyroidism, this is consistent with a adenomatous hyperplasia or adenoma of the thyroid gland and excessive production of thyroxine. The increased thyroxine production by right thyroid results in a negative feed-back loop on the pituitary which in turn reduces thyroid-stimulating hormone, leading to atrophy of the contralateral gland. Autonomy of thyroid production in thyroid glands is caused by dysregulated proliferation of the follicular cells. In cats, the most common manifestation of this is adenomatous hyperplasia and overt adenomas. There are moderate changes in the heart consistent with pathologic hypertrophy. In cats, thyrotoxic hypertrophic cardiomyopathy is a common sequela to hyperthyroidism. The pleural effusion might represent an early manifestation of

6 Page 6 of 7 congestive heart failure related to the cardiomyopathy; however, an inflammatory process cannot be ruled out. The mass in the kidney is most consistent with lymphoma, one of the most common neoplasm of cats. While typically a metastatic lesion, primary renal lymphoma can occur in cats. The large liver mass is most consistent with a biliary cystadenoma, which are common, benign neoplasms of cats. Additionally, hepatic lipidosis is a common sequla to prolonged anorexia. The colonic impaction is relatively moderate, and might have cause some discomfort. Accessory spleens is an incidental finding that might be a congenital anomaly or a sequela to healing from previous splenic insult. Freeze-thawing of this cat prior to necropsy might have obscured subtle gross lesions; however, the gross findings in this cat are not consistent with feline infectious peritonitis. The significance of a positive coronavirus PCR assay result from the thoracic effusion is uncertain. Histopathologic examination is pending and results will follow in a final report. Gabor LG, et al (1998). Clinical and anatomical features of lymphosarcoma in 118 cats. Aust Vet J 76(11): Adler R and Wilson DW (1995). Biliary cystadenomas of cats. Vet Pathol 32(4): Gross Findings: Right thyroid gland: Adenoma Left thyroid gland: Moderate atrophy Heart: Moderate thyrotoxic hypertrophic cardiomyopathy Lung, thoracic cavity: Moderate, generalized, sub-acute fibrinous pleural effusion Peritoneum: Moderate sub-acute peritoneal effusion Left kidney: Lymphoma, presumptive Right and left kidneys: Moderate, generalized, chronic interstitial nephritis and fibrosis Liver: 1. Biliary cystadenoma 2. Mild, generalized hepatic lipidosis Colon and rectum: Moderate fecal impaction Spleen: Accessory spleen Gross Description: Examined is the body of frozen and thawed, 4.1 kg, 12-year old, spayed female, brown tabby Domestic shorthair cat, with a

7 Page 7 of 7 body condition score of 5/9 (Purina scale) and moderate postmortem autolysis. Around the nares and left aspect of muzzle is 2 ml of dark red gelatinous material (partially dried blood). Most of the teeth are missing with teeth 103, 104, 404 and 308 remaining (modified Triadan system). Teeth 103 and 104 are loose in the dental alveoli. All teeth are coated by a thick layer of pale tan to white, hard, amorphous material mostly at the base of the crown (calculus). There is a 4 cm circumferential shaved area on left antebrachium. Adhered to this is a piece of 2 cm circumferential white tape. There is a 7 x 8 cm shaved area on the lateral aspect of the right thorax. The ventral abdomen has a 10 x 14 cm shaved area slightly off center extending 6 cm laterally on the left from the umbilicus and 4 cm laterally on the right abdomen. There is an approximately 0.1 x 0.1 cm round skin defect approximately 2 cm cranial dorsal to the caudal mammary papilla (post mortem renal tru-cut biopsy). This defect is surrounded by faint red crust radiating 0.3 cm from the defect in all directions (dried blood). The abdominal cavity contains approximately 25 ml of orange-red, watery, turbid fluid (peritoneal effusion). On the diaphragmatic, cranial surface of the right lateral liver lobe is a 6 x 5 x 3 cm, well-demarcated, raised, locally extensive, soft, mottled tan to pink, slightly umbilicated mass. On cut section, the mass is slightly bulging, pale tan to white, and cystic (biliary cystadenoma). The liver is generally pale, with an enhanced reticular pattern. There are two spleens, a 7.5 x 3.0 cm and a 7.0 x 1.8 cm (accessory spleen). The larger spleen has a 1.0 x 0.3 cm, raised pink-red, friable, stringy, strand adhered to the serosal surface (fibrin). The aborad descending colon and rectum are moderately distended up to 2.8 cm in diameter with firm, dark green material extending 14 cm cranial from the rectum (fecal impaction). Proximal to this impaction, the contents are dark green and pasty, with green staining of the mucosa. The right kidney is moderately, diffusely pale tan-pink with a 1.0 x 0.2 cm focal depression and white-pink thickening of the capsule that does not peel off the cortex (nephritis). The left kidney is similar to the right kidney with a single round, well-demarcated, bulging 4 mm diameter nodule expanding the cortex. On cross-section, the nodule is pale tan to white, and soft to slightly firm. The renal pelvis is mildly irregular and scalloped. Radiating from the pelvis and dissecting the medulla are approximately a dozen thin, pale, firm striations. The cortex is slightly thinned, with an irregular subcapsular contour. The thoracic cavity contains approximately 15 ml of a watery, red, fluid. The visceral pleura of the lungs, the cranial and caudal mediastinum, and parietal pleura have a ground-glass dull surface with moderate, variable thickening and multiple, slightly adhered strands of a stringy, friable, yellow to tan material (fibrinous effusion). The right thyroid gland is 2.5 x 1.2 x 0.5 cm and weighs 2 g. The left thyroid gland is 1.2 x 0.5 x 0.3 cm. The heart weighs 22 g. The left ventricular free wall is 7 mm and the right ventricular free wall is 2 mm. There is a firm pale pink band extending from a left aortic semilunar cusp to the leaflet of the mitral valve (endocardial fibrosis).