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2006 ACVP Annual Meeting

2006 ACVP Young Investigator Award Recipient Abstracts

Diagnostic Pathology

Diagnostic Pathology – First Place

POLYCYSTIC LIVER AND KIDNEY DISEASE IN TWO RELATED BEAGLES: A PROPOSED PATHOGENESIS. T.P. LaBranche, M. Randolph, C.A. Brown, J.V. Mysore. Pathology Dept., UGA, Athens, GA.

Polycystic Liver and Kidney Disease (PCLKD) is an inherited condition with cystic dilation of hepatic bile ducts and renal tubules in humans and animals. We received two Beagle puppies for necropsy from a dam that produced three successive litters of puppies with non-regenerative anemia and renal failure by 4-weeks of age. Expansion of the extrahepatic biliary system was grossly evident in both puppies, as were many clear fluid-filled cystic spaces that effaced the renal cortex. Microscopically, hepatic portal areas were expanded by dilated, tortuous (10-300x normal size) bile ducts, as well as a mild amount of fibrous connective tissue. Renal cortical and medullary tubules were also tortuous and cystic (10-200x normal diameter), and were lined by a moderately hyperplastic epithelium. Ultrastructural features of the cystic renal tubules include the presence of malformed primary apical cilia, which were either curvilinear, stunted, and / or had variable lengths. We suspect that our case report has close resemblance with the cpk (congenital polycystic kidney) mouse model and the autosomal recessive phenotype of polycystic kidney disease in humans. Recent reports with cpk mice suggest the pathogenesis is due to mutation(s) in proteins (polycystins, cystin and fibrocystin) critical to the mechanosensory function of primary apical cilia, a solitary, nonmotile organelle which is responsible for sensing tubular flow during embryogenesis and regulates tubular epithelial cell apoptosis, growth, and differentiation. Here, we provide ultrastructural evidence to suggest that primary apical ciliary malformation / dysfunction may have lead to formation of cystic dilations of tubules and ducts in these puppies. This is the first discussion of the potential importance of primary apical cilia in the pathogenesis of inherited PCLKD in dogs.

Diagnostic Pathology – Second Place

VASCULAR-ASSOCIATED LYMPHOID TISSUE (VALT) IN SWINE. I.M. Langohr1, H. HogenEsch1, G. Stevenson1, M. Sturek2.1Department of Veterinary Pathobiology, Purdue University, West Lafayette, IN; 2Department of Cellular & Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN.

Focal accumulations of mononuclear cells in the wall of major arteries in young healthy human beings have been described as vascular-associated lymphoid tissue (VALT) at predilection sites for atherosclerotic lesions later in life. Pigs are commonly used as an animal model for the study of cardiovascular disease. To investigate the presence of VALT in this species, samples of major branching sites of thoracic and abdominal aortic, carotid, left anterior descending and right coronary, and brachial and femoral arteries were collected from 10 conventional pigs submitted for necropsy to the Indiana Animal Disease Diagnostic Laboratory. Samples were snap-frozen or fixed in formalin and processed for routine light microscopy and immunohistochemistry. Single or small aggregates of mononuclear cells were noted, predominantly in the intima but occasionally also within the inner portion of the tunica media. The infiltrating cells were mostly CD3+ T cells (with most cells expressing CD4) and some macrophages. MHC class II molecules were commonly expressed by infiltrating leukocytes and overlying endothelial cells. An analogous study was carried out in Ossabaw swine, which are susceptible to obesity, metabolic syndrome, type 2 diabetes mellitus and atherosclerosis. Similar accumulations of T lymphocytes were found in animals on low fat diet at locations where atherosclerotic lesions develop in animals fed a high fat/high cholesterol diet. Since atherogenesis in humans is preceded by infiltration of primarily T cells in the vessel wall, it is possible that the cells forming the VALT play a role in the initiation and progression of atherosclerotic lesions. This study further substantiates pigs as a suitable animal model for studying the development of atherosclerosis.

Diagnostic Pathology – Third Place

TESTICULAR INTERSTITIAL CELL TUMOR AND GYNECOMASTIA IN A RABBIT. K. Maratea, J. Ramos-Vara, M. Miller.Animal Disease Diagnostic Laboratory and Department of Veterinary Pathobiology, Purdue University, West Lafayette, IN.

Unilateral testicular interstitial cell tumor and gynecomastia were diagnosed in an adult rabbit. The interstitial cell tumor was a well-circumscribed, 2 mm diameter, pale tan nodule composed of a uniform population of polygonal cells. The polygonal cells had indistinct borders, ample eosinophilic cytoplasm, round to ovoid hyperchromatic nuclei, and were organized into ill-defined lobules by delicate fibrovascular stroma. Neoplastic interstitial cells exhibited diffuse, granular cytoplasmic staining with Melan A, a marker of steroid-producing cells in humans and dogs. Seminiferous tubules in the surrounding parenchyma and in the contralateral testis were spermatogenically active, with no apparent atrophy. Multiple cystic, cutaneous masses in the caudal abdomen were associated with enlarged nipples and composed of hyperplastic mammary gland tissue with proliferation of ducts and alveoli, prominent lobule formation, and lactational changes. Alveoli were lined by 1-4 layers of cuboidal to low columnar epithelial cells that were sometimes vacuolated or had prominent apical blebbing. In many lobules, ducts and alveoli were distended with pale eosinophilic secretion. In men with functional interstitial cell tumors, gynecomastia results from elevated levels of estradiol and progesterone, and reduced serum testosterone. Administration of either estrogen or androgen, however, may cause gynecomastia characterized by pseudolactactional hyperplasia similar to that observed in the rabbit. Since hormone levels were not assayed in the rabbit, the underlying cause of gynecomastia and its potential association with the interstitial cell tumor are undetermined. To the authors’ knowledge, this is the first report of concurrent testicular interstitial cell tumor and gynecomastia in a rabbit, and also the first description of gynecomastia in this species.

EXPERIMENTAL DISEASE

Experimental Disease – First Place

THE ROLE OF B CELLS IN THE CELL-MEDIATED IMMUNE RESPONSE TO LEISHMANIA AMAZONENSIS. K.N. Gibson-Corley, R.M. Mukbel, C.A. Petersen and D.E. Jones. Veterinary Pathology, Iowa State University, Ames, IA.

C3HeB/FeJ mice infected with Leishmania major develop a CD4+ Th1 response and resolve infection, while infection with Leishmania amazonensis stimulates a poor T cell response, resulting in chronic disease. When C3HeB/FeJ mice are co-infected with both species of Leishmania we observe a healing phenotype similar to infection with L. major alone. In contrast, co-infected C57BL/6 mice have a non-healing phenotype similar to infection with L. amazonensis. We have developed an in vitro assay in which macrophages from C3HeB/FeJ mice infected with L. amazonensis can be activated to kill internalized parasites when co-cultured with lymphocytes isolated from the draining lymph node of a L. major-infected C3HeB/FeJ mouse. Specifically, we have found that CD4+ T cells and B cells from the draining lymph node of L. major-infected C3HeB/FeJ mice are sufficient and necessary for macrophage activation and parasite death. In contrast, when C57BL/6 mouse-derived total lymph node cells or purified CD4+ T cells and B cells infected with L. major are co-cultured in vitro with L. amazonensis infected macrophages parasite killing is not observed. A series of cell transfer studies have demonstrated that B cells from the C57BL/6 mice cannot promote killing of intracellular L. amazonensis. These findings from our in vitro parasite killing assay indicate that B cells play a prominent role in the cell-mediated immune response against L. amazonensis and that B cells from C57BL/6 mice are deficient in promoting killing compared to cells from C3HeB/FeJ mice.

Experimental Disease – Second Place

ROLE OF AIDA-I ADHESIN IN PATHOGENESIS OF E. COLI INDUCED DIARRHEA IN PIGS.M. Ravi1, M. Ngeleka1, S-H. Kim2, C.L. Gyles2, F. Berthiaume3, M. Mourez3, D.M. Middleton1, E. Simko1.1Universities of Saskatchewan; 2Guelph; and 3Montreal, Canada.

Escherichia coli remains a significant cause of diarrhea worldwide, and in recent years a relatively high percentage (7-25%) of E.coli carrying the gene encoding AIDA-I (adhesin involved in diffuse adherence) has been isolated from cases of porcine diarrhea. AIDA-I adhesin and its gene, aidA, were first identified and characterized in E.coli isolated from a human case of infantile diarrhea. We reported previously that experimental infection of pigs with a porcine AIDA-I+ E.coli caused diarrhea. In this study, we investigated the role of AIDA-I adhesin in pathogenesis of porcine diarrhea using the following bacterial strains: i) wild strain: AIDA-I+/STb+ E.coli isolated from a clinical case of porcine diarrhea; ii) mutant strain: AIDA-I-/STb+ E.coli, generated by partial deletion of the aidA gene from the wild strain, iii) complemented strain: AIDA-I+/STb+ E.coli, generated by reintroducing the full length aidA gene into the mutant strain, and iv) non-pathogenic E.coli used as negative control. Firstly, AIDA-1+ strains exhibited in vitro prominent diffuse adherence to HeLa cells, enhanced bacterial auto-aggregation, and biofilm formation, when compared to AIDA-I- strains. Secondly, colostrum-deprived piglets infected with AIDA-I+ wild and complemented strains developed diarrhea 20 to 30 h after inoculation, in contrast, to piglets infected with the AIDA-I- mutant and control strains that did not develop diarrhea. Histologic, immunohistochemical, and electron microscopic examinations showed heavy bacterial colonization with biofilm formation in the large intestine of pigs infected with the two AIDA-I+ strains but not in those pigs infected with the AIDA-I- strains. Thus, AIDA-I may be considered a significant virulence determinant in development of diarrhea caused by porcine diarrheagenic AIDA-I+ E.coli.

Experimental Disease – Third Place

WEST NILE ENCEPHALITIS: SEQUENTIAL HISTOPATHOLOGICAL AND IMMUNOLOGICAL EVENTS IN A MURINE MODEL OF INFECTION.D. Garcia-Tapia1,2, D.E. Hassett1, G.C. Johnson1,2, W.J. Mitchell1,2, and S. B. Kleiboeker1. 1Veterinary Pathobiology Department, University of Missouri-Columbia; 2Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, MO.

West Nile virus (WNV) has emerged as an important cause of encephalitis in humans and horses in North America. Although there is significant knowledge about the pathogenesis of disease caused by this flavivirus and about the immunity against it, no reports exist describing the sequence of pathological changes and their correlation to the immune response in the brain of infected mice. In this report, we describe the major histopathological changes, as well as the major changes in cytokine and chemokine expression, in brains from WNV infected and mock-inoculated C57Bl/6 mice. During the course of infection, skin, spleen and kidney were all sites of WNV replication before virus reached the brain. In brain, increased expression of the chemokines MCP-5 (CCL12), IP-10 (CXCL10), and MIG (CXCL9), preceded the expression of interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha), which have previously been considered to be key early cytokines in the pathogenesis and immune response of WNV encephalitis. The results presented herein provide a more complete assessment of the inflammatory process during WNV infection, suggesting that the chemokines MCP-5, IP-10 and MIG are important triggers of inflammation in brain due to their early up-regulation following WNV infection.

NATURAL DISEASE

Natural Disease – First Place

PATHOGENESIS AND CHARACTERIZATION OF SUBGROUP J AVIAN LEUKOSIS VIRUS-INDUCED HISTIOCYTIC SARCOMAS IN MEAT-TYPE CHICKENS. A.R. Pandiri1,2, I.M. Gimeno1, W.M. Reed2, S.D. Fitzgerald2, A.M. Fadly1. 1USDA ARS Avian Disease and Oncology Laboratory, East Lansing, MI; 2Michigan State University, Department of Pathobiology and Diagnostic Investigation, Lansing, MI.

Histiocytic sarcomas (HS) are variably described in mammals, avian, and other species. The etiology and pathogenesis of these lesions is not clear in many species. However, in chickens, a low incidence (1 - 5 %) of HS is observed in subgroup J avian leukosis virus (ALV J) infections. The effect of ALV J viral factors (strain and dose) and host factors (genetic strain and age at infection) on the incidence of HS was evaluated in 2 experiments involving 449 meat-type chickens and 100 ADOL-Line-0 chickens (white leghorn strain free of endogenous retroviral ev loci). Results indicated that chickens infected at hatch but not in ovo developed HS with no effect of viral strain and/or dose. In addition, these lesions were observed only in meat-type chickens but not in ADOL-Line-0 chickens. In conclusion, these lesions were observed only in meat-type chickens inoculated at hatch and were persistently viremic, irrespective of the antibody response. HS in chickens are primarily of splenic origin with frequent hepatic and renal metastases. Microscopically, the neoplastic cells were pleomorphic histiocytes with moderate to marked anisocytosis and anisokaryosis. Immunohistochemical staining on cryosections indicated that the neoplastic cells were of myeloid origin, and were positive for CD45 and MHC class II. In addition, there were minor infiltrating cell populations that were positive for B cell markers, CD3, CD4, CD8 and ALV J gp85 antigen. HS in chickens appear to share some morphological and immunohistological features of histiocytic splenic sarcomas in humans and canines. This study provides some novel information on retroviral-induced histiocytic neoplasms in chickens.

Natural Disease – Second Place

CORRELATION OF PITUITARY HISTOLOGIC FINDINGS WITH ADRENOCORTICOTROPHIC HORMONE RESPONSE TO DOMPERIDONE IN DIAGNOSIS OF EQUINE PITUITARY PARS INTERMEDIA DYSFUNCTION. I.D. Pardo, M.A. Miller, L.P. Jackson, G.E. Moore, and J.E. Sojka. Departments of Veterinary Pathobiology and Veterinary Clinical Sciences, Purdue University, West Lafayette, IN.

Equine pituitary pars intermedia dysfunction (PPID) is attributed to decreased dopaminergic inhibition of melanotrophs. Diagnosis is complicated in early PPID because clinical signs are not pathognomonic and pituitary lesions may resemble those in aged equids without clinical disease. Domperidone, a dopamine D2 receptor antagonist, causes melanotrophs to express pro-opiomelanocortin-derived peptides. We hypothesized that domperidone administration would not alter plasma adrenocorticotrophic hormone concentration ([ACTH]) in normal horses, but would exacerbate the loss of dopaminergic inhibition of melanotrophs in horses with PPID and result in increased [ACTH]. Fifteen horses, mean age, 21years, some with clinical signs of early PPID, received 3.3 mg/kg domperidone orally. Plasma [ACTH] was determined at 0, 4, and 8 hours. Post mortem pituitary histologic grade and pars intermedia (PI) area in midline sagittal sections correlated with [ACTH] 8 hours after domperidone as follows: Pituitary grade 1 (within normal limits), n=1, 0.23 cm2 PI area, 28 pg/ml [ACTH]; grade 2 (focal hypertrophy or hyperplasia), n=4, 0.36 cm2 mean PI area, 27 pg/ml mean [ACTH]; grade 3 (diffuse adenomatous hyperplasia), n=3, 0.45 cm2 mean PI area, 61 pg/ml mean [ACTH]; grade 4 (adenomatous hyperplasia plus microadenomas [1-5 mm diameter]), n=6, 0.91 cm2 mean PI area, 137 pg/ml mean [ACTH]; grade 5 (adenoma [>5 mm diameter]), n=1, 0.35 cm2 PI area, 67 pg/ml [ACTH]. (The adenoma was in the pars anterior.) We conclude that horses with pituitary histologic grade >2 have PPID and respond to domperidone with increased plasma ACTH concentration.

Natural Disease – Third Place

SUSCEPTIBILITY OF DENDRITIC CELLS TO FELINE IMMUNODEFICIENCY VIRUS INFECTION.W. Sprague1, M. Robbiani2, P. Avery1, K. O’Halloran1, and E. Hoover1. 1Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO; 2Center for Biomedical Research, Population Council, New York, NY.

Feline immunodeficiency virus (FIV), the feline counterpart to human immunodeficiency virus, interacts with dendritic cells (DC) during initial infections. We sought to elucidate the initial interaction of FIV with DC using feline monocyte-derived DC by evaluating the susceptibility of DC to FIV infection in vitro and assessing the potential transfer of infection from DC to CD4+ T cells. Using transmission electron microscopy, we detected FIV uptake into membrane bound vesicles in FIV-pulsed and washed DC within 2 hours of infection. While low concentrations of FIV DNA were found in DC after culture, significantly higher concentrations were found if resting CD4+ T cells were added to the DC cultures, which intensified several log-fold if CD4+ T blasts were added. FIV capsid antigen was detected and steadily increased in DC-CD4+ T blast co-cultures, but was undetectable in DC cultured alone or with resting CD4+ T cells. To determine whether transfer of infection from DC to CD4+ T cells could be initiated by productively infected DC vs bound but not internalized virus, DC were incubated for 2 days to allow for degradation of the virus. Infection of CD4+ T blasts was unobstructed by this procedure, thus T cell infection was likely due to de novo replication of FIV in DC. These results demonstrate that feline DC are susceptible to FIV infection and that there is both immediate and delayed transfer of virus to CD4+ T cells where virus is amplified, and demonstrate that interactions of FIV with DC-T cells are similar to those of HIV and SIV.

TOXICOLOGIC PATHOLOGY

Toxicologic Pathology – First Place

COMPARISON OF THE MAMMARY GLAND EFFECTS IN THE FEMALE RAT OF FOUR SELECTIVE ESTROGEN RECEPTOR MODULATORS AND DIHYDROTESTOSTERONE.J. Lucas1, P. Snyder1, S. Hooser1, A. Peter1, and D. Rudmann2. 1Department of Veterinary Pathology, Purdue University, West Lafayette, IN and 2Department of Toxicology, Eli Lilly and Company, Greenfield IN.

Selective estrogen receptor modulators (SERMs) are non-steroidal compounds that have tissue dependent estrogen receptor (ER) agonist or antagonist characteristics. In previous work, female rats treated with the SERM, LY2066948, had hyperandrogenemia and mammary gland virilism that was blocked by co-treatment with the androgen receptor antagonist, flutamide. The objective of the present study was to compare the mammary gland effects in female rats induced by a structurally diverse group of SERMs or the steroidal androgen, dihydrotestosterone (DHT). Female rats were treated for 30 days with one of four SERM compounds (LY504132, TSE424, Tamoxifen and 117018), all ER antagonists in the rat mammary gland, or DHT. Plasma testosterone and estradiol levels were evaluated along with morphologic alterations of the mammary gland. Three of the compounds (LY504132, TSE424, and 117018) induced hyperandrogenemia and produced ductal and alveolar alterations consistent with mammary gland virilism and similar to those previously reported for LY2066948. Tamoxifen treatment did not result in hyperandrogenemia or in mammary gland virilism. The mammary gland morphologic changes induced by DHT were similar to those induced by the SERMs that caused virilism; however, ductal and alveolar structures were also dilated with secretory material, the latter consistent with hyperprolactinemia, a known effect of steroidal androgen treatment in female rats. Taken together, these data demonstrate that the SERM-mediated virilism observed in female rats is likely independent of the mammary gland ER antagonism of SERMs. Instead SERM-mediated virilism is likely dependent on the ability of the SERM to stimulate the ovary and produce hyperandrogenemia.

Toxicologic Pathology – Second Place

CHRONIC SUBLETHAL MICROCYSTIN EXPOSURE RESULTS IN PROLIFERATION AND CHANGES IN MITOTIC GENE EXPRESSION IN HOMOZYGOUS p53 KNOCKOUT MICE.S. Clark1, S. Hooser2, T. Ryan3, and M. Davis3.1Biomedical Sciences and Pathobiology, Virginia Tech University, 2Department of Veterinary Pathobiology, Purdue University, 3Investigative Toxicology, Eli Lilly and Company, Greenfield, IN.

The homozygous p53 knockout mouse model was used to assess the mechanisms of hepatotoxicity and tumor promotion by the protein phosphatase inhibitor, microcystin-LR (MCLR). Microcystin-LR is a cyclic heptapeptide hepatotoxin produced primarily by the cyanobacteria (blue-green algae) Microcystis aeruginosa. In addition to its acute hepatotoxicity, MCLR is also a tumor promoter and chronic exposure may be associated with the increased risk of liver cancer in some areas of the world. Over 50% of all human cancers bear mutations in the p53 gene that inactivate its function. The p53 protein functions as a transcription factor that regulates downstream genes involved in cell cycle arrest, DNA repair and apoptosis. Mutations in the p53 tumor suppressor gene mutations have been shown to play a major role in hepatocarcinogenesis. Trp53 homozygous mice (inactivated p53) and age-matched wild type control mice were randomly assigned to various dosing groups. Forty micrograms of MCLR per kilogram of body weight or saline control were given daily by intraperitoneal injection for up to 28 days. At 4 hours, 24 hours, 4 days, 14 days and 28 days after the first MCLR dose, the mice were necropsied and liver tissue was collected for histopathology, immunohistochemistry and RNA analysis. Blood was also collected for plasma biochemistry evaluation. The RNA from the 28 day study was processed and hybridized onto Mouse Genome 430A 2.0 GeneChip arrays. The RNA from the other time points was processed for quantitative-PCR. In the 28-day treatment group, there were greater hyperplastic/dysplastic changes morphologically, and an increase in Ki-67 and phospho-Histone H3 (mitotic marker) immunoreactivity in the livers of Trp53 mutant mice. Gene expression analysis revealed differences in cell cycle regulation and cellular proliferation between the mutant and wild-type mice. We conclude that p53 plays a role in microcystin-induced toxicity and tumor promotion. These data suggest that the regulation of the cell cycle by p53 is important in preventing the proliferative response associated with chronic, sublethal microcystin exposure.

Toxicologic Pathology – Third Place

APOPTOSIS IN THE BRAIN OF MOUSE FETUSES BY ETOPOSIDE ADMINISTRATION.C. Nam, H. Yamauchi, H. Nakayama, and K. Doi. Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.

Etoposide (VP-16), a topoisomerase II inhibitor, is an anti-tumor agent which causes embryotoxicity, exencephaly, encephalocele, anophthalmia and major skeletal malformations of fetuses when administered to dams of rodents. Four mg/kg of VP-16 were injected into pregnant mice at day 12 of gestation (GD12), and fetuses were collected from 1 to 48 hours after treatment (HAT). A significant decrease of mitotic figures of neuroepithelial cells was observed in the telencephalic wall from 2 and 4 HAT. The number of pyknotic neuroepithelial cells began to increase at 4 HAT, peaked at 12 HAT, and decreased at 24 HAT. These pyknotic cells were also positively stained by TUNEL and active caspase-3 immunostaining. Immunohistochemically, the number of p53- and p21-protein-positive cells peaked at 4 HAT. Although expression of p53 mRNA was not increased, mRNA expression of p53-target genes (p21, fas, and puma) was significantly increased. Flow cytometric analysis revealed that VP-16 induced S-phase accumulation and G2 arrest at 4 and 8 HAT, and VP-16-induced apoptosis was significantly increased from 4 to 24 HAT. S-phase accumulation was caused by acceleration of the G1/S transition. BrdU-positive signals were observed in some apoptotic cells. The present results suggest that VP-16 might induce cell cycle arrest at G2/M phase and apoptosis in a p53-related manner.

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