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2007 ACVP Annual Meeting

2007 ACVP Young Investigator Awards Poster Competition

Diagnostic Pathology

First Place: $500

PERIANAL SQUAMOUS CELL CARCINOMA IN THREE GENETICALLY RELATED PYGMY GOATS. S. White-Hunt1, D. Thibeault2, H. J. Van Kruiningen1, R. French1. 1Department of Pathobiology and Veterinary Sciences, University of Connecticut, Storrs, CT; 2Brooklyn-Canterbury Large Animal Clinic, Canterbury, CT, USA.
Squamous cell carcinoma is a malignant neoplasm which, in domestic farm animals, is most commonly observed in non-pigmented, sparsely-coated or hairless skin, such as the eyelid, external genitalia, and udder. It is relatively common in cows and horses, less so in sheep, and rare in pigs and goats. This case report describes perianal squamous cell carcinoma in three genetically related, female, white, Pygmy goats (Goats 1, 2 and 3) from a single herd, submitted to the Connecticut Veterinary Medical Diagnostic Laboratory between March 2006 and April 2007. Goats 1 and 2 were siblings, and Goat 3 was an offspring of Goat 1. All three goats had multiple, variably-sized (up to 1.5 centimeters in diameter) foci of epidermal ulceration and crusting of unpigmented perianal skin. Histopathology of the lesions revealed classic SCC with infiltration of the underlying dermis and musculature by numerous islands and trabeculae of neoplastic epidermal cells, sometimes surrounding variably-sized keratin pearls. Although SCC is reported to metastasize only rarely, here in these cases, there was metastatic spread of the tumor to the left lumbar lymph node of Goat 1 and right lumbar lymph node of Goat 3. Goat 1 also had numerous metastases along the omentum and abdominal aspect of the diaphragm as well as a large (17 x 16 x 10 cm), subcutaneous, metastatic mass in the proximal caudolateral right hindlimb. Other lesions observed included a pheochromocytoma in Goat 1 and peritoneal mesothelioma with ascites in Goat 2. This case report may represent a genetic predisposition to SCC in this line of white Pygmy goats.

Second Place: $300

GENOMIC IDENTIFICATION OF BARTONELLA HENSELAE IN AN ABORTED EQUINE FETUS. R. Johnson, J. Ramos-Vara, R. Vemulapalli. Department of Comparative Pathobiology, Purdue University, West Lafayette, IN.
Bartonella henselae is a gram-negative bacterium that often causes an asymptomatic bacteremia, but can cause cat scratch disease, bacillary angiomatosis, bacillary peliosis, and endocarditis. The bacterium has been isolated from numerous mammalian species, but not from equids. An aborted equine fetus exhibited gross and histologic evidence of necrosis and vasculitis in multiple tissues with intralesional spirochete-like organisms. Using universal primers, the bacterial 16S rRNA gene was amplified from DNA extracted from fetal lung and kidney. Nucleotide sequence of the amplified PCR product revealed over 99.9% identity to the 16S rRNA gene of Bartonella henselae. The microscopic and ultrastructural morphology of the intralesional bacteria are not consistent with Bartonella sp., suggesting Bartonella henselae may have been an opportunistic or inapparent copathogen in this animal. The intralesional organism was gram-negative and varied from short rods to wavy, spirilar forms up to 5 um long in periplasmic flagella were not apparent. Leptospira spp. is the most common spirochete that causes abortion in horses. Leptospirosis was excluded by negative Leptospira culture, negative PCR, and negative immunohistochemistry. Borrelia spp. have also been reported as a cause of equine abortion, but multiple attempts to amplify Borrelia-specific DNA via PCR using primers targeting the 16S rRNA and flagellin genes were unsuccessful. EHV fluorescent antibody tests, virus isolation, and bacterial culture were all negative. A definitive identification of the intralesional bacterium is undetermined. To our knowledge, this is the first reported identification of Bartonella henselae in an equid, though its significance as a cause of abortion is unclear.

Third Place: $200

INTERSEX IN A DOMESTIC SHORT-HAIRED CAT WITH MOSAIC KARYOTYPE 38,XX/39,XXY. I.L. Bergin1, U. Prociuk2, S. Mehler1, D. Agnew1. 1Michigan State University, E. Lansing, MI, 2University of Pennsylvania, Philadelphia, PA, USA.
The condition of intersex in domestic cats is known to occur, but histological and genetic documentation of such cases is lacking in the veterinary literature. In this case, we identified intersex in a 6-month-old, domestic shorthair cat with ambiguous external genitalia consisting of an empty scrotal sac and a genital slit. The reproductive tract had a small bicornuate uterus and bilateral small gonads located in the retroperitoneal space. One gonad consisted of an immature, aspermic testis with features of atypia in the germ cell population. Contralaterally, there was an ovotestis that contained both primitive, aspermic, seminiferous tubules and poorly differentiated ovarian stroma. The SRY gene (encoding testis-determining factor) was detected by PCR and the karyotype was determined to be 38,XX (98%)/39,XXY(2%), indicating that the cat was a mosaic. This karyotypic abnormality is a rare reported cause for intersex in humans. Other abnormal karyotypes have been described in phenotypically similar intersex humans and likely exist in veterinary medicine as well. This case illustrates the value of genetic analysis in addition to histopathology in characterizing intersex conditions in animals.

Experimental Disease

First Place: $500

INVESTIGATING THE ROLE OF STAT3 ACTIVATION IN HUMAN AND CANINE OSTEOSARCOMA. S.L. Fossey1, A.T. Liao1, J. Lin2, P.K. Li3, and C.A. London1. 1Department of Veterinary Biosciences; 2Department of Pediatrics; 3Division of Medicinal Chemistry, The Ohio State University, Columbus, OH, USA.
Osteosarcoma (OSA) is the most common malignant bone tumor in children and dogs. Treatment for both involves removal of the primary tumor followed by adjuvant chemotherapy. Approximately 30% of children and over 90% of dogs will develop metastatic disease post treatment. Differences in outcome are likely due to higher dose intensities used in their treatment of children when compared to dogs. Recent evidence suggests that the molecular biology of OSA in dogs is similar to that in children and includes the existence of p53 mutations, aberrant Met expression, and over-expression of ezrin, in addition to indistinguishable transcriptional profiles. Given the similarities in biology and clinical behavior between canine and pediatric OSA, canine OSA represents a relevant animal model in which to investigate the molecular biology of this disease. While evaluating signaling pathways in OSA cell lines,

culture conditions or inhibition of Met signal transduction. Treatment of OSA cell lines with a small molecule STAT3 inhibitor, LLL3, promoted loss of cell viability and apoptosis. Furthermore, treatment with SU6656, a small molecule Src inhibitor, reduced phosphorylation of Src and its downstream target STAT3 in a majority of canine OSA cell lines tested with a corresponding reduction in proliferation. Lastly, OSA cells transfected with STAT3 siRNAs exhibited loss of STAT3 protein and underwent apoptosis. These data support the notion that dysregulation of STAT3 is common in canine OSA and serves as relevant target for therapeutic intervention.

Second Place: $300

CANINE INFLUENZA VIRUS REPLICATES IN CANINE ALVEOLAR MACROPHAGES AND INDUCES TNF-ALPHA EXPRESSION. J.R. Powe, D. Mukhtyar, and W.L. Castleman. Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL., USA.
Immunohistochemical studies on dogs with severe spontaneous disease resulting from canine influenza virus (CIV) infection suggest the possibility that virus replicates in macrophages and contributes to clinical disease pathogenesis. We tested the hypothesis that CIV infects pulmonary macrophages and contributes to the severity of pulmonary disease by inducing proinflammatory cytokines such as TNF-alpha. Canine alveolar macrophages were isolated from mongrel dogs and were inoculated in culture with CIV (Influenza A/Canine/FL/04). Quantitative RT-PCR from 0 to 48 hours following inoculation demonstrated that macrophages supported influenza matrix gene expression that peaked at 6 hours after inoculation. Infectious virus was recovered from macrophage cultures. TNF-alpha mRNA steady-state levels measured by RT-PCR increased by 6 hours. The results are consistent with the hypothesis that CIV replication in alveolar macrophages plays a role in canine influenza virus pathogenesis through induction of proinflammatory cytokines such as TNF-alpha.

Third Place: $200

DECREASED CCL5 CORRELATES WITH HEIGHTENED SUSCEPTIBILITY TO MYCOBACTERIUM TUBERCULOSIS INFECTION, DELATED T CELL TRAFFICKING TO THE LUNGS AND POORLY ORGANIZED GRANULOMAS IN VIVO. G.L. Beamer1,2, D.K. Flaherty3, B. Vesosky1, J. Turner1,2. 1Center for Microbial Interface Biology, The Ohio State University, Columbus, OH; 2Department of Veterinary Biosciences, The Ohio State University, Columbus, OH; and 3Vanderbilt Medical Center, Vanderbilt University, Nashville, TN, USA.
Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb) is a substantial global health problem with 8-10 million newly diagnosed cases and 2-3 million deaths each year. Following infection with M.tb, most people develop protective TH1 immunity within discrete cellular foci called granulomas. Functionally, granulomas benefit the host by focusing TH1 immunity, inhibiting M.tb proliferation and dissemination. The mechanisms governing granuloma formation are not fully understood, but believed to require production of chemoattractant cytokines (chemokines) and appropriate receptor expression. In vitro models have shown that chemokines including CCL5 are secreted by M.tb infected macrophages and exposure to these chemokines induced receptor dependent migration of activated T cells. Using susceptible and resistant mouse models of TB we have correlated these in vitro findings to in vivo granuloma formation and cellular infiltration in the lungs following M.tb aerosol infection. In susceptible mice, decreased CCL5 gene and protein expression correlated with fewer activated CD4 and CD8 T cells in the lungs and was associated with delayed and disorganized granuloma formation. In contrast, resistant mice form compact organized granulomas associated with abundant CCL5 gene and protein expression, and rapidly localized T cells to the lungs. These results suggest that early and abundant CCL5 production mediates T cell recruitment to the lungs and may contribute to M.tb granuloma structure in vivo. Further studies may identify a critical role for CCL5 during M.tb granuloma formation and the development of protective TH1 immunity.

Natural Disease

First Place: $500

EXTRAINTESTINAL PATHOGENIC E. COLI (EXPEC) INDUCED ACUTE NECROTIZING PNEUMONIA IN CATS. R. Sura1, H. J. Van Kruiningen1, C. DebRoy2, L. S. Hinckley1, K. J. Greenberg1, Z. Gordon3, R. A. French1. 1Connecticut Veterinary Medical Diagnostic Laboratory, Department of Pathobiology and Veterinary Science, University of Connecticut, Storrs, CT; 2Gastroenteric Disease Center, The Pennsylvania State University, University Park, PA; and 3Connecticut Animal Shelter, Stamford, Connecticut, USA.
Extraintestinal Escherichia coli (ExPEC) are opportunistic pathogens of humans and animals involved in several disease conditions ranging from urinary tract infection to meningitis. They comprise an epidemiologically and phylogenetically distinct strain, affecting most species, with involvement of any organ or anatomical site. Here, we report fatal cases of necrotizing pneumonia in cats caused by extraintestinal E. coli. Over a one week period, 13 cats from an animal shelter were presented to Connecticut Veterinary Medical Diagnostic Laboratory with a history of respiratory distress. The gross and microscopic findings for all the cats were consistent. E. coli were uniformly isolated from the lungs of all the cats. Serotyping of the E. coli isolates indicated that they were of the O4:H5 serotype. All the isolates were hemolytic and had genes encoding for cytotoxic necrotizing factor 1 (CNF 1) and fimbriae (sfa), and specific adhesin molecules (papG allele I and III) that are characteristic of ExPEC. To date there are only a few reports of similar ExPEC infections in dogs. This is the first report of an ExPEC infection in cats. Since, cats are common domestic pets; this report raises concern regarding zoonotic spread of ExPEC infections.

Second Place: $300

SPONTANEOUS CANINE INTRAEPITHELIAL MAMMARY GLAND LESIONS AS A MODEL FOR HUMAN BREAST PRENEOPLASIA. P. Mouser1, M. A. Miller1, S. S. Badve2, S. I. Mohammed1. 1Department of Comparative Pathobiology Purdue University, West Lafayette, IN and 2Department of Pathology and Laboratory Medicine, Indiana University, Indianapolis, IN, USA.
Early detection of mammary intraepithelial lesions (IELs) in women, through imaging such as mammography, provides the opportunity to intercept the progression of breast cancer. Intraepithelial lesions, especially ductal hyperplasia and ductal carcinoma in situ (DCIS), predict the risk of invasive breast cancer and form the basis for therapeutic decisions. Mammary neoplasia is the most common spontaneous malignancy in female dogs and IELs are common both in tumor-bearing canine mammary glands and in glands that have yet to develop invasive neoplasia. In a cross-sectional post mortem study of 108 female dogs (median age 8 years, predominantly sexually intact hounds retired from breeding) without clinical mammary disease, all mammary glands were evaluated histologically to determine the prevalence and characteristics of ductal intraepithelial lesions. Within this population, 56 dogs (52%) had at least one type of spontaneous mammary IEL. Lesions were classified according to criteria for the human breast as ductal hyperplasia (DH; 49 dogs), atypical ductal hyperplasia (ADH; 14 dogs), low-grade DCIS (19 dogs), intermediate-grade DCIS (12 dogs) and high-grade DCIS (1 dog). Twenty-one dogs had multiple IELs; 24 had atypia (ADH or DCIS). In addition, subclinical benign neoplasms (6 simple adenomas, 12 complex adenomas, 7 benign mixed tumors, 5 basaloid adenomas, 5 ductal papillomas, and 4 sclerosing papillomas) were detected in 11 dogs. The dog shows particular promise as a model for human breast preneoplasia based on its prevalent spontaneous mammary IELs, its large size as compared to a rodent model, and the remarkable histological similarity between canine and human IELs.

Third Place: $200

VESTIBULAR SYNDROME DUE TO BRAINSTEM INFARCTION IN SWISS MICE. T. Southard, S. Trembley, T. Prezioso, K. Gabrielson, C. Morrell, and C. Brayton. Department of Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, MD, USA.
Spontaneous vestibular syndrome in mice, characterized clinically by head tilt, circling or rolling, can be attributed to otitis media, arteritis or central nervous system lesions. Post mortem examination on seven non-inbred Swiss mice (one Hsd:ND4 and six Hsd:ICR(CD-1)) submitted for pathology due to acute onset of vestibular signs revealed similar brainstem lesions. The lesions were characterized by unilateral, well-demarcated areas of necrosis, malacia and gliosis, with variable amounts of hemorrhage, in the lateral aspect of the medulla and caudal pons. The affected area included the medial, lateral and superior vestibular nuclei, the facial nucleus and the spinal trigeminal nucleus. In one mouse, the patency of the vascular supply to the brainstem was investigated. Intracardiac injection of black ink immediately after euthanasia revealed a filling defect in the rostral segment of the vertebral artery, at the level of C1, on the side of the brainstem lesion. These findings suggest that the unilateral brainstem necrosis is secondary to occlusion or rupture of the vertebral artery. Unilateral brainstem infarction represents another potential cause of vestibular phenotype in mice and shares features with Wallenberg's Lateral Medullary Syndrome, the most common brainstem stroke in humans.

Toxicologic Pathology

First Place: $500

THE CALM-AF10 FUSION GENE EXPRESSED IN TRANSGENIC MICE LEADS TO HOXA CLUSTER OVEREXPRESSION AND ACUTE LEUKEMIA. D. Caudell1,2,3, Z. Zhang1, Y.J. Chung1, and P.D. Aplan1. 1Genetics Branch, 2Comparative Molecular Pathology Unit, National Cancer Institute, National Institutes for Health, Bethesda, MD, and 3Department of Veterinary Medical Sciences, University of Maryland, College Park, MD.
To assess the role of the CALM-AF10 fusion gene in leukemic transformation in vivo, we generated transgenic mice that expressed a CALM-AF10 fusion gene. Depending on the transgenic line, at least 40-50% of the F1 generation mice developed acute leukemia, at a median age of 12 months. Leukemic mice typically had enlarged spleens, invasion of parenchymal organs with malignant cells, and tumors with myeloid markers such as myeloperoxidase, Mac1, and Gr1. Although most leukemias were acute myeloid leukemia (AML), many showed lymphoid features, such as CD3 or B220 staining, or clonal Tcrb or Igh gene rearrangements. Mice were clinically healthy for the first 9 months of life, and had normal peripheral blood hemograms, but had impaired thymocyte differentiation, manifested by decreased CD4+/CD8+ cells, increased immature CD4-/CD8- cells in the thymus. Hematopoietic tissues from both clinically healthy as well as leukemic CALM-AF10 mice showed up-regulation of Hoxa cluster genes, suggesting a potential mechanism for the impaired differentiation. The long latency period and incomplete penetrance suggest that additional genetic events are needed to complement the CALM-AF10 transgene and complete the process of leukemic transformation.

Second Place: $300

TIME COURSE COMPARISON OF LY504132 AND DHT ON PROLIFERATION AND APOPTOSIS IN THE FEMALE F344 RAT MAMMARY GLAND. J. Lucas1, S. Hooser1, A. Peter1, D. Rudmann2, and P. Snyder1. 1Department of Veterinary Pathology Purdue University, West Lafayette, IN and 2Department of Toxicology, Eli Lilly, Greenfield IN.
Selective estrogen modulators (SERMS) should suppress proliferation of the mammary gland epithelium (MGE). We previously demonstrated in female F344 rats that some SERMs (including LY504132) induce virilism: characterized by multiple layers of hypertrophied and vacuolated MGE in intralobular ducts and alveoli, hyperandrogenemia and androgen receptor expression, which is morphologically distinct from dihydrotestosterone (DHT) induced lesions. In this study, female F344 rats (n=5) were administered 10 mg/kg oral LY504132, 10 mg/kg subcutaneous DHT, or vehicles, daily for 2, 4, 14 or 30 days. LY504132 and DHT treatment increased serum concentrations of testosterone (radioimmunoassay, Day 2 mean, 2.3 and 5.4 ng/ml, reference range <0.6 ng/ml), but only LY504132 increased serum estradiol (radioimmunoassay, Day 4 mean, 50.3 pg/ml, reference range <30 pg/ml). On MGE morphology (HE), LY504132 induced virilism from Day 4, while DHT induced not only virilism, but also duct dilation with increased secretory product. Similar AR expression (immunohistochemistry) was detected from Day 2 in both LY504132 and DHT treated rats. Over 14 days, significantly less MGE proliferation was observed in LY504132 treated rats (mean Ki-67 positive cells<5%), than controls (mean Ki-67 positive cells 5 to 17.3%) or DHT (mean Ki-67 positive cells 6.6 to 13.8 %). MGE apoptosis (tunel) for DHT and LY504132 was less frequent than control. These data demonstrate that LY504132 decreases apoptosis which accounts for the MGE accumulation, but suppresses MGE proliferation. DHT decreased apoptosis but not proliferation, suggesting some of the differences observed in LY504132 and DHT morphology could be attributed to differing effects on proliferation and apoptosis.

Third Place: $200

MECHANISMS OF DMN-INDUCED HEPATIC NEOPLASIA IN THE F344 RAT VERSUS THE MEDAKA FISH. K.R. Hobbie1,2, A.B. DeAngelo2 and J.M. Law1. 1NCSU College of Veterinary Medicine, Population Health and Pathobiology Department, Raleigh, NC, USA and 2US EPA, Environmental Carcinogenesis Division, Durham, NC, USA.
To determine the roles of transforming growth factor-Beta1 (TGF-Beta1) and hepatic stellate cells (HSCs) in hepatic fibrosis and carcinogenesis, we performed parallel dimethylnitrosamine (DMN) exposures in F344 rats (drinking water) and Japanese medaka (ambient water). DMN, an alkylating carcinogen, is used to model cirrhosis and neoplasia in the rat liver. TGF-Beta1 is associated with hepatic fibrosis and neoplasia in humans and rodents. Liver injury induces HSCs to transdifferentiate to myofibroblast-like cells. TGF-Beta1 stimulates activated HSCs to produce collagen type-1, leading to fibrosis. TGF-Beta1 has tumor suppressor activity through hepatocyte growth-inhibition and apoptosis. TGF-Beta1’s tumor suppressor role has been documented in humans and rodents, and rodent DMN-induced lesions are TGF-Beta1 dependent. DMN is also a potent hepatotoxin and carcinogen in fish.
Mutagenesis studies in DMN-exposed medaka have demonstrated DNA lesions similar to that of rodents, but the mechanisms by which hepatic fibrosis and neoplasia occur in the medaka are unknown. In our study, fish were exposed to 0, 10, 25, 50, or 100 ppm DMN and rats to 0, 0.1, 1, 5, 10, and 25 ppm DMN for 14 days. Post-exposure, specimens were sacrificed for histopathology. The rats were grossly and microscopically unremarkable. Lesions in the fish were confined to the liver. Results included: hepatocellular necrosis, apoptosis, regeneration, and dysplasia; HSC and spindle cell proliferation; and hepatocyte TGF-Beta1 expression. An association between TGF-Beta1 and hepatic lesions in DMN-exposed medaka suggests a similar dependence to that in rodents. Direct comparisons between animal models at different phyletic levels will help facilitate the interspecies extrapolations that are so vital in toxicological risk assessment.

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