Abstract
1: VETERINARY PRACTITIONER USE OF CYTOLOGY AND INTERACTION WITH CLINICAL PATHOLOGISTS.
Cytology is an important diagnostic tool in veterinary practice, yet little information exists regarding practitioner use of cytology and interactions with clinical pathologists. A web-based survey was provided to members of VIN during Sept–Dec 2004. Respondents (n = 871) were in general practice (86.4%), specialty practice (9.4%), and veterinary schools (4.2%). Veterinarians reported obtaining ∼7 samples/week (range 0–100), of which 50% were sent to diagnostic laboratories and 50% were evaluated in-house (with 25% of the latter then sent to a laboratory). Board-certified specialists submitted more samples than others. Cytology was used to guide additional testing (89%), to rule out specific diseases (81%), to obtain a definitive diagnosis (70%) and to stage metastasis (33%). When deciding whether to use cytology, >80% of veterinarians ranked accuracy and availability of cytopathology expertise as very important; <15% ranked cost and availability of ultrasound as very important. Over 75% of respondents felt it was important for the clinical pathologist to know lesion location; history, signalment, and clinical signs; sampling method; and previous cytology or biopsy results. Veterinarians reported consulting clinical pathologists —once/month (range 0–80). Reasons for consults included discrepancy between cytologic and clinical findings (79%), to clarify a diagnosis (69%), and to find out the confidence of the pathologist (62%) or what they really think (41%). Veterinarians were very confident of diagnoses made by board-certified clinical pathologists (95% of respondents), clinical pathology residents (26%), board-certified veterinarians in another discipline (37%), board-certified MD pathologists (6.7%), non-certified veterinarians (<1%), or laboratory technicians (<1%). Fifteen percent of veterinarians reported they did not know the qualifications of the person evaluating their cytology samples. (Reprinted with permission from Vet Clin Pathol 34[Suppl], 2005)
2: NORMAL AND ATYPICAL MITOSIS.
The number and type of mitotic figures found in cytological and histological samples often have a large impact on whether or not the process is considered benign or malignant. This presentation is to review normal mitosis and to describe various abnormal changes in mitosis such as tripolar and other than bipolar mitosis, lagging chromosomes and micronuclei. Morphologic changes are emphasized but significance in the determination of malignancy is also discussed. Studies with pathologists in human medicine have shown a large variation in how different pathologists classify photos of the same cell in terms of if the cell was in mitosis, what stage of mitosis was present and if it was a normal mitosis. The material is assembled from clinical cases and the literature because of curiosity of the author about a part of pathologists’ daily diagnostic activity. It should hopefully provide some insights to others, which should be fun and useful. (Reprinted with permission from Vet Clin Pathol,
3: CHARACTERIZATION OF THE PIGMENTED CYTOPLASMIC GRANULES COMMON IN CANINE HEPATOCYTES.
Microscopic examination of canine hepatic tissue often reveals aggregates of irregular cytoplasmic granules that appear brown on hematoxylin and eosin (H&E)-stained histologic sections and green on Romanowsky-stained cytologic preparations. Pathologists have variably classified this material as lipofuscin or as bile. We assessed these characteristic hepatocellular inclusions in liver samples from 10 dogs of several breeds and 1.5–13.5 (mean 5.5) years of age. A panel of 10 stains (H&E, Stein's, periodic acid Schiff, rhodamine, Giemsa or Wright-Giemsa, luxol blue, Prussian blue, Ziehl-Neelsen, Fontana Masson, and oil red O) was used to assess the material on paired cytologic and histologic samples. Corresponding tissue from 3 of the dogs was assessed by transmission electron microscopy (TEM). All unstained cytologic and histologic samples were assessed for autofluorescence by fluorescence microscopy with excitation at 330–380, 450–490, and 510–560 nm. Copper, bile, hemosiderin, melanin, and neuronal ceroid lipofuscin were similarly assessed by staining and fluorescence microscopy. The target material did not stain like bile and had no consistent positive staining with any of the stains. Like neuronal pigment from a dog with ceroid lipofuscinosis, but unlike bile, melanin, copper, and hemosiderin, it autofluoresced in all unstained histologic and cytologic samples at each excitation wavelength. By TEM, the material was consistent with lipofuscin, appearing as irregular aggregates of variably sized, grey to black granules bounded by single membranes, sometimes in association with lamellar bodies. Staining, fluorescence, and ultra-structural findings in this study provide strong evidence mat this routine pigment is a form of lipofuscin. It should not be regarded as bile or as evidence of cholestasis. (Reprinted with permission from Vet Clin Pathol,
4: FELINE NASAL LYMPHOMA: 46 CASES (1989–2004).
Nasal lymphoma is the most common nasal tumor in cats, yet there are no reports pertaining to the clinicopathologic and cytologic features of this neoplasm. We identified 46 cases of nasal lymphoma diagnosed at our institution by histopathology. Twelve of the seventeen cases in which nasal cytology was performed were diagnosed as lymphoma, while the remaining five cases were inflammatory (3), normal lymphoid tissue (1), or non-diagnostic (1). Cytologically, lymphoma cases displayed a monomorphic population of immature large lymphoblasts, with varying degrees of inflammation and epithelial dysplasia. Interestingly, vacuolated lymphoblasts predominated in 8/12 cytologic lymphoma diagnoses. Six submandibular lymph nodes were aspirated at presentation, none of which showed evidence of lymphoma involvement. Immunohistochemistry was performed on 41 specimens, resulting in 9 T-cell (CD3+, CD79a-), 27 B-cell (CD3-, CD79a+), and 5 mixed populations. Six cases were found to have multiorgan involvement on necropsy, two of which had involvement limited to the cerebellum and frontal cortex, respectively. The mean age of affected cats was 8.8 years and no sex or breed predilection was identified. Mean duration of clinical signs was 6.5 months (range 2 weeks to 60 months). Common clinical signs included nasal discharge (38/46). sneezing (29/46), decreased appetite (28/46), increased upper respiratory noise (27/46), ocular discharge (15/46), increased respiratory effort (14/ 46), facial deformity (14/46), stridor (13/46), wheezing (12/46), and coughing (11/46). Laboratory abnormalities included panhyperproteinemia (23/42), lymphopenia (20/44), hypocholesterolemia (11/42), hyperphosphatemia (14/42), increased aspartate aminotransferase (15/33), and hyperbilirubinemia (13/42). No animals were hypercalcemic and six animals were mildly anemic. Two cats were feline leukemia virus (FeLV) positive, one of which had multiorgan involvement. These findings summarize the diverse cytologic and clinicopathologic findings in cats with nasal lymphoma. (Reprinted with permission from Vet Clin Pathol,
5: EVALUATION OF THE ADVIA 120 CSF ASSAY FOR ANALYSIS OF CANINE CEREBROSPINAL FLUID.
Cerebrospinal fluid (CSF) from animals is an expensive sample to obtain and requires rapid processing with special technical expertise and equipment for appropriate analysis. Current methods incorporating manual cell counts in hemocytometer chambers, cytocentrifugation, and differential cell determinations are laborious and prone to operator variability. Furthermore, most samples are acquired for disease investigation from dogs, where sample volume can be limiting. Introduction of the ADVIA 120 hematology analyzer with specific software adaptations to the veterinary market has enabled reliable assessment of blood leukocytes from dogs and other species. Similarity of canine and human leukocyte morphology, and successful automated CSF analysis of human samples using this instrument, prompted an investigation of its utility for canine CSF. The ADVIA 120 “CSF Assay” uses direct cytometry to enumerate leukocytes and erythrocytes, and provides an automated leukocyte differential from 300 μX of sample. In this study samples from 25 clinically normal dogs and from 25 dogs with evidence of neurological disease were prospectively analyzed with the traditional manual and the new flow cytometric method. Correlation between methods in the leukocyte and erythrocyte concentrations, and the leukocyte differential counts, were assessed. Results indicate that there is good correlation between methods for leukocyte and erythrocyte concentration; however, the correlation for detection of different leukocyte populations was only moderate. Therefore, it was concluded that software algorithms currently employed in the ADVIA 120 CSF assay to identify leukocyte subpopulations might not be suitable for canine CSF samples. (Reprinted with permission from Vet Clin Pathol,
6: ASSESSMENT OF A COMMERCIAL GEL COLUMN TECHNIQUE FOR FELINE AB BLOOD TYPING AND CROSSMATCHING.
The AB blood group system contains three blood types: type A, type B, and type AB. Because of the presence of naturally-occurring alloantibodies, A-B blood group incompatibilities can cause life-threatening acute hemolytic reactions and neonatal isoerythrolysis. Other feline blood incompatibilities, such as those due to the newly identified common red cell antigen Mik, have also been recognized. Hence, blood typing and crossmatching tests are essential in assessing blood compatibility. In this study we evaluated a new gel column method (DiaMed, Cressier, Switzerland) for feline typing and crossmatching, and compared the results to a standard tube method. Both typing methods utilize serum from type B cats and the Triticum vulgaris lectin for anti-A and anti-B reagents, respectively. Comparison of AB blood typing results using both tube and gel column methods for 135 blood samples (in EDTA, citrate, or CPDA) yielded equivalent results for 112 type A, 17 type B, and 6 type AB cats. The gel test typing results were easy to read with unequivocal 3 + to 4+ erythrocyte agglutination reactions and could be photographically archived. The gel agglutination results for blood typing were not adversely affected by anemia, polycythemia, leukocytosis, hyperglobulinemia, hyperlipidemia, hyperbilirubinemia, or hemolysis. When comparing 88 crossmatch test results, AB and other blood incompatibilities were readily detected by both methods, although in few instances discordant results were seen in tube versus gel test (with and without bromelin preincubation). We conclude that the gel test is a useful laboratory method to identify feline blood incompatibilities as has been previously shown for dogs and humans. The standard tube method test relies on the subjective reading of test results, while the gel test appears readily standardized. (Reprinted with permission from Vet Clin Pathol,
7: QUALITATIVE AND QUANTITATIVE COMPARISONS OF CELLULOSE ACETATE AND AGAROSE GEL PROTEIN ELECTROPHORESIS OF CANINE, FELINE AND EQUINE SERA.
Serum protein electrophoresis is commonly used to characterize dysproteinemias in domestic species. The objective of this study was to qualitatively and quantitatively compare results and procedures of serum protein electrophoresis (SPE) using Titan III cellulose acetate membranes (CA) stained with Ponceau S and Titan Gel system (TG) (amido black stain). Canine (78), feline (44) and equine (19) samples were selected from frozen sera previously collected from patients with various disorders during the previous three months. After thawing, total protein concentrations were determined by biuret reaction (Hitachi 911). The serum protein fractions were separated by CA and TG electrophoreses on the same day and scanned with the Quick Scan 2000 with Windows®. Evaluating densitometer scans of each method allowed identification of dysproteinemias such as chronic inflammatory hyperproteinemia, monoclonal gammopathy, and selective and nonselective hypoproteinemias. Major visible differences in the electrophoretic fractions included more α1-globulin staining in TG compared to CA and indistinct separation of β-globulins in TG. For between assay precision (n = 20), the coefficients of variation (CV) for canine albumin concentration was 4% for both assays (mean = 3.7 g/dl for both) and the CVs for globulin fractions ranged from 11–31% and 11–18% for CA and TG, respectively. For canine sera, when TG was compared to CA using Deming regression, there was a positive constant bias for α2-globulins, a positive proportional bias for α1-globulins, a negative proportional bias for γ-globulins, and constant and proportional biases for albumin and β2-globulins. For feline sera, there was a positive proportional bias for γ-globulins and constant and proportional biases for albumin and α2-globulins. Biases were not detected in the equine sera. (Reprinted with permission from Vet Clin Pathol,
8: COMPARISON OF PROTEIN CONCENTRATIONS IN PRECOLOSTRAL AND POSTCOLOSTRAL BOVINE SERA USING SPECTROPHOTOMETRIC, REFRACTOMETRIC, ELECTROPHORETIC, AND RADIOIMMUNODIFFUSION METHODS.
The objective of this study was to compare protein concentrations in precolostral and postcolostral bovine sera using spectrophotometric, refractometric, electrophoretic, and radioimmunodiffusion methods. Serum samples were collected from 30 newborn Holstein calves prior to and 24 hours after ingestion of colostrum (1–2 feedings, 2 qt. for each feeding). Also sera were obtained from 8 healthy 2–3 months old male castrated calves and were pooled to create control serum. Calf samples and control serum aliquots were analyzed in duplicate. Total protein concentrations were determined using two different methods: biuret reaction (Hitachi 911) and refractive index using two refractometers (Leica VET 360 and Leica TS 400). Total globulin concentrations were calculated by subtracting albumin concentrations (bromcresol green) from biuret total protein concentrations. Serum protein fractions were separated by electrophoresis using Titan III cellulose acetate membranes (CA) and Titan Gel system (TG), stained with Ponceau S and amido black stains (respectively), and scanned using Quick Scan 2000 Windows®. Total immunoglobulin G (IgG) concentrations were determined by radioimmunodiffusion (RID, Immunocheck Bovine IgG RID kit, 0.4–3.2 g/dL). By all comparison methods, measured IgG concentrations had an unacceptable positive proportional bias (Deming regression). The differences (postcolostral minus precolostral) in total protein concentrations and differences in the total globulin concentrations compared to differences in γ-globulin concentrations (both CA and TG) had positive proportional biases (Deming regression). Differences in γ-globulin concentrations determined by CA and TG methods had good agreement (average bias <0.1 g/dL). These results indicate that γ-globulin concentration determined by electrophoresis was the best of the evaluated methods for assessing increased IgG concentration after ingestion of colostrum. (Reprinted with permission from Vet Clin Pathol,
9: HAPTOGLOBIN AND SERUM AMYLOID A AS MARKERS OF HEALTH AND WELFARE IN PRODUCTION ANIMALS.
Haptoglobin (Hp) and serum amyloid A (SAA) are acute phase proteins (APP) which increase in plasma following infection, inflammation or trauma. Monitoring their concentration gives valuable diagnostic information in assessing the health and welfare of animals. In dairy cows, both Hp and a mammary form of SAA are secreted in milk from animals with mastitis. Both APP are present in milk as a result of local production by mammary tissues. Monitoring APP in milk has been proposed as an alternative to measurement of somatic cell counts in diagnosis of this economically important disease. The concentration of several APP in the plasma of pigs during production has been investigated. A combination of results from APP that increase in the circulation (positive APP) with proteins that decrease in the circulation such as transthyretin (negative APP) can be used in an index to maximize the diagnostic value of the analyses. Increased APP are related to subclinical infection and hygiene conditions on farm, to pathology of disease in individual animal and to stress inducing conditions. In experimental models of disease the APP can be used to provide quantitative data on host responses. The APP levels in serum and milk in an experimental model of Streptococcus aureus induced mastitis allowed the progress of infection to be monitored. Similarly the use of these analytes to monitor responses to infection in models of Pasturella multocida has provided quantitative data on the infection and on the protection provided by vaccine candidates. Investigation of the APP in animals is still in its infancy and further valuable applications are likely to be discovered in the future. (Reprinted with permission from Vet Clin Pathol,
10: CHARACTERIZATION OF MAJOR SURFACE PROTEIN 5 OF ANAPLASMA PHAGOCYTOPHILUM AND ITS SIGNIFICANCE FOR DIAGNOSTIC USE.
The major surface protein 5 (MSP5) of A. marginale has long been used as diagnostic test antigen for serologic identification of infected cattle. Previously, the msp5 gene of A. phagocytophilum was identified, cloned and recombinant MSP5 (rMSP5) expressed in Escherichia coli. Recombinant MSP5 of A. phagocytophilum was used in an indirect ELISA to detect antibodies in serum samples from naturally infected humans and dogs. In this study, the msp5 gene of A. phagocytophilum was amplified, cloned and sequenced from various geographical isolates in several species of mammals. In addition, cross-reactivity between MSP5 of A. marginale and A. phagocytophilum was evaluated using rMSP5 in an indirect ELISA. There was 100% sequence identity in the msp5 genes in isolates from humans from New York State, Dusky footed wood rats from California, and an isolate from a horse from Sweden. The sequence analysis differed in 3 base pairs in isolates from sheep from Norway. However, there was a 100% sequence identity among the 3 sheep. There is 64% gene sequence identity between msp5 of A. marginale and A. phagocytophilum. Using rMSP5 of A. marginale and A. phagocytophilum in an indirect ELISA, extensive serological cross-reactivity was identified. These results indicate that rMSP5 of A. phagocytophilum is highly conserved and a potential diagnostic antigen for use in identifying infection in various species. Based on the cross-reactivity between rMSP5 of A. phagocytophilum and A. marginale, serological distinction between infections with these two organisms is not possible when rMSP5 is used in an indirect ELISA format. (Reprinted with permission from Vet Clin Pathol,
11: RETICULOCYTE CHANGES AFTER EXPERIMENTAL ANEMIA AND ERYTHROPOIETIN TREATMENT OF HORSES.
Availability of recombinant human erythropoietin (EPO) has facilitated use to enhance red blood cell production, and therefore aerobic performance, in human and equine athletes. Recombinant human EPO promotes growth and differentiation of equine erythroid precursor cells, but in some horses repeat administration induces immune interference with endogenous EPO resulting in fatal anemia. While blood reticulocyte parameters acquire unique changes in humans treated with EPO, with manual enumeration methods, horses were not considered to release reticulocytes from the bone marrow into circulation, even under severe erythropoietic stress. The goals of this study were to determine whether reticulocytes could be detected and characterized in horses that are anemic or have been treated with EPO using a modern hematology analyzer. Anemia was induced in 6 horses by removal of 30 mL of blood/kg of body weight over 24 hours. After 28 days, the horses were treated twice with 55 U/kg of EPO (Eprex®), and after 65 days they were treated thrice with 73 U/kg of EPO. Blood samples were analyzed with the ADVIA 120 instrument every 3 to 5 days, and bone marrow samples 7 days after anemia and EPO treatments. Analysis of blood reticulocyte parameters by ANOVA in a randomized complete block design determined that anemia and EPO induced significant (p < 0.05) increases in red cell distribution width and reticulocyte mean cell volume. Parameters changed only after EPO treatment were cellular hemoglobin concentration mean, mean cell volume, reticulocyte concentration, proportion of macrocytic reticulocytes, and reticulocyte cellular hemoglobin. These findings indicate that horses under erythropoietic stress and after EPO treatment release reticulocytes with unique characteristics into circulation. (Reprinted with permission from Vet Clin Pathol,
12: USE OF A COLLAGEN BINDING ASSAY AND PLATELET FUNCTION ANALYZER IN THE CHARACTERIZATION OF DOGS WITH VON WILLEBRAND DISEASE.
No single test can clearly diagnose or differentiate the three types of von Willebrand Disease (vWD) or predict risk of hemorrhage. The vWF:Ag assay, our current gold standard, results in some indeterminate diagnoses and is ineffective for diagnosis of type II disease, a functional rather than a quantitative deficiency of vWF. Therefore, the purpose of this study was to develop a diagnostic profile that will better evaluate the varied presentations of vWD and to investigate the relationships between profile components and the risk of hemorrhage. Citrated plasma samples were collected from 39 vWD positive, 21 indeterminate and 22 negative dogs. Concentration of vWF was determined using a vWF:Ag assay, and function was evaluated with a collagen binding assay (CBA) and platelet function analyzer (PFA-100). Secondary hemostasis was evaluated with partial thromboplastin times and FVIII activity. Preliminary results support a positive association between the vWF:Ag concentration and the CBA functional analysis. Canine CBA may have use as a screening tool, and when used in conjunction with the vWF: Ag assay, may be able to distinguish Type I and II vWD. Furthermore, the PFA-100 appears to detect more dramatic presentations of vWD (vWF:Ag concentrations less than 26 percent), where the level of factor present is inadequate for normal primary hemostasis. Therefore, our hypothesis that the vWD diagnostic profile will provide more information regarding the various presentations of vWD is supported. It is unclear if associations between the profile results and disease status can be extrapolated for the definitive diagnosis of patients with indeterminate results using the vWF:Ag assay. (Reprinted with permission from Vet Clin Pathol,
13: PERFORMANCE OF TWO CANINE COOMBS’ TESTS AT A VETERINARY TEACHING HOSPITAL.
The Coombs’ test has long been the gold standard for laboratory confirmation of immune-mediated hemolytic anemia (IMHA). However, this test suffers from low sensitivity, leading to discrepancies between clinical diagnoses and laboratory results. The clinical pathology laboratory at the University of Minnesota Veterinary Medical Center utilizes a modified Coombs’ test based on internal research findings suggesting improved performance (poly/monovalent Coombs’). Reagents for this test include both polyvalent and monovalent (IgG, IgM, and C3) antisera, as opposed to the more commonly used VMRD Coombs’ test (VMRD Inc., Pullman, WA) which uses only polyvalent antiserum. The objective of this study was to compare the performance of the poly/monovalent Coombs’ test to the VMRD Coombs’ test. A subset of Coombs’ submissions received by the clinical pathology laboratory from 2/03 to 4/05 was run using both tests in parallel. Fifty-two cases were included, 26 of which met the inclusion criteria for IMHA (e.g., spherocytosis, agglutination, bilirubinemia, etc.), and 26 that were determined not to have IMHA. Of the 26 IMHA cases, 21/26 had positive results by the poly/monovalent Coombs’ test, while only 15/26 were positive by the VMRD Coombs’ test. Five of the 26 IMHA cases were Coombs’ negative by both tests. Sensitivity and specificity of the poly/monovalent Coombs’ test were 81% and 100%, respectively, compared to 58% and 100% for the VMRD Coombs’ test. We conclude that for samples submitted to our clinical pathology laboratory, the poly/monovalent Coombs’ test is a more sensitive test for laboratory confirmation of IMHA than the VMRD Coombs’ test. (Reprinted with permission from Vet Clin Pathol,
14: DEVELOPMENT OF MOUSE MODELS OF HUMAN T-LYMPHOTROPIC VIRUS TYPE-1 ASSOCIATED ADULT T-CELL LEUKEMIA/LYMPHOMA (ATL).
Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus related to bovine leukemia virus that causes adult T-cell leukemia/ lymphoma. About 5% of patients infected with HTLV-1 develop adult T cell leukemia/lymphoma (ATL), which is resistant to therapy and is invariably fatal. To develop a mouse model for ATL, we tested C8166 and MT-2 cell lines (in vitro HTLV-I transformed) and MET-1 cells (derived from a patient). We inoculated 107 cells i.p. into three different immunodeficient strains of mice: NOD/SCID, NOD/SCID 2m(-/-), and NOD/SCID IL2-R(-/-). All strains have reduced macrophage and no detectable B-cell and T-cell activity. While NK cell activity is reduced in NOD/SCID mice, it is absent in NOD/SCID 2m(-/-), and NOD/SCID IL2-R(-/-). NOD/SCID IL2-R(-/-) also have a defective INF-γ production. Both C8166 and MET-1 cells engrafted in all mouse strains with tumor cells present in blood and infiltrating several organs. The time course of engraftment (less than 4 weeks for C8166, 6–8 weeks for MET-1) Was variable perhaps due to difference in the expression of the trans-activator protein Tax. C8166 cells also engrafted with different kinetics in NOD/SCID (3–4 weeks), NOD/SCID 2m(-/-) (2-3 weeks) and NOD/SCID IL2-R(-/-) (1–2 weeks) indicating different levels of immune deficiency between the three mouse strains. MT-2 cells engrafted well in NOD/SCID IL2-R(-/-) but produced only few and small tumors in the other strains. As MT-2 is the only cell line expressing HTLV-I proteins, the differences seen are likely due to expression of viral proteins (other than Tax). Our data indicate that different combinations of cell line/mouse strain can be established as appropriate mouse models for adult T-cell leukemia. (Reprinted with permission from Vet Clin Pathol,
15: CIRCULATING NUCLEIC ACIDS AS A BIOMARKER FOR DOGS WITH CANCER.
In 1975, it was discovered that humans with cancer have higher levels of circulating cell-free DNA than healthy patients. DNA was increased in advanced stages and decreased after therapy. Molecular analysis revealed that the circulating DNA harbors genetic changes identical to those in the primary neoplasm. A recent study in human lung cancer found that increased plasma DNA significantly correlated with poor survival. Literature revealed no investigations of this phenomenon in veterinary patients. We hypothesize that dogs with cancer have higher levels of circulating DNA than healthy dogs. Study objectives include 1) optimizing protocols for DNA detection; 2) establishing reference ranges in normal dogs; and 3) determining if circulating DNA levels are significantly higher in dogs with cancer. Initially, both plasma and serum were tested. Values were often different between the two and standard deviation was high. Our protocol was optimized by adding a second centrifugation to remove residual cells. This resulted in lower DNA levels and higher concordance between plasma and serum. Plasma was used exclusively afterwards. Plasma DNA has been quantified from 25 healthy dogs and 31 dogs with cancers using the improved protocol. DNA was extracted using QIAamp DNA mini kit and quantified using PicoGreen dsDNA fluorescence. Normal values range from 1 to 18 ng/ml (median 4 ng/ml). Dogs with carcinomas and sarcomas had results similar to healthy dogs. However, 5 of 8 dogs with lymphoid neoplasia had higher levels (range 1 to 1019 ng/ml, median 73 ng/ ml, p = 0.0126). Lymphoid neoplasia is common in dogs, and this finding may be useful for prognostication, detection of residual disease, or earlier detection of cancer. Additional samples are being collected to further analyze the utility of circulating DNA as a bio-marker in the canine population. (Reprinted with permission from Vet Clin Pathol,
16: UTILIZATION OF CARDIAC TROPONIN I AND T IMMUNOASSAYS IN DOGS.
Measurement of cardiac troponin (cTn) in veterinary practice and safety assessment studies is becoming common. The purpose of this study was to identify commercial cTn immunoassays suitable for evaluation of troponin in dog sera and to determine the kinetics of the cTn response after isoproterenol-induced cardiac injury. Negative, low, medium and high cTn positive Beagle dog sera were analyzed by the following assays: cTnI Abbott Architect, Bayer Centaur, Beckman Access, DPC Immulite, Dade Behring Dimension, Ortho-Clinical Diagnostics Vitros ECi, Tosoh AIA 600II and Life Diagnostics (ELISA); cTnT Roche Elecsys 2010. All assays, except the Life Diagnostic assay, were optimized for detection of human cTn. Cardiac TnI concentrations were detected for all assays, although the dynamic range of the cTnT assay was much narrower (<0.01 to 0.21 ng/ml). Nine Beagle dogs were treated with 1 mg/ kg isoproterenol (SQ). Serum samples collected at 0, 3, 6, 9, 12, 24, 48, and 96 hrs post dose were evaluated on the DPC Immulite. Troponin concentrations increased at 3 hrs, peaked at 9 to 12 hrs (80.9–91.3 ng/ml) and decreased but were still above baseline levels at 96 hrs. CKMB and LDH1&2 followed a similar response pattern but typically returned to baseline by 48 hours. Cardiac troponin can be successfully measured in dogs with cardiac injury, but the timing of sampling and understanding the dynamic range of the assay utilized are important considerations for interpretation. (Reprinted with permission from Vet Clin Pathol,
17: AN EXPLORATORY STUDY OF MYCOPLASMA SUIS (EPERYTHROZOON SUIS) ON FOUR COMMERCIAL SWINE FARMS IN SOUTHERN BRAZIL.
Detection of Mycoplasma suis (Eperythrozoon suis) in pigs using polymerase chain reaction (PCR) and Southern blots was performed on four farms in southern Brazil. A total of 186 pigs, including 121 sows, 61 piglets and 4 boars were sampled. Following blood collection, packed cell volume (PCV) and total plasma protein (TPP) were determined. DNA was extracted from blood and a 16S ribosomal RNA (16S rRNA) gene fragment of M. suis was amplified by PCR. Southern blot analysis was performed on all samples following amplification. Twenty-two sow samples (18.2%) were positive by PCR, whereas 40 samples (33.1%) were positive using Southern blot. Only one piglet and one boar were positive. PCV, TPP, and M. suis PCR and Southern blot results were not statistically different on these four farms (p > 0.05). However, M. suis results by Southern blot for all pigs were significantly higher than PCR (p < 0.05). PCV and TPP were not statistically different for M. suis positive and negative sows. To the authors’ knowledge, this is the first report in Brazil for detection of M. suis infection in pigs using molecular techniques. Further studies are needed to better establish the prevalence and impact of M. suis on commercial swine production in Brazil. (Reprinted with permission from Vet Clin Pathol,
18: MYCOPLASMA HAEMOFELIS AND MYCOPLASMA HAEMOMINUTUM COINFECTION IN A DOMESTIC CAT (FEUS CATUS) IN BRAZIL.
Mycoplasma haemofelis and Mycoplasma haemominutum coinfection was detected in a suspected cat (Felis catus) in Curitiba, Brazil using Polymerase Chain Reaction (PCR). A female, mixed breed, adult domestic cat was presented with lethargy, anorexia, depression, pallor, prostration and rectal temperature of 40°C. A blood sample was collected for a Complete Blood Cell Count (CBC), showing a regenerative anemia and a leukogram within the reference range. Microscopic observation of a stained blood smear suggested Mycoplasma spp. attached to erythrocytes. DNA was extracted and specific PCR reactions for M. haemofelis and M. haemominutum were performed. PCR products were then run in agarose gel and results were positive for both parasites. The cat was treated with doxycycline (2.5 mg/kg, PO, q12h, 21 days) and sent home. To the authors’ knowledge, this is the first report in Brazil of Mycoplasma coinfection in cats, detected by PCR technique. The anemia and microscopic observation of Mycoplasma were important as screening tests but PCR was the only definitive technique for the specific diagnosis in our case. Since M. haemominutum and M. haemofelis may cause different clinical signs, the co-infection revealed that domestic cats in Brazil may be exposed to both bacteria, demonstrating the importance of specific Mycoplasma detection. (Reprinted with permission from Vet Clin Pathol,
19: BIRTH ABNORMALITIES IN PREGNANT SOWS INFECTED INTRANASALLY WITH PORCINE CIRCOVIRUS 2.
Six pregnant sows were inoculated intranasally at 3 weeks before the expected farrowing date with porcine circovirus 2 (PCV2). The PCV2-inoculated sows showed abortion and premature farrowing, whereas two uninfected negative control sows remained clinically healthy and farrowed normally. PCV2 antigen and DNA were detected by immunohistochemistry and insitu hybridization, respectively, in lymph node, spleen, thymus, lung, tonsil and liver from both stillborn and live born piglets. Simultaneous detection of viral protein and DNA provided molecular evidence of PCV2 infection and replication. The experiment suggested that PCV2 is capable of crossing the placenta, replicating primarily in lymphoid tissues, and inducing reproductive failure. (Reprinted with permission from Vet Clin Pathol,
20: PSEUDOHYPERKALEMIA AND POIKILOCYTOSIS IN A CHINESE SHAR-PEI DOG.
Blood samples (whole blood and EDTA) from an 8 yr F/S Chinese Shar-Pei were submitted to the Atlantic Veterinary College's Diagnostic Laboratory from an outside clinic prior to entropion surgery. Abnormalities included: mild hyperkalemia (6.3 mmol/L; reference interval (RI) 3.6–6.0 mmol/L), mild normocytic (MCV 74 fL; RI 60–77 fL), hypochromic (MCHC 290 g/L; RI 320–360 g/L), non-regenerative anemia (Hct 0.31 L/L; RI 0.37–0.55 L/L, Hb 117 g/L; RI 120–180 g/L) with an increased RDW (26.2%; RI 11.0–14.0%) and poikilocytosis (large cells with increased central pallor resulting in marked drying artifact) affecting a subpopulation of erythrocytes. Follow-up blood samples submitted one week later showed: potassium 10.9 mmol/L (serum) and 8.5 mmol/L (heparinized plasma), Hct 0.43 L/L, Hb 121 g/L, MCHC 280 g/L, and RDW 26.5%. Poikilocytosis persisted. Intra-erythrocyte potassium concentration was measured by ISE (Hitachi 917) after lysing 10 microlitres of saline-washed erythrocytes in 200 microlitres of distilled water in a control dog (0.32 mmol/L) and the patient (0.80 mmol/L). Osmotic fragility was increased. Review of chemistry and hematology submissions (in-house) for this animal over an 8 yr period revealed: serum potassium 3.9–8.5 mmol/L, phosphorus 0.59–2.44 mmol/L (RI 0.82–1.87 mmol/L), Hct 0.34–0.41 L/L, MCHC 283–312 g/L, and RDW 18.2–26.7%. Poikilocytosis was only evident on smears in the previous 2 years. No clinical signs suggestive of hyperkalemia were recorded. High potassium erythrocytes have been reported in Akitas, Shibas, Jindos, other east Asian dog breeds, and in mongrels. Neither littermates nor the parents of the patient were available for evaluation, however, pseudohyperkalemia has been infrequently observed in other Shar-Pei dogs (personal communication). The etiology of the poikilocytosis is undetermined. Spurious hyperkalemia may be an in vitro artifact in some Chinese Shar-Pei dogs. (Reprinted with permission from Vet Clin Pathol,
21: CLINICAL COMPARATIVE STUDY OF IN-CLINIC AND LABORATORY HEMATOLOGY ANALYZERS IN DOGS AND CATS.
Introduced 50 years ago, automated hematology analyzers have evolved greatly. Practicality limited these instruments to reference laboratories; now smaller hematology analyzers have been developed for inclinic use. In this 3-month field study, we compared 7 in-clinic and 2 laboratory hematology analyzers with blood samples from both healthy and diseased dogs (n = 265) and cats (n = 111) from 3 clinics in Florida. The method comparison was limited to the capabilities of the various analyzers. A CBC with a 5-part WBC differential, platelet and reticulocyte count was obtained by the laser-based Advia 120, LaserCyte, and PorCyte. Furthermore, CBC with a 5-part (MS4-5, CellDyn 3500), 3-part (CBC-Diff, VetScan HMT, & ABC), and 2-part (VetAutoRead) WBC differential and platelet count were analyzed and also compared to a manual WBC differential. Compared to the Advia, excellent correlations for PCV/Hct, RBC and WBC counts, and hemoglobin concentrations (r = 0.92) were observed with all analyzers. The correlation for MCV ranged from 0.77 to 0.94 depending on the in-clinic system. The correlation for the platelet count between the Advia and CellDyn was good (r = 0.84) in dogs and fair (r = 0.76) in cats, while the correlations for the platelet count between the Advia 120 and each of the in-clinic systems were fair to good (r = 0.68 to 0.92). Furthermore, the correlations of reticulocyte counts between the Advia and LaserCyte were good (r > 0.79). The differential WBC correlation analyses varied widely between the systems and depended on the comparison used. The number of technical problems with each system was small for all instruments used, while the number of sample reruns requested and flags observed varied by analyzer type. In conclusion, various in-clinic hematology analyzers compare satisfactorily to the current laboratory methods. Although laser-based analyzers can offer more valuable information to assess patients, a microscopic examination of blood is still required. (Reprinted with permission from Vet Clin Pathol,
22: DNA SCREENING FOR THE GLY96GLU MUTATION CAUSING FACTOR VII DEFICIENCY IN COMPANION AND RESEARCH COLONY BEAGLES.
Coagulation factor VII (FVII) deficiency was originally reported in the 60's in Beagles and is inherited as an autosomal recessive trait. Although FVII coagulant activity can be measured, screening for FVII deficiency had been based on finding a prolonged prothrombin time (PT). However, the PT results are not dependable, and FVII coagulant activity measurements do not permit definitive identification of carriers. Recently, a unique G to A missense mutation resulting in the substitution of glycine 96 to glutamic acid in exon 5 of canine FVII was discovered from a deficient Beagle. We describe here the establishment of a DNA-based test to screen Beagles for this disease-causing mutation. Besides applying sequencing of exon 5 from genomic DNA as a test, we developed a Mn11 restriction digestion-based screening test, as the missense mutation abolishes one Mn11 restriction site. We examined FVII-deficient (1–4%) Beagles from two research colonies, show and pet Beagles, as Well as a mixed-breed Beagle, likely originating from another research colony, and showed that all are homozygous for the mutant allele. Clinically post-operative hemorrhage, hemothorax, and/or hematochezia were noted in some of the companion animals, while the research colony Beagles were apparently asymptomatic. Related Beagles were affected, carriers, or normal. The availability of a DNA-based screening test (http://www.vet.upenn.edu/penngen) will readily allow the identification of Beagles which are homozygous or heterozygous for this FVII mutation; the mutant allele appears to be wide-spread in the show as well as research Beagle population. (Reprinted with permission from Vet Clin Pathol,
23: DEFECTIVE PLATELET AGGREGATION IN AN OLDENBOURG FILLY.
This case report presents the investigation of an 18-month-old Oldenbourg filly presented with bleeding diathesis. Laboratory testing included platelet count, buccal mucosal bleeding time (BMBT), PT, aPTT, vWf, clottable fibrinogen, clot retraction, PFA-100 closure time and thromboelastography® (TEG®). Platelet aggregometry was used to assess platelet function. Expression of the platelet receptor for fibrinogen was assessed by flow cytometry using anti-human CD41 (IIb) antibody. Platelet count was within normal range as well as PT, aPTT, vWf and fibrinogen. Abnormal laboratory findings included prolonged BMBT (>12 h), prolonged closure time with the collagen/ADP cartridge (>300 sec, control: 69 sec) and absence of clot retraction after 60 minutes. TEG® analyses were performed on the blood of the patient (P) and a control horse (C) with kaolin and tissue factor as coagulation activators. Reaction times were similar with kaolin (P: 5.3 min, C: 5.6 min) and tissue factor (P: 12.8 min, C: 12.4 min). However, maximum amplitudes were decreased with both activators (kaolin P: 43.7 mm, C: 63.9 mm; tissue factor P: 37.7 mm, C: 57.8 mm). Platelet aggregation studies on platelet rich plasma (PRP) revealed markedly decreased platelet aggregation using 6 μM of ADP (P: 0%, C: 50%), 60 μM of ADP (P: 0%, C: 60%), 2 μg/ml of collagen (P: 3%, C: 59%) and 10 μg/mL of collagen (P: 14%, C: 71%). Flow cytometry studies showed positive CD41 reaction with platelets from the control but not with those from the patient. These results indicate a platelet aggregation defect. Absence of CD41 reacting antigen on platelets supports a diagnosis of Glanzmann's thrombasthenia. (Reprinted with permission from Vet Clin Pathol,
24: EVALUATION OF D-DIMER, THROMBIN-ANTITHROMBIN COMPLEX AND ANTITHROMBIN IN THE PLASMA OF HEALTHY CATS AND CATS WITH HYPERTHROPHIC CARDIOMYOPATHY.
Cats with cardiac disease are at risk for thromboembolism because of activated coagulation. In a clinical setting, molecular markers would be useful in identifying animals with activated coagulation. The purpose of this study was to measure markers of activated coagulation in cats with hypertrophic cardiomyopathy (HCM). Levels of thrombin-antithrombin complex (TAT), D-dimer and antithrombin activity (AT) were evaluated in the plasma of healthy cats and compared to results obtained in cats with HCM. It was hypothesized that these markers could be used to demonstrate that coagulation is activated in cats with HCM. Associations between left atrial diameter (LA) and left atrial to proximal aortic diameter ratio (LA:Ao) with TAT, D-dimer and AT were also evaluated in cats with HCM. Blood samples were obtained from 19 healthy cats and 20 cats with HCM. The reference ranges for D-dimer and TAT in plasma of healthy cats were established between 0.09–0.26 μg/mL and 2.0–20.0 μ/L respectively. D-dimer and TAT were not significantly different between the two groups. However, AT was significantly lower in cats with HCM (p = 0.03). Among cats with HCM, none of the coagulation markers studied correlated with LA or LA:Ao. The results of this study indicate that decreased AT activity in cats with HCM is compatible with coagulation activation. However, it was not possible to use D-dimer and TAT plasma levels to demonstrate a hypercoagulable state in cats with cardiomyopathy. More studies are necessary to evaluate the usefulness of these markers as indicators of activated coagulation in cats. (Reprinted with permission from Vet Clin Pathol,
25: EFFECT OF HYPOPROTEINEMIA ON COAGULATION STATUS OF DOGS WITH HEPATIC DISEASE.
Dogs with liver diseases are at risk for developing coagulopathies because of increased loss, decreased production or altered clearance of proteins involved in hemostasis. The clinical consequences depend on the hemostatic pathway that is most affected. Liver failure causing lack of coagulation protein production may affect pro-coagulant, anti-coagulant and fibrinolytic processes; therefore, the clinical outcome is unpredictable. The purpose of the study was to determine if dogs with hepatic disease and concomitant decreased albumin concentration were more likely to have hemostatic abnormalities than dogs with normal albumin concentration. Citrated plasma samples were collected from nineteen dogs with hepatic disease and normal albumin concentrations (mean 35 g/L, reference interval 29–43 g/L, Group 1) and from eighteen dogs with decreased albumin concentration (mean 24 g/L, Group 2). Disease diagnosis was based on biopsy or post-mortem examination. Diagnoses in Group 1 included neoplasia, steroid hepatopathy, leptospirosis, portosystemic shunt, hepatocellular necrosis, hepatic fibrosis, and chronic active hepatitis. Diagnoses in Group 2 included cholangiohepatitis, cirrhosis, neoplasia, hepatopathy, portosystemic shunt, hepatocellular necrosis, and hepatocutaneous syndrome. Coagulation parameters evaluated included prothrombin time (PT), partial thromboplastin time (PTT), antithrombin, and factors II, V, VIII, IX and X. PTT was significantly (p < 0.05) longer in dogs with hypoalbuminemia and there was significantly (p < 0.05) lower activity of factors II, V, VIII and X than in dogs with normoalbuminemia. PT times and antithrombin and factor IX activity were not significantly different between the two groups. It appears that dogs with hepatic disease and concurrent decreased albumin concentration are more likely to have biochemical hemostatic abnormalities that may predispose to increased risk of hemorrhage. (Reprinted with permission from Vet Clin Pathol,
26: VON WILLEBRAND FACTOR AS A BIOMARKER OF ENDOTHELIAL CELL PERTURBATION IN RATS AND DOGS.
Due to the lack of preclinical or clinical non-invasive biomarkers, the drug development process is complicated when there are pathological findings of drug-induced vascular endothelial injury in dog or rat preclinical toxicology studies. In humans, elevation of plasma von Willebrand Factor (vWF) is considered a marker of endothelial cell (EC) perturbation/injury but this marker has not been thoroughly evaluated in other species. Using different experimental protocols, plasma vWF was measured in rats and dogs to determine if it qualified as a biomarker of EC perturbation. Multiple, repeated venipunctures increased plasma vWF levels in control dogs less than 5% at any time point. The same protocol caused a greater than 30% (p < 0.05) increase in rat plasma vWF. Investigation of species differences determined abundant vWF in rat platelets and low values in dog platelets. We hypothesized that repeated venipuncture in the rat with subsequent EC damage caused platelet activation and aggregation releasing vWF in plasma. To avoid this effect, the Culex automated blood sampling system, which allows for multiple collections through a vascular access port, was evaluated. vWF levels did not increase above baseline in rats when blood samples were collected multiple times using the Culex system. These data suggest that the method of blood collection can affect plasma vWF values in the rat but not the dog. In experimental protocols designed to cause physiological stimulation of the dog vascular EC, plasma vWF levels increased 54% above baseline whereas pathological stimulation resulted in a 128% increase. These protocols have not been conducted in the rat. Therefore, vWF qualifies as a biomarker of EC perturbation in the dog but requires further investigation in the rat. (Reprinted with permission from Vet Clin Pathol,
27: SERUM CHEMISTRY CHANGES INDUCED BY ECONOMICALLY IMPORTANT DISEASES IN COMMERCIAL POULTRY.
Serum chemistries are used to assess health in many mammalian and a few avian species. Little is known about changes in serum chemistries in association with health or disease in poultry. Changes in serum chemistry values observed in commercial broiler and specific pathogen free (SPF) chickens after challenge with virulent Mareks Disease Virus (MDV), Newcastle Disease Virus (NDV), Infectious Bursal Disease Virus or Salmonella typhimurium will be reported. Of particular interest is Alkaline Phosphatase (AP). Unlike the increase seen in mammals with liver or intestinal disease, we observed a consistent decrease in AP levels in MDV infected broiler chickens. Similarly, Gamma Glutamyl Transferase (GGT), was decreased in MDV positive broilers. Vaccination with HVT prevented this decrease. Serum chemistries were analyzed using a Kodak DT-60 dry chemistry analyzer or UCD's wet chemistry analyzer. Five to ten parameters were analyzed for each bird. (Reprinted with permission from Vet Clin Pathol,
28: BIOCHEMICAL AND HEMATOLOGICAL VALUES IN PRE AND POSTPARTUM DAIRY COWS.
Dairy cows have important metabolic changes during the periparturient period, which influence chemical and cellular constituents of blood. It is ideal to have unique reference values for the pre and postcalving periods to accurately interpret laboratory results. More than 1000 Holstein cows from 20 dairy herds in Southern Ontario were sampled at least once prior to parturition and once after calving. Each cow was sampled once from the coccygeal vein between days 8 to 1 prepartum and days 0 to 7 postpartum. Cows which had one or more of the following were excluded: dystocia, milk fever, retained placenta, off-feed, abomasal displacement. clinical ketosis, lameness, metritis, clinical mastitis, culling during the first month after calving. After exclusion, 597 prepartum and 625 postpartum samples remained. Depending on the distribution of the data, parametric or non-parametric analyses were used to determine reference limits, which included 95% of the samples. The values corresponding to the pre-partum and post-partum period, respectively, were: β-hydroxybutyrate 217.9–883.8 and 216.0–1176.8 mmol/L, fatty acids 0.00–0.99 and 0.10–1.40 mmol/L, cholesterol 1.30–2.98 and 1.18–2.86 mmol/L, glucose 2.64–4.75 and 2.30–5.20 mmol/L, urea 2.15–8.02 and 1.86–7.79 mmol/L, calcium 2.18–2.65 and 1.64–2.61 mmol/L, phosphorus 1.48–2.65 and 1.04–2.73 mmol/L, leukocytes (WBC) 4.79–14.62 and 3.90–16.98 × 109/L, neutrophils 1.75–6.22 and 0.73–9.89 × 109/L, lymphocytes 1.89–8.97 and 2.12–9.99 × 109/L, monocytes 0.00–0.27 and 0.00–0.33 × 105/L and eosinophils 0.06–1.19 and 0.00–0.74 × 109/L. All analytes were significantly different (p < 0.0001) between prepartum and postpartum samples with the exception of WBC, neutrophils, lymphocytes and monocytes (p = 0.2817, 0.1592, 0.3006 and 0.06063 respectively). When evaluating laboratory data from cows in the transition period. unique reference limits should be used for pre and postpartum cows. especially for biochemical analytes. (Reprinted with permission from Vet Clin Pathol,
29: EFFECT OF HEMOLYSIS ON BOVINE NON-ESTERIFIED FATTY ACID AND BETA-HYDROXYBUTYRATE CONCENTRATIONS.
Hemolysis is a major source of analytical laboratory error. Our objective was to determine the effect of hemolysis on non-esterified fatty acid (NEFA) and beta-hydroxybutyrate (BHBA) concentrations in bovine serum. These analytes are used for metabolic testing of periparturient dairy cows. Bovine serum samples submitted for routine NEFA (0.10–0.36 mEq/L; n = 5) and BHBA (10–25 mg/dL; n = 9) testing were used in the study. NEFA (Wako), BHBA (Catachem), and hemolytic indices (HI) were determined with a Hitachi 917 chemistry analyzer. A hemolysate was prepared from EDTA anticoagulated bovine blood by freeze-thaw and was added at sequential dilutions to the serum samples to achieve mild to severe hemolysis (HI: 60 to 1,403 units). Median results of hemolyzed samples were compared to baseline values with Kruskall Wallis and Wilcoxon rank-sum tests. NEFA and BHBA concentrations in the hemolysate (HI 20,043 units) were 10.61 mEq/L and 0 mg/dL, respectively. Median NEFA concentrations progressively rose with increasing hemolysis and were significantly higher than baseline at HI 975 and 1,403 units. In contrast, median BHBA concentrations decreased when samples were progressively hemolyzed, up to an HI of 860 units. At the highest HI (1,102 units), BHBA increased to near baseline values. Median BHBA values were significantly decreased from baseline at HI 430 and 856 units. Result interpretation for BHBA and NEFA was affected with moderate (HI > 200 units) and severe (HI > 480 units) hemolysis, respectively. Our results indicate that moderate to severe hemolysis will incur a positive bias with NEFA and a mostly negative bias with BHBA. Therefore, NEFA and BHBA results from hemolyzed bovine samples should be interpreted with caution. (Reprinted with permission from Vet Clin Pathol,
30: A COMPARISON OF CANINE AND FELINE REFERENCE RANGES GENERATED FOR THE I-STAT AND IRMA CHEMISTRY ANALYZERS.
Small animal clinicians at the University of Missouri Teaching Hospital routinely use both the i-STAT portable clinical analyzer and the immediate response mobile analyzer (IRMA) for ionized calcium levels and blood gas analysis. The values obtained from these instruments were often discordant, creating a diagnostic dilemma for the clinician. The purpose of this study was to determine reference ranges in a population of clinically normal dogs and cats using the IRMA and to compare these results to values obtained on the i-STAT using the same blood sample. The study population consisted of twenty-five dogs and twenty-three cats. Venous blood was collected from the jugular vein into lithium heparin vacutainers. IRMA ComboCart and i-STAT CG8+ cartridges were used to assess PH, pCO2, pO2, Na+, K+ and ionized Ca (iCa). Simple linear regression analysis was done to determine the relationship between analyte concentrations measured by the IRMA and those measured by the i-STAT. Reference ranges for each instrument were developed based on a central 95% confidence interval. Results indicated good correlation for all analytes with the exception of iCa in cats. The correlation for iCa in dogs was excellent suggesting the difference in cats was due to sample variations rather than instrument malfunction. The recommendation was made to use only one instrument for each patient during their hospitalization due to different reference ranges generated for each instrument. (Reprinted with permission from Vet Clin Pathol,
31: EFFECTS OF STORAGE TEMPERATURE AND TIME ON THE STABILITY OF THIRTY CHEMISTRY ANALYTES IN RAT AND DOG SERUM ANALYZED BY THE OLYMPUS AU640e.
Batching samples enhances efficiency in the chemistry laboratory through minimizing technician time and reagent use. Our objective was to evaluate stabilities of common analytes under a variety of storage conditions on the Olympus AU640e analyzer. Aliquots of pooled serum from 3 healthy fasted rats and dogs were assayed for activities of ALP, ALT, amylase, AST, CK, GGT, LDH, lipase (LIP), aldolase, and glutamate dehydrogenase, and albumin, calcium, cholesterol, creatinine, direct bilirubin (DBILI), glucose, iron, inorganic phosphorus, total bilirubin, total protein, triglycerides, urea nitrogen, Na, K, Cl, total phospholipids, nonesterified fatty acids, HDL, LDL, and total bile acids (TBA) after storage at room temperature (fresh), 4°C (overnight), or -80°C (1 h, overnight, 7 d, 30 d, 90 d, and 120 d). Mean analyte concentrations were calculated from combined time points along with standard deviations and were used to calculate the coefficient of variation (CV) for each analyte. An acceptable CV was considered 15%. Most CVs were <5% for both species. All analytes for rats had acceptable CVs, except LIP (CV = approx. 15%) and DBILI (CV = approx. 20%). Analytes for dogs had acceptable CVs, except DBILI, TBA, and LDL (CVS ranged from 21–38%). Rat LIP tended to decrease after 30 d at -80°C. The other exceptions exhibited small mean values with disproportionately large CVs; results were sufficiently similar across conditions and were determined to have acceptable stability for clinical and diagnostic purposes. We conclude that other than rat LIP, these serum analytes on the Olympus AU640e were stable for up to 24 h at 4°C or at -80°C for up to 120 d. (Reprinted with permission from Vet Clin Pathol,
32: EFFECT OF SERUM STORAGE, ANTI-INFLAMMATORY DOSAGES OF ORAL PREDNISONE AND SPONTANEOUS HYPERADRE-NOCORTICISM ON SERUM GLUTAMATE DEHYDROGENASE (GLDH) ACTIVITY IN DOGS.
Glutamate dehydrogenase (GLPH) is a mitochondrial enzyme with highest activity in periacinar hepatocytes. It is reported to be a sensitive indicator of hepatic injury, however, studies regarding its tissue specificity are contradictory. The purpose of this study was to examine the effect of 3 factors on serum GLPH activity in dogs: 1) serum storage, 2) antiinflammatory dosages of oral prednisone and 3) spontaneous hyperadre-nocorticism (HAC). Stability of enzyme activity was determined by comparing GLPH activity in 8 serum samples stored at 20C, 4C, and -20C for 4, 24, 48, and 72 hours, 1 week, and 6 months. To determine if there was an effect of prednisone on GLPH activity, the median difference in serum GLPH activity was compared between 5 untreated control dogs and 8 dogs that received a tapering dosage of oral prednisone starting at 1 mg/kg/day for 2 to 3 weeks. The third objective was achieved by comparing GLPH enzyme activity between 17 dogs with HAC and 16 age-matched controls. GLPH activity remained stable for 2 days, 1 week, and 6 months, in serum stored at 20C, 4C, and -20C, respectively. Compared to controls, GLPH activity was not significantly elevated in dogs receiving prednisone, however, dogs with HAC had significantly higher values than age-matched controls. Conclusions: Serum samples should be shipped and stored at 4C if analysis will be delayed by greater than 2 days; serum stored at -20C yields reliable results for up to 6 months. Serum GLPH activity was not increased in dogs receiving anti-inflammatory dosages of oral prednisone over the short term which contrasts to its elevated activity in dogs with HAC. (Reprinted with permission from Vet Clin Pathol,
33: QUALIFYING AN INSULIN ELISA FOR DETECTION OF CANINE HYPOINSULINEMIA USING PANCREATIC STIMULATION TECHNIQUES.
Reduction in basal serum insulin can be due to an autoimmune response, direct cytotoxicity (e.g., alloxan) or interference with insulin synthesis, production or release (e.g., cyproheptadine). Regardless of the mechanism, reduction in basal insulin is an indicator of islet cell injury and in toxicology studies, this observation negatively impacts candidate drug selection. A commercially available porcine insulin ELISA was evaluated to determine basal levels of serum insulin and increases following beta islet cell stimulation by glucose. Serum insulin of control, fasted dogs were at or below the lower limit of detection (0.07 ng/ml) thereby making it impossible to detect compound-induced hypoinsulinemia. Three methods were used to stimulate beta islet cells to increase insulin secretion: standard feeding protocol, oral glucose (4 g/kg) administration and intravenous glucose (0.5 g/kg) administration. Blood samples were assayed for glucose and insulin at predetermined time points after stimulation. Normal feeding did not increase glucose or insulin levels at any time point. Oral glucose administration increased both glucose and insulin levels but the magnitude and timing of maximal insulin response varied dramatically between animals. In addition, one animal did not respond to the oral treatment thereby preventing oral glucose administration from being a reliable method for beta islet cell stimulation and determination of hypoinsulinemia. Intravenous glucose caused a profound increase in plasma glucose and insulin in a narrow range of time (five to ten minutes). Additional studies to further qualify the assay included bolus intravenous glucose administration followed by serial monitoring of serum glucose and insulin. The average intra-assay variability was 0.083 ng/ml (n = 5 assays and n = 5 dogs). (Reprinted with permission from Vet Clin Pathol,
34: COMPARISON OF URINE GLUCOSE MEASUREMENTS FROM THE IRIS AUTION MAX AND YELLOW IRIS (SEMIQUANTITATIVE METHODS) AND THE HITACHI 911 AND OLYMPUS AU640e (QUANTITATIVE METHODS).
Semiquantitative urine chemistry test strips provide convenience and efficiency during routine urinalysis for individual animal patients. However, results often reflect a wide range of concentrations which may limit the application of these methods in other situations (i.e., drug safety studies) where subtle differences between treatment groups might not be identified. The objective of this study was to correlate urine glucose measurements on the IRIS Aution Max and Yellow IRIS (both use semiquantitative test strips), and the Hitachi 911 and the Olympus AU640e (both are quantitative chemistry analyzers). Exogenous glucose was added to pooled urine from healthy dogs to yield concentrations from 0 to 1000 mg/dL and aliquots were analyzed on each instrument. Percent recovery of glucose was acceptable for both the Hitachi (95.7% to 115.0%) and the Olympus (88.9% to 100.2%). The Hitachi and the Olympus results correlated well with each other (r2 = 0.999; 8% ave. difference). Semiquantitative results from the Yellow IRIS and the Aution Max were inaccurate at some concentrations and failed to agree with each other consistently indicating that these two instruments could not be used interchangeably reliably. While the Yellow IRIS and Aution Max results correlated favorably with Hitachi and Olympus results at concentrations ≤50 mg/dL, the test strips tended to yield low results at higher concentrations. At concentrations ≤50 mg/dL results from all methods were considered acceptable. For urine glucose concentrations above 50 mg/dL quantitative methods provide greater accuracy and are recommended when discriminating fine differences is essential and/or to confirm and quantify positive test strip results. (Reprinted with permission from Vet Clin Pathol,
35: PROTEIN DISTRIBUTION PROFILE OF GUINEA PIG AND RAT BRONCHO-ALVEOLAR LAVAGE FLUID.
The objective of this work was to determine the reproducibility of Hartley guinea pig and Sprague-Dawley rat broncho-alveolar lavage (BAL) fluid protein measurements. Broncho-alveolar lavage fluid is a useful biological matrix for evaluating the tolerability and/ or the pharmacological effects of inhalation products. BAL samples were obtained from rats and guinea pigs under anesthesia. Total protein concentration of the BAL samples was measured with a benzethonium turbidimetric method adapted on the Modular Analytics instrument (Roche Diagnostics). Protein fractionation was obtained using the Hydrasis electrophoretic agarose system (Sebia). Intra-assay precision was calculated by testing 8 consecutive measurements of a fresh BAL sample while the inter-assay precision was obtained from 5 different analyses of the same sample. The protein concentration in BAL samples varied from 0.09–0.18 g/L for the guinea pig and 0.06–0.08 g/L for the rat with a coefficient of variation of less than 4.0%. Each protein fraction (albumin, alphal, alpha2, beta, gamma) was well separated with the use of the agarose electrophoretic system. BAL preliminary reference intervals were calculated for Hartley guinea pigs and Sprague Dawley rats; albumin and alpha2 are the major fractions of the guinea pig profile while albumin and beta are the major ones observed in rat BAL profile. In conclusion, the precision and preliminary reference intervals of the measurement of total protein concentration of BAL samples and the electrophoretic agarose protein fractionation in guinea pigs and rats have been shown to be adequate and reliable for research studies. (Reprinted with permission from Vet Clin Pathol,
36: RHABDOMYOSARCOMA OF THE SOFT PALATE IN A DOG.
A 7-year-old, male, Boxer dog was presented with a change in bark intonation, dyspnea and mild dysphagia. On clinical examination, an oral mass, about 4 cm2 in size, localized within the soft palate was found. FNA cytological examination revealed abundant pleomorphic cells, loosely aggregated in clusters or dispersed. The neoplastic cells were small rounded, medium sized triangular, less spindle, with distinct cell borders and scanty or abundant basophilic cytoplasm. Nuclei were rounded or irregular and anisokaryosis was prominent. Many binucleated and multinucleated malignant cells were seen, with nuclei organized in rows in some cells. Few large cells had numerous small dark brown intracytoplasmic granules. Nucleoli were prominent and mitoses were seen. An initial diagnosis of “malignant pleomorphic neoplasm of uncertain histogenesis” was made. Histopathology of incisional biopsy tissue showed a subepithelial, high cellularity growth of pleomorphic cells, ranging from small round to large polygonal with eosinophilic cytoplasm and few spindle cells. Multinucleate cells and many apoptotic bodies were seen and mitotic index was marked. Some areas exhibited cross striation or differentiated immature striated muscle fibers. All above supported the diagnosis of rhabdomyosarcoma. Moreover, immunohistochemistry was performed using a panel of antibodies to confirm the initial histopathological diagnosis. The neoplasm was strongly positive for myoglobin, vimentin, and Ki-67, focally positive for myogenin and MyoD1, weakly reactive for a-sarcomeric actin and desmin, and moreover was negative for pan-cytokeratin, NSE, S-100, EMA and CD99. Neoplasms of striated muscles are rare and few cases of rhabdomyosarcoma within the oral region have been documented using immunohistochemistry. (Reprinted with permission from Vet Clin Pathol,
37: CEREBELLAR PURKINJE CELL DEGENERATION AND HEPATIC MICROVASCULAR DYSPLASIA IN HAVANA BROWN KITTENS.
Two sire-related female Havana Brown kittens developed clinical signs of cerebellar ataxia. One kitten (Kitten A) was five weeks old when clinical signs were first noticed and the other (Kitten B) was less than four weeks of age. Both kittens had progressive ataxia, hypermetria and head-bobbing. Both animals were negative serologically for feline leukemia virus (FeLV), feline infectious peritonitis (FIP), feline immunodeficiency virus (FIV) and feline panleukopenia (FPL). Kitten A was euthanized and necropsied shortly after the onset of signs, while Kitten B was observed clinically, and when the ataxia progressed to frequent falling, she was euthanized at three months of age. Histologically, both kittens had extensive Purkinje cell degeneration. In Kitten A, Purkinje cell degeneration was characterized by vacuolation and increased cytoplasmic eosinophilia. In Kitten B, the predominant cerebellar lesion was Purkinje cell loss with astrocytosis and granule cell apoptosis. Both kittens had hepatic lobular atrophy with mild to moderate microvascular dysplasia. Cerebellar and pontine nuclei were normal in both kittens, as were other areas of the brain, spinal cord and peripheral nerves. Fluorescent antibody testing for FeLV, FIP, FIV and FPL were negative. In light of the lesions in the cerebellum and the common ancestry of the kittens, this is likely an inherited cerebellar abiotrophy. The cause of the hepatic lesions is uncertain, but their presence in both kittens suggests that this may be part of this inherited disorder. A similar disorder has been described in Bernese Mountain dogs.
38: SARCOCYSTIS NEURONA IN A DOG.
A 1.5-year-old male Feist dog was presented for reluctance to stand on the hind legs. Clinical examination detected no injury. Treatment included dexamethasone and the initial response was favorable. Posterior weakness returned and progressed to recumbency by day 10. On day 11, the dog exhibited hyperesthesia and attempts at biting and was euthanized on day 15. All vaccines were current within the past four months. No significant gross lesions were identified at necropsy. FA examination of the brain for rabies was negative. Histopathologic examination revealed multiple foci of encephalitis in both the cerebrum and, particularly, in the cerebellum. Focally, malacic folia in the cerebellar cortex had heavy infiltrates of neutrophils intermingled with macrophages, lymphocytes and plasma cells. The cerebral cortex had scattered similar inflammatory foci. Frequent elongate protozoa occurring singly or as clusters were identified at sites of intense inflammation. Multiple schizonts were identified in areas lacking any inflammation. The protozoa were morphologically consistent with Sarcocystis. Immunohistochemistry by Dr. Bradd Barr of the University of California, Davis, revealed that the protozoa did not react to polyclonal antisera to Toxoplasma gondii or Neospora caninum. The protozoa had a positive reaction to Sarcocystis neurona using a polyclonal antiserum and a monoclonal antibody specific for S. neurona. Since the elucidation of the life cycle of S. neurona and the implication of the opossum as the definitive host, additional aberrant hosts other than horses have been identified. S. neurona infections have been reported in the cat, fisher, skunk, raccoon, mink, Pacific harbor seal and sea otter. Cases of disseminated sarcocystosis in cats have been attributed to immunosuppression and corticosteroid therapy may have allowed rapid multiplication of the organism. To my knowledge, Sarcocystis neurona has not been documented previously in the dog.
39: DESCURAINIA PINNATA (TANSY MUSTARD) INDUCED POLIOENCEPHALOMALACIA IN ARIZONA RANGE CATTLE.
During the late winter/early spring of 2005, increased morbidity and mortality were reported in range cattle on the Navajo Reservation in northeastern Arizona. Clinical signs included: staggering, stupor, blindness and tongue paralysis. Abundant plant growth was reported secondary to above normal rainfall. A field necropsy was performed on a representative animal and no gross lesions were noted. Histologically, sections of the cerebrum exhibited severe laminar cortical necrosis with marked astrogliosis consistent with chronic polioencephalomalacia. Plants identified as Descurainia pinnata (Tansy Mustard) were the significant flora in abundance. Toxicology performed on submitted plants revealed elevated sulfur levels in the plant stems, leaves and roots. Many animals that were removed from the range where plants were in excess and/or were treated with thiamine responded medically and survived the outbreak. Attempts to reproduce this disease by ingestion of Tansy Mustard have been unsuccessful to this point. The association of clinical disease with the plant in abundance, identification of toxic sulfur levels, and clinical improvement of animals removed from infested rangeland suggest that Tansy Mustard is toxic to cattle and results in a syndrome with microscopic lesions suggestive of chronic polioencephalomalacia.
40: WOBBLY HEDGEHOG SYNDROME IN THREE AFRICAN HEDGEHOGS (ATELERIX ALBIVENTRIS).
Three African hedgehogs, two males and a female, from different sources, developed ascending ataxia, which progressed over the period of two to three weeks to the point that the animals were unable to right themselves. All three hedgehogs were euthanized due to poor quality of life. Gross necropsy examinations were unremarkable in all the three cases. Histopathological examination of the nervous system revealed multifocal, often bilaterally symmetrical lesions in the white matter of the cerebrum, cerebellum, cerebellar peduncles, midbrain and the brain stem. Lesions were increased in severity in the brain stem. The lesions consisted of individual or clusters of variably sized vacuoles. The vacuoles were empty or contained pale eosinophilic wispy material and represented individual swollen axonal sheaths or coalescing areas of demyelination with axonal degeneration. Associated with these areas of vacuolation there were astrocytes and gitter cells, the numbers of which varied from rare in some foci to moderate in others. Lesions in all hedgehogs were characteristic of a progressive, degenerative disease affecting the central nervous system of African hedgehogs, commonly referred to as wobbly hedgehog syndrome or progressive paresis. The etiology of this condition is unknown. However, the bilateral symmetrical distribution of the lesion suggests a metabolic, toxic or deficiency condition. The epidemiological and etiological aspects of this interesting disease await further study.
41: CORRELATIONS BETWEEN COMPUTER-ASSISTED IMAGE ANALYSIS AND HISTOPATHOLOGY IN DETECTING EQUINE TESTICULAR LESIONS.
Computer-assisted image analysis (CAIA) was compared with histopathology for detection and assessment of equine testicular lesions. Testes were collected from pubertal stallions (n = 65) at an abattoir in the Spring of 2003. Testes were weighed, measured, and then imaged in a waterbath using a 7.5 mHz linear-array probe (Aloka SSD-900V). All images were recorded using a DVR1000 (Sony). Tissue samples taken within 1 hr. of slaughter from both sides of cranial, caudal poles and equatorial region of each testis were fixed in modified Davidson's solution, stained (H&E) and evaluated microscopically for distribution and severity of testicular lesions. Fifty-eight of 65 stallions (89%) had evidence of seminiferous tubular degeneration, 31 of 65 (48%) had evidence of tubular atrophy and 60 of the 65 (92%) had alterations in the testicular interstitium, primarily consisting of Leydig cell hypocellularity. Ultrasound images of areas concomitant to histological sample sites were acquired at 640 A ∼480 pixel resolution and transferred to a graphics workstation. Quantitative echotextural analyses were performed. Numerical pixel values (gray-scale values from 1 to 255) and pixel heterogeneity (standard deviation of gray-scale values) of sites were measured by CAIA (Synergyne 1©, R. A. Pierson, Saskatoon, Sask., Canada) using 4 circular areas of 20 pixels in diameter placed within the image of the parenchyma. CAIA correlated highly with seminiferous tubule degeneration (p = 0.0002, r2 = 0.2036), seminiferous tubule atrophy (p < 0.0001, r2 = 0.2063) and Leydig cell hypocellularity (p < 0.0001, r2 = 0.2964); however, no correlations were observed with inflammation of the seminiferous tubules (p = 0.3644, r2 = 0.0131). Differentiating cell types was beyond the resolution of the ultrasound instrument. CAIA shows promise as a diagnostic aid in identifying testicular degenerative lesions in stallions.
42: INTERSTITIAL PULMONARY FIBROSIS AND GRANULOMATOUS PNEUMONIA IN HORSES FROM MICHIGAN: ASSOCIATION WITH SILICATE PNEUMOCONIOSIS.
Pneumoconiosis refers to the retention and pulmonary effects of particle (dust) inhalation. Naturally occurring pneumoconioses are rarely reported in domestic animals. In humans, this group of diseases is usually associated with occupational exposure to dusts. Non-occupational forms of pneumoconiosis are generally associated with a geographic distribution due to regional soil compositions or atmospheric contamination. In domestic horses, silicate pneumoconiosis was described in the Monterey-Carmel Peninsula and Carmel Valley in California in 1981. Tissues from three adult horses (mean age 12.7 years) with a history of chronic respiratory disease and lesions consistent with silicate pneumoconiosis were examined at Michigan State University. The findings were restricted to the lungs and bronchial lymph nodes. Grossly, the lungs had severe, diffuse consolidation with numerous pinpoint to 2mm mineralized granulomas. Similar mineralized granulomas were present within enlarged bronchial lymph nodes. Histologically, the lung had interstitial fibrosis and granulomatous inflammation with multiple fibrotic mineralized granulomas. Honeycomb lung formation was common; the affected airspaces were lined by variable epithelium, ranging from hyperplastic type II pneumocytes to metaplastic squamous cells. Mineralized and fibrotic granulomas were present within the bronchial lymph node. Numerous birefringent acicular silicate crystals were found within the lymph node, with fewer associated with the pulmonary granulomas and fibrosis. No infectious agents were detected histochemically or through microbiologic tissue analysis. These findings share features with the diffuse lung fibrosis variant of silicate pneumoconiosis and silicosis reported in humans. To the authors’ knowledge, this is the only report of apparent silicate pneumoconiosis in horses outside of California.
43: OUTBREAK OF MYXOMATOSIS IN DOMESTIC RABBITS IN OREGON: PATHOLOGY AND DETECTION OF VIRAL DNA AND PROTEIN.
In the summer of 2003, western Oregon experienced an outbreak of myxomatosis in domestic rabbits. As typically seen with the California strain, rabbits were either found dead or were culled after showing lethargy. Swelling of the eyelids and/or the genital region was an important and reliable necropsy finding, but was not consistently present. In close to 50% of the grossly examined rabbits, the most severe lesion was hemorrhagic-necrotizing appendicitis and/or ileitis of the sacculus rotundus. None of the rabbits had skin growths (myxomas). The diagnosis of myxomatosis was established by histopathology based on presence of epithelial hyperplasia and ballooning degeneration with eosinophilic, cytoplasmic inclusion bodies at oral, ocular, and genital mucocutaneous junctions. Appendicitis and ileitis were due to necrosis of gut associated lymphoid tissue (GALT). Severe lymphoid depletion was consistently noticed in the spleen and lymph nodes. Bronchial epithelial dysplasia with cytoplasmic inclusion bodies was uncommon. Secondary infections with Gram-negative bacteria were common. There was good agreement between staining results obtained by immunohistochemistry using a polyclonal antiserum and in situ hybridization (ISH) using a PCR-generated probe cocktail (M11L, MGF, and TK genes) based on the German isolate Hasi. Some remaining viable lymphocytes in GALT were stained by immunohistochemistry and ISH, while only viral protein was detectable in macrophages and Kupffer cells. The summer 2003 was exceptionally hot and was likely a successful breeding year for mosquitoes, an important passive vectors of leporipoxvirus. Most private rabbitries in Oregon use outdoor pens where rabbits are neither protected from insects nor from high environmental temperatures.
44: INVESTIGATION OF CARDITIS IN A COLONY OF DEGUS (OCTODON DEGUS).
From January 1998 through February 2005, eighteen Degus (Octodon degus) underwent complete postmortem examinations. Ten had moderate lesions of lymphoplasmacytic carditis. Many had infiltrates of lymphocytes and plasma cells within other tissues. Additional tests failed to reveal a common underlying etiology. In March 2005, a Degu with conjunctivitis presented for postmortem examination. A few other Degus in the colony also had ocular discharge. Histologically, an acute ulcerative conjunctivitis, moderate lymphoplasmacytic carditis, and pneumonia were present. PCR testing of conjunctival samples was positive for Chlamydophila psittaci. The colony was quarantined and treated with doxycycline. Fifty adult Degus from the colony of eighty were euthanized to determine the extent of infection. Forty had histologic lesions of carditis and lymphoplasmacytic infiltrates within other tissues. Conjunctival samples from four of the recently euthanized adults and available frozen tissues from four animals dying previously were positive for C. psittaci PCR. Three neonatal animals that died or were euthanized during the quarantine period had no gross or histologic evidence of inflammation; however, frozen kidney from each was positive. DNA sequencing of the amplified product from the PCR positive cases demonstrated 50% homology with C. psittaci, as well as low homology with any DNA sequences in Gen Bank. Immunohistochemistry for Chlamydia spp. was negative. Eleven adult Degus were culled after forty-five days of treatment. Seven had no heart lesions, three had minimal lesions, and one had moderate lesions. An apparent response to antibiotic therapy suggests a bacterial cause to the carditis. Tests to further characterize the agent involved include in situ hybridization with the amplified DNA product, electron microscopy, and additional cultures.
45: SEAL PARAPOXVIRUS INFECTION IN A NORTH ATLANTIC HARBOR SEAL (PHOCA VITULINA CONCOLAR).
A stranded female harbor seal (Phoca vitulina concolar) was rescued from a beach in Hyannis, MA and transferred to the Marine Animal Rehabilitation Center for evaluation and treatment. The twenty-seven pound, 96 cm long, yearling presented with bilateral nasal discharge, emaciation and weight loss. Multifocal to coalescing, raised and partially ulcerated cutaneous papules and nodules were present on the dorsal surface of the right fore flipper. A surgical biopsy specimen collected from the flipper lesion was fixed in 10% neutral buffered formalin and submitted for routine histopathologic examination. Hematoxylin and eosin stained sections revealed moderate to marked irregular epidermal hyperplasia with erosions and ulcers. Severe, laminar hydropic degeneration of the stratum spinosum and granulosum of the interfollicular and infundibular epidermis was associated with myriad, variably-sized, eosinophilic, intracytoplasmic inclusion bodies. The dermis was expanded by reactive stroma in combination with a moderate to marked, perivascular to interstitial infiltrate of lymphocytes, plasma cells and fewer macrophages and neutrophils. The seal subsequently developed additional nodules on both fore flippers and specimens were collected for histopathology and electron microscopy. Transmission electron microscopy revealed cytoplasmic granular inclusions that contained numerous ovoid viral particles with both partial and complete forms present. Negatively stained preparations revealed regular surface tubule morphology consistent with a parapoxvirus. Virus isolation utilizing bovine turbinate cell cultures was unsuccessful. This appears to be the first confirmed report of seal parapoxvirus infection in a harbor seal on the Eastern coastline of the United States.
46: “STICKY KITS” SYNDROME IN MINK.
Diarrhea and excessive secretion from the cervical apocrine glands in young, suckling mink kits is a well-known, but poorly defined, syndrome, often referred to as “sticky,” “greasy,” or “wet” kits. The primary cause of this syndrome remains unknown. Here we report an outbreak of diarrhea on a mink farm in Idaho affecting approximately 5000 young kits with 1500–2000 (30–40% mortality) kits dying within 12 days. Similar disease outbreaks have occurred at this particular farm at least 3 times during the last 10 years. At necropsy, small intestines of affected kits were distended with large amount of fluid and appeared flaccid. All other organs were grossly unremarkable. Virological testing did not identify an underlying virus infection. More than 1000 colony forming units of Staphylococcus intermedius were isolated from small intestines of multiple mink. Microscopically, large numbers of gram positive coccoid bacteria colonized epithelial cells of small intestinal villi in the absence of morphologic alterations. Ultrastructurally, rows of bacterial cocci were intimately associated with microvilli of villar enterocytes in the absence of morphologic alterations. Based on virology, bacteriology and histopathology results, the diagnosis of a secretory diarrhea associated with colonization of villar epithelial cells by Staphylococcus intermedius was made. Gram positive coccoid bacteria have rarely been reported as primary pathogens in enteric diseases, specifically secretory diarrhea. In the absence of ultrastructural evidence of damage to enterocytes, we hypothesize that Staphylococcus intermedius elicits an exotoxin resulting in hypersecretory diarrhea causing the clinical signs of “sticky kits.” Studies to determine virulence factors of the Staphylococcus intermedius isolates associated with this particular disease syndrome are ongoing.
47: A FATAL OUTBREAK OF AVIAN INFLUENZA H5N1 IN TIGERS (PANTHERA TIGRIS) IN THAILAND.
In January 2004, an epizootic outbreak of highly pathogenic avian influenza (HPAI H5N1 strain) was reported in poultry and various other birds in Thailand. During the second wave HPAI H5N1 also caused severe disease and death in tigers (Panthera tigris). The Sriracha tiger zoo housed 441 tigers in 3 zones: breeder, nursery, and grower. Initially 16 tigers in the grower zone were affected developing high fever, respiratory distress, including serosanguineous nasal discharge and neurological signs. At necropsy of 3 tigers, the lungs were severely congested and hemorrhagic, and there were pleural effusions and multifocal hemorrhages in the heart, thymus, stomach, intestine, liver, and lymph nodes. Microscopically, there was an interstitial pneumonia with severe pulmonary edema, hemorrhages and necrotizing bronchiolitis, a moderate multifocal necrotizing hepatitis and a mild nonsuppurative meningoencephalitis. Infection with HPAI virus was confirmed by multiplex RT-PCR, virus isolation and detection of viral antigen by immunohistochemistry in sections of lung, liver and brain. Animals had been accidentally fed HPAI H5N1 infected raw poultry carcasses. Clinical signs continued within the affected zone despite discontinuing feeding of raw poultry, suggesting possible tiger-to-tiger transmission. By October, a total of 147 tigers had either died or had been euthanized to limit further spread of the disease. Two animal handlers also seroconverted, but did not develop clinical signs. These findings extend the host range of HPAI and have implications for influenza virus epidemiology and wildlife conservation.
48: HEPATIC LIPIDOSIS IN PUPPIES.
Retrospective analysis of case records from the Diagnostic Center for Population and Animal Health, Michigan State University, revealed 30 puppies with pathologic findings consistent with hepatic lipidosis. Twenty-five affected puppies were toy breeds, 3 were small to medium-sized dogs, 1 was a large breed and the breed of 1 was unknown. Affected animals ranged in age from 6 to 16 weeks (mean = 9.1 weeks). The most frequently reported clinical signs were vomiting, anorexia, lethargy, diarrhea and neurological. Limited clinicopathological data were available. The most significant findings were hypoglycemia (6 of 6), hypoproteinemia (5 of 5), low BUN (4 of 5), increased alanine transferase (3 of 3), increased pre- and postprandial bile acid concentrations (2 of 2) and an inappropriate insulin to glucose ratio in one animal (insulin 131 pmol/L, glucose 1.2 mmol/L, ratio = 109). At necropsy, livers were enlarged and pale tan to yellow. Histologically, most had microvacuolar lipidosis, some had both micro- and macrovacuolation and 2 had predominantly macrovacuolation. In 11 puppies, a variety of concurrent diseases were identified: two had intestinal nematodiasis, two had intestinal coccidiosis, two had acute bacterial pneumonia, two had peri-pancreatitis, one had parvoviral enteritis with giardiasis, one had lameness and one had pulmonary adenovirus infection. The remaining 19 animals had no reported evidence of concurrent disease. These findings are consistent with previous reports that suggest hepatic lipidosis in puppies is related to hypoglycemia and anorexia. In a previous report; experimentally induced hypoglycemia in toy breed dogs was associated with decreased insulin and increasing glucagon and Cortisol levels. The hyperinsulinemia identified in one of our animals suggests there may be a more complex underlying metabolic disorder resulting in hepatic lipidosis.
49: POLYMYOSITIS IN THE DOMESTIC FERRET.
Since late 2003, a process tentatively termed idiopathic polymyositis has been diagnosed in 13 ferrets at Northwest ZooPath. It appears to be a disease of young ferrets characterized by rapid onset of clinical signs, high fever, neutrophilic leukocytosis, treatment failure and death (or euthanasia). Necropsy usually revealed no gross lesions, although red and white mottling and dilatation of the esophagus, and white streaks in the heart, diaphragm and intercostal muscles were seen in few ferrets. Histologic changes included moderate to severe suppurative to pyogranulomatous inflammation involving the skeletal muscle and blood vessels at multiple sites, particularly the esophagus (13), Heart (12) and muscles of the hind limbs and lumbar region (11). Myeloid hyperplasia of spleen and/or bone marrow (12), hepatitis (6), pneumonia (6) and mediastinitis (4) were also prominent features. Cultures, electron microscopy and protozoan immunohistochemistry conducted on some of these animals have been negative for infectious agents. The etiopathogenesis of polymyositis in ferrets is not known. The clinical presentation and distribution of histologic lesions, particularly in the esophagus, suggests that this is likely a single distinct entity. Studies are underway to search for other infectious agents, and for other commonalities in affected animals that may provide clues as to the underlying cause.
50: KUDOA-INDUCED MYOPATHY IN SALMON.
A smoked filet of Atlantic salmon was submitted to the AVC Aquatic Diagnostic Services for examination. Grossly, the filet presented with focal and multifocal coalescing opaque discolored areas, distinguishable from the golden brown hue of smoked-processed fish. Histologically, the opaque areas correlated with foci of myofibers that were hypercellular with less distinct endomysial boundaries. Affected myofibers contained large spore aggregates without a wall demarcating them from the sarcoplasm. The aggregates were pseudocysts of Kudoa spp., a myxosporean parasite that infects striated (skeletal and heart) muscles of many marine fishes, including salmon. The parasite stains well with special stains, such as Giemsa, but were best demonstrated by wet mounts of infected freshly cut muscle tissue. By this method, the spores were stellate with 4 valves and 4 polar capsules converging at one end, each containing a polar filament. Severe Kudoa spp. infections have resulted in emaciation and death, particularly in young fish. Muscle tissue damage is greatest after death of the infected fish. Proteolytic enzymes released by dying parasites cause muscle softening and liquefaction (soft flesh disease), which may occur soon after death or after several days of frozen storage.
51: METASTATIC ANAPLASTIC SARCOMA IN A FIFTEEN YEAR OLD MALE CHIMPANZEE.
A fifteen-year-old, experimentally naive, captive born male chimpanzee (Pan troglodytes) presented with a rapidly expanding mass involving the left cubital joint. A CBC revealed moderate anemia and a marked leukemoid reaction (76,000 segmented neutrophils/microliter). A surgical biopsy suggested an invasive neoplasm of undetermined origin. Based on rapid clinical deterioration, a rise in the neutrophil count to 129,000 cells/microliter, and radiographic evidence of metastatic disease in the thoracic cavity, the animal was euthanized. Necropsy revealed widespread metastatic disease throughout the thoracic and abdominal cavities. Histopathology of the elbow and metastatic lesions demonstrated a population of neoplastic cells supported by a fine fibrovascular stroma and associated with prominent necrosis and abundant infiltrating neutrophils. Neoplastic cells exhibited marked anisocytosis and anisokaryosis, varied in shape from spindle to stellate to polygonal and contained minimal to moderate amounts of lightly basophilic cytoplasm. The nuclei were ovoid with open chromatin and one to four angular nucleoli. The mitotic index was considered high at 1–2 per400x field. Immunohistochemistry demonstrated strong, diffuse vimentin staining, faint MITF-M staining, and equivocal cytokeratin staining. Staining for lysozyme, S-100, Melan-A, CD68, desmin and factor VIII related antigen was negative. Based on these findings, the three main differentials were anaplastic sarcoma, melanoma, and anaplastic carcinoma. Further evaluation through electron microscopy revealed no distinguishing features such as melanosomes, premelanosomes, desmosomes and tight junctions. Taken together, the histologic, immunohistochemical and ultrastructural findings supported a diagnosis of anaplastic sarcoma.
52: ECTOPIC THYROID CARCINOMA IN DOGS WITH MEDIASTINAL MASSES.
Biopsy specimens from nine dogs, each with a clinical history of a mediastinal mass and a histologic diagnosis of either neuroendocrine carcinoma or thyroid carcinoma, were reviewed. To assist in differentiation between these two lesions and to assess the original diagnosis, immunohistochemical (IHC) stains including thyroglobulin, calcitonin, thyroid transcription factor-1 (TTF-1) and synaptophysin were performed. Five of the nine specimens examined were consistent with ectopic thyroid carcinoma. Based on IHC results, 4 of these neoplasms were thyroid follicular cell carcinomas and one was a medullary (C-cell) carcinoma. The remaining 4 neoplasms were negative for all thyroid-specific IHC stains and thus interpreted as non-thyroid origin or poorly differentiated (anaplastic) carcinomas. Two of these 4 tumors were synaptophysin positive, supportive of neuroendocrine origin. Of the four confirmed follicular cell carcinomas, all were originally diagnosed as such. The remaining specimens, including the C-cell carcinoma, were originally diagnosed as neuroendocrine carcinomas. Results indicate that thyroglobulin, calcitonin, and TTF-1 are reliable IHC stains for the diagnosis of ectopic thyroid carcinoma that can assist in the differentiation of thyroid C-cell carcinoma from follicular cell carcinoma, and help in differentiating mediastinal masses of thyroid origin from other neuroendocrine derived neoplasms.
53: INVASIVE THYMOMA WITH METASTATIC NEUROENDOCRINE TUMOR IN A CANINE.
Thymomas are rare in domestic animals, but have been diagnosed in multiple species, including the dog. Thymomas can be locally invasive, and rare pulmonary or distant metastasis has been reported in domestic animals. A six-year old, spayed female Australian Heeler presented with dyspnea and hind limb ataxia. Computed tomography and ultrasonography identified a mediastinal mass and multiple liver nodules. The mediastinal mass was diagnosed as thymoma by flow cytometry and PCR for antigen receptor rearrangement (PARR) Assay. Radiation therapy of the mediastinal mass resulted in a mass reduction by 50%. Improvement of hind limb ataxia following irradiation of the neurolocalized region of thoracic vertebrae nine to lumbar vertebrae one, supported suspicion of vertebral canal metastasis. Post-mortem examination revealed the mediastinal mass adhered to and invading the lungs and confirmed the presence of multiple liver nodules. Histologic evaluation of the mediastinal mass revealed haphazard trabeculae of neoplastic epithelial cells that expressed cytokeratin. The mediastinal mass also contained a second, morphologically distinct, population of neoplastic spindloid cells arranged in nests and whorls and containing clear cytoplasm. This population exhibited negative immunohistochemical staining for cytokeratin and vimentin, but was positive for expression of neuron specific enolase, synaptophysin and to a lesser extent chromogranin A. The liver nodules were comprised exclusively of the spindloid cells. Though no discrete mass was found within the vertebral canal grossly, neoplastic spindloid cells were identified within the epidural fat with associated Wallerian degeneration in the spinal cord. We believe that this was a case of thymoma with invasion into lungs and a second disseminated neoplasm with immunohistochemical characteristics consistent with a neuroendocrine tumor.
54: METASTATIC EXTRASKELETAL CHONDROSARCOMA IN THE UVEAL TRACT OF A DOG.
An eleven-year-old, male castrated Border collie dog presented with a history of pulmonary chondrosarcoma with secondary hypertrophic osteopathy. Approximately one and a half months after a right lung lobectomy, this dog was examined for corneal edema and elevated intraocular pressure of the left eye. Primary glaucoma was diagnosed and ocular evisceration was performed with placement of an intraocular prosthesis. Histopathologic evaluation revealed chondrosarcoma of the uveal tract. Extra-skeletal chondrosarcoma is uncommon in the dog and reports of metastasis are extremely rare. This is the first case report of metastatic extraskeletal chondrosarcoma in the uveal tract of a dog.
55: HEMOTHORAX IN A YOUNG DOG DUE TO THYMIC MASS LIKELY HAMARTOMA.
A 4-year female spayed Jack Russell terrier presented with dyspnea, due to hemothorax. Removal of blood revealed a mediastinal mass from which an aspiration cytology was considered indicative of sarcoma. By median sternotomy the mass was partially removed with the largest tissue profile 3 by 2 cm. Histologically, the mass was irregularly encapsulated with alternate areas of thin mesothelial tissue and heavy collagenous bands. Centrally there were multinodular cystic structures lined by flat to columnar epithelium positive with cytokeratin staining and irregularly ciliated. These cysts were surrounded two layers of dense connective tissue suggestive of thymic lobulation. These structures had irregular areas of hemorrhage and were surrounded by stromal proliferation and large cystic areas filled with blood, fibrin and degenerating cells with no evidence of septic involvement. The nuclei of epithelial cells were round to oval, 2 red cells in diameter with a fine uniform chromatin pattern and 1–2 nucleoli of moderate size. Mitoses were rare. The cytoplasm was highly amphophilic and focally ciliated with cell boundaries generally distinct and irregularly infiltrated by neutrophils. The stromal component appeared most atypical, but was considered compatible with benign granulation in response to hemorrhage. There were irregular areas of myxoid ground substance. Areas of hemorrhagic infarction had irregular foci of blood sinus ectasia with normal appearing endothelial nuclei that likely rendered the lesion more prone to hemorrhage. The lesion was considered benign but with unequivocal involvement of thymic anlagen and likely a thymic hamartoma. The animal survived the surgery but had repeated bouts of hemothorax over the following months and was finally euthanized. No necropsy was carried out.
56: BROWN TUMOR-LIKE LESION IN A DOG.
Brown tumors are non-neoplastic lesions seen in middle-aged humans in associated with primary, secondary or tertiary hyperparathyroidism (PTH). Similar cases have not been reported in domestic animals. A 14 month-old female English bulldog with a clinical history of chronic renal failure since 5 months of age was submitted for routine necropsy. Radiological evaluation of the head revealed bilateral destruction of alveolar bone associated with marked mandibular and maxillary osteopenia. Several white, variably sized, smooth surfaced masses were observed at the maxillary and incisive regions of the dental arcade that resulted in complete disorganization and misplacement of teeth. The capsular surface of both kidneys was remarkably scarred and irregular. Histologically, the oral masses were characterized by various multinucleated foreign-body-like giant cells randomly distributed in a stroma of connective tissue predominantly composed of spindle-shaped cells and blood vessels. Marked osetolytic activity was also observed. Chronic renal lesions consisted of severe diffused interstitial fibrosis, marked atrophy and deformation of renal tubules, membranoproliferative glomeruonephritis, multifocal metastatic mineralization, glomerulosclerosis and multifocal interstitial influx of mononuclear inflammatory cells. The gross, histological, and radiographic lesions described in this dog are consistent with those of brown tumors associated with PTH as described in human medicine. In this case, PTH was secondary to chronic renal failure and occurred in a young dog. The central and peripheral giant cell granulomas of the jaw bone must be considered as differential diagnosis, but there is no participation of PTH in these lesions.
57: HETEROTOPIC POLYODONTIA IN A LATE GESTATION OVINE FETUS.
A late term fetal lamb (from a set of triplets) was delivered by caesarian section from a ewe at day 138 of gestation as part of a study investigating novel pulmonary maturation agents in preterm neonates. The lamb died shortly after birth and before treatment due to respiratory distress of immaturity. The fetal lamb had a pedunculated, reddened, alopecic mass (3.0 × 1.5 × 1.4 cm) located on the ventrolateral ramus of the right mandible. The mass was firm to touch with a loose pedunculated stalk attached to the adjacent skin. The lamb was euthanized with sodium pentobarbital and the mass placed in 10% neutral buffered formalin. The mass was demineralized and processed routinely for hematoxylin and eosin staining. Histologically, the mass was centrally composed of stellate to spindle cells on loose connective tissue consistent with dental papillae surrounded by concentric layers of odontoblasts, dentin, enamel, palisading ameloblasts, and a moderately vascularized stellate reticulum that extended to the surface epithelium. The overlying stratified squamous epithelium lacked hair, was continuous with the adjacent haired skin and had a moderate number of random epithelial cells with shrunken hypereosinophilic cytoplasm and pyknotic nuclei. The mass was consistent with a developing tooth outside of the dental arcade, a condition that is termed heterotopic polyodontia. The oral cavity of the lamb was not examined. Heterotopic polyodontia has been described in many species including cattle, sheep, horses, dogs and a cynomolgus monkey. To the authors knowledge this is the first report in fetal lambs and should be considered as a differential for pedunculated masses on the dermis overlying the mandible of fetal lambs.
58: ESOPHAGITIS IN CAMELIDS: REPORT OF THREE CASES. G.
Esophagitis is not well documented in camelids. We report three cases of this condition. All three animals were adults. Animal #1 was a llama with a history of acute recumbency and good appetite. Necropsy findings included emaciation, moderate gastrointestinal parasitism and ulcerative esophagitis, more severe in the cranial third of the esophagus. Histologically, there was extensive ulceration and bacterial colonization. Animal #2 was a camel with chronic weight loss and intermittent diarrhea. Grossly, the middle and caudal portions of the esophagus had multiple ulcers covered by fibrin. Microscopically, lesions in the esophagus were similar to animal #1. Animal #3 was a llama and had severe esophagitis involving the caudal % of the esophagus. Microscopically, numerous bacteria were associated with the ulcerated mucosa. This animal was diagnosed with dysautonomia. Bacterial isolation from the esophagus of animal #1 included alpha-hemolytic Streptococcus and Clostridium subterminale; animal #2 had heavy growth of Arcanobacterium pyogenes; Salmonella spp. was isolated from the esophagus of animal #3. Viral isolation (esophagus, lung, intestine) and FA for multiple respiratory and enteric viruses were negative in all three animals. Immunohistochemistry for BVD in the esophageal lesions was also negative. Camelids are capable of regurgitating gastric contents and also emesis. Emesis can be the result of overloading of C-1, diaphragmatic hernia, esophageal obstruction, and ingestion of poisonous plants. Ingestion of caustic substances was suspected in one case, but the lack of oral lesions argued against it. Gastric reflux/emesis, releasing gastric acid into the esophagus was likely in the animal with dysautonomia (#3) and possibly in another case (#2).
59: T-CELL LYMPHOMA WITH EOSINOPHILIC INFILTRATION INVOLVING THE INTESTINAL TRACT IN 11 DOGS.
Among the intestinal tumors of hematopoietic cell origin, lymphoma is the most common in the dog. Although primary intestinal lymphoma in the dog is thought to originate from B cells, the immunophenotype has rarely been examined. We therefore characterized the clinical and pathological features of 11 dogs (average age, 10.6 ± 2.5 years) with T-cell lymphoma of the intestinal tract. No sex predominance was apparent. All had localized tumor masses in the small intestine. Grossly, the intestinal wall was thickened, and the lumen of the affected intestine was usually narrowed. Microscopically, we observed transmural diffuse invasion by round to pleomorphic neoplastic cells. Neoplastic cells showed varying morphology, from scanty to abundant cytoplasm, and round to ovoid nuclei with scattered to dense chromatin. In 7 of the dogs, neoplastic cells had infiltrated into the epithelium. All showed infiltration of eosinophils and all 11 tumors had a T-cell phenotype (CD3+, CD79-). Only 1 tumor stained positive for the mast cell marker c-kit and none was positive for mast cell tryptase. We did not observe ultra-structurally apparent granules in any of the tumor cells. These results suggest that in dogs, T-cell lymphomas of intestinal origin resemble mast cell tumors of intestinal origin with respect to cell structure and eosinophil infiltration. It is therefore difficult to confirm the differential diagnosis without immunostaining for mast cell and lymphocyte markers, including mast cell tryptase, c-kit, CD3, and CD79.
60: DISEASES IN A COLONY OF PET HAMSTERS WITH “WET-TAIL”.
Enterocolitis is an important cause of morbidity and mortality in hamsters, resulting in substantial economic loss, treatment effort, and loss of life. Wet-tail is a non-specific clinical sign that can result from enterocolitis caused by a number of different enteric pathogens. Although the term wet-tail historically has been associated with the disease proliferative ileitis caused by Lawsonia intracellulars, other bacterial and parasitic diseases may cause diarrhea. We evaluated 9 hamsters from a large commercial breeding facility to determine the pathogens contributing to the enterocolitis. The most significant findings were severe Tyzzer's disease (Clostridium piliforme) and cestodiasis (Hymenolepis nana) in all of the clinically ill hamsters. Eight of nine hamsters were positive for Campylobacter sp. by PCR. Two of nine hamsters had Clostridium difficile toxins, a commonly reported cause of hamster enterocolitis. The protozoa Giardia, Entamoeba, Spironucleus muris, and trichomonads also were detected. The yeast Torulopsis (Candida) heavily colonized the stomach of all hamsters. Rare spiral Helicobacter-like organisms were seen in intestinal wet mounts. Although L. intracellularis has been commonly associated with wet-tail, there was no evidence of proliferative ileitis in our study. A wide variety of potential pathogens are present in pet-quality hamsters. Over the last year, several infections including lymphocytic choriomeningitis virus (LCMV), salmonellosis, and tularemia have been reported in humans as a result of exposure to pet hamsters, but these diseases were not detected in the hamsters in this study.
61: ENCEPHALOMALACIA WITH VASCULAR THROMBOSIS IN NEONATAL CHICKS.
At five days of age, broiler breeder chicks in a flock of 8,700 showed neurological signs, mainly incoordination and twisted necks. Total mortality was 3% over three days. Remaining birds had no clinical signs. No gross lesions were found in any organs. Histopathological lesions included encephalomalacia, vascular thrombosis and gram-positive coccoid bacterial thrombi in the brain stem. The clinical findings, mortality pattern and lesions are suggestive of Enterococcus infection. Experimental reproduction of Enterococcus infection in chicks has not been successful to date. The pathogenesis is poorly understood.
62: CHARACTERIZATION OF ANEURYSM AND RUPTURE OF ABDOMINAL ARTERIES IN DAIRY CATTLE.
Aneurysm and rupture of abdominal arteries in cattle has been recognized by New York practitioners for at least 20 years. To date, there have been only two published reports of individual cows affected with abdominal arterial aneurysm and rupture; one in Minnesota and one in the Netherlands. In this retrospective study, we describe the gross and histologic changes associated with abdominal arterial aneurysm and rupture in 33 adult dairy cattle. Cases were collected from the period between 1982 and 2005 from 29 farms in upstate New York and northern Pennsylvania. All of the affected cattle were female, Holstein, dairy cattle ranging in age from 2.5 to 5.5 years. Grossly, affected cattle exhibited cyanosis and marked hemoabdomen. There was marked dilatation and rupture of the abdominal aorta (2 cases) or one of its branches, including the mesenteric (9 cases), the gastric (9 cases), the celiac artery (4 cases), and the ruminal artery (2 cases). Granulation tissue and hemorrhage were present at the site of arterial rupture. There was mild to marked hyperplasia of the tunica intima that was often irregular and disorderly with adjacent smooth muscle hyperplasia within the tunica media. The internal elastic lamina was often markedly disrupted, fragmented, and coiled and was occasionally completely absent. There was mild to moderate mucinous change and mineralization within the tunica media. To the authors’ knowledge, this is the first description of the gross and histologic changes in cattle with abdominal artery aneurysm and rupture.
63: SPONTANEOUS BONE MARROW EMBOLISM IN A NEONATAL LAMB.
A 3 day-old lamb received an intratracheal inoculation of sterile saline as part of a control group in a parainfluenza virus-3 (PI-3) infection study. The lamb remained normothermic and showed no clinical evidence of disease. After seven days, the lamb was euthanized (as scheduled for the control group) with intravenous sodium pentobarbital for tissue collection. Gross examination was unremarkable and the lungs were pink and free of lesions. Lung sections were placed in 10% neutral buffered formalin, and then processed for routine hematoxylin and eosin staining. Microscopic examination showed accentuation of multifocal large arteries by intraluminal bone marrow emboli. The emboli contained aggregates of erythroid and myeloid precursors in various stages of development with occasional megakaryocytes as well as large clear spaces consistent with lipid. No other significant lung lesions were present and none of the control or PI-3 infected group animals had evidence of embolism. In humans, bone marrow embolism is typically seen following severe trauma, bone fractures, orthopedic surgery, osteomyelitis, or osteonecrosis. Older sheep are frequently utilized as animal models for bone marrow embolism induced by orthopedic surgery/manipulation. The lamb in this case had no clinical evidence of osteomyelitis or antemortem traumatic injury and this finding suggests the bone marrow emboli may occur spontaneously in young lambs.
64: EHRLICHIOSIS RESULTING IN PERIPHERAL PANCYTOPENIA WITH HYPERPLASTIC MARROW DIAGNOSED USING CYTOSPIN PREPARATIONS.
A 7-year-old, spayed female mixed breed dog was presented for anemia of 8 months duration with recent lethargy, weakness, anorexia, and epistaxis. A non-regenerative anemia (Hct-17.9), leukopenia (1,520/μL; neutropenia, lymphopenia, and monocytopenia), thrombocytopenia (41,000/ μL), hypoalbuminemia (1.9 g/dL reference 3.2–4.3 g/dL), hyperproteinemia (7.3 g/dL; reference 5.2–7.1 g/dL), and a mild prolongation in partial thromboplastin time (22.9 s; reference 8.9–18.7 sec) was identified on initial laboratory results. A lymph node aspirate indicated reactive hyperplasia with mild previous hemorrhage. Marrow aspirates prepared by direct and cytospin methods contained hyperplastic particles with adequate megakaryocytes, normal erythroid, and myeloid maturation with increased myeloid series cells with a shift to immature stages. Using cytospin technique preparations Ehrlichia morulae (1 to 4 per cell) were readily observed in myeloid precursors and occasional macrophages when compared to direct marrow smears. Morulae varied in size from 4 μm to 12 μm. Non-regenerative anemia and the presence of pancytopenia meet diagnostic criteria for the chronic phase of ehrlichiosis; however, due to marrow hyperplasia, acute phase infection was preferred. Ehrlichiosis is an infectious disease in dogs caused by one and occasionally more organisms. Since immature myeloid precursors and macrophages contained morulae, a dual infection with E. canis or E. chaffeensis (mononuclear cells), and E. ewingii (granulocytic cells) was considered. Unfortunately, specific PCR testing was not possible. Titers were suggestive of E. canis (1:8192); however, titers do not speciate organisms since cross reaction is common. Two months after treatment with doxycycline the dog has clinically recovered and titers remain moderately elevated (1:512). This case underscores the use of bone marrow aspirates and the cytospin method of specimen preparation in the identification of Ehrlichia morulae versus buffy coat preparation.
65: RETROBULBAR INFLAMMATORY MYOFIBROBLASTIC TUMOR IN A DOG.
Retrobulbar tumors occur rarely in dogs and cats. Of the primary orbital tumors, sarcomas are much more prevalent than epithelial tumors. As is the case in other tissues, precise histological identification of retrobulbar sarcomas is problematic because histopathology cannot distinguish among the many possible tissues of origin; consequently, most of these neoplasms are grouped under the broad heading of retrobulbar spindle cell tumors. Inflammatory myofibroblastic tumors (IMT) are relatively uncommon lesions in humans characterized by discrete masses composed histologically of a mixture of bland-looking fusiform cells, inflammatory cells and histiocytes. In this study, we describe a case of inflammatory myofibroblastic tumor in an 11 year old, castrated male, mixed breed dog presented with a retrobulbar mass caudoventral to the globe. There are several reports of this tumor in horses, but to the best of our knowledge, this is the first reported case in a dog and is in a unique location. The mass was 2 cm in diameter and sharply demarcated from adjacent orbital tissue. Key histologic features were elongate cells arranged in dense, haphazardly whorling or interweaving streams, scattered throughout which were numerous plasma cells and small lymphocytes and dozens of lymphoid follicles, many with germinal centers. The inflammatory infiltrate was confined to the mass. Differential diagnoses included inflammatory meningioma, inflammatory fibrosarcoma and fibrous histiocytoma. The spindle cells were strongly positive for vimentin and calponin, while a subset of the spindle cells expressed smooth muscle actin and desmin. Pan cytokeratin (AE1/AE3), GFAP, S-100 and Melan A immunohistochemical stains were negative. The co-expression of calponin, smooth muscle actin, and desmin was compatible with derivation of this spindle cell neoplasm from myofibroblasts.
66: PAROSTEAL OSTEOSARCOMA IN A FERRET.
An adult ferret presented with a history of motor difficulty followed by a rapid decline. Upon radiologic examination, a radiodense mass within the fourth thoracic vertebra was associated with a ventral deviation of the trachea. The ferret was euthanized. At necropsy, examination of the vertebra revealed a neoplasm that symmetrically enlarged the diameter of the vertebral body to four times normal and extended into the vertebral canal mildly compressing the spinal cord. The neoplasm was composed of small, osteoblastic spindle cells that lined islands and bony trabeculae of osteoid. Parosteal osteosarcoma is a rare variant of osteosarcoma that arises in periosteal connective tissue on the surface of bone, grows slowly, and does not invade the cortex. Metastasis is seen late in the progression of these neoplasms. In humans, this neoplasm has a predilection for the caudal aspect of the distal femur, but in animals it has been reported in various limb bones, skull, and vertebrae. In this case, the neoplasm extends into the medulla of the vertebral body, however, the characteristic histologic appearance of the neoplasm, symmetrical growth and slow progression is strongly suggestive of parosteal osteosarcoma. This is the first case report of this type of neoplasm in a ferret.
67: MEGAKARYOBLASTIC LEUKEMIA IN A DOG: CLINICAL, HISTOPATHOLOGY, AND IMMUNOHISTOCHEMICAL OBSERVATION.
Acute megakaryoblastic leukemia (AML) is a rare form of myeloid leukemia, but due to insufficient specific morphologic description of the disease and lack of specific immunological reagents, it was not fully characterized until now. We have investigated the clinical, hematologic, and histopathologic features of megakaryoblastic leukemia (M7) in a 10-year-old female Shih-Tzu dog. Megakaryoblastic leukemia was diagnosed using anti-human platelet glycoprotein (GP IIIa) and anti-human von Willebrand factor (vWF). We also assessed expression of CD antigen status on megakaryoblasts, using a CD-79a monoclonal antibody. The most consistent and predominant M7 antigen was seen in bone marrow and spleen. Hematological and histological data coupled with immunohistochemical reactivity for platelet GP IIIa, vWF, and CD79a antigen in blast cells confirmed a diagnosis of M7 megakaryoblastic leukemia.
68: PARATUBERCULOSIS (JOHNE'S DISEASE) IN A MOUFLON (OVIS MUSIMON).
A 2-year-old female captive mouflon with a clinical history of chronic diarrhea and emaciation was submitted to NVRQS. Grossly, the animal had a rough hair coat, fecal staining of the tail and hind legs, and large amounts of watery brownish content in the small and large intestines. In addition, the cecal and colonic mucosa were markedly congested. Microscopically, severe villous atrophy was observed in the small intestine and focal to diffuse infiltration of numerous epitheloid cells and Langerhan's type giant cells were detected in the mucosa, submucosa and serosa of the small and large intestines. These granulomatous lesions were present in the cortex and medullary sinuses of mesenteric lymph nodes, the white and red pulps of the spleen, as well as in the liver. Innumerable acid-fast bacilli were found by Ziehl-Neelsen stain in the cytoplasm of epitheloid and Langerhan's type giant cells in these organs. PCR using a primer pair specific for Mycobacterium avium subspecies paratuberculosis (IS 900) was strongly positive on sections of small intestine, although the organism was not isolated from this organ. Based on the results of histology and PCR, the diagnosis of paratuberculosis (Johne's disease) in this mouflon was made. To our knowledge, this is a first report of paratuberculosis in a mouflon.
69: PARELAPHOSTRONGYLOSIS IN A HORSE.
A two-year-old, gray pony gelding with a reported history of slight head tilt, weakness, and four-limb ataxia was euthanized and necropsied. The pony was afebrile and serologically negative for EEE, WNV, and EHV antibodies. No gross lesions were observed. Histopathologic evaluation revealed multifocal, tractlike areas within the white matter of the cerebellum with swollen, often vacuolated axons and swollen axonal sheaths with occasional intralesional macrophages. These foci were associated with moderate to marked lymphocyte and foamy macrophage infiltrates. One section of cerebellum with underlying pons had a single cross-section of an adult male metastrongyle nematode within the neuropil. The parasite was identified as Parelaphostrongylus tenuis based on morphology. Although a commonly described agent of neurologic signs in llamas and alpacas, P. tenuis is a rarely reported cause of ataxia in horses but should be considered when investigating equine neurologic cases.
70: OVINE ABORTION DUE TO YERSINIA ENTEROCOLITICA.
Yersinia enterocolitica was isolated from an aborted fetus from a flock in North Central Indiana. The fetus was approximately four months gestation. The ewe showed no outward signs of illness. Gross lesions were minimal and included a focally extensive area of the umbilical cord that was wet and gelatinous. Histological changes were consistent with necrotizing subacute placentitis with vasculitis and numerous large basophilic gram-negative coccobacillus bacterial colonies, suppurative omphalophlebitis, suppurative pneumonia, and subacute meningitis. Yersinia enterocolitica was isolated from the lymph node, umbilicus, lung, and stomach contents. Yersinia enterocolitica is a rare cause of abortion in sheep. It has only been reported as an experimental infection and in a number of aborted fetuses examined in the North of England. Most of these abortion cases occurred in late pregnancy and the fetuses did not show any distinctive gross lesions. Histologic changes were typical of a severe generalized bacterial infection. Several reports of Yersinia pseudotuberculosis as a cause of ovine abortion have been reported in the United States. However, until now there have been no reports of Yersinia enterocolitica as a cause of abortion in the United States. In a ten year survey of 1,784 ovine abortions and stillbirths by the South Dakota Animal Disease Research and Diagnostic Laboratory, the most common infectious causes of abortion in sheep were Toxoplasmosis, Campylobacter sp. and Chlamydia psittaci. One case in this survey was due to Yersinia pseudotuberculosis, with no reports of Yersinia entertocolitica as a cause of abortion.
71: PNEUMONIA CAUSED BY MYCOBACTERIUM ABSCESSUS IN A CAPTIVE ATLANTIC BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS).
A 26 year-old captive male Atlantic bottlenose dolphin (Tursiops truncatus) was euthanized after prolonged treatment for pneumonia. Prior to euthanasia, cavitating lesions were identified in the right caudal lung lobe by CT scan and Mycobacterium abscessus was isolated from sputum samples obtained from the blowhole. Gross necropsy findings included diffuse severe pneumonia with multiple cavitating lesions and fibrinous pleuritis. The pharyngeal mucosa contained multifocal ulcers and abscesses. Microscopic examination of lung revealed diffuse pyogranulomatous bronchopneumonia without discrete granuloma formation. Rare acid-fast positive bacterial rods were found within macrophages in the lung in both impression smears and in tissue sections. M. abscessus is a rapidly growing, nontuberculous mycobacterium (NTM) found in the environment that can survive normal chlorination of water. This bacterium causes localized dermatitis in numerous species and also has been reported to cause pneumonia in humans, especially those with concurrent chronic respiratory diseases or immunosuppression.
72: CESTRUM INTERMEDIUM POISONING IN SHEEP.
Cestrum intermedium (Solanaceae) is a tree native to South America. This tree occurs in Brazil, Paraguay and Argentina. C. intermedium has been described as a cause of acute hepatic insufficiency and death in cattle in Brazil, but not in sheep. We describe cases of acute poisoning by C. intermedium in sheep observed in a farm in the State of Parana, southern Brazil. Two adult pregnant ewes out of 66 sheep were found dead, and one of these ewes was necropsied. At necropsy, the liver was mildly enlarged, diffusely and moderately orange-brown, and had an accentuated lobular pattern. There was distension of the gall bladder, pulmonary edema, hydropericardium, and petechial hemorrhages on the epi- and endocardium. Histologically, there was marked, acute, diffuse, periacinar, coagulative hepatocellular necrosis associated with congestion and hemorrhage of the hepatic parenchyma. In the paddock where the flock was grazing, there were several green sprouts of C. intermedium, grown from cut trees, with evidence of having been eaten by the sheep. Cestrum spp. poisoning in ruminants can occur when there is shortage of pasture, when animals are moved from a geographic area where Cestrum spp. is not present to a new area where this tree is found, and when the plant is cut, left on the ground to wither. In the present report, poisoning by C. intermedium occurred in pregnant sheep kept in a paddock with scarcity of forage. Also, cut trees produce shoots that are easily accessible to grazing animals. Other Cestrum spp., such as C. parqui, C. corymbosum var. hirsutum and C. laevigatum have also been reported as a cause of poisoning in ruminants in Brazil.
73: HEPATIC ANGIOMATOSIS IN A DOG.
We report an unusual canine hepatic vascular lesion consistent with lymphangiomatosis. A 10-year-old mixed-breed dog presented with decreased appetite. Radiographs revealed a markedly enlarged liver. Ultrasonographically, the liver was hyperechoic with dilated, tortuous bile ducts. Serum chemistry and CBC abnormalities included a mild hyperbilirubinemia, low PCV and hemoglobin concentration, and eosinophilia. Post-meal bile acids were 24.9 mol/L (normal: <20 mol/L). Hepatic biopsy demonstrated a diffuse presence of variably-sized vascular spaces (up to 5 mm in diameter) that contained few erythrocytes and low to moderate numbers mononuclear leukocytes. The thin-walled vessels were supported on a fine to moderate fibrous connective tissue stroma and lined by a single layer of endothelial cells; they surrounded atrophic hepatocytes that were individualized or in short cords, and increased numbers of biliary ductules. Given the scant erythrocytes present intravascularly, and the lack of a mass effect or significant atypia, angiomatosis was the favored diagnosis. In humans, similar hepatic lesions are diagnosed as lymphangiomatosis, and the condition may be limited to the liver or accompanied by lymphangiomatosis of the spleen, skeleton, or other organs. Only the liver is known to be involved in this case. One case of hepatic lymphangiomatosis was previously reported in a 9-month-old Cocker Spaniel. The case exhibited similar hepatomegaly and ultrasound hyperechogenicity. Histopathology revealed a diffuse, ill-defined vascular proliferation.
74: PERIPHERAL AMYLOIDOSIS AND POLYNEURITIS IN A THOROUGHBRED HORSE.
A 14-year-old, male thoroughbred horse was euthanized due to the presence of multiple subcutaneous masses diffusely scattered over its entire body. The masses were firm, present in chains, and ranged from 0.25–5.0 cm in diameter. On cut section, the subcutaneous masses were composed of concentric layers of tissue. Microscopically, severe multifocal to coalescing granulomatous, necrotizing polyneuritis and perineuritis were present in examined sections of sciatic and infraorbital nerves, and the vagosympathetic trunk. On routine hematoxylin and eosin (HE) staining, amorphous orange material was present within the nerves and surrounding tissue. A Congo Red stain was positive for amyloid. Other microscopic observations included severe multifocal to coalescing chronic hepatitis with marked bile duct hyperplasia (etiology undetermined), and mild renal tubular degeneration. In horses, multiple reports have documented the presence of amyloid in the skin and subcutis spontaneously, or in association with malignant lymphoma or plasmacytoma. Other reports have identified amyloid in the nasal meatus and ventral turbinates, the spleen, the gastrointestinal tract, the kidney, the liver, lungs, heart, pancreas, and bone marrow. Hepatic amyloidosis has also been found in horses used for production of antibodies to bacterial antigens. Although the cause of the polyneuritis and amyloid deposition is unknown, to our knowledge, this is the first report of amyloidosis in the peripheral nerves of a horse.
75: UNILATERAL CENTRAL NERVOUS SYSTEM LESIONS IN A LAMB WITH COPPER DEFICIENCY.
A full term, newborn, male, Suffolk lamb weighing 3.22 kg was presented for necropsy following prolonged parturition and assisted delivery. The dam had arrived at Wake Forest University during her third month of gestation from a local vendor with no additional history. Following birth, the lamb was listless, unable to stand or nurse and had a low body temperature. Euthanasia was performed due to the poor prognosis. At gross necropsy, the dorsal and ventral surfaces of the brain were diffusely red. This congestion was more severe on the right side. The right cerebral hemisphere was mildly, diffusely enlarged, displacing midline structures to the left. Histopathologically, marked, diffuse meningeal congestion with multifocal meningeal hemorrhage was seen. Within the right cerebral hemisphere, the white matter extending from centrum semiovale into the corona radiata was markedly pale in locally extensive areas. This locally extensive, white matter pallor was seen in a sharply demarcated, unilateral distribution extending from the right forebrain throughout midbrain, cerebellum, and brain stem. Marked swelling of perikarya with nuclear eccentricity, marked cytoplasmic eosinophilia, and disruption and loss of Nissl substance was locally extensive within the gray matter overlying areas of white matter rarification (neuronal degeneration). Marked expansion of Virchow's space and perineuronal clearing was seen throughout all sections, though most pronounced on the right side in areas of white matter pallor (cytotoxic edema). Capillary endothelial cell membranes were deeply basophilic and granular in appearance multifocally (mineralization). This lesion is consistent with gestational copper deficiency (congenital swayback) in lambs, though unusual in its unilateral distribution. Hepatic copper content was 8 ppm, indicating deficiency.
76: KERNICTERUS IN AN ADULT DOG.
Kernicterus is characterized by yellow discoloration of brain nuclei, accompanied by degeneration of nerve cells. This condition, which is well recognized in neonatal human infants, develops following penetration of the blood brain barrier by unconjugated bilirubin. A seven-year-old, spayed female Wheaton terrier dog was referred to the Veterinary Teaching Hospital at the Western College of Veterinary Medicine following a three day history of icterus, lethargy, anorexia and occasional vomiting. Initial biochemical analysis revealed an extreme hyperbilirubinemia of 609 μmol/L (reference interval: 1.0–4.0 μmol/L) and increased activity of hepatocellular and cholestatic liver enzymes. Following a surgical liver biopsy the dog deteriorated, seizured and died two days later. Necropsy findings included profound icterus and red and yellow mottling of the liver. Yellow discoloration of the thalamic and subthalamic nuclei was detected on subgross examination of the formalin fixed brain. Histological examination of the brain revealed neuronal necrosis within the previously mentioned nuclei in addition to Purkinje cell necrosis and the presence of Alzheimer type II astrocytes, most notably in the cerebrocortical gray matter. Histological examination of the liver revealed extensive necrosis in a periacinar to bridging pattern that often extended to portal triad areas. Kernicterus in human infants is due to a combination of factors involving bilirubin production, conjugation and transport. Rarely, adult humans are reported to acquire this condition secondary to severe hepatic disease. The few reports of this disease occurring in domestic species involve neonates, namely one foal and one kitten. We report a case of naturally occurring kernicterus in an adult dog secondary to extreme hyperbilirubinemia as a result of fulminant hepatic failure.
77: ROUND CELL LIPOSARCOMA IN THE OMENTUM OF JAPANESE MACAQUE (MACACA FUSCATA).
A 5-year-old, female Japanese Macaque (Macaca fuscata) was admitted to the Department of Veterinary Pathology at College of Veterinary Medicine, Seoul National University for postmortem examination after sudden death. At necropsy, multifocal to coalescing, rough reddish tan to white nodules, ranging from 0.5cm to 1cm in diameter, were noted throughout the omentum. Similar neoplastic nodules were also present in the pelvic region, liver, diaphragm and abdominal wall. Histopathologically, the neoplastic mass was composed of round to polygonal cells that were loosely arranged in sheets with little fibrous stroma. The neoplastic cells had abundant eosinophilic or vacuolated cytoplasm and round to oval, eccentric nuclei with one or two prominent nucleoli. One prominent feature of this tumor was the numerous lipid vacuoles without limited membrane, which were identified with Oil red O staining and ultrastructural study. By immunohistochemistry, the neoplastic cells were positive for vimentin and uniformly negative for sarcomeric actin, smooth muscle-specific actin, cytokeratin, and Factor VIII. Based upon the gross, light microscopic, and ultrastructural findings, this case was diagnosed as round cell liposarcoma of omental origin. To the authors’ knowledge, this is the first report of round cell liposarcoma arising in the omentum of nonhuman primates.
78: A NATURALLY OCCURRING WELL-DIFFERENTIATED BETA CELL CARCINOMA IN A RHESUS MACAQUE (MACACA MULATTA).
Pancreatic endocrine tumors rarely occur in rhesus macaques. A clinically normal, 21-year-old female rhesus macaque that was part of a cognitive aging study was necropsied and a firm, well-circumscribed, dark red 3 × 3 × 2 mm smooth nodule was found on the margin of the tail of the pancreas. Histologically, there were multifocal infiltrative masses throughout the sections of acinar parenchyma within the tail of the pancreas. The masses were unencapsulated, moderately well-circumscribed, expansile and locally invasive with multifocal areas of hemorrhage and entrapped acinar cells within the tumoral stroma. The masses were composed of poorly defined lobules, nests and anastomizing cords of large cuboidal and polygonal (islet-type) neoplastic cells supported by a delicate fibrovascular stroma. The neoplastic cells had strongly eosinophilic, finely granular abundant cytoplasm with indistinct cell borders and large oval, euchromatic to occasionally hyperchromatic nuclei with stippled chromatin. Rarely, a single prominent nucleolus was observed. Mitoses averaged 1 per 10 high power fields. There was no histologic evidence of metastasis. Immunohistochemically, the neoplastic cells were diffusely positive for chromogranin A and insulin, sporadically positive for somatostatin, glucagon and Ki-67 and uniformly negative for gastrin. This is the first report of a naturally occurring well-differentiated beta cell carcinoma in a rhesus macaque. (Supported by NIH grants P01-00001 and P51-RR000168)
79: SYSTEMIC PASTEURELLOSIS IN AN AFRICAN GREEN MONKEY (CERCOPITHECUS AETHIOPS).
Few case reports of systemic Pasteurellosis in nonhuman primates are found in the literature, and most are associated with recent shipment and/or poor clinical condition. This report describes a previously healthy 19 year-old male African green monkey (Cercopithecus aethiops) that was found dead with no antemortem clinical signs. Gross necropsy revealed bronchopneumonia, fibrinous pleuritis, and peritonitis consistent with systemic infection. Additional microscopic changes included adrenalitis, nephritis, and splenitis. Intralesional and intravascular bacteria were also observed. A heavy, pure growth of Pasteurella sp. was recovered on culture from the thoracic and abdominal cavity.
80: INTRA-OVARIAN TERATOMA IN A BITCH.
Teratomas are rare tumors composed of structures derived from multiple embryonic germ cells (ectoderm, endoderm, or mesoderm) that may occur within any particular organ; these tumors are more frequently observed in older animals. A teratoma is described in a young dog. A two-year old female German Shepherd dog was submitted for routine necropsy. The right ovary presented a tumor that weighed 3.3 kg and 22 cm in diameter. Grossly, the tumor was well encapsulated and firm. Cross sections revealed an irregular surface that was predominantly covered by haired skin and cystic areas. The cystic structures were imbedded in a white to opaque extensive interconnecting mass that constituted a mixture of soft and firm connective tissue. Histologically, the tumor was composed of a mixture of various embryonic tissues and cystic structures embedded in a muscular background. The outer margin of the tumor was lined by squamous epithelium. Most large cystic areas were irregular-shaped, filled with keratin and demarcated by stratified squamous, keratinized epithelium, with hair-follicles, and sebaceous glands. Smaller cystic areas were lined only by stratified squamous epithelium without adnexal structures. The solid part of the tumor consisted of a dense sheet of a mixture of irregularly oriented, smooth and skeletal muscles fibers and adipose tissue, blood vessels, cartilaginous tissue, intestinal glandular-like structures, nerve fibers, and nervous parenchyma. A diagnosis of teratoma was based on histological evidence of multiple embryonic germ cells within the tumor.
81: ACUTE RESPIRATORY DISTRESS SYNDROME SECONDARY TO BLASTOMYCOSIS IN A DOG.
A four-year-old, castrated male Saint Bernard dog presented to Iowa State University Veterinary Teaching Hospital's ophthalmology service for unilateral uveitis. A diagnosis of panophthalmitis was made following examination, and cytology of the vitreous revealed prior hemorrhage. The patient was sent home on doxycycline and prednisone, with a presumptive diagnosis of rickettsial disease awaiting definitive test results. One week later, the patient developed a cough with purulent nasal discharge. A diffuse interstitial pattern was observed on thoracic radiographs suggestive of fungal or metastatic disease, and a diagnosis of blastomycosis was confirmed with cytology of a sputum sample. Despite aggressive antifungal therapy and intensive supportive care, the patient died and was submitted for necropsy. On gross examination, disseminated small pale nodules were present on the serosal surface of the lungs. Microscopic examination revealed multifocal to coalescing areas of necrosis and inflammatory cell infiltration consisting of degenerate neutrophils, macrophages, and multinucleate giant cells within the parenchyma. Numerous intralesional yeast bodies morphologically consistent with Blastomyces dermatitidis were observed. The adjacent lung parenchyma often had alveoli filled with seroproteinaceous fluid, neutrophils, sloughed pneumocytes, and macrophages, and alveolar walls were lined by hyaline membranes. Alveolar lesions were consistent with those seen with acute respiratory distress syndrome. Acute respiratory distress syndrome secondary to blastomycosis has been previously characterized in human beings, and has been associated with overwhelming proliferation of organisms within pulmonary parenchyma. To our knowledge, this is the first case of acute respiratory distress syndrome secondary to blastomycosis described in the veterinary literature.
82: OSTEOSARCOMA IN A MALIGNANT PILOMATRICOMA.
A small cystic mass from the right hindquarter of a 14-year-old female German shorthaired pointer enlarged after being static for 5 years. At biopsy, the mass was reported to be 8 cm in diameter. Histopathologically, the neoplasm involved the dermis and subcutis and had epithelial and mesenchymal components. The epithelial component consisted of cords and irregular islands of predominantly basaloid cells with fewer squamous cells. There were frequent foci of abrupt, matrical keratinization adjacent to the basaloid cells and fewer areas of gradual, lamellar keratinization adjacent to the squamous cells. Invasive growth, nuclear atypia and high mitotic rate supported the diagnosis of malignant pilomatricoma for the epithelial component. Surrounding and separating the epithelial structures, there were spindloid to polygonal mesenchymal cells arranged in streams. Polygonal mesenchymal cells were multifocally embedded in an eosinophilic matrix consistent with osteoid. The matrix was mineralized in some areas. Osteoid and bone formation together with marked cellular atypia, high mitotic index and bizarre mitotic figures supported osteosarcoma for the mesenchymal component. The specific histogenesis of the osteosarcoma is unknown, but as pilomatricomas are often associated with osseous metaplasia, the osteosarcoma may have arisen in a metaplastic reaction to the hair follicle tumor. Eleven weeks later, the dog presented with a mass at the previous biopsy site, had increased respiratory sounds and died two and a half weeks later. Necropsy was not performed.
83: MEMBRANOPROLIFERATIVE GLOMERULONEPHRITIS TYPE I IN A YOUNG CAT.
A 9-month-old male Japanese domestic cat had anorexia, appendicular edema and diarrhea. At the first examination, pleural effusion, ascites and bilateral renal swelling were revealed by X-ray examination. The results of a blood test showed non-regenerative anemia (PCV: 20%), thrombocytopenia (26000/1), azotemia (BUN: 79 mg/dL, serum creatinine: 1.8 mg/dL) and hypoproteinemia (3.0 mg/dL) indicative of a renal glomerular disorder. Proteinuria (1583 mg/dl) was observed by urinalysis. Tests for FeLV and FIP were positive and negative, respectively. A percutaneous renal biopsy was performed to confirm the diagnosis. Light microscopy of the specimen revealed proliferative glomerulonephritis (GN) characterized by severe mesangial hypercellularity with an increased mesangial matrix and prominent thickening of the capillary walls, resulting in lobular accentuation of the glomerular tufts. The capillary tufts often adhered to Bowman's capsule. By PAS and PAM stains, frequent duplication of the capillary walls was also observed. Unusual cellular localization was seen in the thickened capillary walls at the periphery of the glomerular tufts, indicating mesangial interposition. Interstitial cellular infiltration and fibrosis were slight. Prominent linear or granular deposition of cat IgG and C3 were globally detected along the capillary walls and in the mesangium by immunohistochemistry. Spontaneous immune complex mediated glomerulonephritis has been well reported in cats and membranous nephropathy is the most frequently observed glomerulonephritis. The glomerular lesion of this case may be consistent with human membranoproliferative glomerulonephritis (MPGN) type I. This may be a rare case that developed in young age. In cases of human MPGN, hypocomplementemia is commonly observed in affected patients, however, we could not evaluate it in this feline case.
84: PRIMARY RENAL HEMANGIOSARCOMA IN A DOG.
A ten-year-old neutered female mix-breed dog was examined with a complaint of anorexia and hematuria. On clinical examination, a palpable mass in the right craniodorsal abdomen was detected. Laboratory findings included regenerative anemia, leukocytosis, proteinuria and hematuria. Radiographic examination revealed a large neoplasm involving the right kidney with no evidence of pulmonary metastasis. Six weeks later, gross postmortem examination confirmed the diagnosis of a renal neoplasia and revealed metastases to the lung, regional lymph nodes, right adrenal gland and liver. Microscopic examination revealed that the neoplasm was a renal hemangiosarcoma originating in renal parenchyma. The neoplastic cells were positive for the CD 31 endothelial marker by immunohistochemistry.
85: PROBABLE CHORDOMA IN A MALLARD DUCK (ANAS PLATYRHNCHOS).
A feral adult female mallard duck (Anas platyrhnchos) was found dead. Gross examination revealed numerous firm confluent multinodular non-mobile masses located in the subcutaneous tissue overlying the dorsal synsacrum. The largest of these nodules measured 10 cm in diameter and showed extensive areas of surface ulceration. Histologically, multiple partially-encapsulated multilobular masses were visible within the dermis and underlying skeletal muscle. Cells within the lobules were arranged within poorly-defined nests that were separated by a fine lacy fibrovascular stroma. The neoplastic cells were a pleomorphic population that contained two predominant cell types. The majority of the cells were polyhedral, large and heavily vacuolated. The cytoplasmic vacuoles often displaced the nucleus to the cell periphery and these cells were interpreted as physaliphorous cells. The second population of cells showed chondroid differentiation and were surrounded by well-differentiated cartilage matrix. Occasional small foci of osseous differentiation were visible within the cartilage. Based on the location of the masses and the histological appearance of the cells, a presumptive diagnosis of chordoma was made. Chordomas are neoplastic proliferations of the notochord remnant. They have been previously reported in humans, ferrets, mink, rats, dogs, and a cat; however, to the authors’ knowledge this is the first report of a chordoma developing in a non-mammalian species.
86: LEARNING OBJECTS: EXAMPLES AND A PROPOSAL.
David Wiley, in his 2002 book entitled “The Instructional Use of Learning Objects,” defined learning objects as any digital resource that can be reused to support learning (http://reusability.org/read/chapters/wiley.doc). Examples of learning object pertinent to the field of veterinary pathology will be demonstrated. Some learning objects will be drawn from existing repositories, such as MERLOT (Multimedia Educational Resource for Learning and Online Teaching) and WISC-Online. These repositories can be accessed at http://merlot.org and http://www.wisc-online.com, respectively. Since the early 1980's, veterinary pathologists have been sharing pathology images via Noah's Arkive (formerly known as the International Veterinary Pathology Slide Bank). These resources meet the definition of learning objects in its broadest sense. Is it time to expand our efforts in sharing digital resources and establish a repository for a variety of pathology learning objects?
87: AN UPDATE ON “NOAH'S ARKIVE”—A GROSS PATHOLOGY PICTORIAL DATABASE FOR VETERINARY PATHOLOGISTS.
Organized databases in veterinary pathology are limited. Organizations such as AFIP, Cornell University, C. L. Davis Foundation and The Jackson Laboratory offer databases focusing on different aspects of veterinary pathology. Noah's Arkive (The International Veterinary Pathology Slide Bank), a comprehensive database of gross pathology images developed by our department, is popular among teachers and students of veterinary pathology. This archive is an excellent resource for creating teaching materials and preparing for the ACVP certification examination. The database, conceived in the early 1980's, is a repository of slides contributed by individuals and institutions around the world. More than 250 contributors from 13 countries donated 2 × 2 slides that are identified by contributor, institution, species, system, tissue or organ and diagnosis or keywords. All of this information is entered into a computer database and cataloged for easy retrieval. In 1986, the 3rd edition became available on laser videodisk. Now in its 8th edition, Noah's Arkive consists of digitized images available on compact disc in the following subsets: Clinical Pathology, Canine, Feline, Equine, Porcine, Ruminant and WHEAL (Wildlife, Human, Exotic Animals, Avian and Laboratory Animal). The database currently contains over 30,000 images including gross lesions, histopathology, normal histology, cytology and hematology, parasitology, poisonous plants, animals with/without clinical signs, schematics, radiographs and electron micrographs. Currently, we are soliciting high resolution, digital images of lesions (preferably in TIF format) from different institutions all across the globe; further submission details can be viewed at http://www.vet.uga.edu/vpp/noah/how.php. We appreciate your continued participation as a user and/or as a contributor. This is a non-profit endeavor and all monies are utilized to maintain Noah's Arkive.
88: PATHOLOGY “SHOW AND TELL” IN THE COMPUTER AGE.
Popularized by Dr. John King and others many years ago, “show and tell” or gross pathology rounds have become a staple of pathology training in most veterinary schools. How the lesions are presented is highly variable and may include passing trays of tissues around the room, passing trays between audience participants or having a central location in which to demonstrate the lesions. Regardless of method, the goal remains the same—to allow pathologists, pathology residents, clinicians, clinical residents and interns, and veterinary students the ability to integrate clinical and historical information and antemortem diagnostic tests with gross pathology. While most veterinary curricula have integrated computerized technology to deliver core material, pathologists still rely on a steady flow of necropsy material to teach pathology. Much of the material is archived into kodachrome collections or digital photo libraries for publication and/or didactic pathology training. However, incorporation of archived images and video files with the real-time gross specimen demonstration has been difficult to achieve in the traditional classroom setting. This pedagogical problem has been solved. In coordination with a capital building project, a new Necropsy Demonstration Teaching Theatre (“Show and Tell” Room) was renovated and outfitted with a state of the art Wolfvision Progressive Scan Visualizer, two ceiling mounted DLP data/video projectors, two gray screens, a high resolution demonstration monitor, and a desktop computer. The hardware and software were purchased with the assistance of a Faculty Innovation in Teaching Grant, awarded by The Department of Computer Information Technology (CIT) to individuals who present a pedagogical problem that is best solved through computerized technology. This system has greatly enhanced the pedagogy of gross pathology rounds or “show and tell.”
89: PILOT STUDY OF INTERNET BASED CONTINUING EDUCATION ON LABORATORY DATA INTERPRETATION USING THE VETERINARY INFORMATION NETWORK'S ONLINE CLASSROOM AND THE DIAGNOSTIC PATHFINDER.
The Veterinary Information Network (VIN), provides online continuing education (VIN-CE) courses using real-time classrooms, message boards, and class web-pages. The Diagnostic Pathfinder (dP) is an Internet based program for teaching data interpretation through evaluation and organization of data into pathophysiologic mechanism-oriented diagnostic cascades. The dP has been used successfully in veterinary schools. This project tested the feasibility of using the dP in the VIN-CE environment to teach an online continuing education course in laboratory data interpretation. Eight VIN members started a course on interpretation of blood gases, pH and electrolytes. From the VIN-CE website, they downloaded the dP, an instructional video and 17 cases focusing on the course topic. Didactic information was presented in 5 real-time sessions; the participants worked on dP cases independently. The instructor viewed the participants’ solutions through a separate program supplied with the dP. Participants installed the dP without problems. They indicated having initial difficulty learning to use the dP that could be lessened by a better introduction to mechanism based data interpretation with cases involving commonly seen laboratory abnormalities. They also requested quicker feedback on their solutions to dP cases. In spite of these problems, they indicated that dP use improved their understanding of the course subject and their laboratory data interpretive skills. We concluded the dP can be used for distance learning with VIN-CE once introductory materials specific to online continuing education and better ways for instructor feedback are developed
90: VIRTUAL SLIDE EXAMINATION TECHNOLOGY FOR TEACHING VETERINARY HEMATOLOGY AND CYTOLOGY.
Case-based learning is often used for teaching clinical pathology. When blood smear abnormalities or cytologic findings are important diagnostic aspects of a case, it is advantageous for the learner to be required to recognize the relevant findings on a slide, rather than being shown photomicrographs of key findings. Access to microscopes and sufficient numbers of glass slides limits the amount of actual slide viewing that can be incorporated into learning and testing situations. Virtual slide examination technology provides additional practice in slide evaluation and expands testing capabilities. Large areas of glass slides are digitized at high resolution and image files are incorporated into computer based teaching and testing modules which allow the images to be viewed in a manner emulating viewing of an actual slide on a microscope. A cost-effective and versatile system of creating such teaching aids was devised. Document Express with DjVu image encoder software from LizardTech (TM) (www.lizardtech.com) was used. It dramatically reduces file sizes with minimal effects on resolution, and a freely-downloadable plug-in provides tools which facilitate viewing and manipulation of slides. A software toolset was constructed to enhance the DjVu plug-in and link case data to the slides. It is easy to create new cases and provide the cases to learners via the Web. Slides can be viewed next to corresponding data, next to a normal slide, or independently of the interface. A constrained note-taking tool enables learners to record their findings and interpretations, providing a means of student assessment and allowing students to record any difficulties they encounter.
91: DIGITAL PHOTOGRAPHY IN THE AUTOPSY TRAINING OF PATHOLOGY RESIDENTS.
Proficiency in gross lesion photography is an important, but often neglected component of pathology resident training. Historically, gross photography has utilized 35 mm SLR cameras and color transparency film. Film processing was usually done in an off site laboratory and required several days. Additionally, gross photography with a 35 mm camera is relatively expensive and time consuming since the specimen must be moved to a photography table provided with attached camera and lighting stand. For the past 3 years the consumer market has been flooded with digital cameras that vary markedly in price; however modestly priced digital cameras (∼$200) are capable of producing 3–4 megapixel images which capture gross lesion detail with excellent quality when viewed on a computer monitor. Digital images can be viewed instantaneously, often with better detail than seen in the post mortem room. Pathology residents at TCSVM are assigned a digital camera when on autopsy duty and encouraged to photograph all lesions (real or suspected). These images are used by the resident in preparing the gross description. Subsequently they are edited for usefulness in conveying information to clinicians in addition to teaching value for veterinary students and pathology residents.
92: CONSERVATION NECROPSY: BALANCING A DIAGNOSTIC NECROPSY WITH MUSEUM PRESERVATION.
The preservation of museum specimens is never more important than when a species is endangered or becomes extinct as was recently the case for the Po'ouli, a Hawaiian honeycreeper. One individual of this endangered avian species was brought into captivity in an attempt to begin a breeding program. The only known remaining two individuals in the wild have disappeared and, unfortunately, the captive male died in November of 2004. A necropsy was performed at the San Diego Zoo and the carcass was sent to the Smithsonian National Museum of Natural History (NMNH) for preservation and preparation. The extent of damage to the skin and bones from the necropsy made preservation of the carcass difficult. This prompted a collaborative effort between NMNH staff and National Zoo pathologists to understand our diverging interests in deceased specimens and establish protocols that would allow for both a complete and diagnostic necropsy and a specimen suitable for museum study and display. These techniques may also be used when an owner wishes to submit a treasured pet for taxidermy. A single ventral midline incision from the pubis to the thoracic inlet allows full exposure and access to internal organs. The neck can be disarticulated at the 6th or 7th cervical vertebra and the head and neck pulled from the skin. This minimizes the amount of incision closure that must occur during specimen preparation. Except for the skull, bones are cut and sampled on only one side of the body so that one intact bone may be used to model the other. The tongue and trachea are detached from the laryngeal apparatus, which remains in place with muscular attachments for studies in anatomy.
93: ENHANCING VOLUNTARY PARTICIPATION IN GROSS PATHOLOGY ROUNDS.
Although attendance at gross necropsy rounds is expected of pathology faculty and trainees, student attendance is strictly voluntary. To increase attendance and student participation in gross necropsy rounds, we initiated a Gross Pathology Auction. “Funny money” was printed with the likenesses of departmental pathologists on various denominations and is collectively called “Roger Dollars”, in honor of our emeritus faculty Dr. Roger Panciera. Students receive the “Roger Dollars” for attending gross necropsy rounds and for answering questions, with different denominations awarded according to the difficulty level of the question asked. The students save their money earned from the weekly rounds, and at the end of the year, the students use the money to bid on items at the Gross Pathology Auction. Items include gift certificates from area restaurants and video stores, limited edition art prints, clothing, veterinary books, interactive learning CD-ROMs, anatomy specimens, gasoline from a local service station, pet food and lunch for two with a pathologist. The auction items are donated from area merchants, student clubs, or obtained from cash donations by alumni. The auction has resulted in an increase in student participation, attendance and retention of attendance throughout the academic year.
94: PATHOLOGY AND ROCK ‘N ROLL.
Alternative teaching methods are often employed to break the monotony of lecture and to increase student participation. It has been well established that the use of acronyms and jingles can be successful learning tools. Following this strategy, the lyrics to popular Rock and Roll songs have been altered to reinforce concepts in pathology. Key concepts from a particular topic (such as inflammation, necrosis or neoplasia) are listed and then matched with a song that has similar phonics or syllable arrangement. The song is then played for the students at the end of the lecture that introduces the topic. The songs have become popular enough that the students concentrate or pay attention in lecture so that they will understand the song. Students participate by singing along (and thus repeating the pathology concepts) and by penning lyrics to new songs (reiterating more pathology concepts) themselves. Years removed from freshman pathology courses, senior veterinary students can recall the songs and lyrics when rotating through on the necropsy floor. The pathology Rock and Roll songs are excellent reinforcement for lecture, helpful for student learning and retention, extremely popular with the students and perhaps more fun for participating Rock and Roll pathologists. Lastly, this method shows that other hobbies and interests shared by pathologists, whether arts and crafts, poetry, drama or staging plays can in some way be used for teaching purposes.
95: A NOVEL PEER TEACHING APPROACH USING A JIGSAW METHOD IN A MICROSCOPY LABORATORY.
A combination of limited instructor availability, variation in student microscopy skills, and a lack of student enthusiasm for the material can limit student engagement in large microscopy laboratories. An established method to engage learners in novel material is to have them teach each other in small groups. A jigsaw method of peer teaching requires that each student teach something and that when all topics are combined it forms a coherent body of knowledge. Each laboratory section contained 30 students and covered 8–10 hematology or cytology slides. Five of these slides were used in the peer teaching exercise. The students were divided into permanent groups of five and each student was assigned to be the expert for one slide. The students were given time before class and at the beginning of each class to become familiar with their expert slide. Next all experts for each slide would gather to discuss their findings. An instructor monitored the discussion to ensure each student understood the correct key points for the slide. After the permanent groups were re-assembled, the students worked on the other slides for the day but were also expected to help their peers with questions about their expert slide. Instructors were available to answer questions the expert was not able to address. Outcomes of this approach were: same or better understanding of the material compared to the previous year's class, decreased number of instructors needed per class, and a more pleasant experience in lab for both students and instructors.
96: STUDENT PERCEPTIONS OF CASE-BASED WRITING ASSIGNMENTS AND EFFECTS ON OVERALL PERFORMANCE IN A CORE CLINICAL PATHOLOGY COURSE.
Volunteers (n = 64) from a second year clinical pathology course were matched by overall GPA and randomly assigned to either group A, receiving eight case-based assignments of 2–6 cases for independent use, group B, receiving identical assignments requiring a written response not evaluated by faculty, and group C, receiving identical assignments requiring written responses which were graded and given directed comments evaluating responses. Assignments were evaluated using a zero, check minus, check, check plus scale, but assessments did not contribute to the students’ grades. Assignments required interpretation and synthesis of laboratory data, mechanistic explanations for abnormalities, and justification of differential diagnoses. Mid-term and final course grades based primarily on multiple choice/short answer midterm and final examinations were not significantly different between groups. A final survey evaluated student perceptions of the assignments as a learning tool. Assignment completion rate was highest in C; C more strongly agreed that assignments helped them understand material and feel more prepared for class and exams than A and B. All groups frequently indicated time constraints as a reason for not completing assignments. A and B cited poor motivation for completion of assignments if their responses were not required or evaluated. Almost all respondents agreed that cases should be used in future years. Even though no difference in course grades was detected, we conclude that directed feedback and/or the requirement to produce a written assignment caused students to engage with material more thoroughly. Students perceived this to be helpful in understanding and reinforcing material and it increased learner confidence, allowing students to feel more prepared for class and exams in clinical pathology.
97: THE ILLINOIS CHAPTER OF THE ACVP: ONE PATHWAY FOR SPARKING STUDENT INTEREST IN VETERINARY PATHOLOGY.
Student chapters of the American College of Veterinary Pathologists were created as avenues to increase student interest in pathology training and careers. Since the first charter was accepted in 2002, most American and Canadian schools have established student chapters and use a variety of activities to focus attention on veterinary pathology. Although it is too soon to know what effect these chapters will have on the current and anticipated future shortage of pathologists, it is an appropriate time to examine which activities have successfully created and maintained student interest in pathology. The Illinois chapter has chosen a dual focus of promoting pathology interest among all veterinary students while providing essential, career-building activities for those who will likely pursue a pathology residency. This strategy allows the chapter to reach many students who might not otherwise have considered working in pathology. Activities include weekly histopathology rounds that complement the pathology curriculum, interclub activities to build connections with other veterinary specialties, and frequent opportunities to speak with pathologists from diverse backgrounds. These have increased active club membership and allowed for expanded activity opportunities. Although not all techniques may be successful at an individual school, the activities of the Illinois Student Chapter of the ACVP can serve as one model to increase interest in and respect for pathology in future clinicians while providing career guidance for the next generation of pathologists.
98: MICROSCOPIC FINDINGS IN THE BRAINS OF PSAPP MICE USED AS A MODEL FOR ALZHEIMERS DISEASE.
Transgenic mice expressing APP (Swedish mutation) and mutated presenilin 1 (PS-1) (Tg+ PSAPP mice) were examined. PSAPP mice express higher levels of A beta 1 40 and 1 42, and develop higher numbers of A beta plaques in the cerebral cortex and hippocampus than do single-mutated mice. PSAPP mice also develop more A beta 1 40 positive plaques and more vascular amyloid deposits compared with PDAPP mice. Brain sections (HE, immunohistochemistry specific for A beta [IHC], and thioflavin-S [TS] for amyloid) from 2, 3, 6, 12, and 18-month-old male and female PSAPP and transgene negative mice were examined. A beta plaques were detected in the parenchyma of the brain as early as 3 months of age in Tg+ PSAPP mice using IHC and TS staining. In HE-stained sections of the brain parenchyma, there was an increase in the incidence and severity of microscopically detectable A beta plaques over time. However, using IHC or TS stains, once plaques were evident at 3 months of age there was no further increase in the intensity of staining at 6, 12, or 18 months of age. Using IHC and TS stains A beta plaques were first detected in brain vessel walls at 6 months of age in Tg+ PSAPP mice. However, there were no clear differences in the incidence or intensity of staining of these vascular A beta plaques between the 6, 12, and 18 month old Tg+ PSAPP mice.
99: COMBINED USE OF cDNA MICROARRAYS AND TISSUE ARRAYS TO EXPLORE SIGNAL TRANSDUCTION.
Study of signaling mechanisms in vivo is complicated by interdependence of multiple pathways and structural complexity of whole tissues. The purpose of this study was to compare the utility of cDNA microarrays and tissue arrays to dissect a single pathway (the MAPK pathway) in the context of a well-studied developmental system, the mouse retina. The transcriptional profile of developing mouse retina at 11 time points from E12.5 to P21 was obtained using a 9000-gene retinal cDNA microarray. Tissue arrays were constructed from retinas at 22 time points from E12.5 to P28. These were immunostained with 25 antibodies against proteins of the MAPK pathway and 3 antibodies against markers of proliferation (PCNA), angiogenesis (PECAM) and rod differentiation (rhodopsin). These three markers were highly correlated in both cDNA microarray and tissue array data indicating that the cDNA microarray data reflected the expected biologic processes. 33 known genes involved in the MAPK pathway were identified by cDNA microarray. Apart from 1, (GRB10) all oscillated at relatively low levels throughout development. Of 14 retinal MAPK related genes for which cognate antibodies were available, 8 were detectable by immunohistochemistry. These localized to cell-cycle specific regions in embryonic retina, and to specific cell layers in differentiating retina. cDNA microarrays are a sensitive indicator of expressed genes but provide relatively little functional information in whole tissues. Correlation of transcriptional and immunohistochemical data is good for highly expressed proteins with less posttranslational modification, but poor for phosphoproteins. Tissue array data provides a more functional overview of the role of signaling proteins, and suggests that individual proteins are utilized repeatedly in multiple contexts of proliferation, cell death and differentiation throughout tissue development.
100: THE PATHOGENESIS OF PNEUMOVIRUS INFECTION IN WILD TYPE AND MIP-2 RECEPTOR DEFICIENT MICE.
Pneumoviruses are important pathogens in a variety of species including humans, cattle, and mice. Bovine respiratory syncytial virus (BRSV) is an etiologic agent in the bovine respiratory disease complex, and the efficacy of modified-live and inactivated vaccines in preventing severe clinical disease following infection has been demonstrated. Human respiratory syncytial virus (HRSV) is a leading cause of hospitalization in pediatric patients; however currently there are no vaccines available for this important infection. Pneumonia virus of mice (PVM) is an underutilized model to study the comparative aspects of pathogenesis and immunoprophylaxis in a small animal model, as infection with this natural rodent pathogen mimics the clinical and pathologic sequelae of HRSV and BRSV infections, while genetic manipulation of mice allows the dissection of host immune responses to the virus. The objective of this study was to examine the pathogenesis of PVM infection in wild type mice and in mice with specific defects in primary host defense mechanisms. Macrophage inflammatory protein (MIP)-2, the mouse ortholog of human interleukin-8, is produced locally in response to PVM infection in vivo. Wild type and MIP-2 receptor-deficient mice were infected intranasally with low-titer, in vivo passaged PVM. Mice were sacrificed sequentially after infection and lungs were examined histologically and immunohistochemically. Bronchiolytic and parenchymal lesions were associated with the presence of viral antigens in airway epithelium and in pneumocytes. The target cells of infection did not vary between the strains of mice examined; however, the amounts of virus in the lung and the nature of the inflammatory responses were strain-specific. These results further substantiate the virulence of PVM in the murine lung, and demonstrate the primary target cells for infection. These results also provide evidence for additional immunological mechanisms that may be important in protection from clinical disease following infection.
101: DIFFERENTIAL GLYCOGEN CLEARANCE IN SLOW VS. FAST TWITCH MUSCLE FIBERS IN POMPE KNOCKOUT MICE AFTER RECOMBINANT HUMAN ALPHA-GLUCOSI-DASE TREATMENT
Pompe disease in the Pompe knockout [6neo(-)/6neo(-)](KO) mouse results in the pathologic accumulation of lysosomal glycogen primarily within skeletal myocytes and cardiomyocytes. Intravenous administration of recombinant human alpha-glucosidase (rhGAA) can result in significant glycogen clearance from both cardiomyocytes and skeletal myocytes, however, the amount of clearance varies from one skeletal muscle type to another. We sought to determine what role muscle fiber type predominance played in this variability. To examine this question in the Pompe KO mouse model we delivered intravenous doses of rhGAA once on Day 1, once on Day 14, and harvested a variety of fast- and slow-twitch muscles on Day 28. We measured muscle fiber type content, glycogen clearance and capillary density. Recombinant human-GAA administration resulted in differential clearance of glycogen in the various muscles examined. Slow-twitch muscles cleared glycogen significantly more efficiently than fast-twitch muscles. There was a modest correlation between percent slow twitch fiber content and glycogen clearance. There was a stronger correlation between capillary density and glycogen clearance. Glycogen clearance was linearly related to capillary density, suggesting that at the high doses used in this study the differential glycogen clearance observed between muscles was largely due to differential bioavailability of rhGAA regulated by blood flow.
102: IMMUNOPATHOLOGY OF SIV INFECTION AND AIDS.
Like HIV-infected humans, simian immunodeficiency virus rapidly and selectively infects, replicates in, and destroys memory CD4+ T cells co-expressing CCR5 (viral target cells) in rhesus macaques, resulting in loss of the majority of the body's CD4+ T cell pool within 21 days of infection. The vast majority of these cells reside in the intestinal tract and other mucosal tissues but selective loss of these target cells is detectable throughout the lymphoid system. Restoration of memory CD4+CCR5+ T cells directly correlates with improved clinical course and lower viremia, but these cells are never restored in macaques that progress to AIDS. Continuous and effective antiviral treatment initiated within days of SIV infection can rescue mucosal CD4+ T cells, but delaying therapy for a couple of weeks does not restore these vital helper memory cells, despite effective control of viremia. Similarly, monkeys that appear protected in vaccine challenge studies may in fact harbor smoldering infection in the intestine with continuous CD4+ T cell loss, despite undetectable plasma viremia. The rapidity and severity of the loss of memory CD4+ T cell function is a major reason no cure or vaccine is in sight. In fact, converging evidence suggests that other primate species altered fundamental aspects of their immune system, such as eliminating the need for CD4+CCR5+ T cells (yet maintaining CD8+CCR5+ T cells), to avoid the deleterious effects on the immune system of this infection. This and other data suggest that conventional immune responses simply may not be adequate to control or prevent HIV infection. Regardless, radical thinking in vaccine design and treatment strategies will be required to combat HIV infection.
103: RENAL-HEPATIC-PANCREATIC DYSPLASIA AND PHOTORECEPTOR DEGENERATION IN MICE LACKING ARL3 PROTEIN.
We generate and analyze knockout mice utilizing a high-throughput mutagenesis and phenotyping system that characterizes the function of individual proteins in a systematic search for novel drug targets. Frequently, this screening process identifies genes that, when mutated, result in diseases that may have human counterparts. We have found that mice deficient for ADP-ribosylation factor-like 3 (Arl3) developed severe polycystic disease typical of the renal-hepatic-pancreatic dysplasia found in autosomal recessive polycystic kidney disease in humans. The pathologic lesions in these mice included cystic cortical and medullary renal tubules, dilated bile and pancreatic ducts, and hypoplasia of acinar pancreas. In addition, Arl3 deficient mice exhibited photoreceptor degeneration as early as postnatal day 14. Arl3 protein is one of several Ras-related small GTP-binding proteins thought to be involved in intracellular signaling and vesicular trafficking pathways, including the movement of cellular components within cilia. Interestingly, Arl3 has been localized within the Leishmania flagella, in the ciliary compartment group of several organisms, and to the connecting (primary) cilium of retinal photoreceptors. Primary cilia are present on the surface of most cells but until recently were believed to be vestigial organelles. Although primary cilia are non-motile (except for the embryonic nodal cilium), they have specialized chemosensory (olfactory neurons), photosensory (retinal photoreceptors), or mechanosensory (renal epithelium) functions. In recent years, the defective assembly, maintenance, or function of primary cilia has been linked to the pathogenesis of polycystic kidney disease. Defective primary cilia on renal cells apparently fail to sense urinary flow within the tubules, and loss of this important function leads to aberrant cellular proliferation and cyst formation. Our findings indicate that Arl3 protein is critical for the normal development of the kidney, retina, biliary tree, and pancreatic ducts and suggest that defective function of the primary cilium may be involved in the pathogenesis of the lesions in these mice.
104: EXPRESSION OF INNATE IMMUNE GENES (SURFACTANT PROTEINS A AND D, SHEEP BETA-DEFENSIN-1, TLR4) BY RESPIRATORY EPITHELIA AT PRETERM GESTATION IS LESS THAN AT FULL-TERM.
Respiratory syncytial virus (RSV) infection is the most common cause of respiratory disease leading to hospitalization in children and preterm infants are especially susceptible. Our previous work has shown that preterm lambs, like human infants, have increased RSV susceptibility and severity compared to full-term lambs and older lambs. Respiratory epithelium is an initial site of RSV infection and epithelial cells are vital to innate and adaptive immune responses. However, the extent of innate immune gene expression by epithelia at preterm is poorly understood. In this study, expression of surfactant proteins A and D (SP-AD), sheep beta-defensin-1 (SBD-1), and Toll-like receptor 4 (TLR4) mRNA were determined in whole lung homogenates from five lambs at 115 days of gestation (80% gestation), four lambs from 130 days of gestation (90% gestation) and four lambs at full-term by real-time RT-PCR. Expression was highest at full-term and little expression was seen at 115 and 130 days gestation. In addition, gene expression was determined in type II cells identified by fluorescence using anti-CD208 antibody, non-fluorescent bronchiolar cells (CD208-), and bronchial cells, retrieved by laser capture microdissection and assessed by real time RT-PCR. CD208+ cells had progressively increased mRNA levels of SP-A with gestational and post-gestational age, but not CD208- and bronchial cells; SP-A expression in CD208+ cells was greater than that by bronchial cells. SP-D expression was not consistently changed in any cell type. SBD-1 expression variably increased in CD208+ and CD208- cells with age, but decreased in bronchial cells, while TLR4 expression was variably increased with age in CD208+ and - cells. In addition, SP-A protein production, as determined by fluorescent microscopy, was more intense and widely distributed in bronchiolar and alveolar epithelial cells than SP-D; both SP-A and SP-D protein production increased progressively with fetal and post-partum age. These studies suggest that expression of SBD-1, SP-AD, and TLR4 mRNA and protein production of SP-AD are lower or limited preterm, and this may underlie susceptibility to increased severity of RSV at preterm.
105: A FERRET MODEL OF BACTERIAL SINUSITIS AND OTITIS MEDIA FOLLOWING INFLUENZA.
Because influenza viruses are not natural pathogens of mice, we sought to develop a ferret model of secondary bacterial infections to study differences in the ability of different viral subtypes to prime for these complications. Groups of 6–7 week old ferrets were mock infected with PBS or were infected with 1 × 107 infectious doses of either influenza virus A/Fuji an/411/02 (H3N2), A/Taiwan/1/86 (H1N1), or B/Singapore/222/79 followed 5 days later by 1 × 107 CFU of a type 3 Streptococcus pneumoniae strain which had been transformed to express luciferase. Ferrets had daily nasal washes for viral and bacterial titers and were imaged for bioluminescence at sites of likely secondary infection for 4 days, followed by euthanasia and pathologic examination. Bacteria could be recovered from daily nasal washes at significantly higher titers (p < 0.05 by ANOVA) in influenza-infected ferrets (9.0 × 105, 4.1 × 106, and 4.5 × 106 CFU/ ml nasal wash on day 3 for H3N2, H1N1, and influenza B infected animals respectively, compared to 3.4 × 104 from mock infected animals). Upon bioluminescent imaging colonization of the proximal nasopharynx could be observed in all animals, but 3/5 H3N2 infected ferrets had either otitis media or sinusitis, compared to 1/6 H1N1 infected ferrets, 1/6 influenza B infected ferrets, and 0/6 mock-infected ferrets. Histopathological examination of sinuses and ears supported the diagnosis of the secondary infections. This model provides the unique ability to follow the progression of bacterial infections in influenza infected ferrets. A non-adapted human influenza virus of the H3N2 subtype better supported secondary sinusitis and otitis media than did other influenza viruses, supporting the clinical observation in humans of an increased morbidity and a higher complication rate in years where H3N2 viruses dominate.
106: SYSTEMIC DISEASE IN CATS FROM INFLUENZA A VIRUS (H5N1) INFECTION.
The ongoing outbreak of avian influenza A virus (subtype H5N1) infection in Asia is of great concern because of the high human case fatality rate and the threat of a new influenza pandemic. Case reports in humans and felids suggest that this virus may have a different tissue tropism from other influenza viruses, which are normally restricted to the respiratory tract in mammals. To study its pathogenesis in a mammalian host, domestic cats were inoculated with H5N1 virus intra-tracheally (n = 3), orally (n = 3), or by horizontal transmission (n = 2), and examined by virological and pathological assays. In all cats, virus replicated not only in the respiratory tract but also in multiple extra-respiratory tissues. Virus antigen expression in these tissues was associated with severe necrosis and inflammation seven days after inoculation. In orally inoculated cats only, virus-associated ganglioneuritis also occurred in the submucosal and myenteric plexi of the small intestine, indicating direct infection from the intestinal lumen. All cats excreted virus not only via the respiratory tract, but also via the digestive tract. This study in cats demonstrates that H5N1 virus infection causes systemic disease and has potential novel routes of spread within and between mammalian hosts.
107: EXPERIMENTAL TRANSMISSION OF TRANSMISSIBLE MINK ENCEPHALOPATHY (TME) TO CATTLE BY INTRACEREBRAL INOCULATION.
To compare clinicopathological findings of transmissible mink encephalopathy (TME) with other transmissible spongiform encephalopathies (TSE, prion diseases) that have been shown to be experimentally transmissible to cattle (sheep scrapie and chronic wasting disease, CWD), 2 groups of calves (n = 4 each) were intracerebrally inoculated with TME agents from 2 different sources (mink-derived TME and bovine-derived TME). Two uninoculated calves served as controls. Within 15.3 months post inoculation (PI), animals from both inoculated groups developed clinical signs of central nervous system (CNS) abnormality. CNS tissues of all infected cattle had microscopic spongiform encephalopathy (SE); and abnormal prion protein (PrPres) was detected in their CNS tissues by immunohistochemistry (IHC) and Western blot (WB) techniques. These findings demonstrate that intracerebrally inoculated cattle not only amplify TME PrPres but also develop clinical CNS signs and extensive lesions of SE. The latter has not been shown with other cross-species transmitted TSE agents (scrapie and CWD) similarly inoculated into cattle. The findings also demonstrate that the diagnostic techniques currently used for bovine spongiform encephalopathy (BSE) surveillance would detect TME in cattle should it occur naturally. However, it would be a diagnostic challenge to differentiate TME in cattle from BSE by clinical signs, neuropathology, or by IHC and WB.
108: PATHOLOGY OF NUCLEAR TRANSFER PRODUCED CLONED CALVES.
Current commercial success of cloning is limited by low success rates and health status of offspring. Although healthy animals are born after somatic cell nuclear transfer (NT), the success is low, 2–10%, with significant perinatal losses. Furthermore, there is limited information on the physiological or pathological condition of cloned animals. The aim of this study was to examine late term aborted fetuses and neonatal deaths from several cloning trials to establish causes of mortalities. Calves were derived from traditional NT (n = 10) and hand-made cloning (HMC n = 11). Calves examined were either aborted or euthanised because of illness, congenital abnormalities or environmental accident. There were no macroscopic or histopathological differences between NT and HMC derived calves, therefore the data were pooled. Congenitally abnormal calves had multi-focal bilateral cystic renal calyces (n = 12) with concurrent pyelonephritis and cystitis (n = 2), nodular irregular fibrotic livers (n = 15) with biliary tract hyperplasia and cardiomegaly (n = 15). There was severe lateral flexion of the neck and thorax (n = 4) with marked flexion of the distal limbs. In calves with no congenital abnormalities, additional findings included septicaemia (n = 2), bronchopneumonia (n = 1), renal failure (n = 1) and dislocated hip (n = 1). All calves were negative for bovine viral diarrhoea, Akabane and Aino viruses. Two recipient dams had marked hydroallantosis at parturition. Also some animals showed markedly enlarged placentomes (n = 2), hypoplastic placentomes (n = 1) and excessive adventitious placentation (n = 3). Abnormalities of the internal organs of this kind can be found in non-cloned calves, albeit at a significantly lower rate and usually not in the same animal.
109: ENDURANCE EXERCISE TRAINING REDUCES DIET-INDUCED AORTIC ATHEROSCLEROSIS IN MALE PIGS.
The objective of this study was to examine the influence of exercise training on diet-induced aortic atherosclerosis in male pigs. Mature male Yucatan pigs were fed a normal (NF) or a high fat and cholesterol (HFC) diet and assigned to treadmill exercise (Ex) or sedentary (Sed) groups. After 20 weeks plasma was collected for lipid analyses, the pigs were euthanized, and the abdominal aortas were harvested, fixed in formalin, stained with Sudan IV for fatty streaks, and processed for histopathology. The intima media thickness (IMT) was measured in cross sections of the abdominal aorta stained for elastin. Collagen content of the lesions was measured using Picrosirius red staining. Immunohistochemistry was performed for isoforms of nitric oxide synthase (eNOS, iNOS, nNOS) and cyclooxygenase (COX-1, COX-22). Sections were photographed digitally with an Olympus BX40 photomicroscope and Spot Insight Digital camera. Staining areas were quantified with ImageProPlus image analysis software. Triglycerides, total cholesterol, low density and high density cholesterol, percent Sudanophilia, and IMT were significantly elevated in HFC- as compared to the NF-fed pigs. There was no difference in plasma lipids or Sudanophilia with exercise; however IMT and immundreactive iNOS were significantly reduced, while immunoreactive COX-1 was increased in HFCEx as compared to HFCSed male pigs.
110: THE MOUSE AS AN EXPERIMENTAL MODEL TO MODULATE ANTIBODY RESPONSE TO PORCINE REPRODUCTIVE AND RESPIRATORY VIRUS GLYCOPROTEIN 5 USING ANTIIDIOTYPIC ANTIBODIES.
The idiotype (Id) network theory of immune regulation proposes that the humoral immune response (Ab1) to a given antigen is regulated by a series of Ids. Anti-idiotypic antibody (anti-Id or Ab2), has been found useful for the production of diagnostic reagents and vaccines. Ab2, an internal image anti-Id Ab2, recognizes an Id within the antigen-combining site and bears a structural resemblance to the original antigen. A given idiotypic antibody is under the control of an anti-Id, whereas the anti-Id is regulated by anti-anti-Id (Ab3) that recognizes the original antigen. Porcine reproductive and respiratory syndrome virus (PRRSV) is currently among the most significant pathogens of swine worldwide. Glycoprotein 5 (GPS) is the major envelope protein and serum neutralizing antibody titers are significantly correlated with anti-GP5 titers. To develop more effective vaccines it is imperative to understand the mechanisms that regulate immunity to PRRSV. Therefore, we generated and characterized a monoclonal anti-Id (MAb2) produced against a monoclonal antibody (MAb1) specific for GP5 of PRRSV and determined the structure of the Ab3. The MAb2 was classified as Ab2 preparation because the anti-Id inhibited the binding of MAb1 to PRRSV antigen, and the Id-anti-Id interaction was inhibited by PRRSV antigen. Immunization with MAb2 induced an anti-GP5 response. Serologically, the Ab3 induced with anti-Id MAb2 competed with Ab1 binding to PRRSV antigen, inhibited the Id-anti-Id reaction, and expressed a similar Id present on the MAb1. These results indicated that an Id-anti-Id network may be operational in modulating the immune response to GP5 in PRRSV infection.
111: ABILITY OF CD4+ T CELLS TO INDUCE MACROPHAGE ACTIVATION AND KILLING OF INTRACELLULAR MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS.
The objective of this study was to test the hypothesis that CD4+ T cells from Mycobacterium avium subspecies paratuberculosis (M. a. ptb) infected cattle induce macrophage activation and intracellular killing of M. a. ptb. To test this hypothesis we collected peripheral blood mononuclear cells from blood samples of M. a. ptb infected and non-infected cattle. From these samples we isolated macrophages and CD4+ T cells. Macrophages were infected with M. a. ptb at a multiplicity of infection of 10:1 for 4 hours. Infected macrophages were co-cultured for 48 hours with autologous CD4+ T cells at a ratio of 5 CD4+ T cells/1 infected macrophage. IFN- and nitrite levels were measured in culture supernatants. Macrophages lysates were serially diluted and M. a. ptb colony unit forming units (CFU) determined. Our results demonstrate that infection status of the animal did not significantly affect levels of IFN- or nitrites in supernatants of co-cultured cells. Addition of CD4+ T cells to infected macrophages did not lead to significant increases in nitrites over infected macrophages alone. There was an increase in IFN- in supernatants of co-cultured CD4+ T cells and infected macrophages. Infection status of the animal did influence recovery of CFU. The lowest CFU were recovered from macrophages originating from infected cattle, which were not changed by addition of autologous CD4+ T cells. The highest CFU values were from macrophages originating from non-infected cattle. Addition of autologous CD4+ T cells to these cultures significantly decreased the numbers of CFU recovered. These findings suggest that addition of CD4+ T cells from non-infected animals had the greatest effect on bacterial killing in infected macrophages.
112: MULTIPLE AUTOIMMUNE SYNDROME IN RAG2-/- MICE RECEIVING ADOPTIVE TRANSFER OF T EFFECTOR CELLS.
Recombinase-activating gene-2-deficient mice (Rag2-/-) are unable to undergo V-D-J rearrangement and are left without functional T or B lymphocytes. Adoptive transfer of syngeneic wild-type (WT) lymphocyte subsets into Rag2-/- mice permits evaluation of their specific activities in vivo. Our objective was to characterize phenotypic outcomes in 129S6/ SvEv Rag2-/- mice following adoptive transfer of CD45RBhiCD4+CD25- effector T cells (Teff), alone or in combination with other lymphocyte populations. Histopathological evaluation was completed on 129S6/SvEv Rag2-/- (n = 272) and WT (n = 16) mice. Serum biochemistry, special stains and immunohistochemistry were performed on select cases. Adoptive transfer of Teff cells resulted in site-specific lesions of multiple organs including bile ducts, stomach, and lower urinary tract. Despite severe cholangitis, serum biochemical evidence of cholestatic liver disease was absent. Minor or inconsistent tissue targets included extragonadal sex organs, lacrimal, pancreatic and salivary ducts, oropharynx, lung and skin. Articular synovitis with pannus was also present. Additionally, angiocentric inflammation targeted skeletal muscle, mesentery and gonads. Unlike some other murine models of autoimmunity, endocrine organs and glomeruli were spared. Taken together, lesions in Teff recipient mice resembled human multiple autoimmune syndrome comprising autoimmune exocrinopathy, dermatomyositis, polyarteritis, and rheumatoid arthritis. No lesions developed in non-recipient mice, nor in animals receiving whole splenocytes or CD45RBloCD4+CD25+ regulatory T cells (Treg), alone or in combination with Teff. Thus, Teff were necessary and sufficient to induce autoimmune disease. Lesions were markedly attenuated in animals that received p50-/-p65+/- (NF-kB-hypozygous) Teff, suggesting a requirement for NF-kB in Teff function. In summary, multiorgan autoimmunity results from unregulated Teff activity. Our results are consistent with a model whereby Treg deficiency, host genetics, and environment influence the development of multiple autoimmune syndrome.
113: LYME BORRELIOSIS REGIONAL LYMPHADENOPATHY UNVEILED
Lyme borreliosis has emerged as the most common vector-borne illness in the United States. This disease is caused by the spirochete Borrelia burgdorferi following transmission by ticks belonging to the genus Ixodes. The mechanisms that underlie the regional lymph-adenopathy that ensues the tick bite are unknown. In vitro, B. burgdorferi induces strong B cell proliferation, likely due to stimulation via Toll-like receptor 2 binding of Outer surface protein A. This could suggest that the lymphadenopathy might be the result of nonspecific B cell activation as an early immune evasion mechanism to avoid or delay the induction of effective specific humoral immune responses. Using a murine model of Lyme borreliosis, we confirmed the presence of an infection-induced lymphadenopathy. We furthermore showed that the enlargement was dependent on presence of live spirochetes that have migrated to the cortical areas of the lymph node. The lymph nodes retained normal architecture with all compartments greatly distended. Most prominent were a strong increase in cortical B-cells with infrequent germinal centers by day 7 post infection and increase of plasmablasts in the medullary sinuses. B. burgdorferi-specific antibody-secreting cells appeared in the lymph node as early as day 5 post infection with responses peaking at day 10. Beginning from two weeks post infection, B. burgdorferi-specific B-cells were also detected in the bone marrow. Hybridomas generated from lymph nodes at day 8 after infection showed that 24% of the B-cell hybridomas were reactive against a pool of only eight B. burgdorferi recombinant proteins. These data strongly suggest that lymphadenopathy in early Lyme borreliosis is due to the initiation and development of specific B cell responses that nonetheless fail to provide immune protection from persistence.
114: MEASLES VIRUS REDUCES INTERLEUKIN 12 IN COTTON RAT MACROPHAGES.
Measles virus (MV) infection induces severe immune suppression that results in enhanced susceptibility to bacterial infections making MV a major cause of morbidity and mortality in children in the developing world. Macrophages, important effector and regulatory cells linking innate and adaptive immunity have been shown to be infected during MV infection and seem to play a central role in MV induced immune suppression. The present work tested the hypothesis that MV infection induces decreased IL12 expression in macrophages. The cotton rat was used as the animal model to study MV induced immunosuppression because they are the only rodents where MV replicates in the respiratory tract. To compare in vivo results with in vitro data we established the culture of bone-marrow derived macrophages using mouse M-CSE Macrophages were characterized phenotypically by plastic adherence, morphology, cytoplasmic non-specific esterase staining, and staining for cell surface antigens (i.e. MHCI, MHCII and CD59). Macrophage function was characterized by the ability to phagocytose and kill bacteria and expression of interleukin 12p35 and interleukin 18. IL12p35 expression was evaluated using real-time RT-PCR from macrophages infected with wildtype or vaccine measles virus. Infection of macrophages with wildtype virus decreased IL12p35 expression by two logs, whereas vaccine virus had only a small effect. IL18 expression was not affected. IL12p35 expression was also reduced in ex vivo alveolar macrophages from animals infected with wildtype virus on day 10 after infection. No reduction in IL12 levels were seen after vaccine virus infection. Future work will address the impact of the reduction of IL12 by measles virus on phagocytic capacity of macrophages and the development of a T helper response.
115: TBET AND IFN-GAMMA-INDEPENDENT GASTRITIS DUE TO HELICOBACTER IN MICE.
Background: Gastritis due to H. pylori is induced by a Th-1 mediated response that is CD4-cell and IFN dependent. T-bet is a transcription factor that directs differentiation of and IFNg secretion by CD4+ Th1 T cells. Paradoxically, adoptive transfer of CD4+ T cells from Tbet-knockout mice induces gastritis in H. pylori-infected recipient SCID mice. The goal of this study was to determine if gastritis in recipient mice is Th1 biased. Methods: H. pylori-infected and uninfected SCID mice were given CD4+ splenocytes from C57BL/6 or congenic Tbet-KO mice by intraperitoneal injection. Twelve weeks after transfer, stomachs and spleens were harvested for determination of splenocyte response to H. pylori antigen and quantitation of cytokine expression in gastric mucosa. Results: Gastritis developed in infected mice given T cells from either C57BL/6 or Tbet-KO mice but not in uninfected mice or mice that were not given T cells. In recipients of C57BL/6 CD4 cells gastritis was associated with a delayed-type hypersensitivity (DTH) response to H. pylori antigen and elevated IFNg expression in gastric mucosa, but neither of these was present in H. pylori-infected recipients of CD4 cells from Tbet-KO mice. Gastritis was present in both groups of infected recipient mice. Gastritis, DTH, and IFNg expression were minimal in uninfected mice and in SCID mice that did not receive CD4 cells. Conclusions: In spite of the absence of IFNg expression and consequent failure of Th1 differentiation, CD4 T cells from Tbet KO mice induce gastritis in H. pylori-infected mice. This suggests that Th1-independent mechanisms may contribute to gastric inflammation and damage due to H. pylori.
116: iNOS EXPRESSION WITHIN THE LIVER AND INTESTINES OF ATHYMIC NUDE AND BALB/C MICE INFECTED WITH MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS.
The role of iNOS in the control of Mycobacterium avium subsp. paratuberculosis (M. a. ptb) infection has not been extensively studied. The primary objective of this study was to compare the relative levels of iNOS expression in the livers and intestines of BALB/c mice and athymic nude mice infected with M. a. ptb. Additionally. the relative number of acid-fast bacilli within the hepatic and intestinal granulomas was scored and compared to the relative levels of iNOS expression. Immunohistochemistry was used to detect iNOS expression and the Ziehl-Neelsen method was used to detect acid-fast bacilli. At 3–7 days of age, the athymic and BALB/c mice were given a single intraperitoneal injection with approximately 3 × 107 colony-forming units of M. a. ptb. All of the mice were euthanized 5 months after inoculation. The level of iNOS expression was higher in the BALB/c mice relative to the athymic mice in both the liver and intestines. The elevated levels of iNOS also corresponded to a lower number of acid-fast bacilli both within the hepatic and intestinal granulomas. An unexpected finding was that there was a difference in iNOS expression between the intestine and liver, despite a similar bacterial burden in each of the organs; this difference was more apparent in the athymic nude mice. iNOS expression was higher in the liver relative to the intestine. These data indicate that iNOS production is important in the control of M. a. ptb in our mouse model of M. a. ptb infection, and that expression of iNOS varies between organs following M. a. ptb infection.
117: DELETION OF CRMA FROM COWPOX VIRUS CAUSES ATTENUATION IN AN INTRADERMAL MODEL OF POXVIRUS PATHOGENESIS.
The causative agent of smallpox is variola virus, a poxvirus in the genus Orthopoxvirus. There is interest in developing an animal model of poxvirus infection that will mimic smallpox in humans. There are three established models of Orthopoxvirus infection in mice; ectromelia, cowpox (CPV) and vaccinia virus (VV). Ectromelia causes hepatic disease that is not observed in humans with smallpox. CPV and VV cause dermal and respiratory disease similar to infection with variola. CPV and VV encode three serine protease inhibitors (serpins). In both viruses, one serpin has an aspartic acid at a key site in the serpin (P1). The P1 amino acid determines proteinase specificity. The VV serpin, SPI-2, inhibits pro-inflammatory and pro-apoptotic proteases. The CPV serpin, CrmA, inhibits caspases involved in inflammation and apoptosis and granzyme B. CrmA prevents inflammation and apoptosis during CPV infection of chicken chorioallantoic membranes; but its function during poxvirus infection in animals with a developed cell-mediated immune response is unknown. We have evaluated the role of CrmA using a mouse model of dermal poxvirus infection. We show that CrmA deletion from CPV causes smaller inflammatory lesions and attenuation of the virus. At day 16 post inoculation, virus can be recovered from CPV infected ears but not from ears infected with virus deleted for CrmA. In contrast, intradermal inoculation of VV deleted for SPI-2 causes larger inflammatory lesions and is more virulent than VV. This study illustrates the differences observed with Orthopoxvirus infection in the same animal model and emphasizes the need to examine several models of poxvirus infection in animals before generalizations are made about smallpox in humans.
118: ACINAR CELL APOPTOSIS IN THE SALIVARY AND LACRIMAL GLANDS OF MICE AFTER ENCEPHALOMYOCARDITIS (EMC) VIRUS INFECTION.
EMC virus is a cardiovirus which belongs to the family Picornaviridae, and it has a wide host-range. In mice, EMC virus affects the central nervous system, heart, pancreas and testis, and it has been widely used as a tool for inducing diabetes mellitus since the highly diabetogenic variant (EMC-D) was established. EMC virus is also known to induce sialodacryoadenitis in mice. In the present study, the development of early lesions was examined in the salivary and lacrimal glands of DBN/2 mice for 4 days after intranasal inoculation with 102 PFU/head of EMC-D virus. Pyknotic acinar cells, being positive for TUNEL and cleaved caspase-3 and having ultra-structural characteristics of apoptotic cells, developed earlier and were more frequently observed in the parotid gland than in the lacrimal gland, whereas destruction of acinar cells and interstitial infiltration of macrophages were more prominent in the latter than in the former. In addition, corresponding to the results of the detection of viral RNA signals, small aggregates of virus-like particles having typical size and structure of EMC virus were frequently observed in both the cytoplasm and the nucleus of acinar cells in the lacrimal gland while they were found only in the cytoplasm of a few acinar cells in the parotid gland. Ductal epithelia were free from lesions. In conclusion, it was clarified that EMC virus-induced acinar cell apoptosis was more prominent in the parotid gland showing less viral replication and cellular destruction compared with the lacrimal gland.
119: EXPERIMENTATION WITH SIMIAN HERPESVIRUSES: ASSESSMENT OF BIOSAFETY AND MOLECULAR CONTAMINATION.
Performance of in vivo experiments with viruses highly pathogenic for humans raises concerns regarding the possible persistence of infectious virus in pathology specimens. While fixation of tissues may inactivate infectious virus, there is a concern that fixation may also degrade viral nucleic acid and/or antigens, thereby limiting the ability to detect virus in tissues by PCR or immunohistochemistry. The question then arises as to what is the shortest length of time necessary for formalin fixation to inactivate infectious virus, while having a minimally negative impact on the results of immunohistochemistry or PCR. To address these concerns, this study was performed with these objectives: 1) to see if infectious herpesvirus could be recovered from tissues following short term (<48 hours) formalin fixation, 2) to assess immunohistochemical recognition of viral antigens and PCR detection of viral DNA following long-term (14 days) formalin fixation, and 3) to investigate microtome contamination by DNA carry-over to subsequently sectioned tissues. Tissues from mice infected with the baboon herpesvirus HVP2 were used in these studies. From a biosafety standpoint, concern over formalin inactivation of infectious virus versus the negative impact of formalin fixation upon viral antigen integrity is moot as infectious virus is inactivated after only 24 h of formalin fixation and immunohistochemical staining is sensitive in tissues fixed for up to 14 days. While formalin fixation does degrade viral DNA, viral DNA can still be detected by PCR amplification of very short DNA sequences by real time PCR. If utilizing PCR to detect the presence of virus in tissues, viral DNA can contaminate a microtome knife such that subsequently sectioned uninoculated control tissues exhibit false positive amplification.
120: STUDIES ON TROPISM AND PATHOGENICITY OF CORONAVIRUS VECTOR-BASED VACCINES IN PIGLETS.
The aim of this project is to study the biosafety of recently developed recombinant coronaviral expression systems based on the Transmissible Gastroenteritis Virus (r-TGEV) to establish a DIVA (Differentiating Infected from Vaccinated Animals) vaccines inducing a broad systemic and mucosal immune response. The investigated r-TGEVs are derived from the PUR46-MAD vaccine strain and include either various cloning sites or a deletion of gene 7. Experiments in 3-day-old piglets were accomplished to study the tropism of the vector system with the highest replication efficiency and lowest virulence. After inoculation with 1 × 108 PFU/piglet of each of the rTGEVs, most of the piglets developed symptoms for TGE. Overall mortality was between 25 (r-TGEV-7) and 87.5%. At necropsy there was moderate acute diffuse catarrhal jejunitis and ileitis culminating 36 to 48 hpi. Histopathology showed atrophy and fusion of intestinal villi and submucosal edema with a mild to moderate multifocal bronchointerstitial pneumonia. Plaque assay revealed intestinal tropism of the vectors with higher replication efficiency in the small intestine (5 × 107 PFU/ml) compared to lower titers in the lungs (1 × 104 PFU/ml). Immunohistochemically it was possible to detect TGEV-antigen in numerous enterocytes of the jejunum and ileum including small numbers of M-cells. Electron microscopy demonstrated replication stages of r-TGEV in the enterocytes. These in vivo experiments show that r-TGEV-7 combines the lowest virulence with the highest replication efficiency in the intestine. Therefore this vector is a promising candidate for further vaccine development.
121: THE EFFECTS OF NONCYTOPATHIC TYPE 2 BOVINE VIRAL DIARRHEA VIRUS ON BONE MARROW COLONY FORMING UNIT-GRANULOCYTE-MACROPHAGE (CFU-GM) PROGENITOR CELLS.
The purpose of this study was to investigate the effects of high and low virulent noncytopathic type 2 bovine viral diarrhea virus (ncpBVDV-2) isolates on bone marrow progenitor cell proliferation. Six to seven month old BVDV naive Holstein calves were inoculated intranasally with either a high virulent ncpBVDV-2 (HV24515) isolate, a low virulent ncpBVDV-2 (LV11Q) isolate, or uninfected cell culture medium. Serial bone marrow and peripheral blood samples were collected before and after inoculation. A bovine colony forming unit-granulocyte-macrophage (CFU-GM) assay was optimized and bone marrow mononuclear cells (BMMCs) were isolated and cultured for 5 days before determining the mean number of CFU-GM colonies. 3H-thymidine uptake by BMMCs was determined to indicate the overall proliferative capacity. BVDV virus isolation was done on concurrent BMMCs and peripheral blood samples. Virus was isolated from BMMCs and peripheral blood buffy coat cells as early as day 2 post-inoculation in both viral groups. Both viral groups developed neutropenia, however, in those calves given LV11Q neutrophils rebounded earlier in response to increased proliferation of BMMCs whereas the response was delayed in calves given HV24515. Thymidine uptake was significantly increased in BMMCs post-inoculation compared to pre-inoculation in LV11Q calves, and not in HV24515 or control calves. The mean number of CFU-GM colonies was significantly decreased post-inoculation compared to pre-inoculation in calves given HV24515 calves, but not in calves inoculated with LV11Q. The data support the hypothesis that the prolonged neutropenia observed in calves given HV24515 results at least in part from decreased proliferative capacity of bone marrow progenitor cells.
122: APOPTOSIS IN BONE MARROW CELLS OF CALVES EXPERIMENTALLY INFECTED WITH NONCYTOPATHIC TYPE 2 BOVINE VIRAL DIARRHEA VIRUS.
Bovine viral diarrhea virus (BVDV) continues to be an important complex pathogen of cattle. Recent investigations have examined how BVDV infection affects apoptotic mechanisms. Inhibition of apoptosis may be used to establish persistent infections, and altered apoptosis of virus infected cells could contribute to enhanced virulence of some acute infections. The purpose of this study was to examine the frequency of apoptotic cells in hematopoietic tissue of infected calves since hematological abnormalities tend to be more severe and persist longer with virulent isolates. Six month old BVDV and antibody negative Holstein steers were inoculated intranasally with either a low virulence ncpBVDV-2 isolate (n = 7), a high virulence ncpBVDV-2 isolate (n = 8) or sham inoculated (n = 7). Blood (EDTA) and bone marrow samples were collected on days minus 2, 3, 8 and 12 post-inoculation (pi). Anticoagulated blood was used to determine complete blood counts. Bone marrow was fixed in formalin, embedded in wax and immunohistochemically stained for TUNEL and activated caspase 3. Calves inoculated with the high virulence isolate developed clinical signs of acute BVDV infection, while animals in the other 2 groups exhibited no signs. All animals receiving virus developed leukopenia but it was more severe and persisted longer with the high virulence isolate. There was significantly (p < 0.05) more apoptosis in calves inoculated with the low virulence isolate using the TUNEL method on day 8 pi while there were no differences between groups on any days with the caspase method. It appears that inhibition of apoptosis may be a virulence attribute of some BVDV isolates.
123: EFFECTIVE TREATMENT OF LEISHMANIA MAJOR INFECTION BY NANODISK-BOUND AMPHOTERICIN B.
Treatment of intracellular Leishmania major infection has been difficult, requiring long-term, intravenous drug therapy with significant side-effects. Partially, this is due to the inability of therapeutic drugs to affect the L. major amastigotes protected within the macrophage phagolysosome. Targeting drug treatments to macrophages is one method to reduce toxic effects of anti-parasitic drugs and ensure adequate treatment. Nanodisks (ND) are apolipoprotein-stabilized phospholipid bilayer disc complexes capable of harboring amphotericin B (AMB) and potentially targeting macrophage class A scavenger receptors. L. major infected mice were treated with intra-peritoneal AMB-ND over the course of the first month of infection. Parasite numbers, lesion size progression and cytokine responses were assayed. Mice treated with 7 doses of AMB-ND in 1 to 5 mg/kg doses were significantly protected from L. major, displaying a marked decrease in lesion size and parasite burden, with concomitant increase in survival times. Those treated with AMB-ND completely clear an L. major infection by 150–250 days post-infection, with no lesions and no parasites isolated from either footpad injection sites or lymphoid tissues. In contrast, the commercial liposomal AMB formulation, Am-Bisome, did not clear infections in susceptible BALB/c mice. Cytokine responses were largely unaffected by AMB-ND administration. The marked clearance of Leishmania parasites from susceptible mice without an appreciable change in immune response suggests that AMB-ND represent a potentially useful formulation for treatment of intra-histiocytic organisms susceptible to AMB, including various protozoa and fungi. Nanodisks are potent and highly effective targeted carriers for anti-leishmanial drugs and may be of use in combating other intracellular pathogens, such as Mycobacterium sp.
124: 14-3-3 PROTEIN REGULATES VIRAL-INDUCED NEURODE-GENERATION.
Although the presence of 14-3-3 proteins in cerebrospinal fluid has been documented in several neurodegenerative diseases, the underlying pathophysiologic role of 14-3-3 in mediating neuronal damage in HIV-dementia, as well as other chronic neurodegenerative conditions, remains unknown. 14-3-3 is a ubiquitously expressed adaptor protein that is especially abundant in neurons. By binding and sequestering pro-apoptotic mediators such as Bad, 14-3-3 proteins inhibit downstream mitochondrial death pathways. Conversely, when 14-3-3 dissociates from pro-apoptotic mediators, apoptotic pathways are activated, leading to cell death. To determine whether 14-3-3 plays a crucial role in the regulation of apoptosis in neurons during chronic SIV/HIV infection of the CNS, we examined whether 14-3-3 binding to pro-apoptotic mediators decreased with development of SIV CNS disease when neuronal damage and loss occur. By co-immunoprecipitating 14-3-3- and Bad from brain homogenates and then measuring Bad levels by quantitative immunoblotting, dissociation of 14-3-3 from pro-apoptotic Bad was demonstrated in SIV-infected macaques with CNS disease versus uninfected control animals. To determine whether specific neurotoxic viral proteins disrupted binding of 14-3-3 to pro-apoptotic mediators in neurons, thereby triggering neuronal damage and apoptosis, cultured primary rat neurons were treated with recombinant HIV Tat. Viral induced neuronal damage was measured by MTS assay and Live/ Dead fluorescent assay and then compared with extent of 14-3-3 binding to pro-apoptotic mediators, including Bad measured by co-immunoprecipitation and quantitative immunoblotting. With increasing doses of Tat, neuronal damage correlated with loss of 14-3-3-Bad complex formation, indicating a central role for 14-3-3 proteins in regulating viral-induced neurodegeneration.
125: A HERPESVIRUS TEGUMENT PROTEIN IS ESSENTIAL FOR NEUROVIRULENCE: HISTOPATHOLOGY AND TIME LAPSE RECORDING IN NEURONAL CELL CULTURE.
Pseudorabies Virus (PrV) is an alphaherpesvirus that causes Aujeszky's disease (AD), a severe neurological disorder in a broad range of mammalian species. PrV is also widely used as a neuronal tracer and vector. In our studies we concentrated on the role of viral proteins in neuroinvasion and intraneuronal transport. To this end, PrV mutants unable to express several structural and nonstructural proteins were instilled intranasally into mice. Mean survival times, kinetics of viral spread in the trigeminal circuit, immune response and induction of neuronal apoptosis were examined. All mutant viruses, except five were able to infect first order neurons in the trigeminal ganglion, second order neurons in spinal, pontine and mesenchymal trigeminal nuclei as well as somatocortical neurons, as shown by immunohistochemistry. Deletion of the respective genes indirectly influenced the degree of nonsuppurative encephalitis and ganglioneuritis by longer survival times, whereas no neuronal apoptosis could be detected by immunohistochemistry against active caspase 3. Mean survival times varied from 48 hpi to 140 hpi, confirming the in vitro phenotype of most deletion mutants. However, deletion of the UL37 tegument protein resulted in a nonvirulent virus. To assay the basis of this defect, mutant viruses with a green fluorescent protein were analyzed by confocal time lapse recording. These studies revealed intraneuronal, bidirectional transport of fluorescent capsids after infection of murine primary trigeminal cell culture with wildtype PrV, whereas deletion of the UL37-protein inhibited intraaxonal capsid movement. Therefore, we conclude that the UL37-protein is essential for intraneuronal transport of PrV and might possess connective functions between viral capsids and microtubule-associated motor proteins like dynein and kinesin.
126: IDENTIFICATION AND ULTRASTRUCTURAL CHARACTERIZATION OF LESIONS IN THE VENTRAL HORNS OF SPINAL CORDS FROM WAVE1 KNOCKOUT MICE.
The WASP/WAVE family of proteins are involved in the regulation of the actin cytoskeleton by interacting with actin-related proteins. Actin-related proteins mediate the action of specific GTPases controlling the polymerization of actin filaments. To evaluate the role of WASP/WAVE proteins in tissue morphogenesis, especially apoptosis, a line of mice, in which the WAVE1 gene had been disrupted, were generated for study. Knock-out animals are smaller than wild-type animals and have hindlimb weakness and tremors, and a life span of 30 days. Samples of spinal cord from 20 day-old wild type (wt) and WAVE 1 (-/-) mice were processed for light microscopic examination. Samples from the ventral portions of cervical, thoracic and lumbar segments of spinal cord were processed for ultrastructural evaluation. No lesions were evident in H and E stained paraffin sections of spinal cord evaluated with light microscopy. Axonal dilations were detected in the 1 micron plastic sections. The ultrastructure of these dilated axon and accumulations of organelles are described and discussed in relationship to the possible involvement of the WAVE1 disruption.
127: AN ASTROCYTOMA MOUSE MODEL: A PATHOLOGICAL AND MORPHOMETRIC STUDY OF MICROGLIAL INFILTRATION.
Astrocytomas are the most common primary central nervous system (CNS) tumor in humans. Despite surgical resection, radiotherapy techniques and aggressive chemotherapy, prognosis is usually poor. Both human and experimental gliomas are invaded by macrophages and microglial cells. The interrelations between microglial infiltration and the growth of human gliomas in vivo are complex, so there is a need to dissect the underlying mechanisms of microglial participation in the brain's defense against gliomas. The study of intracranial injected astrocytomas into immunocompetent hosts constitutes a powerful tool to investigate the recently recognized relation between these tumors and the innate immune system of the CNS. Although such models are likely to open novel immunotherapeutic avenues, few reports exist in the literature. To answer this question, we injected the BALB/c derived DBT cell line into the frontal cortex of 7-week old BALB/c mice. Over two weeks, each animal developed a highly aggressive astrocytoma. The tumor contained high numbers of microglia/macrophages (as identified by F4/80 and CD11b staining). The microglial content and distribution in the tumor were analyzed immunohistochemically and morphometrically over time. At week one, there were significantly higher numbers of microglia at the tumor-peritumor border compared to the center of the tumor. At week two, microglia are evenly distributed peri-tumorally and centrally. These murine models offer a valuable tool to investigate the interactions between the microglia response occurring in the CNS in response to astrocytomas. Such study could lead to the identification of novel molecular mechanisms involved in immune recognition of astrocytomas by microgial cells, thus possibly opening novel immunotherapies against these extremely aggressive tumors. Supported by NIH T32 RR07019-23.
128: PATHOLOGY OF CARDIOMYOPATHY IN TRANSGENIC MICE WITH CONTROLLED OVER-EXPRESSION OF ACTIVATED H-RAS IN THE HEART.
Proto-oncogenes are expressed in the heart during embryogenesis, and subsequently in response to cardiac stress. Transgenic mice developed by targeting expression of various proto-oncogenes in the heart recapitulate both human hypertrophic (HCM) and dilative (DCM) cardiomyopathy (CM). In this study, activated H-Ras over-expression was induced in heart using a reverse tetracycline transactivator (rtTA) construct targeted with an alpha-myosin heavy chain (MHC) promoter. This provides ability to control h-ras off-or-on by supplying or withdrawing doxycycline in the food, respectively. All twenty-four MHC-rtTA-H-Ras transgenic mice studied developed CM, compared to twenty-two non-transgenic litter-mates. CM mice had 210 mg mean heart weight (0.008% body weight) compared to 150 mg (0.006% body weight) in controls. Features of both HCM and DCM were observed, in some instances within the same heart, as previously reported for other models. Grossly, eight MHC-rtTA-H-Ras mice had hypertrophic phenotypes with thickened inter-ventricular septa and left ventricular walls with chamber diminution, while seven mice had chamber dilation with global wall thinning, and luminal thrombi. Nine MHC-rtTA-H-Ras hearts were grossly normal. Histology in all MHC-rtTA-H-Ras hearts varied from mild focal to severe diffuse myofiber hypertrophy and disarray, atrophy and loss, with interstitial fibrosis and fatty infiltration. Atria were most severely affected, with fibroplasia, anisokaryosis, and nuclear atypia; architecture was effaced in two CM animals. Immuno- and histochemistry demonstrated loss of cross-striations, accumulation of PAS-positive mucosubstances, and positive immunolabeling for downstream mediators of Ras (activated c-Raf and ERK1/2) in hypertrophic myofibers. In future studies, MHC-rtTA-H-Ras mice with induced CM will be evaluated following h-ras inactivation by utilizing the off-on control mechanism.
129: DEVELOPMENT OF TESTS TO DETECT IL-6 IN DEER: AN APPROACH FOR DEVELOPING ASSAYS FOR UNUSUAL SPECIES.
Epizootic hemorrhagic disease (EHD) is an important disease of white-tailed deer (WTD) caused by EHD viruses. Currently, little is known about the expression of inflammatory mediators in deer with EHD. We hypothesize that IL-6 expression may be important in this disease as it is produced by a wide variety of cells, including the targets of EHD viruses, macrophages and endothelium, and may induce either a pro- or an inhibitory inflammatory response that could modulate disease. Unfortunately, the resources needed to study most inflammatory mediators, specifically IL-6 in our case, are not available for WTD. Thus, the objectives of this study were to develop ELISA and immunohistochemical assays to detect circulating and tissue IL-6, respectively, in WTD. Utilizing WTD IL-6 mRNA sequences that we generated from known sequences of cattle, a synthetic deer IL-6 peptide was designed and produced. This was subsequently used to produce rabbit anti-deer IL-6 antibody. Using the synthetic peptide and polyclonal rabbit anti-IL-6, we developed a direct sandwich ELISA for the detection of IL-6 in WTD serum. Using formalin fixed paraffin embedded WTD tonsil we also developed an immunohistochemical test to detect IL-6 in tissue. Limitations to studying inflammatory mediators in species for which appropriate reagents are unavailable are many. Although we often rely on cross-reaction to antibodies made for closely related species, this approach does not always work. This study illustrates an alternative approach in which antibodies of better specificity are generated with knowledge of very limited genetic sequence.
130: ISOLATION AND CHARACTERIZATION OF CANINE ADIPOSE-DERIVED MESENCHYMAL STEM CELLS (CAD-MSCS).
Stem cells in general and mesenchymal stem cells (MSCs) in particular, are becoming ideal candidates for regenerative and reparative medicine as well as for therapeutic gene delivery. MSCs hold outstanding promise in both veterinary and human orthopedic practice, especially for the restoration of bone defects. Even though MSCs are most commonly derived from bone marrow, they also exist in a variety of other tissues, including different mesodermal tissues and umbilical cord blood. Recently, adipose tissue has been demonstrated as a good source for the isolation of human MSCs, owing to its abundance and ease of isolation. Members of our research group have recently shown that a low calcium medium supplemented with N-acetyl-L-cysteine and L-ascorbic acid-2-phosphate will accelerate the growth and extend the life span of adipose tissue-derived human MSCs (Stem Cells and Development 14:92-102, 2005). Using this method, we have isolated putative MSCs from canine adipose tissue (cAD-MSCs). These cAD-MSCs already underwent more than 25 cumulative population doublings and showed limited cell-cell communication. Osteogenic, chondrogenic and adipogenic differentiation of the cAD-MSCs was noticed in micromass culture, however only osteogenic and chondrogenic differentiation was observed in monolayer, a finding different from the results observed for adipose tissue-derived human MSCs. Characterization of the cAD-MSCs including protein and gene expression analysis pre- and post-differentiation are currently carried out with various techniques, as are studies to further expand the life span of these cells. Studies on adult canine MSCs will evince the emerging usefulness of the dog as a prospective biomedical model system for clinical trials, and will be of significance for both veterinary and human tissue engineering.
131: SODIUM/IODIDE SYMPORTER REGULATION BY tRA, FORSKOLIN, RAS AND PI3K IN MCF-7 HUMAN BREAST CANCER CELLS.
The sodium/iodide symporter (NIS) is a membrane glycoprotein expressed in the thyroid gland and lactating mammary gland. NIS mediates radioiodide imaging and therapeutic ablation of thyroid carcinomas with potential for similar use in breast cancer treatment, as most human breast tumors express NIS. Less is known about the regulation of NIS expression in the mammary gland than in the thyroid. We have identified ras, PI3K and cAMP as potentially important in NIS upregulation in mouse models of breast cancer. MCF-7 human breast cancer cells respond to all-trans-retinoic acid (tRA) with modest NIS expression and radioiodide uptake. In this study, we demonstrated that MCF-7 cells expressing v-Ha-ras or PI3K p110 express significant hypoglycosylated cytoplasmic NIS protein and lack radioiodide uptake. Treatment of MCF-7 cells with tRA leads to NIS expression in less than 10% of cells, but the NIS protein is heavily glycosylated, efficiently trafficked to the cell surface, and functional. Treatment of MCF-7 cells with combined tRA and forskolin leads to a synergistic increase in NIS protein and radioiodide uptake, resulting from an increase in NIS protein in responding cells with no change in the percentage of cells responding. Magnetic antibody cell sorting is underway to isolate MCF-7 cells responding to tRA for analysis of expression levels of RA receptors and related transcription factors, global gene microarray and proteomics analysis. Future studies on NIS regulation in mouse mammary carcinoma cells will include investigation into mechanisms accounting for the lack of radioiodide uptake in certain cells expressing significant NIS protein.
132: CALCIUM-DEPENDENT ENHANCED TRANSCRIPTION OF p300 BY HUMAN T LYMPHOTROPIC TYPE-1 p12I CRITICAL FOR LONG-TERM LYMPHOCYTE SURVIVAL?
Transcriptional co-activators p300 and CREB binding protein (CBP) play a central role in transcriptional control of various cellular and viral DNA binding transcription factors, however transcriptional regulation of p300 is not understood. While p300 and CBP mediate the activities of various transcription factors, their availability in the cell is at limiting concentrations and even small reductions in these co-activators may disrupt transcriptional regulation of the cell. Human T lymphotropic virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATLL), an aggressive CD4+ T lymphocyte malignancy. HTLV-1 encodes various accessory proteins from the pX region of genome. The HTLV-1 accessory protein p12I enhances p300-dependent transcription in a dose-dependent manner. We have demonstrated the critical nature of p12I in HTLV-1 infectivity in vivo and in vitro. This hydrophobic protein localizes in the endoplasmic reticulum (ER) and cis-Golgi apparatus, increases intracellular calcium and activates nuclear factor of activated T cells (NFAT)-mediated transcription. Our gene array analysis indicated that p12I altered the expression of genes associated with a network of interrelated pathways including T-cell signaling, cell proliferation and apoptosis. Expression of several calcium-regulated genes was found to be altered by p12I, consistent with known properties of the viral protein. In the present study, we report that the expression of p300 is regulated by p12I in a calcium-dependent, but calcineurin-independent fashion in T-lymphocytes. We also demonstrate that sustained low magnitude calcium release results in increased RNA and protein levels of p300. In addition, using an ER-localization deficient mutant of p12I, we demonstrate that ER localization of p12I is required for its ability to increase p300. We are the first to demonstrate the calcium-dependent expression of p300. Collectively, our data indicate that HTLV-1 p12I, an essential protein that mediates calcium-dependent transcription in lymphocytes appears also to target a rate-limiting transcriptional co-adaptor critical for long-term cell survival.
133: AN ASSESSMENT OF SPONTANEOUS OVINE INTESTINAL ADENOCARCINOMAS AS AN ANIMAL MODEL OF HUMAN COLONIC CANCER.
Around 7% of old unthrifty sheep in New Zealand have intestinal adenocarcinomas. To determine if these sheep could be used as a model of human colonic neoplasia, the biological behavior and histologic appearance of ovine intestinal adenocarcinomas were compared with that reported of human colonic adenocarcinomas. Fifty intestinal tracts were collected from slaughtered sheep with grossly visible intestinal neoplasia. Neoplasms were assessed using World Health Organization guidelines for assessment of human colonic adenocarcinomas. All ovine adenocarcinomas developed in the small intestine. In contrast, only 4% of human intestinal tumours develop at this location while the majority develop in the colon. Because most human colonic adenocarcinomas develop from colonic adenomas, 89% have a polypoid appearance. Polyps were present in 46% of the ovine neoplasms. Intestinal wall infiltration by the neoplastic cells and rates of lymph node (84% in sheep; 61% in humans) and distant (52% in sheep; 17% in humans) metastases were comparable within both ovine and human adenocarcinomas. However, ovine adenocarcinomas developed more serosal and less hepatic metastases than human adenocarcinomas. Histological grading of ovine tumours revealed similar cell differentiation to that reported within human colonic adenocarcinomas. In conclusion, ovine intestinal adenocarcinomas, like human colonic adenocarcinomas, commonly spontaneously develop and consistently develop widespread metastases. Additionally, tumours within both species appear histologically similar. Therefore, sheep may provide a model of advanced human colonic cancer, allowing evaluation of novel therapeutics and surgical procedures.
134: SLUG-MEDIATED EFFECTS OF CUTANEOUS UVR EXPOSURE.
Slug, a member of the Snail family of zinc finger transcription factors, modulates epithelial-mesenchymal transformation (EMT), the transformation of anchored epithelial cells into motile mesenchymal cells. EMT plays an important role in development, in wound healing, and in carcinoma progression and metastasis. Slug has also been found to have anti-apoptotic functions. We are characterizing the role of Slug in the development of cutaneous squamous cell carcinomas by determining the susceptibility of homo-. zygous and heterozyous Slug knockout mice and their wild type counterparts to skin cancer induced by chronic UVR exposure. Following only two UVR exposures, however, a marked difference in the UVR response of Slug knockout and wild type mice was seen. Slug knockout mice failed to exhibit the skin erythema and peeling seen in wild type mice. This resistance to sunburn was unexpected and prompted an additional study to investigate the role of Slug in the acute response to UVR. Our findings have demonstrated a role for Slug in this process. Although the extent of UVR-induced DNA damage (cyclobutane pyrimidine dimers) was similar in all genotypes, homozygous Slug knockout mice did not show the increase in skin thickness (dermal edema) seen in the wild type mice. Both homozygous and heterozygous Slug knockout mice had markedly reduced dermal neutrophil infiltrates at 48h post-UVR compared to wild type mice. Both homozygous and heterozygous animals also had more cleaved caspase-3-positive epidermal cells than wild type animals, suggesting increased UVR-induced apoptosis. Additionally, the homozygous knockout mice demonstrated delayed expression of keratin 6 and reduced epidermal hyperplasia in response to UVR. These findings indicate an important role for Slug in the apoptotic and inflammatory response to UVR.
135: THE EFFECT OF POLYUNSATURATED FATTY ACIDS ON PROSTATIC ADENOCARCINOMA DEVELOPMENT IN THE PROSTATE-SPECIFIC PTEN KNOCKOUT MURINE MODEL.
The tumor suppressor gene PTEN is frequently mutated in human prostate cancer. Numerous PTEN knockout models have been developed in an attempt to closely mimic disease progression and the lesions seen in the human disease. This study utilizes a prostate-specific PTEN Cre-mediated knockout murine model to determine the effect of polyunsaturated fatty acids on prostate cancer development. Mice were fed three different custom diets composed of varying ratios of omega 3 to omega 6 fatty acids. These diets contained fatty acid ratios that corresponded to a traditional Western diet (1 n-3:20 n-6), a high omega 3 diet (1:1), and a high omega 6 diet (1:40). The wild-type, PTEN heterozygous, and PTEN homozygous knockout mice were sacrificed at 5, 8, and 12 weeks of age. Organ weights and histopathologic examination of the prostate tissues followed. Diet, genotype, and age had profound and significant effects on prostate weight (p less than 0.005). PTEN homozygous animals at 12 weeks of age consuming a high omega 6 diet had a 30% increase in prostate weight in contrast to mice consuming the high omega 3 diet, which had a 16% decrease in prostate weight. The highest numbers of invasive carcinomas were seen in 12 week old PTEN homozygous knockout mice consuming the omega 6 rich diet. There were significant interactions between genotype and age and genotype and diet. This prostate specific model demonstrates that fatty acid ratios can affect the progression of prostatic neoplasms.
136: PANCREATIC PATHOLOGY IN CHICKENS, TURKEYS, AND PIGEONS EXPERIMENTALLY INFECTED WITH NEWCASTLE DISEASE VIRUSES OF DIFFERENT VIRULENCE.
Formalin-fixed, paraffin-embedded pancreatic tissues from birds infected with different Newcastle disease virus (NDV) isolates were compared for the presence of histologic lesions, viral nucleoprotein, and cellular apoptosis. Pancreatic tissues were collected from four-week-old specific pathogen free (SPF) White Leghorn chickens infected separately with NPV isolates of low, moderate, or high virulence and three-week-old SPF Beltsville White turkeys, six-week-old commercial Broad Breasted White turkeys, and 10- to 20-week-old racing pigeons infected with a viscerotropic velogenic NDV isolate. Chickens infected with a virulent NPV of either a neurotropic or viscerotropic type and turkeys infected with a virulent NPV of viscerotropic type had extensive microscopic lesions in the pancreas with the presence of viral nucleoprotein. This was accompanied by induction of apoptosis in exocrine and infiltrating inflammatory cells. Minimum tissue changes, comparable to controls, were observed from infections of chickens with isolates of low and moderate virulence and of pigeons with the viscerotropic isolate.
137: CANINE CONJUNCTIVAL TUMORS OF VASCULAR ENDOTHELIAL ORIGIN.
Conjunctival tumors of vascular endothelial origin are common but under-reported in dogs. We report on a case series of 108 cases from a collection that includes 8300 canine cases submitted between 1989 and 2004. These include 70 hemangiomas and 38 hemangiosarcomas. The age, breed, sex, state of origin, pigmentation of the unaffected tissue, and location on the conjunctiva are recorded. Follow-up information regarding recurrence is available for 49 cases. The cases were matched with two unaffected controls and the two groups were compared by breed, color, and the index of ultraviolet (UV) light exposure in the state of origin. Endothelial tumors tend to occur at the lateral limbus and the leading edge of the third eye-lid. They occur in non-pigmented conjunctival tissue. There is a trend to occur in states with a high index of UV light exposure and in breeds likely to be outdoors. Recurrence is more likely when the diagnosis is hemangiosarcoma (11 of 20) than hemangioma (3 of 29). Three cases were second submissions with radical surgery and none of these recurred.
138: ARGYROPHILIC NUCLEOLAR ORGANIZER REGIONS (AGNORS), P53 AND KI-67 IMMUNOREACTIVITY IN EQUINE OCULAR SQUAMOUS EPITHELIAL TUMORS.
Equine ocular squamous epithelial neoplasms represent a continuum from the benign but pre-neoplastic squamous plaque through the malignant squamous cell carcinoma in situ and invasive squamous cell carcinoma. As such, distinguishing between borderline cases of equine squamous epithelial tumors can be a diagnostic challenge. Since accurate diagnosis determines prognosis and treatment, AgNOR scoring and p53 and Ki-67 immunohistochemistry were evaluated as diagnostic tools to determine the difference histopathologically between borderline cases of archived equine squamous epithelial tumors. AgNOR scoring and p53 and Ki-67 immunohistochemistry were performed on 48 squamous plaques (SP), 46 in situ squamous cell carcinomas (isSCC), and 38 invasive squamous cell carcinomas (ISCC). AgNOR scores proved to be a useful diagnostic tool in discriminating between borderline cases of squamous epithelial tumors. The mean AgNOR score was significantly different between ISCC and SP (p < 0.0001) and isSCC (p < 0.0001), and between isSCC and SP (p = 0.0005). p53 immunoreactivity was helpful in discriminating between SP and isSCC. The mean p53 immunoreactivity score was significantly different between ISCC and SP (p < 0.0001), and isSCC and SP (p = 0.0006). Ki-67 was not helpful in discriminating between ISCC and isSCC, and isSCC and SP, but was useful in discriminating between ISCC and SP. The mean Ki-67 immunoreactivity score was significantly different between ISCC and SP (p < 0.0004), but was not significantly different between ISCC and isSCC (p = 0.0886) or between isSCC and SP (p = 0.0845). We conclude AgNOR and P53, but not Ki-67, can be useful diagnostic tools for discriminating between borderline cases of equine squamous epithelial tumors.
139: AKT PHOSPHORYLATION IN OVINE ENZOOTIC NASAL ADENOCARCINOMAS.
Enzootic nasal tumor virus (ENTV) causes ethmoidal adenocarcinomas in sheep and goats. ENTV belongs to the genus beta-ret-rovirus and is closely related to Jaagsiekte sheep retrovirus (JSRV). The mechanisms of oncogenesis for both viruses are unknown, but it has been found that the Env protein of JSRV is able to transform cultured fibroblasts triggering the phosphatidylinositol 3-kinase (PI3K)/Akt oncogenic signaling pathway. To test the hypothesis that PI3K/Akt pathway is also involved in the development of ovine ethmoidal adenocarcinomas we investigated samples obtained from five sheep with these spontaneously occurring neoplasms. Electron microscopy and PCR-analysis confirmed a beta-retrovirus infection. Sequence analysis of the Gag region revealed ENTV-1, including an ENTV-1-specific pattern of restriction enzyme cleavage sites. In situ hybridization confirmed the presence of ENTV in the cytoplasm of the neoplastic epithelial cells in a multifocal pattern. The immunohistochemical analysis of PI3K/Akt in these ENTV-positive areas revealed that PI3K/Akt was phosphorylated. In addition, the proliferation state of cells in these areas was elevated as shown by staining for PCNA. Inversely, these areas showed a decreased expression of the tumor suppressor protein p53 and the cyclin-dependent kinase inhibitor p21/CIP1. In summary, these results strongly suggest that similar to what has been described for JSRV, ENTV infection also triggers the phosphatidylinositol 3-kinase (PI3K)/Akt oncogenic signaling pathway. The multifocal pattern of increased cellular proliferation and transformation might explain the final development of adenocarcinomas with low malignancy.
140: OCT-4 EXPRESSION IN CANINE NEOPLASMS: A POTENTIAL ROLE FOR EMBRYONIC GENES IN CANCER PROGRESSION.
Cancer cells share many phenotypic characteristics with undifferentiated embryonic cells, both in terms of their morphology and behavior, such as their propensity for proliferation, cellular migration, and tissue invasion. As such, it has been hypothesized that genes involved in the early stages of embryogenesis, namely those genes that are involved in the maintenance of pluripotency of undifferentiated stem cells, may also be involved in cancer. The transcription factor Oct-4 is a POU domain family member that is involved in the maintenance of pluripotency in embryonic cells and germ cells. Recently, Oct-4 has been found to play a role in the pathogenesis of human testicular germ cell tumors, and has been shown to be inappropriately expressed in both human tumors, and human cancer cell lines. In order to test the hypothesis that embryonic genes, such as Oct-4, are involved in carcinogenesis, 128 canine tumors representing 22 different types of neoplasms, including epithelial, mesenchymal, round cell, and germ cell neoplasms, were immunohistochemically stained with anti-Oct-4 antibody and subsequently evaluated for Oct-4 expression. Similar to previous reports in human cancer, Oct-4 was found to be expressed in testicular germ cell tumors. Additionally, moderate to high amounts of Oct-4 was also found to be expressed in other tumors, including mast cells tumors in which nearly 100% of the neoplastic cells expressed Oct-4 in several of the tumors that were evaluated. These results support the hypothesis that embryonic genes, such as Oct-4, are involved in the pathogenesis of neoplastic diseases, and therefore are potential biomarkers and therapeutic targets for cancer.
141: EPITHELIOID VARIANT OF HEMANGIOMA AND HEMAN-GIOSARCOMA IN THE DOG, HORSE AND COW.
Epithelioid hemangiomas, hemangioendotheliomas and angiosarcomas are well recognized histologic variants of human endothelial tumors that in the past have been confused with neoplasms of epithelial or histiocytic origin. We describe 11 epithelioid endothelial vascular tumors in dogs, horses and a cow that show microscopic features that are similar to these tumors in humans. Nine tumors were located within the dermis and subcutis, one in the gastrocnemius tendon and one in the skeletal muscle of the thigh. Key histologic features included the epithelioid appearance of neoplastic endothelial cells, papillary and gland-like acinar arrangements, and infrequent, though striking, cytoplasmic vacuolation of neoplastic cells forming signet ring formations that rarely contained a single erythrocyte. All tumors were positive for von Willebrand factor with variable intensity and distribution and uniformly negative for cytokeratin. Based on conventional morphologic criteria, growth pattern (invasive or not) and metastasis (known in one case at the time of biopsy), we subdivided these tumors into epithelioid hemangiomas (n = 3) and hemangiosarcomas (n = 8); none qualified as epithelioid hemangioendotheliomas. Additional follow up information obtained by written questionnaire was available for 6/11 animals and revealed local recurrence in one epithelioid hemangioma and epithelioid hemangiosarcoma and regional metastasis in one epithelioid hemangiosarcoma. This series represent a novel group of vascular endothelial tumors in domestic animals of which there are only two previous cases reported in the veterinary literature.
142: SPONTANEOUS COLITIS OF TAMARINS KEPT IN A ZOO EXHIBIT IS ASSOCIATED WITH MULTIPLE PHYLOTYPES OF ENTEROHEPATIC HELICOBACTER SPECIES.
All species of New World primates of the family Callitrichidae (tamarins and marmosets) are either threatened or endangered. Spontaneous colitis and wasting are major problems of captive callitrichids worldwide. The cause of colitis is unknown; however, a novel enterohepatic Helicobacter species (EHS), designated cotton-top EHS (CT EHS), has been isolated from the colon of cotton-top tamarins with colitis in a research colony. Histopathological examination of archived necropsy specimens obtained from tamarins belonging to six different species at the Henry Doorly Zoo between 1993 and 2003 revealed 17 with colitis and 8 without significant changes. Amplification of DNA obtained from formalin-fixed and paraffin-embedded colons with oligonucleotide primers specific for a Helicobacter 16S rRNA gene sequence by PCR revealed EHS in all specimens. Phylogenetic analyses of the nucleotide sequences from cloned PCR products amplified from representative colons with and without colitis revealed five phylotypes of EHS with overlapping but distinct complement between each group. Zoo tamarins with colitis had primarily EHS related to either H. aurati, a cause of gastritis and a cecal inhabitant of research hamsters, or H. species MIT biotype 1, an EHS associated with colitis of macaque monkeys kept in research colonies. By contrast, CT EHS was exclusively found in normal colons. The data provides new insight into an important cause of poor welfare and premature death of tamarins kept in a zoological collection in the United States.
143: MYCOPLASMA BOVIS PNEUMONIA AND POLYARTHRITIS IN FEEDLOT BEEF CALVES: PATHOGENESIS AND ASSOCIATION WITH BOVINE VIRAL DIARRHEA VIRUS INFECTION.
Mycoplasma bovis is an emerging cause of mortality in feedlot beef cattle. This study was based on examination of lesions and infectious agents in 99 calves that died or were euthanized within 60 days after arrival in 72 Ontario feedlots. M. bovis pneumonia manifested as cranioventral bronchopneumonia with multiple foci of caseous necrosis. M. bovis was consistently identified in these lesions by culture and immunohistochemistry, but also commonly from healthy lungs and those with pneumonia of other causes. Lesions of M. bovis pneumonia were identified in 53/99 calves, including 30 with concurrent fibrinosuppurative bronchopneumonia typical of pneumonic pasteurellosis. Animals with focal lesions of coagulative necrosis, typical of pneumonic pasteurellosis, were often infected with both Mannheimia haemolylica and M. bovis. Arthritis was present in 25/53 (47%) calves with M. bovis pneumonia, and all calves with arthritis had pneumonia. BVDV infection was more common in calves with lesions of bacterial pneumonia than in those dying of other causes, but BVDV infection was not more common in calves with M. bovis pneumonia than those with fibrinosuppurative bronchopneumonia. Retrospective analysis identified cases of M. bovis pneumonia in the early 1980s, albeit with milder lesions than in current cases. The pathologic and epidemiologic findings suggest that in some calves, lung lesions caused by M. haemolytica become super-infected with M. bovis and M. arginini, leading to chronic antibiotic-resistant pneumonia.
144: AN OVERVIEW OF POST MORTEM FINDINGS OF KILLER WHALES (ORCINUS ORCA) STRANDED 1944–2004.
Killer whales (Orcinus orca) are highly social cetaceans and the largest member of the family Delphinidae. Although cosmopolitan, these animals are most frequently encountered in temperate coastal waters. Through ongoing research efforts, detailed census data of resident populations in the Pacific Northwest have been established. Between 1995 and 2001, there was a dramatic decline in the number of southern resident killer whales (from 97 to 78) and to understand the significance of this decrease from a more global perspective, information on strandings was solicited from a variety of resources. From 1944 to 2004, 222 strandings were recorded. In those cases where post mortem reports were available, morphologic diagnoses were compiled. A total of 309 tissues from 48 killer whales were evaluated. Histopathology of 309 tissues revealed abnormalities in 226 (73.1%) samples from 46 animals. Diagnostic interpretations recorded by pathologists were evaluated and diagnoses assigned according to relative significance: 70 (31%) diagnoses considered the proximate cause of death, 8 (3.5%) as secondary or contributory disease processes, and 17 (7.5%) as incidental findings. Primary disease processes included infectious and parasitic n = 70 (33.6%), traumatic n = 10 (4.4%), metabolic disorders n = 7 (3.1%), neoplastic n = 6 (2.7%), and was undetermined in n = 127 (56.2%) of the cases. Microscopic lesions were most frequently seen in lung (50% of animals with abnormal tissues), lymph node (37%), liver (38%), skin/blubber (30%), and heart (26%). In the Pacific Northwest, each dead animal affords a unique opportunity to better understand the natural history and monitor for emergent and enzootic disease concerns that may impact individual animals, pods or ecotypes.
145: HOOKWORM ENTERITIS BACTEREMIA COMPLEX IN CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS) AND NORTHERN FUR SEALS (CALLORHINUS URSINUS) ON SAN MIGUEL ISLAND.
During an investigation of high mortality of California sea lions (Zalophus califomianus) and Northern fur seal (Callorhinus ursinus) on San Miguel Island, in southern California, hookworm (Uncinaria spp.) enteritis with secondary bacteremia was identified in 65% of the 225 pups examined. Ages ranged from 2 weeks to 9 months. Lesions included parasitic enteritis, peritonitis, myocarditis, hepatitis, encephalitis, nephritis, pneumonia, and arthritis. Adult parasites and even eggs were observed within the peritoneal cavity associated with peritonitis. This severe epizootic of hookworm infection is having an affect on the population of these two species of marine mammals. Over the last 30 years, populations of California sea lions and fur seals have steadily increased causing rookeries to become overcrowded, thus this recent problem with hookworms is considered a density dependent disease.
146: MULTINODULAR EQUINE PULMONARY FIBROSIS: A NEWLY RECOGNIZED HERPESVIRUS-ASSOCIATED RESPIRATORY DISEASE OF HORSES.
Fibrotic lung disease is rare in horses; most cases have an unidentified etiology. Lungs from 4 adult horses with chronic respiratory disease and fever were examined using morphologic assessment, cytology, transmission electron microscopy, PCR, and viral culture. The gross lesions were restricted to the lung, and formed either firm discrete masses, or softer nodules, which blended with the surrounding parenchyma. These changes were found in all lung lobes, but were not found together in a single horse. Histologically, the alveolar interstitium within the masses and nodules was markedly expanded with collagen and the interstitium was lined by hyperplastic type II pneumocytes. The lumen of the airspaces had a mixed infiltrate of primarily macrophages and neutrophils, and the interstitium was infiltrated by lymphocytes. Rarely, there were large macrophages within the airspace with prominent eosinophilic intranuclear viral inclusion bodies. Similar inclusions were found in impression smears from affected lung. Transmission electron microscopy revealed intranuclear viral capsids of a size and shape consistent with herpesvirus. Lesions were excised from frozen lung from 2 horses and DNA was extracted for PCR amplification, using multiple degenerate primer sets derived from conserved regions of the herpesvirus DNA polymerase gene. Sequence analysis of the amplicons indicated that a gamma-herpesvirus, related to equine herpesvirus-5, was associated with the lesions. There is considerable interest in the role of viral infections in human pulmonary fibrosis; to the authors’ knowledge, this is the first report of a spontaneous, virally-associated, fibrotic lung disease in domestic animals.
147: HERPESVIRUS TYPE 1 VAGINTITIS IN POSTPARTUM HOLSTEIN HEIFERS.
A severe necrotizing vaginitis has been observed on several dairies in the Tulare County area of California. All cases involved home raised (not purchased), recently calved (3–10 days post-calving), first calf heifers, with a morbidity rate of up to 80%. Affected heifers were often febrile and/or undergoing treatment for endometritis. Some animals had clinical upper respiratory infections. Clinical examination revealed early development of bright red vaginal mucosa that progressed to the formation of small vesicles and subsequently small ulcers. Vaginal ulcers often coalesced to form larger necrotic regions involving the entire mucosa. Three animals were presented for necropsy and vaginal biopsies from a fourth animal were submitted to the California Animal Health and Food Safety Laboratory for evaluation. At necropsy, all three of the animals had necrotizing lesions of the vagina, with almost complete involvement of the entire surface of the mucosa as well as involvement of the os cervix in two of them. The third animal was less severely affected, with multifocal variably sized areas of necrosis of the mucosa and no involvement of the cervix. All three animals had endometritis (mild in one, severe in the other two), with a mixture of bacteria isolated from the more mildly affected uterus. Histological evaluation of the vaginal mucosa from all samples was characterized by erosion and necrosis of the surface epithelium. Abundant neutrophils were present in the submucosal connective tissue with migration into the necrotic regions and adjacent viable epithelium. Immunohistochemical and FA staining of the vaginal mucosa for Bovine Herpesvirus-1 (IBR) were positive on one animal (biopsy specimens) but were negative in tissues from the necropsied animals. PCR evaluation and virus isolation on the biopsy tissue identified Bovine Herpesvirus type 1. Two of the animals also had a severe tracheitis and bronchopneumonia, with syncytial cells affecting bronchial and tracheal epithelium. Both animals demonstrated IBR antigen in the respiratory epithelium and Bovine Herpesvirus-1 was isolated from one animal. No new cases were identified after the administration of an intranasal MLV IBR vaccine prior to freshening, but one dairy reported recurrence of the vaginitis when they discontinued intranasal vaccination of the heifers prior to calving.
148: VIRULENT-SYSTEMIC FELINE CALICIVIRUSES.
Despite widespread vaccination, or arguably driven by it, pathogenic biotypes of feline calicivirus (FCV) termed hypervirulent FCV or virulent-systemic FCV (VS-FCV) with mortality rates ranging from 33% to 60% have recently emerged. At least 8 distinct outbreaks of VS-FCV have been described since 1998. Histologically detectable lesions were present in the mucosa and skin and visceral lesions were variably present in the lung, pancreas, and liver. By immunohistochemistry, target cells for viral infection include liver, pancreas, endothelium, and epithelium. The viral and/or host determinants of disease caused by the hypervirulent biotypes are unknown. Because vaccination status, age, breed and environment failed to correlate with disease incidence, we postulate that the pathophysiologic events that occur during virulent (VS-FCV) and non-virulent FCV infection of cats reflect inherent genetic differences in these viruses that alter mechanisms of cell pathogenesis and tissue tropism during infection. The periodic emergence of virulent biotypes from relatively benign ancestors is common in RNA viruses. Single-stranded, positive sense RNA viruses like caliciviruses can undergo rapid mutation for several reasons, including polymerase infidelity. We have collected 6 different VS-FCV strains that were each obtained from geographically distinct outbreaks of VS-FCV disease in cats. Four hypervirulent strains have been fully sequenced with no identifiable genetic change in FCV that correlates with biological behavior of individual variants. Because sequence divergence is so high (∼20%) among strains, it is likely that significant sequence changes are masked within divergence noise or that traditional methods of capturing and culturing the virus result in loss of specific sequence or structural information. To date, in vitro assays including growth curves, plaque formation, and thermal stability cannot distinguish hypervirulent from mildly virulent biotypes.
149: THE ATTACHMENT, INTERNALIZATION AND TIME-DEPENDENT, INTRACELLULAR DISTRIBUTION OF CLOSTRIDIUM DIFFICILE TOXIN A.
Toxin A (TcdA), secreted by toxigenic strains of Clostridium difficile, produces lesions typical of C. difficile-associated disease (CDAD) in susceptible mammal species. Colon explants maintained for two hours with TcdA developed severe lesions characterized by cell swelling, swelling of mitochondria and other organelles, distension of cytoplasmic vesicles, expansion of paracellular spaces, apoptosis and necrosis. Severity of lesions was proportional to the dosage of toxin. No lesions were present in uninoculated control tissues after two hours. Receptor-mediated endocytosis is the keystone event in the pathogenesis of the toxin and susceptibility of a given species is thought to depend on the presence of receptors on intestinal epithelial cells. The fate of TcdA applied to viable colon explants was determined by transmission electron microscopy in an anti-toxin-labeled immunogold assay. At five min post-inoculation, the presence of TcdA was indicated at the membrane of microvilli or in the cytoplasm of epithelial cells. TcdA was also indirectly observed within endosomes or attached at their margin. A 30-min inoculation period was associated with many more gold particles labeling structures inside the cell, though some were still attached to microvilli. Within the cell most TcdA was associated with mitochondria of epithelial cells but some gold particles decorated the nuclei. Endothelial cells of the lamina propria had evidence of TcdA at both their luminal and basal aspects, as well as in the cytoplasm and, occasionally, nuclei. Gold particles also labeled the lumen of such vessels as well as leucocytes in blood vessels and in the lamina propria.
150: PEMPHIGUS VULGARIS ANTIBODIES ACTIVATE THE PROTO-ONCOGEN C-MYC.
The autoimmune disease pemphigus vulgaris (PV) manifests by loss of keratinocyte cohesion triggered by autoantibody binding to desmoglein (Dsg) 3, an intercellular adhesion molecule of mucous membranes and epidermis including epidermal stem cells. We recently described a so far unknown signaling cascade triggered by PV antibodies which ranges from degradation of cell surface exposed Dsg3 and associated plakoglobin (PG) to depletion of nuclear PG and abrogation of PG/Lef-1-mediated c-Myc suppression. Here we show that in human and canine PV patients this translates into c-Myc overexpression in all targeted locations including the stem cell compartment. Consequences are keratin 6, 14 and 15 mis-expression, reduced b1 integrin levels and hyperproliferation suggesting lack of desmosomal strengthening, impaired wound healing and stem cell depletion. Furthermore, inhibition of c-Myc by small molecule inhibitors is sufficient to abrogate development of PV-IgG induced lesions in neonatal mice. In conclusion, PV antibodies impose a mitotic signal on keratinocytes that not only impacts on intercellular adhesion but leads to impaired wound healing and disturbance of the homeostatic balance. This study provides a completely novel insight into PV pathogenesis and suggests alternative therapeutic strategies using small molecule inhibitors of c-Myc.
151: PLACENTAL PATHOLOGY IN LATE TERM ABORTIONS OF 2002 MARE REPRODUCTIVE LOSS SYNDROME.
The gross and histological findings of equine placentas associated with mare reproductive loss syndrome late term abortions (MRLSLTA) in 2002 were studied in detail. One hundred and forty nine placentas from aborted fetuses and weak foals were studied. A global assessment score (previously established) was used to identify a fetus and/or placenta as a MRLS specimen. A fetus and/or placenta was considered a probable MRLS-LTA if it was of greater than 268 days of gestation and had no definitive test result (microbiological, toxicological, virological, or other) that identified a likely cause of abortion or foal weakness. A placenta was considered a definite MRLS-LTA if it met the above criteria for a probable case and had at least 2 of the following: positive culture of Streptococcus spp., positive culture of Actinobacillus spp., red bag delivery (premature separation of placenta), marked placental thickening or edema, placentitis or funisitis (inflammation of umbilical cord). Only definite MRLS-LTA cases were used for this study. Of the 149 placentas selected for the study, 126 (84.5%) were submitted as a unit with the fetus/dead foal, and 23 (15.5%) were submitted alone. Funisitis was identified in 95% of the cases. In 33 (22%) of the placentas, inflammation was observed in the coelomic space of the allantochorion, but inflammation of the chorionic villi was not identified in any of the placentas studied. Edema of the allantochorion was observed in 53 placentas (35.5%). The absence of inflammation in the chorionic villi of MRLS placentas suggests that the pathogenesis may be different from the classical equine ascending placentitis, in which the chorionic villi are typically affected. Inflammation of the umbilical cord without accompanying inflammation of the chorionic villi indicates that the pathology of placentitis associated with MRLS is unique.
152: CHONDRODYSPLASIA OF TEXEL SHEEP.
Inherited chondrodysplasia, characterized by autosomal recessive inheritance and variable expression, was diagnosed in Texel sheep in New Zealand. Affected lambs were phenotypically normal at birth, but had reduced growth rate. By 2–4 weeks of age, the animals developed shortened neck and limbs, bilateral varus deformity of forelimbs, wide-based stance and enlarged costochondral junctions. The trachea was often twisted, with thickened, flat cartilage rings; this change reduced the tracheal diameter, sometimes leading to tracheal collapse and sudden death following exercise. In severely affected lambs, erosion of articular cartilage on weight-bearing surfaces of major limb joints occurred within the first few months of life leading to degenerative joint disease. Microscopically, chondrocytes were disorganized and surrounded by a halo of abnormal fibrillar material. The cartilage matrix contained multiple, often coalescing, foci of rarefaction, which coalesced in severe cases to form large areas of chondrolysis traversed by coarse fibrils. Ultrastructurally, haloes surrounding chondrocytes consisted of short, abnormally thick collagen fibrils. Areas of chondrodrolysis consisted of clusters of parallel collagen fibrils which were unmasked by the relative absence of proteoglycans. Biochemical studies revealed undersulfation of cartilage proteoglycans. The distinctive light microscopic and ultrastructural lesions, in addition to the biochemical findings in this chondrodysplasia of Texel sheep are similar to a group of human chondrodysplasias caused by mutations in a sulphate-chloride anti-porter. The pathogenesis of the ovine disease may be similar suggesting that the disease in Texel sheep may be an animal model for this group of human disorders.
153: THE EFFECT OF BOVINE VIRAL DIARRHEA VIRUS ON SECRETION OF LACTOFERRIN IN BOVINE TRACHEAL EPITHELIAL CELLS.
Bacterial pneumonia is the most economically important respiratory disease of cattle. Bovine viral diarrhea virus (BVDV) infection is a risk factor for development of bacterial pneumonia in feedlot cattle, but the mechanisms are not well understood. Observations of BVDV antigen in the airway epithelium of calves with bacterial pneumonia led us to hypothesize that BVDV predisposes to bacterial pneumonia by impairing innate immune responses including Iactoferrin production by airway epithelial cells. Lactoferrin is a mammalian iron-binding glycoprotein secreted by epithelial cells and neutrophils, and has antimicrobial properties against bacteria, fungi, protozoa, and viruses through scavenging of iron, direct binding to pathogens, and regulation of the inflammatory response. The aim of this investigation was to compare the effect of two non-cytopathic BVDV isolates (genotypes 1 & 2) on the LPS-stimulated secretion of lactoferrin in bovine tracheal epithelial cells. Tracheal epithelial cells were isolated from healthy calves, cultured on collagen-coated wells, and divided into four groups in triplicate. For each viral isolate, groups 1 and 2 were infected with BVDV after 90% confluence, and groups 3 and 4 did not receive virus. After 24 hours, groups 1 and 3 were treated with LPS. Sixteen hours later, the lactoferrin concentrations in the medium of all four groups were determined by ELISA. Treatment of tracheal epithelial cells with LPS alone resulted in a 5.5-fold increase in lactoferrin concentration compared to untreated cells (5.5 vs. 29.7 ng/ml), whereas LPS-induced lactoferrin concentrations were significantly lower in cells infected with BVDV type 2 (11.6 vs. 29.7 ng/ml). In contrast, infection with BVDV (type 1) did not affect the LPS-induced secretion of lactoferrin. These data indicate that infection of bovine tracheal epithelium by certain BVDV isolates inhibits the LPS-induced secretion of lactoferrin, suggesting a mechanism by which this virus subverts innate immune responses and predisposes cattle to bacterial pneumonia.
154: ULTRASTRUCTURAL AND FUNCTIONAL ANALYSES OF THE INTERACTION BETWEEN CLARA CELL SECRETORY PROTEIN AND EQUINE NEUTROPHILS.
Clara cells are the predominant epithelial cells in the equine bronchiole and in the transitional zone between the conducting and gas-exchanging regions of the lung. Clara cells are increasingly recognized as custodians of pulmonary homeostasis due to their xenobiotic transforming capacity, and as a source of stem cells for bronchiolar repair. CCSP, the secretory product of Clara cells, is among the most abundant soluble proteins of the epithelial lining fluid, and in vitro, inhibits tumor necrosis factor-alpha, interferon-gamma, interleukin-1, and phospholipase A2 activity by as yet undetermined mechanisms. Accordingly, we hypothesized that CCSP decreases neutrophil function in a cytokine-like manner. To test this hypothesis, we cloned eCCSP into an expression vector and sequenced the expressed protein by Edman degradation. Protein stocks were generated from purification of the 16-kDa protein from non-reducing gels by electro-elution. Neutrophil function was evaluated by flow cytometric measurement of dichlorodihydrofluorescein-diacetate oxidation, phagocytosis of labeled microspheres, and chemotaxis in a Boyden's chamber toward IL-8. Results indicate that pre-incubation with reCCSP diminishes neutrophil function, even if primed with PMA and zymosan-activated serum. We further explored the intracellular interaction of CCSP with neutrophils by immunoelectron microscopy of ultrathin sections labeled with a polyclonal antibody raised against a 21-amino acid region of e-CCSP. The antibody reacted specifically with a 16-kDa protein on Western blots and bound intracellularly to non-ciliated cells in horse lung tissue. Results indicate that CCSP modulates neutrophil movement and function in the airways of horses, and may act in a cytokine-like manner.
155: AN ELISA TO QUANTITATE CLARA CELL SECRETORY PROTEIN IN SERUM AND BRONCHOALVEOLAR FLUID OF NORMAL AND HEAVES-AFFECTED HORSES.
Heaves is a chronic, debilitating respiratory disease of horses characterized by increased end-expiratory effort, cough, nasal discharge and neutrophilic inflammation of the small airways. Recurrent exposure to inhaled antigens and endotoxins from indoor environments is thought to trigger an inflammatory cascade. The pathogenesis of the disease is incompletely understood, but has features of human asthma and hypersensitivity pneumonitis (‘Farmer's Lung’). Currently, a diagnosis of heaves is based on physical exam findings, increased bronchoalveolar lavage (BAL) neutrophils, and, at times, pulmonary function testing. These diagnostic methods do not reliably detect early disease. CCSP, the secretory product of Clara cells in bronchioles, is an abundant protein of the pulmonary fluid that inhibits pro-inflammatory mediators. We hypothesized that CCSP is decreased in the BAL and serum of affected horses as a result of chronic exposure to inflammatory mediators and progressive loss of functional Clara cells. To test this hypothesis, we cloned eCCSP into an expression vector and sequenced the expressed protein by Edman degradation. Protein stocks were generated from purification of the 16-kDa protein from non-reducing gels by electro-elution. We then devised an antigen-capture ELISA for the protein after raising peptide antibodies against two different regions of eCCSP in rabbit and goat. Both antibodies reacted specifically with a 16-kDa protein on Western blots and bound intracellularly to non-ciliated cells in horse lung tissue. Results indicate that horses with heaves have decreased CCSP in BAL, likely as the result of chronic inflammation and loss of functional Clara cells. Quantification of CCSP may be a useful diagnostic assay for heaves in horses.
156: GENETIC AND IMMUNOHISTOCHEMICAL EVALUATION OF NASAL CARCINOMAS IN DOGS.
Tumors of the nasal cavity in dogs account for considerable morbidity and mortality, as over 90% are locally invasive and respond poorly to therapy. Carcinomas are most common and comprise 60%–70% of all canine nasal tumors. No genetic predisposition has been identified in canine nasal carcinomas. In contrast, multiple genetic alterations have been associated with an increased risk for the development of human nasopharyngeal carcinomas (HNPC). Linkage to a region of human chromosome 4 (4p15.1–q12) has been demonstrated in HNPCs in patients from Southern China and deletion, point mutation and/or promoter hypermethylation of tumor suppressor gene p16 are the most commonly observed genetic changes. While HNPC cases are positive for p53 in immunohistochemistry (IHC) studies, few mutations in p53 have been identified. Furthermore, Epstein Barr virus (EBV) has been detected in neoplastic cells of HNPCs. Following microscopic review and selection of 43 archived nasal carcinomas, including squamous cell, transitional cell and adenocarcinomas, we investigated these neoplasms for the loss of heterozygosity (LOH) in the canine genome homologous to human chromosome 4p15.1–q12 and to p16. Tumors were also screened for the presence of herpesvirus nucleic acid using a universal primer as well as specific primers for EBV and bovine herpesvirus 4. Immunohistochemistry for p16 and p53 was performed on 32 selected tumors. For 19 informative cases, there was no LOH in the region homologous to human chromosome 4p15.1–q12, but 2 tumors exhibited LOH in p16. No herpesvirus nucleic acid, including EBV, was detected in the 43 cases. Nine carcinomas had no detectable p16 immunostaining and 10 carcinomas were strongly positive for p53. Current research is aimed on identifying the promoter region of canine p16 to study its hypermethylation and to determine the role of p53 in these tumors.
157: VEGF PRETREATMENT OF NEONATAL LAMBS DOES NOT SIGNIFICANTLY ALTER RSV CLINICAL SIGNS OR GROSS LESIONS.
Preterm infants are susceptible to severe respiratory syncytial virus (RSV) infection. Severe infection is often associated with elevations of tumor necrosis factor-alpha (TNF-alpha) which inhibits surfactant protein A (SP-A) via p38 MAP kinase (p38 MAPK), thus creating a proinflammatory environment. Vascular endothelial growth factor (VEGF) is a growth factor that signals through p38 MAPK and our hypothesis is that pretreatment by VEGF will diminish RSV-associated lesions and clinical disease. Lambs (3–5 days of age) were given betamethasone (2 mg intramuscular), recombinant human VEGF (20 ml intratracheal [IT]) or sterile saline (20 ml IT) followed in 30 minutes by intratracheal (20 ml) RSV or sterile media. Rectal temperatures and respiration rates were monitored during the course of the infection. RSV-infected groups had significant (p < 0.01) elevations in temperature on days 4 and 5 post inoculation and the VEFG/RSV group had elevated mean body temperature compared to RSV controls (p < 0.05, days 1 and 3 PI) throughout the course of infection. Elevations (p < 0.05) in respiration rates were present on days 3, 4 and 5 compared to control groups with no significant differences due to VEGF pretreatment. At necropsy, control lambs were free of RSV lesions while RSV infected lambs had multifocal plum-red consolidation (1–4 mm). Gross lesions were scored and no significant alterations were found between RSV groups. These results show that VEGF pretreatment of lungs does not significantly alter respiration rate or development of gross lesions, but may elevate body temperature. Previous work in our lab showed VEGF induced monocytic recruitment to the lung and this may facilitate or enhance RSV interaction with monocytes for cytokine expression.
158: MICROBE-BINDING FUNCTIONS AND GENE POLYMORPHISMS OF PORCINE FICOLIN.
Ficolins are collagenous lectins that bind N-acetylated glycans including oligosaccharides, lipoteichoic acids, peptidoglycans and chitins that are common constituents on surfaces of many microorganisms. Human ficolins bind various bacteria and initiate the ancestral lectin complement pathway so they are believed to contribute to innate resistance to infections caused by the organisms they bind. We are studying the role of collagenous lectins in disease resistance in young animals. The major ficolin in pig plasma, ficolin alpha, also binds N-acetylglucosamine (GlcNAc) and other N-acetylated targets. We hypothesized that ficolins might be involved in innate resistance of pigs to organisms such as Staphylococcus aureus and fungi that have surface polyGlcNAc patterns and that rarely cause disease in pigs. Plasma concentrations of ficolin were determined by ELISA in young pigs and found to range widely between 10 and 80 mg/1 and did not correlate with acute phase inducible proteins. Ficolin purified from porcine plasma bound to intact Staphylococcus aureus, oligomeric GlcNAc and chitin, from which it could be eluted with GlcNAc. In order to determine if genetic variability might affect levels or functions of porcine plasma ficolin, we prepared cDNA from liver mRNA from healthy and diseased pigs. By single-strand conformational polymorphism (SSCP) analysis we identified several single-nucleotide polymorphisms in the coding sequence of the pig ficolin alpha gene. These results support the hypothesis that pig ficolin alpha has a role in resistance to some microorganisms and that there is genetic variation that might affect its expression and function.
159: GENETIC VARIANTS OF PORCINE MANNAN-BINDING LECTINS.
Mannan-binding lectins (MBLs) are collectins that contribute to the innate immune response and inflammation by binding common glycan patterns on surfaces of various microorganisms. Single-nucleotide polymorphisms (SNPs) in the human MBL2 gene are known to alter plasma concentrations of MBL, either by altered gene expression or by defective assembly of the mature oligomeric protein. Such defects have been implicated in susceptibility to various bacterial and viral infections particularly in the young or immunocompromised because they alter the neutralization, opsonization or complement activation functions of MBL. Pigs have two forms of MBL in plasma. MBL-A, that was found to bind to various pathogenic bacteria, is the product of the MBL1 gene that is a pseudogene in humans. By SSCP analysis and sequencing of cDNA generated from livers of various normal and diseased pigs, we have identified several SNPs in the coding region of pig MBL1. One of these, present in various pig breeds, produces a change in MBL-A analogous to those in human MBL2. A PCR test for this SNP was used to assess the genotype of various pigs submitted for necropsy diagnosis. In contrast, no SNPs were found in the coding region of pig MBL-C that is encoded by the MBL2 gene. However, marked variation in hepatic expression of MBL-C mRNA was observed. Similar reduced expression of the human MBL2 gene is due to SNPs in the promoter region so sequences of the promoter regions of pig MBL genes were analyzed for SNPs that correlate with low and high expression of pig MBL-C and with the occurrence of infections. These studies indicate that pigs have several variants in their MBL genotypes that could contribute to differences in susceptibility to infections and inflammatory diseases.
160: STIMULATION OF BOVINE IMMUNE RESPONSES BY A SALIVARY GLAND PROTEIN OF THE STABLE FLY.
The stable fly (Stomoxys calcitrans) is an economically important pest of livestock. Blood-feeding arthropods have adapted to their hosts by secreting an armory of salivary gland substances at the time of feeding that inhibit host hemostasis, pain-itch and immune responses. Prior studies demonstrated lymphocyte suppressive properties of crude salivary gland extract (SGE) of the stable fly. A dominant protein identified in the SGE with homology to antigen-5 (Ag5) of tsetse flies, was shown to bind to the Fc region of immunoglobulins. The purpose of this study was to determine if Ag5 was responsible for the lymphocyte suppression by SGE observed in vitro and if naïve calves mount an immune response to Ag5. Calves raised in the winter months (when stable flies are not present) were immunized with recombinant Ag5 (rAg5). Control calves were immunized with adjuvant alone. Antibody titers to rAg5 were observed during a 42 day period. Rising antibody titers to rAg5 were detected in rAg5 immunized calves, peaking at 28 days post immunization (DPI), but no increase in titers was observed in calves exposed to adjuvant only. Recall lymphocyte responses to rAg5 were detected in immunized calves at 21 and 28 DPI, but not in control calves. Mitogen-stimulated lymphocyte responses were not suppressed by rAg5. These studies indicate calves immunized with rAg5 produce specific immune responses to Ag5 of the stable fly, and thus other salivary gland substances maybe responsible for in vitro lymphocyte suppression. Studies are ongoing to determine if this immunization strategy can be used to disrupt stable fly feeding and reproduction.
161: SPONTANEOUS NON-HEPATOCELLULAR NEOPLASMS IN THE EASTERN WOODCHUCK (MARMOTA MONAX).
The Eastern Woodchuck (Marmota monax) is used as an animal model to examine the relationship between viral hepatitis and the subsequent development of hepatocellular carcinoma. Spontaneous non-hepatocellular neoplasms have previously been documented in the Eastern Wood-chuck, and include lymphosarcoma, apocrine gland adenoma, digital squamous cell carcinoma, fibrosarcoma, meningioma, and lipoma. In a retrospective study involving 619 woodchuck necropsies, thirteen animals (9 males, 4 females, 2.1%) had spontaneous non-hepatocellular neoplasms. From this cohort of 619 animals, testicular and uterine neoplasms (4 animals) have been previously reported. The neoplasms herein reported are from the remaining 9 animals (7 males, 2 females) and include a disseminated lymphosarcoma, two cases of oral squamous cell carcinoma (one with lymph node and pulmonary metastases), a papillary adenocarcinoma of the mammary gland with metastases to the lungs, a renal tubular adenocarcinoma, a bronchiolar carcinoma with metastases to the regional lymph nodes, a malignant pheochromocytoma, an undifferentiated colonic sarcoma with hepatic metastases, and a pancreatic adenoma. For the neoplasms described here, a habitat (wild caught vs. captive bred) predilection was not evident. Affected animals were generally older (5 years+), but ranged in age from 3 to 9 years old. Woodchucks with neoplasms were either Woodchuck Hepatitis Virus (WHV) free, or were naturally or experimentally infected with WHV. No relationship between viral status and the presence of non-hepatocellular neoplasms was found. Only a single neoplasm was found in each animal. To our knowledge, the majority of these neoplasms have not been previously reported in the Eastern Woodchuck, or they were present in newly described anatomical locations (squamous cell carcinoma, lymphosarcoma).
162: POST MORTEM FINDINGS OF HUNTER-HARVESTED BELUGA WHALE (DELPHINAPTERUS LEUCAS) IN JULY 2004 IN THE MACKENZIE DELTA, NORTH WEST TERRITORIES, CANADA.
Belugas are an integral part of the natural history of the Canadian arctic. To establish baseline information on the health status of hunter harvested beluga whales in the Inuvialuit Settlement Region, a diagnostic assessment of 13 animals, comprised of 9 females and 4 males, was undertaken in July 2004. On necropsy, 6 harvested animals had no gross lesions, 2 were emaciated, 4 had moderate verminous pneumonia, and 1 had massive thyroid nodular enlargement. Significant microscopic lesions included diffuse hyperplastic goiter or adenomatous nodular hyperplasia within virtually all of the examined animals. In 7 of 11 belugas, there were adrenal cortical cysts. Both thyroid and adrenal lesions have previously been recognized in Churchill and St Lawrence beluga stocks and the pathogenesis of these conditions remains unknown. Low grade to intermediate pleuropneumonia and chronic interstitial pneumonia with scattered, secondary suppurative and eosinophilic bronchopneumonia were apparent in 6 of 11 cases. Although no consistent pathogens were recovered from sampled lungs, adult nematodes (presumably Halocercus monoceris) were commonly recognized within examined sections. Myocellular sarcocystosis was evident in 7 animals. Trace mineral analysis disclosed elevated liver mercury and selenium levels in 5 of 7 animals. Four of 13 belugas were seropositive for Brucella spp by competitive ELISA; however, no significant bacterial pathogens were recovered by routine culture. Pooled tissues were negative by polymerase chain reaction for Brucella spp, morbillivirus, canine distemper, Toxoplasma gondii, and Mycoplasma spp. This investigation provides baseline information into the health status of beluga whales harvested for human consumption and potential impact of oil development in this region on the population.
163: ENZOOTIC, SPORADIC AND EMERGENT DISEASES OF PACIFIC NORTHWESTERN MARINE MAMMALS.
Over the past 6 years, there has been an increasing effort to monitor and examine stranded marine mammals from Washington State and British Columbia. Results from systematic postmortem examinations and comprehensive diagnostic evaluations provide baseline information necessary to establish enzootic, exotic and emerging disease concerns. Neonatal and juvenile harbor seals comprise the majority of pinniped diagnostic submissions. Established conditions include localized infections, generalized septicemia, starvation, and neonatal abandonment. Emerging disease concerns of harbor seals include Sarcocystis neurona, Brucella spp., Salmonella spp., multiple antibiotic resistant Escherichia coli and Clostridium difficile-Some of these pathogens may represent exposure to rural or urban surface water discharge or sewage effluent. A similar range of infectious processes have been detected in small cetaceans. In 2004, an incursion of Leptospira pomona was associated with the northward migration of subadult and adult male California sea lions. Ongoing efforts to serologically assess post mortem heart blood have disclosed a number of Brucella spp positive cetaceans along the coast with rare cases of attendant meningoencephalitis. Because of their environmental and dietary sympatry with humans, high trophic levels and relatively longevity, these coastal marine species serve as excellent sentinels of ecosystem health and provide warning of possible emerging disease concerns in the Pacific Northwest.
164: PATHOLOGICAL AND MOLECULAR BIOLOGICAL STUDIES OF BOVINE PAPILLOMA IN KOREA.
Clinical bovine papilloma-like infection has been reported frequently in Korea. To investigate its prevalence, pathology and molecular epidemiology, teats of 880 cows (432 from Holstein and 448 from Korean native cows) were collected from local abattoirs of 9 different provinces in Korea during the period from January 2001 to November 2002. Suspected bovine papilloma lesions were observed in 296/880 (33.6%) teats. grossly and histologically. The prevalence of papilloma was 8 times greater in Holsteins (263/432) as compared to native Korean cows. Histologically, teat papillomas were characterized by varying degrees of hyperkeratosis, hyperplasia of granular and prickle cell layers, and large, irregular keratohyaline granules in granular cells. Immunohistochemical staining for bovine papilloma virus (BPV) antigen revealed scattered staining in the nuclei of degenerated granular and cornified cells. Twenty-three percent of papillomas in Holsteins were positive for BPV by immunohistochemistry (IHC), as compared to only 3.8% positive in Korean native cows. Using electron microscopy, BPV particles were found in 39.2”″ of papillomas in Holsteins, but were not found in any of the samples from Korean native cows. To increase the sensitivity and specificity ° BPV detection, a PCR assay was developed, using one primer pair to detect any BPV type and six primer pairs specific to BPV types 1 through 6, respectively. BPV DNA was detected in 71.4% of Holstein teat papillomas with the BPV universal primer pair, compared to a 21.4% positivity rate for Korean-native samples. Of ten samples positive using the universal primer pair, only two were positive using the primers for types 1 through 6 (one was positive for type 1 and the other for type 6). The nucleotide sequences between each PCR amplified product of field teat papilloma were compared with the other known BPV types. All 21 tea papillomas showed 36.776.0% nucleotide sequence homology in comparison with that of BPV 16 reference strains. In conclusion, bovine papilloma infection is prevalent in Korea and it is speculated that variants of BPV circulate in Korea.
165: APPLICATION OF NESTED PCR TO BODILY FLUIDS FOR THE DETECTION OF CATTLE INFECTED WITH MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS (MAP).
The objective of this study was to determine the potential of nested PCR (nPCR) on blood, milk, semen and placental fluid to detect Mycobacterium avium subsp. paratuberculosis (Map) in clinical and subclinical infected cattle. A nPCR probing for IS900 was developed and its sensitivity and specificity in detecting MapDNA in peripheral blood and milk samples was compared with that of ELISA and AGID serology in a cohort of 11 clinical and 46 subclinical lactating Holstein cows from a dairy herd with confirmed Johne's disease. In addition, placental fluid from 8 pregnant cows from the same herd and of 3 pregnant cows from a beef herd with confirmed Johne's disease was analyzed for evidence of MapDNA via nPCR. Blood and semen from a Holstein bull from the same dairy herd with clinical signs of Johne's disease was examined for evidence of MapDNA via nPCR. Map was confirmed in all blood and milk samples (100%) from all 11 clinical cows from the dairy herd by nPCR as well as by ELISA, but only 55% of samples were positive by AGID. A total of 52% of the subclinical cows tested positive for Map by nPCR, compared to 39% positve animals by ELISA and none by AGID. Two (11%) of the placental fluid samples tested positive by nPCR as did the blood and semen samples of the tested bull. nPCR has a potential as an antemortem test applied to various bodily fluids to detect clinical and subclinical animals infected with Map.
166: COLITIS ASSOCIATED WITH CLOSTRIDIUM DIFFICILE IN THE SPECIFIC PATHOGEN FREE C3H-SCID MICE.
Colitis associated with Clostridium difficile in gnotobiotic mice is characterized by decreased cecal size, polymorphonuclear cell infiltration and edema of the lamina propria in the large intestine. Inflammatory edema of the cecal submucosa occurs in mice in association with moderately toxinogenic C. difficile strains. Soft feces and reduced reproduction occurred in specific pathogen free C3H-SCID mice, Grossly, the ceca of these mice were smaller than those of normal mice and contents were decreased. Histologically, severe edema was observed in the cecal submucosa, and neutrophils and mononuclear cells infiltrated the lamina propria and submucosa of the ceca and colon. Clostridium difficile was isolated from cecal contents and toxin A was detected from the isolate. Evidence of other murine enteric pathogens, i.e., Salmonella spp., C. rodentium, P. aeruginosa, C. kutscheri, C. piliformis, and mouse hepatitis virus was not detected by bacteriological or serological tests. The findings in our case are similar to those of mice described in previous reports. Based on pathologic findings and results of microbiological examination, our case was diagnosed as C. difficile-associated colitis in SCID mice.
167: THE DISTRIBUTION AND DENSITY OF CLOSTRIDIUM DIFFICILE TOXIN RECEPTORS ON THE INTESTINAL MUCOSA OF NEONATAL PIGS.
Clostridium difficile is an enteric pathogen affecting a variety of mammals and has recently been diagnosed as a cause of neonatal typhlocolitis in pigs. Toxin A (TcdA) is a large AB-like exotoxin and an important virulence factor of C. difficile. It has been clearly established that the effects of TcdA on host tissues are dependent upon the receptor-mediated endocytosis of the intact toxin. One hypothesis to explain the resistance of most species as neonates (e.g. humans and hamsters) is that they may lack significant numbers of toxin receptors. The susceptibility of neonatal pigs suggests cells of the gastrointestinal mucosa in this species, express sufficient numbers of toxin receptors for lesion development. Immunohistochemical (IHC) assays documented specific binding of toxin A, but not toxin B, to epithelium of the small and large intestine. The carbohydrate Galalphal-3betal-4GlcNAc-R, has been described as an important receptor for TcdA. However, IHC indicated a distribution on cell surfaces much different than that of TcdA binding, suggesting a specific interaction of toxin with an alternative receptor.
168: PHYLOGENETIC CHARACTERIZATION OF CANINE DISTEMPER VIRUSES DETECTED IN NATURALLY INFECTED NORTH AMERICAN DOGS.
In 2004, six puppies and one adult dog from four separate premises underwent necropsy evaluation. Five of seven dogs had clinical signs consistent with canine distemper virus (CDV) infection. I In all dogs, a diagnosis of CDV was established based upon gross and histologic lesions, immunohistochemical labeling for CDV antigen and detection of CDV RNA by RT-PCR. To further characterize the CDV strains detected in the four cases, complete gene sequences were determined for the hemagglutinin (H) and fusion (F) protein genes while partial gene sequencing was performed for the phosphoprotein (P) gene. A total of 4,508 bases were sequenced for the CDV strains detected from the four cases. Two cases were found to have identical sequence except for two bases in the intergenic region of the F and H genes. Phylogenetic analysis strongly suggested an evolutionary relationship between sequences detected in these two cases with those of Phocine distemper virus 2 and two other strains of CDV not previously detected in the continental United States. Clear phylogenetic relationships were not established for viruses detected in the two additional cases, however one strain showed similarity to CDV strains detected in pandas from China. The three CDV strains detected were demonstrated to be genetically distinct from known vaccine strains and strains previously reported in the continental United States.
169: DETECTION OF CLOSTRIDIUM BOTULINUM TYPES B AND C IN TISSUES BY IMMUNOHISTOCHEMISTRY.
Equine toxicoinfectious botulism is a neuromuscular disease caused by toxins from Clostridium botulinum types B and C. Current diagnosis requires identifying toxin in serum or gastrointestinal contents by mouse bioassay or ELISA. Because horses are so susceptible to the toxins, levels below detection limits of these tests may be sufficient to cause disease. We believe equine toxicoinfectious botulism is currently underdiagnosed, and more appropriate tests are needed. Additionally, the high prevalence of equine gastric ulcers warrants investigation of their role in the pathogenesis of this disease. Our objective was to develop immunohistochemical tests to detect Clostridium botulinum types B and C in equine gastric ulcers. We tested three commercial antibodies, one each to type B and C neurotoxin (Metabiologics), and one to type B neurotoxin complex (Biodesign). We tested each antibody against preparations of known C. botulinum types B and C, other clostridial species, and paraffin-embedded sections of equine gastric ulcers. Each antibody correctly identified its respective type of C. botulinum in known bacterial preparations, with minor cross-reaction between types and some cross-reaction with other clostridial species. Of the 34 equine gastric ulcers examined, 7 contained Clostridium-like bacteria on hematoxylin and eosin stained sections. Of these, 4 tested positive for C. botulinum type C, 5 tested positive for C. botulinum type B, and 3 tested positive for both types. Three ulcers without evidence of bacterial rods also tested positive for C. botulinum type C. These results suggest that immunohistochemistry with currently available antibodies may be a useful screening tool for equine toxicoinfectious botulism.
170: FATAL MELIOIDOSIS IN TWO RECENTLY IMPORTED CYNOMOLGUS MACAQUES (MACACA FASCICULARIS).
Two adult male wild-caught cynomolgus macaques (Macaca fascicularis) were received from Indonesia four and seven months prior to death. Clinical and pathological findings in both cases were similar. The animals presented with acute lethargy and depression and died before thorough diagnostic evaluations could be completed. Gross findings included suppurative hepatitis, splenitis, and peritonitis in both animals. Nephritis and seminal vesiculitis were also noted in one animal, and thoracic and abdominal abscessation was present in the other. Microscopic changes included widespread pyogranulomatous inflammation with extensive necrosis, often with demonstrable intralesional bacteria in liver, spleen, kidney, lungs, stifle, seminal vesicle, epididymis, prostate and meninges. A pure growth of Burkholderia pseudomallei, the causative agent of melioidosis, was recovered on culture. Given the zoonotic nature of this organism, the potential for laboratory transmission, and its classification as a Category B agent of bioterrorism by the Centers for Disease Control and Prevention's Strategic Planning Workgroup, melioidosis should be considered a critical differential diagnosis in Asian macaques with suppurative visceral infections. Current bioterrorism concerns highlight the importance of veterinary medicine in public health, and warrant heightened awareness of the role of potential bioterrorism agents in exotic animal diseases.
171: SIMILAR SCRAPIE PRION PROFILES IN SHEEP WITH DIFFERING PRION GENOTYPES AND STRAINS.
Ovine scrapie is a transmissible spongiform encephalopathy endemic in the United States. Scrapie susceptibility is controlled, in part, by a combination of host genotype (coding polymorphisms in the prion precursor gene PRNP) and scrapie strain (probably associated with poorly defined conformational changes in the disease-associated prion protein PrP-d). Two strains of scrapie have been described in experimental and field studies in the UK and Europe and in field cases in the US. In spite of differences in incubation time, transmission efficiency, and susceptible genetic groups between the scrapie strains, PrP-d molecules from the two US strains were indistinguishable in apparent molecular weight, glycoform profile and proteinase K sensitivity. The distribution of PrP-d and the extent and type of spongiform change in the medulla of affected sheep were also similar between PrP-d strains. Sheep with the PRNP genotype AVQQ are susceptible to both strains. In the absence of definitive criteria for strain differentiation, diagnosis of scrapie in sheep of this genotype may result in removal of all flockmates susceptible to either strain.
172: ENDOCRINOLOGICAL EFFECTS OF SPARGANOSIS IN THE BABOON.
The second stage larva (Sparganum) of the pseudophyllidean cestode of the genus Spirometra causes larval diphyllobothriasis or sparganosis in intermediate hosts, including humans. Here we describe a case of sparganosis in a 12 year old, captive, pregnant, wild-caught (Tanzania) baboon that had multiple subcutaneous nodules. During a term caesarean section the nodules were biopsied and sparganosis was diagnosed histologically. Blood samples for hematology/biochemistry, estradiol/progesterone analysis, and anti-Sparganum antibodies were taken from mother and fetus. Morphometries were performed on the fetus and placenta. Necropsy of the mother revealed disseminated sparganosis, involving skeletal muscle, lymphatics, mesentery, serosa, pleura, pericardium, kidney, liver and diaphragm. Larvae were not found in central nervous system, mammary glands, spleen, placenta or fetus. Placental weight and fetal hematological/biochemical/morphometric/hormonal parameters were within normal ranges. Antibody titers to Sparganum were not different in maternal (1.08 ng/ml) compared to fetal (0.91 ng/ml) circulation. In addition, antibody titers to Sparganum were determined in 76 additional wild-caught colony baboons. Twenty eight of the 76 baboons had positive antibody titers to Sparganum. Since the growth factor produced by Sparganum is very similar to mammalian growth hormone, the hormonal status of Sparganum-infected baboons was investigated. Estradiol/progesterone analysis in 8 of these animals (antibody titers range: 0.71–1.7 ng/ ml) showed no statistically significant difference with age/cycle phase-matched parameters compared to antibody-negative females. Insulin-like growth factor and growth hormone values in these 8 baboons were within the normal range. In this case of disseminated baboon sparganosis, placental and fetal development were not impaired. Baboons with high antibody titers to Sparganum had normal endocrine values.
173: HEMORRHAGIC DISEASE IN DOGS INFECTED WITH AN UNCLASSIFIED INTRAENDOTHELIAL PIROPLASM.
A disease affecting dogs in Brazil, referred to popularly as “nambiuvú” (bloody ears) and believed to be transmitted by ticks, has been observed in animals infected with an unclassified organism described originally in 1908 as a protozoan parasite, and known locally as Rangelia vitalli. In a series of 9 cases, the disease was characterized clinically by anemia, jaundice, fever, lymphadenosplenomegaly, hemorrhage in the gastrointestinal tract, and bleeding from the nose, oral cavity and tips and external surface of the pinnae. The ixodid ticks Rhipicephalus sanguineus and Amblyomma aureolatum infested affected dogs from suburban and rural areas, respectively. Laboratory findings included regenerative anemia, spherocytosis, icteric plasma and bilirubinuria. Cytology revealed zoites in the bone marrow. Histologically, numerous round, 2.5 micrometer zoites were seen within the cytoplasm of blood capillary endothelial cells which were positive immunohistochemically for vWF. Langhans-type multinucleate giant cells were found in the lymph nodes and choroid plexus. There was erythrophagocytosis in the lymph node sinuses and infiltration of the medullary cords by plasma cells. Ultrastructurally, this organism had an apical complex with a polar ring and rhoptries, and was contained in the cytoplasm of capillary endothelial cells, within a parasitophorous vacuole which had a trilaminar membrane with villar protrusions. The organism tested positive by immunohistochemistry for Babesia microti, and was positive by in situ hybridization for B. microti. The disease was reproduced by intravenous inoculation of blood from a naturally infected dog into an experimental dog. This study shows that this organism is a piroplasm. It seems to be different from Babesia as it has an intraendothelial stage.
174: PROGNOSTIC SIGNIFICANCE OF INTRATUMORAL MICRO-VESSEL DENSITY IN CANINE SOFT-TISSUE SARCOMAS.
Canine soft-tissue sarcomas (STS) are a heterogenous group of neoplasms that are classified collectively, since they have similar histologic features and clinical behavior. Prognosis has been traditionally based on histologic grading, but the prognostic value of certain cellular proliferation markers has recently been demonstrated. Another method of predicting the behavior of neoplasms is intratumoral microvessel density (IMD), which is a measure of the tumor angiogenesis (intratumoral new vessel growth). The general consensus regarding IMD and tumor prognosis is that higher IMD values correlate with faster growing tumors and increased tumor metastases. The prognostic significance of IMD has been documented in many human neoplasms and in a limited number of canine and feline neoplasms. To evaluate the prognostic value of IMD in canine STS, we studied 57 STS and compared IMD with histologic grade. Using immunohistochemistry, the STS were labeled with anti-Factor VIII-related antigen (FVIII-RA) antibodies to determine their IMD. There is positive association between increasing mean and highest microvessel counts based on FVIII-RA expression with increasing histologic grade. Mean microvessel counts were 112 vessels/mm2 (grade I), 163/mm2 (grade II), and 186/mm2 (grade III). Highest vessel counts were 134 vessels/mm2 (grade I), 201/mm2 (grade II), and 230/mm2 (grade III). Determination of IMD in canine STS using FVIII-RA immunohistochemistry is positively associated with histologic grade. Additional studies are ongoing, investigating relationships between IMD and clinical parameters, and the use of anti-CD31 antibodies to determine IMD.
175: HISTOPATHOLOGICAL STUDY ON THE ADRENAL GLANDS OF BOVINE CLONES.
Bovine clones derived from somatic cell nuclear transfer have been produced since 1998. We have examined aborted and stillborn calves to evaluate the phenotypic characteristics of the bovine clones. We previously reported abnormal retraction of umbilical vessels, hyperplasia of placental trophoblasts, and decreased ACTH-positive cells in the pituitary of these calves. In this study, we demonstrate the pathological findings of the adrenal glands. Nineteen clones (Japanese Black; 11 males, 3 females, Holstein; 5 females) that were aborted (1), stillborn (13), died as neonates (3), or euthanized (1) were studied. Three calves produced by artificial insemination served as controls. Tissues were processed as usual and the Cherukian Schenck stain was performed to identify chromaffin cells in the adrenal medulla. Macroscopically, enlargement of the adrenal glands were observed in 13 animals, including all 6 clones that had “large offspring syndrome”. The adrenal glands of 6 clones had an irregular surface. Enlargement was due to increased cortical area in most cases. Histologic abnormalities were detected in 4 clones. In these glands, epinephrine-producing cells, normally located in the outer zone of the medulla, were not detected. There were also decreased norepinephrine-producing cells located in the inner zone of medulla. Nodular hyperplasia of the cortex was observed in 3 out of the 4 clones. Hyperplasia of the cortex cells were recognized in the other 2 clones. These findings in the bovine clones might be related to the large offspring syndrome and/or prolonged gestation of the bovine clones.
176: OCULAR MELANOSIS IN CAIRN TERRIERS.
Ocular melanosis (OM) is an inherited condition that occurs predominantly in Cairn terriers. The purpose of this study was to describe the clinical phenotype, histopathologic features and mode of inheritance of this disease. Ophthalmological examination records of 169 affected Cairn terriers were reviewed and a pedigree of affected families was constructed. 18 enucleated globes were examined by light microscopy, immunohistochemistry, and transmission electron microscopy. Bilateral ophthalmoscopic changes were first noted between 4 and 12 years of age. Initially the iris root was thickened and pigmented patches appeared in the sclera/ episclera overlying the ciliary body. Pigmented particles were released into the aqueous and deposited ventrally to obscure the drainage angle. Eventually secondary glaucoma developed. Microscopically, large plump pigmented cells infiltrated the entire uvea, obliterated the iridocorneal filtration angle, and transversed the sclera into the episclera. The infiltrating cells were negative for melan A, synaptophysin, SMA, MSA, chromogranin A/B, PGP9.5, CD45, CD18, cytokeratin AE1/AE3, and MNF116. Variable numbers of these cells were positive with MITF and vimentin, but most were positive for HMB45. Ultrastructurally, pigment laden cells had a single distinct, cleaved, centrally located, euchromatic nucleus and contained stage 3 and 4 and occasionally stage 2 melanosomes. Few mitochondria, but no intermediate filaments were observed. Pedigree analysis indicates that the condition is inherited in an autosomal manner, but the exact mode of inheritance was not defined. OM in Cairn terriers is characterized by slowly progressive infiltration or proliferation of pigment laden cells primarily affecting the anterior segment. Immunohistochemical staining and ultrastructural features suggest that these cells are melanocytes, although the exact cell lineage remains to be determined.
177: IMMUNOHISTOCHEMICAL CHARACTERIZATION OF RETINAL CHANGES IN THE ROD CYCLIC GMP PHOSPHODIESTERASE ALPHA (PDE6A) MUTANT DOG MODEL OF RETINITIS PIGMENTOSA.
The retinal dystrophy in the Cardigan Welsh corgi (CWC), known as rod cone dysplasia type 3 (rcd3) is a model for autosomal recessive retinitis pigmentosa due to mutations in the gene encoding rod cyclic GMP phosphodiesterase alpha subunit (PDE6A). This study characterizes the retinal changes in CWC due to the PDE6A mutation. The posterior eye cups from PDE6A mutant dogs and normal controls between 3 and 60 weeks of age were processed and stained with antibodies against rhodopsin, red/green cone opsin, PKCalpha, calbindin, Hu, calretinin and GFAP. Development of rhodopsin positive outer segments of mutant dogs was halted at 4 weeks of age and followed by a rapid loss of the rod cells. Cones positive for red/ green opsin degenerated at a much slower rate with remaining cells showing altered localization of opsin immunoreactivity suggesting they were perturbed by the loss of surrounding photoreceptors. PKCalpha staining rod bipolar cell loss and altered pattern of cell processes and synapses followed the loss of rod photoreceptors. Staining for inner retinal neurons using anti-Hu (ganglion and amacrine cells), calbindin (horizontal cells) and calretinin (amacrine, ganglion and horizontal cells) demonstrated relative preservation of the inner retina early in the disease process. Activation of GFAP-positive Müller cells was apparent from an early age, prior to substantial loss of photoreceptors. Based on these observations, the number of neuronal processes remaining in the inner retina appeared proportionally greater than the number of surviving cell bodies indicating sprouting of neuritis and rewiring, including misdirection of neuronal processes.
178: A COMPARATIVE STUDY ON THE ACTIVITY OF MATRIX METALLOPTOTEINASES (MMPS) AND TISSUE INHIBITORS OF METALLOPROTEINASES (TIMPS) IN MAMMARY TUMORS OF DOGS AND RATS.
The ability to degrade extracellular matrix is important in the biology and behavior of neoplasms. Using spontaneous canine mammary neoplasms, and chemically-induced (intragastric 7,12-dimethylbenz(a)anthracene) mammary neoplasms from rats, we conducted zymography and reverse zymography to detect the activity of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), respectively. Film in situ zymography (FIZ) was used for tissue localization of gelatinolytic activity. MMP-9 and MMP-2 was higher in canine neoplasms than the rat tissues. The activity of latent MMP-2 and latent and active MMP-9 was significantly higher in malignant canine neoplasms than in benign tumors. The activity of latent MMP-2 was higher in benign and malignant rat tumors compared to normal tissues. The results of reverse zymography indicated that the activities of TIMP-1, -2 and -3 were significantly higher in rat neoplasms compared to the canine tumors. TIMP activity was higher in malignant canine neoplasms than benign. FIZ results correlated with MMP-2 activity in canine cases; the digested area was the same in both benign and malignant rat mammary tumors. These results suggest that the more aggressive behavior of the malignant canine mammary neoplasms (compared to the rat) is, in part, the result of high MMP and low TIMP activity.
179: MARE REPRODUCTIVE LOSS SYNDROME: IRRADIATED EASTERN TENT CATERPILLARS INDUCE ABORTION IN MARES.
Late instar Central Kentucky Eastern tent caterpillar (ETC, Malacosoma americanum) larvae were irradiated at 25, 30 and 40 kilo Grays to evaluate the ability of radiation to destroy viruses and bacteria but to conserve enzymatic activity. Based on the results of this initial study, ETC larvae were irradiated in bulk at 30 kiloGrays and administered at 100 g/day by oral gavage to six late term pregnant mares until they aborted or for 10 days. Non-irradiated larvae and normal saline were administered to late term pregnant mares and served as positive (n = 6) and negative (n = 6) controls, respectively. Three of the six mares receiving irradiated ETC aborted on experimental days 12,14, and 24, respectively. Mares receiving non-irradiated ETC (positive controls) aborted within a period of 1.2–5 days. No negative control mare aborted. Umbilical cords from two of the mares receiving irradiated ETC had funisitis (inflammation of umbilical cord) similar to that observed in naturally occurring Late Fetal Loss/Mare Reproductive Loss Syndrome (LFL/MRLS). Actinobacillus spp. and Streptococcus spp., the same bacteria which were isolated in naturally occurring MRLS abortions, were isolated from multiple organs and placenta of these fetuses. Irradiation of ETC substantially reduced the abortifacient potential of ETC larvae in pregnant mares. Abortions induced by irradiated ETC closely resembled field LFL/MRLS and the data suggest that bacterial or viral agents associated with ETC are not the primary cause of MRLS. This is also the first experimentally induced funisitis in any animal species.
180: IMMUNOHISTOCHEMICAL DEMONSTRATION OF PROTEIN GENE PRODUCT 9.5 IN CANINE CUTANEOUS EPI-THELIOTROPIC LYMPHOMA (MYCOSIS FUNGOIDES).
Protein gene product 9.5 (PGP 9.5) has been identified in neurons and used as a marker of human neuroendocrine tumors. Studies have demonstrated PGP 9.5 in a variety of neuroendocrine and non-neuroectodermally derived normal tissues and tumors. In an immunohistochemical evaluation of a canine cutaneous epitheliotropic round cell tumor, suspected of being melanoma, neuroendocrine carcinoma, or lymphoma, we found strong immunoreactivity for PGP 9.5 along with similar immunoreactivity for CD3, CD45, and no staining for melanoma markers (Melan A and S100). The diagnosis was cutaneous epitheliotropic T cell lymphoma (mycosis fungoides; CETL). We subsequently examined the immunoreactivity of PGP 9.5 on 10 formalin fixed, paraffin-embedded canine CETLs. PGP 9.5 immunoreactivity was observed in 4 cases (40%). The staining was chiefly cytoplasmic and mildly nuclear in >70% of neoplastic cells. To the best of our knowledge, this is the first report of detection of PGP 9.5 in CETLs in dogs. In cases of canine round cell tumors with PGP 9.5 immunoreactivity, leukocytic markers (e.g. CD45, CD3, CD79a, CD20) should be used for accurate diagnosis.
181: CHARACTERIZATION OF P63 EXPRESSION IN SPONTANEOUS EPIDERMAL TUMORS OF DOGS, CATS, HORSES AND CATTLE.
A member of the p53 gene family, p63 is a marker for keratinocyte stem cells and has a critical role in normal epithelial development. Several studies have implicated p63 in the pathogenesis of epidermal tumors in humans. The goal of this project was to characterize p63 expression in spontaneous epidermal tumors of domestic animals. Immunohistochemistry (IH) for p63 was performed on biopsies of naturally occurring canine (n = 45), feline (n = 45), equine (n = 53) and bovine (n = 29) squamous cell carcinoma (SCC), and on basal cell tumors (BCT) and basal cell carcinomas (BCC) of dogs (BCT: n = 10, BCC: n = 6) and cats (BCT: n = 10, BCC: n = 4). In all species, nuclear staining in the basal and parabasal cells was present in the normal epidermis. Staining in the tumors was assessed and compared to that present in normal and hyperplastic epidermis. Increased nuclear expression of p63 was observed in SCC from all species (p < 0.05). Terminally differentiated squamous cells were negative, as in normal epidermis, or only focally expressed p63. Less differentiated cells were generally positive. Carcinoma in situ in horses was also associated with increased nuclear expression of p63 compared to both normal and hyperplastic epidermis (p < 0.05). Canine and feline BCT and BCC had increased nuclear expression of p63 compared to normal epidermis (p < 0.05). BCT and BCC of both dogs and cats had increased and more diffuse expression of p63 than SCC (p < 0.05). These results are similar to those found in studies of human epidermal tumors, and indicate that p63 may have an important role in the cellular differentiation of epidermal tumors in animals.
182: EXPRESSION OF BCL-2 IN THE GLOMERULAR PODOCYTES AND TUBULAR CELLS IN THE KIDNEY OF OSBORNE-MENDEL RATS.
Progressive glomerulonephropathy with severe proteinuria spontaneously develops in Osborne-Mendel (OM) rats. The disease develops earlier and more severely in male than in female rats. The most characteristic and important glomerular change is the loss of podocytes from the glomerular tufts resulting in capsular adhesion and focal segmental sclerosis. The podocyte loss might be due to detachment of these cells from the underlying glomerular basement membrane and/or apoptosis. The podocyte is a terminally differentiated long-life cell and may be strictly protected from apoptosis. It may be difficult to detect podocytes undergoing apoptosis in the glomeruli because of flushing into urine, however, we occasionally find apoptotic bodies in the glomeruli of proteinuric OM rats. To assess the change in expression of Bcl-2 protein in the kidneys of OM rats in association with development of the glomerulonephropathy, immunohistochemical examination was performed in this study. Bcl-2 protein was localized within the podocytes of the renal glomeruli in OM rats as well as other strains of rats (Sprague-Dawley and F344 rats). However in the kidneys of male OM rats aged 15 weeks or more with the progressive disease, some podocytes showed no or decreased expression of Bcl-2 protein. On the other hand, proliferating epithelial cells of Bowman's capsules and epithelial cells of atrophic tubules in the cortex showed distinct expression of Bcl-2 protein. The results suggest that Bcl-2 protein may contribute to podocyte survival and loss of Bcl-2 might partially contribute to loss of podocytes leading to focal segmental sclerosis and progressive glomerulonephropathy.
183: CELLULAR LOCALIZATION OF THE P-GLYCOPROTEIN, ALPHA FETOPROTEIN, AND VON WILLEBRAND FACTOR IN CANINE HEPATOCELLULAR CARCINOMA AND LIVER CIRRHOSIS TISSUES.
The objective of the study was to utilize immunohistochemistry to compare expression of P-glycoprotein (multi-drug resistance gene, MDR-1), Alpha fetoprotein (AFP), and von Wille-brand factor (vWF) in canine hepatocellular carcinoma (HCC) and canine hepatic cirrhosis. Increased expression of MDR-1 was observed in the HCC and cirrhotic livers. Major sites of localization were found on biliary canaliculi and small ductules. AFP was detected exclusively in the cytoplasm of the HCC and a few of the cirrhotic liver tissues. In both cases, the AFP-positive cells were morphologically similar to hepatocytes and had an even distribution of AFP in their cytoplasm. Expression of vessel density (vWF) was significantly higher in cirrhotic liver than HCC. This is the first comprehensive immunohistochemical study of MDR-1, AFP, and vWF expression in two hepatocellular proliferative disorders (neoplastic and non-neoplastic) in the dog. We have demonstrated the presence of a co-regulated receptor system in vivo furthering our understanding of these two diseases in dogs.
184: UBIQUITIN EXPRESSION IN MUSCLE FROM HORSES WITH POLYSACCHARIDE STORAGE MYOPATHY.
Serial sections of formalin fixed, paraffin embedded muscle biopsy samples from twenty-eight Quarter horse, Paint, and draft-related breeds aged 0.5–23 years were stained with periodic acid-Schiff stain (PAS) for glycogen and immunostained to detect ubiquitin expression. Based on PAS stained sections, a diagnosis of equine polysaccharide storage myopathy (EPSSM) was made in twenty-two horses aged 2–23 years (mean 9.4 years); six horses aged 0.5–15 years (mean 7.3 years) were normal. Ubiquitin expression was detected in all but a 2-year-old EPSSM horse and was not detected in the normal horses. Ubiquitin expression was greater than the degree of PAS positive, amylase resistant material and ubiquitin was expressed by amylase sensitive glycogen aggregates as well as by amylase resistant material. Findings support the hypothesis that glycogen storage precedes development of amylase resistant inclusions. Results indicate that the abnormal polysaccharide in EPSSM muscle is ubiquitinated and that ubiquitin expression precedes development of amylase resistant inclusions. Ubiquitin immunostaining was most useful for diagnosis of EPSSM in horses with only amylase sensitive glycogen aggregates and in horses with very early amylase resistant inclusions. But ubiquitin immunostaining is no more sensitive for diagnosis of EPSSM than is PAS stain for glycogen.
185: PATHOBIOLOGY IN THE MMP AND ADAM FAMILIES DETERMINED BY INDUCED MUTATIONS.
The metzincin superfamily of metalloproteases includes matrix metalloproteinases (MMPs, secreted or membrane-type [MT-MMPs]), and disintegrin and metalloprotease (ADAMs and ADAMs with thrombospondin motif [ADAMTSs]) subfamilies that function in cell-extracellular matrix and cell-cell interactions. Targeted deletions and gene insertions have helped to define the involvement of MMPs, MT-MMPs, ADAMs, ADAMTSs and the tissue inhibitors of matrix metalloproteinases (TIMPs) in normal processes and disease, and their potential for therapeutic modulation. Some members of the MMP and ADAM subfamilies use overlapping substrates and have functional redundancy that compensates; resulting in no or subtle abnormalities after changes in expression of these proteins. Changes, most frequently a loss of other proteases in these subfamilies results in distinct structural and functional abnormalities including lethality, reproductive failure, altered host defense and immunity, and defects in the genesis of blood vessels, bone, muscle, adipose tissue, and epithelia. Phenotypic alterations in representative targeted deletion and insertional mutation mouse models such as MMP-9 and ADAM17 (tumor necrosis factor-alpha converting enzyme; TACE) that have contributed to our understanding of the pathobiology of proteases will be discussed.
186: ORAL AND ESOPHAGEAL PATHOLOGY IN BALB/C MICE FOLLOWING CHRONIC ADMINISTRATION OF TGF-BETA ANTAGONIST.
Transforming growth factor-beta (TGF-beta) is a pleiotropic family of growth factors participating in such cellular processes as embryogenesis, extracellular matrix and bone formation, wound healing, immune/inflammatory processes, and carcinogenesis. TGF-beta has a bifunctional role in the immune system, regulating both proinflammatory and immunosuppressive actions. In exploratory toxicology studies, BALB/c mice were treated intraperitoneally for 12 weeks or longer with cumulative doses of at least 30 mg/kg TGF-beta neutralizing antibody biweekly. During treatment, some mice in the highest dose groups lost weight, and several animals lost the clinical crowns of incisor teeth. The incisors re-grew but were discolored and misshapen. At necropsy, several mice had glossal swelling. Histologically, mice exhibited glossal cystic epithelial hyperplasia, neutrophilic and lymphocytic esophagitis, and hyperplasia of the esophageal mucosa. In addition, severe hyperplasia of the proximal gingival and anchoring epithelia, suppurative periodontitis, and dental dysplasia were present in some animals. These findings demonstrate dysregulation of epithelial cell proliferation, which may be due to the blockade of TGF-beta's inhibitory role in epithelial cell growth. The inflammatory responses also suggest that the normal suppressive actions of TGF-beta on other proinflammatory cytokines, such as IL-1, IL-12, and TNF-alpha may be muted by neutralizing antibody. These histologic findings are dose dependent and have not been observed in doses below 5 mg/kg TGF-beta neutralizing antibody biweekly for treatment periods of up to 6 months. This particular spectrum of lesions has not been seen to date in other species and therefore may represent a mouse-specific phenomenon.
187: PHYTOL-INDUCED HEPATOTOXICITY IN MICE: IMPLICATIONS FOR PEROXISOMAL OXIDATION OF LIPIDS.
Degradation of chlorophyll by ruminal bacteria produces phytol which is readily converted to phytanic acid, a branched chain, saturated fatty acid present in high concentrations in dairy products and ruminant fat. Other dietary fats, including vegetable oils, contain lower levels of phytol which is readily converted to phytanic acid after absorption. Phytanic acid undergoes oxidation in peroxisomes. Hereditary defects in some peroxisomal enzymes cause massive accumulation of phytanic acid and its metabolites. Phytanic acid is a peroxisome proliferator, binding the nuclear transcription factor PPAR alpha to induce expression of genes encoding enzymes of fatty acid oxidation in peroxisomes and mitochondria. In mice, sub-chronic (12–18 days) administration of phytol at supraphysiological levels (0.5–1% w/w diet) caused accumulation of high levels of phytanic acid and its metabolites in serum and liver and predictable PPAR alpha-mediated responses such as peroxisome proliferation and increased expression of catalase. Microscopically, it also caused hepatocellular necrosis that appeared to have a midzonal distribution as well caused hepatocellular fatty change, hypertrophy and hyperplasia (confirmed by PCNA immunohistochemistry). Female mice were more susceptible to dietary phytol than male mice, presumably because of their 5-fold lower expression of sterol carrier protein-x (SCPx), a lipid-binding protein. These findings support the postulated role of SCPx in the peroxisomal oxidation of branched chain fatty acids. Dietary phytol can be used to metabolically challenge mice lacking genes implicated in peroxisomal uptake and oxidation of lipids.
188: INVESTIGATION OF BIOMARKERS AND MECHANISMS OF P-AMINOPHENOL-INDUCED NEPHROTOXICITY.
Early detection of compound-induced toxicity during drug development offers cost savings and insight into potential mechanisms of toxicity. p-Aminophenol (PAP) is a metabolite of acetaminophen and is known to cause damage to the S3 segment of the nephron. Using PAP-induced nephrotoxicity as a model, this study aimed to identify early biomarkers of nephrotoxicity and to investigate the pathogenesis of acute tubular injury. PAP was given intraperitoneally to rats at a dose of 150 mg/kg. Control and PAP-treated rats were necropsied at 6, 12, 24 and 48 hours post-injection. Histopathologic examination of kidneys revealed acute necrosis of proximal tubules, tubular dilation, and protein casts at all 4 time points. Tubular regeneration was evident starting at 24 hours. Blood chemistry revealed only mild elevations in blood urea nitrogen (BUN) and creatinine. In contrast, PAP caused marked glucosuria, proteinuria, and increased urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG), gamma-glutamyl transferase (GGT), and alpha glutathione S-transferase (alpha-GST) at as early as 6 hours. Of these three urinary enzyme biomarkers, alpha-GST was the most sensitive, being >800-fold elevated at 6 hours. For the most part, changes in urinary biomarkers were greatest at 6 hours, followed by a decline to near pre-treatment levels over time. Dysregulation of known and putative kidney biomarkers, including KIM-1, lipocalin, and heme oxygenase-1, was detected by oligonucleotide microarray analysis of the kidney cortex from control and PAP-treated animals. These results were largely corroborated by microarray analysis of affected and normal proximal tubules isolated using laser capture microdissection (LCM). Sensitive urinary and gene-based biomarkers thus hold promise for detecting early proximal renal tubular injury and providing insight into mechanisms of PAP nephrotoxicity.
189: ANTI-RAT ERBB2 ACCELERATES DOXORUBICIN CARDIAC TOXICITY IN RATS.
Anti-erbB2 (Herceptin) given with doxorubicin induced cardiac toxicity in cancer patients preventing its combined use. We hypothesized that protection from doxorubicin toxicity requires a functional cardiac erbB2/Akt pathway. Female Sprague Dawley rats were group randomized, given six weekly intra-jugular injections of doxorubicin (2.5 mg/ kg) (n = 20), anti-rat erbB2 monoclonal antibody (7.16.4) 2 mg/kg (n = 16), both doxorubicin and anti-erbB2 combined (n = 16), IgG2a (n = 15) or saline (n = 12) and underwent serial echocardiography. Pre-dosing through week 6, 2-D ejection fraction (EF) was similar in all groups. During week 8, in the combined therapy group, the EF was significantly reduced to 50 + 4.6% (p = .0001), compared to 74 + 2.3% (doxorubicin), 86 + 1.4% (anti-erbB2), 85 + .9% (IgG2a) and 86 + 1.2% (saline). Rats were euthanized at week 8 and left ventricle sections frozen. A subgroup of doxorubicin treated rats (n = 5) was followed by echocardiography until week 13 (versus week 8) and euthanized when EF reached 50% suggesting anti-erbB2 accelerated EF decline. Modulation of cardiac erbB2 expression and Akt phosphorylation was analyzed by western blotting and normalized by Coomassie blue. Paradoxically, 2 weeks after the final injection, anti-erbB2 treatment potentiated erbB2 expression (10.3 fold increase over controls in doxorubicin/anti-erbB2 combination treatment) versus (6.1 fold increase in doxorubicin alone). With total Akt remaining constant, doxorubicin alone induced a 4.4 fold increase in pAkt that was paradoxically reduced to 2.8 fold increase in the combined drug group suggesting that anti-erbB2 treatment, given with doxorubicin, attenuates erbB2 pathway and Akt activation. In conclusion, anti-rat erbB2 given with doxorubicin accelerates EF decline and attenuates Akt activation in the face of elevated erbB2 protein. The status of perbB2 and erbB2 cellular location will be helpful in determining the physiological disconnection of this pathway.
190: ANDROGEN DEPENDENT MAMMARY GLAND VIRILISM IN RATS GIVEN A SELECTIVE ESTROGEN RECEPTOR MODULATOR (SERM).
Selective estrogen receptor modulators are nonsteroidal compounds with tissue specific estrogen receptor (ER) agonist or antagonist activities. SERMs modulate the hormonal homeostasis of rodents and may produce morphologic changes in hormonally-sensitive tissues. The rat mammary gland is sexually dimorphic and its morphologic appearance is modified with hormonal perturbations. Ducts predominate over alveoli in normal females and are lined by one or two layers of cuboidal cells with scant cytoplasm (tubuloalveolar morphology). Alveoli predominate over ducts in normal males and are lined by multiple layers of large, vacuolated epithelial cells (lobuloalveolar morphology). The morphology of mammary glands from female rats given the SERM, LY2066948 HC1, for one month was composed mostly of ducts but had intralobular ducts and alveoli lined by multiple layers of vacuolated, hypertrophied epithelial cells, the latter characteristic of male rats. We therefore hypothesized that these SERM-mediated changes represented an androgen-dependent virilism of the female rat mammary gland. To test this hypothesis, the androgen receptor antagonist flutamide was co-administered with LY2066948 HC1 (175 mg/kg) to female rats for 1 month. Female rats given SERM alone had hyperandrogenemia and the duct and alveolar changes described above. Flutamide completely blocked the SERM-mediated mammary gland change despite hyperandrogenemia. In the mouse, a species without a sexually dimorphic mammary gland, SERM treatment resulted in hyperandrogenemia but did not alter mammary gland morphology. These studies demonstrate that LY2066948 HC1 produces an androgen-dependent virilism of the female rat mammary gland that is unique to the rat because of its mammary gland sexual dimorphism.
191: COMPREHENSIVE GENE PROFILING OF THE LIVER IN A LIEBER PECARLI RAT MOPEL TO PREPICT PROGRESSION TO ALCOHOLIC STEATOHEPATITIS.
Alcoholism is a major public health problem in the United States that costs more than $185 billion and results in about 100,000 deaths per year. Chronic ethanol (EtOH) ingestion induces a sequence of hepatic pathologies ranging from steatosis (fatty liver) on one extreme, to the final development of cirrhosis. Alcoholic steatosis (AS) and alcoholic steatohepatitis (ASH) represents the early phase alcoholic liver disease (ALP), which eventually progress to fibrosis/cirrhosis, if not diagnosed and treated early. Currently, the precise mechanisms for progression from AS to ASH are unknown. Thus, identifying genes unique to AS and ASH is of priority to identify mechanistic basis underlying progression from AS to ASH. We hypothesize that a unique set of hepatic genes are expressed in alcoholic patients during alcoholic steatosis that may predict the development of steatohepatitis. The in vivo rodent Leiber PeCarli diet model of AS and ASH was employed to investigate the transcriptome profiles from the liver tissues using the CodeLink® Rat Whole Genome Bioarray which contains approximately 34,000 genes and expressed sequence tags (ESTs). Hierarchical clustering was employed to better characterize the gene expression data in liver tissues from different groups (AS, ASH and respective controls). While the model of AS resulted in both micro- and macrovesicular steatosis, the ASH model showed histopathologic evidence of steatosis, multifocal neutrophilic infiltration and oncotic necrosis. Microarray analysis showed the presence of 4 distinct clusters in which genes were either (a) upregulated in both AS and ASH (b) down regulated in both (c) upregulated in AS and down regulated in ASH (d) down regulated in AS and upregulated in ASH. The protein signatures corresponding to the differential gene expression between AS and ASH will also be presented. To conclude, this study illustrates the value of comprehensive gene profiling to understand the novel mechanisms for progression of pathology from AS to ASH.
192: EARLY ONSET OF SPONTANEOUS RENAL TUBULAR ADENOMA AND MAMMARY GLAND ADENOCARCINOMA IN YOUNG SPRAGUE-DAWLEY RATS (RATTUS NORVEGICUS).
Spontaneously occurring renal tubular adenoma and mammary gland adenocarcinoma in our facilities were identified in control male and control female Sprague-Dawley rats (Rattus norvegicus) in three separate subchronic toxicity studies. Renal tubular adenoma was present in a 30-week-old male, while the mammary gland adenocarcinomas were detected in 10-week-old females. In all these cases, the neoplasms were detected at the conclusion of their respective studies. The renal tubular adenoma was unilateral, subcapsular, multilobulated, formed acini containing debris, and was partially necrotic. Spontaneous renal tubular neoplasms are relatively uncommon in both subchronic and chronic rodent toxicity studies; however, a male predisposition does exist. Histologically, one mammary tumor was expansile, unencapsulated, formed irregular acini, and exhibited both papillary and solid patterns. The second mammary tumor was multifocal and fairly well demarcated. It formed irregular acini and exhibited a solid pattern. Mammary tumors, in female rats, are present much more commonly in rats greater than one year of age, but are infrequently documented in younger animals. Two reports of mammary adenocarcinoma have been found in young Sprague-Dawley rats in Japan; one was seen in a 12-week-old female, the other in a 10-week-old female. To our knowledge, this current report is the first description of a renal tubular adenoma in a relatively young male rat on a subchronic toxicity study. It is also the first report characterizing mammary gland adenocarcinoma in young rats outside of Japan. Such documentation of the early onset of spontaneous neoplasms in the rat will help facilitate the interpretation of tumor data in both subchronic and chronic rodent toxicity studies.
193: LIVER LESIONS IN CATTLE EXPERIMENTALLY POISONED BY SENECIO BRASILIENSIS: QUANTITATIVE AND SEMI-QUANTITATIVE STUDY OF EXTRACELLULAR MATRIX AND SINUSOIDAL CELLS.
Extracellular matrix (ECM) plays an important role in chronic hepatic lesions and has been studied in experimental intoxication models. Since to the authors knowledge there is no specific study in ECM to date in either normal or chronically damaged bovine liver, an experimental model using cows orally fed with Senecio brasiliensis, which contains pyrrolizidine alkaloids that cause either acute or chronic progressive liver damage was designed. Five animals were orally given 0.38 g of dry leaves of Senecio brasiliensis/kg of body weight per day during 24 days. Clinical signs of digestive complications started at the 3rd week. Liver biopsies were performed in five fortnightly moments during 60 days. One animal died at the 45th day. Light microscopy demonstrated, starting on 30th day, hepatocellular degenerative changes, necrosis, portal apoptosis and megalocitosis, centrilobular and pericellular fibrosis. Immuno-histochemistry showed that total collagen and collagen types I and III were progressively increased in every liver compartment. Changes in location, amount and disposition of elastic fiber system were also observed. Damage, necrosis and apoptosis of nuclear membrane and organelles of hepatocytes were seen under transmission electron microscopy. Significant increase of Kupffer cells in 30 days and total sinusoidal cells in 45 to 60 days were observed. Many of sinusoidal cells were similar to transitional hepatic stellate cells previously described. Liver damage was progressive and irreversible even following interruption of plant toxins. Earlier and more severe fibrotic lesions were located on portal and peri-portal tract, followed by veno-occlusive and pericellular lesions. Collagens types I and III were present in every compartment of bovine normal liver, with predominance of type I. Progressive increase of total collagen in every compartment did not change the predominance of type I collagen. Collagen augment was parallel to the increase in elastic fiber system and the number of total sinusoidal cells.
194: INCIDENCE OF SPONTANEOUS MYOCARDIAL VALVULAR LESIONS IN RATS AND MICE ON SUBCHRONIC AND CHRONIC TOXICITY STUDIES.
Spontaneous myocardial lesions are relatively common in sub-chronic and chronic rodent toxicity studies. The most common of these is a microscopic observation characterized as spontaneous cardiomyopathy. Rodent myocardial valvular lesions, however, are infrequently identified in the literature. A retrospective study was conducted to determine the incidence of valvular lesions in an effort to develop an historical control database. Sections of myocardium from control animals and treated animals from the last 18 years were evaluated for lesions including valvular endocardiosis, endocarditis, degeneration and cysts. The incidence of valvular lesions was noted from 3,880 rats and 416 mice. Total incidence of valvular lesions for rats was 0.33% with incidences of 0.13% for valvular degeneration, 0.10% for cysts, and 0.03% for valvular endocardiosis and endocarditis. There was no apparent predilection of gender in rats to develop these lesions. Total incidence of valvular lesions in mice was 1.92% with percentages of 0.24% for valvular degeneration and cysts, and 1.44% for valvular endocarditis. A male predilection was found in mice (88% of lesions noted). Valvular lesions were more commonly seen in the left AV valves; however, lesions were also present on the right AV valves as well. The age range for valvular lesions was between 5 weeks old to 2 years old. No increase in incidence was found between controls and those animals receiving test articles that included contraceptives, anti-inflammatories, and anti-depressants. The data reported here help quantify the spontaneous incidence of myocardial valvular lesions in rats and mice, and help to establish an historical control base for sub-chronic and chronic rodent toxicity studies.
195: PATHWAY OF P53-DEPENDENT TROPHOBLAST CELL APOPTOSIS IN CYTOSINE ARABINOSIDE-TREATED PLACENTA.
Increased placental apoptosis is thought to be related with intrauterine growth retardation or other abnormal pregnancies in human. We previously reported that cytosine arabinoside (Ara-C), a teratogenic DNA damaging agent, induced trophoblast cell apoptosis in rat placenta. In the present study, we injected Ara-C into pregnant rats and mice on day 13 and 12 of gestation, respectively, to investigate the mechanisms of Ara-C-induced placental apoptosis in relation to p53, a tumor suppressor gene which is involved in the onset of apoptosis after DNA damage. In Ara-C-treated placenta, p53 protein expression was elevated and phosphorylation of p53 at serine 15 indicating activation of p53 protein was also increased. Increased expression of pro-apoptotic p53 target genes, noxa and puma, was detected with a reverse transcription-polymerase chain reaction. In p53-deficierit mice, induction of apoptosis was almost completely abrogated. In addition, cDNA microarray analysis detected up-regulation of other p53 target genes such as p21 and reactive oxygen species related genes such as heat shock protein. These results indicate that Ara-C-induced placental apoptosis requires p53 activation, and they also suggest that oxidative stress mediates induction of DNA damage in Ara-C-treated placenta. Because oxidative stress is thought to affect the pathogenesis of abnormal human pregnancy, it is possible that p53 would contribute to placental dysfunction through induction of trophoblast cell apoptosis in response to DNA damage.
196: EFFECTS OF PREGNANCY AND LACTATION ON CYPs PROTEIN AND GENE EXPRESSION PROFILING DURING PREGNANCY IN RAT LIVER.
In the present study, the alterations of CYPs expression during pregnancy and postpartum in the F344 rat liver were investigated by Western blot analysis and immunostain. Total nine anti-rat CYPs antibodies (CYP1A1, CYP2B1/2, CYP2C6, CYP2C12, CYP2D1, CYP2D4, CYP2E1, CYP3A1, and CYP4A1) were used. In comparison with age-matched non-pregnant control rats, there were significant decreases in hepatic levels of CYP2B2, CYP2C6 and CYP4A1 in mid-pregnancy (day 13) and CYP2B2, CYP2C6, CYP4A1, CYP1A1, CYP2B1 and CYP2E1 in late-pregnancy (day 19). By postpartum 28 day (PPD 28), however, the hepatic CYPs down-regulated during pregnancy reverted to the same level of control rats. The expression of CYP2C12, CYP2D1 and CYP3A1 remained unchanged even by pregnancy. CYP2D4 was not detectable in all the liver samples examined. The results of immunostain revealed that CYP1A1 was expressed in endothelial cells of both sinusoids and veins in the liver. The other CYPs were mainly expressed in centrilobular hepatocytes. There were no differences in the distribution and intensity of the expression of the CYPs between pregnant and non-pregnant rats. cDNA microarray analysis was used to identify the changes in the gene expression profile in rat liver during pregnancy (mid- and late pregnancy). Of the approximately 16,000 gene transcripts interrogated, the expression of 394 (257 up-regulated and 137 down-regulated) and 1092 (513 up-regulated and 579 down-regulated) was significantly modified in mid-and late pregnancy, respectively. Real-time PCR analysis was used to confirm the important changes of drug metabolism-related genes detected by microarray analysis. This characterization of the gene expression profile in rat livers during pregnancy will provide a basis for investigating toxicity of extraneous chemicals to fetus and mother and their metabolism during pregnancy.
197: DEFINING THE SAFETY OF DRUG-ELUTING STENTS SYSTEMS IN PORCINE CORONARY AND RABBIT ILIAC ARTERIAL MODELS.
In the forum of interventional cardiology, drug-eluting stent (DES) systems have revolutionized the treatment of coronary artery disease (CAD) and transpired above balloon angioplasty and bare metallic stents to become the current standard of care. DES systems consist of an intravascular implant device that mechanically returns patency to stenotic, diseased coronary arteries and releases a pharmaceutical and/or bioactive agent to reduce the proliferation of neointimal tissue to prevent restenosis at the implant site. Thus, preclinical evaluation for safety of DES systems consists of a fusion of vascular pathology to metallic and polymer-based implant devices with the local and systemic pathology of the pharmaceutical and/or bioactive agents. To date, DES systems have primarily consisted of a base metallic stent with a drug-polymer coating; however, current trends show a rapid and progressive increase in the complexity of these devices, their components, and their targeted biomechanical actions. The projected technological advancement of these DES systems has necessitated the development of standardized preclinical models with standardized means for evaluation of the relative safety of these devices. Herein, the current widely used preclinical models of porcine coronary arteries and rabbit iliac arteries are compared, and a proposed standardized means for routine histopathological assessment of these models is presented.
198: EVALUATION OF THE EFFECT OF A VEGFR-2 INHIBITOR ON ALANINE AMINOTRANSFERASE GENE EXPRESSION AND CONCENTRATION IN THE RAT LIVER.
Traditional assessment of potential drug-induced hepatotoxicity includes gross and microscopic examination of the liver and evaluation of liver enzymes. The aim of this study was to determine the origin of drug-related elevation in serum alanine aminotransferase (ALT) in the absence of hepatic morphologic changes by utilizing molecular and immunohistochemical techniques. Three liver lobes (caudate, right medial, and left lateral) were examined. Eight female Sprague-Dawley rats were divided into 2 groups (n = 4 per group) and dosed orally twice daily with 200 mg/kg of AG28262, a VEGFR-2 inhibitor, for seven consecutive days. Following treatment, ALT serum concentration was significantly (p < 0.05) elevated on day 3 and further elevated on day 8. Histologic changes were not observed in the liver lobes examined. Increases in TUNEL and caspase 3 positive cells in the AG28262-treated group were not observed. ALT gene expression in the caudate lobe following treatment was up-regulated by 63% (p < 0.05) compared to the control caudate lobe. There was also an upward trend in the right medial lobe but no statistically significant change in the left lateral lobe. Correspondingly, significant increases in liver ALT concentration occurred in the caudate (96%; p < 0.01) and right medial (41%; p < 0.01) lobes, and an upward trend was detected in the left lateral lobe. This study demonstrates that AG28262 causes an increase in serum ALT, which could be due in part to both gene up-regulation and increased synthesis of the enzyme in the liver. Differences between liver lobes may be attributable to differential distribution of blood from portal circulation. The results suggest that when evaluating potential drug-induced hepatocellular changes, it is important to sample multiple liver lobes. In addition, because of the complexity of the liver, screening strategies for the detection of hepatotoxicity should include serum chemistry in conjunction with morphology and gene/protein expression data.
199: ADVANCING THE STATE-OF-THE-SCIENCE OF IMMUNOTOXICOLOGY.
The mission of the HESI Immunotoxicology Technical Committee (ITC) is to identify and address scientific issues related to the development and application of immunotoxicology to public health and human health risk assessment; to promote the understanding and appropriate use of immunotoxicologic data to protect human health; and to contribute substantively to the scientific decision-making processes relative to the development of guidelines and regulations for immunotoxicology testing at the local, national and international levels. This mission has been realized through the support of global member companies reflecting the agrichemical, chemical, consumer products, petrochemical and pharmaceutical industries, and significant input from academic and government colleagues. The focus of this poster will be to highlight some of the recent activities of the ITC including the following: a better understanding of how to increase research opportunities to address immune-mediated drug hypersensitivity reactions (IDHR); a proposed testing framework for developmental immunotoxicology; an identification of the priority research needs in respiratory allergy reflecting protein-specific, chemical-specific, and drug-specific issues; an assessment of preclinical data needs for human immunogenicity of large molecules; an appreciation of clinical immunotoxicology; liabilities and bridges between nonclinical and clinical approaches; and an update on the progress of creating a nonhuman primate immunotoxicology database. The success of the HESI ITC is based on an approach where international experts from academia, industry and government come together in a neutral forum and a cooperative atmosphere to engage in an open and transparent exchange of scientific ideas.