Vet Pathol 2011 2011 ACVP Annual Meeting E1 E51

Veterinary Pathology Online
http://vet.sagepub.com/ 2011 ACVP Abstracts

Vet Pathol 2011 48: E1 DOI: 10.1177/0300985811425342 The online version of this article can be found at: http://vet.sagepub.com/content/48/6/E1
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2011 ACVP Abstracts

Clinical Pathology: 146* Diagnostic Pathology: 47129 Education: 130138 Experimental Disease: 139199 Industrial and Toxicologic Pathology: 200211 Natural Disease: 212247 *ASVCP Abstracts 146 are being published in Veterinary Clinical Pathology 2011;40(4) and online at www.wileyonlinelibrary.com/journal/vcp. cells and lymphocytes are also present in the lamina propria. Conclusions: The finding of mild inflammation within the very superficial lamina propria of the rectum in biopsies from horses with vague gastrointestinal signs is common. Does this interfere with enterocyte function and is this a form of IBD in the horse? The superficial location in the lamina propria of these subtle lesions is reminiscent of human microscopic/collagenous/lymphocytic colitis. Further comparative studies with rectal samples from horses lacking gastro-intestinal disease is needed to establish the specificity of these findings.
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203 Cases of Equine Lymphoma Classified According to the World Health Organization (WHO) Classification Criteria
A. C. Durham1, C. A. Pillitteri2, F. Scott3, M. San Myint4, and V. E. Valli5. 1 Department of Pathobiology, University of Pennsylvania University of Pennsylvania, School of Veterinary Medicine, Philadelphia, Pennsylvania; 2Department of Pathobiology, University of Tennessee, College of Veterinary Medicine, Knoxville, Tennessee; 3Graduate Group in Epidemiology, University of California Davis, Davis, CA; 4Department of Clinical Medicine College of Veterinary Medicine University of Illinois, Urbana IL; and 5VDx Veterinary Diagnostics, Davis, California The Veterinary Cooperative Oncology Group (VCOG) of the Veterinary Cancer Society Lymphoma is the most common malignant neoplasm in the horse. Single case reports and small retrospective studies of equine lymphomas are reported infrequently in the literature. Ranges of clinical presentations, tumor subtypes and outcome have been described and the diversity of the results demonstrates the need to better define lymphomas in horses. As part of an initiative of the VCOG, 203 cases of equine lymphoma have been gathered from 11 institutions. Hematoxylin and eosin slides from each case were reviewed and 185 cases were immunophenotyped and categorized according to the WHO classification system. Data regarding signalment, clinical presentation and tumor topography were also examined. The goal of this project is to provide a comprehensive characterization of lymphoma in the horse. Ages ranged from 2 months to 31 years (mean10.7years). Twenty-four breeds were represented; quarterhorses were the most common breed (n55), followed by thoroughbreds (n33) and standardbreds (n30). Lymphomas were categorized into 13 anatomic sites. Multicentric lymphomas were common (n83), as were skin (n36) and gastrointestinal tract (n25). A total of 16 lymphoma subtypes were identified. T-cell rich large B-cell lymphomas were the most common subtype, diagnosed in 87 horses. Peripheral T-cell lymphomas (n44) and diffuse large B-cell lymphomas (n26) were also frequently diagnosed.
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An Outbreak of a Low-Virulence Calicivirus in a Rabbit Colony
J. Gary1, D. Desjardins1, J. Patterson1,2, M. Kiupel1, A. Lim1, and S. Bolin1. 1 Diagnostic Center for Population and Animal Health, Michigan State University, Lansing, MI, 2NIH Comparative Biomedical Scientist Training Program, National Cancer Institute, Bethesda, MD (JG) The well-known rabbit caliciviruses, Rabbit Hemorrhagic Disease Virus (RHDV) and European Brown Hare Virus (EBHV), can have a high morbidity and mortality in susceptible populations. Less virulent caliciviruses have been detected following outbreaks of mild disease in apparently healthy rabbits. Recently, a calicivirus associated with a disease outbreak of moderate morbidity and low mortality in a colony of 324 rabbits was detected at the Diagnostic Center for Population and Animal Health at Michigan State University. Rabbits presented for necropsy early in the outbreak had mild bile duct hyperplasia, mild periportal hepatitis, and eosinophilic, cytoplasmic hepatocellular inclusions (Councilman bodies). Eight subsequently submitted rabbits had pale livers with an accentuated lobular pattern. Histologically, livers from these rabbits were characterized by submassive hepatocellular necrosis and variable bile duct hyperplasia. A calicivirus in the Lagovirus genera was detected in nine rabbits using PCR primers targeting the viral capsid gene. Tissues submitted to the Foreign Animal Disease Diagnostic Laboratory at Plum Island, NY, were negative by viral species specific PCR for RHDV and were inconclusive for antibody against RHDV by ELISA. The sequence for a 344 base segment of the viral capsid gene was derived and nucleic acid sequence homology was less than 70% with EBHV, about 80% with RHDV, and 90% with the low virulence Michigan Rabbit Calicivirus. The corresponding amino acid sequence homology was less than 70%, about 85%, and 92%. The identification of a rabbit calicivirus with low mortality suggests that low-virulence caliciviruses may circulate in rabbit populations.
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Aortic Rupture Associated With Increased Mortlaity in Male Turkeys
H. L. Shivaprasad and G. Rimoldi. California Animal Health and Food Safety Laboratory SystemTulare Branch, University of California, Davis During 2001 2010 and 2011 about 20 outbreaks of aortic rupture occurred in male commercial turkeys between the ages of 14 and 20 weeks in many ranches in the central valley of California. History included sudden death of turkeys and excessive mortality ranging from 0.75% to 1.5% per week. There were also increased numbers of dead on arrival of turkeys at the processing plants. Female turkeys from the same breeder source and on the same diet as the males were not affected. About 60 turkeys were necropsied. The carcasses were pale and they had large amounts of clotted blood in the abdominal cavity. Most of the turkeys had a 0.5 to 2.0 cm linear or semi-linear tear in the aorta at the origin of the coeliac artery. A few birds had similar linear tears in the aorta between the coeliac and the ischiatic arteries. Birds with rupture in the posterior abdominal aorta had severe perirenal hemorrhage. Histologically, most of the aortas had severe subintimal thickening, degeneration of media, necrosis, cleft formation and hemorrhage. Stain for elastin revealed severe displacement of the internal elastic lamina and lack of elastic fibers in the tunica media. Trichrome stain revealed increased collagen especially in the subintima. Analysis of the livers from most of the turkeys revealed normal levels of copper and zinc. The cause of aortic rupture could not be determined. Diet or body weights of the turkeys did not appear to have contributed to the aortic rupture. The occurrence of aortic rupture only in male turkeys and its simultaneous occurrence on many ranches suggests agenetic basis.
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A Retrospective Study of Rectal Biopsies in Horses With Vague Signs of Gastrointestinal Disease: Something New?
M.J. Pinilla and B.A. Summers. Royal Veterinary College, North Mymms, Hatfield AL9 7TJ, United Kingdom Study objectives: Rectal biopsy, to potentially elucidate the cause of inappetance, mild weight loss and occasional diarrhoea, is a common procedure in equine practice. Our impression is that such samples often show a very subtle pattern of inflammation, an apparent equine idiosyncrasy. As these findings appear not to be reported in the literature, this study describes the microscopic findings in biopsies from affected horses. Material and methods: From submissions of equine rectal biopsies over 3 years, 19 were identified from horses with mild clinical signs as described. The samples were processed for routine histopathologic evaluation and stained with hematoxylin and eosin. Results: The defining histological findings were always located within the lamina propria of the rectum, immediately adjacent to the superficial plexus below the apical enterocytes. There, close inspection reveals individual inflammatory cells undergoing necrosis (apoptosis?), typified by nuclear pyknosis and karyorrhexis plus low numbers of intact macrophages containing small amounts of tan-brown intracytoplasmic pigment. Less regularly, mildly increased numbers of plasma
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Bone Formation Within a Canine Insulinoma
E.M. Pieczarka, D.S. Russell, K.S. Santangelo, F. Aeffner, M.J. Burkhard. Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio Mature bone formation has been described within a variety of epithelial and mesenchymal neoplasms other than those derived from primary osteogenic origin in both humans and animals. However, intratumoral bone has not been previously described within a pancreatic islet cell carcinoma. Herein, we present the first case of an islet cell carcinoma (insulinoma) with bone formation in a mammal. An 11-year-old male neutered mixed-breed dog was presented for exercise intolerance, tetraparesis, and persistent hypoglycemia. Abdominal ultrasound revealed two nodules within the right limb of the pancreas. Cytology from one nodule was consistent with a carcinoma of neuroendocrine origin, with a primary differential of insulinoma. Histopathology revealed a partially encapsulated, multilobulated mass of polygonal cells, arranged into packets delineated by delicate fibrovascular stroma. Positive staining with synaptophysin confirmed a neuroendocrine origin of these cells, and subsequent positive staining for insulin was consistent with a diagnosis of insulinoma. Additionally, composing approximately 60% of the mass, there was a solitary focus of mineralized compact bone. The bone had broad and irregularly arranged, anastomosing trabeculae lined by a single layer of osteoblasts. To the authors knowledge, this is the first report of bone formation within an insulinoma.
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Cerebral Coenurosis in a Cat
J. Ragsdale and N. Takacs. New Mexico State University, New Mexico Department of Agriculture, Veterinary Diagnostic Services, Albuquerque, NM A 2-year-old, spayed female, domestic shorthair cat (Felis catus) was euthanized after a two month history of clinical signs of vestibular disease that progressively worsened over time. The cat developed anisocoria and eventually became nonambulatory. At necropsy, the cat had a 12 millimeter diameter cyst in the brainstem in the area of the pons just ventral to the cerebellum. On cross section, the cyst was filled with clear slightly gelatinous fluid with at least 20 opaque, white scolices that tended to occur in linear rows and appeared similar to grains of sand. The lateral ventricles were dilated. Microscopically, the cyst was a cestode bladder lined by an internal membrane with multiple scolices characterized by a rostellum armed with hooks, suckers, and calcareous corpuscles suspended in a parenchyma covered by a tegument. The adjacent neuropil of the brainstem contained perivascular infiltrates of lymphocytes, plasma cells, and eosinophils. Due to the presence of multiple scolices within the cestode bladder, the larval cestode was classified as a coenurus. Thus, the diagnosis of cerebral coenurosis was made. Based on the arrangement of the scolices in the bladder as well as the disappearance of Taenia multiceps from the United States, the coenurus was most likely the result of Taenia serialis infection, which has a canid-rabbit life cycle. Isolated cases of cerebral coenurosis of cats believed to be caused by an aberrant infection of T. serialis have been reported.
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CD34 Immunohistochemical Labeling in Feline Nonvascular Tumors
R.N. Jennings, J.A. Ramos-Vara, M.A. Miller. Department of Comparative Pathobiology, Purdue University CD34 is a molecule expressed in normal endothelial cells, hematopoietic stem cells, nerves, hair follicles, muscle bundles, sweat glands, meninges, and also in neoplasms such as meningioma, gastrointestinal stromal tumor, fibrous tumors, and pleomorphic sarcoma. In a previous study, we observed that CD34 had greater average immunolabeling intensity than either Factor VIII-related antigen or CD31 in feline vascular tumors. As part of the validation of CD34 in feline vascular tumors, we examined its expression in onehundredthirtyone feline nonvascular tumors, which included sixty-three epithelial tumors, forty-three mesenchymal tumors, twenty-three round cell tumors, and two melanomas. Ninety-one percent of mesenchymal tumors (39/43), 35% of round cell tumors (8/23), 22% of epithelial tumors (14/63), and 100% of melanomas (2/2) labeled positively for CD34. Many soft tissue sarcomas examined including leiomyosarcoma (8/10), vaccine-site sarcoma (8/8), peripheral nerve sheath tumor (2/2), and fibrosarcoma (1/1) labeled positive for CD34. Soft tissue sarcomas are likely to be in the differential diagnosis for poorly differentiated vascular tumors, and therefore, the widespread CD34 labeling of soft tissue sarcomas makes the sole use of this marker of equivocal value when hemangiosarcoma is considered in the differential diagnosis. In addition to CD34, complementary markers such as CD31 and Factor VIII-related antigen may be necessary to confirm a diagnosis of hemangiosarcoma. Our results are similar to those observed in human tumors in which many nonvascular mesenchymal tumors express CD34.
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Characterization of Posttransplantation Lymphoma in Feline Renal Transplant Recipients
A. Durham, E. Holmes, A. Donahue, and L. Aronson. Departments of Clinical Studies and Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA The development of malignant neoplasia following solid organ transplantation and immunosuppression is well-recognized in humans. Posttransplantation malignant neoplasia (PTMN) includes non-melanoma skin cancers, non-Hodgkin lymphoma and Kaposi sarcoma, and many of these cancers have a known or suspected viral cause. More specifically, posttransplantation lymphoproliferative disorders (PTLD) in humans comprise a spectrum ranging from polyclonal proliferations to lymphomas. A similar increased incidence of cancer is seen in cats who have received a renal transplant and lymphoma may be the predominant PTMN in this population. This study examines a population of cats that have received renal transplants at the University of Pennsylvania School of Veterinary Medicine and subsequently developed neoplasia. From 1998 to 2010, 114 cats were transplanted and of these, 25 cats developed neoplastic disease (22%). 14 cats were diagnosed with lymphoma (56%), making it the most common neoplasia in this patient population. The average interval between transplantation and diagnosis of lymphoma was 997days (median617days) and the average survival time following the diagnosis of lymphoma was 34 days (median2days). Tissues from 7 of these cats were available for histopathologic review as either necropsy samples (n5) or biopsy submissions (n2). Five of these cats had multiorgan involvement with sites including the liver, spleen, peripheral and mesenteric lymph nodes, small intestine, urinary bladder, heart, mesenteric fat and body wall. Four cats had involvement of the renal allograft and two of these cats also had lymphoma of the native kidney. All lymphomas were classified as mid to high grade, diffuse large B cell lymphomas, which is also the most common lymphoma subtype in human cases of PTLD.
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Classification of Feline Mammary Tumours: Prognostic Morphology and Markers Expression
V.E.G. Zappulli , D. Caliari , R. Rasotto , M.H. Goldschmidt , M. Castagnaro1. 1Department of Public Health, Comp. Pathology and Vet. Hygiene, University of Padua, Italy, 2Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, PA Most feline mammary tumours are aggressive carcinomas (80-90%) with poor prognosis. When compared to canine tumors, they are less heterogeneous, with few complex and mixed tumours. A new histologic classification of canine mammary tumours was recently published. Our study analyzes the morphology of feline mammary neoplasms and compares them with their canine counterparts. We evaluated phenotypic and prognostic markers (pancytokeratin panCK, CK8_18, CK5, CK14, calponin, alpha smooth muscle actin, vimentin, p63, beta-catenin, E-cadherin, estrogen and progesterone receptors, Ki-67, HER-2, p53), which were compared with morphologic subtypes, postsurgical survival and normal/hyperplastic glands. In normal feline mammary glands, undifferentiated basal/myoepithelial cells (CK5, CK14, p63, vimentin) were confined to proximal ducts; vimentin expression was luminal and ductal, with pronounced coexpression with CK14 exclusively in terminal intralobular ductules. In interductal lobules, luminal cells were negative for both these markers. We identified several less aggressive morphological subtypes of mammary tumors, including ductal adenomas/carcinomas and intraductal papillary adenomas/carcinomas that exhibit specific immunomarker expression (vimentin- and CK14 basal/myoepithelial cells), with prolonged patient post-surgical survival. Mammary non-ductal neoplasms expressed vimentin (95% of tumours, 10-90% positive cells). There was a significant correlation between vimentin and CK14 (90% of tumours, 6-100% positive cells) expression, which was inversely related to the expression of estrogen and progesterone receptors, CK8_18 and improved prognosis. This suggests a possible role of vimentin/CK14 cell population of the terminal end intralobular ductular structures in aggressive feline neoplasms. Thus the newly proposed canine mammary tumour classification may be applicable to feline mammary tumors, with a significant role for specific immunomarkers in determining prognosis.
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Cutaneous Metastasis of Transitional Cell Carcinoma in 11 Dogs
L.T. Reed1,3, D. Knapp2, M.A. Miller1. Departments of 1Comparative Pathobiology and 2Veterinary Clinical Sciences, Purdue University, West Lafayette, IN; and 3NIH Comparative Biomedical Scientist Training Program, National Cancer Institute, Bethesda, MD Cutaneous metastasis of transitional cell carcinoma (TCC) is uncommon and has been attributed to retrograde lymphatic dissemination, hematogenous spread, or surgical implantation. Theoretically, TCC could also invade urine-scalded skin transepidermally. Skin was evaluated from eleven dogs with cutaneous TCC and a history of urinary incontinence. None had undergone abdominal surgery for diagnosis or treatment. In 10/11 dogs, the cutaneous metastases were localized, though spread over a broad area, to perineal, inguinal, or abdominal skin. Lesions ranged from papules to ulcerated plaques or nodules, and, except in one dog, were in perineal, inguinal, or ventral abdominal skin. Epidermal erosion or ulceration was detected in 4/8 dogs with epidermis in the histologic section; two of these had a clinical diagnosis of pyoderma. Uroplakin-III immunoreactivity confirmed urothelial origin of the carcinoma in 5/5 dogs. Dermal and subcutaneous lesions were graded for percent tissue invasion by TCC as 0none, 1 <25% or only microscopic aggregates, 225-50% or 1-2-mm nodule(s), or 3>50% or >2-mm nodule. The number of vascular profiles with TCC emboli was graded as 0none, 1one, 2<25%, or 3>25%. All seven dogs with dermal TCC had grade 3 invasion. Of ten dogs with subcutaneous TCC, two had grade 2 invasion; eight, grade 3. Of nine with intravascular TCC, one, three and five dogs had Grades 1, 2, and 3, respectively. The proximity of cutaneous metastases of TCC to the vulva or prepuce in dogs with urinary incontinence raises the possibility of transepidermal spread, although lymphatic and hematogenous spread remain possible as well.
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Cutaneous Lymphangioma in a Donkey (Equus africanus asinus)
A. J. Castillo-Alcala, F. Page, R. Bidwell and L. Ross. University School of Veterinary Medicine, West Farm, Saint Kitts West Indies A case of cutaneous lymphangioma affecting the right tarsus of a 10-yearold female donkey (Equus africanus asinus) is described. Over a 12-month period the donkey developed a progressive subcutaneous swelling over the right tarsus. Radiologic findings of the right tarsal area indicated severe extra-capsular soft tissue swelling that extended proximally over the distal right tibia and over the proximal metatarsal region with no additional osseous abnormalities and/or degenerative joint disease detected. Ultrasonographically, the subcutaneous tissue was thickened and diffusely contained a matrix of innumerable cavitations filled with anechoic fluid. Histological examination of the subcutaneous tissue in the affected area demonstrated the presence of multiple, variably-sized cavernous spaces lined by a single layer of attenuated, oval to elongated endothelial-like cells in the dermis and the subcutis. The cavernous spaces were generally empty or contained few erythrocytes. The adjacent subcutis was characterized by the presence of fibroblasts, fibrocytes, mature collagen fibers and numerous small blood vessels. Multifocally, discrete aggregates of lymphocytes and plasma cells were scattered throughout the tissue. The microscopic features of the mass were consistent with cutaneous lymphangioma. Fluid samples obtained from the affected area were cultured and no growth was observed after 72 hours. Cutaneous lymphangiomas are benign, locally invasive tumors of the lymphatic system. These lesions occur rarely in both human and veterinary patients. These tumors are reported to account for only 0.1% of all equine cutaneous neoplasms, and to the authors knowledge this is the first case of cutaneous lymphangioma reported in a donkey.
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Cutaneous Rhabdomyosarcoma With Pulmonary Metastasis
K.A. Koistinen1,2, E.L. Stevens2, M. Fleetwood3, and C. Lowery4. 1Veterinary Pathology Service, Joint Pathology Center, Silver Spring, MD; 2Department of Veterinary Pathology, Armed Forces Institute of Pathology, Washington, DC; 3 New Hampshire Veterinary Diagnostic Laboratory, University of New Hampshire Durham, NH; and 4South Plains District Veterinary Command, Ft Hood, TX A cutaneous mass on the tip of the left pinna from a 12-year-old, spayed female Labrador retriever mix breed was removed and evaluated; the mass recurred locally two months after initial excision. Histologically the infiltrative neoplasm was composed of pleomorphic spindle cells arranged in vague haphazard streams within a moderate eosinophilic to myxomatous matrix. By immunohistochemistry neoplastic cells were positive for desmin, muscle specific actin, myoglobin, and S-100 protein, and negative for glial fibrillary acidic protein and smooth muscle actin. Tumor histomorphology and the immunohistochemical features were consistent with rhabdomyosarcoma. Metastasis to the lungs was discovered nine months post-excisional biopsy. Complete necropsy did not identify gross evidence of primary rhabomyosarcoma at a more typical anatomic location, including the tongue, heart, or in association with a large skeletal muscle. This is an uncommon case of metastatic primary cutaneous rhabdomyosarcoma.
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Description of Gross and Histopathologic Lesions Associated With Phenylpropanolamine Toxicity in a Dog
A. DeFrancisco and K. Singh. Department of Pathobiology, University of Illinois, Urbana , IL Phenylpropanolamine (PPA) or d, l norepinephrine is used for the treatment of urinary incontinence in dogs; however, the description of gross and microscopic lesions associated with this toxicity are lacking. A 30 kg, 5-year 10-month old, male castrated, mixed breed dog presented to a local emergency clinic with a history of consuming approximately 86 mg/kg of PPA, secondary tachycardia, and hypertension. Hematemesis was noted post vomition with hydrogen peroxide, and resolution of clinical signs did not occur post treatment. The dog was then transferred to the UIUC emergency clinic, where ventricular premature contractions (VPC), hemoglobinuria, ascites, and injected mucous membranes were also observed. Five days post PPA ingestion, the dog went into cardiac arrest and died. On gross examination, the pleural surface, tracheal mucosal surface, and gastric serosal surface contained multifocal to coalescing acute hemorrhages. The urinary bladder contained thick, dark red to black urine with a specific gravity of 1.020, glucose, and blood. Mild hepatomegaly with a prominent reticular pattern and moderate pulmonary edema were also present. The distal right forelimb, at the location of catheter placement, was approximately 1.5 times the normal diameter, and oozed a red, translucent fluid on cut surface. Histologically, the liver, kidney, heart, adrenal glands, and dermis contained multifocal acute hemorrhages. The left and right cardiac ventricles and interventricular septum contained areas of myocyte necrosis characterized by condensed and hypereosinophilic sarcoplasm with pyknotic nuclei. Small numbers of neutrophils, lymphocytes, and macrophages were also observed associated with areas of necrosis. Mild vacuolar hepatopathy with rare hemosiderin laden Kupffer cells were also observed. The dermis, from the right forelimb, was markedly expanded by edema, neutrophils, and macrophages.
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Dual Infection With Mycobacterium avium Complex and Hammondia heydorni in a Basset Hound
K.A. Bradford1, L.J. Rich2, E.M. Johnson1, and R.W. Allison1. 1Department of Pathobiology, Center for Veterinary Health Sciences, Oklahoma StateUniversity, Stillwater, OK; 2Antech Diagnostics, Irvine, CA A 2-year-old female spayed Basset Hound with a >1 year history of intermittent coughing, fever, lameness, lethargy, vomiting and diarrhea was diagnosed with systemic mycobacteriosis (Mycobacerium avium complex, MAC) based on cytology of peripheral and mesenteric lymph nodes and confirmed by bacterial culture. Repeated cytology of mesenteric lymph nodes and small intestine several months after initial diagnosis revealed numerous free and intracellular protozoal organisms that were 10x2 um, crescent shaped, and lightly basophilic with a round eccentrically placed pink nucleus. The morphology of these organisms was consistent with several different nintestinal coccidians that reproduce in the dog with an enteroepithelial cycle including Cystisospora spp., Neospora caninum, Hammondia heydorni, and Sarcocystis spp. Examination of a fecal flotation revealed 12x10 um oocysts suggestive of Neospora caninum or Hammondia heydorni, whose oocysts are microscopically indistinguishable. Differentiation of these organisms is important as they carry vastly different prognoses. Neospora caninum can be associated with high morbidity including severe neurological and muscular disease as well as cardiopulmonary and hepatobiliary pathology. Conversely, Hammondia heydorni is considered nonpathogenic. PCR performed on DNA extracted from intestinal aspirates was negative for Neospora caninum and Toxoplasma gondii, and serum antibody assays for Neospora caninum and Toxoplasma gondii were negative. PCR analysis of feces was positive for Hammondia heydorni and negative for Neospora caninum and Toxoplasma gondii. Disseminated MAC infection is rare in dogs, but Basset Hounds appear to be overrepresented due to a suspected immunodeficiency. This is the first reported canine case of concomitant infection with disseminated mycobacteriosis and Hammondia heydorni diagnosed initially by cytology.
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Diplomyelia and Myelodysplasia in a German Shephard Dog
A.C. McCalla-Martin, M. Asakawa, and N.J. Olby. North Carolina State University College of Veterinary Medicine, Raleigh, NC and NIH/NCI, Bethesda, MD Reported is a 2-year-old, male castrated, German Shephard dog that presented at one year of age with bilateral coxofemoral joint luxations, symmetric hind limb musculature atrophy and inability to flex tarsal or stifle joints. On post mortem examination the spinal cord was found to abruptly terminate in the cranial lumbar region.Three centimeters prior to termination, a second cord arose within the same sheath continuing caudally. Histologic changes observed in the caudal cord included: paramidline location of the ventral median fissure, paracentral location of the central canal, absence of dorsolateral sulci, and absence of identifiable grey matter horns with swirling and abnormal arrangement of grey and white matter. Within the caudal lumbar segments, two cords were seen which were fused by a region of shared white matter. One cord had irregularities of white and grey matter and the other cord had a normal presentation with identifiable dorsal and ventral horns. Both cords in the caudal lumbar region contained central canals. Post mortem and histologic evaluation revealed significant developmental spinal cord disease. Hind limb motor function within this animal was likely impaired as a result of the spinal abnormalities resulting in significant skeletal muscle atrophy and secondary joint and bone degenerative changes. Myelodysplastic disease and dysraphism has been reported in the Weimaraner breed, and is due to a co-dominant mutation with variable penetration that is lethal in the homozygous animal. These animals are not reported to have joint flexion abnormalities (as in this case) but alternatively, present with arthrogryposis-like lesions. To the best of our knowledge, this specific spinal lesion and associated degenerative changes have not been reported in the canine species.
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Equine Giant Colonic Diverticulum and Leiomyomas
Kali Holder,1 Mustajab Mirza,2 and Nobuko Wakamatsu1. Departments of 1 Pathobiological Sciences and 2Veterinary Clinical Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA 70803 This case details the occurrence of a very large (1.5 x 1 x 0.2 meter) mass associated with the large colon of a term pregnant, mature American Quarter Horse that presented to LSU Veterinary Teaching Hospital with a short history of colic. The gross and histological examination revealed a massive diverticulum with leiomyomas at the attachment site. Leiomyomas have been reported with low frequency in horses, and diverticula are welldescribed in this species. To our knowledge, however, association of smooth muscle/stromal tumors and equine diverticula has not been described in the literature. In addition, the size of this diverticulum far exceeds the dimensions of typical diverticula. A rare entity known as giant diverticulum of the colon (GDC) is reported in humans, with the most common presentation in geriatric patients. In the infrequent reports of tumors in individuals with GDCs, investigators have proposed that occlusive colonic masses may provide a ball-valve mechanism for increasing intraluminal air pressure to induce a diverticulum or expand an existing diverticulum. This diverticulum was not appreciably gas distended at necropsy; however, the wall of the structure had ruptured antemortem, and would have released any trapped gas. The location of the firm masses at the stalk/orifice of the diverticulum supports a similar occlusive mechanism with regards to vasculature, thus explaining the profoundly edematous nature of the mural tissue seen.
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Equine Renal Adenocarcinoma With Metastasis to Cervical Musculature and Brain
L.T. Reed , B. Toth , S. Taylor , M.A. Miller . Departments of Comparative Pathobiology and 2Veterinary Clinical Sciences, Purdue University, West Lafayette, IN and 3NIH Comparative Biomedical Scientist Training Program, National Cancer Institute, Bethesda Primary renal tumors are rare in horses, with most diagnosed as renal adenocarcinoma (RAC). Metastatic lesions are reported mainly in perirenal tissues, liver or lung. A nineteen-year-old, Quarter Horse gelding had a 3-day history of ataxia, lethargy and intermittent urinary incontinence. The left kidney was mostly replaced by an 11.2 kg, roughly spherical, brown-yellow mass that was red to black and friable on cut section. The contralateral renal lymph node was enlarged. A tan mass in the neck was deep to the right brachiocephalicus muscle. A red-brown, friable, subdural mass was between the cerebrum and cerebellum. Two separate dark red nodules were in the left frontal and occipital lobes of the cerebrum. Histologically, the renal mass was classified as tubular RAC, and consisted of nests of neoplastic epithelial cells, arranged in tubules (some with intraluminal mucus), with a moderate amount of eosinophilic cytoplasm, an ovoid hypochromatic nucleus, a prominent nucleolus, and distinct cellular borders; some cells had a distinct brush border. The differential diagnosis for the intracranial masses included ependymoma, choroid plexus carcinoma, and metastatic RAC. However, none of the intracranial masses were contiguous with the ependymal lining or choroid plexus, eliminating ependymoma and choroid plexus carcinoma. Furthermore, the neoplastic cells in all extrarenal sites resembled those in the renal tumor. In addition, neoplastic cells at all sites were positive for cytokeratin, but negative for vimentin (further eliminating ependymoma), consistent with RAC and metastasis to the renal lymph node, neck, and brain.
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Female Adnexal Tumor of Probable Wolffian Origin in a Baboon (Papio sp.)
L.J. Dickson1,2, W.E.Culp2, C.W.Schmitt1,2, M.A. Owston3, and E.J. Dick Jr3. 1 Veterinary Pathology Service, Joint Pathology Center, Silver Spring, MD, 2 Department of Veterinary Pathology, Armed Forces Institute of Pathology, Washington, DC, and 3Texas Biomedical Research Institute, San Antonio, Texas A 21-year-old adult, ovariectomized, female baboon (Papio sp.) with a history of severe spondylosis and inguinal rash presented at necropsy with a hypoplastic uterus. The uterus had two small grey-white, ovoid, bilateral masses attached by a thin stalk to the cranial-dorsal aspect, and a large pedunculated mass at the cranial aspect which was diagnosed histologically as a adenomyoma with adenofibromatous component. Histologic examination of one of the bilateral masses revealed a 5 mm, unencapsulated, well-circumscribed neoplasm composed of cuboidal to polygonal cells arranged in closely packed tubules, acini, and cords which expanded the oviduct. The contralateral mass, which was surrounded by fibrovascular tissue, had similar cellular morphology, but was poorly circumscribed, infiltrative, and densely cellular, with fewer tubules and acini. Immunohistochemically, neoplastic cells of the bilateral masses were diffusely positive for cytokeratin and vimentin, and negative for inhibin and epithelial membrane antigen. Histologic and immunohistochemical findings of the bilateral masses are highly suggestive of a female adnexal tumor of probable wolffian origin (FATPWO). This tumor occurs rarely in humans and to our knowledge has not been reported in the veterinary literature.
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Esophageal Gland Adenocarcinoma in a 53-Year-Old Mata Mata Turtle (Chelus fimbriatus)
A.V Desoutter1, E.D. Lombardini2, R.J. Montali3, and F. Del Piero1. 1Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 2Armed Forces Radiobiology Research Institute, Uniformed Services University of the Health Sciences, and 3Department of Molecular and Comparative Pathobiology, Johns Hopkins University The Mata mata turtle (Chelus fimbriatus) is a freshwater turtle which inhabits stagnant pools in the northern part of South America. Translated from Spanish, mata mata means "I kill, I kill". The Mata mata hunting technique is unique among the chelonians. While opening their mouth and expanding their throat, the Mata mata extends the head up and catch their prey. These movements, create a suction action that draws the prey to their throat; the pray is swallowed whole and the water is expelled out. A 53-year-old male, captive Mata mata turtle was examined following its unexplained death. The animal was in good nutritional, muscular and post-mortemcondition. The esophageal wall was circumferentially expanded by 6 discrete, oblong, irregular, tan, and soft contiguous gland-like structures, each measuring about 7-10 cm in length and 1.5 cm in diameter. Histologically, the masses were comprised of irregular, sometimes cavitated islands of pale polygonal cells arranged in variably sized glands. Neoplastic cells expressed pancytokeratins while vimentin, chromogranin, synaptophysin and thyroglobulin were not detected. The neoplasm was interpreted as an esophageal gland adenocarcinoma. Little is known about the role of the esophageal gland but it is assumed to aid in the feeding behaviors of sucking down whole fish, and does not seem to play a role in enzymatic digestion. The literature has very few reports of lesions in this endangered species, especially in older animals. To our knowledge this is the first report of an esophageal gland adenocarcinoma in this genus.
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Genital and Extragenital Canine Transmissible Venereal Tumor in Dogs in Grenada, West Indies
A. Chikweto, S.M. Kumthekar, M.I. Bhaiyat, H. Larkin, and K. P.Tiwari. Pathobiology Academic Program, School of Veterinary Medicine, St. Georges University, Grenada, West Indies This retrospective study conducted from 2005 and 2011 sought to evaluate the total number of transmissible venereal tumor (TVT) cases, and to report extragenital TVT lesions in dogs from the small tropical island setting of Grenada, West Indies. Evaluation of biopsy and necropsy specimens from dogs with TVT was carried out by cytological and histopathological methods. In the present study, we observed a total of 74 dogs with TVT, comprising 39 male and 35 female dogs. Most cases were from dogs between 3 and 6 years; less frequently from older dogs (over 6 years) or younger dogs (1-2 years of age). There are very few reports on extragenital lesions of canine TVT. In the present study, we report 13 cases (17.6 %) of dogs with TVT in extragenital sites, most (60%) of which were males. Tumors were noted in the nasal cavity, eye orbit, spleen, liver, skin and subcutaneous tissue, ovaries, and submandibular, cervical and inguinal lymph nodes. The most interesting finding was the presence of extragenital lesions without primary genital involvement in 4 cases (5.4%). This is consistent with the possibility that the transmission of tumor cells occurs not only by genital contact but also by transplantation through other muco-cutaneous surfaces. Our findings emphasize the need to consider TVT on the list of differential diagnosis for masses in extragenital sites in dogs from geographic regions where TVT is prevalent.
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Hemolytic Anemia Compatible With Copper Toxicosis in a Ferret
L.A. Garcia-Camacho , C. Faz-Basurto C , Y. Romero-Sanchez , and I.C. Rangel-Rodriguez1. 1 Facultad de Estudios Superiores Cuautitlan, Universidad Nacional Autonoma de Mexico and 2Private practice, Mexico A female, 3 year-old domestic ferret was examined because of hemorrhagic diarrhea, ascitis, CNS depression and lethargy. The animal was euthanatized and necropsy revealed ascites, hydrothorax, hemorrhagic gastritis and segmental enteritis, mottled pale liver, dark and red friable kidneys with hydronephrosis. CNS was not examined. Microscopically, gastric and intestinal mucosa was replaced by abundant necrotic tissue. The crypt remnants were either distended by leucocytes or had proliferation of enterocyte that contained intranuclear basophilic inclusion bodies. The lamina propria was moderately distended with lymphocytes. In the liver, centrilobular and paracentral degeneration and necrosis with mild hemorrhage wereseen. The hepatocytes were markedly vacuolated displaying fine golden pigment, and the Kupffer cells were laden with coarse golden-brown granules or eosinophilic material in the cytoplasm. Splenic macrophages also contained cytoplasmic golden-brown pigment. In the kidney, hemoglobinuric nephrosis with a few hemoglobin casts in the renal tubules was observed. Epithelial cells of proximal renal tubules had hyalin droplets and fine golden granules. Special staining revealed iron in liver, spleen and kidney and copper in the liver. The microscopic findings in the gastrointestinal tract were compatible with parvoviral gastroenteritis similar to what it has been described in cats and chinchillas. No lesions of Aleutians disease such as lymphocytic and/or plasmacytic infiltration of multiple organs, including hepatic portal triads were seen. The hemoglobinuric nephrosis in conjunction with liver, splenic, and renal hemosiderosis are evidence of an intravascular hemolytic crisis most likely caused by copper toxicosis on the basis of the findings from liver histologic examination and special staining. Both conditions, parvovirus infection and copper toxicosis, promote neurological disorders. Unfortunately, CNS tissues were unavailable.
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Histological and Immunohistochemical Characterization of Adenocarcinoma of the Dorsal Glands in Two European Ground Squirrels (Spermophilus citellus)
C. Nassuato, A. Carminato, F. Mutinelli, E. Bozzato, and M. Vascellari. Istituto Department of Histopathology, Zooprofilattico Sperimentale delle Venezie, Legnaro, Italy Two cases of adenocarcinoma of the dorsal glands (specific integumentary glands localized in the dorsal region) in two different European ground squirrels (Spermophilus citellus) are presented. Histologically, the surgically removed tumors were demarcated, but unencapsulated subcutaneous masses, characterized by proliferation of pleomorphic cells arranged in tubules, acini and sheets. Diffuse infiltration of neutrophils was also observed. In both lesions, nests of cells were present within the lumen of blood vessels. By immunohistochemistry the neoplastic cells were positive for CKAE1-3 and CK19. PAS stain confirmed the glandular origin of the neoplasia. Three months after surgery the squirrels are well, without any sign of recurrence. Authors speculate about the biological behavior of this type of tumors affecting the dorsal gland. The immunohistochemistry and histochemical results supported the histological diagnosis of dorsal gland adenocarcinoma.
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Hepatocellular Carcinomas in Vietnamese Pot-Bellied Pigs
J.L. Haddad , P.L. Habecker . Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC and 2Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA Neoplasia has uncommonly been reported in Vietnamese pot-bellied pigs with rare reports of hepatocellular tumors. Twenty-two pot-bellied pigs diagnosed with hepatocellular carcinoma at necropsy over a 3 year period at one institution are described, representing 31% of the total pot-bellied pigs necropsied. The average age of affected pigs was 16.6 years with 15 males and 7 females. The most common clinical signs were decreased appetite (16/22) and weight loss (7/22); 3 pigs had no documented signs related to the liver tumor. Grossly, the majority were massive tumors (13/22) with fewer nodular tumors (8/22) and one diffuse tumor. Massive tumors were typically multilobulated, very large and encompassing one or more adjacent liver lobes, and were soft to firm and tan-yellow to orange-brown. Nodular tumors had multiple 1-15cm diameter, discrete nodules in multiple liver lobes. Gross evidence of necrosis, cysts, abscesses or hemorrhage associated with the tumor was often described (9/22). Half of the cases had possible intrahepatic metastasis, and extrahepatic metastatic disease was identified in 3 cases, including to the hepatic lymph node (1/3), lung (2/3), spleen (1/3) and kidney (1/3). Histologically, all tumors had trabecular or solid pattern, or a combination. An adenoid pattern was only identified in small regions of few tumors. The neoplastic cells were relatively well-differentiated with moderate pleomorphism and a low mitotic index. Other histologic features within the tumors included extramedullary hematopoiesis, bile stasis, intracellular glycogen or lipid accumulation, and foci of coagulative necrosis. Aged pot-bellied pigs may be predisposed to hepatocellular carcinomas, which are locally aggressive and may metastasize within the liver and to other organs.
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Idiopathic Follicular Bronchiolitis in Neonatal Beef Calves
J.B. Stanton1, G.J.Haldorson1, A.W. Layton2, T.E. Besser1, J.D. Ramsay1, D.D. Nelson1, J.F. Munoz1, and K.K. Lahmers1. 1Department of Veterinary Microbiology and Pathology and Washington Animal Disease Diagnostic Laboratory, Washington State University, Pullman, WA and 2Montana Veterinary Diagnostic Laboratory, Bozeman, MT Several cases of chronic follicular bronchiolitis in neonatal beef calves from western states have been identified in the past several years. Affected animals are typically born normal, but then steadily worsen with signs of respiratory distress. Animals typically die within 1224 hours. No diagnostic gross lesions are observed. Histologically, these cases are defined by peribronchiolar hyperplastic lymphoid aggregates, often with mature lymphoid follicle development. Other pulmonary pathology, including intra-airway multinucleated cells and bronchointerstitial pneumonia are variably present. Testing for known and unknown pathogens to date has failed to demonstrate any causative organisms. The histopathology of these cases shares features with human follicular bronchiolitis (a.k.a. BALT hyperplasia) and lymphocytic interstitial pneumonia; however, no human neonatal cases have been identified within the literature. Additional testing on these cases is ongoing. These cases represent an interesting pulmonary change that must have developed in utero based on the chronicity of the lymphoid hyperplasia.
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Idiopathic Hypothyroidism in a Canadian Lynn (Lynx canadensis)
A. DeFrancisco and A.W. Stern . Department of Pathobiology, University of Illinois, Urbana, IL and 2Veterinary Diagnostic Laboratory, University of Illinois, Urbana, IL A 1.5-year-old intact male Canadian Lynx (Lynx canadensis) presented to the referring veterinarian with a 1 year history of poor appetite, lethargy, and weakness with eventual development of neurologic signs (vestibular signs, ataxia). At necropsy, gross lesions were few and non-specific. The lynx was moderately emaciated with little subcutaneous adipose stores and decreased skeletal muscle mass. The serosa and mucosa of the urinary bladder contained multifocal to locally extensive regions of hemorrhage, and focally, a left corneal ulcer was present. Histologically, the thyroid glands were bilaterally decreased in size and approximately 75% of the thyroid gland was replaced by mature adipocytes. The remaining thyroid follicular cells are cuboidal and form follicles of varying sizes (10 um 100 um) with a mild amount of colloid. No other significant histologic abnormalities were identified. The histopathologic findings are consistent with idiopathic follicular thyroid atrophy. This is the first report of idiopathic hypothyroidism in a young Canadian Lynx.
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Immunohistochemical Evaluation of Tyrosine Kinase Receptor and Ligand Expression in Feline and Canine Soft Tissue Sarcomas
E. Howey1, R. Amorim2, I. Langohr1, M. Kiupel1. 1Department of Pathobiology and Diagnostic Investigation, Michigan State University, Lansing, MI and 2 Faculdade de Medicina Veterinaria e Zootecnia, Universidade Estadual Paulista, Botucatu, SP, Brazil Soft tissue spindle cell sarcomas are common subcutaneous neoplasms in cats and dogs. The pathogenesis of these tumors has yet to be fully elucidated and treatment is commonly limited to surgical excision; however, recurrence is common due to their highly infiltrative nature. Tyrosine kinase receptors (TKRs) play a major role in extracellular signal transmission in normal and neoplastic cell activity. Recently, tyrosine kinase inhibitors (TKIs) have been established as effective therapeutic agents for canine mast cell tumors. KIT/ SCF interaction is known to occur in various types of neoplasms and is responsible for deregulation of neoplastic growth, while VEGFR/VEGF and PDGFR/ PDGF play a role in neovascularization and mesenchymal cell proliferation within neoplastic populations. In this retrospective study, paraffin-embedded tissues from 50 canine peripheral nerve sheath tumors (PNST) and 47 feline vaccine-associated sarcomas (FVAS) were evaluated immunohistochemically for expression of tyrosine kinase receptors: KIT, vascular endothelial growth factor receptor (VEGFR), and platelet derived growth factor receptor (PDGFR), and their respective ligands: stem cell factor (SCF), VEGF and PDGF. Thirty-six of the PNST were positive for PDGF, 48 were positive for PDGFR, 44 were positive for VEGF. Forty-three PNST were positive for VEGR and SCF. Only 7 PNST were positive for KIT. All FVAS were positive for PDGF, PDGFR and VEGF. Thirty-three FVAS were positive for VEGFR, 45 were positive for SCF, and 10 were positive for KIT. The high percentage of canine and feline soft tissue sarcomas expressing TKRs warrants further investigation of the therapeutic benefits of TKIs in the treatment of these neoplasms.
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Iliac Arteriovenous Fistula in a Cynomolgus Monkey (Macaca facicularis)
L.D. Schmidt, M.K. Gee, and J.M. Cline. Department of Pathology/Comparative Medicine, Wake Forest University School of Medicine, Winston Salem, NC A 4-year old, male cynomolgus monkey presented with unilateral fremitus and tachycardia during a routine intake physical examination at the Wake Forest University Primate Center. Electrocardiography showed a tall R wave and an elongated T wave (left ventricular enlargement suspected) and blood pressure was normal. At necropsy, the left iliac artery was 3mm in diameter (twice the size of the right iliac artery). The lungs had multifocal hemorrhages. Histologically, the intima of the left iliac vein was irregularly thickened up to 500 microns by fibroblasts, smooth muscle cells and loose, pale eosinophilic extracellular matrix. Verhoeff van Gieson staining demonstrated a sharp transition from an arterial to venous morphology in transverse sections of the lesion, consistent with an arteriovenous fistula. The arterial side of the fistula had internal and external elastic lamina that ended sharply and the other half of the vessel had an irregularly thickened intima. The lungs had multifocal interstitial pulmonary hemorrhages with erythrophagocytosis, fibrosis and histiocytic and neutrophilic inflammation, consistent with chronic/repeated embolism. The intimal thickening of the iliac vein is consistent with jet lesions through the arteriovenous shunt. Since this monkey was experimentally naive, the arteriovenous shunt is believed to be congenital.
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Immunohistochemical Labeling of Two Canine Dysgerminomas With Protein Gene Product 9.5 and C-Kit
K.N. Corps1,2, J.M. Cullen1. 1Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC and 2NIH Comparative Biomedical Scientist Training Program, National Institute of Neurological Disorders and Stroke, Bethesda, MD Dysgerminomas are rare ovarian tumors arising from primordial germ cells and are considered the ovarian correlate of the more common seminoma. Dysgerminomas have been reported in most domestic species and humans, with canines highly represented among veterinary patients. Mitotic rate and pleomorphism do not correlate with clinical behavior. Immunohistochemical features are not well characterized. A recent report described immunohistochemical findings in one bitch with a dysgerminoma, characterized by positive staining for vimentin and ALP and a lack of staining for CD3, CD79a, CK, inhibin-a, and S-100. C-Kit was also recently suggested as a diagnostic marker for dysgerminoma and has shown positive staining in seminomas in several species. Protein Gene Product (PGP) 9.5 has been detected immunohistochemically in healthy neuronal tissue as well as human oocytes and spermatogonia and in neoplastic testicular germ cells. PGP 9.5 is a member of the ubiquitin carboxyl-terminal hydrolase family. PGP 9.5 staining has previously been demonstrated in neuroendocrine tissues and in testicular germ cells in a study of mixed germ cell sex-cord stromal tumors. PGP 9.5 and c-Kit immunohistochemical stains were applied to sections from two canine dysgerminomas. We demonstrate diffuse, intense cytoplasmic staining of neoplastic cells with PGP 9.5 and multifocal, granular, cytoplasmic staining with c-Kit. S-100, applied to these sections as a negative marker for comparison, had only rare cytoplasmic staining of neoplastic cells and strong, cytoplasmic staining of stromal spindle cells. PGP 9.5 is a potential diagnostic marker for canine dysgerminomas. C-Kit may provideadditional support for a diagnosis of dysgerminoma.
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Improving the Sensitivity of a Canine T Cell Molecular Clonality Assay
S.M. Keller and P.F. Moore. Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA, USA Molecular clonality assays are an important adjunctive tool for the diagnosis of lymphoma. Recently, we developed a new canine T cell clonality assay based on the full description of the canine T cell receptor gamma (TRG) locus. The assay covered all rearranged genes through a sophisticated multiplexing strategy. This led to the detection of multiple clonal rearrangements unique to each case and improved sensitivity over existing assays. However, routine use in clonality diagnostics revealed a potential shortcoming of the new assay. In lesions with a high proportion of non-neoplastic lymphocytes, the polyclonal background can potentially obscure clonal populations. The current study explored different strategies to further improve the sensitivity of the assay in situations with a high polyclonal background. We found that the fewer primers used, the less dominant the polyclonal background. Given that canine T cell lymphomas generally have multiple TRG rearrangements, a reduction of primers is tolerable if at least one clonal rearrangement is detected. However, omission of primers has to be done based on V/J gene usage to avoid exclusion of frequently used genes. Clonal rearrangements in a series of T cell lymphomas were sequenced. Genes in cassettes 2, 3 and 7/8 were most frequently rearranged. V gamma 2 genes preferentially rearranged to 3 J gamma genes and V gamma 3/V gamma 7 genes rearranged to 5 J gamma genes. Based on these findings, the current primer set was modified. Additional strategies included the development of V gene specific primer sets and the splitting of primers into multiple tubes. Together, these approaches further increased the sensitivity of the clonality assay.
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Medium-Grade (Anaplastic) Astrocytoma in a Cougar (Puma concolor)
H. Kondo1, A.M. Leone1, J. Gary2, M. Kiupel, L.L. Farina1, and J.R. Abbott1. 1 Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL, 2Diagnostic Center for Population and Animal Health, Department of Pathobiology and Diagnostic Investigation, Michigan State University, Lansing, MI A 17-year-old, male castrated cougar (Puma concolor) presented with lethargy, depression and neurologic signs, including inability to stand, dilated pupils and lack of pupillary light reflex. Significant gross findings included severe, degenerative, coxofemoral joint disease, bilateral thyroid adenomas, and moderate endocardiosis of the mitral and tricuspid valves. Sectioning of the fixed frontal lobe revealed a tan to gray, invasive, cavitated mass on the ventral aspect of the left cerebral hemisphere, rostral to the caudate nucleus. Histologically, the mass was composed of poorly-demarcated, infiltrative sheets of polygonal to fusiform neoplastic cells supported by a fine, fibrillar stroma, with islands of residual neuropil. The neoplastic cells had moderate eosinophilic cytoplasm and ovoid nuclei with coarsely-clumped chromatin and an occasional basophilic nucleolus. Rare large, polygonal cells with abundant, eosinophilic cytoplasm and ovoid nuclei with coarse chromatin were scattered throughout the mass. Other findings included moderate cellular pleomorphism and nuclear atypia, as well as a high mitotic rate. The cytoplasm of approximately 80% of the neoplastic cells had abundant strong labeling for glial fibrillary acidic protein (GFAP). Scattered cytoplasmic processes expressed S-100 protein and neurofilament. The neoplastic cells did not express CD3, CD79E or synaptophysin. These findings were consistent with a medium-grade astrocytoma, also known as an anaplastic astrocytoma. To our best knowledge, central nervous system tumors have not been previously reported in cougars, and this is the first report of an astrocytoma in a cougar or other large felid.
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Leukodystrophy in a Juvenile Labrador Retriever
C. Bradley , J. Cross , and T. Van Winkle . Department of Pathobiology, Anatomic Pathology and 2Department of Clinical Studies, Neurology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia PA A 9 month old Labrador retriever dog presented for progressive ataxia of 1 month duration, characterized by intention tremors, mild tetraparesis and a dysmetric bouncing gait, with normal mental status and postural reactions. Pertinent history included a presumed seizure in association with distemper vaccination at 4 months of age, although subsequent vaccinations were not associated with any neurologic changes. Magnetic resonance imaging of the brain revealed diffuse, symmetrical, hyperintense lesions within white matter tracts on T2 and diffusion weighted images, consistent with a leukoencephalomyelopathy. Serum chemistry, complete blood cell count, and cerebrospinal fluid analysis were within normal limits. Due to progression to nonambulatory status, euthanasia was elected. Gross necropsy was unremarkable. Histopathology revealed disease isolated to the central nervous system. Throughout the brain and spinal cord, confined to the white matter, there was moderate to severe bilateral symmetrical demyelinaton with extensive gliosis, myelin sheath dilation with myelomacrophages, and vascular prominence with perivascular globular and fibrillar eosinophilic material. Representative slides were stained with Massons trichrome and luxol-fast blue and for glial fibrillary acidic protein (GFAP) and neurofilament. Luxol blue confirmed the myelin loss and the perivascular material did not stain with trichrome or luxol blue. GFAP demonstrated a marked astrocytosis in affected areas with intracytoplasmic spherical structures (GFAP positive-globular eosinophilic structures) and prominent, thickened cytoplasmic foot processes (GFAP positive-eosinophilic fibrillar structures). Neurofilament staining demonstrated axons were intact in affected white matter. Sections of brain and spinal cord were negative for canine distemper virus antigen. To the authors knowledge this form of leukodystrophy has not been previously characterized and may represent a novel canine leukodystrophy.
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Megalocytivirus in Ornamental Fishes
B. Manickam1, T. Waltzek2, and A. Camus1. 1University of Georgia College of Veterinary Medicine, Department of Pathology, Athens, GA and 2University of Florida, Department of Environmental and Global Health, Gainesville, FL The family Iridoviridae comprises a diverse group of large ds DNA viruses that infect mainly invertebrates and vertebrate species, such as fish, amphibians and reptiles. Members of the Lymphocystivirus, Ranavirus, and Megalocytivirus genera represent a group of well known and emerging fish pathogens. In particular, megalocytiviruses (MCV) are important pathogens producing systemic disease and mortalities up to 100% in over 112 species of fresh and saltwater ornamental and cultured food fish. All isolates are currently considered strains of the same viral species, infectious spleen and kidney necrosis virus (ISKNV). This report details histopathology, electron microscopy and PCR results from a pearl gourami Trichogaster leeri that died from megalocytivirus infection. The fish was one of a group experiencing chronic low level mortalities while in quarantine following shipment from SE Asia. Microscopic examination revealed moderate numbers of severely hypertrophied cells in multiple organs characterized by large, smudgy to finely granular basophilic cytoplasmic inclusion bodies that peripherally displaced nuclei. Inclusions were typically found immediately adjacent to vascular lumens. Tissue changes were limited to mild necrosis of hepatocytes, the pancreatic interstitium, and skeletal muscle. More significant necrosis was associated with widespread splenic hemorrhage and hilar fibrin deposition. Transmission electron microscopy revealed icosahedral, 125-135 nm virus particles consistent with iridoviridae. PCR and sequence results were identical to a 156 bp segment of the ISKNV major capsid protein. Similar diseases are emerging in increasing numbers of fish species, due in part to the global shipping of infected fish; therefore, it is important to recognize typical signs and lesions. There are no effective treatments and depopulation followed by disinfection is recommended.
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Mesenteric Torsion in a Harbour Porpoise (Phocoena phocoena)
L. Begeman, S. Hiemstra and R.I. Fleis. Department of Pathology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands An adult, female harbour porpoise (Phocoena phocoena) in good body condition presented for necropsy in November 2010, part of a large comprehensive study of stranded harbour porpoises in the Netherlands. The animal stranded alive but died with no clinical signs during transport to a rehabilitation facility. At necropsy, four hours post death, the animal had a mesenteric torsion of 360? counterclockwise from the ventral aspect, involving the aboral third of the intestines. The mesenteric lymph nodes and the intestines were hyperemic and edematous with dark red or brown to black fluid contents. With a sharp demarcation, the uninvolved intestines were pale tan to pink and contained deep green fluid (bile). The mesenteric lymph nodes involved in the torsion were moderately enlarged, and bulging, wet and mottled on cut surface. Approximately five liters of turbid, malodorous, red-brown fluid was within the abdominal cavity. Histologically, the intestines involved in the torsion were characterized by severe congestion, edema, and variable necrosis of the mucosa with mild to moderate numbers of neutrophils. In some areas, a layer of bacteria, predominantly large bacilli, covered the mucosa. In other areas, there was complete loss of mucosa with expansion of the underlying tunica muscularis by variably sized optically empty spaces lined by rod shaped bacteria (emphysema). From the intestinal contents Clostridium perfringens was cultured and PCR identified it as type A. Suspecting this was most likely secondary overgrowth, a definitive underlying cause for the torsion could not be determined. Data from our study cohort and the current literature suggest that mesenteric torsions are rare lesions in harbour porpoises. The unusually short postmortem interval makes this case remarkable.
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Naked Mole-Rat Skin and Soft Tissue Metastatic Mineralization
A.J. Crews, E. Ramsay and S. J. Newman. Departments of Biomedical and Diagnostic Sciences and Small Animal Clinical Sciences, University of Tennessee, Veterinary Medical Center, Knoxville, TN This is a report of skin and/or soft tissue metastatic mineralization which is occuring in naked mole-rats (NMRs), Heterocephalus glaber, at the Knoxville Zoological Gardens (KZG). In a recent poll of 14 zoological parks, 6 (48%) zoos are seeing similar mineralizations. The objective of this report is to characterize lesions grossly and histologically and to briefly discuss the theoretical pathogenesis for the development of these lesions. Pathologic lesions in KZG NMR necropsy and biopsy cases show a distribution varying from focal to multifocal with disseminated dermal and visceral foci. Grossly, lesions are irregularly round, multifocal to coalescing, white to tan, chalky nodules which range in size from 1mm to 5mm in diameter. Histologically, nodules are small to large lakes of deeply amphophilic material (mineral) that are surrounded by minimal granulomatous to pyogranulomatous inflammation. We theorize the development of mineralization seen in the NMR from KZG is due to excess levels of vitamin D and/or calcium in their captive diets. In the wild, NMR are underground dwellers and are naturally deficient in the prohormone cholecalciferol (vitamin D3), which is a precursor of 1, 25 dihydroxyvitamin D3 (calcitriol) and have no obvious dietary source of vitamin D. However, even with an impoverished vitamin D status, studies have shown that naturally vitamin Ddeficient NMR are able to independently maintain mineral homeostasis without appreciable levels of cholecalciferol. We hope to better characterize the mechanisms of calcium and vitamin D metabolism in this unique species with ongoing captive diet changes and future measurement of serum vitamin D and calcium levels.
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Nonfunctional Thyroid Carcinoma in a Captive Cougar (Felis concolor)
B. Lewis1, W. McGee2, A. Ambrus3, and S.Jaques1. 1Texas Veterinary Medical Diagnostic Laboratory, College Station, TX, 2Bridgeport Animal Hospital, Bridgeport, TX, and 3Department of Veterinary Pathobiology, Texas A&M University, College Station, TX A 21-year-old, intact female cougar (Felis concolor) in good body condition was examined by the submitting veterinarian for a non-painful, subcutaneous mass in the right caudal cervical area. The mass had been present for several years, but recently was enlarging dramatically. An exploratory surgery revealed an approximately 14 cm diameter, fluid-filled mass that incorporated the cervical neck musculature, blood vessels and nerves. Grossly, the fixed mass was firm and multiloculated with encapsulated cystic spaces ranging from 0.5-1.0 cm in diameter. Protruding into the lumina were multiple firm, white to grey, closely packed, granular to nodular masses up to 0.5 cm in diameter and variably covered with clotted blood. Histologically, the nodular masses were composed of tall cuboidal cells forming closely packed trabeculae and variably sized follicles containing brightly eosinophilic colloid. The mass infiltrated at least one lymph node. Colloidal thyroglobulin immunoreactivity was strong and there was strong nuclear immunoreactivity for thyroid transcription factor-1 (TTF-1). Serum thyroid concentrations measured T3 0.610 ng/ml, T4 1.97 ug/dl and free T4 17.70 pmol/L, and appeared to be in the normal range for cougars, according to previously published data. Thoracic radiography indicated metastatic disease; however, the cougar is alive to date.
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Necrotizing Enteritis Caused by Clostridium perfringens Type AE in Foals
K.A. Potter, J.L. Oaks, R.H. Mealey, and T.E. Besser. Washington Animal Disease Diagnostic Laboratory and Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA Clostridium perfringens is a gram positive anaerobic bacterium that causes enteric disease in a variety of species. The species is divided into types A, B, C, D and E dependent upon their repertoire of exotoxins. C. perfringens type C (CPC) is the type most commonly associated with necrotizing enteritis in foals, although another species, C. difficile (CD), is also associated with necrotizing enterocolitis in horses of all ages. C. perfringens type A (CPA) is present as normal flora in most domesticated mammals. CPA that also produces enterotoxin (CPAE) has been associated with simple diarrhea in humans. During 2000-2010, CPAE was isolated from 12 cases of necrotizing enteritis in neonatal foals submitted to the Washington Animal Disease Diagnostic Laboratory. In 3 of these cases CD was also detected. The age distribution, clinical signs and gross lesions were similar to those reported for CPC infections in foals. When one pathologist (KAP) blindly reviewed histologic lesions, CPAE, CPC and CD cases could not be distinguished by histopathology. We conclude that CPAE may be a significant cause of necrotizing enteritis in neonatal foals and that routine genotyping of C perfringens should include the enterotoxin gene.
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Nonspecific Rabies-like Inclusions in the Brain of a Mixed Breed Dog
C.J. Ramirez, G.C. Johnson, D.Y. Kim, and K. Kuroki. Veterinary Medical Diagnostics Laboratory, University of Missouri, Columbia, MO A 7-year-old Poodle/Yorkshire terrier mixed breed canine died unexpectedly three days after an episode of seizure activity and paraparesis following administration of annual vaccinations. On post-mortem examination no significant gross pathologic finding were observed. Histopathologically, Negri-body like intraneuronal inclusions were observed in the brain along with ischemic necrosis of the spinal cord between T11 and L3. Bilaterally, cell bodies in the lateral and medial geniculate nuclei of the thalamus had round to oval, granular, eosinophilic, intracytoplasmic inclusions. A few Purkinje cells also had elongate, eosinophilic inclusions. The inclusions were periodic acid Schiff (PAS) negative. PCR and immunohistochemistry for canine distemper virus were also negative. There were no notable microscopic lesions in the heart, lung, liver, spleen, kidneys, or intestines. Although the animal had a history of annual rabies vaccination and there was no history of wild animal exposure, rabies could not be definitively ruled out. Paraffin blocks were sent to the Centers for Disease Control and Prevention and found to be negative for rabies antigen by immunohistochemistry. The cause of this animals spinal cord infarction and death could not be determined. Previous reports have demonstrated that rabies-like intracytoplasmic neuronal inclusions can be observed in nonrabid dogs; however, the nature or the etiology remains undetermined. Ten of the 22 reported cases, including the animal in the present study, were Poodles or Poodle mixed breeds suggesting a breed predisposition. Veterinary pathologists should be aware that rabies-like inclusions can occur in nonrabid dogs, and fluorescent antibody testing or immunohistochemical testing is important to rule in or rule out rabies when intraneuronal inclusions are present in histologic sections of brain.
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Polyglandular Autoimmune Syndrome Type II in a Dog
V.E. Watson and E.W. Howerth. Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA Polyglandular autoimmune syndromes are rare endocrine disorders that include type I, II, and III in human medicine, and each type is associated with two glandular immune-mediated diseases. Polyglandular autoimmune syndrome type II (PAS type II) has been documented in humans and dogs, and is characterized by autoimmune inflammation and destruction of two of the following three organs: adrenal gland, thyroid gland, and pancreas. This causes hypoadrenocorticism, hypothyroidism, and/or diabetes mellitus. Additionally, in human medicine, PAS type II occurs more frequently in females and between the ages of 20 and 60. We report a case of a four-year-old, spayed, female, Great Dane that presented severely hypothermic and minimally responsive. An ante-mortem thyroid profile was performed and revealed low T3, low T4, and high TSH, with subsequent diagnosis of hypothyroidism. A necropsy was performed and both the thyroid and adrenal glands were bilaterally atrophied. Histopathologically, the thyroid glands were diffusely effaced by an infiltrate of lymphocytes and plasma cells, with few macrophages and neutrophils. The adrenal glands were severely atrophied and there were few, small aggregates of lymphocytes and plasma cells within the cortices. Immunohistochemistry using antibody against cluster of differentiation (CD) 3 to detect T cells and CD79 for B cells established that the lymphocytes in the thyroid gland were 40% T cells and 60% B cells, whereas in the adrenal gland percentages were 50% each. Due to the lymphoplasmacytic inflammation and destruction of the thyroid and adrenal glands, a diagnosis of PAS type II was made. This represents a recently recognized, immune-mediated and endocrine disease in dogs.
84
Osteogenic Sarcoma in a Guinea Pig
M.R.S. Ilha. Veterinary Diagnostic and Investigational Laboratory, University of Georgia, Tifton, GA A 2-year-old male guinea pig had its right front leg and an axillary subcutaneous mass removed and submitted for histopathology. The distal front leg was swollen and a 2cm in diameter and 3.5cm long mass was present at the level of the ulna and radius extending to the carpus. The mass was surrounding and partially effacing these bones. On cut section, the mass was gray to white, hard and gritty. An axillary subcutaneous mass was also present and 1 x 0.5 x 1 cm, hard and white. Microscopically, the leg mass was composed of multiple thin islands and trabeculae of osteoid and bone intermingled with variable amounts of osteoblastic neoplastic cells and loosely arranged connective tissue stroma. The mass infiltrated through the medullary cavity of pre-existing bone. Bone resorption, periosteal new woven bone formation, invasion and compression of adjacent skeletal muscle, and muscle atrophy were additional findings. The subcutaneous mass from axillary region was consistent with a lymph node almost completely replaced by a similar neoplastic mass (metastasis). The guinea pig was apparently well in a follow up clinical evaluation 3 months after surgery. No radiographs of the thorax were taken at that time. Osteogenic sarcomas (osteosarcomas) are rarely reported in guinea pigs. Two cases of osteogenic sarcomas originating from the lumbar vertebrae have been reported in inbred strains of guinea pigs. Metastatic disease to multiple organs was described in one of the affected animals. A case report of an extraskeletal multicentric osteogenic sarcoma in a guinea pig that consisted of multiple subcutaneous submandibular, cervical, and thoracolumbar masses, with involvement of liver, spleen, kidneys, and omentum was alsoreported. A few additional reports are also referred in older literature.
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Polymyositis and Addisons Disease in a Dog
A.G. Cino Ozuna and G.A. Andrews. Department of Diagnostic Medicine/ Pathobiology, Kansas State University, Manhattan, KS An eight-year-old male castrated Labrador Retriever dog was presented for necropsy to the Kansas State Veterinary Diagnostic Laboratory (KSVDL) with a history of regurgitation and megaesophagus. The dog was previously diagnosed with Addisons disease. Gross findings included megaesophagus, right sided cranial ventral pneumonia, and severe atrophy of the left adrenal gland. The right adrenal gland was not found. Microscopic examination revealed polymyositis characterized as a histiocytic and lymphoplasmacytic myositis with myofiber necrosis, regeneration and fibrosis in the hind and fore limb muscles and similar myositis with eosinophils in the tunica muscularis of the esophagus. There was also histiocytic and lymphoplasmacytic myocarditis with cardiocyte degeneration in the heart. The left adrenal gland had severe cortical atrophy with mild lymphoplasmacytic adrenalitis. In addition, there was acute suppurative bronchopneumonia. The histologic findings in the muscle are those of a generalized inflammatory myopathy (polymyositis). Generalized inflammatory myopathies can be classified as infectious or immunemediated. Infectious myositis can be caused by protozoal organisms, bacterial infections, and migrating parasites. No infectious agent was identified by routine histopathology. Immunohistochemistry for Neospora caninum and Toxoplasma gondii were both negative in all the tissues. PAS, GMS, and Gram stains did not reveal the presence of infectious agents. The findings in skeletal muscle, heart, and esophagus are similar to those reported for immunemediated polymyositis in dogs and humans. The lesions in the adrenal glands are consistent with Addisons disease, which is commonly caused by immunemediated adrenalitis in dogs. There are no reports linking Addisons disease with polymyositis in dogs; however, individuals affected by more than one autoimmune disease are described in dogs and humans. An overlap of immune-mediated polymyositis and adrenalitis is suggested.
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Renal Cystic Disease in Dolphins From Brazil: Report of Three Cases
O. Gonzales-Viera , V. Ruoppolo , J. Marigo , C.P. Bertozzi , V.L. Carvalho4, and J.L. Catao-Dias1. 1Laboratorio de Patologia Comparada de Animais Selvagens (LAPCOM), Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo, Brasil, 2International Fund for Animal Welfare (IFAW), Yarmouth Port, MA, USA, 3Projeto BioPesca, Brasil, and 4 Associacao de Pesquisa e Preservacao de Ecossistemas Aquaticos (Aquasis), Brasil Renal cystic disease (RCD) is a term comprising hereditary, developmental or acquired kidney disorder uncommonly reported in cetaceans. This study reports RCD in two adult males, a Rough-Toothed dolphin Steno bredanensis (Sb) and a Franciscana dolphin Pontoporia blainvillei (Pb), and an adult female Striped dolphin Stenella coeruleaoalba (Sc). These animals were studied during the pathological assessment of 158 cetacean kidneys from the Marine Mammal Tissue Bank - LAPCOM. Grossly, kidneys of Sb were markedly enlarged (35 cm long), and appeared as a solid mass composed of multiple cysts (1-4 cm diameter) with sparse renal parenchyma. Pb presented simple or multiple renal cysts (0.3-0.5 cm diameter) in the cortex of each reniculus, filled by a homogeneous, red-brownish substance. Sc had few renal cysts (0.3-0.4 cm diameter) with multifocal distribution. Microscopically, the cysts present in all three animals shared characteristics such as uniform lining of flattened to cuboidal cells with variable amount of acidophilic serous fluid with hyperplasia of the tubular epithelia. Individual characteristics included, markedly large cysts and disorganization of renal architecture (Sb), large cysts (cortex) and small cysts (medulla) with connective tissue thickened (Pb), and small cysts (mostly in medulla), accompanied by chronic tubule interstitial nephritis and glomerulosclerosis (Sc). Regarding gross and histopathological features, RCD was classified as polycystic kidney (Sb), simple cysts (Pb) and acquired RCD (Sc). These are the first records of renal cystic disease in these dolphin species. Acknowledgement: FAPESP/CNPq.
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Reovirus-Induced Meningoencephalomyelitis in Baboons
S. Kumar1,2, M.A. Owston2, Y.R. Bommineni3, G.B. Hubbard2, and E.J. Dick Jr2. 1Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, 2Southwest National Primate Research Center at Texas Biomedical Research Institute, San Antonio, TX, and 3Division of Veterinary Diagnostic Services, New Mexico Department of Agriculture, Albuquerque, NM Spontaneous viral encephalitis is rare in baboons (Papio spp.). During 1993-94 an outbreak of viral meningoencephalomyelitis (MEM) occurred among the captive baboon population at Southwest National Primate Research Center (SNPRC), San Antonio, TX. The etiologic agent was identified as a novel orthoreovirus: baboon reovirus (BRV). Sporadic cases of suspected BRV MEM were observed at SNPRC, but could not be confirmed due to a lack of diagnostic assays. We developed a novel immunohistochemistry-based assay to detect the presence of BRV. An internal database was searched for diagnoses of encephalitis, meningoencephalitis, encephalomyelitis, myelitis and meningitis from the years 1989 to 2010. All spontaneous cases with no other specific etiology were selected (n 71; includes 4 virus isolation positive controls). Using standard immunohistochemistry techniques and monoclonal antibodies we tested for BRV, Alphavirus and Flavivirus. Out of the 71 cases tested, 62 were positive for BRV, 1 was positive for Flavivirus (but negative for West Nile and St. Louis Encephalitis viruses by PCR), and 8 tested negative for all three. One of the positive BRV controls failed to test positive by immunohistochemistry. Our results indicate that BRV is endemic in the SNPRC baboon colony and accounts for the majority of viral MEM cases in baboons. This assay would be a valuable tool in confirming the presence of BRV in baboons with MEM.
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Renal Leiomyosarcoma in a Cat With Chronic Renal Failure
N. W. Fowlkes1, B. A. Berryhill2, D. E. Evans1. 1Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA and 2Staring Plaza Veterinary Center, Baton Rouge, LA A 10-year-old, spayed female, domestic shorthaired cat presented for necropsy after a 3-year history of managed, chronic renal disease and sudden death immediately preceded by a brief episode of mental dullness and confusion. Grossly, the left kidney was enlarged and rounded compared to the right, and measured 4.0 x 4.0 x 4.0 cm. On the cut surface, the renal cortex and medulla were effaced and replaced by a single, large, cavity containing approximately 15 ml of clear, slightly viscous, mucinous fluid. An approximately 5.0 mm diameter nephrolith was present in the pelvis of the right kidney with mild pyelectasis. On histopathology of the left kidney, the renal cortex and medulla were effaced by a poorly demarcated, infiltrative, unencapsulated, poorly differentiated, spindle cell neoplasm. Invasion beyond the capsule was present with neoplastic cells surrounding the left adrenal. Immunohistochemical staining was consistent with a leiomyosarcoma. Evidence of chronic, lymphoplasmacytic interstitial nephritis and glomerulosclerosis were present with nephrolithiasis and peripelvic fibrosis in the right kidney. Renal leiomyosarcoma is an uncommon neoplasm in humans and has also been reported rarely in the dog. To these authors knowledge, there are no published reports of renal leiomyosarcoma in cats. The origin of primary renal leiomyosarcoma is thought to be the capsule, pelvis, or vasculature of the kidney. The role of chronic renal disease in the development of renal leiomyosarcoma in this patient is uncertain.
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Safe for Humans, Toxic for Dogs: A Report of Xylitol Toxicosis
P.S. Malhi, B. Bawa and B. DeBey. Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS A 6-year-old, male, neutered Golden Retriever/Standard Poodle mix dog was presented to the referring veterinarian with a history of ingesting an unknown amount of xylitol sweetener and an acute onset of vomition and diarrhea of 48 hours duration. A blood sample was submitted for CBC and serum chemistry. The dog did not respond to therapy, and died approximately 45 minutes after presentation. Necropsy conducted by the referring veterinarian revealed free blood in the abdominal cavity, and multifocal haemorrhages subcutaneously on the ventral trunk, on the intestinal serosal surface, cranial lung lobes, and on the cranial pole of the right kidney. The liver was congested. The stomach contained thick, slimy, dark-brown substance. Tissues were submitted for histopathology. The dog had elevated serum alanine aminotransferase (22862 IU/L; reference range 12-118), elevated alkaline phosphatase (364 IU/L; reference range 5-131), elevated total bilirubin (2.3 mg/dL; reference range 0.1-0.3), elevated hematocrit (64%; reference range 36-60), elevated BUN (43 mg/dL; reference range 6-31), slightly elevated creatinine (2.1 mg/ dL; reference range 0.5-1.6) and decreased glucose (31 mg/dL; reference range 70-138) concentrations. The hepatic parenchyma had diffuse, massive hepatocellular necrosis and loss. The necrotic hepatocytes were characterized by homogenous, vacuolated, eosinophilic cytoplasm with lack of nuclei, pyknotic nuclei or presence of karyorrhectic debris. The hepatic lobules were diffusely filled with erythrocytes. There were occasional viable hepatocytes remaining around portal areas. Multifocal haemorrhages were also observed in the heart, lungs, small intestine, colon and mesenteric adipose tissue. The capsule of the spleen was wrinkled (splenic contraction). A diagnosis of massive hepatocellular necrosis secondary to xylitol toxicity was made based on history, clinical pathology and histopathology findings.
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Splenic Marginal Zone Lymphosarcoma in an Asian Small Clawed Otter (Aonyx cinerea)
N. L. Stedman, P. A. Black, M. S. Burton, and A. Cole. Busch Gardens Tampa, Tampa, FL Severe splenomegaly was found during routine examination of a clinically normal 7 year old male Asian small clawed otter. The spleen and three adjacent enlarged splenic lymph nodes were immediately removed. An enlarged pancreaticoduodenal lymph node that could not be resected was left in the abdomen. Grossly, the spleen weighed 310 g (approximately 7% of the otters body weight). The spleen and resected lymph nodes were infiltrated by coalescing nodules of grey-white smooth soft tissue. Tissue imprints of the spleen and nodes were consistent with lymphosarcoma. Histopathology revealed severe effacement of the splenic and lymph node architecture by coalescing sheets of neoplastic medium-sized lymphocytes forming expansive nodules reminiscent of follicles but without any distinct follicular architecture. Some nodules of neoplastic lymphocytes surrounded a central round core ofnonneoplastic lymphocytes and occasional hyaline material suggestive of remnants of follicles. Most neoplastic cells had a vesicular chromatin pattern with a single large nucleolus. Immunohistochemically, the neoplastic cells exhibited cytoplasmic reactivity with antibodies against CD79a and BLA36, but not with antibodies against CD3. Histopathologic and immunohistochemical findings were consistent with primary splenic marginal zone lymphosarcoma (MZL), an indolent form of B cell lymphosarcoma that spreads slowly to the abdominal and extra-abdominal lymph nodes. Despite residual disease, the otter has remained clinically normal four months after splenectomy. To the authors knowledge, although primary alimentary lymphosarcoma has been rarely reported in other otter species, primary splenic lymphosarcoma has not been reported in any otter species.
93
T CellRich B Cell Lymphoma With Mott Cell Differentiation
T. Kurotaki, A. Royal, K. Banajee, L. Gaschen, and R. Bauer. Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA A 4-year-old female, Labrador Retriever, was presented with one week history of right sided head tilt and severe vestibular signs with right sided cranial nerves 5, 7, and 8 deficits. MRI revealed multiple lesions in brain stem and cerebellum along with fluid in the right tympanic bulla. Aspiration of right tympanic bulla revealed chronic mixed inflammation. CSF tap revealed lymphocytic pleocytosis withunusual number of Mott cells. At necropsy, there was a space occupying lesion on the right caudoventral side of the brain covering the ventral brainstem, right ventral cerebellum, right pyriform lobe, pituitary, and extending to the right ventral cerebrum. The space occupying lesion obscured the view of cranial nerves 5 through 9 on the right side. The ileoceal lymph node was enlarged. Microscopically, the brain is characterized by an expansile, infiltrating, unencapsulated, densely cellular round cell tumor with features indicative of a lymphosarcoma. Occasionally, there are small numbers of Mott cells that have intracytoplasmic eosinophilic globules (Russell bodies). On immunohistochemistry, the majority of the cells are positive for CD 20 with a minor percentage which are CD3 positive. Therefore, the tumor is consistent with a T cell rich B cell lymphoma that also has Mott cell differentiation. T cell rich B cell lymphoma with Mott cell differentiation and extension to the brain and cranial nerves has not been reported previously.
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Suspected Primary Immunodeficiency in Pigs
A.G. Cino Ozuna , R.R. Rowland , J.C. Nietfeld , and J.C. Dekkers . Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS and 2Department of Animal Science, Iowa State University, Ames, IA Severe lymphoid hypoplasia/depletion was identified in four 5 to 7 weeksold pigs that died unexpectedly or were euthanized because of illness during a porcine reproductive and respiratory syndrome virus (PRRSV) challenge study. Two of the pigs had been inoculated at 4 weeks of age with PRRSV, and two had not. At necropsy, all four were thin, had rough hair coats, and were smaller than their pen mates. One of the PRRSV inoculated pigs displayed antemortem clinical signs of respiratory disease and had severe fibrinosuppurative bronchopneumonia from which Streptococcus suis was isolated. The thymus was not found in any of thefour. Microscopically, there was diffuse, severe lymphoid hypoplasia/ depletion with a complete lack of follicles in lymph nodes, tonsils, and Peyers patches, and an absence of perivascular lymphoid sheaths in the spleen. Immunohistochemical staining for porcine circovirus type-2 was negative for all animals. Immunohistochemical staining revealed scattered T lymphocytes and an absence of B lymphocytes in lymph nodes, spleen, tonsil, and Peyers patches. The affected pigs were part of a group of 100 purebred Yorkshire pigs selected over 7 generations for increased feed efficiency. All parents were related, resulting in the offspring having 12-14% inbreeding. Each affected pig was from a different litter, but three had the same sire. Lymphoid tissues from the other 96 pigs in the group, including littermates, were normal. The lesions resemble descriptions of primary immunodeficiencies in other species. This suggests a possible genetic basis for the lymphoid hypoplasia/depletion in these pigs, possibly caused by a recessive mutation.
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Aberrant Heartworm Disease in Two Cats
B. Baughman and A.J. Cooley. College of Veterinary Medicine, Mississippi State University, Pearl, MS Although the incidence of heartworm disease in cats is considerably less than in dogs, aberrant migration of larvae to sites such as the central nervous system is believed to be more common. Cats have fewer adult heartworms than dogs and pulmonary arterial inflammation can be marked. Sudden death syndromes are a possible sequela. Different disease manifestations in cats are attributable to cats not being a natural host for heartworms versus dogs that are the definitive host. Two ectopic migrations of Dirofilaria immitis to brain and spinal cord are reported. A 2.5-year-old female spayed DSH cat presented for acute onset of lethargy, anorexia and ataxia. The cat was FeLV/FIV negative and current on vaccinations including rabies. Occult heartworm test was positive. The condition progressively worsened, the cat was euthanized and complete necropsy was performed. Cross sections of brain revealed an irregular hemorrhagic malacic tract spanning the right and left cerebral cortex, thalamus and caudal cerebellum. A single heartworm was present within the cerebral lateral ventricle. The lungs were expanded by edema and multifocal pulmonary arteries were enlarged by smooth muscular hypertrophy and marked intimal thickening. An 11-month-old female spayed DSH cat presented for acute onset of lethargy. The cat had a normal CBC, current vaccines including rabies, and was FeLV/FIV negative. The cat developed difficulty walking which progressed to paraplegia. Neurologic signs progressed to seizures and the cat died. Selected tissues including thoracic spinal cord were examined. A cross-section of a heartworm was identified compressing dorsal funiculus of the spinal cord and was associated with localized meningitis. Scattered inflammation, necrosis and gliosis were in the region of the cerebellum and cerebellar peduncle. The lungs had prominent pulmonary arterial hypertrophy and villous endarteritis.
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Arsenic Toxicosis in a Beef Herd
L.J. Baseler , , F.R. Bertin , J.E. Sojka-Kritchevsky , and S.D. Lenz . Departments of 1Comparative Pathobiology and 3Veterinary Clinical Sciences, Purdue University, West Lafayette, IN. 2National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, MT Twelve beef cows had access to a pasture where construction lumber had been burnt. Within 24h of exposure, one animal (cow 1) became recumbent and died without overt clinical signs. A second cow (cow 2) died but was not necropsied. Cows 3-6 subsequently developed ataxia, hind limb weakness, dehydration, hematuria and diarrhea, and were admitted to the Purdue University Veterinary Teaching Hospital for treatment. Lesions in the first cow included abomasal erosions and acute renal proximal tubular necrosis. Cow 6 was euthanized on day 5 post-exposure. Gross lesions included abomasal erosions and submucosal edema, segmental jejunal mucosal congestion, and urinary bladder mucosal petechiae. Histologically, there was proximal renal tubular necrosis with regeneration. Cow 3 developed hemolysis and icterus, and died 27 days post-exposure. At necropsy, the subcutis and perivisceral adipose was diffusely icteric. Arsenic was detected in wood and ashes, and toxic concentrations were detected in whole blood from sick cows (747 ppb) and kidney from cow 1 (15 ppm). High concentrations of arsenic were detected in urine, kidney, and liver from other animals. Arsenic toxicosis in these cattle was attributed to ingestion of ashes from burnt copper-chromium-arsenic treated lumber. Four deaths occurred 1 (2 animals), 5, and 27 days post-exposure; three animals were necropsied. Cows 1 and 6 had a spectrum of lesions that represented different post-exposure stages of tissue damage and regeneration. The lesions in cow 3 could not be directly attributed to arsenic ingestion.
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Cardiac Inflammatory Myofibroblastic Tumor in a Cat
D.K. Ajithdoss1, W.P. Sims1, B.S. Perry2, and B.F. Porter1. 1Department of Veterinary Pathobiology, 2Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station TX A two-year-old, spayed female, American domestic medium hair cat was presented to the Veterinary Medical Teaching Hospital at Texas A&M University with a three-day history of lethargy and sudden onset of dyspnea. On auscultation, the cat had muffled heart sounds and decreased ventral lung sounds. Ultrasound revealed a severe pericardial effusion and a mild pleural effusion. Due to poor prognosis, the cat was euthanized. At necropsy, a 1.5 x 1.2 x 2.4 cm, pink to dark red, round, firm mass was found protruding from the right auricle. Microscopically, the mass was composed of spindle-shaped cells arranged in interweaving fascicles and was uniformly infiltrated by mixed inflammatory cells (mostly CD3- positive lymphocytes with fewer plasma cells, neutrophils, and eosinophils). On immunohistochemistry, the spindleshaped cells were strongly positive for vimentin and smooth muscle actin. Only a few scattered cells were positive for other muscle markers (myoglobin, desmin, muscle specific actin, and sarcomeric actin). The cells were negative for both factor-VIII related antigen and cytokeratin. Based on the cellular morphology, inflammatory infiltrate, and immunohistochemistry results, a diagnosis of inflammatory myofibroblastic tumor (IMT) was made. This unusual lesion has been described in humans and a variety of animal species, including cats. While cardiac IMT is well documented in the human literature, to our knowledge, this is the first case report of cardiac IMT in animals.
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Canine Cutaneous Mast Cell Tumor: Correlation Between Histologic Grades and Metastasis to Regional Lymph Node
A.V. Desoutter, M.D. Sanchez, and M.H. Goldschmidt. Department of Patho biology, School of Veterinary Medicine, University of Pennsylvania Currently, several grading systems of canine cutaneous mast cell tumors are used by veterinary pathologists in an attempt to provide accurate prognostic information. However, the prognostic value of these grading systems is still under debate. The aim of this study was to determine the prognostic significance of the Patnaik grading system (grade I, II or III) and the recently described 2-grade histologic classification system (high or low grade). The histologic grades of 171 cutaneous canine mast cell tumors submitted with their regional lymph nodes to the Surgical Pathology Service at the University of Pennsylvania, School of Veterinary Medicine from 1990 to 2011 were evaluated using both histologic grading systems. The presence of metastasis in the regional lymph node submitted with the primary tumor was also assessed. 62% (107/171) of the tumors were given a high grade using the 2-grade system, and all of the high grade tumors were also given a grade III using the Patnaik system, except one case which was given a grade II. 96% (103/106) of the high grade/grade III tumors had metastasis in the regional lymph node. 37% (63/ 171) of the tumors examined were diagnosed as low grade/grade II, and 28% (18/63) of the latter showed metastasis to the regional lymph node. The one tumor that was given a high grade/grade II did not show metastasis to the lymph node. In conclusion, almost all high grade/grade III tumors metastasized to regional lymph nodes and approximately one third of the low grade/grade II tumors metastasized to the regional lymph node.
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Cardiac Necrosis in a Horse Secondary to Rattlesnake Envenomation
J. Trybus. Texas Veterinary Medical Diagnostic Laboratory, Amarillo, TX A two-year-old female, 400 kg Quarter Horse presented to the clinical veterinarian five days following a rattlesnake bite on the distal left front limb. Despite supportive care the animal developed a worsening fever, cardiac abnormalities (arrhythmia, tachycardia) and dependent edema. The animal was euthanized 18 days post-envenomation because of the worsening clinical condition and poor response to treatment. Tissues were submitted for evaluation following necropsy. Gross findings included widespread, coalescing foci of myocardial pallor of the left and right ventricular free walls and interventricular septum. There were petechial to ecchymotic endocardial hemorrhages. The liver had a prominent reticular pattern. Histologic findings include extensive, coalescing foci of myocardial degeneration and necrosis involving the endocardium, myocardium, and epicardium. Additionally, small myocardial arteries occasionally exhibit fibrinoid necrosis of the tunica intima and tunica media. Diffusely centrilobular to midzonal hepatocytes are necrotic with extensive mineralization. Snake bite envenomation is common in domestic animals. It is a complex poisoning generally associated with varying degrees of local tissue swelling and necrosis, hemorrhage, hypotension, and hematologic abnormalities (hemolysis, thrombocytopenia, and clotting disorders). These effects are mediated by various enzymes, polypeptides, and nonenzymatic proteins (cardiotoxins, neurotoxins, and myotoxins) contained in the venom. Severe cardiac disease has been previously reported in up to 16% of horses following rattlesnake envenomation.
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Classification of Canine Lymphoplasmacytic Lichenoid Dermatosis of the Nasal Planum by Immunohistochemistry
K.A. Mietelka , B. Steficek , E. Rosser , and I.M. Langohr, . Diagnostic Center for Population and Animal Health, 2Department of Pathobiology and Diagnostic Investigation, and 3Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, Lansing, MI Canine mucocutaneous pyoderma (MCP) and lupus-like disease of the nasal planum (LLDNP, formerly canine discoid lupus erythematosus) are lichenoid and/or interface dermatoses in dogs with overlapping clinical and histopathologic presentations, but different treatment protocols. The purpose of this study was to use archived cases of lymphoplasmacytic lichenoid dermatoses of the canine nasal planum to develop a prospective diagnostic tool using immunohistochemistry and histomorphologic criteria. Nine cases met all the selection criteria. The majority were ultimately diagnosed with lupus-like disease of the nasal planum (LLDNP) based on resolution of the lesions following treatment, generally with doxycycline and niacinamide. One case of LLDNP received steroids alone with clinical resolution. A single case of mucocutaneous pyoderma responded to cephalexin, mupirocin and niacinamide. The intralesional distribution and density of T-cells and complement (C3c) was evaluated utilizing immunohistochemistry. Apoptosis of basal cells was observed in 77% of LLDNP cases. In animals that did not receive steroids, 44% had multifocal to diffuse distribution of C3c in the epidermis, compared to similar staining in 22% of animals that received steroid therapy. Of animals with the highest density of CD3-positive cells (50-80% of the cell population, or grade 2 on a scale of 0-3), all were treated with cephalexin at some point. Nevertheless, none of these differences reached statistical significance. Future studies should evaluate a greater number of cases, which may be achieved by expanding selection criteria.
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Degenerative Encephalopathy in an Ibizan Hound
D.B. Whitley1, J.M. Levine2, S.C. Kerwin2, and B.F. Porter1. Texas A&M University, College of Veterinary Medicine and Biomedical Sciences, 1Department of Veterinary Pathobiology and 2Department of Small Animal Clinical Sciences, College Station, TX A ten-month-old, intact female Ibizan Hound dog was evaluated for a progressive gait abnormality of several months duration. Multiple relatives of the animal had displayed similar signs, and a hereditary neurologic disease was suspected. Clinically, the dog exhibited pronounced cerebellar ataxia in all limbs, bilaterally absent menace response, and intention tremors associated with the head and trunk. The neurologic exam findings localized the lesion to the cerebellum. On T2-weighted magnetic resonance imaging, there was severe, bilateral, symmetrical hyperintensity of the caudate nuclei. The cerebellar parenchyma was atrophied with enlargement of the sulci. On necropsy, the only gross lesion observed was a slight reduction in cerebellar size. Histologically, the cerebellar folia were atrophied with narrowing of the molecular layer, granular cell depletion, and moderate to severe loss of Purkinje cells. Degenerative changes were observed in the caudate, cerebellar, cochlear, and vestibular nuclei, and consisted of neuronal depletion, reactive astrocytosis, microgliosis, and spongiosis. The histologic changes were similar to those of hereditary striatonigral and cerebello-olivary degeneration of Kerry Blue Terriers. The findings in this case may represent a unique neurodegenerative disease in the Ibizan Hound, clinically similar but pathologically distinct from the degenerative myelopathy previously reported in this breed.
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Cutaneous Metastasis of a Malignant Pheochromocytoma in a Dog
C.E. Hoover, G.L. Mason, and E.J. Ehrhart. Department of Microbiology, Immunology, and Pathology and Veterinary Diagnostic Laboratory, Colorado State University, Fort Collins, CO Pheochromocytomas originate from the catecholamine-secreting cells of the adrenal medulla. In dogs, these tumors can be either benign or malignant with approximately 50% of malignant tumors reported to metastasize to regional lymph nodes, liver, lung, kidney, spleen, and bone. In this report, a 2 year old female spayed dog presented for necropsy with a 5 month history of intermittent vomiting and diarrhea and palpable tissue masses in the abdominal cavity and dorsal cervical skin. Postmortem examination revealed an approximately 14.0 x 10.0 x 12.0 cm multinodular abdominal mass in the region of the right adrenal gland with invasion into the caudal vena cava and an approximately 3.0 x 3.0 x 0.5 cm skin mass in the dorsal neck. Microscopically, neoplastic cells of both the primary abdominal mass and skin metastasis had similar neuroendocrine cell morphology. Examination of the contralateral adrenal gland revealed an additional microscopic focus of neuroendocrine cell neoplasia effacing the medulla. Neoplastic cells in all locations demonstrated positive immunoreactivity to synaptophysin and chromogranin A, confirming the diagnosis of malignant pheochromocytoma. This is the first report of malignant bilateral pheochromocytoma with metastasis to the skin in a dog. Pheochromocytoma with cutaneous metastasis is rarely reported in human medical literature and is only described in association with the genetic syndrome of multiple endocrine neoplasia (MEN) type II.
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Demodicosis in Free-Ranging White-Tailed Deer (Odocoileus virginianus) and Other Mammals in the Southeastern United States
N. Nemeth1, M. Ruder1, J. Brown1, B. Munk1, R. Gerhold2, and K. Keel1. 1 Southeastern Cooperative Disease Study, University of Georgia, Athens, GA and 2The Center for Wildlife Health, University of Tennessee, Knoxville, TN Demodex spp. are generally host-specific and have been well documented in many domestic animal species. Demodex odocoilei is a widespread, common, cutaneous commensal of white-tailed deer. We retrospectively reviewed cases of demodectic mange in free-ranging white-tailed deer (Odocoileus virginianus) and other mammals submitted to the Southeastern Cooperative Wildlife Disease Study. Demodectic mange infestations occurred in hunterharvested or vehicle-killed deer in eleven states from 1977-2011. Among 36 deer with demodectic mange, 13 (36%) were female and 22 (61%) were male (sex was undetermined in one individual). Most deer (32/36; 89%) were from 1.5-6.5 years of age and in adequate nutritional condition. Gross lesions varied from mild to generalized alopecia with raised nodules, crusting, scaling, and hyperpigmentation. Lesion distribution also varied but often included head, neck, limbs, and trunk. Microscopically, hair follicles were distended with mites, keratin, and karyorrhectic debris with perifollicular, lymphoplasmacytic inflammation, and occasional furunculosis, fibrosis, and secondary infections. Most deer with demodicosis were observed in fall and winter, coinciding with the rut and breeding season when hormone levels are elevated and food is relatively scarce. These factors could induce immunosuppression, which may predispose to, or be exacerbated by, mite infestations. Generalized demodicosis may reflect individual immunologic or genetic disorders. Demodicosis was also documented in four black bears (Ursus americanus) and two big brown bats (Eptesicus fuscus). Demodex ursi infestations in bears led to similar lesions to those observed in deer. Demodex sp. were in the skin over the muzzle of both bats. Population-level implications in all three species are likely minimal.
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Disseminated Gastric Squamous Cell Carcinoma in an Alpaca (Vicugna pacos)
S. Spagnoli, W.J. Mitchell, D. Nagy, and D.Y. Kim. University of Missouri College of Veterinary Medicine, Columbia, MO An eight year old female alpaca (Vicugna pacos) was presented to the veterinary teaching hospital in lateral recumbancy, and was euthanized due to poor prognosis. Post mortem examination revealed peritoneal and thoracic effusion with multifocal white nodules (ranging in diameter from 1-2mm) firmly adhered to the body wall, omentum, and to the serosal surfaces of abdominal viscera. Within the forestomachs, at the level of the C1-C2 junction, arising from the mucosa, invading transmurally, and surrounding the proximal aspect of C3, there was a firm, white, multilobulated, roughly dome-shaped mass (measuring approximately 10x14x14cm at its widest point). Similar dome-shaped to umbilicated masses (ranging in diameter from 2-7cm) were present multifocally throughout C3. Microscopically, the neoplasms were composed of sheets, islands, nests, and trabeculae of polygonal cells with distinct cell borders, frequent intercellular bridges, and abundant granular eosinophilic cytoplasm. Islands of neoplastic cells frequently demonstrated squamous differentiation, forming central accumulations of lamellated keratin. Neoplastic cells were strongly positive for cytokeratin by an immunohistochemical staining assay. The morphologic features and immunohistochemical staining of the neoplasm were consistent with a diagnosis of squamous cell carcinoma. Metastases were observed in the lung, liver, lumbar, and hepaticopancreatic lymph nodes. Gastric masses were negative for papilloma virus via immunohistochemical staining assays. Gastric squamous cell carcinoma is a rare neoplasm in domestic bovids. Among South American camelids, gastric squamous cell carcinomas have been reported in llamas (Lama glama) and guanacos (Lama guanicoe). To the authors knowledge, this is the first reported case of gastric squamous cell carcinoma in an alpaca.
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Equine Odontoclastic Tooth Resorption and Hypercementosis Syndrome
R. C. Smedley1, E. T. Earley2, S. Galloway3, R. M. Baratt4, and J. Rawlinson5. 1 Diagnostic Center for Population and Animal Health, Michigan State University, Lansing, MI, 2Laurel Highland Farm & Equine Service, LLC, Williamsport, PA, 3Animal Care Hospital, Somerville, TN, 4Salem Valley Veterinary Clinic, Salem, CT and 5College of Veterinary Medicine, Cornell University, Ithaca, NY Equine odontoclastic tooth resorption and hypercementosis (EOTRH) syndrome is a painful condition of older horses that involves both the canines and incisors. EOTRH is uncommonly recognized by veterinary pathologists and in some cases misdiagnosed as a cementoma. The etiology is unknown. The goal of this descriptive study was to classify the histopathological features of EOTRH in 11 affected horses from the United States and to increase awareness of this syndrome. Submissions ranged from one affected tooth to the entire rostral mandible and maxilla. All horses had cemental lesions which included hyperplasia, proliferation of irregular cementum, and lytic lesions with necrotic debris; bacteria (7) and plant material (5) were noted in some. Periodontal ligament lesions, primarily inflammation, were noted in 10 horses. Gingival lesions were noted in 4 out of 7 horses. All horses exhibited at least one dentinal lesion which included lysis (11) and necrotic debris (8). In one horse there was a large lytic area that extended from the cementum into the dentin that contained bacteria and plant material. Ten out of 10 horses had at least one lesion in the pulp cavity which included inflammation (6), lysis (2), necrotic debris (2), and fibrosis. In 2 of these 10 horses, a distinct pulp cavity was not seen due to a large lytic area that contained inflammatory cells, necrotic debris, bacteria, and plant material. Lesions in the alveolar bone were present in 4 out of 7 horses.
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Epizootic Hemorrhagic Disease in a Holstein Cow
R.B. Moeller Jr , P. Blanchard , B. Crossley , E. Henry , and A. Clavijo . 1 California Animal Health and Food Safty Laboratory, University of California, Tulare, CA, 2California Animal Health and Food Safety Laboratory, University of California, Davis CA, 3California Department of Food and Agriculture, Animal Health Branch, Tulare, CA, 4Texas Veterinary Medical Diagnostic Laboratory, College Station, TX Epizootic hemorrhagic disease virus (EHDV) is an Orbivirus closely related to bluetongue virus (BTV). Epizootic hemorrhagic disease is known to cause significant death losses in white-tailed deer and occasionally causes a bluetongue-like disease in cattle. In early September 2010, an adult (3.5 year old) Holstein cow was presented to the California Animal Health and Food Safety Laboratory with a history of sore feet and painful mouth with oral lesions on the dental pad. On clinical examination, the animal had excessive salivation, slight photophobia with conjunctivitis and ocular discharge. Further examination of the nose identified bilateral erosion of the nasal antrum where nose tongs had been used earlier. In the oral cavity ulcerative lesions up to *2 cm diameter and roughened eroded regions with hyperemia and hemorrhage involving the dental pad, particularly the ridges and lower lips were present. Also, erosion and necrosis of the conical papilla of the buccal mucosa was observed. Bilateral fetlock edema and edema and petechial in the coronary band and dewclaws were present. The right front foot had a linear ulcer running along the interdigital region. Testing for foot and mouth disease, bluetongue virus, malignant catarrhal fever, vesicular stomatitis virus, bovine papular stomatitis, bovine viral diarrhea virus, and Infectious bovine rhinotracheitis virus by PCR techniques was negative. Testing for epizootic hemorrhagic disease virus by RNA rtPCR was positive. Further testing by rtPCR using type specific primers, revealed the presence of EHDV-type 2.
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Fatal Canid Herpesvirus-1 Infection in an Adult Dog
B. Gadsden, I. Langohr, R. Maes, A. Wise, and M. Kiupel. Diagnostic Center for Population and Animal Health, Michigan State University Canid herpesvirus-1 (CHV-1) is a well known cause of fatal hepatic and renal necrosis in neonatal puppies. In adult dogs infected with CHV-1, papulovesicular genital lesions may be observed. CHV-1 infection during pregnancy can lead to embryonic resorption, abortion and stillbirth. In high density dog populations CHV-1 can also contribute to kennel cough. Furthermore, recent literature has clearly documented that CHV-1 can induce ocular disease both in immature and adult dogs. Here we report a case of fatal CHV-1 infection in a 9-year-old, spayed female Bichon Frise dog. Following a history of vomit ing and diarrhea the dog deteriorated and subsequently died. The main lesions were multifocal areas of necrosis with intranuclear inclusion bodies in the liver, lung, adrenal gland and small intestine, similar to those lesions observed in CHV-1-infected puppies. Infection with CHV-1 was confirmed on samples of liver by PCR, direct fluorescent antibody testing, and in situ hybridization. There was no indication of immunosuppression in this dog. CHV-1 should be included in the list of differential diagnoses of hepatic necrosis in adult dogs.
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Fibroadenoma of the Apocrine Sweat Gland in a Dog
A. Stern. Veterinary Diagnostic Laboratory, University of Illinois, Urbana IL A six-year-old, spayed female, mixed breed dog presented to the referring veterinarian with a 2 year history of a focal nodular mass in the skin overlying the cranial aspect of the left stifle joint. An excisional biopsy was performed and was submitted to the Veterinary Diagnostic Laboratory at the University of Illinois for examination. Histologically the dermis was expanded by a mass composed of bundles of thick collagen and multiple ducts lined by a single to double layer of flattened to cuboidal epithelial cells. There was minimal cellular pleomorphism. Histochemical results with Massons trichome highlighted the collagenous stroma. Neoplastic epithelial cells exhibited positive cytoplasmic immunoreactivity for cytokeratin 7 and cytokeratin 20. The cytokeratin profile of this case is similar to that of normal apocrine glands and the neoplasm is histologically similar to fibroadenomas of the mammary gland of the dog. Based on the findings of this case a fibroadenoma of the apocrine sweat gland was diagnosed. To the authors knowledge this report documents the first case of a fibroadenoma of the apocrine gland in a dog.
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Glomerular Lipidosis in Eighteen Dogs
A.E. Ellis and C.A. Brown. Athens Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Georgia, Athens, GA Following sporadic identification of glomerular lipidosis in brachycephalic dogs, a search of the archives of the Athens Veterinary Diagnostic Laboratory was performed. Initially included were all necropsy or mail-in necropsy cases from the preceding 10 years with a histologic finding of glomerular lipidosis. Based on the overrepresentation of certain breeds, all necropsy/mail-in necropsy cases involving a Boston terrier or bulldog were also reviewed. Eighteen dogs with glomerular lipidosis were identified including 5 Boston terriers, 11 bulldogs (8 English, 2 French, 1 American), 1 Dachshund, and 1 Great Dane. Ages of these dogs ranged from 11 months to 12 years with a mean of 5.8 years. Twelve males (2 intact) and 6 females (3 intact) were affected. Lipidotic lesions generally involved a small percentage of glomeruli and consisted of irregular, segmental to global expansion of the capillary tuft. Lesions consisted of clear vacuoles, yellow-brown pigment, large round to polygonal cells with foamy cytoplasm, and homogeneous eosinophilic material. Ultrastructurally, affected glomeruli contained segmental endocapillary accumulations of vacuolated cells. The majority of dogs died of nonrenal diseases, and other significant renal lesions were noted in only 4 dogs. Although glomerular lipidosis has been previously reported in dogs as a presumed incidental finding, this study provides further description at the light and electron microscopic levels and suggests a potential breed predisposition.
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Focal Small Intestinal Lipogranulomatous Lymphangitis in Six Dogs
V.E. Watson , M.M. Hobday , and A.C. Durham . University of Georgia College of Veterinary Medicine, Veterinary Pathology Department, 2Veterinary Specialty & Emergency Center, 3University of Pennsylvania School of Veterinary Medicine, Department of Pathobiology, Philadelphia, PA Lymphangiectasia is a diffuse lymphatic disease associated with malabsorption and protein-losing enteropathy (PLE). Lesions in the small intestine include dilation of lacteals and mural lymphatic vessels. Lipogranulomatous lymphangitis is occasionally seen in these cases and is likely caused by chronic leakage of lipid-laden chyle. Grossly, lipogranulomas typically present as disseminated small masses on the serosa and lymphatic vessels. Herein, we report six dogs with focal, small intestinal, lipogranulomatous lymphangitis without diffuse lymphangiectasia and PLE. All dogs were presented with intermittent abdominal discomfort, vomiting, and/or diarrhea. Routine blood work showed no abnormalities and diagnostic imaging revealed focal to regional small intestinal masses, often with involvement of the adjacent mesentery. All cases had resection and anastomosis surgeries performed. On histopathologic evaluation, there was severe mural and mesenteric lipogranulomatous lymphangitis with foamy epithelioid macrophages, acicular clefts, necrosis, and fibroplasia. Lymphangiectasia was noted in five cases, but only in sections with the masslike lesion; tissue without lipogranulomas had no to minimal lymphangiectasia, suggesting a localized phenomenon. The outcome of 5 cases was available. Three dogs showed remission of clinical signs with no subsequent treatment; one dog had partial alleviation of clinical signs and receives nutritional management; one case developed regional intestinal thickening distal to the surgical site and is undergoing medical and nutritional management. This case series describes a unique manifestation of intestinal lipogranulomatous lymphangitis that should be considered as a possible differential diagnosis in dogs with intestinal masses.
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Halicephalobus gingivalis in Two Horses from Costa Rica and Honduras
A. Berrocal1, JB. Oliveira2. 1Histopatovet, A.P. 904-3000-Heredia, Costa Rica and 2Catedra de Parasitologia, UFRPE, CEP 52171-900, Pernambuco, Brasil Halicephalobus gingivalis (formerly Micronema deletrix) is a free-living nematode commonly found in soil and organic matter. It has been considered a facultative parasite in horses and humans. The route of infection is believed to be oronasal, causing tissue damage during its migration. To our knowledge, these are the first two cases reported from Central America. Case 1: Six year old, Arabian stallion. The animal was disorientated, circling left, apparently blind and incoordination. He was euthanized after treatment with antiinflammatory drugs and antibiotics for five days with no improvement. The brain, both kidneys and a piece of liver were sent for examination. Grossly, both kidneys showed large white nodules (0.10 to 2.50 cms). The samples were routinely processed for histopathologic examination and scraping of the kidney nodules were collected and stained with Giemsa. Several nematodes were found mixed with mononuclear cells, epithelial cells and fibrocites. Microscopically, both kidneys showed similar changes, consisting of multiple necrotic foci with longitudinal and transversal sections of nematode larvae. In the brain, there were several foci with similar parasites, surrounded by lymphocytes and gitter cells. Case 2: Eight year old, Spanish stallion. The trainer indicated that six days before death, the horse was depressed and anorexic. It was treated with antibiotics and analgesics, without response and died three days after onset clinical signs. Only two small (0.50 and 1.50 cms) renal biopsies were submitted for histopathological examination. The microscopic findings were similar to those of case 1. Morphologic features of the parasites were consistent with the description of H. gingivalis. Although uncommon, H. gingivalis should be considering in the differential diagnosis of horses with neurological signs.
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Immunohistochemical Prognostication of Canine Diffuse Large B-Cell Lymphomas
C. J. Amuzie , B. E. Kitchell , H. (Rita) Ho , and M. Kiupel . Department of Small Animal Clinical Sciences, 2Department of Pathobiology and Diagnostic Investigations, Michigan State University, East Lansing, MI In human medicine, immunohistochemical algorithms are used to more accurately prognosticate diffuse large B-cell lymphomas (DLCBL). Most commonly, DLBCL are immunohistochemically divided into two prognostically distinct classes: germinal center B-cell-like (GCB) and activated B-cell-like (ABC) based on expression of GCET1, CD10, MUM1, BCL-6, and FOXP-1. Patients with GCBs have been shown to have better survival times than patients with ABCs. However, to our knowledge, no similar algorithm exists for canine DLCBL, one of the most common lymphoma subtypes in dogs. We hypothesized that a similar IHC algorithm can be applied to canine DLCBL for more accurate prognostication. In a case controlled study with a treatment regimen containing cyclophosphamide, doxorubicin, vincristine, and prednisone, 10 chemo-sensitive and 10 chemo-resistant canine DLCBL were identified and labeled immunohistochemically with antibodies against GCET1, CD10, BCL-6, BCL-2, MUM1, Cyclin D2 and FOXP1. GCET1, CD10, BCL-6, and MUM1 did not show strong labeling in up to 30% of the neoplastic cellular population for both groups of canine DLBCL. The majority ofneoplastic cells (over 80%), in both groups, had strong nuclear labeling with FOXP1, suggestive of the human Chemo-resistant ABC subtype. However, labeling with BCL-2 resulted in better discrimination. Chemo-resistant canine DLBCL tended to be strongly positive for BCL-2. Our results indicate that chemosensitive and chemo-resistant canine DLCBL cannot be discriminated with the immunohistochemical algorithms established for human DLBCL. . Furthermore, FOXP1 is not a marker of chemo-resistance in canine DLCBL, and IHC for BCL-2 is a potential prognostic marker for canine DLCBL and may represent a useful marker for targeted anti-Bcl-2 therapy.
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Left Atrial Thrombosis in Two Owl Monkeys (Aotus griseimembra)
C.P. Drew1, L.G. Livingston2, B.L. Skinner2, and S.R. Zaki1. 1Infectious Diseases Pathology Branch and 2Animal Resources Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, 30333 Cardiovascular disease is a common cause of morbidity and mortality in owl monkeys (Aotus spp.). The etiology and pathogenesis of this disease are incompletely described, although hypertension has been shown to play a major role. This report describes the pathological features of left atrial thrombosis in two adult owl monkeys housed in a large research colony. The first case, an 8-year-old female, presented clinically with subcutaneous edema, a cardiac arrhythmia, and a heart murmur, and rapidly decompensated despite therapy. Gross examination revealed anasarca and a thrombus that filled the left atrium and was adhered to the endocardial surface adjacent to the entry of the pulmonary veins. The intrathoracic esophagus adjacent to the left atrium was compressed, and the mucosa was circumferentially ulcerated. The second monkey, a 10-year-old male, went into cardiopulmonary arrest during a routine physical examination. Gross examination revealed pleuropericardial effusion, cranial mediastinal edema and a thrombus predominantly within the left atrial appendage. The thrombi in both monkeys ranged in character from partially organizing to acute aggregates of fibrin, erythrocytes, and intact and fragmented leukocytes. The lungs of both monkeys showed widespread evidence of left heart failure, characterized by abundant hemosiderin laden macrophages within alveolar spaces and pulmonary interstitial fibrosis. Other evidence of underlying cardiovascular disease included varying degrees of myocardial fibrosis, rare myocardial necrosis, and arteriosclerosis. Left atrial thrombosis has not been described in owl monkeys and these lesions likely represent another manifestation of the complex cardiovascular disease syndrome in this species.
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Initial Diagnosis of Protothecosis in a Dog via Cerebrospinal Fluid Cytology
L.V. Lane , J.H. Meinkoth , J. Brunker , S.K. Smith II , T.A. Snider , J. Thomas3, D. Bradway4 and B.C. Love1,3. 1Department of Veterinary Pathobiology and 2Clinical Sciences, Oklahoma State University, Center for Veterinary Health Sciences, Stillwater, Oklahoma; 3Oklahoma Animal Disease Diagnostic Laboratory, Stillwater, Oklahoma; and 4 Washington Animal Disease Diagnostic Laboratory, Pullman, Washington A five-year-old, spayed female, Shetland sheepdog mixed-breed dog was evaluated for a history of recent seizure activity with progressive hind limb ataxia and polyuria/polydipsia. There was no history of gastrointestinal signs. Physical examination findings included conscious proprioceptive deficits, ataxia, and anterior uveitis along with a hypermature cataract in the right eye. Results of a CBC, chemistry profile, T4, urinalysis and computed tomography scan of the brain were unremarkable. Cerebrospinal fluid (CSF) analysis revealed a marked eosinophilic pleocytosis (total nucleated cell count 1,106/ microliter; 41 percent eosinophils, 39 percent large mononuclear cells, 11 percent large granular lymphocytes, and 9 percent non-degenerate neutrophils). Cytologic examination demonstrated rare organisms consistent with Prototheca species within neutrophils and macrophages. Postmortem histologic examination revealed mononuclear inflammation and numerous intralesional algal organisms in the brain, eyes, kidneys, and heart similar to those seen on the cytologic preparations. Cerebrospinal fluid and sub-dural/olfactory bulb swab cultures yielded growth of Prototheca species and polymerase chain reaction with DNA sequencing confirmed the organisms as Prototheca zopfii. This was an unusual case of disseminated protothecosis in that the dog presented primarily for neurologic signs with an absence of gastrointestinal signs and initial diagnosis of the causative agent was made on CSF cytology.
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Malignant Peripheral Nerve Sheath Tumor in a Cat With Metastasis to Regional Lymph Nodes and Lung
E. Buza, R. Menzies, M. Goldschmidt, and A. Durham. Departments of Clinical Studies and Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA Peripheral nerve sheath tumors (PNST) in domestic felines are infrequently reported. A recent study of feline PNSTs showed that the majority of tumors were benign and that both benign and malignant PNSTs can recur, although recurrence in those tumors with features of malignancy is more likely. An 11-year-old domestic short hair cat was presented to the University of Pennsylvania School of Veterinary Medicine with a malignant peripheral nerve sheath tumor of the right rostral maxillary gingiva diagnosed by incisional biopsy. The neoplasm recurred over the next six months to a much larger size and the cat was euthanized two months later. Metastatic spread to the regional lymph nodes (left and right mandibular and retropharyngeal) and lung was discovered at necropsy. Histologically, the neoplasms contain two distinct regions: neoplastic spindle cells arranged in dense interwoven bundles with Antoni A areas and Verocay bodies, and Antoni B regions with loosely arranged neoplastic spindle cells separated by a mucinousmatrix. Immunohistochemically, the neoplastic cells within the right maxillary mass and right mandibular lymph node are diffusely and strongly positive for vimentin, S-100, and glial fibrillar acidic protein (GFAP). The neoplastic cells within the lung are positive for vimentin and weakly positive for S-100 and GFAP. All tumors were negative for smooth muscle actin and neurofilament, although neurofilament-positive nerves were noted adjacent to the primary neoplasm. This is the first known report of a metastatic malignant peripheral nerve sheath tumor in a domestic feline.
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Malignant Renal Nephroblastoma in a Dromedary Camel (Camelus dromedarius)
S. Hazarika, N. Gurfield, and B. Rickman. Department of Agriculture, Weights, and Measures, San Diego County Animal Disease Diagnostic Laboratory, San Diego, CA Necropsy of a 6-month old intact male dromedary camel (Camelus dromedarius) with a 3-month history of inappetance and weight loss revealed a large well-demarcated firm multilobular renal neoplasm of the left kidney. Histological evaluation of the affected tissue exhibited substantial proliferation of primitive nephrogenic structures and was principally comprised of epithelial, blastemal, and stromal components. In addition, other congenital abnormalities found in this camel include a dysplastic and polycystic right kidney, as well as a patent ductus arteriosus. In view of the macroscopic and histological findings gathered, the primary tumor was identified as an embryonic renal nephroblastoma. Also known as a Wilms tumor, this malignant neoplasm most commonly occurs in young children and is associated with a genetic mutation of the Wilms tumor suppressor gene (WT1). In contrast to humans, nephroblastomas are quite rare in non-human mammalian species and have been infrequently recognized in canine, feline, swine, rodent, and non-human primate species. Renal neoplasms of the camelid species are not well documented. This report represents the first documented case of a renal nephroblastoma observed in a camelid. A relationship between the development of a Wilms tumor and the other congenital defects found in this camel remains unidentified, however the possibility of such an association cannot be excluded.
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Naturally Occurring Neoplasms in Pigeons in Research Colonies: A Retrospective Study
N. Shimonohara, T. L. Lin, and W. L. Wigle. Animal Disease Diagnostic Laboratory and Department of Comparative Pathobiology, Purdue University, West Lafayette, IN The present study was undertaken to characterize and summarize gross and histopathologic findings of naturally occurring neoplasms in 79 pigeons (predominantly 5 to 20 years old with 9 unknown-aged adults) that were necropsied at Purdue Animal Disease Diagnostic Laboratory from 2001 to 2011. Pigeons were homing pigeons (including mixed breed and White Carneau) used in behavioral studies at Purdue University. Fourteen types of neoplasms were detected and identified in 23 pigeons (29%). Four pigeons had 2 or 3 types of neoplasms. Of the 79 pigeons, 10 (12.7%) had seminoma, 4 (5.1%) had lymphoma, 4 (5.1%) had thyroid adenoma, 2 (2.5%) had pulmonary carcinoma, and 2 (2.5%) had cutaneous hemangioma. Also identified were single incidences of oviductal cystadenocarcinoma, oviductal adenocarcinoma, ovarian adenocarcinoma, granulosa cell tumor, mesothelioma, liposarcoma, cloacal adenocarcinoma, cloacal papilloma, and ventricular carcinoma. The most frequently occurring tumor was seminoma; 7/10 cases effaced both testicles and 3/10 cases had metastasis to the liver or kidney. The relatively high incidence of neoplasms in pigeons in the present study seemed related to the advanced ages of pigeons kept in the research colonies. The results indicated that in addition to naturally occurring higher numbers of seminomas, carcinomas and sarcomas were present in the hemolymphatic, endocrine, pulmonary, female reproductive and gastrointestinal tissues of aged laboratory pigeons.
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Metastatic Melanoma in a Lop Eared Rabbit (Oryctolagus cuniculus)
E.D. Lee , W.E. Culp , and E.L. Carroll . Veterinary Pathology Service, Joint Pathology Center, Silver Springs, MD, 2Department of Veterinary Pathology, Armed Forces Institute of Pathology, Washington, DC, and 3Patrick Air Force Base, South Atlantic District Veterinary Command, Fort Stewart, GA There are few reports of melanoma in lagomorphs, and the biological behavior of the tumor in these species is not well characterized. A 51/2-year-old spayed, female lop-eared rabbit (Oryctolagus cuniculus) presented with a 2x1x0.5 cm round, raised, firm, black, freely-movable cutaneous mass near the left coxofemoral joint that had been present for one month and had doubled in size since initial observation. Cytologic evaluation by needle aspirate revealed spindle to polygonal cells with abundant intracellular and extracellular greenbrown pigment, interpreted as melanin. Tumor histomorphology included an expansile and invasive neoplasm in the dermis composed of polygonal to spindle cells arranged in interlacing streams, whirls and solidly cellular areas, with foci of junctional activity and cells containing melanin; the features were diagnostic for malignant melanoma. The mass recurred at the surgical site two months following excision and the rabbit died one month later. At necropsy, tumor metastases were identified in the liver, lung, and sublumbar and left popliteal lymph nodes. This case report supports the previous literature that melanoma in rabbits tends to be aggressive and readily metastasizes.
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Newly Developed Immunohistochemical Detection Using Pre-Adsorbed Enzyme-Conjugated Polymers Permits Rapid Single and Multiplex Stains on Canine, Feline, Bovine, Equine, and Porcine Tissues
M. Lobo. J. Vargas, and D. Tacha. Biocare Medical, Concord, CA A routine and simple method for immunohistochemistry (IHC) staining on canine, feline, bovine, equine and porcine tissues was developed using preadsorbed enzyme-conjugate polymers. The polymer detection was exceedingly sensitive, eliminated biotinylated antibody steps, and required no avidin-biotin blocking. The pre-adsorbed polymers demonstrated minimal cross-reactivity of endogenous IgGs on all tissues including normal spleen. A wide variety of clinical primary antibodies were tested on various normal and neoplastic tissues and provided excellent staining. Double and triple staining techniques on animal tissues were also developed using a cocktail of primary mouse and rabbit antibodies, and sequentially detected with a cocktail of pre-absorbed polymer enzyme-conjugates (horse radish peroxidase and alkaline phosphatase). Visualization was achieved using DAB (brown) and fast red (fuchsia red) chromogens. A third antibody was applied for sequential staining and either a purple, green or blue chromogen was used to achieve a triple stain. The procedure was performed both manually and on an automated IHC stainer. In conclusion, the biotin-free pre-adsorbed polymer detection assays may provide a valuable diagnostic or research tool for the veterinary pathologist.
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Novel Multiplex Serological Profiling Assay for Rhesus Macaques Measures Exposure to Viral Pathogens by Protein Microarray
K. Luke1, J. Brink1, K. Andrews2, and D. Clutter1. 1Gentel Biosciences, Madison, WI and 2Oregon National Primate Research Center, Beaverton, OR Protein microarrays are useful for studying biological responses in a multiplex and high-throughput manner. We successfully designed a serological screening tool that detects specific antibodies to viral pathogens in sera of rhesus macaques. We evaluated several types of antigens for use on this platform including recombinant proteins, peptides, and virus-infected cell lysates. Pathogens represented included Herpes B virus (Cercopithecine herpesvirus 1), STLV (simian T lymphotropic virus), SRV/D (simian retrovirus type D), SIV (simian immunodeficiency virus), Measles (morbillivirus), LCV (lymphocryptovirus), rhesus CMV (cytomegalovirus), SFV (simian foamy virus), and RRV (rhesus rhadinovirus). Viral antigens were arrayed onto ultrathin nitrocellulose-coated glass slides, and serum samples applied onto individual subarrays. SilverQuant1 (SQ) protein detection reagents were used to visualize bound immunoglobulin complexes through non-enzymatic silver deposition method. Correct identification of positive or negative sera and overall signal-to-noise ratio was evaluated for each antigen. The best performing reagents were used to further screen serum samples from several specific pathogen-free (SPF) NHP colonies, to validate the assay performance. We found that our expanded assay identifies serological status for a broad spectrum of infectious agents in NPHs in a novel and economical multiplex assay with high levels of sensitivity and specificity, and will be a useful tool for easy and rapid monitoring of super clean colonies. To our knowledge, this assay is the first multiplex serological test to include LCV, RRV, and rhesus CMV in addition to the standard SPF agents. This assay also demonstrates the potential of protein array and SilverQuant detection system for rapid and high-throughput tool for screening potential vaccine candidates and associated antibody response.
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Postmortem Findings in 54 Cases of Anesthetic Associated Death in Cats From Two Spay-Neuter Programs
J. Gerdin1, M. Slater2, K. Makolinski3, A. Looney4, L. Appel5, N. Martin6, and S. McDonough 7. 1Department of Biomedical Sciences, division of Anatomic Pathology, Cornell College of Veterinary Medicine, Ithaca, NY, 2American Society for the Prevention of Cruelty to Animals, Schaumburg, IL, 3American Society for the Prevention of Creulty to Animals, New York City, NY, 4Department of Clinical Sciences, Division of Anesthesiology, Cornell College of Veterinary Medicine, Executive Director,5Shelter Outreach Services, Ithaca, NY, American Society for the Prevention of Creulty to Animals, New York City, NY, 6Department of Biomedical Sciences, division of Anatomic Pathology, Cornell College of Veterinary Medicine, Ithaca, NY Anesthetic associated death (AAD) in cats is infrequent, but occurs far more frequently than in people. Post-mortem investigations of AAD in cats are uncommon, and their results only sporadically published. Here we report the findings in 54 cases of AAD in cats. Significant gross and/or microscopic disease, including pulmonary, cardiac, and systemic disease, was detected in 33% of cases. Pulmonary disease was most frequently diagnosed (n13), and included 5 cases of Aelurostrongylus abstrusus. Heart disease, including 2 cases of hypertrophic cardiomyopathy, was less frequent (n6). Surgical complications were infrequent (4% of cases; n2). No significant gross or microscopic disease was detected in 63% of cases. Additional studies are needed to determine if the findings in these cases are similar to those of cats with regular access to veterinary care. This study demonstrates that post-mortem investigation of AADs is an important and worthwhile endeavor.
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Pathologic Findings in the Thyroid Glands of Three Captive Diplodactylid Geckos
L. Mangus , S. Beck , R. Montali , C. Hatfield , L. Clayton , and M. Clancy . 1 Johns Hopkins University School of Medicine, Department of Molecular and Comparative Pathobiology, Baltimore, MD, 2The National Aquarium in Baltimore, Baltimore, MD The reptilian thyroid gland plays important roles in ecdysis, reproduction, growth, oxygen consumption, hematopoiesis, endocrine function, and tail regeneration. Although thyroid dysfunction is not well documented in reptiles, reported conditions include hyperplasia (goiter) secondary to iodine deficiency and toxicosis, hyperthyroidism, hypothyroidism, and neoplasia. Here we present three examples of proliferative thyroid lesions in captive, diplodactylid geckos, all of which were submitted to the animal pathology service at Johns Hopkins University within a three-month period from September to December 2010. Lesions in two animals, a smooth knob-tailed gecko (Nephrurus levis) and a rough knobtailed gecko (Nephrurus amyae), were histologically consistent with thyroid carcinoma, while a marbled velvet gecko (Oedura marmorata) was found to have adenomatous hyperplasia (goiter). The rough knob-tailed gecko also showed evidence of metastases to the liver and lungs. Clinical signs in the geckos included intraoral mass, swelling of the ventral neck, regurgitation, and weight loss.
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Preputial Demodex sp. in Four Big Brown Bats (Eptesicus Fuscus)
J.S. Lankton, K.M. Newkirk, S.A. Kania, and E.C. Ramsay. Departments of Biomedical and Diagnostic Sciences and Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN In this report, we describe Demodex mites in the preputial skin of 4 adult male big brown bats (Eptesicus fuscus). All bats were wild-caught in East Tennessee and housed at the Knoxville Zoological Gardens. Bats died of unrelated causes and underwent gross and microscopic examination. Four of 11 bats (36%) examined between 2008 and 2010 had mites within hair follicles and sebaceous ducts of the prepuce, including ducts of preputial glands. Mites ranged in size up to 112 x 25 um and had a thin chitinous exoskeleton, striated muscle, short jointed appendages, and a hemocoel. In 3 of 4 cases, there was concurrent folliculitis, dermatitis or preputial adenitis. There were no clinical signs directly attributable to the mites, although penile prolapse was present in one case. In addition to the 4 bats reported with preputial demodicosis, a single bat had similar mites in hair follicles of the ventrolateral abdomen. Demodex spp. have been reported in the meibomian glands and body hair follicles of various species of bats but have not been reported in preputial skin. The true prevalence and significance of preputial demodicosis in captive and wild populations of big brown bats is unknown. Additional prevalence and morphological studies are planned and a PCR primer for identification of the genus Demodex is under development.
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Pseudorabies Virus Infection in Feral Swine Hunting Dogs
S.D. Cramer , G.A. Campbell , B.L. Njaa , S.E. Morgan , S.K. Smith , W.R. McLin IV4, B.W. Brodersen5, A.G. Wise6, G. Scherba7, I.M. Langohr6, and R.K. Maes6. 1NIH Comparative Biomedical Scientist Training Program, National Cancer Institute, Bethesda, MD, 2Oklahoma State University, 3Oklahoma Animal Disease Diagnostic Laboratory, Stillwater, OK, 4McLin Veterinary Services, Elmore City, OK, 5University of Nebraska-Lincoln, Lincoln, NE, 6 Michigan State University, Lansing, MI, and 7University of Illinois, UrbanaChampaign, IL Pseudorabies is caused by suid herpesvirus 1. Pigs are the natural host of pseudorabies virus (PRV), but the virus has a broad host range and causes fatal encephalitis in many species. PRV was eradicated from domestic swine herds in the United States in 2004; however feral swine populations remain infected. This report describes PRV infection in three Oklahoma dogs used for hunting feral swine. Clinical signs included facial pruritus, dyspnea, vomiting, diarrhea, ataxia, and death. Main histologic changes included trigeminal ganglioneuritis with neuronophagia and equivocal intranuclear inclusion bodies. Pseudorabies virus was isolated from fresh brain from two dogs. Immunohistochemistry detected the virus in the brain of the third dog. Partial gC gene sequencing and phylogeny based upon available GenBank sequences revealed that the virus was likely a field strain closely related to a cluster of PRV strains previously identified in Illinois. Though PRV was eradicated from domestic swine in the United States, the virus is present in feral swine and should be considered a possible cause of disease in dogs and other domestic animals.
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Small Intestinal Leiomyositis in a Young Mixed Breed Dog
T.A. Snider, S.D. Cramer, and J. Chang. Oklahoma State University, Stillwater, OK A 1-yr old, spayed female, mixed breed dog was euthanized and necropsied following a two-week history of intestinal foreign body, surgical removal, ensuing ileus, 13-day failure to pass intestinal contrast media, and mild, localized peritonitis. At gross necropsy, localized peritonitis was confirmed with notation of several mild, small intestinal serosal adhesions and omental adhesions, apparently extending from the previous enterotomy site at the ileocecocolic junction, which appeared intact. The entire small intestine was markedly dilated and frequently appeared thin-walled. Salient histologic findings in the small intestine are as follows: segmental and severe loss of the tunica muscularis with the smooth muscle replaced by fibrous connective tissue infiltrated moderately by lymphocytes, plasma cells, and fewer macrophages; affected areas often exhibited numerous neocapillaries within the fibrous stroma; some foci exhibited segmental retention of clusters or islands of smooth muscle cells and fibers characterized by degenerative changes including hypereosinophilia and vacuolated sarcoplasm. These changes also extended into a single section of the proximal large intestine, at the ileocecocolic junction. Observed autonomic ganglia appeared within normal limits. Small intestinal mucosal changes were mild and reflective of dilation and ileus. Histochemical and immunohistochemical techniques applied to selected intestinal sections included Massons trichrome, smooth muscle actin, and neuron specific enolase. Results confirmed loss of smooth muscle, replacement by collagen, and retention of autonomic neural elements. Intestinal leiomyositis is an uncommon condition in dogs, and prior reports are few. It has been correlated clinically with a condition known as chronic intestinal pseudo-obstruction. Intestinal leiomyositis in man is reported more frequently and, though the pathogenesis is poorly understood, is thought to be a manifestation of autoimmune illness.
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Pulmonary Arterial Myxoma in a Dog With Extensive Intravascular Embolization and Vasculopathy
R. Weiss1, R. Dillon2, L. Reid2, and J. Hathcock2. 1Department of Pathobiology and 2Department of Clinical Sciences, College of Veterinary Medicine, Auburn University, AL A 10 year old castrated male mixed breed dog with a recent history of coughing and hemoptysis was presented dyspneic to the Auburn University Small Animal Teaching Hospital Critical Care Unit on February 3, 2011. Blood evaluations revealed arterial hypoxemia unresponsive to oxygen and a persistently elevated hematocrit (61-70). Thoracic radiology showed increased opacity in the hilar area. Angiography revealed multiple filling defects suggestive of pulmonary emboli, with minimal enhancement of the right pulmonary vasculature and a portion of lung lacking perfusion. Following elective euthanasia requested by the owner, a complete necropsy was performed. Grossly, a poorly circumscribed, slightly firm, approximately 2 x 3 cm, intrapulmonary mass partially occluded the right pulmonary artery just below the bifurcation of the pulmonary trunk. Throughout the right lung numerous intravascular emboli consisting of tissue grossly similar to the mass in the major artery were in variably-sized pulmonary artery branches. Histologically, the primary arterial mass and intravascular emboli consisted of loosely adherent paucicellular sheets of mildly pleomorphic hyperchromatic spindled to small stellate cells interspersed within abundant Alcian blue positive stroma incompletely separated into lobules by delicate fibrovascular trabeculae. Anisocytosis and anisokaryosis were mild to moderate and mitotic figures were lacking. In a number of affected vessels there was moderate irregular thickening of the intima and subintimal degeneration with increased fibrocollagenous to myxoid matrix. Myxomas are the most common cardiac tumor in humans arising most frequently in the left atrium (75% of reported cases). In dogs, there are few reports of cardiac myxoma all involving the tricuspid valve later extensively embolizing throughout the lungs.
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Spectrum of Histopathological Lesions in the Distal Third Metacarpal Bone in Racehorses
M.J. Pinilla, C.A. Tranquille, K.C. Smith, T. Parkin and R.C. Murray. Royal Veterinary College, University of Glasgow, Glasgow Catastrophic condylar fractures of the third metacarpal bone (MCIII) are the most frequent catastrophic fracture in racehorses. There is evidence that these are training-associated repetitive overload injuries. Aim: To describe and grade the histopathological lesions of MCIII in cadaver limbs from Thoroughbred racehorses. Methods: 38 MCIIIs were obtained from racehorses euthanased at the racecourse. Limbs were selected if they had magnetic resonance imaging (MRI) changes that are seen in condyles detected in racehorses in training with or without lameness. MCIIIs were classified into groups based on distribution of MRI signal intensity and uniformity of the lesion present. Limbs were sectioned targeting the lesion seen on MRI. Sections were examined under light microscopy and scored using an adapted scoring system based on the OARSI system for osteoarthritis. Scores were compared between groups. Results: Characteristic histopathological features detected included cartilage degeneration and alteration of the calcified layer in heavily remodeled (sclerotic) bones. SCB plate ischaemic necrosis and SCB collapse were associated with alterations in both SCB and cartilage (hyaline and calcified). Cartilage hypertrophy and bone trabecular thickening were frequent features. Trabecular bone ischaemic necrosis tended to occur associated with microcracks. Conclusions and practical significance: These findings highlight adaptive responses of the entire osteochondral unit that may appear to be exceeded in failure of distal MCIII. Acknowledgements: The HBLB for funding and Mr Ray Wright for histological preparation.
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Survey of Goat Tumors, Department of Pathology and Athens Veterinary Diagnostic Laboratory, College of Veterinary Medicine, UGA, From 2007-2011
E.W. Howerth and A. Butler. Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia In recent years, interest in goat production has expanded across the country and goats are now a common necropsy submission at many diagnostic laboratories. Although there are multiple case reports of tumors in goats and larger studies on potentially viral-induced tumors such as nasal carcinoma and fibropapilloma of the udder, tumor surveys in goats are limited and relatively old. We surveyed goat biopsy or necropsy submissions to the Department of Pathology and Athens Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Georgia from 2007-2011. Twenty tumors in 19 goats from a total of 188 animals (11%) were identified. Affected breeds included pygmy, Nigerian dwarf, Nubian, Boer, KiKo, Saanen, and mixed. Tumors occurred in animals 2-16 yrs (average age 9 yr) and most were females (74%). Tumors included, in decreasing order of prevalence, squamous cell carcinoma (lung, perineal, eyelid, vagina), pheochromocytoma, thymoma, sebaceous epithelioma, unidentified carcinoma, uterine adenocarcinoma, multicentric lymphoma, mesothelioma, hemangioma, and gingival fibroma. Tumor prevalence was low and all types observed, except sebaceous epithelioma, have been reported previously in goats.
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Unusual Ocular Mass in a Commercial Layer Chicken
S. Williams, A. Ellis, G. Zavala, and S. Hafner. Department of Population Health, Athens Veterinary Diagnostic Laboratory, University of Georgia, FSIS Eastern Laboratory, USDA, Athens, GA Seven commercial layer chickens (56 week old hens) were submitted to the Poultry Diagnostic and Research Center for necropsy with the complaint of decreased egg production, ocular lesions, and increased mortality. Grossly, several chickens exhibited irregular pupil shape and corneal discoloration. Microscopically, one eye was partially replaced by a mass that focally perforated the scleral cartilage and extended into the retina adjacent to the optic disc. The cells composing this mass appear to be of neuronal origin. IHC was negative for myelin basic protein, glial fibrillary acidic protein, and pancytokeratin and positive for neurofilament, S100, and NSE.
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Tissue MicroarrayBased Analysis of Prognostic Markers in Cutaneous Mast Cell Tumors
R. Laufer Amorim1, R. Torres Neto1, R.B. Ferioloi1, M.M. Vidale1, and J. Wer ner2. 1Veterinary Medicine and Animal Science Schooll, University of Sao Paulo State, Botucatu, SP, Brazil and 2Werner & Werner Private Pathology Practice, Curitiba, PR, Brazil Cutaneous mast cell tumors (MTC) are one of the most common neoplasms in dogs. Histological grade has consistently been demonstrated as a strong predictor of prognosis. The most widely used grading system for cutaneous MCT has been the Patnaik system. However, several studies have shown that grading concordance is poor between pathologists. A novel two-tiered grading system was recently proposed by Kiupel et al. 2011.The aim of this study was to evaluate different proteins expression in tissue microarray slides and correlate them with histological grading systems. Tissue microarray (TMA) slides, with 110 MTCs were used for immunohistochemical of KIT (CD117), Ki67, caspase3, triptase, PGE2, VEGF, p53, and apototic evaluation by TUNEL. Mitotic figure (1mm2) in HE-stained TMA slide and Ki67 positive cells (0,7mm2) were counted. KIT pattern, triptase and Ki67 scores showed correlation with both grading systems (p<0,05). Gender of the dogs also correlated with both histological grading systems, and females had lower grade tumors than males. Mitotic figures (HE-stained) and Ki67 positive cells were significantly different in Patnaik (p<0.001) and Kiupel (p<0.001) grade systems. Survival time was achieved in 21 Boxer dogs with MCTs. Survival curves by Kaplan-Meier plots were significantly different using the 2-Tier System (p<0.0001), Patnaik system (grade 2 and grade 3, p0.02), mitotic figures (p0.02) and Ki-67 positive cells (p<0.0001). Tissue microarray can be a method for evaluating prognostic markers in MCTs. Quantification of mitotic figures, analysis of KIT pattern and cellular proliferation by Ki-67 in TMA slides were found to have clinical value.
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Why Is the North American Veterinary Medical Education Consortium Important for Us?
A. Alcaraz1, C. Crocker2 and J.M. Peralta3. 1Pathology, 2Physiology and 3Welfare and Ethics, College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA The North American Veterinary Medical Education Consortium report, Roadmap for Veterinary Medical Education in the 21st Century: Responsive, Collaborative, Flexible (http://www.aavmc.org/data/files/navmec/navmecdraftfinalreport.pdf), emphasizes the different challenges for veterinary education. The report speaks about the specific competencies that need to be developed by a future veterinarian. Some of these competencies, such as comparative medicine, disease prevention, diagnosis and control of zoonotic diseases, are related to pathology. Some of the non-clinical competencies, such as critical thinking, problem solving, life-long learning, and acquisition of interdisciplinary knowledge, are also integral skills for pathologists. The way we teach pathology, or any other subject, will have an impact on all of these competencies. We can help the students reach to the new challenges of the profession. What is the best way to teach our students? Are self-directed learning models applicable to the solution? Pathologists need to create opportunities to design practical exercises or training tools that will improve the application of knowledge. We need to understand the technology that will generate resources that can be measured, shared and used in a flexible way. Such opportunities could be web based courses or cases, short case identification through macro and microscopic images, interdisciplinary activities where mechanisms are combined in a morphologic setting with anatomy or physiology through pathophysiological mechanisms. Those experiences will broaden the horizon of our students. Pathology has a central role connecting the clinical and non-clinical disciplines with a practical spin and the opportunity to use interdisciplinary knowledge. Our pathology curriculum needs to adapt to the fast changing world of the future veterinarian.
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Developing Diagnostic Skills Using Enquiry-Based, Interactive Computer Modules
B.H. Herrin, C.G. Lamm, B.L. Njaa, and R.W. Allison. Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK and School of Veterinary Medicine, University of Glasgow In Veterinary Medicine, we face two distinct challenges: providing students with the skills to apply their knowledge within the clinical setting and adapting our teaching styles to accommodate their ever growing technological capabilities. Most of the instruction is through didactic lectures with minimal use of technology. The purpose of this tool is to teach students how to apply the knowledge they have gained in lecture to real-world cases through interactive, computer-based modules. The computer module was designed to allow students to work through the diagnosis of veterinary diseases while encouraging them to think critically about each case. Each module presents a case, including history and physical exam, followed by a list of diagnostic tests. The students select from the given tests, are provided the results, and are asked to interpret them in order to come to a final diagnosis. Following each case, there is a series of case-specific questions used to reinforce key concepts about the diagnostic tests used and disease processes involved. For assessment purposes, the students are asked to keep track of the money they spend as they order tests and provide a statement to the owner discussing the results of those tests and the final diagnosis. Initial student feedback on the computer modules is overwhelmingly positive and these programs are now regularly used in veterinary pathology education at the University of Glasgow and Oklahoma State University. We currently plan to continue to expand these modules to incorporate other aspects of the veterinary medicine curriculum.
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Exposing College Undergraduates to Alternative Careers in Veterinary Medicine: Experiences From a Medical School Comparative Medicine Department
D. M. Bouley. Department of Comparative Medicine, Stanford School of Medicine, Stanford, CA Stanford University attracts some of the brightest undergraduate students in the country. The Department of Comparative Medicine (DCM) within Stanford University School of Medicine makes it a high priority to encourage Stanford undergraduate pre-veterinary students to consider alternative careers in veterinary medicine, well before they ever apply to veterinary schools. By offering unique undergraduate classes, freshman and sophomore seminar courses, independent study, and research opportunities, we expose these highly motivated undergraduates to careers of the DCM faculty whose credentials include, ACVP, ACLAM, ACVA, ACVPM, ACVIM, MPH and Ph.Ds. The Stanford Undergraduate Pre-Veterinary Club (SUPVC) has been in existence since 2003, and receives support from the DCM and several main campus undergraduate programs to fund summer research projects, educational dinner meetings, honoraria for invited speakers, field trips, and an informational website. SUPVC members participate in community outreach to regional high school and college students, academic counselors, and parents, in the form of day-long symposia (Pre-Vet Expos) that offer a glimpse into the diversity of the veterinary profession and guidance on maneuvering the pathway to veterinary school. It is in part these unusual opportunities bestowed upon our SUPVC members that have resulted in an exceptionally high first time applicant acceptance rate to veterinary schools. Currently, several SUPVC alumni are employed or engaged in the following disciplines: research compliance, anatomic pathology/Ph.D residency programs, DVM/Ph.D, DVM/ MPH combined programs, and laboratory animal and primate medicine tracks. Overall, this early, focused exposure of Stanford undergraduates to the DCM faculty, combined with existing undergraduate programs, creates an innovative approach to address the future needs of the veterinary profession in our current climate of One Medicine, One Health.
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Development of a Web-Based Searchable Archive for Gross Pathology Images
K. Newkirk1, H. Pang2, A. Nelson1, and D. Carnduff2. 1Department of Pathobiology, College of Veterinary Medicine, and 2Innovative Technology Consulting, University of Tennessee, Knoxville, TN Interpretation of gross pathology is crucial to the training of veterinary students and pathology residents. Although there are several web-based or subscription based gross image databases available, most veterinary institutions have large private collections of images that may be difficult to access or search. To help make these images more accessible, a website was developed to showcase these images. Archival gross images on 2x2 Kodachrome slides were screened; the highest quality images were selected and scanned to create digital images. Digital images from routine biopsy and necropsy specimens presenting to the University of Tennessee were also screened for high quality images of classic or unique disease entities. The relevant case information: signalment, diagnosis, system, tissue, and disease process were entered into a spreadsheet. With the help of University of Tennessee Innovative Technology Consulting and a Faculty First Grant, a searchable website was developed to easily view the images. The website is freely accessible, but password protected. Faculty can use the website to retrieve images for use in teaching. A quiz feature is also available on the website. Most images have difficulty ratings, so that residents preparing for boards can choose a more difficult quiz; whereas veterinary students in the general pathology course can take a more basic quiz. New images are easily added to the website by anyone with an administrator password; only basic computer training is required.
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Outcomes Assessment and Student Perceptions of the Use of Case-Based Writing Exercises in Veterinary Clinical Pathology
H.T. Michael1, L.C. Sharkey1, B.A. Center2, and D.A. Wingert3. University of Minnesota, 1Departments of Veterinary Clinical Sciences, 2Veterinary Medical Student Affairs, and 3Educational Psychology The Clinical Pathology course in the spring semester of second year is one of the first clinically-oriented courses in the veterinary professional curriculum and assesses student mastery using multiple choice and essay exams, quizzes and seven case-based free-response assignments that require integration of clinical history and laboratory abnormalities to provide pathophysiologic explanations for data and generation of differential diagnoses. A previous study demonstrated that students found case-based assignments to be valuable learning tools. This study was designed to determine if casebased writing assignments better predict success later in the curriculum than multiple-choice assessments and if positive student perceptions of case-based assignments persist into the clinical practicum. At the end of the course, students reported the case-based assignments were a valuable part of class (100%), helped them learn factual information (94%) and increased their understanding (82%) compared with traditional lecture and multiple choice assessments. In years 3 and 4, most students continued to report that casebased assignments facilitated retention of information, clinical reasoning and communication of information better than preparing for multiple-choice assessments. Second year cumulative GPA and clinical pathology course grade strongly correlated with the 3rd year GPA (0.966 and 0.90, respectively) while case-based assignments were less predictive of 3rd year academic success. Student performance data from the clinical practicum are under analysis. In conclusion, the case-based writing assessments did not improve prediction of 3rd year grades. However, student perception that case-based writing assignments in the clinical pathology course were important to learn information and to improve clinical reasoning and medical communication persisted throughout their later didactic and clinical education.
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Survey Results From Employers of New Veterinary Graduates: Role of Core Skills
J.A. Danielson1,2, T-F Wu2, A. Fales-Williams1, R. Kirk2, V. Preast2, and K. Kuehl2. 1Department of Veterinary Pathology, 2Office of Curricular and Student Assessment, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50011-1250 Employers (N 75) of 2007, 2008 and 2009 ISUCVM graduates were surveyed regarding (a) their overall satisfaction with their supervised employees and (b) their employees preparation in a series of medically-focused veterinary knowledge and skills (core) and non-medically-focused veterinary knowledge/ skill areas (non-core) one year following graduation. Response rates for the three years were 51%, 78%, and 47%, respectively. This abstract addresses feedback on core skills, including nine items and four subscales: Data Collection (2 items, e.g., Collect a medical history; alpha0.85), Data Interpretation (2 items, e.g., Interpret relevant diagnostic tests; alpha0.73), Planning (2 items, e.g., Select relevant diagnostic tests and procedures; alpha0.80), and Taking Action (3 items, e.g., Perform common surgical procedures and implement after care; alpha0.87). All items were rated on a five point scale (1 very unprepared to 5 very prepared). Correlation results indicated a strong positive relationship between the four types of core veterinary skills and employer satisfaction (correlation coefficients ranging from r .45 to r .52; p < .001). Further, results of the simultaneous regression analysis indicated that Taking Action (implementing diagnostic and treatment plans) was the most important core veterinary skill to employers (beta .40, p < .05). In sum, our results indicate that employers are more likely to report a high level of satisfaction with their employees, if their employees are well prepared on the four types of veterinary core skills. In particular, employers recognize new graduates who are able to implement diagnostic and treatment plans.
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The Electronic Book Project: The Diagnostic Pathfinder/Thinkspace Software Linked to the Textbook Fundamentals of Veterinary Clinical Pathology: Development and Results After Four Years of Implementation
R. Di Terlizzi1, H.S. Bender2, and J.A. Danielson2. 1University of Pennsylvania, School of Veterinary Medicine, Philadelphia, PA and 2Iowa State University, College of Veterinary Medicine, Ames IA The purpose of this project was to allow veterinary students immediate, just-in-time, and focused access to the electronic version of the clinical pathology textbook: Fundamentals of Veterinary Clinical Pathology (FVCP e-book) through direct links from Diagnostic Pathfinder and ThinkSpace software programs. Veterinary students are often pressed for time and become overwhelmed by the amount of information and competing demands during the four years of veterinary school. Veterinary Clinical Pathology, often a second year core course, contains extensive amounts of complex information. We linked the electronic FVCP text book to the Diagnostic Pathfinder software (years) and most recently to the new version of the software called ThinkSpace (years). The electronic links are designed to focus students attention on key concepts and help them to gain a better understanding of course objectives. After solving a complex clinical pathology case, students click on FVCP e-book hyperlinks that are embedded in the case solutions. Any given link directs them to the section of the e-book that explains that specific concept, providing a more efficient and less overwhelming learning experience. Portions of the textbook that are relevant to second year veterinary students are highlighted to help focus the students attention on the most important and basic concepts of the course. The e-book project was introduced in February 2009 at Iowa State University and at the University of Pennsylvania in January 2010. Student satisfaction data collected during the e-book implementation (2009-2011) at ISU and UPenn will be discussed during the ACVP educational session.
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Survey Results of Participants of a Mock Clinical Pathology Certification Exam
J.A. Neel1, C.B. Grindem1, R. Patel2, H.L. DeHeer3, A.L. MacNeil4, C.J. LeBlanc5. 1NCSU-CVM, Raleigh, NC, 2U Penn-CVM, Philadelphia, PA, 3 Antech Diagnostics, New York, NY, 4UI-CVM, Urbana, IL, and 5UTCVM, Knoxville, TN Participants of a multi-institute mock exam were surveyed regarding aspects of the exam. Response rate92.3% (36/39 participants). A Likert scale from 1/most positive -5/most negative was used. Average ratings are listed parenthetically and included: value as a training tool (1.14), utility in tracking board preparation progress (1.75), and exam stressfulness (2.44). 52.8% of participants had good (1-2) and 47.2% had moderate to no (3-5, 2.42) understanding of the certifying exam structure prior to taking the mock exam, although 80.6% participants found the mock very helpful (1.22) in better understanding the structure. 77.8% didnt consider mock exam availability in selecting residencies (4.39), however 100% now consider a mock exam experience critical (77.8%, 1) or very important (22.2%, 2, 1.22). 97.2% were likely/very likely (12, 1.33) to recommend prospective residents choose a program offering a mock experience. 97.2% would be very likely (1, 1.03) to recommend this mock exam for individuals preparing for boards. Participants having sat the exam (n26) were asked additional questions including: realism of the mock exam (1.54), difficulty of the mock compared with the certifying exam (2.65; 34.6% mock harder/much harder, 53.4% exams similar, 11.5%, certifying exam harder), usefulness in preparing for fatigue (2.08) and stress (2.00) during the certifying exam, usefulness for practicing timing (1.64), and helpfulness in identifying weak areas (1.46). Forced ranking of test aspects showed greatest value of the mock in practice with timed write-ups, as a marker of board preparation progress, and for better understanding of exam sections. Results indicate trainees consider a mock exam experience a critical training program component.
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Visual Diagnostic Reasoning Skill Assessment Utilizing Eye-Tracking Technology: Novice-Expert and Educational Interventions in Clinical Pathology
A.L. Warren1, T.L. Donnon2, N.J. Fernandez1, and C.R. Wagg1. 1Department of Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine and 2Department of Community Health Services, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada Visual diagnostic reasoning skills are paramount to competency in veterinary pathology. Little or no research has been conducted in either the medical or veterinary fields on effective teaching methods for visual diagnostic reasoning. Studies in human radiology, dermatology and pathology have utilized eye movement tracking (gaze-tracking) as a measure of visual diagnostic reasoning capability. Eye-tracking (eye-position) analysis uses the position of the viewers eye as a representation of visual attention and provides a unique opportunity to understand the learners perceptual processing during learning. During visualization of a diagnostic image, saccade (rapid eye movements between focal points) and fixation points (static eye-position points that correlate to an area of cognitive interest) are not random but have a highly replicable scan path between viewing sessions. In medical pathology studies, in visual diagnostic reasoning, novices tend to use hypothetico-deductive reasoning where eye-movement patterns are laborious and complicated, and experts tend to use pattern recognition skills with highly efficient and rapid eye-movement patterns. Using eye-tracking technology, we aim to see if there is a difference between novice (veterinary student) and expert (board-certified) pathologists in eye-movements when reading and diagnosing a virtual microscopic slide. We hypothesize that expert participants will take less time to scan a slide, be more accurate in identifying diagnostically useful points of interest and spend more visual dwell time on fixed points correlating to their points of interest. A series of educational interventions and use of eye-tracking as a measure of DVM student visual diagnostic reasoning development will be discussed.
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A Comparative Study of Experimental Salmonella dublin Infection in Newborn Calves and Buffalo Calves
D.G. Silva, A.M. Santana, L.J.L. Pizauro, K.M.M.G. Simplcio, V. Clemente and J.J. Fagliari. Veterinary Clinics and Surgery Department, FCAVUNESP, Jaboticabal Campus, SP, Brazil To evaluate the effects of experimental salmonellosis in newborn calves and buffalo calves, a total of eleven 10 to 26-day-old healthy calves were randomly allotted in the following groups: six Hostein calves infected orally with 108 CFU of Salmonella Dublin (group 1) and five Murrah buffalo calves infected orally with 108 CFU of Salmonella Dublin (group 2). All animals were submitted to physical examination before inoculation and every 12 hours during the seven days after the experimental infection. Rectal swabs were collected for Salmonella Dublin isolation. In group 1, all calves had severe diarrhea 12 to 84 hours after inoculation, accompanied by fever (40.1 C), dehydration and respiratory signs. Salmonella Dublin was isolated 12 hours after inoculation. Two out of the six Holstein calves died during the experimental period. In group 2, all buffalo calves had mild to severe diarrhea 24 to 96 hours after inoculation. Four buffalo calves had fever 60 to 96 hours after experimental infection (40.5 C) and two buffalo calves showed mild dehydration 72 and 96 hours inoculation. Salmonella Dublin was isolated from rectal swabs in group 2 at 24 hours after the experimental infection and no death was observed in this group. In conclusion, the oral administration of 108 CFU of Salmonella Dublin induced clinical signs of salmonellosis in newborn 10 to 26-day-old calves and buffalo calves, but in calves the experimental infection produced severe systemic disease.
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Aerosolized Nucleotide Synthesis Inhibitor Therapy for Influenza A (H1N1) Infection in Mice
F. Aeffner, Z. Traylor, and I.C. Davis. Department of Veterinary Biosciences, The Ohio State University Influenza virus causes highly contagious acute respiratory disease with significant morbidity and mortality. We have shown that H1N1 influenza A virus infection of BALB/c mice induces nucleotide-mediated inhibition of alveolar fluid clearance (AFC) at 2-6 days post-infection (d.p.i.). We hypothesize that ion transport inhibition underlies development of pulmonary edema and hypoxemia in influenza. Furthermore, we propose that influenza-induced AFC inhibition will be amenable to therapeutic blockade with FDA-approved nucleotide synthesis inhibitors. 8 week-old BALB/c mice were infected intranasally with mouse-adapted influenza A/WSN/33 (10,000 FFU/mouse), and were treated at 1 or 5 d.p.i. with the pyrimidine synthesis inhibitor A77-1726 (5 E M/kg), delivered by nose-only aerosol (Scireq, InExpose). Body weight, peripheral O2 saturation (SpO2) and survival were compared to saline-treated, influenza-infected controls. AFC and lung function parameters were measured by the BSA instillation method and the forced oscillation technique (Scireq, flexiVent), respectively. Lung water signals were detected by magnetic resonance imaging. Carotid PaO2 was measured using an iSTAT blood gas analyzer. Aerosol administration of a single dose of A77-1726 at 1 d.p.i. did not significantly alter infection-associated weight loss, viral replication, or BAL inflammatory cell counts, but alleviated influenza-induced AFC inhibition and hypoxemia (SpO2 and PaO2:FiO2 ratios) at 2-4 d.p.i., and had a moderate beneficial effect on survival. A77-1726 treatment also reversed pulmonary edema and alterations in lung function. A77-1726 treatment at 5 d.p.i. had similar beneficial effects on lung function at 6d.p.i. Pyrimidine synthesis inhibitors may represent novel therapeutics to ameliorate influenza-associated acute lung injury. Importantly, these agents are inexpensive, stable, and amenable to stockpiling, and might therefore be of particular value in pandemic situations.
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A Viral-Vectored RSV Vaccine Induces Robust Humoral Immunity and Protects Against Viral Challenge
J. Grieves1,2, R. Durbin2, L. Martinez-Sobrido3, A. Garcia-Sastre4 and J. Durbin2. The Ohio State University College of Veterinary Medicine1 Columbus, OH; New York University2 New York, NY; University of Rochester3, Rochester, NY; Mount Sinai School of Medicine4, New York, NY Human Respiratory Syncytial Virus (RSV) is the leading cause of lower airway disease in infants worldwide. Severe lower airway RSV infection during infancy is not only life-threatening, but has also has been associated with development of recurrent wheezing in later childhood. Furthermore, RSV repeatedly infects the upper airway of healthy, immunocompetent individuals throughout life. The inadequate, short-lived humoral immune response to RSV infection is well-recognized but poorly understood. Recent evidence suggests that type I interferons (IFNs), in addition to their critical role in induction of the anti-viral state, are essential for downstream development of robust, long-lived B cell immunity. Thus, the active inhibition of IFN production that is observed following RSV infection may be a major mechanism by which RSV dampens the host B cell response. Based on the hypothesis that an RSV vaccine that induces robust IFN production would be protective, we constructed a viral-vectored vaccine that expresses the F glycoprotein of RSV. Compared to controls, RSV challenge of vaccinated cotton rats resulted in significantly reduced upper and lower airway viral load and more robust F-specific antibody production. Additionally, pulmonary inflammation observed in response to RSV challenge was of shorter duration in vaccinated animals, which could have important implications considering the mounting evidence that lower airway RSV infection is associated with subsequent development of recurrent wheezing. Overall, these data demonstrate the feasibility of our approach and support progression of our RSV vaccine toward clinical trials as well as additional dissection of mechanism of protection.
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Beta Cell Distribution, but Not Density, Is Altered in the Neonatal CF Pig Pancreas
D.K. Meyerholz1, A.K. Olivier1, C.M. Hochstedler1, P.W. Naumann1, D.A. Stoltz2, P.B. McCray Jr.3, M.J. Welsh2,4,5, A. Uc3. Departments of 1Pathology, 2 Internal Medicine,3Pediatrics, 4Molecular Physiology and Biophysics and 5 Howard Hughes Medical Institute, University of Iowa, Iowa City, IA In cystic fibrosis (CF), endocrine pancreas disease is increasingly recognized and leads to CF related diabetes. We studied neonatal CF and non-CF pig pancreas using insulin immunostaining to determine beta cell distribution and density. In both groups, beta cells were organized as varying sized small islets to solitary cells within the parenchyma. Most of the CF beta cells were restricted to within or adjacent to diminutive remnant lobules, whereas the interlobular space lacked beta cells as well as endocrine islet tissue. Beta cell tissue density did not differ between CF and non-CF groups (NS, MannWhitney test) in whole pancreas sections; however, CF pancreas had increased beta cell tissue density within diminutive lobules (P<0.05, Mann-Whitney). The results of this study give insight into CF pathogenesis. 1) Beta cell density does not appear to be altered at birth and suggests morphologic changes may be a result of postnatal pathophysiology. Importantly, this study does not rule out pathophysiologic differences at birth. 2) Consistent with islet ontogeny, beta cells are restricted to the diminutive lobules of the CF pig pancreas. Accordingly, generous sampling of pancreas is essential to prevent artifactually low or high estimation of beta cell populations. 3) Increased beta cells in lobular tissue may reflect the findings of early CF autopsy studies where infant pancreas was described to have increased islet tissue. Future fetal and postnatal studies combined with clinical analysis of endocrine pancreas function will further define the role of beta cells in the pathogenesis of CF related diabetes.
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COX-2 Positive Cells in Comedo-Type Necrosis: Comparison of Two Canine Mammary Carcinomas Mouse Xenograft Models
J.C. Illera1, L. Camacho1, A. Gonzalez-Gil1, M.J. Illera1, M.D. Perez-Alenza2, L. Pena2. 1Department of Animal Physiology, 2Department of Animal Medicine, Surgery and Pathology, Veterinary School of Complutense University, Madrid, Spain Mammary comedocarcinoma is a histological type of mammary neoplasm, recently included in the histological classification of canine mammary tumors. Similarly to human mammary comedocarcinoma, it is characterized by the presence of necrotic areas within the center of the neoplastic cell aggregates. The aim of this study was to investigate the formation and evolution of the canine mammary comedo-type necrosis and the immunoexpression of COX2-positive cells in comparison with non comedo-type necrosis, using two mouse model xenografts. Samples of a canine mammary comedocarcinoma grade III and solid carcinoma grade III were obtained immediately after surgery. Serial transplanted xenografts were established in BALB/c SCID female mice. Mice were sacrificed at 4,6,8,10 and 12 weeks after xenotransplantation. At necropsy, tumor samples were taken for histopathology and immunohistochemistry. Immunohistochemistry of cytokeratin AE1/AE3, CK14, vimentin, actin, ERE, ERE, PR, AR, Her-2, COX-2 and Caspase-3 (apoptotic marker) were performed on the primary canine mammary tumors and the mice xenografts. Histology and immunohistochemistry of tumor xenografts were similar to canine primary mammary tumors. Comedo-type formations were first seen at 6 weeks of development of the neoplasms. All comedocarcinomas were negative for COX-2 except some strongly stained cells typically located in the necrotic limit area. In solid mammary tumors, necrosis areas were first seen at 4 weeks of development. These tumors presented isolated or grouped strongly stained COX-2 cells, evenly distributed. The comedo-type formation is a mixture of karyorrhexis and apoptosis in which COX-2 may have an important role (probably via apoptosis).
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Experimental Salmonella dublin and Salmonella typhimurium Infection in Buffalo Calves
A.M. Santana, V. Clemente, D.G. Silva, K.M.M.G. Simplcio, L.J.L. Pizauro and J.J. Fagliari. Veterinary Clinics and Surgery Department, FCAVUNESP, Jaboticabal Campus, SP, Brazil Clinical signs and bacterial isolation in buffalo calves infected with experimental Salmonella Dublin and Salmonella Typhimurium were studied. Calves were allotted into three groups (n5): control (group 1); experimentally infected with 108 CFU of Salmonella Dublin (group 2) and 109 CFU of Salmonella Typhimurium (group 3). Physical examination was performed before inoculation and every 12 hours for seven days. Rectal swab samples were collected for isolation before the inoculation and daily until to get fifteen consecutive negative results.Cardiac and respiratory rates of infected groups were within normal range. 4/5 calves of group 2 had fever at 60-96 hours after infection (40.0-41.0 C), returning to normal 148 hours after infection. Only 1/5 animals of group 3 had fever at a single time point 72 hours after infection (40.0 C). All animals of infected groups had diarrhea with mucus and/or blood in the feces starting 24-96 hours (group 2) and 72-108 hours after infection (group 3). Spontaneous recovery occurred at 168-240 hours(group 2) and 192-336 hours (group 3) after infection. Mild dehydration was observed in 2/5 calves from group 2 (72-96 hours after infection) and 1/5 calves from group 3 (120-144 hours after infection). The isolation of S. Dublin and S. Typhimurium from rectal swabs of infected groups first occurred 24 hours after infection, and the bacteria could be isolated until 16 days after infection. Death was not observed in the infected groups. In conclusion, oral administration of Salmonella induced clinical signs of salmonellosis with S. Dublin being more severe since there was a higher incidence of fever in this group.
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Effects of Paraffin Section Ageing in Immunohistochemistry
J.A. Ramos-Vara , J.D. Webster , D. DuSold , and M.A. Miller . Department of Comparative Pathobiology, Purdue University, West Lafayette, IN, 2Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD Environmental stresses, such as light and temperature, can alter the reactivity of certain immunohistochemical markers. We evaluated the effect of temperature and lighting on the immunoreactivity of fifty cellular or microbial antigens. Four serial, 4-mm-thick, paraffin sections were cut 12, 10, 8, 5, 3, and 1 month, and 3 and 1 day before immunohistochemistry. Slides were stored at (A) room temperature (RT) in the dark, (B) 4 C in the dark, (C) RT in direct cool-white fluorescent light (4100-4200 Kelvin), or (D) RT with window-sill exposure to sunlight and ceiling-mounted fluorescent light. Simultaneous immunohistochemistry runs were performed in an automated immunostainer using standard immunoperoxidase methods. Immunoreactivity scoring: 4highest, 310-15% reduction, 216-60% reduction, 1>60% reduction, or 0no reactivity. Any loss of immunoreactivity was proportional to the tissue section age, and was greater in sections exposed to light than in those kept in the dark. Immunoreactivity was eliminated completely only in lightexposed sections as early as 1 month post-sectioning (CD45). Other markers with complete loss of immunoreactivity were bovine viral diarrhea virus, canine and feline CD18 (only with treatment C), CD31, CD45, CD68, canine parvovirus, chromogranin A, thyroid transcription factor-1, and vimentin (only treatment C). Markers with complete loss after light exposure also had reduced immunoreactivity when kept in the dark, as early as day 3. Six markers (Bartonella spp., CD11d, E-cadherin, high molecular weight cytokeratins, p63, progesterone receptor) had no decrease in immunoreactivity during the study, regardless of treatment. In conclusion, light-induced antigen decay (tissue section ageing) is antigen-dependent, and should be considered in trouble-shooting unexpectedly weak or negative immunohistochemical reactions in stored paraffin sections.
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Gastric Tumor Development in SMAD3-Deficient Mice Initiates From Forestomach/Glandular Transition Zone Along the Lesser Curvature
K.T. Nam1,2,5, R.ONeal2,5, R.J. Coffey1,3,4,5, and J.R. Goldenring1,2,4,5. 1 Nashville Department of Veterans Affairs Medical Center, and 2Departments of Surgery, 3Medicine and 4Cell and Developmental Biology, 5Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, Tennessee SMAD proteins are downstream effectors of the TGF-b signaling pathway. Smad3 null mice develop colorectal cancer by 6 months of age. In this study, we have examined whether loss of Smad3 promotes gastric cancer in mice. Pathology of stomachs from Smad3-/- mice at 4, 6, and 10 months of age was compared with age-matched Smad3 heterozygous and wild type mice. E-cadherin, BrdU, Ki-67, pSTAT3, and TFF2/SP expression was analyzed by immunohistochemisty. In addition, we examined DCLK1 expression and investigated whether tuft cell markers identify these cells. Examination of Smad3-/mouse stomachs at 6 months of age revealed the presence of cauliflower-like growths along the lesser curvature in the proximal fundus. Histologically, 6month-old Smad3-/- mouse stomachs showed metaplastic columnar glands initiated from the transition zone junction between the forestomach and the glandular epithelium along the lesser curvature of the stomach. 10-month-old Smad3-/mice all exhibited invasive gastric neoplastic changes with increased Ki-67, pSTAT3 expression, and aberrant cytosolic E-cadherin staining in papillary glands within the invading submucosal gland. DCLK1 expressing cells were observed in 10-month-old Smad3-/- mice within tumors and in fundic invasive lesions. DCLK1 staining cells co-localized with the tuft cell marker acetylated-a-tubulin. Smad3 null mice develop gastric tumors in fundus, which arise from the junction between the forestomach and the glandular epithelium. These lesions progress to prominent invasive tumors. Tuft cells labeled by DCLK1 were found throughout the gastric fundic tumors. These findings suggest Smad3 null mice represent a novel model of proximal fundic gastric cancer.
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Immortalization of Elephant Umbilical Vascular Endothelial Cells for Elephant Endotheliotropic Herpesvirus Infection
S.Y. Long, C.J. ap Rhys, S.Y. Heaggans, and G.S. Hayward. Viral Oncology Program Johns Hopkins School of Medicine, Baltimore, MD Elephant endotheliotropic herpesvirus (EEHV), which causes a rapid acute hemorrhagic disease in young Asian elephant calves (Elephas maximus), was first reported in 1999 and has resulted in more than 75 reported young captive Asian elephant deaths in the zoos, circuses and wild. Seven different species of EEHVs have been identified to date. Research on these viruses has been inhibited due to the inability to grow these viruses in cell cultures. Past attempts to establish a stable elephant umbilical vascular endothelial cell line (EUVEC) from Asian elephant umbilical cords as a reagent for viral propagation were restricted by a limited number of cell passages before replicative senescence or spontaneous transformation into spindle cells . We transduced EUVEC with Simian virus 40 Large T protein and Human Telomerase Reverse Transcriptase (hTERT) using a vesicular stomatitis virus-G protein pseudotyped retrovirus with drug selection. A separate green fluorescent protein pseuotyped retrovirus was used to confirm transduction with EUVEC . Transduced EUVECs continue to proliferate after numerous passages with no spontaneous transformation into spindled cells when compared to non-transduced EUVECs. Polymerase chain reaction with specific primers designed for inserted genes will be used to confirm cellular transduction.
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Lymphocryptovirus-Associated PTLD Viral Transcript Expression in 8 Xenotransplanted Cynomolgus Macaques
Laura Cavicchioli1, Susan Westmoreland2, Serena Ferraresso1, Martina Carniel1, Andrea Danesi1, Giuseppe Palmisano1, Sarav Kaliyaperumal2, Marta Vadori3, Emanuele Cozzi3,4, Massimo Castagnaro1. 1Faculty of Veterinary Medicine, University of Padova, Italy, 2New England Primate Research Center, Southborough, MA, United States, 3CORIT, Padova, Italy, 4Padova General Hospital, Padova, Italy The aim of this work is to characterize posttransplant lymphoproliferative disorders (PTLD), frequently related to Epstein Barr Virus. PTLD was observed in 8 adult immunosuppressed cynomolgus macaques, transplanted with neural precursors from CTLA4Ig transgenic pigs to treat pharmacologically-induced Parkinsons disease. Mean time of occurrence of clinical signs of PTLD was 177.62 days and mean survival time was 199.87 days. In 7/8 cases (87.5%) PTLD occured extranodally either in the nasal cavity (50%), intestinal tract (50%), or both (12.5%); In 4/8 cases (50%), lymph node involvement was present. Histologic diagnosis and immunohistochemical profile for CD3, CD5, CD20, CD79cyt, and Ki-67 were compatible with a monomorphic PTLD, most frequently, high grade diffuse large B-cell lymphoma. Double-labeled immunohistochemistry for Epstein-Barr virus nuclear antigen (EBNA)-2 and CD20 revealed a high percentage of double positive CD20/EBNA-2 neoplastic cells, while CD3 cells in the tumors were negative for EBNA2. RT-PCR of transcripts EBNA-1 and Latent Membrane Protein-1 (LMP-1) were performed in neoplastic cells from 5/8 PTLD primates and 2 hyperplastic lymph nodes from 2 PTLD primates. Consensus-PCR with specificity for EBNA-2 gene was performed on PTLD samples with 92% sequence identity between Rhesus and cynomolgus viruses. EBNA-1 and EBNA-2 expression was demonstrated in all PTLD specimens (no expression in controls). Four different RT-PCR assays failed to detect LMP-1 in all specimens. This report identifies pathologic features of PTLD in cynomolgus monkeys in the setting of neuronal transplantation and a specific pattern of viral transcript expression (EBNA-1 EBNA-2 LMP-1-) in Lymphocryptovirus-associated PTLD.
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Innate Immune Response in Cache Valley VirusInfected Ovine Fetuses
A. Rodrigues , P. Dorniak , J. Filant , K. Dunla , F.W. Bazer J. Welsh , A. de La Concha3, P. Varner1, and J.F. Edwards1. 1Department of Veterinary Pathobiology and 2Center for Animal Biotechnology and Genomics, Department of Animal Science, Texas A&M University, College Station, TX, and 3Texas Veterinary Medical Diagnostic Laboratory, College Station, TX Cache Valley virus (CVV) is a mosquito-borne bunyavirus distributed throughout the United States. CVV causes in utero malformations mainly affecting the musculoskeletal and central nervous system of fetal lambs. These lesions are associated with direct viral infection and destruction of neurons and myocytes as well as infection of placental membranes and a reduction of placental fluid volumes. The virus is cleared from most infected tissues between 4-6 weeks after infection and before production of antibodies. We hypothesized that antiviral molecules of the innate immune system clear virus from infected tissues. Gene expression associated with this innate, immune response was quantified by real-time, quantitative PCR. Upregulated genes in infected fetuses included ISG15, Mx gene, IL-1, IL-6, TNF-E, TLR-7 and TLR-8. The amount of Mx protein, an interferon stimulated GTPase capable of restricting growth of bunyaviruses, was elevated in the allantoic and amniotic fluid in infected fetuses. These results demonstrated that the ovine fetus is able to stimulate an innate immune response to CVV and this response presumably contributes to viral clearance in infected animals. This is the first demonstration of the ovine fetal, antiviral, innate immune response against bunyaviruses.
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Progesterone Receptor Gene Up-Regulation: A Screening Tool to Detect Estrogen Illicit Use in Beef Cattle
S. Divari1, C. Mulasso1, F. Uslenghi1, F.T. Cannizzo1, F. Spada1, R. De Maria1, N. Brina2, and B. Biolatti1. 1Department of Animal Pathology, Faculty of Veterinary Medicine, University of Turin, Italy, 2COOP Italia, Italy The monitoring of gene regulation via mRNA level to detect anabolic sex steroids administration in cattle is a new approach to trace illicit treatments of livestock in the meat production industry. Although sensitive and accurate analytical methods, as GC/MS or LC/MS, are applied to detect residues in tissues and other matrices from animals, often problems arise and it may be difficult to identify new synthetic steroids or their metabolites. Our previous study showed that progesterone receptor (PR) gene expression increases in bulbourethral glands and prostate of prepubertal calves treated with 17b estradiol, indicating its role as specific molecular biomarker of estrogen treatment. The aim of this study was to verify the applicability of the PR biomarker in sexually mature beef cattle treated with 17b estradiol alone or in combination with other growth promoters. Accessory sex glands were sampled from 42 male beef cattle, divided in six experimental groups, including two control groups K1 and K2. Group A was treated with 17b estradiol (20 mg i.m., weekly, for 5 times), group B was treated with dexamethasone (0.7 mg/per os/day/animal for 40 days), group C was implanted with Revalor-200 and group D was implanted with Revalor-200 plus dexamethasone (0.7 mg/per os/day/animal). 17b estradiol, either alone or in combination, induced up regulation of PR gene expression even when histological changes of sex accessory glands were absent, confirming the high sensitivity of PR biomarker as an indirect diagnostic screening tool to detect illicit estrogen treatment in beef cattle.
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Renal Glomerular Changes in 5/6 Nephrectomized Common Marmoset Monkeys
Y. Suzuki1, I. Yamaguchi1, K. Myoujou1, N. Kimoto1, K. Saeki1 M. Imaizumi1, C. Takada1, K. Takaba1, and J. Yamate2. 1Toxicological Research Laboratories, Kyowa Hakko Kirin, Co., Ltd., Shizuoka, Japan, 2Veterinary Pathology, Osaka Prefecture University, Osaka, Japan Chronic kidney disease is emerging as a worldwide public health problem. We are investigating the relevancy and availability of five sixthnephrectomized common marmoset monkeys (Callithrix jacchus) as a renal failure model. As a part of the investigation, in this study, renal glomerular changes in the nephrectomized marmosets were histopathologically and immunohistochemically evaluated, and compared with those in the nephrectomized rats. In four female marmosets aged 45-63 months at 13 weeks after the nephrectomy, enlargement of Bowmans capsule and atrophy of glomeruli were observed in all animals and other slight changes were present in 1 or 2 animals. However, there were no significant changes in mesangial matrix injury score, vimentin positivity, desmin positivity and the number of WT1 positive cells between the control and partial nephrectomy groups. On the other hand, in rats at 13 weeks after the nephrectomy, various histological changes were observed; mesangial matrix injury score, vimentin positivity and desmin positivity were increased and the number of WT1 positive cells was decreased. As a result, we concluded that impacts on the renal glomerulus at 13 weeks after five sixth-nephrectomy in marmosets were weaker than those in rats. We have to conduct further studies about the reason why renal glomeruli in marmosets were not affected by nephrectomy, and about tubular injury, interstitial cellular infiltration and fibrosis. In the upcoming meeting, we will provide more detailed similarities/differences of glomerular lesions between humans and marmosets.
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The Domestic Ferret Model of Influenza Virus Infection and Disease Pathogenesis
Ian N. Moore1,2, Kurt J. Williams2, and Kanta Subbarao1. 1Laboratory of Infectious Disease National Institute of Allergy and Infectious Diseases, NIAID, NIH, Bethesda, MD, and 2Diagnostic Center for Population and Animal Health, Michigan State University East Lansing, MI In 2009, a newly emerged swine-origin influenza A virus caused a pandemic associated with severe disease and mortality in humans. The ferret model was used to investigate the kinetics of virus replication, disease pathogenesis and anti-viral and vaccine efficacy for this strain of influenza. However, disease outcomes following experimental infection with the pandemic virus varied widely. We hypothesize that dose of virus, volume of inoculum, age of ferrets, and viral subtype contribute to the clinical and pathologic severity of experimental influenza A virus infection in ferrets. We evaluated the effect of these variables on disease outcomes by administering virus to young (8-12 week old) ferrets at doses of 106 TCID50/ml or 107TCID 50/ ml. Each dose of virus was administered in one of three different inoculum volumes (0.2, 0.5 and 1.0ml). At days 1, 3 and 6 animals were humanely euthanized and tissues from the upper and lower respiratory tract were collected for virus titration and histopathology. The virus replicated to high titer in the upper and lower respiratory tract but was associated with minimal clinical illness and no mortality. We observed consistently higher virus titers in the right caudal lung lobe. We did not observe a significant effect of virus dose on viral replication. Similar experiments in older (6 month old) ferrets are in progress. Based on our findings, we will propose parameters to reduce inter and intra-laboratory reproducibility for ferret studies.
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Rhesus Cytomegalovirus-Associated Facial Neuritis in Simian Immunodeficiency VirusInfected Rhesus Macaques (Macaca mulatta)
B. T. Assaf and A. D. Miller. New England Primate Research Center, Harvard Medical School, Southborough, MA 01772 Cytomegalovirus (CMV) is a ubiquitous Beta-herpesvirus that has multiple pathologic manifestations in immunocompromised individuals. Central and peripheral nervous system pathology is a common feature in individuals chronically infected with human immunodeficiency virus (HIV). Although human CMV (HCMV)-associated disease is common in HIV infected individuals, HCMV neuritis is less common, with the exception of HCMV-associated optic neuritis. Rhesus cytomegalovirus (RhCMV) is similarly a common opportunistic infection in simian immunodeficiency virus (SIV) infected rhesus macaques; however the incidence of RhCMV-associated neuritis in optic and facial nerves is unknown. A retrospective analysis of the SIV positive rhesus macaques necropsied at the New England Primate Center identified 115 cases of histologically confirmed RhCMV infection. Of the 115 cases, 10 cases of RhCMV-associated neuritis of facial nerves were identified. Multiple nerves within facial tissues were involved, including nerves adjacent to the lacrimal glands (2/10), parotid and submandibular salivary glands (2/10), labial and buccal mucosa (3/10), tongue (5/10), and peri-tonsillar soft tissue (2/10). Histologically, all lesions were similar and characterized by moderate to marked infiltration by non-degenerate neutrophils admixed with fewer histiocytes associated with myelin vacuolation and degeneration. The inflammation frequently invaded into the surrounding tissue and was associated with variable degrees of degeneration and necrosis. Within all lesions, numerous cytomegalic cells with marked karyomegaly were scattered throughout the inflammation and contained prototypical cytomegalovirus inclusion bodies. Involvement of facial nerves emphasizes the neurotropic potential of RhCMV and illustrates another potential site of RhCMV infection in immunocompromised patients. Further exploration of RhCMV-associated facial neuritis is indicated to determine if this is an appropriate animal model for human facial neuritis syndromes.
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A Common Marmoset Model of Nonalcoholic Steatohepatitis
J.A. Kramer1, J. Grindley2, L. Makaron1, R. Kohli3, M. Kirby3, K.G. Mansfield1, and L.M. Wachtman1. 1New England Primate Research Center, Southborough, MA, 2Cummings School of Veterinary Medicine, N. Grafton, MA, 3Division of Gastroentrology, Hepatology, and Nutrition, Cincinnati Childrens Hospital Medical Center, Cincinnati, Ohio Non-alcoholic fatty liver disease (NAFLD), one aspect of metabolic syndrome, is a growing public health concern worldwide. NAFLD may progress to nonalcoholic steatohepatitis (NASH) and can ultimately cause cirrhosis or hepatocellular carcinoma. Animal models of NAFLD/NASH are needed to investigate their causes, progression, and pathogenesis. Unfortunately, many of the current rodent models fail to naturally recapitulate one or more features of the human disease. We have previously shown that common marmosets fed either a high-fat or glucose-enriched diet develop many features of metabolic syndrome including dyslipidemia, atherosclerosis, hyperglycemia, diabetes mellitus, and increased body mass. To further assess this model, we compared serum chemistry results, absolute amounts of hepatic triglyceride and glycogen, and assessed hepatic triglyceride and glycogen content and hepatocellular inflammation in liver samples obtained at necropsy. Our data shows that animals fed modified diets develop increased total body weight, liver weight, cholesterol, AST, and GGT and have increased hepatic inflammation including CD3 lymphocytes and HAM56 macrophages. 4/8 animals fed a glucose-enriched diet and 5/8 animals fed a high-fat diet developed NASH which was associated with a variety of clinical findings including increased body weight, liver weight, AST, ALT, GGT, and serum triglycerides. In addition, animals that developed NASH had increased hepatic inflammation and hepatic triglycerides. A nonhuman primate model of NASH has benefits over currently used rodent models including a closer genetic relationship to humans, more analogous physiology, and as a large animal model in drug development and discovery.
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Abnormal Glucose Tolerance in Neworn Cystic Fibrosis Ferrets Alterations in Islet Morphology and Function
A.K. Olivier1*, Y Yi2*, X Sun2*, H Sui2, B Liang2, D Lei2, J.T. Fisher2, N.W. Keiser2, W Zhou2, G Li3, Z Stewart3, A.W. Norris4, and J.F. Engelhardt2. Departments of 1Pathology, 2Anatomy and Cell Biology, 3Surgery, and 4Pediatrics, University of Iowa, Iowa City, Iowa 52242. *Equal contribution Cystic fibrosis related diabetes (CFRD) leads to worsened lung disease, decreased nutrition and decreased survival. We have developed a CF transmembrane conductance regulator knockout ferret model that demonstrates abnormal glycemia at birth. Our study goals were to define structural and functional characteristics of newborn CF and non-CF ferret endocrine pancreas. Pancreas sections were immunostained for insulin (beta cells) and glucagon (alpha cells) to quantify structural differences. The percentage of lobular area expressing insulin or glucagon was not different between genotypes. However, newborn CF pancreata had fewer large islets with significantly more islet cell clusters and isolated insulin positive cells. There was no difference in beta-cell replication by insulin/ PNCA dual staining. These results demonstrate morphologic differences in size and number of islets in the newborn CF pancreas without altered total insulin and glucagon expression. Progressive loss of insulin and glucagon staining was observed as CF animals aged, associated with extensive exocrine pancreas pathology. Glucose tolerance tests (GTT) and L-arginine tolerance tests (ATT) were performed on newborn animals to assess endocrine function. Compared to non-CF, CF kits demonstrated elevated peak blood glucose levels during GTT with a 4-fold blunting of first phase insulin secretion. By contrast, CF newborn second phase insulin secretion was elevated 4-fold. These results suggest that abnormalities in endocrine pancreas function occur prior to overt exocrine pancreas destruction. Future studies utilizing the CF ferret model should aid in understanding the multiple factors that contribute to development of CFRD.
157
Additional Characterization of a Carbonic Anhydrase-1 Promoter/Enhancer Cre-Recombinase Transgenic Mouse Model of Colon Cancer
R.L. Johnson, J.C. Fleet, and P.W. Snyder. Departments of Comparative Pathobiology and Nutrition Science, Purdue University, West Lafayette, IN We previously reported the use of a modified carbonic anhydrase-1 promoter/enhancer to drive cre-recombinase (Cre) expression in approximately 10 percent of epithelial cells of the large intestine of mice (CAC mice). This model provides unique advantages for studying the molecular biology of the colon and colorectal carcinogenesis when crossed with mouse strains containing floxed alleles of interest. For example, when we used the CAC mouse to conditionally delete the tumor suppressor gene Apc from the large intestine, heterozygous and homozygous mice developed tumors limited to the large intestine. This was enhanced by inducing colitis with dextran sulfate sodium. Initial characterization of this transgenic mouse also revealed weak, multifocal expression of Cre in hepatocytes. Here we further examined transgene expression by crossing CAC mice with B6.129S4-Gt(ROSA)26Sortm1Sor/J (ROSA) mice, whose genome contains an E. coli beta-galactosidase transgene preceded by a floxed stop codon. Cells expressing Cre remove this stop codon and allow transcription of beta-galactosidase, making the ROSA mouse a reporter of Cre activity. Beta-galactosidase was detected by immunohistochemistry and by observing the histochemical conversion of one of the enzymes substrates (X-gal) to a blue crystalline precipitate. Using this approach, we found evidence of weak Cre activity in neurons, gastrointestinal interstitial cells of Cajal, seminal vesicle epithelium, seminiferous tubules, and ovarian stroma. We observed no gross or histologic phenotype in these tissues when the CAC mouse was crossed with a floxed Apc mouse strain. None-the-less, this model is still a valuable tool for investigating the molecular biology of the colon and colorectal carcinogenesis because large intestinal transgene expression is much more robust than in other tissues.
156
Activation of the TGF-Beta Pathway in a DMN-Induced Fish Model of Hepatic Fibrosis
A.J. Van Wettere1,3, S.W. Kullman2,3, D.E. Hinton4, and J.M. Law1,3. 1College of Veterinary Medicine, 2Department of Toxicology, 3Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, NC, and 4Nicholas School of the Environment, Duke University, Durham, NC Despite a wealth of information in humans and rodents, the mechanism of hepatic fibrosis is poorly understood in fish. In mammals, hepatic stellate cell (HSC) transdifferentiation into a fibrogenic myofibroblast-like phenotype is a key event and transforming growth factor beta (TGF-b) pathway is a critical mediator of this process. In this study, we examined the changes in the mRNA expression of tgfb1, tgfb receptor I (tgfbr1) and II (tgfbr2), and smad3 during development of hepatic fibrosis in a Japanese medaka fish model of hepatic injury. Three-month-old medaka were exposed to dimethylnitrosamine (DMN) in the ambient water for two weeks and euthanized 2, 4, 6, or 10 weeks after exposure. Gene expression was determined using quantitative RT-PCR and correlated with histology. Levels of tgfb1, tgfbr2 and smad3 mRNA were significantly upregulated during development and progression of fibrosis. Expression level of tgfbr1 remained unchanged. We also assessed hepatic stellate cell activation using muscle specific actin immunohistochemistry and collagen content using Massons trichrome-stained liver sections. Increase in tgfb1, tgfbr2 and smad3 mRNA expression coincided with activation of HSC and deposition of extracellular matrix. Collectively these data support a role for TGF-b pathway in mediating the development of fibrosis during chronic liver injury in the medaka fish model.
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An Orthotopic Xenograft Model of Osteosarcoma With Metastasis
B.K. Chaffee, F. Xu, and M.J. Allen. College of Veterinary Medicine, The Ohio State University, Columbus, OH Osteosarcoma (OSA) is the most common primary malignancy of bone in humans and dogs, and the second most frequent cause of cancer related deaths in children. Despite improvements in the management of the primary disease, many patients eventually succumb to distant metastasis, most often involving the lungs. We have developed an orthotopic xenograft model that recapitulates the growth of the primary bone tumor followed by metastasis to the lung. Nude mice were injected subcutaneously with 106 canine osteosarcoma cells. The subcutaneous tumors were allowed to grow and then were aseptically harvested and frozen or implanted as fresh tissue. Additional mice underwent intra-tibial implantation of the fresh or frozen harvested solid tumor tissue. Mice were monitored weekly by digital radiography. Beginning one week postimplantation, two mice were euthanized every week to evaluate the time course of the tumor growth in the tibia. At 5 weeks post-implantation, remaining mice underwent hind limb amputation at the coxofemoral joint to completely remove the primary tumor. Micro-CT imaging of the tibias was performed, followed by routine histologic processing. At 12 weeks post-implantation, the mice were euthanized and the lungs harvested for histology. All mice developed lytic and proliferative radiographic lesions in the proximal tibia and the diagnosis of osteosarcoma was confirmed by histology. At the 12 week time point, 7 out of 16 mice had gross evidence of lung metastasis with many more showing microscopic metastasis. The successful growth of the osteosarcoma at the primary site in bone, followed by a relatively high rate of metastasis, demonstrates that this is a promising model that can be used to further characterize the disease and investigate therapeutic interventions.
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Antigen 85 Vaccination Protects CBA/J Mice Against Aerosol Challenge With Mycobacterium tuberculosis Despite Low AntigenSpecific InterferonGamma Responses
G. Beamer1,2, J. Cyktor1, D.K. Flaherty3, P.C. Stromberg2, B. Carruthers1, and J. Turner1,4. 1Center for Microbial Interface Biology, The Ohio State University, Columbus, OH 43210, 2Department of Veterinary Biosciences, The Ohio State University, Columbus, OH, 3Vanderbilt Medical Center, Vanderbilt University, Nashville, TN, 4Division of Infectious Diseases, Department of Internal Medicine, The Ohio State University, Columbus, OH CBA/J mice are more susceptible to aerosol infection by Mycobacterium tuberculosis (M.tb) than are the commonly used C57BL/6 mice. We are interested in understanding differences in mouse strain-dependent immune responses during primary M.tb infection and M.tb challenge, because the results may provide insight into the responses of M.tb-susceptible humans. CBA/J mice had poor M.tb Ag85-specific interferon-gamma responses during the prolonged symptom-free phase of primary M.tb infection, as compared to C57BL/6 mice. These differences were most pronounced during early infection, and were also apparent from blood samples. In both mouse strains, Ag85 vaccination followed by M.tb challenge increased Ag85-specific interferon-gamma responses, and this was associated with a significant reduction of the pulmonary M.tb burden. Interestingly, Ag85 vaccination protected CBA/J mice equally well as C57BL/6 mice, yet the levels of the Ag85specific interferon-gamma were still significantly less than that of C57BL/6 mice. These results suggest that pre-exposure Ag85 vaccination may protect both M.tb-resistant and -susceptible individuals, and that functional M.tb vaccination needs only to increase antigen-responsiveness by a small amount. Further studies are needed to determine whether disease outcome is improved by Ag85 vaccination and whether post-exposure vaccination can protect CBA/J mice.
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Characterization of the Effects of EBI2 Gene Disruption in the Immune Response and the Formation of Germinal Centers During B Cell Response to T CellDependent Antigens
O. Mendes, S. Dwyer, S. Shimshock, D. Chen, L. Li, R. Diao, El-B. Haddad, M. Taurino, and S. Thurairatnam. DSAR, F&WH TSU, I&I TSU, Biologics, MIT Departments, Sanofi, Bridgewater, NJ There is increasing evidence that EBI2 (GPR183) has an important role in B lymphocyte migration and formation of germinal centers (GC). We disrupted EBI2 by targeted mutagenesis to create Balb/c Tac - EBI2 N7 (EBI2-KO) mice to morphologically and functionally evaluate differences between wildtype (WT) and EBI2-KO immune response and formation and function of GCs. EBI2-KO mice had lower white blood cell and lymphocyte counts. MRI of EBI2-KO mice showed no evidence of an altered spleen volume. Although spleen morphology was similar to WT, fewer active less defined germinal centers were observed in EBI2-KO mice along with alterations in mounting and development of immune response to a variety of stimuli including B cell response to T Cell-Dependent antigens. EBI2 expression was increased with anti-CD40/IL4 stimulation; EBI-2 KO had reduced LPS-induced NF-kB response; and reduced Delayed Type Hypersensitivity after tuberculin challenge. Additionally, EBI2 KO inoculated with T-cell antigen showed a less significant increase in spleen weight, smaller follicle size, less follicle definition and fewer numbers of active follicles and germinal centers with less GC B cells and Dendritic Cells. We conclude that the data offers additional detail to the growing evidence that EBI2 is a modulatory molecule in mounting and development of B cell response to T Cell-Dependent Antigens observed here with a different array of in vivo and ex vivo stimulants and in vivo EBI2 disruption alone is sufficient to significantly change the morphological appearance of the spleen architecture after T Cell-Dependent Antigen stimulation.
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Cellular Tropism of Orthopoxviruses in the Nonhuman Primate Ovary
J.A. Cann, V. Wahl-Jensen, R.F. Johnson, A.J. Johnson, and P.B. Jahrling. Integrated Research Facility, Division of Clinical Research, National Institute of Allergy and Infectious Diseases, NIH, Frederick, MD It is well known that orthopoxvirus infections commonly result in abortion, however, the pathologic mechanisms responsible are not known. We performed a retrospective immunohistochemical study to determine the incidence and cellular tropism of orthopoxviruses (OPV) in the nonhuman primate ovary. Cynomolgus macaques were inoculated with 109 pfu variola virus (VARV; n3), 105-107pfu monkeypox virus (MPXV; n14), or 105-107pfu cowpox virus (CPXV; n11). Ovaries were collected and immunostained with a panOPV marker (VACV) alone or in conjunction with the cellular markers steroidogenic factor (SF)-1 and SF-2. Colocalization was quantified using multispectral imaging analysis. VACV staining was found in 3/3 VARV-infected, 7/14 MPXV-infected, and 11/11 CPXV-infected animals, and incidence was independent of inoculation dose. VACV staining localized to the theca interna, theca externa, and corpora lutea. Of the VACV-positive pixels, 39.4 /8.7% also stained for SF-1, and 47.6 /- 4.4% also stained for SF-2. Transmission electron microscopy confirmed active viral infection of thecal and luteal cells. These results demonstrate that orthopoxviruses target steroidogenic cells (granulosa-thecal and luteal) in the ovary and suggest that virus-associated impairment of progesterone production may be one mechanism by which fetal loss occurs in pregnant females infected with orthopoxviruses.
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Chemokine Receptor 5 Inhibition Prevents SIV-Induced Cardiac Dysfunction
K. Kelly, C.G. Tocchetti, A. Lyashkov, S.E. Queen, R.J. Adams, D.R. Graham, N. Paolocci, and J. L. Mankowski. Departments of Molecular and Comparative Pathobiology and Medicine, Johns Hopkins University School of Medicine SIV-infected rhesus macaques develop echocardiographic abnormalities significantly correlated with myocardial viral load that closely resemble cardiac dysfunction in HIV patients. To define the mechanism underlying HIV/ SIV cardiac dysfunction, we performed complementary in vitro and in vivo studies focusing on the potential role of chemokine receptor 5 (CCR5), an important receptor mediating both viral infection and inflammation. As signaling subsequent to interaction between receptor and HIV/SIV envelope glycoprotein or cognate chemokines presents a mechanism mediating viral damage in the absence of productive infection, CCR5 is an attractive target for further characterization in the heart. In our studies, we established that isolated rhesus macaque cardiomyocytes express CCR5 and that cognate chemokine ligands of CCR5 such as CCL5 alter sarcomeric contractility. The addition of CCL5 to cardiomyocytes significantly decreased contractility (p<0.0001, paired t-test) that was reversed by addition of the small molecule CCR5 inhibitor Maraviroc (MVC). CCL5 significantly increased contraction time (p0.03, paired t-test) without altering calcium flux, suggesting that CCL5 signaling through CCR5 desensitizes the sarcomere to calcium. To confirm the role of CCR5 in the development and progression of SIV cardiac dysfunction, cardiac function in six MVC-treated, SIV-infected macaques were compared to 22 untreated, SIV-infected macaques. MVC-treated, SIV-infected macaques did not develop cardiac dysfunction and hadpreservation of diastolic parameters (MV DT, E/A, and myoRT) and ejection fraction compared to untreated, SIV-infected macaques. Thus, CCR5 inhibition may represent an attractive new avenue to treat or prevent systolic and diastolic dysfunction in HIV-infected individuals.
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Clinical and Pathologic Features of H-Type Bovine Spongiform Encephalopathy Associated With E211K Prion Gene Polymorphism
J.J.Greenlee1, J.D. Smith1, M.H.West Greenlee2, and E.M. Nicholson1. 1Virus and Prion Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, Iowa, 2Department of Biomedical Sciences, Iowa State University, Ames, Iowa The majority of cases of bovine spongiform encephalopathy (BSE) have been ascribed to the classical form of disease that is transmissible to humans in the form of new variant Creutzfeldt-Jakob disease. Since 2004, forms of BSE have been described that have atypical molecular profiles (H- or L-type, high and low, respectively). Most cases of atypical BSE arise spontaneously, but one case of H-type BSE was associated with a heritable mutation in the prion protein gene (prnp) called E211K. The purpose of this work is to describe features of transmission of E211K H-type BSE including clinical signs, antemortem test results, histopathology, and tissue distribution of PrPSc by western blot (WB) and immunohistochemistry (IHC). In a calf with E211K polymorphism inoculated with E211K H-BSE, there was rapid progression to disease with onset of clinical signs at 9.4 months post-inoculation (PI). Signs of depression, head pressing, reluctance to rise and accentuated licking and chewing movements that were not associated with feeding increased in frequency and severity until the calf was euthanized at 9.8 months PI. BSE-H was confirmed by WB and widespread distribution of abnormal prion protein (PrPSc) within neural tissues was demonstrated by IHC. Preclinical deficits in retinal function were demonstrated by electroretinogram and an antemortem decrease in retinal thickness was demonstrated by optical coherence tomography. A rare genetic form of BSE may represent a potential origin of classical BSE. Future studies will assess potential for transmission to cattle with wild type prnp rather than prnp with E211K polymorphism.
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Cutaneous Papillomatosis due to a Novel Laboratory Mouse Papillomavirus (MusPV) Infection
J.P. Sundberg1, C.S. Potter1, K.A Silva1, A. Ingle3, J. Joh2, S.J. Ghim2, and A.B. Jenson2. 1The Jackson Laboratory, Bar Harbor, ME, 2James Graham Brown Cancer Center, University of Louisville, Louisville, KY, and 3Tata Memorial Centre, ACTREC, Navi Mumbai, India Spontaneous, florid papillomatosis arose in a colony of NMRI-Foxn1nu/ Foxn1nu (nude) mice and were transmissible to both immunocompetent and immunodeficient mice. Lesions appeared at the mucocutaneous junctions of the nose and mouth affecting hair follicles. Histologically, lesions were classical papillomas with epidermal hyperplasia on thin fibrovascular stalks in a verrucous pattern. Koilocytotic cells were observed in the stratum granulosum of the papillomatous lesions. Koilocytes were positive for PV groupspecific antigens. Virus particles were observed in crystalline intranuclear inclusions within keratinocytes. The MusPV genome is 7510 bp in length, is organized similar to other PVs, and has at least 7 open reading frames (E1, E2, E4, E6, E7, L1, and L2). Phylogenetic analysis indicates that MusPV belongs to the pi-genus together with 4 other rodent PVs (McPV2, MaPV1, MmiPV, and RnPV1). Of the rodent PVs, MusPV appears most closely related to Mastomys coucha PV (McPV2), with 65% genomic homogeneity and 80% L1 amino acid similarity. Subsequent transmission studies to B6.Cg and NU/J mice, both homozygous for Foxn1nu/Foxn1nu resulted in papillomas on the face but basal cell carcinomas on the dorsal skin in B6.Cg nudes but no lesions in NU/J nudes, indicating strain specific modifier genes are present modulating infectivity. MusPV provides a potentially valuable novel mouse model to study mechanisms of infection, oncology, and preventive and therapeutic approaches in mice that can be translated to diseases caused by human PVs.
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Comparison of the Pathogenicity of Chinese and Low Virulent US Porcine Reproductive and Respiratory Syndrome Viruses
J.N. Henningson1, K.S. Faaberg1, B. Guo2, S.N. Schlink1, L.C. Miller1, M.A. Kappes1, M.E. Kehrli, Jr.1, S.L. Brockmeier1, T.L. Nicholson1, A.C. Vorwald1, and K.M. Lager1. 1National Animal Disease Center ARS-USDA, Ames, IA, 2 Iowa State University, Ames, IA Recently, a new strain of porcine reproductive and respiratory syndrome virus (PRRSV) has resulted in huge economic losses in the Chinese pig industry. We imported a cDNA clone of the rJXwn06 Chinese strain from which infectious virus was obtained to test the hypothesis that novel Chinese PRRSV strain would not induce severe clinical disease and pathology as seen in Asia when inoculated into U.S. high-health swine. Under ABSL3 biocontainment following NADC IACUC guidelines, 10-week-old swine were infected by intranasal inoculation with either 2 ml of 106 TCID50/ml rJXwn06 (n12 challenge, n4 contact) or the North American prototype strain VR-2332 (n8), or a sham inoculum (n8). On necropsy consistent findings were severe thymic atrophy, lymphadenopathy, and extensive interstitial pneumonia in the Chinese PRRSV inoculated pigs; these lesions were not as severe in the VR-2332 inoculated group and were not present in the sham inoculated pigs. On histopathology, a majority of Chinese PRRSV inoculated pigs had more severe and extensive, lymphoid depletion, interstitial pneumonia, lymphohistiocytic perivascular meningoencephalitis, myocarditis, and interstitial nephritis as compared to the sham and VR-2332 inoculated pigs. Bacterial copathogens likely played a contributory role to development of lesions in Chinese inoculated pigs. Immunohistochemistry labeled PRRSV antigen in pulmonary macrophages in both the Chinese and VR-2332 inoculated pigs. Preliminary results confirm that the Chinese PRRSV is a virulent virus that could be a threat to the US swine industry. Further histopathology examination and immunohistochemistry for PRRSV, CD79a, CD3 and macrophages in select tissues are planned.
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Development of Liver Tumor Models Using Transgenic Zebrafish
Z. Gong1, A.T. Nguyen1, Z. Li1, H. Zhan1, X. Huang1, C. Li1, C.H.V. Koh1, A. Emelyanov2, S. Parinov2 and J.M. Spitsbergen3. 1Department of Biological Sciences, National University of Singapore, Singapore, 2Temasek Life Sciences Laboratory, Singapore, 3Department of Microbiology, Oregon State University, Corvallis, OR The zebrafish has become a premier animal model for high-throughput low cost studies as a model for human disease. To generate zebrafish models for liver cancer, we created transgenic fish which over-express selected oncogenes under a liver-specific promoter. We have generated inducible and noninducible transgenic lines by expression of kras, cmyc or Xmrk. All lines exhibit liver neoplasia. In the kras transgenic lines, in which a constitutively active form of kras mutant (G12V) was expressed as a GFP fusion protein, abnormal over-growth of liver could be observed within one week of fertilization and the lesion of liver became increasingly severe with time, from hyperplasia to adenoma then to carcinoma. Microarray and western blot analyses confirmed upregulation of the Kras pathway. A similar trend was also observed in Xmrk transgenic fish but not in cmyc transgenic lines. The phenotype of cmyc transgenic fish was relatively mild and produced only liver adenoma. We have applied two inducible transgenic systems using the Tet-on and mifepristone induction. In both inducible systems, we demonstrated that production of liver tumors could be induced at any developmental stages and even in adult fish. These tumors were transplantable to adult fish and survived, propagated and migrated in the host fish. To confirm utility of this model in anticancer drug discovery, we showed suppression of liver hyperplasia by a MEK1 inhibitor, PD98059, in kras transgenic fish. Thus, we are now positioned for highthroughput chemical screening for anti-cancer drugs using our transgenic zebrafish models.
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Dissecting the Role of Sharpin in Idiopathic Hypereosinophilic Syndromes
C. S. Potter , Z. Wang , K. A. Silva , V. Kennedy, L. D. Shultz , H. HogenEsch2, and J. P. Sundberg1. 1The Jackson Laboratory, Bar Harbor, ME, and 2 Department of Veterinary Pathobiology, Purdue University School of Veterinary Medicine, West Lafayette, IN Two allelic mutations of the mouse Sharpin gene result in an idiopathic hypereosinophilic syndrome similar to that found in humans. Mutant mice develop a systemic disease including scaly skin characterized by hyperproliferation with marked apoptosis of the epidermis and a granulocytic, primarily eosinophilic, dermal infiltrate. To dissect the mechanisms behind this disease, compound mutants, Rag1tm1Mom (lack T and B cells) and Sharpincpdm-Dem, develop a mildly attenuated disease. Th2 cytokines play a contributing role in the Sharpin mutant phenotype. Il4ratm1Sz (diminished TH2 helper T cell response, unresponsive to both IL4 and IL13) and Sharpincpdm-Dem compound mutants develop a very severe skin disease with liver failure due to necrosis and mineralization. SHARPIN null mice have reduced secretion of IL12, possibly due to Sharpin expression in dendritic cells. Mutant mice treated with recombinant IL12 had diminished disease in a dose response manner. We previously demonstrated that treatment of Sharpin mutant mice with anti-IL5 reduced eosinophilia but not the severity of skin lesions. This work further supports those findings suggesting that eosinophils are part of the chronic proliferative disease mutant mouse phenotype, but eosinophils are not the primary effecter cells.
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Elevated Expression of Toll-like Receptor 2 in Krabbes Disease
E.R. Snook1,2, B.A. Bunnell1,2,3,4. 1Division of Regenerative Medicine, Tulane National Primate Research Center, Covington, LA, 2Graduate Program in Biomedical Sciences, 3Center for Stem Cell Research and Regenerative Medicine, 4 Department of Pharmacology, Tulane University School of Medicine, New Orleans, LA Early infantile Krabbes disease (globoid cell leukodystrophy) is a lysosomal storage disease characterized by the lack of galactocerebrosidase to adequately metabolize galactosylceramidea glycosphingolipid primarily found in myelin. In the presence of the enzyme, galactosylceramide is metabolized into galactose and ceramide, but in its absence it is deacylated into psychosine and a fatty acid through an alternative metabolic pathway. Globoid cell leukodystrophy is histologically characterized by large numbers of globoid cells distended with lysosomal storage product, prominent astrogliosis, oligodendrocyte cell death, and demyelination. Our hypothesis is that Toll-like receptors (TLRs) play a role in the pathogenesis of the globoid cell leukodystrophy through the recognition of a ligand produced during the early stages of the disease, which leads to the terminal constellation of histopathologic findings. Real-time PCR data in the twitcher mouse (murine model for Krabbes disease) demonstrates a marked upregulation of TLR2 mRNA early in the disease process and remains elevated through the terminal stages of the disease. Additionally, TLR2 has been identified by immunofluorescent histochemistry on microglia and oligodendrocytes in affected animals with increased frequency. Based on the PCR and immunohistochemical results, it appears that TLR2 is upregulated by microglia and oligodendrocytes during the course of the disease. Age-matched controls demonstrate rare TLR2 positive cells throughout the brain. Research is underway to elucidate whether TLR2 plays a functional role in the upregulation of cytokines and chemokines and which molecule(s) is responsible for its upregulation.
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Effects of Rennin-Angiotensin System on Podocyte Injury in Osborne-Mendel Rats
K. Yasuno1, H. Sakashita1, S. Araki1, R. Kobayashi1, K. Ogihara2, J. Kamiie3, and K. Shirota1,3. 1Research Institute of Biosciences, 2Laboratory of Environmental Pathology and 3Laboratory of Veterinary Pathology, Azabu University, Sagamihara, Kanagawa, Japan Osborne-Mendel (OM) rats develop mild hypertension and progressive glomerular injury with early-onset proteinuria. Our previous study suggested that podocyte injury was a key determinant for the glomerulopathy in OM rats. In this study, we tested the hypothesis that the rennin-angiotensin system (RAS) may be involved in the pathogenesis of the podocyte injury in OM rats. Male OM rats were administrated antihypertensives which included RAS blockers (angiotensin-converting enzyme inhibitor and angiotensin II type 1 receptor (AT1R) blocker) or vasodilator, hydralazine (HYD) from 3 weeks to 13 or 20 weeks of age. Age-matched untreated male OM rats were used as controls. In all treated rats, systolic blood pressure remained normotensive during the experiment. Urinary protein excretion and podocyte injury were distinctly prevented in RAS blocker-treated rats, but not in HYD-treated rats. Protein and mRNA expression of nephrin, a podocyte specific protein and a major component of slit diaphragm, reduced with age in untreated rats, but the reduction was inhibited in all antihypertensive-treated rats. However, the inhibitory effect of nephrin reduction was less in HYD-treated rats as compared to those of RAS blocker-treated rats. Additionally, we analyzed the expression of AT1R mRNA in primary-cultured podocytes of OM and F344 rats, and found that the expression of AT1R mRNA was significantly higher in OM rat. These results suggested that the glomerulopathy of OM rats might be blood pressureindependent and associated with the high-sensitivity of podocytes of the rats to angiotensin II.
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Experimental Infection of Capillaria hepatica Induces Type II Cryoglobulinemia With Glomerulopathy in ICR Mice
N. Aihara, J. K. Chambers, K. Hosono, J. Kamiie, and K. Shirota. Laboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, Sagamihara, Japan Type II mixed cryoglobulinemia is characterized by systemic vasculitis with deposition of cryoprecipitatable-immunoglobulins containing rheumatoid factor. Pathogenesis of type II mixed cryoglobulinemia has not yet been completely clarified and no animal models have been established. Here, we report an experimental model of type II mixed cryoglobulinemia that is induced by Capillaria hepatica (C. Hepatica) infection in ICR mice. In this study, oral infection of 8,000 C. hepatica eggs induced cryoglobulinemia in ICR mice at 20 and 30 days post injection (DPI). Using immunological analysis, cryoglobulinemia in infected mice was classified as type II mixed cryoglobulinemia by detection of monoclonal IgM rheumatoid factor and IgA in the cryoprecipitate of serum. Immunofluorescence revealed an increase in the number of double-positive cells for mu heavy and kappa light chains of immunoglobulin in the spleens of infected mice. The serum tests showed a marked increase in AST and ALT levels in all mice at 10 DPI. No animals showed azotemia and proteinuria during the experiment. Histologically, C. hepatica infection in the liver was established in all infected mice. Characteristic glomerulopathy associated with intense deposition of IgM and IgA filling in capillary lumina developed in the kidneys at 30 DPI. Ultrastructural analysis showed that glomerular deposits consisted of stacks of twisted microtubular structures. These serological and histological features resembled those of type II mixed cryoglobulinemia in human. This is the first experimental animal model of type II mixed cryoglobulinemia that will enable detailed studies on the pathogenesis of cryoglobulinemia.
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Expression of Secreted Mucins (MUC2, MUC5AC, MUC5B, and MUC6) and Membrane-Bound Mucin (MUC4) in the Lungs of Pigs Experimentally Infected With Actinobacillus pleuropneumoniae
H. W. Seo, K. Han, Y. Oh, C. Chae. Seoul National University, Korea The expression patterns of different secreted (MUC2, MUC5AC, MUC5B, and MUC6) and membrane-bound (MUC4) mucins were determined immunohistochemically in the lungs of pigs experimentally infected with Actinobacillus pleuropneumoniae. Forty 7-week-old colostrum-deprived pigs were randomly allocated to infected (n 20) or control groups (n 20). Five infected and uninfected pigs were euthanized at 0, 6, 12, and 48 hours postinoculation (hpi). In the infected pigs, the expression of both types of mucins, which were invariably observed, was associated with bronchiolar and respiratory bronchiolar lesions. Strong positive mucin signals were seen on the surface of bronchiolar and respiratory bronchiolar epithelium with neutrophil infiltration. The mean mucin-positive area peaked at 6 hpi and decreased significantly to control levels by 48 hpi on the surface of the bronchiolar and respiratory bronchiolar epithelium. The present study clearly demonstrates that the respiratory mucosa of uninfected pigs has weak secreted and membrane-bound mucin expression, whereas bronchopneumonia induced by A.pleuropneumoniae increases the expression of MUC2, MUC5AC, MUC5B, MUC6, and MUC4. Although the secreted and membrane-bound mucins examined in this study are significantly expressed by the airway epithelium, the expression of MUC5AC and MUC5B is significantly increased in the lungs of pigs experimentally infected with A. pleuropneumoniae compared to any other mucins examined in this study. Further studies are needed to establish the functional relationship between mucin expression and the host defense mechanism against A. pleuropneumoniae in the lungs of infected pigs.
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Fetal Obstruction in the Exocrine Pancreas Is a Primary Lesion Leading to Sequential Organ Destruction in Cystic Fibrosis Pigs
D.K. Meyerholz1, D.A. Stoltz2, A. Uc3, P.B. McCray Jr.3, M.J. Welsh2,4,5. Departments of 1Pathology, 2Internal Medicine, 3Pediatrics,4 Molecular Physiology and Biophysics, and 5Howard Hughes Medical Institute, University of Iowa, Iowa City, IA Cystic fibrosis (CF) produces lesions in multiple organs and the pancreas is prone to disease. In humans, pancreas lesions begin in fetal life. We morphometrically examined early fetal pancreas development in CF and non-CF pigs (day 41-82 of gestation - term *114 days). As early as day 41, both CF and non-CF groups had qualitative detection of amphiphilic to eosinophilic, slightly fibrillar material within lumens; however, CF pancreata had an increased incidence of this luminal material compared to non-CF pancreata (P<0.01, unpaired T-test). This increased incidence in CF pancreata remained significant through the time course. By day 54, CF pancreata had increased mean lumen diameter (P<0.05, unpaired T-test) with rare detection of dilated acini filled with eosinophilic material. This remained significantly increased in CF acini through the remainder of the time course. CF pancreata had an increased lumen to whole acinus ratio that was first detected at day 60 (P<0.01, unpaired T-test) and this continued through the remainder of the time course. Our data together with previous work shows that accumulation of luminal material is one of the earliest observed changes in the CF pig pancreas and this begins as early as day 41 of gestation. Accumulation of luminal material leads to obstruction and sequential pathologic changes including ectasia with secondary inflammation, mucous cell change and duct proliferation. Future studies aim to define the composition of this lumen material as well as the environmental conditions and cellular processing in acini that may contribute to lesion formation in CF pancreata.
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Fetal Long Bone Lesions Following Experimental Persistent Infection With Bovine Viral Diarrhea Virus
B.T. Webb1,2, R.W. Norrdin2, N.P. Smirnova1, A.Q. Antoniazzi1, H Van Campen2, C. M. Weiner2, and T.R. Hansen1. Departments of 1Biomedical Sciences and 2Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO Persistent infection (PI) with Bovine Viral Diarrhea Virus (BVDV) has been associated with osteopetrosis and other skeletal lesions in cattle. This study was undertaken to characterize the morphogenesis of the fetal long bone lesions. Forty six, BVDV nave pregnant heifers were inoculated either with non-cytopathic BVDV2 or media on Day (d) 75 of gestation to produce PI and control fetuses, which were collected via cesarean section on d82, 89, 97, 192 and 245 of gestation. No radiographic or histomorphometric abnormalities were detected in fetal long bones prior to d192. Radiographs of d192 PI fetal long bone metaphyses contained focal densities (3/7 fetuses) and multiple, alternating transverse radiodense bands (3/7 fetuses). D245 fetuses were similarly affected radiographically. Extrapolation of metaphyseal elongation rates from d185 to 192 suggests the radiodense bands are separated by approximately 15 days of longitudinal growth. Histomorphometric analysis of proximal tibial metaphyses revealed transverse zones of increased calcified cartilage core (Cg.Ar/Tb.Ar), and trabecular bone (Tb.Ar/T.Ar) areas in regions corresponding to radiodense bands (P<0.05). Numbers of tartrateresistant acid phosphatase-positive osteoclasts (N.Oc/mmB.Pm) and bone perimeter occupied (Oc.Pm/B.Pm) were both decreased (p<0.05). The greater radiodensity of calcified cartilage cores relative to fetal trabecular bone is thought to be responsible for the radiographic appearance. Collectively, these results suggest that periodic abnormal trabecular modeling is secondary to reduced numbers of osteoclasts. The factors responsible for the periodicity are unknown but may also be related to the time required for osteoclast differentiation and the known ability of BVDV to infect early myeloid cells. USDANIFA-AFRI #2008-35204-04652.
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Gastric Regulatory and Effector T Cell Engraftment and Gastritis Due to Helicobacter pylori Depends on T Cell Transfer Dose
K.A. Eaton, C.A. Fontaine, D.M. Loeffler, B.M. Gray. University of Michigan Medical School, Ann Arbor, MI Background: H. pylori causes mild, slowly progressive gastritis in C57BL/ 6 mice, but severe, rapidly progressive gastritis in immunodeficient recipients of CD4 T cells. In order to determine the role of regulatory T cell engraftment in extent of gastritis, we evaluated T cell engraftment in recipients of different populations of CD4 T cells. Methods: H. pylori-infected RAG-KO mice were given 0.5, 1.0, or 10x106 unfractionated congenic CD4 T cells, or 0.5 or 1.0x106 CD25 enriched CD4 T cells by adoptive transfer. Eight weeks later, splenocytes and gastric lamina propria lymphocytes were isolated, and effector and regulatory T cells were quantified by flow cytometry. Extent of gastritis was determined by histologic scoring. Results: Gastric lamina propria engraftment of effector T cells was greatest in recipients of 0.5 or 1.0x106 unfractionated cells. Transfer of 10x106 cells or of populations enriched in CD25 CD4 cells resulted in lower gastric CD4 cell engraftment. In contrast, gastric engraftment of CD25 FoxP3 regulatory T cells was negligible in recipients of 0.5, or 1.0 x 106 unfractionated CD4 T cells, but was up to 100% of engrafted T cells in recipients of 10x106 cells and in recipients of CD25 enriched T cells. Splenic T cell engraftment was not affected by transferred cell population. Severity of gastritis was proportional to gastric CD4 cell engraftment and inversely proportional to regulatory T cell engraftment. Conclusions: Severe gastritis in H. pylori-infected recipient mice is likely due to preferential gastric engraftment of effector vs regulatory T cells. Regulatory T cell engraftment and suppression of gastritis is enhanced by larger T cell inocula.
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Histopathology of Aging Alpha-Galactosidase A-Deficient Mice: Comparison With Histopathologic Changes in Fabry Disease Patients
D. Bangari, J. Marshall, K. Ashe, C. Maloney, J. Pacheco, S.H. Cheng, R.K.Scheule, R.J. Desnick, and B.L. Thurberg. Genzyme Corporation, Framingham, MA Fabry disease (FD) is an X-linked genetic disorder caused by mutations in the alpha-galactosidase A (a-gal A) gene. Deficient a-gal A activity causes progressive accumulation of globotriaosylceramide (GL-3) and related glycosphingolipids within lysosomes in multiple cell types including capillary endothelial cells (ECs), vascular smooth muscle cells (VSMCs), renal tubular cells (RTCs), podocytes, mesangial cells, cardiomyocytes and neurons. FD has been modeled in mice through targeted disruption of a-gal A gene. These knockout (KO) mice completely lack a -gal A activity, accumulate GL-3 and serve as useful models for evaluation of various therapeutics. However, unlike Fabry patients, KO mice do not exhibit clinical disease and have minimal tissue pathology and normal survival, thus limiting their relevance as a disease model. In this study we examined tissue pathology of 10-18 months old Fabry KO mice to determine whether aging mice would produce advanced pathology that more closely resembled human disease. We compared light and electron microscopic changes in KO mice to those reported in Fabry patients. Compared to the age-matched wild-type mice, Fabry KO mice exhibited age-dependent substrate accumulation in various tissues including kidney and dorsal root ganglia (DRG). Most affected cell types included RTCs, DRG neurons, VSMCs and interstitial cells. Unlike FD patients GL-3 accumulation was not observed in ECs and mesangial cells; renal podocytes were rarely affected. DRG neuron involvement was associated with abnormal nociceptive response. Overall, a -gal A-deficient mice phenocopy some but not all aspects of human FD pathology. While this model has been useful for efficacy evaluation by biochemical tissue measurements, its utility for assessing efficacy by histopathology requires additional study.
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Hypoxia Upregulates Stem Cell Markers in a Canine Glioblastoma Cell Line
J.W. Koehler, N.E. Morrison, and N.R. Cox. Department of Pathobiology and Scott-Ritchey Research Center, Auburn University, Alabama Malignant gliomas currently represent the most lethal and poorly treatable primary brain cancers in both humans and dogs. Canine patients suffer from all four WHO grades of astrocytic gliomas, as well as malignant oligodendrogliomas, mixed glial tumors, and ependymomas, a fact that presents a valuable opportunity to 1) study glioma biology and therapeutics to the direct benefit of canine patients, 2) contribute to the broader scientific knowledge base by studying a naturally-occurring large animal model of the human disease, and 3) enroll dogs in clinical trials as a prelude to human trials. High-grade gliomas are invasive tumors that often have extensive areas of necrosis and are composed of a heterogeneous population of neoplastic cells expressing varied surface and cytoplasmic markers of differentiation. Within the bulk of the tumor, a subpopulation of cells with stem-like behavior appears to be responsible for the chemoresistance, radioresistance, persistent regrowth, and aggressively infiltrative behavior that are the hallmarks of these tumors. All currently available methods for prospectively isolating this subpopulation of stem-like cells for further study are imperfect, with each having its own benefits and drawbacks. In contrast to standard 20% oxygen cell culture environments, hypoxic culture conditions more closely mimic the in vivo microenvironment and have been demonstrated to have potent effects on in vitro growth, cell signaling, and up-regulation of certain key stem cell markers in human glioma cell cultures. In this work we show that canine glioma cell lines grown under hypoxic (2%) oxygen conditions have upregulation of several putative stem cell markers, including CD133 and nestin, and have upregulation of vascular marker CD31 and hypoxia marker HIF2a as measured by q-PCR.
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Human Respiratory Syncytial Virus Memphis 37 Strain Causes Moderate to Severe Disease in Perinatal Lambs
R. Derscheid, A. Van Geelen, J. Gallup, and M. Ackermann. Department of Veterinary Pathology, Iowa State University College of Veterinary Medicine, Ames, IA Respiratory syncytial virus (RSV) is a leading cause of hospitalization due to lower respiratory tract illness in infants and children of industrialized countries. Despite its ubiquity and potential severity, efficacious prophylactic drugs and treatments remain inadequate partly due to lack of satisfactory models of severe disease in the highest risk group: infants. Memphis 37 strain of human RSV (hRSV) (M37) has been used to produce mild to moderate upper respiratory disease in healthy adult volunteers. Our group has previously shown mild to moderate disease in perinatal lambs with both bovine RSV and A2 strain of hRSV. We hypothesized that M37 would produce enhanced disease in perinatal lambs. To test this, 3-5 day old lambs (n 5) were inoculated intranasally with 2ml/nostril of 1 x 107 PFU M37 and clinical signs, gross and histological lesions, immune and inflammatory responses were assessed at six days post-inoculation. Clinically, infected lambs had moderate to severe increase in expiratory effort and consistent moderate to severe gross lesions. Lambs had bronchiolitis with neutrophils and syncytial cell formation along with increased mRNA expression of inflammatory mediators such as interleukin-8 (IL-8) and interferon gamma (IFN g) with reduced expression of IFN b. The lesions and immune responses parallel those observed in infants with RSV and demonstrate infection capacity greater than hRSV A2 strain in lambs which causes mild to moderate disease when administered intrabronchially and minimal infectivity when administered intranasally. This work introduces a model with potential to test environmental, host, and clinical outputs affecting disease severity as well as provide opportunities to test new treatment modalities and therapies.
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In Vitro and In Vivo Fitness of a Cloned SIV CTL Escape Mutant in a Pigtailed Macaque Model of HIV CNS Disease
S.E. Beck, S.E. Queen, and J.L. Mankowski. Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore MD Pressure exerted by major histocompatibility complex (MHC) class-Imediated cytotoxic T-lymphocyte (CTL) control drives escape mutations in human immunodeficiency virus (HIV). These HIV CTL escape mutations have a variable impact on HIV fitness depending on their specific location in the viral genome. Some MHC class I alleles are associated with low plasma viral load and long-term non-progression in humans, but other MHC class I alleles are associated with rapid progression to AIDS. We have demonstrated that expression of the MHC class I allele Mane-A*10 confers resistance to SIV-induced CNS disease in a pigtailed macaque model of HIV CNS disease. However, resistance to SIV CNS disease is not absolute in macaques, likely because escape from ManeA*10 control develops in some animals (*25%) leading to CNS disease. In these animals, cloning and sequencing of viral RNA from the CNS shows emergence of a consensus K165R escape mutation in the capsid region of SIV gag. To characterize in vitro and in vivo fitness of this escape mutation, we used sitedirected mutagenesis to insert K165R into the backbone of the neurovirulent molecular clone SIV/17E-Fr and then produced infectious viral stocks. The SIV/17E-Fr K165R clone, like parental SIV/17E-Fr, was shown to be replication competent in vitro in both pigtailed macaque primary macrophage cultures and in the CEMx174 cell line. These studies have set the stage for evaluating in vivo fitness of SIV/17E-Fr K165R. As therapeutic vaccination strategies to enhance CTL responses against HIV could promote HIV escape, it is important to understand the relevance of viral escape, especially in CNS disease progression.
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Isolation and Culture of Feline Adipocytes
E. Graff , D. Wanders , and R. Judd . Dept. of Pathobiology, Dept. of Anatomy, Physiology & Pharmacology, Auburn University, College of Veterinary Medicine Obesity is an epidemic in both people and domestic animals. Cats have been established as an excellent model for examining the pathophysiology and complications of obesity and type 2 diabetes mellitus. However, isolation and culture of feline adipocytes has not been performed, which limits the advancement and use of this animal model. There are notable differences in adipocyte biology of various adipocyte depots (subcutaneous adipose tissue vs. visceral adipose) in both humans and rodents. Evaluations of the various depots in primary culture have not been evaluated in cats. Culture of primary adipocytes allows for investigation of the biological differences in a controlled in vitro setting. Therefore, the first objective of this pilot study was to optimize methods of feline adipocyte isolation and culture and evaluate their lipolytic response. An additional goal was to evaluate differences between subcutaneous and abdominal adipose tissue depots. Adipose tissue was isolated from the falciform and inguinal fat pads of healthy, lean female cats. Adipocytes were placed in a collagen matrix and treated with isoproterenol, insulin or niacin. Media samples were collected at 3, 6 and 24 hours and glycerol was measured as a marker of lipolysis. Addition of isoproterenol resulted in marked stimulation of lipolysis in both abdominal and subcutaneous adipocytes. Treatment with insulin or niacin trended towards inhibition of lipolysis in abdominal adipose tissue; however, changes were not statistically significant. There was no evidence of inhibition of lipolysis by either insulin or niacin in the subcutaneous adipose tissue. This pilot study demonstrates the application of feline adipocytes in primary culture.
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Optimization of Histologic Pattern Recognition Algorithms for Automated Tissue Image Analysis
J.D. Webster, S.B. Hoover, J.E. Dwyer, B.R. Wei, and R.M. Simpson. Laboratory of Cancer Biology and Genetics, National Cancer Institute, Bethesda, MD Histologic pattern recognition image analysis software provides the ability to automate segmentation of tissue classes within histologic sections based on unique spatial-spectral features. Users define tissue classes of interest and provide representative training set examples. Subsequently, iterative software training identifies unique spatial-spectral features that discriminate each tissue class. Software applications include quantifying complex tissue areas, identifying regions of interest for downstream morphometric analyses, and potentially screening tissues for diagnostically significant histologic lesions. To understand how to maximize the image segmentation capability of pattern recognition software, we developed individual algorithms, using commercially available software, to segment and quantify areas of (1) pulmonary metastases in murine models, (2) tumor and stroma in canine tumor biobank specimens, and (3) features in teratomas derived from stem cells grown in immunodeficient mice. Consideration of the spatial-spectral features of each tissue class, including the range of features within the class, was important when selecting representative examples of each class for training. Algorithms performed best when segmenting 2 highly divergent tissue classes (e.g. metastatic carcinoma and lung). Variations in tissue processing, including lung insufflation, tissue fixation, post-mortem handling, and histochemical staining, influenced the ability to consistently and accurately segment all images within a set of unknowns or multiple similar tissues sets processed at different time points. In order to optimize tissue class segmentation across all images within given study sets, it was necessary to create multiple tissue subclasses or, at times, multiple algorithms for tissues with highly variable histomorphologies, such as spontaneous melanomas from dogs.
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Masculinization of the Distal Tubular and External Genitalia in Female Dorest Sheep Following Prenatal Exposure to Androgens
C. Lamm, P. Hastie, N. Evans, and J. Robinson. School of Veterinary Medicine, University of Glasgow, Glasgow , UK Prenatal exposure to endogenous or exogenous androgens has a negative effect on reproductive development of the fetus and poses a risk to human and animal health. Previous studies have described the masculinization of ovaries and external genitalia in androgenized sheep and the present study is the first to characterize the changes within the tubular genitalia. Thirty two adult ewes were given intramuscular injections of 100 mg of either testosterone propionate (TP) or dihydrotestosterone (DHT) twice weekly in vegetable oil from 3090 days of pregnancy. The ewes lambed normally and the reproductive tracts were examined from 37 post-pubertal female offspring at 10 months of age. The gonads were ovarian and did not have any male structures such seminiferous tubules or Leydig cells. The oviducts and uteri were grossly and histologically normal in both TP- and DHT-treated sheep; however, the uterus connected to a malformed, sacular vagina. The malformed vagina opened into a masculine urethera in the DHT-treated and ended in a blind sac in the TP treated sheep. In both TP- and DHT-treated animals, the urethra was approximately 5 times the length of the control sheep, resembling a male urethra, and two sets of male accessory genital glands were present. The urethra terminated in a fully developed penis in both TP- and DHT-treated sheep and a scrotal sac was present. These results show that prenatal exposure of genetic female sheep to exogenous androgens results in masculinization of the internal and external genitalia.
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Oral Bacteriophage Treatment to Reduce Fecal Shedding of Salmonella enterica Serotype Newport From Calves
J.E. Hyland, S.B. Price, J.C. Wright, P.H. Walz, and S.R. Kitchens. Department of Pathobiology, College of Veterinary Medicine, Auburn University, AL Antibiotic resistance in pathogenic bacteria is a growing problem in food-producing animals. In particular, Salmonella enterica serotype Newport (S. Newport) strains are eighteen times more likely to be multi-drug resistant when obtained from bovine sources, and multi-drug resistant Salmonella strains have greater virulence when compared with drug-susceptible strains in humans. Thus, finding alternatives to antibiotic use is a critical need. Bacteriophage treatment has many advantages and oral administration of Salmonellatargeted bacteriophage was hypothesized to decrease the duration of clinical signs, the duration of fecal shedding, and the bacterial numbers in the feces. Wild-type bacteriophages were obtained from Salmonella positive fecal samples and a cocktail containing five bacteriophages was designed. Calves were administered S. Newport and subsequently treated with the bacteriophage cocktail when a fever spike was noted. An immediate decrease in fecal S. Newport shedding was noted with a concomitant reduction in fever and a return to normal body temperatures. Bacterial cultures became negative for S. Newport within 2-3 days of instituting bacteriophage treatment. Treatment with bacteriophage is a viable alternative to antibiotic use in cattle, both for treatment and just before slaughter to decrease fecal contamination of meat by Salmonella. This would reduce the incidence of human infection obtained from bovine sources and the development of further antibiotic resistance in these isolates.
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Pancreatic Duct Ligation Results in Differential Apoptosis and Proliferation Responses in Laboratory Mice and Rats
D.K. Meyerholz1, Z. Yuan2, I. Samuel2,3. Departments of 1Pathology and 2Surgery, University of Iowa Carver College of Medicine; and Surgical Services, Iowa City, 3Veterans Affairs Medical Center, Iowa City, IA In humans, gallstone obstruction of the terminal pancreatic duct may result in severe pancreatitis and in some cases death. Both laboratory mice (B6) and rat (Sprague Dawley) have been used as surgical ligation models for this condition; however, mice exhibit more severe clinical response with necropurulent pancreatitis and a high mortality rate. To identify potential mechanisms for these differences, we studied pancreas in sham and ligated groups from both models at 5, 24 and 48 hours duration. We morphometrically examined for proliferation (PCNA) and apoptosis (TUNEL and activated caspase-3) in immunostained sections. In both species, PCNA immunostaining was similar among treatment groups at 5 hours, but by 24 and 48 hours duct ligation caused decreased PCNA staining in mice (P<0.001, vs. shams) while rats had increased staining (P<0.01, vs. shams). In rats, increased PCNA staining was seen mostly in centroacinar, ductular and less duct epithelium with corresponding anisocytosis and mitotic figures consistent with proliferation. We then examined apoptosis through activated caspase-3 and TUNEL immunostaining. Mice from both groups had nominal detection of apoptosis in all time points. In contrast, duct ligation in rats had increased caspase-3 staining (P<0.05, vs. shams, 24hr) detected in acinar cells with a similar (albeit non significant) trend for TUNEL. These results suggest the rat model has multiple protective pathways to mitigate injury associated with duct ligation and these pathways are lacking in the mouse model. Future studies of cellular and hormonal regulation of these apoptotic and proliferative mechanisms may lead to potential therapies to prevent severe consequences of obstructive pancreatitis.
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Persistent PCV2 Infection and PCV2 Vaccination Suppress PCV2-Mediated Regulatory T Cell Induction in Pigs
T.E. Cecere1, X.J. Meng1, T. Opriessnig2, S.M. Todd1, N.M. Beach1, and T. LeRoith1. 1Department of Biomedical Sciences and Pathobiology, Virginia Tech, Blacksburg, VA and 2Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, IA Porcine circovirus associated disease (PCVAD) is currently one of the most economically important diseases in the global swine industry. Porcine circovirus type 2 (PCV2) is necessary for the development of PCVAD; however, co-infection with other pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV), is required to induce clinical disease. While it is not fully understood why co-infection is necessary to produce disease, immune modulation likely plays a critical role in the pathogenesis of PCVAD. We have shown that PCV2 alone can induce regulatory T cells (Treg ) in vitro, and this effect is enhanced by PRRSV/PCV2 co-infection. To determine if PCV2 infection prior to PRRSV infection is associated with more severe immunosuppression, we evaluated Treg induction in vivo and in vitro in pigs chronically infected with PCV2 or challenged with PCV2 following vaccination with a live or inactivated PCV2 vaccine. There was no significant difference in Tregs in peripheral blood mononuclear cells among negative control, PCV2 vaccinated or chronically infected pigs. However, following in vitro infection of monocyte-derived dendritic cells with PCV2, PRRSV or both viruses, cocultured lymphocytes from the chronically infected and live vaccine challenge pigs had decreased Treg expression in the virus infected groups compared to the negative controls. Cytokine analysis revealed no up-regulation of IL-10 or TGF-E following virus inoculation in vitro. These results suggest that Treg activation is suppressed in chronically PCV2 infected or PCV2 vaccinated pigs.
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Pathology and Immunohistochemical Study in Mallards Intranasally Infected With Low Pathogenic Avian Influenza Virus
M. Franca, D.E. Stallknecht, R L. Poulson, J. Brown, and E.W. Howerth. Department of Pathology and Southeastern Cooperative Wildlife Disease Study, University of Georgia, Athens, GA Mallards are important natural hosts for low pathogenic avian influenza viruses (LPAIVs). LPAIVs are mainly transmitted by a fecal-oral route and are excreted in high concentrations in the feces. The objective of this study was to investigate the pathology and viral antigen distribution of LPAIV in intranasally infected mallards at different time points after infection. Upper and lower respiratory tract, the entire gastrointestinal tract and the bursa of Fabricius were collected from a total of 11 mallards infected with 106 median embryo infectious dose of A/Mallard/MN/199106/99 (H3N8) on days post infection (dpi) 1, 2, 3, 4, 5 and 6. Oropharyngeal and cloacal viral shedding were analyzed by virus isolation and Real Time Reverse Transcriptase Polymerase Chain Reaction (RRT-PCR). Oropharyngeal and cloacal viral shedding were detected from dpi 1with higher cloacal viral shedding detected by RRT-PCR on dpi 2 and 3. Gross lesions were not observed. Moderate lymphocytic tracheitis and laryngitis were observed in the birds euthanized on dpi 1 and 2. Avian influenza virus (AIV) antigen was detected in rare epithelial cells of the larynx and trachea only on dpi 1. In the gastrointestinal tract, mild to abundant expression of AIV antigen was detected only in the lower intestinal tract from dpi 1 to 4. AIV antigen was also expressed in epithelial cells of the bursa of Fabricius on dpi 2 and 3. This study shows that LPAIV is associated with early upper respiratory tract pathology in mallards and that the main site of AIV replication is the lower intestinal tract.
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Proliferative Squamous Epithelial Lesions In C57BL/6 Mice With Dextran Sodium Sulfate-Induced Colitis
K.A. Maratea1, N.P. Clayton2, and B.L. Thurberg1. Departments of 1 Pathology and 2Applied Discovery Research, Genzyme Corporation, Framingham, MA Dextran sodium sulfate (DSS)-induced colitis is a widely used experimental model of inflammatory bowel disease. In late 2008, oral administration of DSS to C57BL/6 mice according to our standard protocols of acute and chronic DSS-induced colitis resulted in more severe ulcerative colorectal lesions than observed historically. The glandular mucosal epithelium of the distal colon and rectum was obliterated and replaced by hyperplastic stratified squamous epithelium in 53% of mice with acute DSS-induced colitis and 100% of mice with chronic DSS-induced colitis. Additional studies were conducted to re-optimize the models and characterize the squamous epithelial lesions. Female C57BL/6 mice (n5) were orally administered 0.5, 1.0, 2.0 or 3.0% DSS in drinking water for 7 days (acute model) or 3 cycles of 1, 1.5, or 2.0% DSS followed by a variable recovery period (chronic model). Dose-dependent increases in the severity of DSS-induced colitis and incidence of proliferative squamous epithelial lesions were observed in both studies. In the acute study, squamous epithelialization of the colorectal mucosa was present in 25% and 100% of mice treated with 2% or 3% DSS, respectively. In the chronic study, squamous epithelialization of the colorectal mucosa was observed in all DSS dose groups, and appeared to be irreversible at doses 1.5%. Between Days 63-91, 5% of mice treated with 1.5% DSS and 33% of mice treated mice treated with 2.0% DSS developed squamous cell carcinoma within areas of squamous proliferation, and squamous epithelium acquired features of non-haired skin, including keratohyaline granules and superficial keratin layer. Squamous epithelialization is believed to result from direct ingrowth of the anal mucosa with proximal extension into the rectum and distal colon.
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Prolonged and Enhanced Efficacy of Pegylated Feline Granulocyte Colony-Stimulating Factor in FIV-Infected and Uninfected Laboratory Cats, and in Cats With HuG-CSF Induced Neutropenia
H. Kondo, Y. Sakagawa, J.K. Coleman, M.J. Offner, J.G. Coisman, J.K. Yamamoto, and J.R. Abbott. Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL Neutropenia in cats is caused by various conditions including hematopoietic disorders, chemotherapy, and immunodeficiency diseases such as FIV infection, as well as neutralizing antibodies induced by repeated administration of human drug, such as human granulocyte colony-stimulating factor (HuGCSF). Compared to HuG-CSF, feline G-CSF (FeG-CSF) is more effective at elevating neutrophil levels without inducing neutralizing antibodies but is not commercially available. Our laboratory has recently developed a novel pegylated FeG-CSF that has a more potent efficacy than FeG-CSF. In current studies, short- and long-term therapeutic efficacies of PegFeG-CSF were investigated with 10 FIV-infected and 11 uninfected laboratory cats, and with cats with HuG-CSF-induced neutropenia. Treatment with PegFeG-CSF accelerated the neutrophil differentiation with higher magnitude and longer duration of efficacy than treatment with either FeG-CSF or HuG-CSF in both infected and uninfected groups. No evidence of neutralizing antibodies against PegFeG-CSF was detected despite long-term treatment of over 1 year. In addition, PegFeG-CSF was effective at restoring normal neutrophil levels in both infected and uninfected cats with HuG-CSF-induced neutropenia. Overall, PegFeG-CSF has an enhanced, rapid efficacy; longer duration of activity (i.e., longer half-life) requiring less treatment; no adverse effects including neutralizing antibodies to the drug; less frequency of treatment which is less stressful to the animals and their owners; and is thus a safe drug to treat feline neutropenia found in a wide variety of clinical conditions, even in cats nonresponsive to HuG-CSF.
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SIV Infection Alters Lipid Metabolism Resulting in Steatohepatitis
S.K. Shanmukhappa1, J.M. Billingsley2, L. Wachtman1 R. P. Shannon3, R.P. Johnson2 and K.G. Mansfield1. 1Division of Comparative Pathology and Medicine, 2Division of Immunology, New England Primate Research Center. Southborough, MA 01772, and 3Department of Medicine University of Pennsylvania School of Medicine, Philadelphia, PA 19104 Introduction: Metabolic disorders commonly encountered in HIVinfected patients include dyslipidemia, insulin resistance, and visceral fat deposition. In such patients the risk of developing insulin resistance is 65% and strongly correlates with the degree of hepatic lipidosis or nonalcoholic steatohepatitis (NAFLD). A clear mechanism has not been identified for the development of hepatic lipidosis but likely involves host, viral and environmental factors. Methods: We utilized the SIV rhesus macaque model of AIDS. Rhesus macaques were fed a high fat/high cholesterol diet for 6 months to induce hepatic lipidosis. The animals were then challenged with SIVmac239 and were euthanized 2 months after infection. RNA was isolated from the livers and microarray was performed using the Affymetrix Rhesus Macaque chip. The data was analyzed using the GeneSpring and Ingenuity Pathway Analysis software. Results: SIV-infected animals developed severe hepatic lipidosis compared to non-SIV-infected animals. High fat diet resulted in increased expression of genes regulating the lipid metabolism. When the diet was superimposed with SIV infection, it resulted in increased expression of genes involved in inflammation (CXCL10, IL1B & perforin 1 ) and hepatic fibrosis (STAT1, IGF1 & TGFb) along with corresponding decreased expression of genes regulating lipid metabolism (fatty acid desaturase2, lipase, subtilisin). Conclusion: SIV-infection perturbs the lipid homeostasis by altering genes responsible for lipid metabolism and inducing others responsible for inflammation & hepatic fibrosis. We hypothesize that increased infiltration of immune cells may contribute to the induction of lipidosis through the production of cytokines and other inflammatory mediators.
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Promotion of a Functional B Cell Germinal Center Response After Leishmania spp. Co-infection Is Associated With Lesion Resolution
K.N. Gibson-Corley1,2, P.M. Boggiatto2, C.A. Petersen2, T.J. Waldschmidt1, and D.E. Jones2. 1Department of Pathology, University of Iowa, Carver College of Medicine, Iowa City, IA, and 2Department of Veterinary Pathology, Iowa State University, College of Veterinary Medicine, Ames, IA Co-infection of C3HeB/FeJ (C3H) mice with both Leishmania major and Leishmania amazonensis leads to a healed footpad lesion, while coinfection of C57Bl/6 (B6) mice leads to chronic, non-healing lesions. This inability to heal corresponds to a deficiency in B cell stimulation of macrophage-mediated killing of L. amazonensis in vitro. We have also characterized that the antibody response in B6 mice to Leishmania infection is less robust than the antibody response in C3H mice. Although the mechanism that leads to healingof these lesions is not completely known our studies implicate the B cell response as having an important effector function in killing L. amazonensis. To more completely understand this disparate clinical outcome to the same infection we describe the draining lymph node germinal center B cell response between co-infected C3H and B6 mice. There are more germinal center B cells, more antibody isotype-switched germinal center B cells, more memory B cells and more antigen-specific antibody-producing cells in co-infected C3H mice compared to B6 mice as early as 2 weeks post-infection. However, we also show that IL-21 production and IL-21 receptor expression in both mouse strains is similar at 2 weeks, suggesting that the difference in the anti-Leishmania response in these mouse strains may be due to differences in T follicular cell commitment or intrinsic B cell differences. Furthermore the data supports the idea that functional B cells are important for healing L. amazonensis in this infectious disease model.
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SV40 Infection of Cortical and Hippocampal Neurons in Immunosuppressed SIV-Infected Rhesus Macaques
S. Kaliyaperumal1, H.L. Knight1, C. Wuthrich2, X. Dang2, I.J. Koralnik2, K.G. Mansfield1, and S.V.Westmoreland1. 1Harvard Medical School, New England Primate Research Center, Division of Comparative Pathology, Southborough, MA, 2Beth Israel Deaconess Medical Center, Division of Neuro Virology, Department of Neurology, Boston, MA Simian virus 40 (SV40) belongs to the polyomaviridae family, a group of small non-enveloped DNA viruses, and can cause fatal disease in immunocompromised animals. Viral reactivation or primary infection in immunosuppressed animals may cause a number of clinical conditions with CNS, pulmonary, or renal involvement. SV40 is highly homologous to the human JC polyomavirus (JCV). Reactivation of SV40 in immunosuppressed rhesus monkeys can cause a progressive multifocal leukoencephalopathy (PML)-like illness, which is the prototypical disease caused by JCV in immunosuppressed humans. Recently, we have shown that JCV can infect cerebellar granule cell neurons and cortical pyramidal neurons in immunosuppressed individuals. In addition, a neurotropic SV40CNS1 when experimentally infected in two immunocompromised rhesus monkeys caused neuronal infection. To examine whether SV40 neuronal infection occurs spontaneously in nonhuman primates, we analyzed archival brain specimens from 15 SIV-infected rhesus macaques with AIDS and SV40 brain infection from 1997 to 2011. Unlike classical PML cases, some animals had unique SV40 virus distribution of periventricular and subpial cortex. Retrospective analysis of archival brain tissue by double-label immunohistochemistry for SV40 and MAP2 identified 8 cases (53.3%) with SV40 neuronal infection. SV40-infected neurons were detected in parietal, occipital, and temporal cortices, hippocampus and dentate gyrus, thalamus, and brain stem. The presence of SV40 DNA in cortical samples from these animals was verified by PCR and sequencing. These observations indicate that spontaneous SV40 neuronal infection in rhesus macaques is not rare, and suggests the same may be true for JCV-neuronal infection in people.
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The Detection of Immunoglobulin Producing Cells in Teleost Fish Tissues by In Situ Hybridization
S.A. Aschenbroich1, J. Zhang1, A. C. Camus1, H. W. Dickerson2, and C. C. Brown1. 1Department of Pathology, University of Georgia, College of Veterinary Medicine, Athens, GA and 2Department of Infectious Diseases, University of Georgia, College of Veterinary Medicine, Athens, GA Various tissues were harvested from two distinct groups of channel catfish (Ictalurus punctatus) for the purpose of determining the location of immunoglobulin producing cells. Formalin-fixed, paraffin embedded tissues from both control, non-infected channel catfish and from channel catfish infected with the protozoan parasite, Ichthyophthirius multifilis (Ich), were subjected to in-situ hybridization (ISH) with a single riboprobe corresponding to the messenger RNA for fish immunoglobulin. A perinuclear positive signal within a cell was taken as an indication that there was active transcription of the gene and therefore, likely production of antibody. Positive cells were quantified using a 0 to 3 scale, and in general, the numbers of positive cells were similar for the infected vs. non-infected fish in the organs tested via ISH. Immunoglobulinproducing cells were localized, in approximate order, from most to least numbers of positive cells: head kidney, trunk kidney, spleen, gill, intestine, liver. The only exception noted was that Ich-infected fish had relatively more positive antibody-producing cells in gill than the control, non-infected fish. However, this finding is not altogether surprising as gill is one of the target organs of Ich. Overall, this study demonstrates that in-situ hybridization can be used for the localization of immunoglobulin producing cells in channel catfish formalin-fixed, paraffin-embedded tissues. Further, our data highlights the immunological differences between mammals and teleost fish by demonstrating that immunoglobulin-producing cells can be found in large numbers in the renal interstitium, which assumes the role of the bone marrow in fish.
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The Gallbladder as an Ecological Niche for Campylobacter Jejuni Following Bacteremia in Guinea Pigs (Cavia porcellus)
E.R. Burrough1, O. Sahin2, P.J. Plummer1, Q. Zhang2, and M.J. Yaeger3. Departments of 1Veterinary Diagnostic and Production Animal Medicine, 2 Veterinary Microbiology and Preventive Medicine, and 3Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA Highly virulent Campylobacter spp. are a frequent cause of septic abortion in nave sheep following oral exposure, and pregnant guinea pigs have been described as a useful model for studying abortifacient strains. In our previous work with this model, we have observed a high frequency of Campylobacter-positive bile samples from aborting guinea pigs inoculated with Campylobacter jejuni IA3902, a field isolate of an emergent clone of C. jejuni that is associated with the preponderance of Campylobacter-associated sheep abortions in multiple states. Histologic evaluation of gallbladders from guinea pigs where Campylobacter was recovered from the bile revealed varying degrees of suppurative cholecystitis, and ultrastructural evaluation of representative tissue samples identified low numbers of unattached spiral-shaped rods consistent with Campylobacter spp. admixed with erythrocytes adjacent to luminal epithelia with variable microvillus degeneration. Histochemical and lectin histochemical evaluation of guinea pig gallbladder epithelia revealed abundant mucoprotein and L-fucose containing surface glycans, two substances previously shown to enhance growth and drive chemotaxis of C. jejuni. These results suggest the mucous layer of the guinea pig gallbladder epithelium provides an ecological niche for C. jejuni allowing colonization and persistent carriage. Given that slaughter surveys have demonstrated a high number of apparently healthy sheep that harbor C. jejuni in the gallbladder, this subclinical carriage may play a critical role in the epidemiology and pathogenesis of ovine campylobacteriosis.
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The DNA Binding Domain of Meq, a Mareks Disease Virus Oncoprotein, Plays a Major Role in the Transformation Ability of the Virus
D.K. Ajithdoss, S.M. Reddy and B. Lupiani. Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station TX We have previously demonstrated that Meq, a bZIP protein of Gallid herpesvirus 2 (GaHV-2) or Mareks disease virus serotype-1 (MDV-1), is essential for the development of T cell lymphomas in chickens. Interestingly, CVI988/Rispens (an attenuated vaccine strain) does not induce tumors despite expressing Meq. Here, we studied the role of CVI988 Meq and its DNA binding domain (DBD) and transactivation domain (TAD) in lymphomagenesis. We replaced the meq gene in an oncogenic strain, Md5, with parental CVI988 or chimeric meq genes generating rMd5-CVI-Meq, rMd5-Md5/CVI-Meq (DBD of Md5 and TAD of CVI), and rMd5-CVI/ Md5-Meq (DBD of CVI and TAD of Md5) viruses. All recombinant viruses replicated to similar levels both in vitro and in vivo, and were transmitted horizontally to sentinel birds. rMd5 (parental virus), rMd5-CVI-Meq, rMd5-Md5/CVI-Meq and rMd5-CVI/Md5-Meq viruses induced lymphomas in 100%, 6%, 100% and 46% of chickens, respectively. No disease was detected either in CVI988/Rispens inoculated birds or uninoculated controls. In diseased birds, 100% lymphomas were found in the sciatic nerves. Interestingly, rMd5, rMd5-CVI-Meq, rMd5-Md5/CVI-Meq and rMd5-CVI/ Md5-Meq viruses induced 33%, 0%, 60% and 0% visceral lymphomas, respectively. rMd5-Md5/CVI-Meq induced tumors were observed in the kidney (60%), gonads (6%), spleen (46%) and heart (33%). Collectively, our data suggests that CVI988 Meq is a very weak oncoprotein even in the context of Md5 genome, and the DBD plays a more important role than the TAD in the transformation ability of Meq.
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The Potential for the Therapeutic Application of Bone MarrowDerived Adult Equine Mesenchymal Stem Cells (EMSCs) in an Ex Vivo Model of Corneal Healing
L. Breickner1, K. Newkirk2, D. Hendrix3, N. Neilsen3, and M. Dhar1. Departments of 1Large Animal Clinical Sciences, 2Pathobiology, and 3Small Animal Clinical Sciences, University of Tennessee Veterinary Medical Center, Knoxville TN There has been extensive investigation in the potential therapeutic applications of adult equine mesenchymal stem cells (EMSCs). Much research has focused on EMSCs as an adjunct in the treatment of equine musculoskeletal injuries. The goal of this project was to investigate the potential therapeutic benefits of EMSCs in an in vitro model of corneal healing. Normal equine corneas were sterilely harvested immediately following euthanasia from 11 donated research horses. Prior to removal from the globe, each cornea was divided into quadrants and a trephine was used to create a 5 mm diameter wound of consistent depth in each quadrant. A total of 75 quadrants were cultured in standard media with cholera toxin (with or without cultured EMSCs). On days 1, 2, 3, 4 and 5, 12%, 50%, 7%, 10% and 21% of samples respectively were formalin fixed and processed routinely. Histologic sections were scored by a blinded anatomic pathologist (0, not healed to 3, healed). Only 58/75 sections had properly oriented, identifiable wounds for scoring; 31 (53%) of these had been treated with EMSCs. Irrespective of day of harvest, 50%, 83.3%, 75% and 48% of sections scored 0, 1, 2 and 3 respectively had been cultured with EMSCs. Challenges included bacterial contamination, which occurred in 75% of the samples, and in developing a scaffold to encourage attachment of the EMSCs to the corneal surface. Although these results suggest EMSCs may assist in corneal wound healing, more ex vivo studies are required to explore this potential.
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The Role of Type 2 Turkey Astrovirus in Poult Enteritis Syndrome
S. K. Mor, M. Abin, G. Costa, A. Durrani, N. Jindal, S. M. Goyal, and D. P. Patnayak. Department of Veterinary Population Medicine, University of Minnesota, St. Paul, MN An experimental study was conducted to determine the comparative pathogenicity of type 2 turkey astroviruses (TAstV-2) obtained from turkey flocks afflicted with poult enteritis syndrome (PES) and those with inapparent infection with TAstV-2. Seven-day-old poults, negative for astrovirus, rotavirus, coronavirus, and reovirus by reverse transcriptase-polymerase chain reaction (RT-PCR), were divided into three groups (A, B, and C) of 30 poults each. Group A was inoculated orally with turkey astrovirus-positive intestinal contents from birds affected with PES. Group B received turkey astroviruscontaining intestinal contents from apparently healthy flocks. Group C served as a negative control and received phosphate buffered saline (PBS). Clinical signs of diarrhea, depression and dullness were observed in group A. Group B also showed clinical signs similar to those in group A but the signs were milder in nature. Birds in group C did not show any clinical signs. At 16 DPI, the gain in body weight of birds in group A was significantly lower than those in groups B and C. In addition, the bursa size was reduced in group A but not in groups B and C. Birds in groups A and B, but not group C, were found to shed turkey astrovirus in feces as detected by RT-PCR. These results provide preliminary indication that TAstV-2 from PES birds may be more pathogenic than those from apparently healthy poults. Further studies are needed to determine if pathogenic and non-pathogenic strains of TAstV-2 exist in the environment. These results also reinforce our previous observations that astrovirus is involved in PES causing significant retardation in growth/ weight gain.
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Unexpected Lesions in Nontarget Tissues in Transgenic Zebrafish Expressing Mutant Kras Targeted to Liver
J.M. Spitsbergen1, A.T. Nguyen2, C.H.V. Koh2, A. Emelyanov3, S. Parinov3 and Z. Gong2. 1Department of Microbiology, Oregon State University, Corvallis OR, 97311, USA, 2Department of Biological Sciences, National University of Singapore, Singapore, and 3Temasek Life Sciences Laboratory, Singapore We have developed transgenic lines of zebrafish with either constitutively active or inducible mutant (G12V) kras targeted to liver using the liver-specific promoter of the liver fatty acid binding protein gene. In one of these transgenic lines, in addition to liver neoplasia, we have also observed profound lesions in other organs. In fish in which mutant kras is induced following completion of organogenesis, by 2-3 months of age, when foci of hepatocellular alteration and benign and malignant neoplasia have developed in the liver, we also observed lesions in kidney and endocrine pancreas. These lesions were absent in wildtype fish from the same genetic background. In several transgenic fish we observed severe diffuse global membranoproliferative glomerulonephritis. Other transgenic fish showed less severe lesions with mild to moderate multifocal segmental membranoproliferative glomerulonephritis. The endocrine pancreas showed focal to diffuse, mild to severe ballooning degeneration of islet endocrine cells. Real time PCR assays of endogenous and transgenic kras expression conducted on liver, kidney, and pancreatic tissue of adult transgenic fish confirmed that mutant kras was overexpressed only in liver. Also in situ hybridization studies indicated no detectable expression of the transgene in tissues other than liver. Microarray analysis of gene expression in transgenic fish indicated that overexpression of mutant kras in liver leads to strong activation of the complement system and elevation of inflammatory mediators. These elevated inflammatory mediators may underlie the lesions observed in nontarget tissues.
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The Roles of Osteopontin in Gastritis Induced by Helicobacter pylori in Mice
J-W Park1, B-R Lee1 H-J Kwon2, S-H Lee1, H-J Kim2 and D-Y. Kim1. 1Department of Veterinary Pathology, Seoul National University and 2Korea Research Institute of Bioscience and Biotechnology, Ochang, Korea Osteopontin (OPN) is a multi-functional protein involved in many physiological and pathological processes including inflammation and tumorigenesis. In immune system, it has been considered that OPN is a major amplifier of Th1-immune responses. Growing evidence from recent studies demonstrates that Th-1 mediated immune mechanisms may be critical in the development of gastritis associated with H. pylori infection. Thus, we hypothesized that OPN may be a critical mediator in regulating Th-1 mediated gastritis in response to H. pylori infection. In the present study, we tested this hypothesis using mice lacking OPN. Mice were orally inoculated with H. pylori (mouseadapted Sydney strain) and were euthanized 8 and 16 weeks later. OPN was upregulated in H. pylori infected wild-type mice compared non-infected wild type mice, as well as the development of moderate to severe gastritis at 8, 16 weeks, as determined by a significant infiltration of neutrophils, T cells, and macrophages. In contrast, OPN-deficient mice showed significant attenuation of inflammation. Additionally, we found more prominent H. pylori colonization in OPN-deficient mice, as revealed by quantitative real-time PCR. Quantitative real-time PCR analyses also revealed substantial reduction of IFN-gamma, TNF-alpha, IL-1beta in OPN-deficient mice. Moreover, H. pylori infection in OPN-deficient mice was associated with significant decreased epithelial proliferation and apoptosis compared with wild-type mice, but there were no significant differences in proliferation / apoptosis ratio between each group. These results indicate that OPN exerts considerate influences on the host defense, acting through Th-1 mediated immune response. Moreover, OPN might finally influence gastric carcinogenesis in mice treated with the combination of H. pylori infection and carcinogen.
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Vitamin E Deficiency Causes Degenerative Myopathy and Impaired Swimming Behavior in Zebrafish
K.M. Lebold1,2,5, C.V. Lohr5,6, C.L. Wright1,5, E.M. Labut1,5, C.L. Barton4,5, G.W. Miller1,5, R.L. Tanguay3,4,5, and M.G. Traber1,2,5. Linus Pauling Institute1; Departments of Nutrition and Exercise Sciences2, Environmental & Molecular Toxicology3; Sinnhuber Aquatic Research Laboratory4; Environmental Health Sciences Center5; College of Veterinary Medicine6; Oregon State University, Corvallis, OR Zebrafish (ZF) were used to assess the behavioral and pathological consequences of long-term dietary vitamin E deficiency. ZF were fed a defined diet with (E) or without (E-) vitamin E for 1 year. ZF were analyzed for a-tocopherol (AT), ascorbic acid (C), and malondialdehyde (MDA, a measure of oxidative stress); pathology was assessed using H&E staining of fixed whole-mounted ZF. To assess swimming behavior, ZF (n91) were placed in individual tanks and monitored following a startle-response stimulus (single or multiple tap on tank). E- compared with E ZF contained 300 X less AT (p<0.0001), half the C (p0.0025) and 3 X more MDA (p<0.0001); swimming speed was halved (p<0.05) in response to either stimuli. E ZF responded to both the single and multiple-tap stimuli with characteristic escape behavior and increased swimming speed (p<0.05 compared with baseline). E- ZF did not respond to the single-tap stimulus, but did increase swimming speed following multiple taps (p<0.05 multiple tap vs. baseline). The differential response to a single vs. multiple tap in E- ZF is indicative of neurosensory deficits, while the reduced swimming speed compared with E ZF is largely due to severe skeletal muscle pathology indicated by multifocal, polyphasic myopathy. The myopathy severity ranged from acute necrosis to complete fiber loss. Thus, vitamin E deficiency causes increased oxidative stress and a secondary depletion of C, resulting in a decreased neurologic response, severe damage to muscle tissue and impaired swimming performance.
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Whole-Slide-Imaging and Histopathology Evaluation in the Assessment of Fibrosis Progression in a Xenobiotic-Induced Mouse Model of Sclerosing Cholangitis and Biliary Cirrhosis
O. Mendes, N. Rogacki, X. Ying, X. Ren, T. Taurino, and A. Subramanian. DSAR and F&WR Departments, Sanofi US, Bridgewater , NJ Mice fed 3, 5-diethoxyxycarbonyl-1,4-dihydrocollidine (DDC) develop increased biliary porphyrin secretion associated with a pronounced pericholangitis and activation of periductular myofibroblasts that lead to biliary type fibrosis. Whole slide imaging allows quantitative evaluation of research endpoints and provides the investigator with insights in the progression of disease in vivo. Here we assess hepatic fibrosis formation in five different mouse strains (CD1, FVB/N, 129/SV, Swiss Webster and C57/BL6) treated for 8 weeks with a 0.1% DDC diet. We used conventional histopathology evaluation with a modified Knodell-Ishak biliary fibrosis scoring system and whole slide imaging for total collagen quantification. Histopathology evaluation revealed the presence of periportal fibrosis with scoring grades of 2 to 3 in CD1 and FVB/N mice and 3 to 4 in 129/SV, Swiss Webster (SW) and C57/BL6 mice. This lesion was accompanied with pigment accumulation, biliary epithelium hyperplasia and portal inflammation. Quantification of collagen by whole slide scanning of sections stained with trichrome blue showed a correlation between the histopathology scoring of increased fibrous connective tissue and total collagen in livers of the five mice strains studied, with CD1 and FVB/N mice having comparatively lower collagen content and Swiss Webster and C57/BL6 having higher collagen content. We conclude that correlation between parameters obtained by digital slide evaluation and histopathology scoring systems is a valuable tool in animal model development that needs to be interpreted according to its biological significance within the animal model used.
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Cyp1b1-Deficiency Is Associated With Trabecular Meshwork Extracellular Matrix Ultrastructural Abnormalities
L.B.C. Teixeira1, Y. Zhao2, R.R. Dubielzig1, C.M. Sorenson3, and N. Sheibani2. 1Comparative Ocular Pathology Laboratory of Wisconsin,2Ophthalmology Visual Sciences, and 3Pediatrics, University of Wisconsin, Madison, WI The Cyp1b1 gene is expressed in several non-hepatic tissues, including the eye. Recent studies have identified CYP1B1 as a causative gene in human primary congenital glaucoma. The study goal was to determine the ultra-structural morphology of the iridocorneal drainage angle of Cyp1b1-deficient (Cyp1b1-/-) mice. Eyes from 10 C57BL/6J wild-type (Cyp1b1/) and 10 Cyp1b1-/- mice (6 weeks[n6], 3[n6], 7[n6], and 8.5 month[n2]) were enucleated and processed for transmission electron microscopy. The iridocorneal angle, trabecular meshwork (TM), Schlemms canal and aqueous veins were analyzed. The 6 weeks, 3 and 7-month-old C57BL/6Cyp1b1-/- mice presented marked disruption of anterior and posterior TM, multifocal atrophy of trabecular beams with accentuation of intertrabecular spaces. Collagen of the beam cores was markedly fragmented and multifocal accumulations of nodular elastic tissue were observed. The number of endothelial cells covering trabecular beams was diminished in Cyp1b1-/- mice, and the basement membranes were multifocally disrupted. An atrophic solitary trabecular beam extending from the anterior to mid-meshwork contacting a collapsed posterior meshwork with fragmented collagen and elastic fibers surrounded by few typical endothelial cells was observed in 8.5-month-old Cyp1b1-/- mice. Schlemms canal and the aqueous veins were normal. The Cyp1b1-/- mice presented ocular drainage ultrastructural abnormalities at 6 weeks, 3, 7 and 8.5-months that became more severe with age. Different arrays of structural lesions observed suggest an age related evolution. Thus, Cyp1b1 expression and/or activity contribute to the integrity and function of the TM. We hypothesize that, based on the known interactions of Cyp1b1, its deficiency interferes with the expression and function of TM matricellular proteins impairing endothelial cells attachment and extracellular matrix turnover.
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A Novel Transcriptomic Biomarker of PPAR-Gamma Activation
W.R. Buck, J.M. Maher, R. Ciurlionis, T.A. Sharapova, Yi Yang, E.A.G. Blomme. Departments of Cellular, Molecular, and Exploratory Toxicology and Investigative Toxicology and Pathology, Abbott, Abbott Park, IL Undesirable side effects of off-target activation of peroxisome proliferator activator, receptor-gamma (PPAR-g) nuclear receptor, include fluid retention, edema, cardiac hypertrophy, and increased adiposity. Robust in vitro methods exist to determine affinity and functional activity of compound interaction with PPAR-g. However, in vitro assays cannot predict PPAR-g -associated side effects without complex modeling incorporating target organ drug level exposures. Here we report a single gene, fatty acid binding protein 3 (FABP3), as a biomarker of PPAR-g activity in adipose tissue from rats dosed orally with rosiglitazone and pioglitazone and dogs dosed orally with rosiglitazone. In rats dosed for 1 and 5 days, treatment with PPAR-g agonists at doses which induce undesirable side effects in the rat (>50X induction at 50 mg/kg) produced >100-fold induction of FABP3. The magnitude of change was much less at doses that do not cause side effects (6-20X at 5 mg/kg). In dogs, FABP3 was induced <2.5X at 5 mg/kg for 7 days, but at 28 days 2 out of 3 dogs had induction > 4X. FABP3 was induced in rat adipose and differentiated human preadipocytes with PPAR-g agonists rosiglitazone and pioglitazone, but not with compounds classified as partial agonists, indicating potential use across preclinical species. The magnitude of change was greater than that observed with plasma adiponectin and NT-proANP. In rats, FABP3 expression increases of 12X and 30X were detectable 24 hours after single dose rosiglitazone at 5 and 50 mg/kg, respectively. In summary, FABP3 expression in rat adipose or cultures of human adipocytes is a suitable biomarker of PPAR-g activation to mitigate risk associated with low level activation of PPAR-g.
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Investigative Studies on the Effects of an Antisense Oligonucleotide on Kidney Glomeruli in Mice
V. Castillo2, T. Gales1, D. Mullins1, R. Mirabile1, J. Kane1, L. Tierney1, and K. Frazier1. 1Department of Safety Assessment, GlaxoSmithKline, King of Prussia, PA and 2Pathology, Microbiology and Immunology, University of California-Davis, CA In a 6-month toxicology study using CD1 mice, a proprietary antisense deoxyoligonucleotide resulted in glomerulopathy and renal amyloidosis. An investigative study was performed to explore the nature and mechanism of glomerular toxicity. Kidneys collected from mice given 10 doses of drug for 56 days were examined using HE, trichrome, Congo Red, PAS, GMS, von Willebrand Factor (vWF), and CD68 stains and electron microscopy. Microscopically, minimal glomerulopathy including increased mesangial matrix and glomerular cellularity with occasional inflammatory cells or nuclear debris, and rare intraglomerular basophilic granules was observed. GMS and PAS stains demonstrated thickened basement membranes and increased mesangial matrix. Staining for vWF within glomeruli suggested areas of endothelial hypertrophy, degeneration, or pooling of vWF protein in cytoplasm of affected cells. CD68 immunostaining indicated inflammatory cytokine upregulation. Ultrastructurally, endothelial cells were hypertrophied with expanded cytoplasm and/or pyknotic nuclei. Endothelial cells, mesangial cells, and podocytes had numerous membrane-bound vesicles suggestive of lysosomes, containing electron-dense material. Glomerular basement membranes had multifocal thickening with deposition of electron-dense and electron-lucent material. RT-PCR using Taqman1 Gene Signature Mouse Immune Array demonstrated at least a two-fold increase in RNA levels of 29 immune response related genes, including CD68. In summary, after 56 days of compound, glomerular changes include ultrastructural alterations in the glomerular basement membrane and specific effects on glomerular endothelial cells associated with inflammatory cytokine upregulation. Inflammatory cytokine release causes increased stimulation of amyloid production and glomerular injury. Inflammatory mechanisms are thus critical to progressive deposition of amyloid and mesangial matrix within affected glomeruli in mice given deoxyoligonucleotide therapies.
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Lateral Nasal Gland Necrosis in Mice Administered Systemic Naphthalene
A.K. Olivier , W. Xie , and J.F. Engelhardt . Departments of Pathology , Anatomy and Cell Biology2, Molecular & Cellular Biology Program3, University of Iowa, Iowa City, Iowa Naphthalene is an aromatic hydrocarbon that causes necrosis of nonciliated Clara cells in the trachea and bronchioles following systemic administration. This report documents novel histopathologic findings of acute naphthalene toxicity in a mixed C57B6/FVB/NJ background. Approximately 24 hours following naphthalene administration (275 mg/kg intraperitoneal) mice were lethargic with a hunched appearance, at which time they were euthanized and a complete necropsy was performed. Histopathology of the nasal tissue showed necrosis of olfactory epithelium with diffuse edema and vascular congestion of the lamina propria. There was extensive bilateral coagulative necrosis of the glandular epithelial cells of the lateral nasal glands, with a sharp line of demarcation between the affected lateral nasal glands and maxillary glands. The thymus had prominent lymphoid necrosis. Epithelial cells of the bronchus and bronchioles were multifocally swollen and vacuolated with sloughed cells in the lumen. The toxic effects of naphthalene are predominantly due to toxic metabolites produced by the CP450 monooxygenase system. The toxicity of systemic naphthalene on olfactory epithelium has previously been documented however mice in this report had toxicity at a lower dose. The necrosis of the lateral nasal glands was striking and is not well documented in naphthalene toxicity. Thymic lymphocyte depletion has been reported in mice administered higher doses of naphthalene in chronic studies; however, the acute changes have not been well documented. The severity of the nasal changes was more severe than expected for the dose administered and may be related to strain variation in nasal cavity expression of CP450.
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Toxicopathology of Sodium Fluoride in Wistar Rats and its Amelioration by Calcium Carbonate
P. Sureshkumar, D.V. Joshi, B.J. Patel, and S.S. Chaudhary. College of Veterinary Science and Animal Husbandry, S.D.Agricultural University, Saradrkrushinagar-385 506, Gujarat, India In the present study, an attempt has been made to study the symptomatology, hemato-biochemical alterations, fluoride estimation and pathological changes induced by sodium fluoride (NaF) toxicity and ameliorative action of calcium carbonate in rats. Accordingly, thirty young albino Wistar rats were divided uniformly into three equal groups. Group A served as control while Group B was given 200 mg/L NaF through drinking water for 90 days. Group C was given 200 mg/L NaF along with 50 mg/kg calcium carbonate orally for 90 days. The clinical symptoms were characterized by yellowish discoloration of teeth, altered behavior, ruffled fur coat in rats receiving NaF alone. No significant difference was observed in any of the hematological parameters in any groups. A significant (P < 0.05) rise in mean values of plasma ALT, plasma AST and plasma alkaline phosphatase was observed in all the treated groups on 90th day post treatment as compared to control rats. Group C rats showed significant decrease in ALT, AST and ALP at 90th day post treatment as compared to Group B. Group C rats also showed significant decrease in serum fluorine concentration as well as kidney and bone fluorine concentration as compared to Group B rats. The severity and distribution of pathological lesions were greater in rats of Group B than that of Group C. The main target organs affected were bone, teeth and liver. From the present study, it is concluded that dietary incorporation of calcium had ameliorative effect on sodium fluoride toxicity as evident by biochemical and histopathological alterations.
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Promotion of Helicobacter pyloriInduced Gastric Carcoinogenesis in Mice Lacking VDUP1
H-J Kwon1, Y-S Won1, I-P Choi2, H-C Kim1, and D-Y Kim3. 1Korea Research Institute of Bioscience and Biotechnology, Ohcang, 2Cell Therapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Taejon and 3 Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University, Korea Expression of Vitamin D3 up-regulated protein 1 (VDUP1), a tumor suppressor gene, is markedly reduced in some human cancers, including gastric cancer. However, mechanisms underlying tumor development remain unclear. In the present study, we investigated the roles of VDUP1 on Helicobacter pylori-induced gastric carcinogenesis in mice. Gastric tumors were generated in VDUP1 knockout (KO) and wild-type (WT) mice using a combination of N-methyl-N-nitrosourea (MNU) treatment and H. pylori infection. Fifty weeks after treatment, gastric tissues were examined by histopathology, immunohistochemistry, and immunoblotting. An In vitro study using a human gastric cancer cell line, AGS, was also performed to identify the underlying mechanisms of cancer development. Overall gastric cancer incidence was significantly higher in VDUP1 KO than WT mice. Similarly, VDUP1 KO mice showed more severe chronic gastritis, glandular atrophy, foveolar hyperplasia, metaplasia, and dysplasia. Although no differences in apoptotic index were apparent, lack of VDUP1 increased the rate of gastric epithelial cell proliferation in non-cancerous stomachs, with corresponding rises in TNF-a level, NF-kB activation, and COX-2 expression. An in vitro study showed that H. pylori-associated cell proliferation and induction of TNF-a, NF-kB, and COX-2 were inhibited in cells transfected with VDUP1. Additionally, overexpression of VDUP1 in AGS cells suppressed TNF-a-induced NF-kB activation and COX-2 expression. Our data demonstrates that VDUP1 negatively regulates H. pylori-associated gastric carcinogenesis, in part by disrupting cell growth and inhibiting induction of TNF-a, NF-kB, and COX-2. These findings provide important insights into the role of VDUP1 in H. pylori-associated gastric cancer development.
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Characterization of Hepatic Lipidosis in the Zucker Obese Diabetic Rat
E.A.G. Blomme, M. Liguori, J. Lai-Zhang, A. Lisowski, and W.Buck. Preclinical Safety, Abbott Laboratories, Abbott Park, IL Hepatic lipidosis predisposes to drug-induced liver injury (DILI). Several models of hepatic lipidosis have been proposed for DILI prediction, such as obese Zucker rats which are insulin-resistant and develop hepatic lipidosis. The objective of this study was to evaluate the onset and progression of hepatic lipidosis in Zucker rats using histopathology, serum chemistry, transcriptomic analysis and biochemical assays. Data were compared to values from lean Zucker rats fed a standard diet (i.e. normal control group). Male Zucker rats (6-7 week-old upon arrival from Charles River) were fed a certified high-fat rodent diet (Purina diet # 5008) for at least 2 weeks and sacrificed weekly (n 2-3/week) for 6 successive weeks. All rats had moderate hepatic lipidosis with only a marginal progressive increase in severity, as assessed by histopathology, Oil Red O staining, and hepatic triglyceride (TG) concentrations. Compared to lean Zucker rats, serum glucose, TG, cholesterol, ALT, AST and GLDH concentrations/activities were mildly to moderately increased at all time points with no significant changes over time. Transcriptional analysis of the liver confirmed significant perturbations in pathways related to cholesterol, TG and lipid biosynthesis and homeostasis (e.g. up-regulation of the Ampk pathway and down-regulation of the leptin receptor pathway), but also demonstrated activation of pathways related to cholestasis and inflammation and evidence of decreased mitochondrial function. Overall, there were limited differences in transcriptional profiles during the time course. Altogether, these data indicate that male obese Zucker rats aged 9-15 weeks have similar degree of hepatic lipidosis and functional liver impairment and that using rats as young as 9 weeks of age after 2 weeks on a high-fat diet is appropriate for DILI investigations.
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Lesions Associated With 28-Day Oral Administration of a Potential Chemopreventative Agent, Phospho-Ibuprofen, in Rats
J.F. Mann1, R.W. Watkins1, C.V. Rao2, I.M. Kapetanovic3, and C.D. Hebert1. 1 Southern Research Institute, Birmingham, AL, 2University of Oklahoma 3 Health Sciences Center, Oklahoma City, OK, and National Cancer Institute, Bethesda, MD Phospho-ibuprofen was administered via daily oral gavage to Sprague Dawley rats at 0, 20, 100, and 500 mg/kg. All core animals survived to scheduled euthanasia, however there were test article-related unscheduled deaths in the satellite group of animals (500 mg/kg) that were used for toxicokinetics studies. Macroscopic and microscopic examinations were performed on 10 animals per sex per dose group. The observed test article-related lesions were consistent with class characteristic lesions induced by nonsteroidal antiinflammatory drugs, including ibuprofen. Test article-related macroscopic lesions, which corresponded to the microscopic lesions of ulceration, inflammation, and epithelial hyperplasia, were observed in the forestomach of the 500 mg/kg dose group. Test article-related microscopic lesions were observed in the glandular stomach (acute, chronic-active and granulomatous inflammation, necrosis, ulceration, erosion, and edema); forestomach (acute, chronic-active, and granulomatous inflammation, mesothelial cell hyperplasia, epithelial hyperplasia, necrosis, ulceration, epithelial cytoplasmic vacuolization, and edema); cecum (chronic and chronic-active inflammation and edema); colon (chronic and chronic-active inflammation and ulceration); liver (acute and chronic-active inflammation and multifocal necrosis); and kidney (granular and hyaline casts, transitional epithelial hyperplasia, acute inflammation, acute and chronic-active inflammation of the papilla and renal pelvis, papillary necrosis and edema, renal tubular necrosis and dilatation, focal tubular dilatation, and hyaline droplet accumulation). Test articlerelated lesions were observed in all dose groups and the highest incidence and severity of these lesions were observed in the 500 mg/kg dose group. Funded wholly with federal funds from National Cancer Institute Contract HHSN26120043307C.
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Phosphate Dysregulation Following Pan-FGFR Inhibition in Rodents
A. Vitsky, G. Li, D. Sace, L. Liu, A. John-Baptiste, J. May, T. Wong, P. Venne and E. Blasi. Drug Safety Research and Development and Cancer Biology, Pfizer, San Diego, CA The fibroblast growth factor (FGF) family is composed of 22 proteins that regulate numerous processes in embryonic development andadult physiology. These proteins are desirable targets for the development of oncologic therapeutics, as they play a major role in angiogenesis. In an exploratory efficacy study, Scid beige mice bearing neoplastic xenografts were treated orally once daily for 14 days with vehicle, 0.5, 1.25, 5, or 20 mg/kg of a small molecule panFGFR kinase inhibitor. Mice treated with 20 mg/kg of compound required early sacrifice on day 5 due to moribundity. Dose-related efficacy was seen in all remaining dose groups, and no adverse effects were noted in animals treated with 0.5 or 1.25 mg/kg FGFR inhibitor. In mice treated with 5 or 20 mg/kg of compound, serum chemistry revealed marked, dose-dependent elevations in serum phosphorus. Histologically, mice in these two dose groups exhibited vascular and parenchymal tissue mineralization affecting the heart, great vessels, stomach, and kidneys. To further examine this effect, WistarHan rats were treated orally once daily for 4 days with 5 mg/kg of the same pan-FGFR inhibitor. Dosed rats demonstrated clinicopathologic and histologic changes similar to those in mice, as well as progressively increasing FGF23 levels. FGF23, a signaling protein that interacts with FGFR1c, has been demonstrated to play a critical role in renal phosphate reabsorption and vitamin D metabolism, and inactivating mutations in FGF23 are one cause of familial tumoral calcinosis in humans. Our data suggest that pan-FGFR inhibition may interfere with the FGF23 pathway, predisposing to hyperphosphatemia and a tumoral calcinosis-like syndrome in rodents.
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Long-Term Effects of Intravascular Exposure to Gold Nanoparticles in Mice
B.M. Brenseke1, J. Bahamonde1, and M.R. Prater1,2,3. 1Department of Biomedical Sciences and Pathobiology, Virginia Tech, Blacksburg, VA, 2Department of Biomedical Sciences, Edward Via College of Osteopathic Medicine, Blacksburg, VA, 3Institute for Critical Technology and Applied Science Center for Systems Biology of Engineered Tissues, Virginia Tech, Blacksburg VA Nanotechnologythe science of manipulating matter on a near-atomic scale to produce materials, structures and deviceshas revolutionized many industries. Nanomedicine, the medical application of nanotechnology, offers great promise in novel diagnostics and treatment modalities. However, use of nanoparticles in living systems also presents potential dangers including tissue damage, inflammation, and carcinogenesis. Gold nanoparticles (GNP) are emerging candidates for diagnostic contrast imaging and cancer radiation therapy enhancement, but long-term effects of GNP are unknown. The objective of this project was to study long-term effects of intravascular exposure to GNP in the mouse model. At 42 days of age C57BL/6 mice were exposed via tail vein injection to either 0.1 mL phosphate buffered saline (PBS) or 0.1 mL PBS containing 20 mg monodispersed 15 nm GNP. The GNP appeared dark red in suspension, and caused persistent purple cutaneous discoloration following intravenous infusion. Mice were monitored daily and weighed weekly, with no apparent adverse effects on appetite, activity, or body habitus. Mice were euthanized at 6 months of age, for whole-body evaluation by microcomputed tomographic image analysis, and light and electron microscopic imaging of individual tissues such as liver, kidney, and lung for evidence of GNP distribution, inflammation, fibrosis, and metaplasia/neoplasia. This new knowledge may be applied to human medicine, and is critical to determine long-term safety of GNP for diagnostic purposes.
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Respiratory Syncytial Virus Vaccine Development in the Cotton Rat Model
K.E. Bowenkamp1, C.A. Shaw2, J-R. Galarneau1, J.E. Markovits1, G.A. Palmer2, N.M. Valiante2, P.R. Dormitzer2, and G.R. Otten2. 1Preclinical Safety, Novartis Institutes for Biomedical Research and 2Novartis Vaccines and Diagnostics, Cambridge, MA In the 1960s, vaccination of seronegative infants and children with formalin-inactivated human respiratory syncytial virus (FI-RSV) led to vaccine-enhanced respiratory disease (v-ERD) and deaths upon communityacquired RSV infection. The cotton-rat model of FI-RSV-induced v-ERD has been used to identify the pathogenesis of ERD in the original vaccine trial and to characterize the safety of novel vaccine candidates. Imbalanced Th2 immune responses were hypothesized to contribute to v-ERD after FI-RSV inoculation. In a series of experiments, cotton rats were vaccinated on days 0 and 21 with FI-RSV or formalin-inactivated culture components (mock FI-RSV) combined with alum or novel Toll-like receptor (TLR) agonist adjuvants to augment immunomodulatory activity. RSV-neutralizing antibody titers and RSV F protein-specific IgG in serum were measured on days 14, 35 and 48, and rats were challenged intranasally with crude or purified RSV preparations on days 49-50. Lung histopathology 5 days post-challenge was scored using a modification of published criteria, and antigen-specific cytokine production was evaluated by ELISA from harvested splenic lymphocytes. Cell culture contaminants in immunogen and challenge virus preparations were required for robust alveolitis, a hallmark feature of ERD, which correlated best with enhanced bovine serum albumin (BSA)-specific IL-4 and IFN-gamma production by splenic T-cells rather than with immune responses against RSV antigens. TLR9 agonist adjuvants generally reduced crude FI-RSV induced v-ERD and decreased cytokine production post-challenge while maintaining neutralizing antibody titers. This suggests that v-ERD is associated with imbalanced Th2 responses to non-viral antigens in crude vaccine preparations and that novel immunomodulating adjuvants may favorably modulate immune responses in new vaccine candidates.
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STP Best Practices for Evaluation of Nonclinical Safety Biomarkers
J. E. Burkhardt , K. Pandher , P.F. Solter , S.P. Troth , R.W.Boyce , T.S. Zabka6, and D. Ennulat7. 1Abbott Laboratories, Abbott Park, IL, 2Pfizer, Groton, CT, 3College of Veterinary Medicine, University of Illinois, Urbana, IL, 4Merck Research Laboratories, West Point, PA,5Amgen, Thousand Oaks, CA, 6Roche Pharmaceuticals, Nutley, NJ, 7Glaxo SmithKline, King of Prussia, PA The Society of Toxicology Pathology (STP) has recently endorsed a set of Best Practices for the evaluation of non clinical safety biomarkers. Best practices for histopathology evaluation for safety biomarker qualification studies are generally similar to those for non clinical safety studies, specifically that the pathologist be unblinded or have access to all study data with the exception of data specific to the candidate biomarker undergoing qualification. While histopathology evaluation in biomarker qualification studies must be performed without knowledge of novel biomarker data, the study pathologist(s) should be involved in the attendant meta-analyses of these data. Blinded evaluation is an experimental tool in biomarker qualification studies that is only appropriate when well-defined criteria for specific histopathologic findings are identified prior to blinded review. In addition to the important question of blinding, this paper also considers the management of bias, the use of a tiered evaluation approach, the importance of using qualified pathologists and standard reporting, and the management of spontaneous findings.
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An Overview of Avian Bornavirus and Proventricular Dilatation Disease in Psittacines and Other Birds
H. L. Shivaprasad. California Animal Health and Food Safety Laboratory System Tulare Branch, University of California, Davis Proventricular Dilatation Disease (PDD) was first recognized in psittacines in the late 1970s. PDD has been observed in more than 80 species of psittacines but also in some non psittacine species such as canaries, Canada geese and others. PDD is one of the most common and often fatal diseases of psittacines. Clinical signs of PDD include anorexia, regurgitation, passing of undigested seeds in the feces, diarrhea, loss of weight, lethargy, neurological signs and sudden death. PDD is characterized by dilation of the proventriculus in most cases, with associated microscopic changes such as lymphoplasmacytic ganglioneuritis involving the gastrointestinal tract, nonsuppurative encephalomyelitis, peripheral neuritis and ganglionitis, myocarditis, adrenalitis, etc. A novel Bornavirus was identified from psittacines with PDD in 2008. The virus was named Avian Bornavirus (ABV) because it was quite distinct and shared only 65 % nucleotide sequence with the well known Bornavirus disease virus (BDV) of mammals. Bornaviruses are negative sense, enveloped, singlestranded spherical medium-sized 70-130 nm in diameter RNA viruses that are members of the family Bornaviridae, order Mononegavirales. Based on nucleotide sequence analysis of numerous ABV isolates from psittacines, six distinct genotypes have been identified. Other distinct ABV genotypes have been identified in canaries and Canada geese with PDD pathology. Both Polymerase reaction (PCR) and Western blot analysis have demonstrated that positive results for ABV can occur in normal and non-symptomatic PDD birds as well false negatives in PDD birds. PCR and IHC have demonstrated ABV not only in the neural tissues but also in non neural tissues. PDD has been successfully reproduced in cockatiels (Nymphicus hollandicus) and Patagonian conures (Cyanoliseus patagonus).
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A Forensic Investigation Into the Etiology of Bat Mortality at a Wind Farm: Barotrauma or Traumatic Injury?
D.K. Meyerholz1, K.E. Rollins3, G.D. Johnson2, A.P. Capparella3, and S.S. Loew3. 1Department of Pathology, University of Iowa, Iowa City, IA, 2Western EcoSystems Technology Inc., Cheyenne, WY, 3School of Biological Sciences, Illinois State University, Normal, IL Migrating bats experience mortality associated with moving blades at wind farms. We evaluated the competing hypotheses of barotrauma and traumatic injury to determine the cause. First, we studied environmental influences on the development of postmortem lung artifacts using laboratory mice as a model. We found that time and temperature significantly influenced the development of lung artifacts including hemorrhage and edema. Because these findings mimic diagnostic criteria for barotrauma, lungs are not useful to diagnose barotrauma in salvaged bats. Next, we examined wind farm bats and, as a control, bats that died following collision with buildings. Wind farm bats had increased incidence and distribution of bone fractures with more external lacerations. Wind farm bats showed internal injuries consistent with traumatic injury including diaphragmatic hernia and bone marrow embolism. Most wind farm bats (73%) had lesions consistent with traumatic injury. We also examined bats for ruptured tympana, as this is a common sequela in barotrauma; we predicted this to be a sensitive marker because bats have thin tympana. Building collision bats had only one case of rupture (2%), but this was attributed to concurrent cranial fractures, whereas wind farm bats had a 20% incidence. When cases with concurrent traumatic injury were excluded, this yielded a small fraction (6%) of wind farm bats that had lesions possibly consistent with barotrauma etiology. Our data suggest that traumatic collision with moving turbine blades is the principal cause of bat mortality at wind farms and, at best, barotrauma is a minor etiology.
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Anaplastic Sarcoma in an Intersex Madagascar Tree Boa (Sanzinia madagascariensis)
S. Sharpe1, C. Lamm1, and R. Killick2. 1School of Veterinary Medicine, University of Glasgow, Bearsden, Glasgow, UK, 2Bristol Zoo, Clifton & West of England Zoological Society Bristol Zoo Gardens, Clifton, Bristol, UK An adult Madagascar tree boa (Sanzinia madagascariensis) underwent coeliotomy for investigation of a coelomic mass. At surgery, a large mass originating from the pancreas and peri-pancreatic tissue and invading the gall bladder was removed. The snake did not recover from general anaesthesia. A complete post-mortem was performed and samples were submitted to the University of Glasgow for histopathology. On histological examination, the mass was composed of adipose tissue infiltrated with a poorly demarcated spindle cell neoplasm. The neoplastic cells were highly pleomorphic with abundant cytoplasm and frequent clear cytoplasmic vacuoles, suggestive of adipocyte origin. The neoplastic cells were immunoreactive for MHC II but negative for vimentin, and lysozyme. Metastatic neoplastic cells were present in the liver, lung and brain. As an incidental finding, the gonads contained both maturing ovarian follicles and seminiferous tubules with intact germinal epithelium and evidence of spermatogenesis, along with other features of male and female gonad anatomy. This report describes a rare neoplasm in snakes and is the first to report an intersex condition in a Madagascar tree boa.
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Angular Limb Deformity in Farmed Deer Stags
K.G. Thompson , N.S. Beatson , and K.E. Dittmer . Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand and 2Rural Veterinary Services South Island, Ltd, Timaru, New Zealand Over several seasons, angular limb deformity was reported in a percentage of young, growing stags, commencing at approximately 13-15 months of age, on a large deer farm in the mid-Canterbury region of New Zealand. The property included a mix of purebred Wapiti (Elk), Red deer and Wapiti/Red deer crossbreds farmed commercially for venison and velvet. The limb deformity occurred predominantly in Red and Wapiti/Red crossbred stags, seldom in purebred Wapiti and never in hinds. The problem was confined to stags being reared for velvet production rather than venison, and according to the owner, stags with heavy, wide-beam antlers were predisposed. The deformities were confined to the forelimbs, were usually bilateral, and predominantly varus in nature. Clinical signs included lameness, swollen carpi, reduced weight gain and a harsh hair coat. By 2 years of age, affected deer were no longer lame but the angular deformity persisted. Gross lesions were confined to the distal radial physes and varied in severity. In some cases there was mild, segmental thickening or duplication of physeal cartilage. In others there was segmental destruction and hemorrhage involving physeal cartilage and the adjacent metaphysis. Histologically, lesions varied from mild thickening and dysplasia of physeal cartilage to extensive physeal necrosis with cleft formation and hemorrhage. In such cases, there was loss of trabecular bone in the adjacent primary spongiosa and replacement with granulation tissue. Copper deficiency during a period of rapid skeletal growth, leading to reduced strength of newly-formed bone and cartilage, thus predisposing to traumatic damage, is considered the most likely pathogenesis.
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Detection of Porcine Genogroup 1 Torque Teno Virus in Tissues From PCVAD Cases in Mexico: Preliminary Results
L.A. Garcia-Camacho, A. Vargas-Ruiz, I.C. Rangel-Rodriguez, Y. Romero, and V. Quintero-Ramirez. Departamento de Ciencias Biologicas, Facultad de Estudios Superiores Cuautitlan, Universidad Nacional Autonoma de Mexico, Mexico Recently, co-infection of porcine circovirus type 2 (PCV2) with porcine genogroup 1 torque teno Virus (pTTV-1) has been widely reported in association with PCV2 associated disease (PCVAD). In order to assess the presence of pTTV-1 in positive cases of PCVAD recorded from 2001 to 2008 in Mexico, paraffin embedded tissues with characteristic microscopic lesions and PCV2positive by in-situ hybridization (ISH) were evaluated for porcine TTV genogroup 1 by nested PCR. Selected cases were representative of each year and particular PCVAD clinical form. PMWS cases from 2001 were consistently negative for pTTV-1 but they were positive since 2002. Regarding cases associated with granulomatous enteritis (GE), they were pTTV-positive from 2005 which correlates with the first descriptions of GE in Mexico. Cases with fetal and neonatal non-suppurative myocarditis were positive from 2003-2008 as well. PDNS cases were scarce but positive to TTV-1 in years 2003 and 2005. Our preliminary findings are compatible with co-infection of genogroup 1 pTTV in the majority of PCVAD forms currently described in Mexico. Therefore, it might be feasible that TTV-1 participates in the development of PCVAD as previous reports have proposed. The lack of positivity for pTTV in 2001 cases of PCV2 associated PMWS suggests that at that time, pTTV was not indispensable for the development of this syndrome. However, an exhaustive retrospective search for pTTV from PMWS affected and PMWS nonaffected pigs must be performed to determine its precise emergence in our farms. At present, the aim is to implement ISH protocols to detect of TTV-1 in tissues from our complete case archival.
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Chemokines Improve Anti-neurotropic Virus Immune Response by Attracting Antibody-Secreting Cells to the CNS
H. Lee, Y. Sunden, K. Ochiai, and T. Umemura. Lab. of Comparative Pathology, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Japan The CNS is a relative immunosuppressive organ, therefore, immune cell access to the CNS is restrictive. In this study, we used intracerebral chemokine injection to attract antibody secreting cells (ASCs) to the brain for control of neurotropic virus. Inactivated pseudorabies virus (PRV) or rabies virus (RV) was used for immunization. After secondary immunization, lymphocytes were collected from spleen and bone marrow (BM) to investigate kinetics of Agspecific ASCs, and an in vitro chemotaxis assay was performed with splenocytes. Intracerebral virus challenge after chemokine injection to the brain was performed. The PRV-specific ASCs in BM increased gradually, though they were less than in spleen, and exceeded that in spleen at 21-25 dpi. At 24 dpi, CXCL12 and cocktail chemokine (mixture of CXCL9, 10, 12 and 13) attracted PRV-specific ASCs most strongly, compared to CXCL9, 10 and 13 in in vitro chemotaxis assay and in vivo intracerebral chemokines injection experiment. Intracerebral injection of CXCL12 did not affect the survival rate in live PRV challenge, though it promoted long-term survival. But in live RV challenge CXCL12 increased the survival rate. On the other hand, intracerebral injection of cocktail chemokine increased the survival rate in both PRV and RV challenge. Chemokine injection to the brain led to the chemotaxis of ASCs to the brain and contributed to the suppression of neurotropic viruses (PRV and RV).
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Evaluation of Capillary and Myofiber Density in the Pectoralis Major Muscles in Rapidly Growing, High-Yield Broiler Chickens During Increased Heat Stress
K. S. Joiner1, G.A. Hamlin1, R. J. Lien2, and S.F. Bilgili2. 1Department of Pathobiology and 2Department of Poultry Science, Auburn University, Auburn, AL Skeletal muscle development proceeds from early embryogenesis through marketing age in broiler chickens. While myofiber formation is essentially complete at hatching, myofiber hypertrophy can increase after hatch by assimilation of satellite cell nuclei into myofibers. As the diameter of the myofibers increases, capillary density peripheral to the myofiber is marginalized, thereby limiting oxygen supply and its subsequent diffusion into the myofiber, thereby inducing micro-ischemia. The superficial and deep pectoralis muscles constitute 25% of the total body weight in a market age bird, thus compromise of those muscle groups can have profound economic impact on broiler production. We hypothesized that marginal capillary support relative to the hypertrophic myofibers increases the incidence of micro-ischemia and therefore myofiber damage, especially in contemporary high-yield broilers under stressing conditions such as high environmental temperatures. We evaluated the following parameters in four different broiler strains at 36 and 49 days of age when reared under thermoneutral (20 to 25 C) versus hot (30 to 35 C) environmental conditions: capillary density, myofiber density and diameter, and degree of myodegeneration. Our data demonstrate that myofiber diameter significantly increased with age (p<0.0001), but decreased with hot environmental temperatures (p0.0041) in both age groups. The incidence and degree of myodegeneration also increased in hot conditions. Effects of age and temperature on capillary density were similar (p0.0388 and p0.2054, respectively), but not statistically significant. Differences among strains were not observed, although a single strain showed slight differences in hot temperatures. Further studies evaluating capillary to fiber ratios are being performed.
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Expression and Localization of Podocyte-Associated Molecules in Normal and Diseased Glomeruli in Dogs
R. Kobayashi1, K. Yasuno1, K. Ogihara2, M. Mishina3, T. Watanabe3, J. Kamiie4, and K. Shirota1,4. 1Research Institute of Biosciences, 2Laboratory of Environmental Pathology, 3Department of Nephrology and Urology, Teaching Animal Hospital and 4Laboratory of Veterinary Pathology, Azabu University, Sagamihara, Kanagawa, Japan Podocytes play a crucial role in ultrafiltration by the renal glomeruli as a barrier to macromolecules. Since the podocyte has poor regenerative capacity, injury leads to development and progression of glomerular damage and renal dysfunction. To clarify podocyte involvement in pathogenesis of canine glomerular diseases, we examined expression of podocyte-associated molecules which localize in foot process and slit diaphragm in canine glomeruli. Firstly, by immunofluorescence we revealed expression and localization of nephrin, podocin, alpha-actinin-4 and beta3-integrin, which are main podocyteassociated proteins for maintaining podocyte morphology and function in canine normal glomeruli . Western blot analysis and reverse transcriptionPCR were also performed using isolated glomeruli. Furthermore, we observed expression of these molecules in 16 renal biopsies from dogs with various glomerular and tubular diseases including membranous glomerulopathy, membranoproliferative glomerulonephritis, mesangial proliferative glomerulonephritis and other types of glomerulopathy, and tubulointerstitial diseases. Semiquantitative analysis in immunofluorescence showed decreased expression of podocyte-associated proteins in glomerular diseases, in particularly nephrin and podocin. Localization of nephrin and alpha-actinin-4 evidently changed in glomerular diseases. Generally, the linear and covering glomerulus pattern of nephrin was shifted to a granular pattern, and alpha-actinin-4 expression was aggregated along glomerular basement membranes. The degree of expression of these molecules was not correlated with urinary protein/creatinine ratio, but tended to reflect the severity of ultrastructural podocyte damage. In conclusion, the expression of podocyte-associated molecules was decreased and localization was altered in canine glomerular diseases. Nephrin may be the most sensitive and useful marker for podocyte injury in dogs.
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In Situ Characterization of Productive Cytotoxicity in Tumor Infiltrating Lymphocytes of Canine Cutaneous Histiocytoma
M. Neta1, P. Katavolos2, R.D. Wood1, D. Bienzle1, D. Hodgins1, P.F. Moore3, and R.M. Jacobs1. University of Guelph1, Abbott Laboratories, USA2, and University of California, Davis3 Perforin (Perf), the cytolytic missile of the cellular immune system, is a tightly regulated effector molecule. It is a valuable marker for peripheral differentiation of cytotoxic lymphocytes (CL) and the most reliable predictor for granule-dependent killing. Canine cutaneous histiocytoma (CCH), a benign, naturally-regressing tumor, offers a unique model for successful immune-mediated tumor regression. The present study characterized expression of Perf and granzyme B (GrzB) in CL participating in successful antitumor immune response. A peptide antibody targeting a conserved region of canine Perf was generated. Immunohistochemical studies demonstrated Perf expression in CD3 lymphocytes and co-localization of Perf and CD63 confirmed sub-cellular localization of perforin to secretory lysosomes. Consistent expression of Perf and GrzB in tumor infiltrating lymphocytes (TIL) of CCH opposed contrasted with lack of expression in resting lymphocytes. Perforin expression was found in a minute fraction of TIL and was markedly less than GrzB. Proportions of cells showing polarization of Perf and GrzB immunoreactive granules were high suggesting frequent engagement of CL in immunological synapses (IS). Marked relative abundance of GrzB polarizing cells implied frequent assembly of perforindeficient CL in IS. By overcoming the challenges of probing perforin in tissue, we have collected novel data about the physiology of TIL within the tumour microenvironment. We provide in situ evidence for the differential expression of Perf and GrzB as well as support for the presence of a perforin-independent killing mechanism during tumor rejection. Outlining morphologically identifiable parameters indicative of productive antitumor immunity provides a model for comparison to commonly encountered, ineffective TIL, which confounds effective cancer immunotherapy.
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Histopathologic Findings Based on Disease Chronology of Hansen Type I Intervertebral Disk Disease in Dogs
K. Kuroki1, C.L. Vitale2, P. Pithua2, and J.R. Coates2. 1Veterinary Medical Diagnostic Laboratory, University of Missouri and 2Veterinary Medical Teaching Hospital, University of Missouri, Columbia, MO Intervertebral disk disease (IVDD) is a common cause of spinal cord injury in dogs. The present study was designed to elucidate the relationship between clinical chronology of thoracolumbar Hansen type I IVDD and histopathologic changes of extruded disk material removed during surgical decompression of forty-five privately-owned dogs. A presumptive diagnosis of Hansen type I IVDD was based on neurologic examination and spinal imaging, and the diagnosis was confirmed at surgery. Time course for onset of disease was classified into four groups: 1-peracute (< 1 day), 2-acute (1-3 days), 3-subacute (4-7 days), or 4-chronic (> 7 days), based on time elapsed from initial clinical signs to surgical intervention. Number of cases per group included 11 peracute, 13 acute, 9 subacute and 12 chronic (range from 8 to 45 days; mean days + SEM, 17.1 + 3.1 days). The extruded disk material removed at surgery was subjectively evaluated by a pathologist blinded to clinical history and surgical findings. Chronicity of histopathologic changes was characterized based on the pattern of cellular infiltrates and reparative changes as the following: acute (no cellular infiltrates with or without hemorrhage or neutrophil predominant infiltrates with or without fibrin); subacute (macrophage predominant infiltrates with or without hemosiderin); or chronic (fibrosis with or without hematoidin). Twenty-three specimens were acute, 12 were subacute and 10 were chronic. There was a positive correlation between disease chronology and histopathological characterization of lesion chronicity (r0.529, p0.0002) suggesting value for histopathology examination on otherwise discarded disk material and this classification scheme as a diagnostic tool for comparison studies.
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In Vivo Reduction or Blockade of Interleukin-1Beta in Primary Osteoarthritis Influences Expression of Mediators Implicated in Pathogenesis
K.S. Santangelo, G. Nuovo, and A.L. Bertone. Departments of Veterinary Biosciences and Clinical Sciences, The Ohio State University, Columbus, Ohio Osteoarthritis (OA) is the leading cause of physical disability and results in significant burdens on health care and society. Interleukin-1beta (IL-1beta) is considered a major cytokine involved in OA-related pathology but in vivo characterization of its function in primary disease remains unclear. We recently described temporal expression and tissue distribution of IL-1beta through progression of spontaneous OA in two strains of guinea pigs with varying propensity for disease development. OA-resistant animals demonstrated significant reduction in immunostaining for IL-1beta in knee joints at 120 and 180 days while OA-prone animals exhibited persistent expression, which may correlate to early disease incidence. Here, we report the results of in vivo studies using select viral vectors to reduce or block IL-1beta-mediated pathways. Successful reduction was achieved: via RNA interference (RNAi) using a novel adeno-associated viral (AAV) vector containing an IL-1beta knockdown sequence or administration of an adenovirus (Ad) vector encoding interleukin-1 receptor antagonist protein (IRAP). Contralateral stifle joints of 16 male OA-prone guinea pigs were injected with either experimental or control viral vectors at 60 days of age for data collection at 120 and 180 days. Diminution of IL-1beta signaling was demonstrated over this period by RNAi and IRAP with no statistical difference in reporter gene expression from either viral vector. Importantly, this reduction was associated with decreased expression of three inflammatory mediators and one catabolic agent implicated in OA, and increased expression of a key anabolic molecule. Thus, we provided biological evidence that IL-1beta serves a crucial role in primary OA in vivo and long-term studies to evaluate disease-modifying potential of these translational tools are warranted.
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Microgranuloma Formation Is Not Enough for Control and Survival Following Infection With the Novel Murine Pathogen Brucella microti
A. Alcaraz1, M.P. Jimenez de Bagues2, A. de Martino3, J.F. Quintana2,4, and J. Pardo4,5. 1College of Veterinary Medicine, Western University of Health 2 Sciences, Pomona, CA, Unidad de Sanidad Animal, Centro de Investigacion y Tecnologa Agroalimentaria (CITA), Gobierno de Aragon, Zaragoza, Spain, 3 Unidad de Anatoma Patologica, Instituto de Investigacion Sanitaria de Aragon. Zaragoza, Spain, 4Dpto. Bioquimica y Biologa molecular y Celular, 5 Facultad de Ciencias, Universidad de Zaragoza, Spain, and Fundacion Aragon ID (ARAID), Spain Brucella microti, a novel murine pathogen isolated from wild rodents, was used to experimentally infect wild type and immunodeficient mice that lack B (Jh), T and B (SCID) or T, B and NK (SCID.Beige) cells. Infection was studied at 3, 7 and 21 days post infection (dpi). Wild type mice developed microgranulomas predominately at 7 dpi and completely cleared bacteria 21 dpi from liver and spleen. SCID.Beige mice were most affected, with animals dying 16 dpi. Severe inflammation with numerous microgranulomas was observed in liver and spleen with bacterial counts > 108 cfu/liver. SCID mice had less severe hepatic pathology compared to SCID-Beige and had higher bacterial load in spleen and liver than WT and Jh mice after 1 week and maintained the same level during the next two weeks. In contrast, bacteria levels progressively increased in SCID.Beige mice until death at 16-18 dpi. Our results indicate that mice infected with B. microti develop characteristic granulomatous hepatitis. The early granuloma formation is critical to clear infection. In addition to granuloma formation, to obtain complete elimination or control systemic spread of B. microti, our data supports the need of T and/or B cells and established a central role of NK cells during experimental brucellosis.
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Mortality in Cetaceans Collected Along the Northeast Atlantic Coast of the US Between 1995 and 2011: 176 Cases
A. Berkowitz1, P. Habecker1, R. Schoelkopf2, F. Del Piero1. 1University Of Pennsylvania, School of Veterinary Medicine, Department of Pathobiology, New Bolton Center, Kennett Square, PA and 2The Marine Mammal Stranding Center, Brigantine, NJ The objective of this study was to examine trends in mortality and significant pathologic findings in cetacean species collected along the New Jersey USA coast over a period of 16 years (1995-2011). A total of 176 cases were submitted to the University of Pennsylvania at New Bolton Center for necropsy and additional diagnostics. The tested population consisted of 153 dolphins representing 6 species (50 common dolphin, 41 bottlenose dolphin, 32 harbor porpoise, 14 striped dolphin, 11 Rissos dolphin, 5 white sided dolphin) and 23 whales, representing 5 species (14 pygmy sperm whale, 1 dwarf sperm whale, 4 beaked whale, 3 short fin pilot whale, 1 melon headed whale). Fifty eight percent (102) of cases presented between February and June. Juveniles and neonates constituted 34% (60) and 6% (10), respectively, of the tested population.The cause of death was determined in 118 cases (67%). The most frequently affected systems were the central nervous system (brain) and pulmonary system, comprising 32 cases (18%) each. Other documented causes included: emaciation 18 (10%), gastrointestinal disease 6 (4%), hepatic disease 3 (1.5%), hematolymphatic disease 3 (1.5%), dermatitis 4 (2%), trauma 10 (6%), cardiomyopathy in sperm whales 6 (4%), renal disease 2 (1%), and neoplasia 2 (1%). The most common single cause of death, was emaciation (18 cases, 10%), followed by encephalitis associated with Nasitrema spp. (16 cases, 9%). As populations of cetaceans are declining, it is crucial to continue monitoring trends in mortality and improve diagnostic techniques to improve disease detection.
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Morbilliviral Encephalitis in a Striped Dolphin (Stenella coeruleaoalba) Calf Stranded on the Italian Coastline
G. Di Guardo1, L. Leonardi2, C. Cocumelli3, F. Scholl3, C. E. Di Francesco1, R. Speranza1, M. Pennelli1, D. Venditti4, C. Eleni3. 1University of Teramo, Faculty of Veterinary Medicine, Department of Comparative Biomedical Sciences, Piazza Aldo Moro, 45 - 64100 - Teramo, Italy, 2University of Perugia, Faculty of Veterinary Medicine, Department of Biopathological Sciences and Animal and Food Product Hygiene, Via S. Costanzo, 4 - 06126 - Perugia, Italy, 3Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Via Appia Nuova, 1411- 00178 - Rome, Italy; 4Servizio Veterinario Azienda USL RM/D, Rome, Italy A young male striped dolphin (Stenella coeruleoalba) found dead in November 2009 had a chronic, bilateral, multifocal, non-suppurative meningo-encephalitis, characterized by prominent perivascular mononuclear cell cuffs, neuronal degeneration, nodular and diffuse microgliosis, astrogliosis/astrocytosis, neuronophagia, and occasional multinucleate syncytia. Immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR) of the brain were positive for Morbillivirus in the diencephalon, mesencephalon, pons, and medulla oblongata, with the cerebellum, lung, spleen, pulmonary and mesenteric lymph nodes being negative. Viral antigen was detected in neurons and, to a lesser extent, astrocytes. A low neutralizing antibody titer (1:10) against Morbillivirus was also found, with no simultaneous presence of serum antibodies to Brucella spp. or Toxoplasma gondii. Furthermore, no pathogenic bacteria were isolated from any tissue. This is the second report of morbilliviral encephalitis in a striped dolphin stranded along the Italian coastline within the past 18 years. These 2 cases were found more than 10 years after the last report of morbillivirus infection in dolphins. This case is unusual because only the brain from this very young animal was positive for morbillivirus suggesting possible postnatal or transplacental infection.
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Multiple Malformations Associated With Choanal Atresia in Alpaca (Vicugna pacos)
A.G. Armien and K. Reed. College of Veterinary Medicine, University of Minnesota Choanal atresia (CA) is a common malformation in new world camelids that is characterized by an obstruction of the airflow from the nose to the larynx. We have accumulated evidence that CA in alpacas is part of a multiple malformation syndrome (MMS) and share similarities with the human CHARGE syndrome. We provide a detailed characterization of 23 alpacacria with CA and test a candidate gene for association with this syndrome. Choanal atresia was bilateral in all the cases. Four major presentations of the syndrome were identified: 1) Alpacas that present CA only, 2) Alpacas with CA associated with skull, brain and visceral malformation, 3) Alpacas with CA associated only with skull and brain malformation, 4) Alpacas with significant macroscopic skull, brain and visceral malformation but WITHOUT Choanal atresia. Six animals that fulfill all criteria of CHARGE syndrome in human were identified and the complete CHD7 coding sequence on these animals was assembled. The CHD7 gene is large and variable in alpaca. Over 50 nucleotide substitutions were identified among our sample set including a polymorphic region test for association with CA in all affected animals.With one possible exception, mutations that indicate a causative association with CA were not identified. The results of this work do not however, rule out a role for CHD7 in CA
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Myocarditis and Myofasciitis in a Series of Gray Foxes (Urocyon cinereoargenteus) From California
J.A. Luff1, D.L. Clifford3, S.M. Keller1, P.A. Pesavento1, D. Famini4, L.W. Woods2. 1Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA, 2California Animal Health and Food Safety Laboratory System, University of California, Davis, CA, 3California Department of Fish and Game, Rancho Cordova, CA and 4Sonoma County Wildlife Rescue, Petaluma, CA This report describes a non-suppurative myofasciitis and myocarditis in eleven gray foxes from Sonoma County, California examined between 2008 and 2010. Affected foxes were all less than 6 months old and admitted to a local rehabilitation center between late June and September. These foxes exhibited an abnormal bunny hopping gait characterized by flexion of the hind limbs and decreased mobility. The most severely affected foxes were euthanized and submitted for necropsy examination. Significant macroscopic findings included atrophy of skeletal muscle, most significant in the hind limbs, and generalized enlargement of lymph nodes. Histologically, there were plasma cells, histiocytes, and lymphocytes throughout the skeletal muscle and fascia at multiple sites, often surrounding and tracking along nerves and blood vessels. In the heart, a similar inflammatory infiltrate was present throughout the myocardium and underlying the endocardial and epicardial surfaces. Myofiber necrosis was not a feature of the disease. Additionally, there was interstitial pneumonia, ranging from mild to severe, characterized by lymphocytes and histiocytes surrounding blood vessels and expanding alveolar septa. There was reactive lymphoid hyperplasia within the lymph nodes and spleen. Immunohistochemistry, polymerase chain reaction, transmission electron microscopy, bacterial culture, and virus isolation were negative for a variety of infectious agents. The characteristic clinical presentation and pathological features described in this report suggest that this myofasciitis is a distinct entity within gray foxes in California. The underlying etiology for this condition remains unknown.
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Quali-quantitative Investigation on Follicular Dendritic Cells of Ileal Peyers Patches From Sarda Breed Sheep Carrying Different PrP Genotypes
G. Marruchella1, C. Ligios2, and G. Di Guardo1. 1University of Teramo, Faculty of Veterinary Medicine, Department of Comparative Biomedical Sciences, Teramo, Italy and 2Istituto Zooprofilattico Sperimentale della Sardegna G. Pegreffi, Sassari, Italy Peyers patches (PPs) are known to play a key role in sheep scrapie infections pathogenesis, with PrPSc early accumulating inside PP follicles, particularly in macrophages and follicular dendritic cells (FDCs). In ruminants, ileal PPs function as a primary lymphoid tissue, undergoing involution at sexual maturity. The present study was aimed at quali-quantitatively evaluating FDCs residing within ileal PPs from Sarda breed sheep, with special emphasis on the following variables: age, PrP genotype, and scrapie infection. Thirty animals were investigated, 12 of them being scrapie-free lambs, 12 scrapie-free sheep, and 6 scrapie-affected ovines. FDCs were detected using a commercially available primary monoclonal antibody (clone CNA-42, DAKO) and quantified by image analysis, with statistical analysis being subsequently performed. The average number of PP follicles ranged from 15 in adult sheep to 65 in lambs. The FDC network was significantly more dense and wider in lambs when compared with adults, independently from PrP genotype and PrPSc deposition. Additionally, no evidence of FDCs was found within intrafollicular microgranulomas. Therefore, age-related ileal PP involution and FDC network reduction could both modulate hosts resistance/susceptibility to natural scrapie infection in sheep.
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Putative Biomarkers of Feline Pancreatic Neoplasia
M.D. Meachem , E.C. Snead , B.A. Kidney , M.L. Jackson , V. Larson , and E. Simko1. 1Western College of Veterinary Medicine, Saskatoon, SK and 2 C.A.R.E. Center Animal Hospital, Calgary, AB As in people, the early diagnosis of feline malignant exocrine pancreatic neoplasia is challenging, and delayed identification typically results in a paucity of treatment options, widespread metastasis, and a high mortality rate. The objective of this study was to characterize the proteome in cats with malignant and inflammatory pancreatic disease in comparison to age-matched healthy adult cats, in order to identify differences in protein expression and putative biomarkers for pancreatic carcinoma. Plasma from 6 cats with pancreatic neoplasia, 6 with pancreatitis, and 6 healthy adult cats was subjected to standardized 2-dimensional SDS PAGE electrophoresis (iso-electric focusing with subsequent separation based on molecular weight). Resultant gels were stained with Coomassie Blue to visualize separated proteins and comparatively analyzed with specialized software (Image Master 2D Platinum 7.0, GE healthcare). The relative volume of each protein spot was calculated as a percentage of the total volume of all the protein spots in each gel to minimize variations due to protein loading and staining. Two proteins were significantly over-expressed (p<0.05) in the plasma proteomes of cats with pancreatic carcinoma when compared to healthy cats: P20 (MW20kDa; iso-electric point4.8), and P53 (MW53kDa; iso-electric point5.2). Furthermore, 5 proteins were over-expressed in cats with pancreatic neoplasia and those with pancreatitis when compared to healthy controls, and an additional 6 proteins were under-expressed in cats with pancreatic neoplasia. Results of our study indicate that there are significant differences in the proteomes of cats with malignant pancreatic neoplasia, pancreatitis, and healthy controls, some of which may act as putative biomarkers of disease. Proteins of interest will be identified by liquid chromatography mass spectrometry.
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Tumor Infiltrating Lymphocytes in Canine Hemophagocytic Histiocytic Sarcoma Exhibit Defective Polarization and De-granulation of Cytotoxic Granules
M. Neta1, R.D. Wood1, P.F. Moore2, and R.M. Jacobs1. 1University of Guelph and 2University of California, Davis Familial Hemophagocytic Lymphohistiocytosis (FHLH) constitutes a complex of inherited immune-deficiencies in humans. FHLH manifests as uncontrolled proliferation and activation of lymphocytes and histiocytes resulting in hypercytokinemia and severe peripheral cytopenias. Defective genes in FHLH encode proteins involved in granule-dependent killing (GDK), a fundamental apparatus of cellular immunity. Canine hemophagocytic histiocytic sarcoma (CHHS), an aggressive proliferative disease of macrophages has similar clinical and pathological presentation to FHLH and un-elucidated genetic background. We hypothesized, similarly to FHLH, defective GDK underlies CHHS. Intercellular interactions between T-cells and tumor cells were analyzed to identify GDK-structurally manifested defects. CHHS tissue sections were compared to those of canine cutaneous histiocytoma (CCH), a benign naturally-regressing tumor and an established model of effective anti-tumor immunity. Immunofluorescence and three-dimensional imaging were applied to outline the microanatomy of T cell-tumor cell intercellular communication, delineating key components of GDK. Formation of an immunological synapse (IS), TCR signalling, granule polarization and de-granulation were evaluated. Similarly to CCH, cytotoxic T cells (CTL) infiltrate CHHS tumor parenchyma, form IS with tumor cells and exhibit intact TCR signal transduction manifested as ZAP70 phosphorylation and cytolytic enzyme expression. Conversely, stark differences in polarization and de-granulation of cytotoxic granules was identified as these were frequently recorded in CCH, but rarely detected in CHHS. We uncovered a morphologically identifiable defect in CTL infiltrating CHHS. Our results suggest that failure of cell-mediated target cell killing in the tumor microenvironment might be a component in development of CHHS. We propose that CHHS may serve as a promising natural disease model for FHLH, as well as for investigating possible mechanisms behind success and failure of anti-cancer immune responses
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Tumor Size as a Prognostic Indicator of Postexcisional Survival Time in Cats Diagnosed With Mammary Carcinoma: A Retrospective Study of 210 Cases
J.L. Davies1, C.G. Duncan4, B.K. Wobeser1, S. Hendrick2, B.A. Kidney1, V.S. MacDonald3, and E. Simko1. 1Departments of Veterinary Pathology, 2Large Animal Clinical, and 3Small Animal Clinical Sciences, Western College of Veterinary Medicine, Saskatoon, Saskatchewan, 4Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Fort Collins, Colorado Mammary gland carcinoma is a relatively common and frequently fatal tumor in domestic cats. Tumor size at the time of diagnosis is reported to be the most important prognostic factor and is inversely proportional to survival time. However, the reported median survival times for small tumors (<2 cm in diameter) are highly variable, and this variability diminishes the prognostic value of this data. To determine whether tumor size is a reliable prognostic factor for feline mammary carcinomas, we examined the postexcisional survival time of 210 cats diagnosed with mammary carcinoma. Median survival times for tumors <2 cm, 2-3 cm and >3 cm in diameter were 20, 12, and 12 months, respectively. Survival periods for cats with tumors <2 cm ranged from 1 to 67 months. Using the Kaplan-Meier product limit, a statistically significant difference was observed between survival curves for cats with tumors <2 cm and tumors >3 cm. Thirty-five percent of cats with tumors < 2 cm, 53% of cats with tumors 2-3 cm, and 54% of cats with tumors > 3cm died of tumor related disease within 1 year of excision. These results suggest that tumor size alone is of limited prognostic value for small tumors given the broad range of survival times and high 1 year mortality rate. Further, the data highlights the need to identify additional prognostic factors for feline mammary carcinomas.
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Clinical and Histomorphometric Correlations in Canine Adrenalitis and Adrenocortical Atrophy
C.B. Frank1, S.Y. Valentin2, J.C.R. Scott-Moncrieff2, M.A. Miller1. Departments of 1Comparative Pathobiology and 2Veterinary Clinical Sciences, Purdue University, West Lafayette, IN Canine primary hypoadrenocorticism (Addisons disease, AD) results from idiopathic adrenocortical failure. The adrenocorticotrophic hormone (ACTH)stimulation test is useful in clinical diagnosis, but associated lesions have not been well described, and postmortem diagnostic criteria have not been established. Adrenal glands from 37 dogs with adrenalitis, adrenocortical atrophy, or metastatic neoplasia were compared to 45 control glands without lesions to establish a grading scheme for inflammation and cortical atrophy. Clinically, the dogs included 6 confirmed AD cases, 12 with suspected AD, and 19 with other systemic disease. Histologically, 33 dogs had adrenalitis: 17 lymphoplasmacytic, 3 granulomatous, 8 neutrophilic, 4 lymphocytic, 1 lymphohistiocytic. Ten dogs had diffuse cortical atrophy; all but 2/10 had moderate to severe lymphoplasmacytic adrenalitis. Four dogs had diffuse atrophy of the zonae fasciculata (ZF) and reticularis (ZR), with sparing of the zona glomerulosa; in all but 1/4, lymphoplasmacytic adrenalitis was severe. Of the 6 confirmed AD cases, 4 had moderate to severe lymphoplasmacytic adrenalitis with diffuse cortical atrophy; one long-term treated dog had diffuse cortical atrophy, but only mild lymphoplasmacytic adrenalitis; one had moderate lymphocytic adrenalitis with atrophy of ZF and ZR, but segmental ZG sparing. Twelve dogs (6/6 confirmed AD cases, 4/12 suspected AD cases, and 2/19 with other systemic disease) had corticomedullary cross-sectional area ratios <1.10 (zero percentile of controls). Because the area ratio correlated (r0.94) with the corticomedullary thickness ratio, a thickness ratio <1.12 could also indicate severe adrenocortical atrophy. In conclusion, the inflammation in idiopathic adrenocortical atrophy/AD was usually lymphoplasmacytic. Othertypes of inflammatory or neoplastic infiltration were more commonly part of systemic disease, and less commonly associated with severe adrenocortical atrophy.
232
Cartilage Lesions of the Canine Humeral Head
L.E. Craig1 and A. Reed2. 1College of Veterinary Medicine Department of Pathobiology and 2Research Computing Support, University of Tennessee, Knoxville, TN All dogs submitted to the University of Tennessee College of Veterinary Medicine for complete necropsy during 2010 had their humeral heads measured and examined for articular cartilage lesions. 309 humeral heads from 155 dogs were examined (there was one amputee). Eighty (52%) of the dogs had gross lesions of articular cartilage, including fibrillation, erosion, or eburnation. The presence and severity of articular cartilage lesions were positively correlated with age, humeral head size, and body weight, but not body condition score. A Bonferroni corrected alpha of .002 was used to evaluate correlations. The average age of dogs with cartilage lesions was 8.8 years. Fifty dogs were 3 years of age or younger and only 3 (6%) of these had cartilage lesions, none of which were grossly or histologically consistent with osteochondrosis. Cartilage erosion of the caudal humeral head in dogs is a degenerative lesion acquired in older large breed dogs; osteochondritis dissecans does not precede the lesion in the vast majority of cases.
234
Cystic Mucoid Enteropathy in a Dog With Chronic Vomiting
S. Warner and E.W. Uhl. Department of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, GA A 9 year old castrated male Shih Tzu presented with a one month history of melena, vomiting, and diarrhea. Exploratory laparoscopy and biopsies were unremarkable and the dog was eventually euthanized for unrelenting vomiting that did not respond to therapy. Gross examination revealed multifocal ulcers and erosions within the proximal duodenum with adjacent areas of thickened mucosa. The ulcers and erosions were covered by accumulations of mucus. Histologically, the epithelium near the erosions and ulcerations contained increased numbers of goblet cells with pockets of cystic mucoid dilation immediately adjacent to the epithelial defects. Examination of the epithelium revealed the increased goblet cell differentiation began at the mid-level of the crypt and eventually led to destruction of the crypt through cystic distension with mucus. Based upon these findings a diagnosis of cystic mucoid enteropathy (CME) was made. CME has been described as a cause of protein losing enteropathy in dogs. The cause is unknown. As in this case the segmental lesions are multifocal and thus easy to miss by surgical biopsy. The lack of inflammation suggests this disease may be induced by a response to changes in the GI microenvironment that results in multifocal stimulation of mucus production.
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Disseminated Toxoplasmosis (Suspected) in a Juvenile Ring-Tailed Lemur (Lemur catta)
J. L. Romero and R. J. Montali. Department of Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, MD A juvenile female ring-tailed lemur (Lemur catta) living in a privately owned sanctuary died following a 10-day history of lethargy and anorexia. The lemur was the third animal of the group to die in 2.5 weeks. Gross necropsy findings included hepatomegaly, pneumonia, and splenic capsular petechiae. Histopathologic findings were multi-organ inflammation and necrosis intermingled with numerous intralesional bradyzoites and tachyzoites predominantly in the liver. Given that Toxoplasma gondii and Neospora caninum produce histologically similar tachyzoites and are unable to be differentiated by light microscopy alone, immunohistochemistry was performed. Immunohistochemistry confirmed the diagnosis of Toxoplasma gondii, an obligate intracellular protozoan that can infect many warm-blooded species. Toxoplasmosis is a ubiquitous disease in which felids can serve as both definitive and intermediate hosts. Sporadic reports of T. gondii infection have been noted in ring-tailed lemurs elsewhere, and experimental data coupled with spontaneous disease suggest that ring-tailed lemurs might have a predilection for toxoplasmosis. It is possible the two sanctuary lemur mates may have also succumbed to toxoplasmosis, but the animals were not available for necropsies. All of these three lemurs were housed outside in a covered cage with a dirt bottom. The source of this fatal disease is considered to be food and/or water contaminated by feral cats or intermediate hosts. This study emphasizes the importance of good husbandry practices to prevent infection in these particularly susceptible species.
237
Feline Lanceolate Hair-like Disease: Clinical, Histological, and Ultrastructural Features
A. Rostaher1, C.S. Potter2, J. Chen3, S. Bettenay1, K.A. Silva2, L. Specht1, R.S. Mueller1, and J.P. Sundberg2. 1Medizinische Kleintierklinik Munich, Germany, 2The Jackson Laboratory, Bar Harbor, ME; 3University of Colorado, Denver, CO; and Tierdermatologie Deisenhofen, Germany Hair follicle dystrophies resulting in structural abnormalities of the hair shaft are rare, often genetic based diseases affecting many mammalian species. Laboratory mice with hair and skin abnormalities were maintained for decades to centuries as pets and provided valuable tools for modern biomedical research. Likewise, domestic animals were created and maintained as specialty breeds with follicular dystrophies due to their pleasant aesthetic appeal for the owners. We describe here a unique, spontaneously occurring lanceolate hairlike phenotype in cats which resembles the laboratory mouse lanceolate hair (Dsg4lahJ) and keratin 75 targeted mutant (Krt75tm1Der) mutant phenotypes.
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Equine Metabolic Syndrome, Pituitary Disease, Mesenteric Lipomas and Old Horses: What Does It All Mean?
K. Newkirk1, K. Chameroy2, E. Tadros2, B. Rohrbach3, and N. Frank2. Departments of 1Pathobiology, 2Large Animal Clinical Sciences, and 3Comparative Medicine, College of Veterinary Medicine, University of Tennessee, Knoxville, TN Fifty-one horses were donated for research purposes. A frequently sampled intravenous glucose tolerance test was performed on 40/51 (78%) of horses. Based on those results and concurrent clinical signs, 15/40 (38%) were diagnosed with equine metabolic syndrome (EMS) and 25/40 were considered negative for EMS; the EMS status of the remaining horses was not known. Horses were necropsied immediately following euthanasia; at necropsy mesenteric lipomas were counted and measured. The pituitary and both adrenal glands were collected, fixed in formalin, processed routinely and then evaluated histologically. The pituitaries were scored in accordance with previously published criteria (0, normal to 5, macroadenoma). The right and left adrenal cortex and medulla were measured with an ocular micrometer. Pituitary scores had a significant and positive correlation with age (r 0.67, p 0.0001) and with higher total lipoma areas (r 0.48, p 0.002). Horses with EMS had significantly higher pituitary scores (p 0.007) and body condition scores (BCS) (p 0.0001), when compared with non-EMS horses; but they did not have an increased total area of lipomas. Across the total population, BCS was not correlated with an increased total area of mesenteric lipomas. Neither EMS nor increasing pituitary scores correlated with an increased adrenal corticomedullary ratio. These data suggest that although EMS is associated with pituitary pars intermedia disease and these horses had significantly higher body condition scores, EMS alone does not account for the frequency of mesenteric lipomas in these horses.
238
Gross and Histological Study on Lipomatous Muscular Dystrophy in Piedmontese Cattle
E. Biasibetti1, G. Rubiola1, L.Di Stasio2, A. Brugiapaglia2, S. Amedeo1, F. Valenza1, and M.T. Capucchio1. 1Faculty of Veterinary Medicine, 2Faculty of Agriculture, University of Torino, Italy This study investigated the pathology of lipomatous muscular dystrophy in Piedmontese cattle. This disease is a substitution of muscle tissue with adipose tissue, which can appear as large white veins or as a normal marbling. The authors examined 39 muscle samples from animals slaughtered in different herds of Piemonte Region, 36 beef cattle equally distributed about males and females, and 3 cows (8,10 and 11 years). None of the animals showed clinical signs of muscular disease and all had an excellent state of nutrition. Muscle specimens were frozen and submitted to histological and enzymatic investigations.Gross pathology revealed a different grade of infiltration of adipose tissue, involving multiple or single muscles. The most affected regions were the ventral abdomen and the shoulder, especially the cutaneous muscles and the muscles of the thoracic group.Morphological staining revealed an infiltration of adipose tissue varying in distribution and severity, changes in muscle fiber size, and increased numbers of fibers with centrally located nuclei, suggesting muscle degeneration-regeneration. Necrosis and non suppurative inflammatory cells were also seen. Furthermore, proliferation of connective tissue and nonspecific myopathic changes, were present. Chemical and physical characteristics of the affected tissue were also evaluated. Proximate analysis showed a low protein and a very high ether extract percentage. Due to the high fat content, Lightness and Hue values were high. Also the rheological properties were markedly influenced. The histological lesions were similar to those reported in human dystrophy such as Duchenne or limb-girdle. In next step the authors will delving into genetic factors, as vascular and deficiency causes seem not involved in pathogenesis.
Abstracts
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Histologic and Immunohistochemical Characterization of Myocardial Fibrosis in Rhesus Macaques (Macaca mulatta)
S. Macri, K. Mansfield, and A.D. Miller. Division of Comparative Pathology, New England Primate Research Center, Harvard Medical School, Southborough, MA Myocardial fibrosis is a pernicious process that ultimately leads to loss of normal cardiac architecture, increased cardiac stiffness, and dysrhythmias. A variety of conditions including hypertension, diabetes mellitus, coronary artery disease, myocarditis, and cardiomyopathy can result in fibrosis of the myocardium. Rhesus macaques are being used as an animal model to study cardiomyopathies and aging; however spontaneous cardiac pathology is poorly understood in this species. Histologically and immunohistochemically we characterized cardiac fibrosis in 34 rhesus macaques that were divided into three distinct age groups. The degree of fibrosis was determined using scoring criteria for both global myocardial fibrosis as well as midmyocardial interstitial/perivascular fibrosis which positively correlated with age (p<0.0001 and p0.0048 respectively). Immunophenotypic characterization of antigen presenting cells revealed differential expression of CD163 and DC-SIGN between the two younger groups as compared to the advanced age cohort (p<0.0001). HAM-56 expression was similar between the young and middle aged cohorts but decreased significantly in the advanced age cohort (p0.0021). The expression of CD8, CD163, and DC-SIGN correlated positively with age (p 0.0319, p0.0016, p0.0033 respectively). These results indicate marked alterations in macrophage and dendritic cell phenotype in rhesus macaque myocardium associated with age. The work confirms the importance of myocardial fibrosis as a common background lesion in rhesus macaques and illustrates age associated alterations in macrophage phenotypes that may alter myocardial disease susceptibility and contribute to fibrotic cardiac remodeling.
241
In Vitro Assessment of Influenza Virus Attachment to Swine Respiratory Epithelial Cells
S. Detmer, M. Gramer, S. Goyal, and M. Torremorell. Veterinary Population Medicine, University of Minnesota, College of Veterinary Medicine, St. Paul, MN As influenza A viruses continue to emerge and evolve in the North American swine population, strains have arisen that are associated with increased virulence. Direct assessment of viral binding in the respiratory tract using virus histochemistry will enhance our understanding of the pathogenesis of these new viruses. Selected viruses represented the four genetic clusters of North American swine H1 (SwH1) viruses. Additionally, a group of viruses containing a two amino acid insertion in the binding site of the hemagglutinin gene and their presumed ancestral viruses were selected. A/CA/04/2009 and a vaccine virus were the positive controls and virus label, fluorescein isothiocyanate (FITC), was the negative control. Viruses were bound to formalin-fixed paraffin-embedded, 6-week-old (6w) and adult pig tissues. Virus histochemistry scores per respiratory zone ranged from to for all viruses, with bronchioles having the highest and most consistent scores, regardless of animal age. Virus attachment was quantified in the digital images of five bronchioles per 6w tissue section using image analysis software. Although variation was found between four staining runs of one virus (p0.0094), no variation was found among four 6w pigs (p0.3757) for the same virus. Significant differences were found in the SwH1 viruses (p0.0397) and the ancestral and insertion viruses (p0.0007). The present study provides new insights on the binding of swine influenza viruses to porcine respiratory epithelial cells. The information provided in this study combined with the information derived from the genetic analysis of these viruses will provide a benchmark for evaluating future changes in viral binding, as well as aid our understanding of swine influenza virus persistence in pigs.
240
Histological Grading in Canine Malignant Mammary Tumors Is an Independent Prognostic Factor
L. Pena and M.D. Perez-Alenza. Department of Animal Medicine, Surgery and Pathology. Veterinary School. Complutense University of Madrid, Spain The histological variability of canine mammary tumors makes difficult their diagnosis and gives little prognostic information. A histological grading system, adapted from human Oncology to canine mammary cancer, has been proposed as a useful method to further classify canine malignant mammary tumors according to their histological malignancy. Nevertheless, the prognostic value of this canine-adapted grading system needs to be investigated. This is a prognostic prospective study in which 66 female dogs with at least one malignant tumor were included. Animals were presented throughout 2008 at the Complutense Veterinary Teaching Hospital of Madrid (Spain), were clinically evaluated, surgically treated and followed-up (min 28 months maximum 38 months). Tumors were histologically diagnosed and graded (grade I, n29; grade II, n20; grade III17). Statistical analyses were performed including epidemiological (age, breed, previous hormonal treatments, regularity of heats), clinical (number of malignant tumors, size, ulceration, adherence to underlying tissues, clinical stage), histological (histological type, grade, lymph node involvement) and follow-up variables (recurrences, metastases, diseasefree period, overall survival). P value<0.05 was considered statistically significant. Kaplan-Meyer survival curves and multivariate analyses (Cox regression) were performed for disease-free period and overall survival. The histological grade was significantly associated with clinical stage, lymph node affectation, histological type of tumor, development of recurrences and metastases, disease-free survival and overall survival. Multivariate analyses selected clinical stage and histological grade as independent variables related to overall survival and disease-free period. According to our results, histological grading of canine malignant mammary tumors provides further information regarding malignancy and prognosis of canine mammary tumors.
242
Infestation of Brown-Headed Cowbirds (Molothrus ater) With Harpirhynchid Mites (Harpirhynchus quasimodo): Three Cases From the Southeastern United States
N. Nemeth1, J. Brown1, B. Munk1, M. Spalding2, and K. Keel1. 1Southeastern Cooperative Wildlife Disease Study, University of Georgia, Athens, GA and 2 Department of Infectious Diseases and Pathology, University of Florida, Gainesville, FL Mites within the diverse Harpirhynchidae family feed on the skin of birds and snakes. A newly described species of North American mite, Harpirhynchus quasimodo, was found in the skin of three, free-ranging, brown-headed cowbirds (Molothrus ater) in Florida, Tennessee, and Arkansas from 2004-11. The mites formed single or multiple, 0.3- to 1.5-cm diameter, cutaneous, raised masses that varied in location (e.g., cervical, pectoral, axillary, inguinal, and interscapular). Grossly, the masses consisted of yellow-orange, homogenous, friable material and were comprised of countless mites. Histologically, the feather follicles were markedly expanded by multi-loculated cysts filled with mites, free keratin fragments, and sporadic eggs. The cysts were lined by disorganized and hyperkeratotic epithelium and compressed the adjacent fibrous connective tissue. Lymphocytes and plasma cells were scattered in the underlying dermis. H. quasimodo are distinguished from congeneric mite species by a tripartite, hump-like formation on the anterior propodonotum in males, and setae number, length and location. Mite-associated lesions in cowbirds were confined to the skin with no invasion into underlying tissue and no apparent health effects. However, lesions were numerous and extensive in one of the three cowbirds, and likely affected some natural behaviors and activities. The prevalence and severity of H. quasimodo infestations among cowbirds and other passerine species, and the potential effects on the general health status and productivity of these birds, is unknown. Behavioral, physiological, or immunological factors may predispose birds to severe mite infestations. Bird-tobird transmission likely occurs through close contact following rupture of mite-containing masses.
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Interpreting Lesions in Zooarcheological Specimens: Facilitating Comparisons to Bone Diseases in Modern Animals
E.W. Uhl and C. Kelderhouse. Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA Interpretation of lesions in zooarcheological material is dependent upon what is known about disease in modern animals. However very few veterinary pathologists are involved in animal paleopathology, and, partly because of this, the field is not as well established as its human counterpart. One important issue is that the nature of most ancient animal remains i.e.: dried disarticulated skeletons or skeletal fragments, has made it difficult for veterinary pathologists to easily apply their expertise. To enhance understanding of animal disease in ancient times, examples of dried bone specimens from animals with definitively diagnosed lesions are being characterized. This characterization has greatly facilitated interpretation of lesions in a collection of 13th century Native American dogs. For example, it has been used to establish criteria for differentiating chronic coxofemoral luxation from hip dysplasia that could potentially even be used on pelvic fragments. In addition, one of the challenges in animal paleopathology is differentiating a neoplastic lesion from chronic osteomyelitis, as both lesions can be severely proliferative and lytic. Although more specimens need to be collected and analyzed, initial observations are that some common neoplasias induce more irregular and spiculated bone lesions than inflammatory or reactive processes. Thus it may be possible to differentiate tumor from chronic osteomyelitis based upon the characteristics of lesions in macerated bone specimens. Other such comparisons would establish the criteria for using the gross features of dried specimens to diagnose lesions and thus facilitate both animal paleopathology and veterinary forensics.
245
Quantitative and Qualitative Evaluation of Laminitis-Associated Distal Phalangeal (P3) Osteopathology
J.B. Engiles1, D. McDonald2, M. Dishowitz3, H. Galantino-Homer2, and K.D. Hankenson3. 1Department of Pathobiology, New Bolton Center, 2Clinical Studies- New Bolton Center, 3Animal Biology, School of Veterinary Medicine, University of Pennsylvania Laminitis remains an incurable disease of ungulates, which often results in humane euthanasia due to poor prognosis or intractable pain. In horses, the anatomic structures suspending the distal phalanx (P3) from the hoof wall are disrupted, leading to separation of the sensitive epidermal and dermal lamellae of the inner hoof wall, rotational and/or distal displacement of the distal phalanx with eventual prolapse of P3 through the sole. Certain biomechanical, inflammatory, and endocrine/metabolic disease associated osteopathologies are well described in human and veterinary medicine. Although radiographic descriptions of laminitis-associated solar margin fractures and modeling of P3 are well described in cases of chronic laminitis, the onset and progression of laminitisassociated P3 osteopathology has neither been systematically characterized, nor quantified. Given the unique and intimate anatomic connection between the sensitive lamellae and distal phalanx, which includes a shared blood supply, we hypothesize that P3 osteopathology occurs in both earlier (acute and subacute) stages of laminitis, and progresses in chronic laminitis. Utilizing microcomputed tomography (microCT) of non-decalcified paragagittal sections of P3, as well as histopathology of sensitive lamellar tissue and decalcified parasagittal sections of P3, we correlate progressive quantitative and qualitative distal phalangeal osteopathologies with sensitive lamellar pathologies in a representative cross-section of necropsy specimens from clinically laminitic and non-laminitic horses.
244
Pathological Prion Protein Deposition in Chronically Inflamed Organs of Scrapie-Affected Sheep
G.M. Cancedda, G. Marruchella, G. Di Guardo, C. Maestrale, M. Masia, C. Santucciu, S. Macciocu, M.P. Vargiu, and C. Ligios. Istituto Zooprofilattico Sperimentale della Sardegna, G. Pegreffi Sassari Universita degli Studi di Teramo, Facolta di Medicina Veterinaria, Dipar` ` timento di Scienze Biomediche Comparate, Teramo During sheep scrapie, pathological prion protein (PrPSc) first accumulates in the lymphoreticular system (LRS) and finally in the central nervous system (CNS). It has been demonstrated that, both in mouse models and in natural host, PrPSc deposition may expand to a range of other unsuspected organs affected by chronic lymphoproliferative inflammation. Here, we investigated PrPSc distribution in scrapie-affected sheep with granulomatous or lymphoproliferative chronic inflammations, which are very common disorders in small ruminants. To do this, PrPSc detection was carried out by immunohistochemistry (IHC) and western blotting (WB), with immuno-phenotyping of the inflammatory cells being also performed, in sheep with clinical and preclinical natural scrapie (n. 38). Our data demonstrate that ectopic PrPSc accumulates exclusively in well-organized neoformed lymphoid follicles with follicular dendritic cells (FDCs). On the contrary, no PrPSc deposition was immunohistochemically found in the granulomas, even when closely located to the above-mentioned neoformed lymphoid follicles. These findings suggest that, in sheep with coexistence of natural scrapie and inflammatory conditions, there may be a risk of prion contamination in unsuspected organs only in presence of neoformed lymphoid follicles.
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Seneca Valley Virus Associated With Vesicular Lesions in a Pig With Idiopathic Vesicular Disease
S. Corner1, K. Singh1, S.G. Clark2, and G. Scherba1. 1Veterinary Diagnostic Laboratory and 2Department of Animal Sciences, University of Illinois, Urbana-Champaign, IL Swine idiopathic vesicular disease (SIVD) syndrome is a relatively new disease, which is characterized by the formation of non-debilitating ulcers, erosions and vesicles on the skin, coronary bands and in the oral cavity of pigs. The clinical importance of SIVD is its resemblance with vesicular foreign animal diseases. Although the etiology of SIVD remains unknown, Seneca Valley virus, which belongs to the family Picornaviridae, was previously identified in such pigs. Here we report gross and histopathologic findings in a 6-month-old intact male Chester White boar presented with a history of anorexia, lethargy and lameness. Intact and ruptured vesicles, erosions and ulcers were clinically observed. These lesions were present within the oral cavity, around the nares, coronary bands, and all 4 limbs. In addition, gross pathology revealed serofibrinous peritonitis and pericarditis, locally extensive hemorrhagic jejunitis, and afocal gastric ulcer. Microscopically, chronic ulcers with numerous degenerate and intact neutrophil infiltration, edema, hemorrhage, fibrin and granulation tissue were observed along with orthokeratotic and parakeratotic hyperkeratosis and epidermal hyperplasia. Various diagnostic tests were negative for swine vesicular disease virus, foot-and-mouth disease virus, vesicular exanthema of swine virus and vesicular stomatitis virus infection. However, vesicular scrapings and oral pharyngeal fluid were positive for the presence of Seneca Valley virus by RT-PCR.
Abstracts
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Tubuloreticular Inclusions in Human, Canine, and Feline Renal Diseases
N.D. Jackson1, K. Robinson1, G. Lees1, B. Berridge2, R. Cianciolo3, and F. Clubb1. 1Texas A&M University, College of Veterinary Medicine and Biomedical Science, College Station, TX, 2GlaxoSmithKline, Research Triangle Park, NC, and 3College of Veterinary Medicine, North Carolina State University, Raleigh, NC Ultrastructural tubuloreticular inclusions (TRIs) in humans are associated with autoimmune disease, Systemic Lupus Erythematosis (SLE), Human Immunodeficiency Virus (HIV) and other families of viruses, as well as specific neoplasms. Frequently, these disorders cause renal disease ranging from Focal Segmental Glomerulosclerosis to End Stage Renal Disease. In these human diseases, TRIs predominate in lymphocytes, monocytes, and glomerular endothelial cells. TRIs consist of 20-30 nanometer branching tubules located within the endoplasmic reticulum cisternae and perinuclear envelope. They form via reorganization of internal cell membranes, and have been linked to the upregulation of interferon alpha. TRIs have not been investigated extensively in animals. Recent studies have shown TRIs in non-human primates with Simian Immunodeficiency Virus and Ebola Virus. From 2005 to May 2011, ten canine renal biopsy specimens (2.0%) and a single feline renal biopsy specimen evaluated by the Texas Veterinary Renal Pathology Service contained TRIs in glomeruli. Animal cases had ultrastructural TRIs within glomerular endothelial cells similar to humans, with the exception of two dogs having TRIs within mesangial cells (first documented cases of mesangial cell involvement). During the same time period, 124 human renal biopsies (13.4%) had TRIs in glomerular endothelium and had glomerulopathies associated with SLE, HIV, Hepatitis C, and other viral infections that upregulate interferon alpha. Though the incidence of TRIs in domestic animal species appears lower than in humans, the presence of TRIs may be useful as an ultrastructural marker for disease states, similar to their use in humans.

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Veterinary Pathology 00(0)

Downloaded from vet.sagepub.com by guest on April 12, 2012

Downloaded from vet.sagepub.com by guest on April 12, 2012

Veterinary Pathology 00(0)

Downloaded from vet.sagepub.com by guest on April 12, 2012

Downloaded from vet.sagepub.com by guest on April 12, 2012

Veterinary Pathology 00(0)

Downloaded from vet.sagepub.com by guest on April 12, 2012

Downloaded from vet.sagepub.com by guest on April 12, 2012

Veterinary Pathology 00(0)

Downloaded from vet.sagepub.com by guest on April 12, 2012

Downloaded from vet.sagepub.com by guest on April 12, 2012

Veterinary Pathology 00(0)

Downloaded from vet.sagepub.com by guest on April 12, 2012

Downloaded from vet.sagepub.com by guest on April 12, 2012

Veterinary Pathology 00(0)

Downloaded from vet.sagepub.com by guest on April 12, 2012

Downloaded from vet.sagepub.com by guest on April 12, 2012