NECROPSY REPORT Accession Number: NA2009-31 Hospital Name: Some Animal Hospital Hospital Address: N. Los Angeles Street, Los Angeles CA Hospital Telephone #: 555-555-4900 Antech ID#: 00000 Doctor’s Name: Dr. Jones Owner’s Name: Wilson Pickett Pet’s Name: Delilah Sex: F/S Age: 2 years Species: Dog Weight: 20 lbs

HISTORY (LIMITED) Delilah was admitted for a possible squamous cell carcinoma of the face. On physical examination it was found that Delilah was very emaciated and in poor condition. She was euthanized on 9/26/09. The body was frozen prior to submission for necropsy.

GROSS EXAMINATION The animal submitted for necropsy is a 2 year old female spayed Cocker Spaniel canine. The animal measures approximately 27 inches from nose tip to tail-base and weighs approximately 20 pounds. The hair coat is long, thick with mats, and is diffusely black in color (Figures 1 - 2). Body condition score is approximated at 1.5 out of 5. Figure 1 Figure 2 1

Integumentary and Musculoskeletal System: The hair is shaved over the radius of the front right forelimb. The left half of the nasal planum is ulcerated, characterized by a moist red appearance, and there is a 0.5 cm circular ulcer on the right upper lip (Figure 3). The first digit of the right front paw (Figure 4) and the first digit of the left hind paw (Figure 5) are characterized by similar ulcerative masses which have a moist reddened appearance. A similar mass is noted on the tail. The subcutaneous tissue of the thoracic and abdomen feature multifocal to coalescing firm, smooth, nodular, subcutaneous, yellow and white masses that ranged from 1 mm to 1.5 cm in diameter (Figure 6 - 7). The right axillary lymph node is markedly enlarged (4 x 3cm) and has an irregular, cream to yellowish appearance on cut surface (Figure 8). Gross Diagnoses: 1) Moderate emaciation 2) Multiple cutaneous, digital, and nasal ulcerations and ulcerative masses 3) Marked multifocal subcutaneous masses 4) Marked right axillary lymphadenopathy

Figure 3 Figure 4

Figure 5 Figure 6

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Figure 7 Figure 8

Eyes: Figure 9 The third eyelid of the right eye has a raised pink mass approximately 1.5 x 1 cm in size (Figure 9). The conjunctiva around both eyes is reddened. Inside the globe of the left eye is some prominent, irregular whitish tissue (Figures 10 - 11). Gross Diagnoses: 1) Bilateral conjunctivitis with focal right third eyelid mass 2) Focally extensive and proliferative uveitis, O.S.

Figure 10 Figure 11

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Gastrointestinal tract and pancreas: Figure 12 Within the oral cavity, the tongue, gingival surfaces, and the oro-mucosal surfaces of the lips all feature multiple, slightly raised, occasionally coalescing soft circular ulcerative and proliferative lesions ranging from 1mm to 2.5 cm in size (Figure 12-15). Several teeth are loosened or missing subsequent to these proliferative lesions. No masses or ulcers noted in the esophagus. The small intestines feature moderate postmortem reddening and gaseous dilation (Figure 16). The stomach is essentially empty except for a small foreign body; an approximately 2 cm square piece of hard plastic (Figure 17). The foreign body is not lodged or obstructing the sphincters, and there is no evidence of gastric inflammation, ulceration, or neoplasia. The small and large intestines features moderate postmortem autolysis with no evidence of masses, nodules, ulcers, necrosis, or neoplasia. The pancreas features postmortem reddening but appears to be of normal size and conformation with no gross pathologic changes (Figure 18). Gross Diagnoses: 1) Severe multifocal ulcerative and proliferative gingivitis, glossitis, and chelitis 2) Focal gastric foreign body 3) Small and large intestinal autolysis; no significant gross lesions observed 4) Pancreatic autolysis; no significant gross lesions observed

Figure 13 Figure 14

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Figure 15 Figure 16

Figure 17 Figure 18

Figure 19 Liver: The parenchyma of the liver has a diffuse reddish appearance (Figure 19). The surface is smooth and glistening and there are no fibrinous or fibrous tags are noted. The edges are sharp, and no necrosis or neoplastic masses are noted on serial incision. Gross Diagnosis: Grossly normal liver

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Spleen: The spleen is contracted, measures approximately 15 cm in length, and has no elevated nodules or masses. Gross Diagnosis: Grossly normal spleen Adrenal glands and thyroid glands: No significant gross lesions are noted in the adrenals and or thyroid glands. Both sets of glands appear to be of normal size and conformation with no nodules or masses noted. Gross Diagnosis: Grossly normal adrenals and thyroid glands Cardiopulmonary System: The lungs are characterized by patchy, dark red regions throughout all lobes (Figure 20). No masses or areas of consolidation are detectible in the parenchyma. There is an adhesion between the left thoracic wall and the left cranial lung lobe. Approximately 11cc of serosanguinous fluid is present in the thoracic cavity (Figure 21). A 7x10 cm firm botryoid mass is present in the cranial mediastinal region (Figures 22 – 24). The cut surface of the mass had an irregular white, tan, and yellow color appearance (Figure 25). The tracheobronchial lymph nodes located dorsally at the junction of the lungs and trachea are enlarged (Figure 26). The cut surface of these nodes is similar in appearance to the mediastinal mass. There is very prominent frothy edema located at the tracheal bifurcation and distal trachea (Figure 27). The heart measures approximately 9 cm from its base to its slightly rounded apex (Figure 28). The left ventricle is moderately dilated (Figure 29). No valvular changes are noted. Gross Diagnoses: 1) Mild serosanguinous pleural effusion 2) Moderate to marked pulmonary congestion and edema 3) Focal cranial mediastinal mass 4) Enlarged tracheobronchial lymph nodes 5) Moderate left ventricular dilation

Figure 20 Figure 21

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Figure 22 Figure 23 Figure 24 Figure 25

Figure 26 Figure 27

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Figure 28 Figure 29

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Figure 30

Figure 31

Urogenital System: Both of the kidneys are of normal size and conformation (Figure 30). They are both moderately pale and feature no evidence of inflammation, necrosis, or neoplasia. The bladder is empty and there are no significant gross lesions on the mucosal surface. GROSS DIAGNOSES: 1) Bilateral renal pallor 2) Grossly normal bladder Brain: The brain is characterized by a soft mushy consistency and a diffuse reddening of the surface due to postmortem autolysis (Figure 31). It was serially sliced transversely at 5mm intervals and no hemorrhage, malacia, or neoplasia was observed. Gross Diagnosis: Grossly normal brain

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HISTOPATHOLOGY SKIN, NASAL, SUBCUTANEOUS, AND DIGITAL MASSES/LESIONS: Examined samples all featured similar morphologic changes. All featured prominent autolysis and very mild inflammation of mostly histiocytes and lymphocytes. The majority of the lesional expansion is due to the presence of large numbers fungal yeast organisms. These organisms often have clear ovoid to tear-drop nuclei with wide nonstaining capsules. The organisms measure approximately 10-15u in diameter and the capsules often coalese to forming large clear spaces. Several organisms are observed to be undergoing narrow-based budding. Similar inflammation and organisms are observed in the enlarged right axillary lymph node. Microscopic Diagnosis: Severe multifocal granulomatous dermatitis, cellulitis, and lymphadenitis with numerous intralesional Cryptococcus organisms. RIGHT EYE AND LEFT THIRD EYELID MASS: The third eyelid is expanded primarily by edema, scattered histiocytic inflammation, and large numbers of fungal organisms. These organisms are similar in appearance to those described in the skin. Similar inflammation and organisms are observed in the anterior and posterior chambers of the right eye. Microscopic Diagnoses: 1) Granulomatous third eyelid conjunctivitis with numerous intralesional Crytococcal organisms 2) Granulomatous uveitis (O.S.) with numerous intralesional Cryptococcal organisms TONGUE, GINGIVAL, AND ORO-MUCOSAL LIP MASSES/LESIONS: Examined samples all featured similar morphologic changes. All featured mild inflammation of mostly histiocytes and lymphocytes. The majority of the lesional expansion is due to the presence of large numbers fungal yeast organisms. These organisms often have clear ovoid to tear-drop nuclei with wide nonstaining capsules. The organisms measure approximately 10-15u in diameter and the capsules often coalese to forming large clear spaces. Several organisms are observed to be undergoing narrow-based budding. Microscopic Diagnosis: Severe multifocal granulomatous glossitis, gingivitis, and chelitis with numerous intralesional Cryptococcus organisms. SMALL INTESTINE: Sections of small intestine features focally extensive, often diffuse luminal autolytic change. In less autolyzed areas there is no evidence of blunting, ulceration, necrosis, or inflammation. There is very mild edema in the lamina propria, but no lacteal dilation. There is no evidence of rupture of the bowel wall and no evidence of peritonitis. Microscopic Diagnosis: Moderate luminal autolytic changes, with mild to moderate submucosal edema and congestion

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COLON: There is no evidence of ulceration, necrosis, or inflammation. There is very mild edema in the lamina propria and mild vascular congestion. There is no evidence of rupture of the bowel wall and no evidence of peritonitis. Microscopic Diagnosis: Within normal limits SPLEEN: Examination of the splenic sections reveals contraction of the parenchyma with prominence of the fibroleiomatous septae. The vascular red pulp is minimal and there is mild depletion of the white pulp follicles. No evidence of inflammation, necrosis, or neoplasia. Microscopic Diagnosis: Normal splenic contraction with mild lymphoid depletion PANCREAS: Pancreas sections feature prominent autolytic changes with mostly normally arranged acini, and normal numbers of well-spaced pancreatic islets. Focally is observed a region characterized by mild granulomatous inflammation and the presence of fungal organisms. These organisms are similar in appearance to those described in the oral cavity. Microscopic Diagnosis: Focal granulomatous pancreatitis with intralesional Crytococcal organisms and marked postmortem autolysis LUNG: All of the pulmonary tissue vasculature is markedly congested. Most of the alveoli contain alveolar macrophages, along with variable amounts of edema fluid and RBCs. No evidence of overt inflammation or necrosis is noted. Microscopic Diagnosis: Marked, diffuse pulmonary congestion, edema, intra-alveolar hemorrhage. CRANIAL MEDIASTINAL MASS: Examined multiple samples from the mass featured similar morphologic changes. All samples were characterized by autolysis and mild inflammation of mostly histiocytes and lymphocytes. No recognizable normal tissue was observed. The majority of the lesional expansion is due to large numbers fungal yeast organisms. These organisms often have clear ovoid to tear-drop nuclei with wide nonstaining capsules. The organisms measure approximately 10-15u in diameter and the capsules often coalesce to forming large clear spaces. Several organisms are observed to be undergoing narrow-based budding. Similar inflammation and fungal organisms are observed in the tracheobronchial lymph nodes as well. Microscopic Diagnosis: Severe multifocal granulomatous inflammation and lymphadenitis with numerous intralesional Crytococcal organisms. HEART: Examined sections of heart musculature feature no significant histologic lesions beyond autolysis and mild vascular congestion and edema. Myocardial fibers

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are intact, organized, and feature no hyalinization, degeneration, or inflammatory changes. Microscopic Diagnosis: Moderate autolysis and mild congestion LIVER: examined sections of liver are characterized by marked autolysis with moderate sinusoidal and vascular congestion. The accumulation is most notable in some of the central veins and periacinar regions. There is mild periacinar hepatocellular cloudy swelling and hydropic change. No evidence of periacinar fibrosis is noted. Microscopic Diagnosis: Autolysis with subacute passive hepatic congestion with mild periacinar hepatocellular swelling ADRENAL GLAND: Both adrenal glands features a normal cortical and medullary architecture. No hyperplastic or neoplastic growth is observed. No evidence of inflammation or necrosis is noted. Microscopic Findings: Histologically normal left and right adrenals THYROID AND PARATHYROID GLANDS: Examined sections of thyroid glands featured normal follicular developing with no hyperplastic or neoplastic growth observed. There is no evidence of inflammation or necrosis. The observed parathyroid tissue is normal. Microscopic Diagnosis: Histologically normal thyroid and parathyroid glands KIDNEYS: Both kidneys featured similar morphologic changes. Sections features prominent autolysis but well proportioned cortical and medullary tissue. Glomeruli are adequate in number and are not distended or sclerotic. Bowmans capsules are not thickened. No inflammation, crystaluria or proteinuria is noted in the renal tubules. Microscopic Diagnosis: Autolytic but histologically normal kidneys BLADDER: Sections of bladder are characterized by mild to moderate mucosal and submucosal congestion. There is no evidence of significant inflammation, necrosis, or neoplasia. Microscopic Diagnosis: Mild to moderate bladder congestion BRAIN: Brain sections feature prominent autolytic changes with mostly normal brain parenchyma. Multifocally are noted several regions characterized by mild granulomatous inflammation and the presence of fungal organisms. Similar organisms are noted in the meninges as well. These organisms are similar in appearance to those described in the oral cavity. Microscopic Diagnosis: Multifocal meningoencephalitis with intralesional Crytococcal organisms and marked postmortem autolysis

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COMMENTS AND CONCLUSIONS:

The cause of death in this case, as indicated from the history, was humane euthanasia. There was no gross and microscopic evidence of trauma or physical abuse. Numerous significant proliferative and ulcerative lesions were present both externally and associated with several internal organs. All of these lesions had in common the presence of fungal yeast organisms consistent with Cryptococcus spp., making this a severe case of systemic disseminated Cryptococcosis. Cryptococcosis is caused by a fungal yeast that has a worldwide distribution, though an increased incidence in temperate regions such as southern California and Australia. It can affect a wide variety of mammalian species, including cats, dogs, and humans. Cryptococcus neoformans is the species that primarily causes disease in domestic pets. These organisms are saprophytic in nature and generally have a round, to oval, to teardrop nucleus measuring 10 - 20 microns in diameter, and a large non-staining heteropolysaccharide capsule. It also has a characteristic "soap-bubble" appearance due to the capsule that protects the organism from desiccation and immune attack when invading tissues. Cryptococcosis is the most common fungal disease in cats, affecting them 7 to10 times more often than dogs. The normal presentation is usually sneezing and nasal discharge due to the tendency to affect the nose and upper respiratory system. There is a predilection for infection in male cats, most likely due to the increased roaming of toms. Siamese cats are also over-represented in most studies. The four primary systems affected are the respiratory, central nervous, ocular, and cutaneous systems. The clinical signs, therefore, depend on the system(s) affected. Dogs are not infected by C. neoformans nearly as often as their feline counterparts. The average age of infected dogs is 3.5 years and, unlike cats, there is no gender predisposition. Over-represented dog breeds include American Cocker Spaniels and Labrador Retrievers. Cryptococcosis affects the same four organ systems as with cats, but the CNS and eyes are more commonly involved in dogs than in cats. Pathologist: R.E. Moreland BS, DVM Antech Necropsy Coordinator