Abstract
1: EVALUATION OF PLASMA ANTITHROMBIN III AND D-DIMER CONCENTRATIONS IN POPULATIONS OF HEALTHY AND CLINICALLY ILL CATS.
Current coagulation tests lack sensitivity and detect only severe coagulopathies. Measurement of antithrombin III (ATIII) and D-dimer concentrations permit earlier diagnosis and more precise classification of coagulopathies in some species. To determine the utility of these assays in cats, we evaluated an ATIII chromogenic assay (Stachrom AT III, Diagnostica Stago) and a quantitative D-dimer micro-latex immunoassay (STA-Liatest D-Di, Diagnostica Stago) using the automated STA Compact Hemostasis System (Diagnostica Stago). Citrated plasma samples were collected from 30 healthy, 30 ill, and 13 cardiomyopathic cats. Both assays were linear (ATIII, r = 0.938; D-dimer, r = 0.968). The lower limit of detection was 30% for ATIII and 0.20 mcg/mL for D-dimer. A feline standardization curve for ATIII was similar to the human curve provided by the assay manufacturer. Intra-assay and inter-assay variability for ATIII were 2.68% and 7.48%, respectively. Reference intervals of 96%-142% for ATIII and < 0.20 mcg/mL for D-dimers were established using 30 healthy cats assessed clinically and with CBC and coagulation tests. Neither ATIII nor D-dimer was highly sensitive or specific for the diagnosis of coagulopathies in ill feline patients. Ten patients had elevated D-dimer concentrations. Concentrations of ATIII were increased in twelve patients and decreased in two patients. Although concentrations of ATIII in cardiomyopathic patients were significantly higher than healthy patients (p = 0.01), all were within developed reference intervals. D-dimer concentrations in cardiomyopathic patients were not significantly different from healthy patients (p = 0.69). Further evaluation of these tests will be necessary before they can be used clinically for the early detection of coagulopathies in this species.
2: HEMATOLOGIC AND BIOCHEMICAL ABNORMALITIES INDICATING IRON DEFICIENCY ARE ASSOCIATED WITH DECREASED RETICULOCYTE HEMOGLOBIN CONTENT AND RETICULOCYTE SIZE (CHR & RMCV) IN DOGS.
Background: The Advia 120 automated hematology system uses low- and high-angle light scatter to determine individual RBC or reticulocyte volume and hemoglobin (Hgb) concentration. This allows for the measurement of Hgb content of individual RBCs and reticulocytes. Currently available hematologic and biochemical markers of iron status in the canine patient are insensitive or highly variable, especially in the presence of concurrent disease (i.e. inflammation, neoplasia). The reticulocyte hemoglobin content (CHr) has proven useful in detecting early iron deficiency (ID) and ID masked by concurrent disease in human patients. Objectives: We investigated the association of low CHr and reticulocyte mean cell volume (rMCV) with hematologic and biochemical abnormalities indicative of ID in canine patients. We also established reference intervals for CHr and rMCV and determined the prevalence of decreased CHr and rMCV in the canine population at Colorado State University Veterinary Teaching Hospital (CSU-VTH). Methods: CBC and biochemistry results from the 833 dogs at the CSU-VTH were evaluated and separate groups for low CHr and low rMCV were generated. Results: 7% of all dogs had low CHr and 6% had low rMCV. These dogs had a higher frequency of microcytosis, anemia, low serum iron (Fe) and low percent saturation of transferrin (% sat) than control dogs. Dogs with low CHr had lower median HCT, MCV, serum Fe and % sat than control dogs. Conclusion: Low CHr or rMCV are associated with hematologic and biochemical abnormalities indicative of ID. CHr and rMCV hold promise as non-invasive, cost-effective measures of iron status in the dog.
3: CHRONIC LYMPHOCYTIC LEUKEMIA IN CATS IS PRIMARILY A T HELPER CELL DISEASE.
There is a lack of information in the veterinary literature regarding Chronic Lymphocytic Leukemia (CLL) in cats. Twenty cats with CLL were identified. Clonality assessment by PCR was employed to help confirm the diagnosis in those cases where the diagnosis remained equivocal on the basis of more routine testing. Information pertaining to signalment, history and clinical signs was collected, along with routine CBC information. The immunophenotype of the proliferating lymphocyte population in each instance was determined via flow cytometry and immunocytochemical staining of smears utilizing a panel of 21 monoclonal antibodies. CLL occurred in older cats (mean age 12.2 years; range 6–19 years). The female: male ratio was 1.5. Blood lymphocyte counts ranged from 25,000/ul to 575,130/ul with a mean of 143,157/ul. Fifteen of twenty cats (75%) had T helper lymphocyte (CD3+/CD4+/CD5+) proliferations. Two cats had cytotoxic T cell (CD3+/CD5+/CD8+) proliferations, 2 had a double negative T cell phenotype (CD3+/CD4-/CD8-/CD5 variable) and a single cat had a double positive T cell phenotype (CD3+/CD4+/CD8+/CD5+). Of those cats tested, all were negative for feline retrovirus infection. On initial presentation, lethargy was noted in 9/15 (60%) cats with CLL, splenomegaly or hepatomegaly was noted in 7/15 (47%) and hepatosplenomegaly was noted in 5/15 (33%). Weight loss was noted in 13/15 (86%) of cats and pale mucus membranes were noted in 4/15 (27%). Hematological abnormalities included anemia (Hct < 30%) in 11/18 (61%) cats. Seven cats of 18 (38%) were thrombocytopenic (plts < 180,000/ul). While 3/18 (16%) of were neutropenic (neutrophils < 2,000/ul), 9/ 18 (50%) had neutrophilia (neutrophils > 9,000/ul).
4: NOVEL LIPOSOME-BASED VACCINE FOR ELICITING CYTOTOXIC T-LYMPHOCYTE RESPONSES.
Effective vaccination for many tumors, viruses and intracellular bacteria requires stimulation of cytotoxic T lymphocytes (CTL) through antigen presentation by MHC class I molecules. Historically, it has been difficult to enable exogenous antigens to be presented through the MHC class I pathway. Use of peptide antigens requires that the peptide be restricted to a specific MHC class I haplotype which precludes their use in out-bred species where different individuals express different MHC haplotypes. Also, the antigenic epitope of most protein antigens is unknown, precluding the use of a peptide antigen in many vaccines. A vaccine that incorporates a whole protein antigen and allows the protein antigen to enter the cells own antigen processing pathway would be able to produce peptides specific for its own MHC haplotype. This process is called cross-presentation. Researchers have used various adjuvants and delivery systems to target cross-presentation pathways that elicit CTL responses. We have previously shown that cationic liposomes complexed with plasmid DNA are extremely immunogenic, and thus, may serve as potential adjuvants. We hypothesized that liposome DNA complexes combined with protein antigens would elicit a strong, functional CTL responses. The purpose of the studies presented here was to determine if liposome antigen nucleic acid complexes could elicit antigen specific CTL responses to a protein antigen. Using flow cytometry and tetramer analysis, we demonstrated that vaccination with liposome-antigen-nucleic acid complexes (LANAC) elicited large numbers of antigen specific, CD8+ T cells compared to controls. An in vivo CTL assay confirmed that the antigen specific CD8+ T cells are functional CTLs. These findings suggest that LANAC warrant further investigation for their use as cross-priming vaccines.
5: EVALUATION OF KITS FOR DETECTION OF FIBRIN(OGEN) DEGRADATION PRODUCTS AND D-DIMER IN HORSES.
Our objective was to compare the ability of commercially available latex-agglutination kits to detect equine fibrin(ogen) degradation products (FDPs) and D-dimer. The study population consisted of 30 healthy horses and 20 horses with severe colic and laboratory evidence of DIC, i.e abnormalities in 2 of: prothrombin time (PT), activated partial thromboplastin time (aPTT), Clauss fibrinogen (FIB) and antithrombin activity (AT) within 24 hours of admission. Samples were collected into specialized tubes for serum FDP (Thrombo-Well-cotest) and into 3.2% citrate for hemostasis assays, plasma FDP (Diagnostica Stago), and D-dimer. Four D-dimer kits were evaluated: 3 latex-agglutination (D-Di, Diagnostica Stago; Murex D-dimer, Murex; Accuclot, Trinity Biotech) and 1 immunofiltration assay (Nycocard, Nycomed). At admission, 40% of colic horses had prolonged PT, 55% had prolonged aPTTs, 30% had low FIB and 75% had low AT. Serum (>10 ug/ml) and plasma FDPs (> 5 ug/ml) were 37% and 15% sensitive in colic horses and 57% and 100% specific in healthy horses, respectively. At a 1 ug/ml D-dimer cut-off, kit sensitivities were 20%, (D-Di), 40% (Murex) and 50% (Accuclot, Nycocard). Kit specificities were 100% (D-Di), 57% (Murex), 97% (Accuclot) and 90% (Nycocard). Assay (FDP, D-dimer) and kit results were poorly correlated (Spearman rank < 0.5). Non-surviving colic horses had significantly longer aPTT and lower AT than surviving colic or healthy horses. Accuclot and Nycocard D-dimer were higher in non-surviving colic than healthy horses. Our results indicate that healthy horses have detectable levels of serum FDP and D-dimer and a high D-dimer (Accuclot, Nycocard) supports the diagnosis of DIC in horses with colic. In this case series, prolonged aPTT and low AT were negative prognostic indicators in horses with severe colic.
6: PHENOTYPIC DIFFERENCES WITHIN THE AB BLOOD TYPE OF THE FELINE AB BLOOD GROUP SYSTEM.
Flow cytometry immunolabeling, tube agglutination tests and thin layer chromatography immunostaining with two different anti-A monoclonal antibodies (mAbs) and one anti-B mAb were used to demonstrate differences in expression of the A antigen between erythrocytes from type A and AB cats, and in the A and B antigens between erythrocytes from four different type AB cats. Although the flow cytometric patterns of reactivity and agglutination scores for erythrocytes from type A and B cats with the anti-A and anti-B mAbs were consistent within each type, reactivity between erythrocytes of different type AB cats was variable. By flow cytometric analysis, one anti-A mAb bound 100% of type A erythrocytes but only 2.5 to 4% of erythrocytes from 3 of 4 type AB cats and 61% of erythrocytes from 1 of 4 type AB cats. Another anti-A mAb bound 20–38% of erythrocytes from the same 3 type AB cats and 68% of erythrocytes from the remaining type AB cat. The binding of anti-B mAb varied inversely with the amount of A antigen expressed, binding 83–97% of erythrocytes from the same 3 type AB cats and 73% of erythrocytes from the remaining type AB cat. Agglutination scores of type AB cats were comparable to the percent binding on flow cytometry, and thin layer chromatography immunostains confirmed differences in the amount of A antigens between erythrocyte glycolipids of type A and AB cats and of two different type AB cats. These results demonstrate that at least two different phenotypes exist within the feline AB blood type, that differ in the amount of A antigen expressed on the erythrocyte surface.
7: THE EFFECT OF MULTIPLE DOSES OF HETASTARCH AND IN VITRO HEMOLYSIS ON SERUM COLLOID OSMOTIC PRESSURE IN HYPOALBUMINEMIC DOGS.
The purpose of this study was to determine if multiple doses of hetastarch have cumulative effects in hypoalbuminemic dogs like those seen in hypoalbuminemic people. Data were collected from fourteen dogs (albumin < 2 g/dL), but a complete set of samples was not available from all dogs. Colloid osmotic pressure (COP) was measured with a Wescor 4420 colloid osmometer in sera collected before and after administration of hetastarch. Non-parametric statistics (Wilcoxon signed ranks test) were applied to determine statistical differences between the groups at the different time points. There was a significant increase in the COP both after the first and after the second administration of hetastarch compared to pretreatment values along with a significant decrease in COP by 12 hours after both treatments. The COP values were significantly higher after the second dose than after the first dose, and also the increase in COP was greater with the second dose. However, there were no significant differences between the COP at baseline, by 12 hours after the first, and by 12 hours after the second dose of hetastarch. Total protein (TP) concentrations significantly decreased after both treatments. By 12 hours after both doses of hetastarch, the TP concentrations were not different. Based on the COP data, the second dose of hetastarch was more effective in increasing the COP; however we did not detect a difference in the duration of this effect between the first and second doses. The decreases in TP concentrations were considered to be mostly due to intravascular volume expansion. In vitro hemolysis resulted in a significant increase in COP values.
8: VALIDATION OF THE NOVA CRT8 FOR THE MEASUREMENT OF IONIZED MAGNESIUM IN FELINE SERA.
Evaluation of serum magnesium concentrations is becoming important in human and veterinary critical care and emergency medicine. Using an ion-selective electrode, it is possible to measure the physiologically active ionized magnesium fraction. The purpose of this study was to validate an ion specific electrode and assay for the measurement of ionized magnesium in feline sera and to determine a reference interval for this analyte. Venous blood samples were anaerobically collected from clinically healthy cats and serum obtained was utilized for the validation. With minimal exposure to air, serum ionized magnesium concentrations appear stable after storage at 20° C for up to 24 hours, at 4° C for up to 72 hours and at -20° C for up to 4 weeks. The intra-assay and interassay precision had coefficient of variations of 1.23% and 1.98%, respectively. Good linearity was achieved using sera (r = 0.998) and manufacturer supplied linearity solutions and quality control materials (r = 0.999). The apparent analytical sensitivity was 0.02 mmol/L. There were good mean recoveries for ionized magnesium in samples with up to 1+ icterus (104%), 4+ lipemia (99.3%) and 1–4+ hemolysis (98.6%). No significant difference (p = 0.522) was detected in the ionized magnesium concentrations between sera collected in tubes containing no additives and sera collected in glass separator tubes. The serum ionized magnesium reference interval for healthy cats was 0.47 to 0.63 mmol/L. The Nova CRT8 system provides a precise and reliable method of measuring ionized magnesium concentrations in feline sera. Strict adherence to suggested blood sampling, sample handling and storage techniques are recommended to obtain reliable ionized magnesium concentrations.
9: CELL BLOCK ANALYSIS—A NEW LESS INVASIVE DIAGNOSTIC ALTERNATIVE TO SURGICAL BIOPSY.
Cell block preparation of fine needle aspirate tissue biopsies with immunocytochemical analysis may be an excellent, less-invasive, economical, and equally diagnostic alternative to evaluation of tissue specimens by surgical biopsy. A cell block is a condensed group of cells created from a fine needle aspirate biopsy that is fixed and embedded in paraffin and evaluated using traditional stains, histochemistry or immunocytochemistry. The purpose of this study is to optimize and validate the cell block technique using normal canine lymph node and liver aspirates and determine the diagnostic sensitivity and specificity of the cell block technique using lymph node and liver aspirates from canine patients with lymphoma and liver neoplasia, respectively. All tissue samples will be evaluated using traditional hematoxylin and eosin stains as well as a panel of immunocytochemical markers, which includes: vimentin, cytokeratin, CD3 and CD79a. Cell blocks were prepared using two techniques commonly used in human diagnostic laboratories: thrombin clot technique and agar gel technique. Samples were evaluated for preservation of cell morphology, sample adequacy of cellularity, and quality of immunocytochemical markers for each technique. Nine cell blocks have been evaluated and thus far the thrombin clot technique has proven superior to the agar gel technique. This is an ongoing study and neoplastic tissues are currently being evaluated using the cell block technique with the panel of immunocytochemical markers previously mentioned.
10: CIRCULATING NUCLEIC ACIDS AS A BIOMARKER FOR DOGS WITH CANCER.
In 1975, scientists discovered that humans with cancer have higher levels of cell-free DNA in serum than healthy patients. DNA levels were increased in advanced stages and decreased after successful therapy, especially in lymphomas and carcinomas. Since then, the circulating DNA was demonstrated to have genetic and molecular changes identical to the neoplastic cells. A literature search has not revealed any investigations of this same phenomenon in veterinary cancer patients. We hypothesize that dogs with cancer have higher levels of plasma or serum DNA than healthy dogs or dogs with non-malignant disease. Specific study objectives include 1) optimizing protocols for sample collection, DNA extraction, and DNA detection; 2) establishing reference ranges of cell-free DNA in normal dogs; and 3) determining if there are significant differences in cell free DNA levels from healthy dogs, dogs with nonneoplastic disorders such as autoimmune and inflammatory diseases, and dogs with cancer. To date, DNA has been quantitated from 13 healthy dogs and 23 dogs with cancers. DNA was extracted using QIAamp DNA mini kit and quantitated using PicoGreen dsDNA fluorescence method. DNA quantities using this method correlate well with PCR from the same samples. Values from normal dog plasma ranged from 45–114 ng/ml, with a median of 63. Normal serum ranged from 39–146 ng/ml, with a median of 60. Due to small sample sizes, differences between normal and cancer dogs are not significantly different, but median plasma DNA values for dogs with lymphoma, carcinomas, and anaplastic cancers were 188 (range = 47–422), 97 (range = 37–200) and 96 (range = 86–105) ng/ml, respectively. As more samples are collected and measured a more accurate reference range will be calculated and statistically significant differences between sample groups may emerge.
11: SELECTED EFFECTS OF PARENTERAL BASIC FIBROBLAST GROWTH FACTOR ADMINISTRATION TO OSTEOPENIC OVARIECTOMIZED RATS.
Basic fibroblast growth factor (bFGF) is a pleiotropic mitogen, which shows promise as an osteoporosis therapy. Five month old, osteopenic ovariectomized rats were injected subcutaneously with vehicle (n = 9) or bFGF (n = 10) at 1 mg/kg daily for 3 weeks. Hematologic and biochemical analyses were performed; and kidneys, livers, and proximal tibiae were processed for histomorphometry. Basic FGF treatment resulted in a 6-fold increase in osteoid surface (P < 0.0004). There was no significant difference in body weight, but other non-bone effects occurred in bFGF-treated rats. Basic FGF-treated rats became anemic with a 2.5-fold decrease in PCV (P < 0.0001). The bone marrow myeloid: erythroid ratio was higher in bFGF-treated rats (P = 0.0010), though there was no significant difference in the hematopoietic area of tibial marrow. Hepatic extramedullary hematopoiesis (EMH) that was predominantly myeloid arose in bFGF-treated rats. There was a 19-fold increase in the number of EMH foci (P = 0.0001) and a 27-fold increase in the area of EMH foci (P < 0.0001). The parietal layer of Bowman's capsule was thicker (P ≤ 0.0001), and the glomerular diameter was greater (P = 0.0023) in bFGF-treated rats. This was associated with a miniscule (0.06 mg/dL), but significant (P = 0.0419) increase in serum creatinine. There were no significant differences between groups in serum albumin, calcium, and phosphorus, nor in urine protein: creatinine ratio and urine fractional excretion of phosphorus. Basic FGF strongly stimulates bone formation. However, bFGF-treated ovariectomized rats develop anemia due erythroid hypoplasia. A relative increase in myelopoiesis occurs in the marrow and liver. The mild glomerular hypertrophy linked with bFGF injection is not attended by strong biochemical evidence of renal dysfunction or a decrement in general health status. Results indicate that rats tolerate short-term bFGF administration relatively well.
12: CLONING AND EXPRESSION OF THE GENE ENCODING THE MAJOR ANTIGENIC PROTEIN 2 HOMOLOG OF ANAPLASMA PHAGOCYTOPHILUM AND POTENTIAL APPLICATION FOR SERODIAGNOSIS.
Anaplasma phagocytophilum (formerly known as the Human Granulocytic Ehrlichia, Ehrlichia equi and Ehrlichia phagocytophilum) is an obligate intracellular organism causing clinical disease in humans and various species of domestic animals. The major antigenic protein 2 (MAP2) homolog of A. phagocytophilum was sequenced, cloned and expressed in Escherichia coli. Sequence homology of the various map2/msp5 genes was compared among the various Anaplasma and Ehrlichia species. Recombinant MAP2 of A. phagocytophilum was used in an ELISA to detect antibodies in serum samples from humans and dogs infected with the organism. The map2 gene of A. phagocytophilum is 582 bp in length. There was 63.9% sequence identity between the map2 gene of A. phagocytophilum and the msp5 gene of A. marginale, 59.3% sequence identity with the map2 gene of E. canis and 57.9% sequence identity between the map2 gene of A. phagocytophilum and the map2 gene of E. chaffeensis. Serum samples from 47 individuals, naturally infected with A. phagocytophilum, were tested in an ELISA format using the rMAP2 protein. Forty-two of 47 samples contained antibodies to the rMAP2 resulting in a test sensitivity of 89%. Serum samples from three dogs experimentally infected with a Swedish strain of A. phagocytophilum tested positive at 17, 28 and 97 days post-inoculation. These results indicate that the rMAP2 of A. phagocytophilum has potential for use as a diagnostic test antigen and that sequence variation among the agents tested may provide sufficient dissimilarity to distinguish between infections.
13: HYPOPHOSPHATEMIA AS A PROMINENT ABNORMALITY OF POST RENAL OBSTRUCTION OF GOATS.
We reviewed the clinical laboratory results of caprine samples from the UC-Davis VMTH collected from January 1990 through May 2003 to determine whether any patterns of laboratory abnormalities could be associated with post renal obstruction (PRO). Four hundred fifty-six goats were identified as being first time admissions to the Large Animal Service. The first food animal chemistry profile from those visits was entered into a database containing the visit's discharge diagnosis, signalment, and discharge status. Non-renal diseases (NRD) were diagnosed in 329 goats, while PRO occurred in 85 goats. Renal disease was relatively uncommon (n = 16). Results from 26 healthy goats, tested for herd health surveillance were used to confirm the validity of reference intervals. As expected, most of the PRO goats were azotemic (82%). Surprisingly, their mean phosphorus (Pi) was 4.1 mg/dl, below the reference range (4.2–9.1), with 71% being hypophosphatemic, 20% normophosphatemic, and 9% being hyperphosphatemic. Hypophosphatemia was also relatively common (40%) in NRD goats. Azotemic goats with PRO had significantly lower Pi and anion gap and significantly higher calcium and TCO2 concentrations, compared to NRD goats with prerenal azotemia. Serum Na, K and Cl were not significantly different between the groups. Hypophosphatemia was not associated with any specific clinical outcome or the occurrence of anemia. Although PRO was more common in male goats of small African breeds compared to NRD, neither of these variables could explain the differences in Pi. Hyperphosphatemia has been associated with PRO in other domestic animals. It is unknown what difference in phosphorus metabolism in goats could explain the marked difference found in this study.
14: RATE OF DETERIORATION OF CANINE CEREBROSPINAL FLUID.
The objective of this prospective clinical study was to determine the effects of multiple variables including time between sample collection and analysis, initial sample composition, and presence or absence of additives on standard analysis of canine cerebrospinal fluid (CSF). We divided 30 abnormal CSF samples into aliquots, either unaltered or with added fetal calf serum (FCS) or Hetastarch (HS), and stored them at 4°C. Unaltered aliquots were analyzed immediately and at 2, 4, 8, 12, 24, and 48 hours post-collection; aliquots with added FCS or HS were analyzed at 48 hours. To look for time-dependent changes in cellular composition of unaltered samples, we analyzed data both globally to detect differences in values between any of the various time points and for trends to detect patterns of increasing or decreasing values. We found significant (p < 0.05) differences in the percentages of most cell types, as well as in cellular morphology, and significant trends in the absolute numbers of different cell types. We also looked for effects of initial sample composition on stability, and detected significant or marginally significant (0.05 < p < 0.10) interaction between initial protein concentration and time-dependent linear decrease in the number of some cell types. Addition of FCS or HS resulted in significantly lower percentages of unrecognizable cells, and significantly higher percentages of some recognizable cell types. Some deterioration of canine CSF is evident within 2 hours of sample collection, but delaying analysis is unlikely to affect diagnostic interpretation in most cases. Adding FCS or Hetastarch to canine CSF improves the stability of the sample.
15: COMPARATIVE SEQUENCES OF THE CANINE AND FELINE ARGININE-VASOPRESSIN PROHORMONES.
Vasopressin plays a key role in cardiovascular homeostasis, and is synthesized as a prohormone containing 4 peptide domains: a signal sequence, vasopressin nonapeptide, neurophysin II, and a C-terminal glycoprotein. Our laboratory is investigating the hormonal effects of heart failure in dogs and cats and has been attempting to verify, when it is not known, the nucleotide and amino acid sequences of relevant peptide hormones as tools for research. For that reason we sought to determine the structure of canine and feline vasopressin prohormone. Initially, PCR amplification of cDNA from feline and canine hypothalamus was done using consensus primers designed to regions upstream and downstream from the vasopressin sequence. This resulted in a 222 bp feline and canine cDNA product. Analysis of these sequences revealed that the nucleotides of feline and canine vasopressin nonapeptide differ slightly, but the amino acid sequence (CYFQNCPRG) is identical to that of most other mammalian species, except swine. Reconstruction of the complete canine prohormone from the newly available canine whole genome shotgun database at NCBI, was subsequently done. Sequence homology of other canine and feline prohormone domains was not as well conserved as that of vasopressin nonapeptide. The results of this study corroborate the use of vasopressin immunoassays developed for other species with canine and feline vasopressin nonapeptide. This sequence data will also allow the design of specific vasopressin nucleotide probes for molecular studies and will be useful in the design of antibody probes to other vasopressin prohomone domains.
16: COMPARISON OF TWO METHODS TO DETERMINE HEMOGLOBIN IN MAMMALIAN, REPTILIAN, AND AVIAN BLOOD.
The cyanmethemoglobin method is the standard for determining blood hemoglobin values in human and veterinary medicine. Disadvantages of this protocol include laboratory safety concerns in handling the potassium cyanide-based reagent and the need for a large bench-top spectrophotometer. The azide-methemoglobin method, employed by the HemoCue B-Hemoglobin photometer (HemoCue AB, Ängelholm, Sweden) is an approved waived test method, and provides a non-toxic alternative that is easy to use, portable, and requires a smaller sample volume than the standard method. Although both methods have been validated for human blood, they have not been compared in exotic animals. Hemoglobin values were measured concurrently using both methods on 82 samples: 41 mammals, including bottlenose dolphin, golden lion tamarin, pygmy sperm whale, two-toed sloth, and three species of seal; 38 reptiles, including terrestrial and sea turtles, lizards, and snakes; and three yellow-headed Amazon parrots. The data were analyzed in two groups, mammals and non-mammals, due to the differences in anticoagulants used and red blood cell morphology for each group. Statistical analysis of the paired data using Pearson product moment correlation coefficient showed good correlation (r = 0.979 and 0.984, respectively) and the Student's t-test analysis showed no significant difference (P > 0.05). These results indicate that the HemoCue offers reliable, easy-to-use, rapid hemoglobin analysis for mammals, birds, and reptiles.
17: ALTERATIONS IN ERYTHROCYTE MORPHOLOGY CAUSED BY LOSS OF ALPHA HEMOGLOBIN STABILIZING PROTEIN.
Thalassemias are a group of genetically inherited hematologic disorders characterized by reduction in synthesis of one or more globin chains in the hemoglobin molecule, ineffective erythropoiesis, hemolysis and anemia. For example, in beta thalasssemia, beta globin chain biosynthesis is reduced and the excess unpaired alpha globin chains produced are very unstable leading to red blood cell membrane deficiencies, inclusion bodies and shortened erythrocyte half-life. Alpha Hemoglobin Stabilizing Protein (AHSP) is an erythroid-specific protein that binds free alpha hemoglobin to prevent its precipitation in vitro and in heterologous cells. To further characterize the functional and biological significance of AHSP in vivo we studied the morphological and physiological effects on mature erythrocytes following genetic disruption that causes partial or complete loss of the AHSP gene in mice. Blood samples from wild type (+/+), heterozygous (+/-) and homozygous null (-/-) mice were evaluated on the hematology analyzer and microscopically. In AHSP (-/-) animals, changes in MCV, MCH, RDW, HDW and reticulocytes were noted in hematology. Peripheral smear examination revealed widespread abnormal erythrocyte morphology. Additionally, numerous polychromatophilic erythrocytes containing eosinophilic, crystal violet positive inclusions suggestive of denatured globin were noted specifically in ASHP -/- or +/- mice and biotin labeling studies revealed markedly shortened erythrocyte half-life in AHSP -/- animals. These data demonstrate that AHSP is essential for normal hemoglobin stability and erythrocyte physiology.
18: REDUCED BLOOD VOLUME REQUIREMENTS FOR A MOUSE CBC ON THE BAYER ADVIA 120 HEMATOLOGY ANALYZER.
Limited mouse blood volume obtained at terminal vena cava bleeds (∼0.5 mL) creates a challenge when performing both hematology and chemistry analyses. We investigated a technique of diluting mouse blood for CBC analysis on the Bayer Advia 120. Blood was collected into EDTA pediatric microtainer tubes (Ram Scientific, Needham, MA) optimized for low volumes (65 uL-250 uL). Samples from 50 CD-1 mice were diluted, by two different technicians, 1:3 in 0.9% NaCl and the results were compared to their respective undiluted counterpart. A total of 400 uL of blood was drawn from each mouse, a 250 uL aliquot (minimum needed for CBC) was dispensed into a microtainer tube which was run undiluted, and a 125 uL aliquot was delivered to another tube, which was used for the dilution (100 uL of blood and 200 uL saline). The correlation coefficients for this method to method comparison study are as follows: WBC, r = 0.9411; RBC, r = 0.7366; HGB, r = 0.7997; HCT, r = 0.5043; MCV, r = 0.9474; MCH, r = 0.3063; MCHC, r = 0.7316; CHCM, r = 0.9975; RDW, r = 0.9817; PLT, r = 0.9107; MPV, r = 0.6578; RETIC, r = 0.9840; %LYMPH, r = 0.8305; %NEUT, r = 0.8545; %MONO, r = 0.2211; %EOS, r = 0.5388; %BASO, r = 0.1466; %LUC, r = 0.5180. In addition to providing reasonable correlation with most parameters in undiluted samples, platelet clumps appeared to be less frequent in diluted samples, thus possibly providing a more accurate platelet count. The main disadvantage of this technique is the time it takes to make the dilution and in some cases, not an ideal (i.e. Hgb, r value = 0.7997) correlation to the results with an undiluted sample. Exploration of a different diluent (5% BSA in saline) and precision studies are ongoing to further optimize and characterize this technique.
19: IDENTIFICATION OF A HAEMOMYCOPLASMA SPECIES IN ANEMIC REINDEER (RANGIFER TARUNDUS).
During an 18 month period, 9 animals in a herd of 19 reindeer experienced episodes of anemia. These animals had histories of weight loss, unthriftiness, and occasionally edema of dependent parts. CBCs from the affected animals revealed moderate anemia characterized by microcytosis, hypochromasia, schistocytosis, keratocytosis, acanthocytosis, and dacryocytosis. Numerous basophilic punctate to ring-shaped bodies which measured less than 1.0 micron were found on the surface of RBCs and were often observed encircling the outer margins of the cells. Based on these cytologic findings, DNA preparations from selected affected animals in the NADC herd and one animal from a private herd which was experiencing similar episodes of anemia were assayed by PCR for the presence of hemolytic bacteria. Primers from the 16s rRNA genes of Mycoplasma (Eperythrozoon) suis, Mycoplasma haemofelis, Anaplasma marginale, Anaplasma spp., and Ehrlichia spp. were used. Seven animals had positive reactions using the M. haemofelis primers. No other primers produced products. Internal primers to the M. haemofelis 16s rRNA gene segment were used to amplify the initial product. The product from the nested PCR assay was used for DNA sequencing. BLAST searches were performed on the resulting sequences. Six animals had an organism which was most closely related to M. ovis (90–98%), M. wenyonii (91–96%), M. haemolama (89–91%), and M. suis (88–89%). One animal had an organism which was most closely related to M. haemofelis (94%) and M. haemocanis (94%). This represents the first identification of haemomycoplasma species in reindeer. Although several of these animals were also infected with abomasal nematodes, the presence of this organism may have contributed to the anemic syndrome.
20: EVALUATION OF HEMATOLOGY AND CLINICAL CHEMISTRY OF GOATS UNDER THERMAL STRESS.
The adaptive capacity of the goat is determined by its ability to dissipate heat by increasing skin temperature and respiratory rate and increasing body temperature without inhibiting its activity. When mammals are exposed to environmental temperatures above their zone of thermal comfort, many physiological and biochemical changes happen that constitute thermal stress. Changes in the hormonal equilibrium due to the environmental temperature are responsible for decreases in growth, reproduction and milk production and interfere with the ability of a species to live in a certain region. This study evaluated in the hemogram and serum levels of T3, T4, Cortisol, total serum proteins, albumin, globulin, calcium, phosphorus and magnesium, sodium, potassium and aspartate aminotransferase caused by thermal stress in young female goats, both pure Alpine and half-breed Alpine and Boer. Eight five-month old goats, including 4 Alpine and 4 half-breed, were kept in a bioclimate chamber at 35° to 40° C, for 5 hours a day for fourteen days with food and water available ad libitum. The highest mean values of hematocrit, hemoglobin, and total plasma proteins were observed in the pure Alpine goats. All the goats, when subjected to thermal stress, became clinically dehydrated, however, the highest values of erythrocytes, hemoglobin, hematocrit and total plasma proteins were seen in the pure Alpine goats. The half-breed goats were shown to be better adapted to the effects of heat, showing fewer increases in Cortisol, T3 and T4.
21: DETECTING ACTIVATED PLATELETS IN CANINE WHOLE BLOOD USING FLOW CYTOMETRY.
Platelet activation is essential for thrombus formation and hemostasis and may be potentially useful to evaluate in patients with suspected thromboembolic disease. In canine patients, thromboembolic disease can occur with autoimmune hemolytic anemia, nephrotic syndrome, inflammatory bowel disease, and other inflammatory conditions. Activated platelets undergo a complex process which includes shape change, secretion of granules, and adhesion. Coagulation is promoted on the surface of these platelets. Prior to a thrombotic state, a prothrombotic state may exist where only a small number of platelets are activated. Identification of a prothrombotic state using activated platelets may help direct medical intervention to prevent a thromboembolic episode. Flow cytometry using whole blood can be a valuable tool to assess activated platelets in vivo. This study evaluated platelets in whole blood from 10 normal dogs. Changes in specific markers of platelet activation in unstimulated versus stimulated samples were assessed, using varying concentrations of two platelet agonists, PAF and ADP. Flow cytometry was used to detect platelet CD61 (gpIIIa), CD62P (P selectin), and the leukocyte marker CD45. Annexin V was used to identify exposed phosphatidylserine. Platelet activation was determined by measurement of P selectin expression, platelet-leukocyte aggregates, platelet microparticles, and phosphatidylserine exposure. A significant difference (P < 0.05) was observed in the number of P selectin events, platelet leukocyte aggregates, microparticles, and Annexin V positive events in the samples stimulated with 10 nM PAF versus the unstimulated samples, with platelet leukocyte aggregates showing the greatest difference. Activated platelet markers can be measured in whole blood of canine patients and may be a useful diagnostic test for thromboembolic disease.
22: VALIDATION OF THE ACL 9000 COAGULATION ANALYZER FOR EVALUATION OF HEMOSTASIS AND FIBRINOLYSIS IN CANINE AND FELINE PATIENTS.
The ACL 9000 (Instrumentation Laboratory) is a fully automated microcentrifugal analyzer that has the capability to perform a wide range of tests for assessment of hemostasis and fibrinolysis. Assay methods are based on evaluation of clot formation through the change in light scatter over time (coagulometric), measurement of formation of a product that absorbs light at a particular wavelength (chromogenic) or change in light scatter resulting from agglutination of latex beads (immunoturbidometric). We have validated the use of this instrument for measurement of the following parameters in cats and dogs: prothrombin time (PT), partial thromboplastin time (PTT), antithrombin (AT), D-dimer and fibrinogen. Intra- and inter-assay precision for each analyte were assessed using pooled plasma from clinically healthy cats and dogs. Correlation between results obtained with this instrument and our previous analyzer (ACL 3000) was evaluated to assess accuracy, using samples obtained from patients of the TAMU Veterinary Teaching Hospital. Reference intervals were established for the ACL 9000 using plasma obtained from clinically healthy cats and dogs (30 each). The methods for D-dimer and fibrinogen measurement on this instrument are quantitative, and are performed using citrated plasma. This allows the construction of a comprehensive panel to assess coagulation status that requires only a single blood sample. We have also completed preliminary studies showing that the methodology available for this instrument is directly applicable to the measurement of von Willebrand factor (vWF), and that accurate measurement of protein C levels can be accomplished with slight modification of the assay method. The ACL-9000 is a versatile instrument and has the potential to greatly expand our ability to routinely characterize coagulation abnormalities in veterinary patients.
23: EVALUATION OF FACTOR VIII ACTIVITY IN DOGS WITH NEOPLASIA USING COAGULOMETRIC AND CHROMOGENIC METHODS.
Hemostatic abnormalities are common in canine cancer patients. Since studies in human medicine have indicated that altered Factor VIII (FVIII) activity contributes to coagulopathy in neoplasia, and this has not been investigated in dogs, our goals were 1) to compare the utility of canine reference plasma (CRP) against human reference plasma (HRP) for standardization, 2) to compare chromogenic and coagulometric assays for FVIII activity, and 3) to assess differences in FVIII activity between canine cancer patients and healthy dogs. Citrated plasma was obtained from canine cancer patients and healthy blood-donor dogs. A PTT-based FVIII coagulometric assay and a commercially available chromogenic assay that relies on the ability of FVIII to activate FX were used. Standard curves were obtained by evaluating FVIII activity in serial dilutions of CRP and HRP. A positive correlation (r2 = 0.97) was observed between CRP and HRP for the coagulometric assay (n = 69). Perfect correlation (r2 = 1.00) was observed between the CRP and HRP for the chromogenic assay (n = 36). A comparison of the 2 methods using CRP indicated a positive correlation (CUSUM of >0.1, n = 36). No significant difference in FVIII activity was found between cancer patients and controls within the small sample size. The use of CRP and HRP as standards correlated well within individual assays and there was a positive correlation between the coagulation and chromogenic assays. The chromogenic assay might be more sensitive to species differences. There was no difference in FVIII activity between cancer patients and normal dogs in this sample population.
24: DEVELOPMENT OF ARC183: A NOVEL ANTICOAGULANT COMPOSED ENTIRELY OF DNA.
Although heparin is the standard anticoagulant used for coronary artery bypass and graft (CABG) surgeries, the long half-life, the need to use a reversal agent (protamine) and occasional cases of heparin-associated thrombocytopenia provide rationale for the development of more effective anticoagulants. ARC183 is a novel DNA aptamer composed of 15 nucleotides that binds specifically to thrombin in a variety of species (rat, dog, pig, macaque, human) and inhibits the conversion of fibrinogen to fibrin. Although the molecule has an in vitro serum half-life of less than 15 minutes, extensive renal filtration and other clearance mechanisms further shorten the in vivo half life to two to seven minutes, depending upon the species. In the rat, pig and cynomolgus macaque, the short in vivo half life of ARC183 allows one to fine-tune the ACT, PT and APTT by simply adjusting the infusion rate. Following cessation of treatment, clotting parameters in each species return to baseline within 5 minutes and subsequent administration of heparin still effectively prolongs clotting times. ARC183 may be a useful anticoagulant for CABG surgery and other cardiovascular procedures.
25: EVALUATION OF ARGYROPHILIC NUCLEOLAR ORGANIZER REGIONS (AGNORS) AS A PROGNOSTIC INDICATOR FOR CANINE LYMPHOPROLIFERATIVE DISEASES.
A prospective study was conducted to evaluate the prognostic value of argyrophilic nucleolar organizer regions (AgNORs) for 60 cases of lymphoproliferative disease in dogs. Nucleolar organizer regions reflect both DNA ploidy and proliferation activity. The purpose of this study was to evaluate three counting methods for AgNORs on cytologic specimens of lymphoproliferative disease relative to survival time, clinical substage, immunophenotype, and WHO classification. Mean AgNOR (mAgNOR), proliferative AgNOR (pAgNOR), and nucleolar AgNOR (nAgNOR) indices were counted from silver-stained samples. The m-AgNOR count was defined as the mean number of AgNORs per nucleus in 100 cells. The percentage of cells exhibiting 5 or more AgNORs per nucleus was defined as pAgNOR. Proliferative AgNOR was statistically similar to mean AgNOR and neither was statistically different relative to survival duration categories (< 50 days and > 50 days) or median survival. B cell immunophenotype displayed a higher pAgNOR (mean = 38.1%, median = 38.0%) than T cell cases (mean = 23.2%, median = 13.0%). The percentage of cells displaying a large single or a cluster of small AgNORs within nucleoli was defined as nAgNOR. There was only suggestive evidence of a relationship between nAgNOR and survival duration (P = 0.0679) and between nAgNOR and substage. Median differences in nAgNOR for combination therapy-treated diffuse large B-cell (DLBC) cases were statistically significant (P = 0.0164) relative to sub-stage category. While differences were noted in nAgNOR mean and median values between different WHO classification groups, for example, peripheral T lymphomas (mean = 83.9%, median = 89.5%), DLBC (mean = 88.8%, median = 95.5%), and large granular leukemia (mean = 96.0%, median = 97.0%), these were not statistically significant. The conclusion is that nAgNOR is more reliable prognostically than mAgNOR or pAgNOR for canine lymphoid neoplasia especially when applied within a single WHO classification category.
26: IMMUNOCYTOCHEMICAL STUDY OF P53 AS A PROGNOSTIC MARKER IN CANINE MAMMARY NEOPLASIA.
Canine mammary tumors are challenging for clinicians and pathologists because of complex histologic classification, low specificity of cytologic diagnosis, and unpredictable biological behavior. In histologic specimens, expression of the tumor suppressor gene P53 has been shown to have prognostic value for canine mammary tumors and to correlate with malignancy and low survival rates. The objective of this study was to determine the expression of the tumor suppressor gene in canine mammary tumors by immunochemical detection of P53 in cytologic specimens and to determine its relationship to clinical and pathologic variables and patient outcome. Spontaneous mammary tumors from 30 female dogs were surgically excised. Imprint specimens for cytologic evaluation were wet-fixed in ethanol; histologic specimens were prepared routinely. Immunostaining was performed with the NCL-240 polyclonal antibody against P53; it was graded from negative to + + +. Dogs were followed for 18 months. Multivariate logistic regression analysis was used to determine correlations between immunocytochemical results, tumor and clinical variables, and patient outcome. P53 in cytologic specimens were significantly lower for nonmalignant tumors than for malignant tumors. High index values of P53 were positively correlated with metastasis, death from neoplasia, low disease-free survival rates, and low cytologic specimens correlated with that of histologic specimens. The prognostic value of the P53 index in canine mammary tumors by using wet-fixed cytology imprint specimens was similar to that observed previously for histologic specimens. Immunocytochemical detection of P53 could improve the accuracy and value of cytology by providing safe and rapid information about malignancy and patient outcome.
27: EFFECT OF ANTICOAGULANT, STORAGE TEMPERATURE AND TIME ON NON-ESTERIFIED FATTY ACID AND BETA-HYDROXYBUTYRATE CONCENTRATIONS IN DAIRY COWS.
Non-esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHBA) concentrations are used as indicators of peripartum negative energy balance and subclinical ketosis in dairy cows, respectively. The objectives of this study were to compare NEFA and BHBA concentrations in EDTA-anticoagulated or non-anti-coagulated (NA) blood at different storage temperatures (4° C, 24° C) and storage times (up to 72 h). Blood was collected from 10 cows into EDTA or NA tubes. Samples were immediately centrifuged and baseline values measured. The remaining whole blood (WB) or separated serum or plasma samples were analyzed daily after storage at 4° C or 24° C for 24 (WB) to 72 h (separated samples). NEFA (Wako) and BHBA (Catachem) concentrations were measured using colorimetric enzymatic methods (Hitachi 917). Median baseline NEFA concentrations were similar for EDTA (0.19 mEq/L) and NA (0.20 mEq/L) (p = 0.11). At 24 h, median NEFA concentrations in WB were not significantly different from baseline values at 4° C (EDTA, 0.20 mEq/L; NA, 0.19 mEq/L, p > 0.05), but were increased at 24° C (EDTA, 0.21 mEq/L; NA, 0.23 mEq/L, p < 0.05). Median NEFA concentrations were stable in separated EDTA plasma or serum for 72 h at 4° C (72 h plasma and serum, 0.18 mEq/L), but progressively increased after 24 h at 24° C (72 h plasma, 0.23 mEq/L; 72 h serum, 0.24 mEq/ L). BHBA concentrations were unaffected by sample type, storage temperature or time. For dairy herd evaluation of negative energy balance or subclinical ketosis, blood can be collected into EDTA or NA tubes. However, for optimal NEFA results, samples should be separated within 24 h of collection and maintained at 4° C until analysis.
28: EVALUATION OF IONIZED AND TOTAL MAGNESIUM CONCENTRATIONS IN HYPERTHYROID CATS.
Hyperthyroidism can increase the renal excretion of magnesium, thus leading to hypomagnesemia; this has been reported in various species. Anaerobically collected serum samples from 15 hyperthyroid and 40 normal healthy cats were analyzed on an ion selective electrode instrument (Nova CRT8, Nova Biomedical, Mississauga, ON, Canada) and a serum biochemical analyzer (Hitachi 917 analyzer, Roche Diagnostics, Indianapolis, IN, USA). The serum ionized and total serum magnesium concentrations of the hyperthyroid cats were compared to those from the healthy cats. The relationship between ionized and total magnesium serum concentrations and other biochemical parameters in hyperthyroid cats was evaluated. There was no significant difference for ionized (p = 0.271) and total magnesium (p = 0.436) concentrations between healthy and hyperthyroid cats. A significant negative correlation (r = -0.670, p = 0.006) was detected between ionized magnesium and logarithmically transformed thyroxine concentrations in hyperthyroid cats. The ratio of ionized/total magnesium concentrations for hyperthyroid cats with higher thyroxine concentrations was significantly lower (p = 0.004) than that of healthy cats. Hyperthyroid cats had a significantly lower total protein concentration (p = 0.002) than healthy cats. There was a significant correlation (r = 0.894, p = 0.000) between ionized and total magnesium concentrations in hyperthyroid cats. The negative correlation between ionized magnesium and logarithmically transformed thyroxine concentrations in hyperthyroid cats suggests that the duration or severity of hyperthyroidism may contribute to a decrease in ionized magnesium. Further research is warranted involving larger numbers of hyperthyroid cats.
29: BRAZILIAN PEPPERTREE SAWFLY LARVAE TOXICITY IN BOVINES.
Brazilian peppertree (BP) has become a problem in Florida because it competes with advantage over native plant species. BP sawfly Heteroperreyia hubrichi is a primitive wasp that does not sting and one of the BP host-specific natural enemies. For that reason, BP sawfly has been indicated for BP biological control in Florida. However, since BP sawfly likely contains lophyrotomin and pergidin hepatotoxins, its accidental ingestion may be toxic, particularly for herbivorous vertebrates such as cattle. In order to evaluate BP sawfly possible hepatotoxic effects, BP sawfly larvae were obtained, disinfected, mixed to food and orally given to twelve female, nine months old calves at the Veterinary Hospital of the Universidade Federal do Paraná. Three calves were randomly assigned to each of the four treatments, with two recurrences each group. Treatment A received 900 larvae (126g), three doses in a period of 96 hours. Treatment B received 450 larvae (42g), with three doses in a period of 72 hours. Treatment C received 225 larvae (36g), with three doses in a period of 48 hours. Treatment D, used as control, received zero larvae. Following acute larvae ingestion, all twelve animals were clinically evaluated for 90 days. After physical examination, blood samples were obtained from jugular vein as follows: one day before, one day after and two days after the first larvae administration. All biochemical assays were performed using commercial diagnostic kits and spectrophotometric methods, and plasma proteins determined by refractometry. Clinical signs of hepatotoxicity were not observed in the calves within the study period. Hematological parameters (RBC, WBC, hemoglobin, PCV, MCV and MCHC) were within normal range for all calves with no statistical differences between treatment and control groups. AST values were also within normal range and varied from 45 to 110 U/L for groups A, B and C and from 60 to 80 U/L to group D (control). GGT were normal as well and values ranged from 10 to 25 U/L for all groups. All calves presented BUN and plasma protein levels within normal range. Based on our results, we were able to rule out cholestatic or hepatocellular disease, including severe hepatic failure, insufficiency or necrosis. In conclusion, no acute hepatotoxicity or cholestasis was observed in calves following three different doses of orally given Brazilian Peppertree Saw-fly larvae. Our data indicate that Brazilian Peppertree Sawfly larvae are unlikely to produce hepatotoxicity to cattle and therefore can be safely used as biological control of Brazilian Peppertree population in Florida.
30: A NOVEL CALICIVIRUS IN MICHIGAN RABBITS: SIMILARITIES WITH RABBIT HEMORRHAGIC DISEASE.
In January 2001, a fatal disease outbreak in a Michigan rabbitry, that contained 200 New Zealand White rabbits, resulted in a death rate of 35%. There had been no animal introduction into the rabbitry over the past 18 months. Pregnant does appeared primarily susceptible and developed clinical signs and lesions that closely resembled rabbit hemorrhagic disease (RHD), including severe pulmonary hemorrhages and hepatocellular necrosis. Some dead does had a severe suppurative meningoencephalitis secondary to bacterial septicemia and most dead does were pregnant. As previously reported, livers of affected rabbits tested positive by RT-PCR using published RHD virus-specific primers. However, diagnostic investigations conducted by APHIS ruled out RHD. Initial sequence analysis demonstrated a 76.8% nucleotide sequence identity of the RT-PCR amplicon with RHDV and a 78.5% identity with the nonpathogenic rabbit calicivirus. Following complete genomic sequencing and phylogenetic analysis, a novel rabbit calicivirus that appears to be a member of the genus Lagovirus, was identified and named Michigan rabbit calicivirus (MRCV). Interestingly, immunohistochemistry using antibodies against RHDV and in-situ hybridization using oligoprobes against MRCV revealed a staining pattern in severely affected livers similar to lesions reported in rabbits experimentally infected with RHDV. When healthy adult rabbits were inoculated with MRCV, no clinical signs, gross or microscopic lesions were observed, except for rare single cell degeneration of MRCV-positive hepatocytes. We speculate that MRCV primarily causes subclinical infection of healthy rabbits and may induce overt disease similar to RHD under certain field conditions. These data indicate the diagnostic challenge posed in detecting nonpathogenic viruses that are closely related to foreign animal disease-causing viruses.
31: BOVINE SPONGIFORM ENCEPHALOPATHY (BSE): PHENO-TYPES, AGENT STRAINS AND CASE DEFINITIONS.
Studies of BSE in the UK and other countries have indicated geographical and temporal uniformity of the pathology of BSE. Together with epidemiological data and the biological properties of the BSE agent on transmission these observations underpin the major hypothesis that BSE worldwide is sustained by a single, stable, cattle adapted strain of a transmissible spongiform encephalopathy (TSE) agent. Recent reports of possible phenotypic variants of the disease therefore may have implications for the existence of different TSE agents in cattle. This paper reviews the ways in which TSEs or prion diseases are defined with particular reference to BSE. Components of phenotypic characterization, including the species naturally affected, geographical and epidemiological occurrence (e.g. age), clinico-pathological features, transmissibility and host genomic differences, are currently the basis of differentiation of TSE types among different hosts and within a specific host. Classification or subclas-sification of phenotype also has a basis in the biological characterization of disease on transmission to rodents (so-called strain typing). Biochemical methods of typing TSEs from different sources are based upon molecular properties of the host's disease specific protein (PrPD). Definition of a case, or particular instance of disease, is conceptually distinct from the definition of phenotype and is specific to each epidemiological study. Thus a case definition may include some or all of the characteristics of a single phenotype, more than one phenotype, or, indeed, all phenotypes of the disease or disease group. Surveillance for BSE is not invariably conducted utilizing a case definition that would allow confirmation of a BSE phenotype. Reports of atypical BSE have reinforced the importance of reviewing the phenotypic definition of BSE and ensuring appropriate sampling methods to investigate phenotype diversity of TSE in cattle.
32: BOVINE RESPIRATORY SYNCYTIAL CELL PNEUMONIA IN A CALF.
Fresh and formalin fixed tissues were received from a five-month-old Angus calf that had died acutely without clinical signs. Microscopic findings were restricted to the lung. The lung is diffusely hypercellular with widening of the interlobular septa by congestion, edema and increased numbers of neutrophils and macrophages. The inflammation is intensified around bronchi and bronchioles with flooding of adjacent alveolar sacs by edema. There is necrosis of bronchiolar epithelium and the bronchioles are partially occluded by large foamy macrophages, intact and degenerate neutrophils, edema, fibrin and scattered multinucleate (syncytial) cells. There are also follicular type aggregates of lymphocytes and plasma cells around some bronchioles. The histologic lesions and conspicuous syncytial cells suggested a viral pneumonia. The PCR test on DNA collected from lung tissue was positive for bovine respiratory syncytial cell virus (BRSV). In addition, bacterial culture of lung tissue produced moderate growth of Mycoplasma sp. This case represents an acute bronchointerstitial pneumonia secondary to BRSV infection in a young calf. The histologic lesions are unique compared to normal field cases submitted to our laboratory, as significant lesions from a secondary bacterial infection are not present. BRSV typically affects young, late-weaning age calves to those up to one year of age. BRSV can infect bronchiolar ciliated and non-ciliated epithelial cells as well as type II alveolar cells and I. Although usually a prelude to secondary bacterial infections as part of the “bovine respiratory disease complex”, BRSV can produce outbreaks of respiratory disease and occasional deaths without contribution from secondary bacterial infections.
33: BRONCHIAL-ASSOCIATED LYMPHOID TISSUE (BALT) B-CELL LYMPHOMA IN A CAT CONFIRMED USING PCR ASSESMENT OF ANTIGEN RECEPTOR GENE CLONALITY.
Lymphoma confined to the mucosa-associated lymphoid tissue (MALT) is a rare disease, usually affecting the gastrointestinal tract. In humans MALT lymphoma involving the bronchial-associated lymphoid tissue (BALT) is an indolent disease of B-cell origin. A 12-year-old, castrated-male, domestic shorthaired cat developed respiratory difficulty over a 5-month period before being euthanized. The lung was investigated using morphologic assessment, immurtohisto-chemistry, and PCR-based DNA clonality studies. Grossly, 2–3mm individual and coalescing nodules were visible beneath the pleural surface. The pulmonary infiltrates formed cuffs around the bronchioles; the lumen of the bronchiole was often visible within the center of the infiltrate. Histologically, the neoplastic cells surrounded and infiltrated the bronchi and bronchioles, effacing the wall of the conducting airways. The cells expanded the submucosa and infiltrated the mucosal epithelium; this epithelium was often comprised of metaplastic squamous cells. The neoplastic cells were moderately pleomorphic with pale amphophilic cytoplasm, and heterochromatic nuclei. Interspersed at the periphery of the neoplastic infiltrates were small numbers of mature lymphocytes. Immunohistochemistry for the B-cell receptor antigen CD79a, revealed intense signal along the cell membrane of the neoplastic cells; the mature lymphocytes expressed the T-cell receptor molecule CD3. PCR analysis of the immunoglobulin heavy chain gene (IgH) revealed that the B cell infiltrate originated from a single clone, while analysis of the T-cell receptor gene (TCRG) indicated that infiltrating T cells had a polyclonal origin. This is the first report of BALT lymphoma in domestic animals; further, PCR is an effective means of determining the clonality of B-cell neoplasms in the cat.
34: LITTORAL CELL ANGIOMAS OF THE CANINE SKIN AND SPLEEN.
Littoral cells or lining cells of sinuses occur in the splenic sinus or red pulp areas and in the medullary areas of lymph nodes. Littoral cells are characterized by a dual function and react positively in immunohistochemical preparations for both endothelial cells (Factor VIII, CD31) and for phagocytic cells (lysozyme). They are credited with the multifunctionality that gave rise to the concept of the reticuloendothelial system of Aschoff. The phagocytic nature of these cells is evident in oversight stains where cellular debris and hemosiderin can frequently be found in the cytoplasm of sinus lining cells. Recently, it has been reported that these littoral cells of the human spleen may become neoplastic and form cavernous angiomatous tumors. These lesions are not commonly recognized and only 11 human cases are reported to date. Littoral cell angioma of the spleen is described in the 9th edition of Rosai's Surgical Pathology. Littoral cell angioma occurs in the skin of the dog and differs from the usual benign hemangioma by lining cells having vesicular nuclei and much more cytoplasmic volume and by ingested cytoplasmic granules. They mark with both CD31 and lysozyme. The splenic lesions are characterized by clusters of 20 or more sinuses with wide dilation that appear as solidly congested areas against the more mixed cellular background of sinus areas and are seen in spleens with actual angiosarcoma of the usual type in adjacent areas. Because endothelial neoplasms are commonly seen in the canine spleen while they are relatively rare in humans it maybe that a benign littoral cell neoplasm may underlie the genesis of some canine hemangiosarcomas.
35: NECROTIZING PROTOZOAL DERMATITIS DUE TO A TOXOPLASMA GONDII-LIKE ORGANISM IN A DOG.
Severe generalized necrotizing dermatitis and vasculitis with intralesional protozoal organisms was diagnosed in a 10 year old female mixed breed dog. The dog was being treated for lymphoma and immune-mediated thrombocytopenia with a standard chemotherapy protocol (cyclophosphamide, doxorubicin, vincristine, prednisone). Small nodular dermal lesions developed after 11 treatments with progression to severe exudative dermatitis following the last treatment. The lesion is characterized by extensive epidermal ulceration and necrosis that includes the follicular epithelium. Necrosis extends from the epithelium to the superficial hypodermis with edema, hemorrhage and an inflammatory infiltrate of neutrophils, histiocytes, small lymphocytes and plasma cells. Small l–2u oval organisms were found free in the necrotic tissue debris, within macrophages and, occasionally, within endothelial cells. Immunohistochemistry was performed and the organisms reacted strongly with Toxoplasma gondii antibody and did not react with Neospora caninum antibody. Necrotizing dermatitis in the dog due to a Toxoplasma gondii–like apicomplexa organism and Neospora caninum has been previously described. Predisposing factors such as chronic ehrlichiosis, cardiomyopathy and immune-suppressive therapies for immune-mediated disease and neoplasia were reported in most cases.
36: CUTANEOUS ANGIOMATOSIS IN A YOUNG DOG.
A 1 year-old, female spayed, mixed-breed dog had two reddish-purple cutaneous lesions, one on the right dorsal antebra-chium and the other on the right shoulder. The lesions consisted of approximately 13 × 3 cm and 15 × 10 cm irregular patchy regions of 0.5–3.0 cm circular, sometimes raised, reddish-purple swellings resembling ecchymoses. The lesion on the antebrachium had been noticed since the dog was adopted at 6 months of age and appeared to have increased in size over an 11 week period, at which time skin punch biopsy revealed an infiltrative pattern of well-differentiated blood vessels leading to an interpretation that the lesion was a well-differentiated hemangiosarcoma. The second lesion was revealed when the dog had its fur shaved in that area during surgical preparation to excise the antebrachial lesion. No other skin lesions were found on the dog. Microscopically, there was a widely disseminated and infiltrative-like pattern of benign-appearing small blood vessels, which were throughout the superficial and deep dermis and subcutis. Although the disseminated nature suggested malignancy, the histological appearance of well-differentiated small blood vessels and non-progressive clinical features indicate that the lesions were benign. The dog has been followed for 6 years and to date has no evidence of progression of the antebrachial lesion or shoulder lesion. To the authors’ knowledge, this is the first report of a congenital angiomatosis-like lesion in a young dog with extensive involvement of the forelimb.
37: CANINE CUTANEOUS CLEAR CELL ADNEXAL CARCINOMA: HISTOPATHOLOGY, IMMUNOHISTOCHEMISTRY AND BIOLOGICAL BEHAVIOR.
Thirty tumors including 27 distinctive cutaneous neoplasms and 3 metastatic tumors from 26 dogs were collected from diagnostic submissions to three laboratories. Characteristic histopathologic features included location in the subcutis and/or dermis; lobular, nodular and nest-like architecture; and a component of epithelioid cells with clear cytoplasm. Additional features present in most cases included follicular papilla-like structures, mitotic activity, nuclear pleomorphism, necrosis and mineralization. Cytoplasmic PAS-positivity, which was abolished by pretreatment with diastase, indicated the presence of glycogen in all cases. The oil red O stain did not demonstrate cytoplasmic lipid. Melanin granules, accentuated by the Fontana-Masson method, were observed infrequently. A pilosebaceous mesenchyme-like stroma and stromal cartilaginous differentiation were uncommon. By immunohistochemistry, neoplastic cells stained positively for cytokeratin (29 of 29), vimentin (28 of 28), S-100 protein (24 of 29) and melan A (8 of 12); results were negative for smooth muscle actin and calponin in all cases. Clinical follow-up information was obtained on all twenty-six dogs. One recurred and two metastasized to regional lymph nodes. In another case, possible pulmonary metastasis was noted radiographically. The findings are consistent with a poorly differentiated, low-grade, adnexal carcinoma of the skin. Similar canine cutaneous neoplasms have been reported as “clear-cell hid-radenocarcinoma” and “follicular stem cell carcinoma”. We propose the designation “cutaneous clear cell adnexal carcinoma.”
38: IMMUNOHISTOCHEMICAL STUDY OF THE PCNA AND E-CADERIN AS PROGNOSTIC MARKERS IN CANINE MAMMARY NEOPLASIA.
Canine mammary tumors are challenging for clinicians and pathologists because of complex histologic classification and unpredictable biological behavior. In histologic specimens, expression of the tumor proliferation marker PCNA and the protein which provides adherence to the cells called e-caderin have been testing to provide prognostic value for canine mammary tumors and to correlate with malignancy and low survival rates. The objective of this study was to determine the value of the PCNA and e-caderin as prognostic markers in canine mammary tumors and to determine its relationship to clinical and pathologic variables and patient outcome. Spontaneous mammary tumors from 60 female dogs were surgically excised. Histologic specimens were prepared routinely. Immunostaining was performed with the PC-10 monoclonal antibody against PCNA and NCH-38 monoclonal antibody against e-caderin; they were graded from negative to + ++. Dogs were followed for 18 months. Multivariate logistic regression analysis was used to determine correlations between immunohistochemical results, tumor and clinical variables, and patient outcome. PCNA in histologic specimens was significantly lower for nonmalignant tumors than for malignant tumors. On the other hand, e-caderin was significantly lower for malignant tumors than for nonmalignant tumors. High index values of PCNA and low index values of e-caderin were positively correlated with metastasis, death from neoplasia, low disease-free survival rates. The prognostic value of the PCNA and e-caderin could improve the accuracy and value of immunohistochemistry by providing corroboration about malignancy and patient outcome.
39: OSTEOPETROSIS IN A NEWBORN PERUVIAN PASO FOAL.
Osteopetrosis is a disease of bones characterized by the accumulation of primary and/or secondary spongiosa within the medullary cavity of long and flat bones. In mammals, it is most often secondary to defective osteoclast resorption. The disease is well described in numerous breeds of cattle as an autosomal recessive condition. A full-term, newborn, male Peruvian Paso foal was euthanized less than 6 hours after birth for progressive weakness, failure to rise, and craniofacial abnormalities. Gross abnormalities included brachygnathia inferior, dental deformities, multiple rib fractures, and generalized osteopetrosis. Osteopetrosis was characterized by filling of the medullary cavity by densely packed, parallel trabeculae of metaphyseal bone originating at the physis and tapering to a point at the mid-diaphysis. Radiographs demonstrated increased medullary opacity in the extremities, axial skeleton, and skull. In long bones, this was characterized by an hour-glass arrangement of uniform columns of trabecular bone. Bone ash evaluation demonstrated increased percent mineral in metaphyseal trabeculae as compared to an age-matched control. Preliminary histologic evaluation revealed filling of the marrow cavity by mineralized trabeculae of woven, and occasionally lamellar, bone, persistence of primary spongiosa in the proximal metaphyses, and irregular invasion of the physeal cartilage. There were sparse marrow elements and multinucleate osteoclasts were present in normal to increased numbers. Osteoclasts were identified both free and intimately associated with bone surfaces, and had occasional, discrete, cytoplasmic vacuoles suggesting active phagocytosis, but ruffled borders were indistinct to absent. A literature search reveals three previous reports. In three previous reports of foal osteopetrosis, four of the five horses were of Peruvian Paso breed, and share similar gross and microscopic findings.
40: HYPERNATREMIC POLIOENCEPHALOMALACIA SECONDARY TO DEHYDRATION IN A DOG.
Hypernatremia is a potentially life-threatening electrolyte abnormality. This condition develops most often because of loss of water from an animal or from gain of sodium chloride. A three-year-old, neutered male German Short Haired Pointer was presented for evaluation of uncontrollable seizures after being left unattended outdoors for approximately 12 hours on a hot day with no access to water. Diagnostic evaluation revealed that the levels of serum sodium (163 mEq/L; reference range, 142–152 mEq/L) and chloride (131 mEq/L; reference range, 108–120 mEq/L) were elevated. The dog was euthanized due to poor prognosis of uncontrollable seizures despite aggressive therapy. Necropsy findings included pulmonary congestion and edema, increased cerebrospinal fluid and mild heartworm infestation. Histological analysis of the brain revealed neuronal necrosis of the frontal, parietal, temporal and occipital lobes of the cerebral cortex. Specific lesions include neuronal necrosis (hypereosinophilia, cellular shrinkage and nuclear pyknosis), vacuolation of neuropil, prominent and reactive blood vessels, and perivascular accumulations of leukocytes in cerebral capillaries, and perineuronal and perivascular edema. Hypernatremic polioencephalomalacia has previously been reported in dogs in association with abnormal salt intake or abnormalities in osmoregulation. We report a case of hypernatremic polioencephalomalacia due to dehydration resulting from inaccessibility to water.
41: ANAPLASTIC MENINGEAL SARCOMA IN A DOG.
A three year old, neutered male, Sheltie was presented to a nearby small animal clinic with a complaint of fever (106.6F). The dog collapsed during the initial examination. The CBC, clinical chemistry and electrolyte panels and urinalysis were normal. Thoracic and abdominal radiographs and EKG also were normal. The animal was vomiting blood in the terminal stages of the illness. At necropsy, a 2.0 × 3.5cm intracranial mass was located along the lateral aspect of the left temporal lobe. The mass was firmly attached to the overlying dura mater, tan, firm with a smooth external surface and was easily separated from the adjacent cerebrum. Histologically, the tumor was well-demarcated, non-encapsulated, and composed of a densely cellular sheet of highly pleomorphic polygonal to spindle cells. Multinucleated tumor giant cells and atypical mitotic figures were numerous. The neoplasm was diffusely infiltrated with a mixed population of inflammatory cells, contained variably sized randomly distributed foci of coagulative necrosis and was infiltrating the underlying neuropil. Neoplastic cells were strongly positive for vimentin and negative for GFAP. A diagnosis of anaplastic meningeal sarcoma (malignant meningioma) was made on the basis of cellular pleomorphism, the presence of numerous atypical mitotic figures and foci of necrosis, infiltrative behavior, and immunohistochemical staining properties.
42: FELINE OBESITY IS ASSOCIATED WITH INCREASED MYOCARDIAL LIPID AND OXIDATIVE STRESS.
The current epidemic of human obesity emphasizes the need to identify animal models in which to study the mechanisms of obesity-related cardiovascular disease. Obesity in rats and humans has been associated with the accumulation of lipid droplets within cardiomyocytes. These lipids may be predisposed to peroxidation by hydroxyl radicals resulting in the production of 4-hydroxy-trans-2-no-nenal (HNE). HNE has been shown to inhibit cardiomyocyte contraction. Immunoreactive HNE is increased in skeletal muscle from obese humans and in endomyocardial biopsies from human cardiomyopathy. We collected formalin-fixed samples of left ventricular myocardium from 8 adult cats that died of unexplained causes, were free of macroscopic and microscopic evidence of endocrine or renal disease, had massive accumulations of visceral and subcutaneous adipose tissue, and a body weight greater than 2 standard deviations above the mean for cats presented to the Veterinary Medical Diagnostic Laboratory. These were compared with left ventricular samples from 8 normally fleshed (body weight within 2 standard deviations of the mean for cats in our laboratory) age-matched cats that died without evidence of cardiopulmonary disease. Frozen sections were stained with oil-red-o for evaluation of lipid accumulation. Formalin-fixed paraffin-embedded sections were stained with hematoxylin and eosin (H&E) and immunohistochemistry was performed for HNE. There were no differences in H&E-stained sections; however the cardiomyocytes from obese cats contained more lipid droplets and HNE than those from normal cats. These findings suggest that the routine histopathology may not be sufficient to detect cardiac disease in the obese cat and that the cat may be a useful model for the study of cardiac oxidant stress in obesity.
43: CYTAUXZOONOSIS IN A KENTUCKY DOMESTIC SHORT-HAIRED CAT.
A locally born and raised cat was presented exhibiting lethargy, anorexia, hyperthermia (105°F) and harsh respiratory sounds. In clinic FeLV and FIV tests were negative. Results of all in house lab work were within normal limits. The cat declined despite antibiotic treatment and IV fluids. The last 12 hours preceding death it became very agitated and aggressive. Clinical diagnosis was unknown. Histopathologic evaluation revealed multifocal intravascular protozoal schizonts in the brain, heart, lung, intestine, lymph nodes, spleen and kidney that were consistent with Cytauxzoon.
44: OSSIFIED YOLK SAC REMNANT IN AN EQUINE PLACENTA.
The yolk sac develops from an outgrowth of embryonic midgut entoderm intimately associated with the umbilical cord. In mammals, the sac normally involutes by the time of parturition. Incomplete involution of the vitelline duct produces a Meckel diverticulum. This report describes incomplete involution of the yolk sac resulting in a placental mass and abortion. A 300-day gestation thoroughbred fetus was aborted from a 14-year-old multiparous mare. Necropsy of the fetus revealed no significant gross or microscopic lesions. The placenta was expelled 48 hours later. It weighed 6.8 kg and had a pedunculated mass extending from the amniotic umbilical cord. The stalk of the mass was 40 cm long and consisted of a pair of 3 mm diameter tubes enveloped within a connective tissue sheath. The ensheathed tubes anastomosed proximally with vessels in the umbilical cord and distally with the surface of a 14 cm diameter globoid cyst. The cyst had been incised by a curious equine practitioner and thus, the contents were not available for examination. The cyst wall was 4 mm thick, smooth and had approximately 20 mottled white and red, hard, 4–43 mm diameter mural plaques. Microscopic examination of the wall revealed loose fibrovascular connective tissue with haphazard foci of cartilage, lamellar bone and bone marrow. Blood vessels were present in greater numbers on the outer 1/4 of the cyst than on the inner surface. The tubes in the stalk consisted of multiple vessels lacking muscular tunics embedded within fibrous connective tissue. Differentials included amorphus globosus and teratoma, however, the structure was concluded to be a remnant yolk sac with metaplastic bone. Compression of the umbilical cord or placenta by the yolk sac may have resulted in abortion.
45: NECROPSY WHAT? BASIC ARACHNID AND INSECT PATHOLOGY.
Tarantulas, scorpions, millipedes, and other charismatic invertebrates are increasingly popular in the pet trade, zoological collections and research. This popularity has created a demand for veterinary care and diagnostics including necropsy evaluation. Such procedures are invaluable to the management of collections and in some cases, conservation of a species. Necropsy evaluation of arachnids and insects has received the stigma of being a fruitless endeavor. However, at the Zoological Society of San Diego, routine gross necropsy and histologic evaluation provided insight into important disease conditions in the majority of invertebrates examined. Species examined included Goliath bird-eating spiders, Mexican red-legged tarantulas, giant African millipedes and emperor scorpions. Gross findings included exoskeletal and limb fractures, distal limb desiccation, cuticular ulcers and thickening of book lung ostia. Histologic evaluation revealed well-developed and clearly identifiable nervous, respiratory, circulatory, digestive, reproductive, excretory and musculoskeletal systems. Sites of exoskeletal damage frequently correlated histologically with entry points for secondary systemic fungal and bacterial infections. More importantly, histologic evaluation revealed primary bacterial and fungal infections in cases where no gross lesions were initially identified. Inflammation demonstrated histologic patterns that bore remarkable similarity to the responses of vertebrates to injury and infection. Systemic infections resulted in multiorgan infiltration by hemocytes, the basic blood cells of invertebrates, with the formation of granuloma-like structures in many organs. Hemocytes also demonstrated phagocytic capacity with numerous intracellular bacteria. At sites of exoskeletal trauma, hemocytes infiltrated the hypodermis and underlying skeletal muscle creating a barrier between the body cavity and the external environment. Necropsy evaluation of the invertebrate can be as powerful a diagnostic tool as the examination any vertebrate species.
46: BILATERAL NOCARDIAL ENDOPHTHALMITIS IN A PROTHONOTARY WARBLER (PROTONOTARIA CITREA).
A 7-year-old captive female Prothonotary warbler (Protonotaria citrea) died following chronic feather and weight loss. At necropsy the right eye had a 2 × 2 × 1 mm corneal plaque of inspissated yellow-tan material, edema of the lower eyelid, and a hyper-reflective retina. Microscopically, both eyes had chronic, diffuse, severe pyogranulomatous endophthalmitis with retinal necrosis and detachment. Numerous intralesional branching, gram-positive, beaded, filamentous bacteria formed a thick mat attached to the retinal pigmented epithelium and extending into the pecten. Bacteria were strongly acid-fast positive by Fites stain, but only occasionally acid-fast positive by Ziehl-Neelsen staining, a characteristic consistent with a Nocardia spp. There was no evidence of primary bacterial infection in the other organs examined.
47: SUSPECTED ZYGOMYCOSIS IN A FREE-RANGING ATLANTIC BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS).
Infectious disease can be difficult to assess in free-ranging marine mammals due to a limited assessable cohort population. Viral, bacterial, protozoal, and fungal diseases have been reported in individuals and in epizootics. We report a case of disseminated fungal infection in a free-ranging, 114 kg, 222.8 cm, male, Atlantic Bottlenose Dolphin (Tursiops truncatus) (Field # WAM 591) found stranded on the coast of North Carolina. This individual was emaciated with a markedly thinned blubber layer, serous atrophy of subcutaneous fat, focally extensive ulcerative and proliferative dermatitis, panniculitis, and myositis involving the right tailstock, cerebellar cavitary hemorrhagic necrosis, and multifocal granulomatous pneumonia. Histologically, there was severe pyogranulomatous mycotic dermatitis, panniculitis, myositis, pneumonia, and meningoencephalits. Fungal organisms were present within necrotic vessels in the blubber, lung, aorta, and brain. Fungi were 8 to 15 microns in width with irregular walls, indistinct septa, irregular branching, and bulbous projections. Entry was likely through the cutaneous wound as has been reported in captive cetaceans. Zygomycoses have been reported infrequently in different species of free-ranging cetaceans and occasionally in captive marine mammals.
48: DIAGNOSIS OF NECROTIZING MYOCARDITIS AND MYOCARDIAL INFARCTION DUE TO NEOSPORA CANINUM IN AN ADULT MASTIFF DOG BY MULTIPLEX PCR AND IMMUNOHISTOCHEMISTRY.
A three-year-old male castrated Mastiff dog died suddenly with locally extensive, severe, acute necrotizing myocarditis and myocardial infarct. A novel multiplex polymerase chain reaction (PCR) using fresh myocardial tissue provided rapid preliminary results positive for Neospora caninum and negative for Toxoplasma gondii. PCR results were confirmed by identification of tachyzoite organisms on hematoxylin and eosin stained histologic sections and by immunohistochemistry with a murine monoclonal antibody. Immunohistochemistry for Toxoplasma gondii was negative. Myocarditis is one of several presentations of neosporosis in adult dogs, but sudden death secondary to myocardial infarct has only been previously published once. As in this case, the previously reported case involved a young adult Mastiff, which may suggest an innate susceptibility to Neospora caninum myocarditis in this breed.
49: ULCERATIVE AND HEMORRHAGIC TYPHLOCOLITIS IN AN ANGUS HEIFER ASSOCIATED WITH NATURAL BOVINE ENTEROVIRUS TYPE-1 INFECTION.
A 2-year-old, 7 months pregnant Aberdeen-Angus bovine presented with acute, progressive abdominal pain and died within 10 hours of hospitalization. Significant post mortem findings were limited to the intestines. The serosa of the distal jejunum, ileum, cecum, and spiral colon were diffusely hyperemia The contents of the spiral colon were bloody; its mucosal surfaces were dark red, with widely scattered foci of mucosal ulceration. At the ceco-colonic junction, there were multiple, discrete white foci covered by fibrinohemorrhagic exudate. Microscopically, the colon had multiple foci of hemorrhage, necrosis, and ulceration. There was moderate to severe lymphocyte necrosis of the submucosal lymphoid tissues of the colon and small intestine. The splenic white pulp was moderately depleted. Virus isolation from the intestinal lesions yielded a cytopathic virus, which was classified by electron microscopic examination as a member of the Picornaviridae family. Further characterization classified the virus isolate as bovine enterovirus type 1 (BEV-1). Additional testing of the colonic lesions by PCR was positive for BEV-1. No other significant pathogens (viral or bacterial) were detected. Our results suggest that BEV-1 isolated was associated with the lesions described; however, a direct cause-and-effect relationship was not established. Nevertheless, in the absence of other detectable pathogens, the clinical history, the pathological lesions, and the laboratory results obtained suggest that the BEV-1 isolated from this case could represent a virulent strain. To the best of our knowledge, this is the first report of intestinal lesions and localization in BEV-1 infection, and the first report of BEV-1 from North America.
50: LANGERHANS CELL HISTIOCYTOSIS IN A CAT.
A 9-year old, female spayed, domestic shorthaired cat that presented with a chronic history of clinical airway disease was euthanized after the development of progressive respiratory signs and anorexia despite symptomatic treatment. At necropsy, the lungs were effaced by myriad, small, 0.5 to 1.5 cm diameter, irregularly shaped, tan masses. Throughout the renal cortices there were similar multifocal, variably sized, tan masses. Histologically, the lungs contained multifocal to coalescing, intraalveolar and intrabronchiolar sheets of oval to pleomorphic cells that resembled histiocytes, and which were surrounded by bands of fibrous connective tissue. Rare mitotic figures and few foci of necrosis were evident. Sheets of similar cells multifocally infiltrated the renal cortices. The proliferative cells in the lungs and kidneys expressed vimentin and CD18; they were negative for cytokeratin, CD45R, CD79a, and CD3. The cells in the lung, but not in the kidney, also expressed E-cadherin. This pattern of CD expression in combination with ultrastructural demonstration of Birbecks granules in some of the proliferating cells in the lungs and kidneys demonstrates their Langerhans cell origin and supports a diagnosis of Langerhans cell histiocytosis (LCH). The presence of large aggregates of lesional cells distinguishes LCH from reactive dendritic cell proliferative diseases. To the authors’ knowledge, this report describes the first conclusive case of Langerhans cell histiocytosis in a cat.
51: IMMUNOHISTOCHEMICAL CHARACTERIZATION OF EQUINE CUTANEOUS MAST CELL TUMORS WITH AN ANTIBODY TO CD117 (KIT).
The KIT protein is a tyrosine kinase receptor and the product of the c-kit proto-oncogene. This protein is expressed in numerous tissues including interstitial cells of Cajal, mast cells, term placenta, brain, erythroid precursors, melanocytes, basophils and mast cells. The ligand for the KIT receptor, stem cell factor, is involved in mast cell proliferation and maturation and KIT has been detected in mast cell tumors of dogs and cats. The purpose of this work was to detect KIT in equine cutaneous mast cell tumors by immunohistochemistry. Twenty-six cutaneous mast cell tumors were used. Samples were fixed in 10% formaldehyde and routinely processed. For IHC, heat antigen retrieval, by immersing slides in citrate buffer (pH 6.0) at 90–95 C for 20 min., was used. A rabbit polyclonal antibody to CD 117 (KIT) at a dilution of 1/100 was incubated for 60 minutes at room temperature. A peroxidase-DAB, polymer-based detection system (EnVision+) was used to demonstrate the immune reaction. All tumors were positive for KIT. The staining was in the plasma membrane and cytoplasm (with a diffuse, paranuclear or perinuclear patterns) in more than 80% of cells in all tumors examined. Nuclear staining was not apparent. The most common cytoplasmic staining pattern was diffuse, with 8 cases having paranuclear, and 3, perinuclear staining. Nonneoplastic dermal mast cells were also strongly positive for KIT, but other dermal leukocytes did not react with KIT antibody. Apocrine gland epithelial cells were strongly labeled in four cases. The tumors we examined had benign morphologic features so IHC reactivity could not be correlated with cellular atypia. Immunohistochemistry for KIT in equine mast cell tumors may add in the characterization of these tumors.
52: CUTANEOUS EQUINE MAST CELL TUMORS: IMMUNOHISTOCHEMICAL DETECTION OF TRYPTASE.
Mast cell tumors are uncommon, usually benign neoplasms of horses. Histologically, mast cell tumors consist of single to multiple and coalescing nodules and sheets of well-differentiated mast cells admixed with mature eosinophils in the dermis and subcutis. Areas of necrosis containing degenerated eosinophils and foci of collagen degeneration are frequent. Tryptase is one of the most common neutral proteases of mast cells in numerous species. Immunohistochemistry for tryptase is a very specific and sensitive method to identify mast cells in humans and dogs. In dogs, poorly differentiated mast cell tumors tend to have fewer granules that are difficult to detect with metachromatic stains. We have examined the expression of tryptase by IHC in 29 equine mast cell tumors fixed in formalin and embedded in paraffin. We used as primary antibody a mouse monoclonal antibody to tryptase at 1/100 dilution, incubated for 30 min. Tissue sections were heated at 90–95 C in citrate buffer, pH 6.0, for 20 min for antigen retrieval. A peroxidase-DAB, polymer-based detection system (EnVision+) was used to demonstrate the immune reaction. All tumors yielded a strong reaction. The reaction was diffuse, cytoplasmic and granular without membrane or nuclear staining. Areas of necrosis, eosinophilic infiltrates or degenerated collagen were consistently negative. Our results confirm that tryptase can be consistently detected by IHC in equine mast cell tumors. Although equine mast cell tumors are not difficult to diagnose with routine stains, tryptase IHC may aid in the detection of small clusters of neoplastic cells.
53: NEOSPOROSIS IN A DOG WITH AUTONOMIC FAILURE.
Pandysautonomia has been described in domestic animals, including horses, dogs, cats and hares, as well as human beings. Although the etiology of most cases is not known, degeneration, necrosis, and loss of neurons, and gliosis are the hallmark histologic findings in this autonomic system disease. Neospora caninum is an apicomplexan protozoan that causes encephalomyelitis, polyradiculitis, polymyositis and abortion in multiple animal species. A 10-week-old, female Siberian husky was submitted to the VMDL of the University of Missouri for a cosmetic necropsy examination. The animal had a history of megaesophagus, gastrointestinal distention, aspiration pneumonia, loss of anal tone, decreased papillary light reflexes and bladder distension. Dysautonomia was suspected based on the unresponsiveness of heart rate and ocular reflexes to pharmacological testing and radiography. Grossly, the cranioventral lung lobes were diffusely dark red, wet and heavy and the gastrointestinal tract was severely distended by ingesta and gas. Histologically, multifocal granulomas were found adjacent to blood vessels of the brain, especially around the medullary vagal nuclei. Oval cysts, approximately 40 to 50 μm in diameter with PAS+, eosinophilic hyalinized walls were present in the center of some of the granulomas. Within the cysts were numerous basophilic, oval bradyzoites, ranging from 1 to 2 μm in diameter. Immunohistochemical staining and serologic analyses were positive for Neospora caninum. Neuronal degeneration was observed in the cranial mesenteric ganglion and myenteric plexus of the gastrointestinal tract, and a single Neospora cyst was visible in the myenteric plexus of the jejunum. Although it is uncertain if this parasite is responsible for the dysautonomia, this is the first observation of this parasite in the myenteric plexus. Trypanosoma cruzi infection is not infrequently associated with Chagas-induced dysautonomia in human patients. However, it is possible that Neospora may occasionally be involved in canine dysautonomia, producing lesions in the peripheral and central autonomic nervous systems.
54: CANINE HETEROBILHARZIASIS IN NORTHEAST ALABAMA: A CASE REPORT.
A 10-year-old intact male Labrador Retriever was presented to a veterinarian in Etowah County, Alabama, with an approximately one-month history of lethargy, weight loss, and intermittent diarrhea. The dog was reportedly housed outdoors and had frequent access to local rivers and streams. Initial physical exam findings included poor body condition, weakness, mild dehydration, and mucous membrane pallor. Pertinent clinicopathologic results included a moderate leukocytosis (22,800/mm3; reference range 6000–17000/mm3), increased alanine aminotransferase levels (510 IU/L; reference range 5–65 IU/L), mildly elevated blood urea nitrogen (57 mg/dl; reference range 10–30 mg/dl) and marked anemia (PCV 12.7%; reference range 37–55%). Fecal examination was positive for occult blood, and negative via sodium nitrate flotation for parasite ova. Due to deteriorating condition, the owner elected for euthanasia. A necropsy was performed and sections of small intestine were submitted to the Auburn University Diagnostic Pathology Service. Mucosal crypts, lamina propria, and the submucosa contained numerous cross-sections of 50–60 micron diameter, round to ovoid, parasitic ova with a thick, refractile, yellow-brown to unstained, partially collapsed shell, consistent with Heterobilharzia americana. These ova, which often contained a developing miracidium, were surrounded by intense granulomatous inflammation and mild fibrosis. Canine schistosomiasis in North America, caused by the digenetic trematode Heterobilharzia americana, occurs primarily along the Gulf Coast in the Southeastern United States. There have been previous reports of canine infection ranging from North Carolina to as far west as central Texas and Kansas. Fresh water snails, Lymnaea cubensis or Pseudosuccinea columella, are intermediate hosts necessary for completion of the life cycle, and are thought to limit the geographic range of heterobilharziasis.
55: AMYLOID PRODUCING ODONTOGENIC TUMOR IN A 10 YEAR OLD TIGER (PANTHERA TIGRIS).
Epithelial odontogenic tumors are uncommon in domestic animals and most of the reported cases being dogs. One subtype, the amyloid producing odontogenic tumor (APOT, previously called calcifying epithelial odontogenic tumor) is generally located in the mandible with tendency to be slow glowing but occasionally causes bone destruction and displacement of teeth. Histologically, the neoplasm is characterized by epithelial proliferation, mineralization in the epithelium and stroma, and deposition of amyloid. In this report, mandibular APOT is described in a 10-year-old male tiger (Panthera tigris) that has been kept in the Everland Zoological Garden, Korea. The tiger was euthanized due to poor prognosis after suffering from rapidly growing mass at the gingiva. At necropsy, the gingiva of mandibular symphysis was markedly enlarged and hard. Secondary destruction of mandibular bone was evident on radiographs. Histologically, the neoplastic mass consisted of odontogenic epithelial cells with multiple nodular amyloid deposits as stained by Congo Red. In the areas of cyst formation, lining epithelium showed squamous metaplasia with keratin production. Dystrophic calcification, smaller foci of irregular basophilic material with spheroidal and laminated structure, so called Liesengang rings were also noted. The diagnosis was based upon the results of light microscopy and special staining. This appears to be the first reported APOT in tiger.
56: OMPHALOCELE ASSOCIATED ABORTION AND CHRONIC AMNIONITIS IN A FOAL.
Omphalocele is a congenital malformation in which variable amounts of abdominal contents protrude into the base of the umbilical cord. A full term aborted fetus submitted for necropsy examination had the posterior half of small intestine, entire cecum and large colon protruding into the amnionic cavity through the base of umbilical cord. The amnion covering this area had diffuse thickening of approximately 3–4 cm. The amnionic segment of the umbilical cord had adhesion to this prolapsed intestinal mass. Histopathological examination of the amnion showed diffuse thickening by proliferating fibrous connective tissue indicating that this is a chronic process. Unlike the more common umbilical hernia where the sac is covered by overlying skin, covering of the hernial sac in an omphalocele is the epithelium of the umbilical cord, a derivative of amnion. To the authors’ knowledge, this report is the first case of omphalocele in a foal leading to abortion.
57: THE USE OF MOLECULAR APPROACHES IN THE DIAGNOSIS OF CANINE TRANSMISSIBLE VENEREAL TUMOR IN BRAZIL.
The canine transmissible venereal tumor (TVT) is a naturally occurring neoplasm common in tropical regions like Brazil. TVT mainly affects the external genitals of dogs and has the ability of being transplanted to other sites such as the nose or mouth. These transplantations occur during coitus or other habits like sniffing or licking. According to the literature, TVT do not metastasize. In Brazil, many of the TVT cases attended in our university's hospital show signs of metastasis and morphologic disparity with classical TVT. In some cases it is hard to determine the origin of the tumor, making the diagnosis by cytology and histology unclear. In the present work, we used the PCR technique to detect a translocation that probably occurs only in the TVT cells in which the c-myc oncogene is rearranged by the insertion of a transposable genetic element in its first exon, in order to confirm the diagnosis of twenty TVT samples. All PCR reactions were performed using a negative control for TVT (DNA from canine blood) and a negative control for contamination. All samples evaluated were positive for the presence of the translocation, confirming the previous diagnosis. It is suggested that this method could be used as a chemotherapy control, as it can detect the presence of small amounts of tumor cells in normal tissue. The high sensitivity of this PCR approach indicates that it may be used routinely to achieve reliable TVT diagnosis.
58: MIXED GERM CELL-SEX CORD STROMAL TUMOR WITH RENAL METASTASIS AND MAMMARY GLAND ADENOMA.
A mixed breed, male, year-old canine was presented with a firm gray multilobar testicular mass that was totally effacing normal parenchyma, invading the surrounding tissues and extending into the epididymis. The contralateral testicle is atrophic. Microscopically, the tumor is composed of polyhedral and spindle shaped cells displaying marked anisocytosis disposed in a tubular and a diffuse pattern intermixed with ovoid and polygonal cells with large vesicular to hyperchromatic nuclei and multiple prominent nucleoli. The tumor cells are arranged into nodules and separated by dense collagenous fibrous tissue. Mitotic figures are common in the seminoma-like areas. In addition, a renal metastasis showing similar microscopic features was observed. There was no clinical evidence of skin lesions but a mammary gland papillary adenoma was diagnosed. The gross and histopathologic findings are compatible with a Mixed germ cell-sex cord stromal tumor. Although the metastasis trend for both tumor types (Sertoli cell tumor and seminoma) is low, renal involvement has been reported. Moreover, the metastatic Sertoli tumor cells are capable of producing hormones. In the present case, the mammary gland tumor is most likely due to hyperestrogenism caused by the Sertoli tumor cells from primary and secondary sites.
59: MALIGNANT TRANSMISSIBLE VENEREAL TUMOR WITH MULTI ORGAN METASTASES IN A MASTIFF
Transmissible venereal tumor (TVT) is a well-documented transplantable tumor in dogs, with no breed or sex predilection. Extragenital TVT has been found in the oral/nasal cavity after scratching, sniffing, or licking of either preputial or vaginal discharge of infected dogs. TVT is reported to have a low metastatic rate. In this report, a 2-year-old, intact female Mastiff dog was presented with numerous, rapidly growing, firm, tan nodules throughout the subcutis, particularly along the dorsal body plane and the caudal half of the ventral abdomen. The dog was euthanized due to poor prognosis. At necropsy, neoplastic nodules similar to those seen in the subcutis were also noted in the lung, anterior mediastinum, liver, spleen, kidney, and superficial and deep lymph nodes in both abdominal and thoracic cavities. Histologically, proliferation of monomorphic round cells with solid pattern was noted. The neoplastic cells had round nuclei, moderate amounts of pale eosinophilic to vacuolated cytoplasm, and distinct cell borders. The neoplastic masses from multiple sites as well as metastatic foci were examined and revealed similar histologic findings. On immunohistochemistry (IHC), the neoplastic cells were positive for lysozyme but were negative for vimentin, CD3, CD79a, and CD18. No diagnostic ultrastructural features were noted on electron microscopy (EM). The results of toluidine blue stains were also negative. Based upon the results histopathology, EM, and IHC, the diagnosis of an malignant TVT was made. This case is unique in that it is accompanied by wide multiorgan metastasis.
60: BILATERAL ABNORMAL ERUPTION OF DECIDUOUS TEETH IN A CYNOMOLGUS MONKEY (MACACA FASCICULARIS).
History/Presentation: An adult male cynomolgus monkey (Macaca fasicularis) had bilateral gingival masses (0.5 × 0.5 cm right and 1.0 × 1.0 cm left) ventral to a gap between the first and second mandibular premolars. Both masses exuded purulent material. All bloodwork was normal. The right mass had a fistulous tract that was continuous with the gingiva adjacent to the second premolar. Radiographs revealed small, sparsely mineralized structures bilaterally. The right second premolar and associated mass were surgically removed, antibiotic therapy was initiated pending preliminary histopathology findings, and two weeks later the left second premolar and associated mass were also removed and were submitted for microscopic evaluation. Findings: The right and left mandibular second premolars were unremarkable. Each mass contained a single small, unerupted deciduous tooth. The right deciduous tooth had marked suppurative pulpitis and chronic gingivitis with fibrosis. Dense mats of gram-positive and gram-negative bacteria and fungal hyphae were present on the tooth surface, and the fungal hyphae extend into the enamel. The left deciduous tooth, which was extracted following antibiotic therapy, had a slight to mild pulpitis and gingivitis, and was covered by a dense matt of mixed bacterial colonies. Conclusions: The bilateral masses were unerupted deciduous teeth complicated by gingivitis, pulpitis, and colonization with bacteria and fungi. Surgical removal was curative. Abnormal eruption of deciduous teeth is not uncommon and the bilateral presentation in this case suggests a genetic influence. Contributing factors can include trauma (such as hard pelleted food), abnormal position or orientation of the tooth germ root, and disturbances in the bones of the jaw. Radiographic visualization of these teeth can be difficult.
61: DABSKA-LIKE TUMOR IN A CHIMPANZEE (PAN TROGLODYTES).
In humans, the Dabska tumor (malignant endovascular papillary angioendothelioma, papillary intralymphatic angioendothelioma) is considered an extremely rare, low-grade malignancy that most often affects infants and children. It shows a wide anatomic distribution, with predilection for the skin. The medical literature suggests that these lesions recur locally and rarely spread to lymph nodes without systemic metastasis. Close clinical monitoring for regrowth or metastasis is recommended. This 4 year-old male chimpanzee (Pan troglodytes) had a rapidly appearing, approximately 75 × 50 mm in diameter, raised, cutaneous, sparsely haired, plaque-like dermal nodule in the mid-lumbar region of the back. Microscopically, within the dermis, neoplastic cells variably occlude dilated lymphatics and extend as papillary structures from the lymphatic lining. When not visibly intravascular, the cells form nodules and nests, and infiltrate the adjacent dermis. Neoplastic cells are spindled to polygonal and are supported by a moderate fibrovascular stroma that is often centrally hyalinized. Cells have variably distinct borders, scant to small amounts of eosinophilic fibrillar to amorphous cytoplasm, and oval nuclei with finely stippled chromatin and 1–2 distinct nucleoli. There is mild anisokaryosis and mitotic figures average 1–2 per high power field. Immunohistochemically, neoplastic cells are positive for vimentin, CD31, CD34, Factor VIII related antigen, and S100; and negative for kermix, keratin 903, cytokeratin 7, cytokeratin 20, epithelial membrane antigen (EMA), Ber EP4 epithelial antigen, and Melan A. To date, this lesion has not recurred after removal. To our knowledge, there are no published reports of a similar primary lymphatic neoplasm in a non-human primate.
62: PATHOLOGY SURVEILLANCE OF AVIAN RELEASE PROGRAMS ON MAURITIUS.
Mortalities experienced during the breeding and release of Pink Pigeons (Columba mayeri) into the wild were examined according to a standard protocol over a six month period. Birds ranged in age from 7 days to 11 years. Trichomoniasis was the most important cause of mortality (9/14), followed by idiopathic hemorrhage (2/14), pyogranulomatous cloacitis (1/14), and handling trauma (1/14). A range of parasites were present, including sarcosporidiosis (2/14), acariasis (1/14) and the first record of the renal fluke Paratanaisia (1/14) in Pink Pigeons in Mauritius. Incidental findings included idiopathic granulomatous myositis (2/14), aspiration pneumonia (2/ 14), capture myopathy (1/14), pyogranulomatous bursitis (1/14) and pustular epidermitis (1/14). An outbreak of disease in young Mauritius Parakeets (Psittacula echo) at a release site caused rapid weight loss, lethargy and regurgitation. Four animals died and were submitted for necropsy, and bacterial swabs were taken from others. Minimal histological findings included renal tubular necrosis, bursal depletion, and visceral hyperemia. Testing for chlamydia by PCR was negative. Moderate or heavy growths of gram negative organisms (Klebsiella sp., Pseudomonas aeruginosa and Acinetobacter baumannii) were isolated from a range of tissues from multiple birds. The coliform count of boiled stream water supplied to birds during the breakdown of a mobile sterilizing unit was found to be markedly elevated, and Acinetobacter baumannii of similar antibiotic sensitivity was cultured from it. Despite the success of such release programs, the congregation of birds at release sites promotes parasite transmission and increases the risks of hygiene breakdown, particularly where amenities are basic. Furthermore, the host-parasite interactions in released birds often remain unknown.
63: SYSTEMIC MYCOSES IN THREE CAPTIVE JUVENILE REINDEER (RANGIFER TARANDUS).
Over a period of one year, three juvenile reindeer from five to nine days of age were presented to the Animal Diagnostic Laboratory at the Pennsylvania State University for postmortem examination. All reindeer calves exhibited acute onset of weakness progressing to death within a one to two day period. Microscopically, severe meningoencephalitis, interstitial nephritis, myocarditis and bronchopneumonia were present in each animal. The lesions were characterized by large accumulations of neutrophils, macrophages, lymphocytes, fibrin and necrotic cellular debris intermixed with variable numbers of fungal hyphae. Marked intravascular thrombosis was also a prominent feature present within the majority of the inflammatory foci. Additional lesions included severe myocardial infarction, splenitis and ulcerative abomasitis in one calf, and necrotizing hepatitis and suppurative omphalitis in a second animal. Aspergillus sp. was recovered from affected lungs of two calves. Rhizopus sp. was recovered from the affected abomasum of the remaining reindeer. Factors that may have contributed to the systemic mycoses include inadequate passive immunity due to poor quality colostrum or inadequate colostrum ingestion and absorption by the calf, poor maternal care, calving areas heavily laden with fungi and inadequate neonatal management techniques.
64: OSSIFYING FIBROMA IN AN ADULT RABBIT.
In humans and animals, ossifying fibroma is a benign neoplasm that most frequently affects the mandible, often resulting in cosmetic deformities and malocclusion. It is considered rare in animals and most frequently affects young horses. Ossifying fibroma has also been reported in cats, dogs, a llama, a greater kudu and a sheep. A surgical biopsy from a six-year-old miniature Rex rabbit was submitted to the Department of Veterinary Pathology, Armed Forces Institute of Pathology for consultative light microscopic examination. The biopsy was obtained from an approximately 1 × 2 × 2 centimeter, solitary mass located beneath the gingiva in the right maxillary region, which had overgrown teeth and expanded the adjacent hard palate. The submitted incisional biopsy specimen was pale pink, firm and nodular. Histopathologically, the neoplasm was composed of fibroblastic cells separated by abundant collagen. The neoplastic cells were interwoven with osteoblasts surrounding islands of mineralized, bony matrix containing few, widely spaced, often empty, lacunae. Minimal inflammation was present. Based on the histopathologic features, the tumor was diagnosed as an ossifying fibroma. To our knowledge, this is the first report of an ossifying fibroma in a rabbit.
65: LEARNING BENEFITS TIED TO SPECIFIC CHARACTERISTICS OF THE DIAGNOSTIC PATHFINDER SOFTWARE.
According to learning theory, during problem solving the individual constructs a problem space, or mental representation of all the information known to him/her that is relevant to addressing the problem. Diagnostic problem solving is particularly difficult for veterinary students because much of the knowledge that constitutes an adequate problem space is relatively new to them. Based on recent work, this difficulty can be compensated for by providing learners with tools for constructing and manipulating representations of the problem space. The Diagnostic Pathfinder (DP) is such a tool. Earlier studies suggest that using the DP improves students’ problem solving ability; the purpose of this study was to determine specifically how the DP produces this result. Between the fall of 2003 and the spring of 2004, 289 students used the DP to complete between 14 and 93 cases as homework assignments at 4 colleges of veterinary medicine. Each student completed a related questionnaire, the responses to which were classified and analyzed according to the related specific characteristic of the DP and perceived impact. Students found benefit in the DP's requirement of completeness in data considered, interface for data manipulation, and availability of expert feedback in the same format as the learner rationale. The results of this study provide further evidence that allowing students to manipulate tools that facilitate problem space representation can support the learning of problem solving.
66: A GLIMPSE INTO WEBCT VISTA 3.0 AND EYESPY HIGH RESOLUTION IMAGE SERVER.
In 2002, WebCT launched Vista, a new enterprise version of its course management software. From a technical perspective, Vista is built on a platform architecture that allows for integration into existing campus computing systems and authentication programs. Institutions have the ability to customize Vista to meet their needs. A single installation of Vista can support multiple institutions or multiple colleges within one institution. Each individual institution or college, however, can retain control of their local system. Content that meets the SCORM (Sharable Courseware Object Reference Model) standard can be imported into Vista. Likewise, content created in Vista can be packaged for export to other courses. The faculty member using Vista for his/her course will see a new interface designed for greater efficiency than in previous versions of WebCT. A different paradigm for creating content has been adopted; various components (such as a listing of interesting websites or pages of informational content) are created and then inserted into the course at the appropriate places for optimized learning. Vista also enhances the ability to integrate and share course content. Another key improvement is the creation of different roles for course personnel; individuals are classified as designers, instructors and/or teaching assistants, each with the ability to handle specific tasks. These key features of Vista will be demonstrated. EyespyTM Image Server (licensed by AXD Technologies, Inc.) is able to deliver high resolution images very quickly over the internet. Parts of a high resolution image can be magnified to reveal the details of the image. We have experimented with converting our 2 × 2 teaching slide set into high resolution images and results of this endeavor will be demonstrated.
67: PREVENTING PUBLICATION ERRORS: THE NEED FOR A PATHOLOGIST IN THE EVALUATION OF GENETICALLY ENGINEERED MICE.
During the past 15 years, numerous publications on genetically engineered mice (induced mutants, transgenics, conditional mutants, etc.) have appeared in the biomedical literature. Veterinary and medical pathologists have been co-authors on many papers but biochemists and geneticists often provide their own interpretation of pathological findings without assistance of a pathologist or anyone familiar with normal gross anatomy, histology or pathology of the mouse or any other species in a surprisingly large number of papers. Although these publications can provide accurate information on histology and pathology without expert input, they often lack expertise in these important areas of medical research. Journal reputation is no barrier. Lack of pathology knowledge or expertise has been evident with erroneous journal cover illustrations, recognition of normal histological structures as lesions, publication of low quality illustrations, diagnosis of non-neoplastic lesions as tumors, and unusual or unaccepted pathology terminology. Examples will include normal structures and lesions of the skin, tumors in various tissues, interpretation of spontaneous tumors as induced tumors in aging mice, lack of statistical evaluation of tumor incidence, failure to understand normal histological variations in organs that normally cycle, and diagnosis of nonneoplastic lesions as neoplastic. As pathologists, we need to educate these investigators, as well as follow up publications with critical letters to the editor to correct mistakes before they become dogma.
68: STUDENT RESPONSE TO A CASE-REPORT BASED PEER ASSESSMENT ASSIGNMENT.
Efforts to establish and maintain valid and effective mechanisms for assessing learning outcomes in higher education are frequently complicated by the amount of faculty time they consume. In order to obtain high-quality assessment data while simultaneously respecting limited faculty time, the Veterinary Pathology Department at the Iowa State University College of Veterinary Medicine (ISUCVM) implemented an assessment project that utilizes junior students as peer reviewers of senior students’ work. During the spring semester of 2004, 94 junior students each reviewed 3 randomly assigned Case Correlation Assignments (CCA) that had been completed by seniors in the necropsy rotation. To complete the CCA adequately, seniors must thoroughly explain relationships between ante- and post-mortem data for a hospital case. The resulting reports reflect the students’ understanding of the pathophysiology of relevant diseases, as well as their ability to explain clinical laboratory data, cytology, and gross lesions. Juniors use a rubric to review and score the CCA's in a number of categories, including description of the pathogenesis of relevant disease(s), correlation between ante- and post-mortem data, and other similar criteria. After reviewing the CCA's, 93 juniors responded to a questionnaire regarding the experience. Most students indicated approval. They specifically reported that the peer review assignment made them more appreciative of and attentive to good record keeping, and that it helped them better understand relationships between ante- and post-mortem data. Several students also made comments that will be used to improve the assignment for future years. For instance, some students requested cases dealing with certain species of interest to them, indicating a need to more thoroughly explain the requirement that cases be randomly assigned.
69: EVALUATING STUDENT DEVELOPMENT: USING ASSESSMENT TO ENCOURAGE CRITICAL THINKING SKILLS.
Pathology is a bridge discipline between basic and clinical sciences and hence we have a unique position within the professional medical education that extends from general principles of pathologic processes through the clinical application of the study of disease. Consequently, our curriculum must then extend across the taxonomy of educational development from simple knowledge acquisition, comprehension, and application to information synthesis and evaluation. Despite lofty goals, a strong motivator for the student is the form of assessment that is used to generate the course grade. Many different forms of assessment can be utilized and can make markedly different demands upon the students. Thoughtful use of different assessment tools can become an integral part of the learning experience for the student and when appropriately designed have the power to challenge students to reach for more complex understanding and use of course material. Different forms of assessment used throughout the pathology courses at Michigan State University will be discussed with a consideration of strengths and weaknesses of each form of assessment. The coordinated use of different assessments to develop and encourage student critical thinking skills will be emphasized.
70: DESIGNING COURSES/LECTURES BY STARTING AT THE END AND ENDING WITH THE START.
Designing your lectures, laboratories, or courses can be a challenging endeavor; especially during your first few attempts. Where do you start? What do you need to know? When do you schedule instructional activities? How do you help students learn? Where do you end? Specific answers for these questions will vary, but there are basic strategies and methods that may help you find the answers that pertain to your teaching activities. One method is to begin at the end, determine how you are to help students prepare for the ending, and then end at the beginning. This method will be presented along with related instructional strategies.
71: AN INTEGRATIVE AND CASE-BASED APPROACH TO THE TEACHING OF GENERAL AND SYSTEMIC PATHOLOGY.
In the professional veterinary curriculum at the Michigan State University College of Veterinary Medicine, General Pathology is a 3-credit course taught in the spring semester of the first year, and Clinical and Systemic Pathology is a 5-credit course taught in the subsequent semester i.e., the fall semester of the second year. Each course includes concepts in both anatomic and clinical pathology, and certain topics (e.g., inflammation, neoplasia, digestive system pathology, urinary pathology, lymphoid system pathology) are taught from both specialty perspectives in an integrated fashion. In addition, selected diseases which may affect multiple body systems are presented in the Clinical and Systemic Pathology course in a manner which gives students the big picture, and integrates concepts covered under the specific body systems. There is also integration of pathology course material with material covered in other courses of a given semester (e.g., Microbiology, Parasitology, Pharmacology), through case presentations and case discussions in courses called Veterinary Integrative Problem-Solving (VIPS). Most case discussions in the VIPS courses are conducted in problem-based learning format, with students in small groups in the presence of a facilitator. Within the individual pathology courses, many instructors teach with a case-based approach, and involve the students in the case discussion, even in a room containing the whole class of more than 100 students. We believe that the integration of course material, both within the pathology courses and with other courses, and the demonstration of important pathology concepts through case discussion enhance students deep understanding of the subject matter and make it enjoyable to learn. Students comments on course evaluation forms support our opinions.
72: DEVELOPING AND FOSTERING A DYNAMIC PROGRAM FOR TRAINING IN VETERINARY PATHOLOGY FROM VETERINARY STUDENTS TO POST GRADUATE EDUCATION.
The Department of Veterinary Biosciences offers outstanding pathology and clinical pathology training for post doctoral veterinarians and professional veterinary students. The department has a successful program in experimental and applied veterinary pathology demonstrated by sustained extramural funding and an excellent record of graduates who pass the certifying examination of the American College of Veterinary Pathologists (ACVP). To prepare veterinarians for the ACVP certifying examination, individual 12 month preparation plans are developed with goals for didactic reading, review of clinical materials, mock histopathology and cytology exams, and intensive one-on-one instruction. This training is supplemented with seminars and intensive student-faculty interactions in the necropsy, surgical pathology, clinical chemistry, cytology, and hematology. The program has competed successfully for NIH-funded training grants to develop veterinary scientists with skills in comparative medicine and in the use of murine models of human disease. Trainees develop skills in clinical, gross, and histologic pathology, coupled with molecular and immunologic techniques, to identify and characterize disease models. A primary goal of program is to also provide an environment for veterinary students to attain technical and conceptual pathology skills to perform hypothesis-based research. This is done through targeted recruitment, elective courses, and support of student-based clubs. The program is coordinated through an established graduate program and is supported by an interdisciplinary group of basic and clinical scientists. Trainees interact with our multidisciplinary faculty to identify the range of research problems related to comparative medicine. The overall goal of the program is to provide trainees modern skills in pathology needed to work in research teams to fulfill national needs.
73: GLOBAL PERSPECTIVES—THE VETERINARY PATHLO-GISTS ROLE.
The veterinary community, including students and faculty, needs to be aware of global issues related to veterinary medicine. The diverse avenues of the profession provide opportunities at an expanding rate and a need for global issue awareness. A number of pathologists have been involved in global veterinary education, probably since numerous global issues are related to disease pathogenesis, where pathologists are conversant in research, education, and outreach. Relevant issues include emerging, zoonotic, and exotic diseases; wildlife and domestic animal disease transmission; environmental impact and conservation; animal welfare related to food animal production; and wildlife production for food, to name a few. Several objectives are important for the educational experience of students. Individual student experiences are worthy, but a shared experience can have a beneficial impact for a broader audience. We have found the creation of websites, papers, seminar presentations, and peer-reviewed publications by students will result in a shared product that will be beneficial. Two examples are listed:
http://www.vetmed.iastate.edu/academics/international/recenttrips/sweden2003/
http://www.vetmed.iastate.edu/academics/international/recenttrips/brazil2003/
Our veterinary students will be decision makers and leaders for global issues and influence what laws and regulations are adapted for animal welfare, possibly influenced by the EU; the impact among wildlife, domestic species and environmental conservation; and have opportunities to directly contribute to veterinary medical care in other countries. Increasing shared experiences among the veterinary community can provide access to this information.
74: USES OF WEBCT IN THE PATHOLOGY CURRICULUM AT IOWA STATE UNIVERSITY.
Many universities have access to one of several web-based course management systems, such as WebCT or Blackboard. These programs allow for automation of teaching while generating student performance data for assessment studies. Web-based teaching programs facilitate instructor efficiency through ease of grading, management of assignments, and communication with students. Exams and papers can be graded from home or on the road, easing location or schedule restrictions for the professor. The Department of Veterinary Pathology at Iowa State University uses WebCT for a variety of purposes within the four years of professional curriculum courses. At its most basic function, WebCT stores and reports scores to students. More advanced uses include quizzes, exams, and submission of written papers. Assignments can be submitted through WebCT, graded, and uploaded back to the server for student viewing. WebCT is uniquely adapted for pathology education as it facilitates inclusion of images that can easily be used for a variety of exam questions. Development of question sets with answers facilitates automatic grading of exams. This in turn leads to immediate feedback for students and reduced repetition for instructors. WebCT also has high potential for distance learning. Additional benefits of WebCT include course chat-rooms, message boards, and calendars. Conversion of traditional exam material for use on WebCT is relatively simple through programs such as Respondus. WebCT is particularly helpful for short, repetitive courses such as senior clinical rotations, electives, or self-taught modules. WebCT tracks student effort data, such as time spent on exams or performance on individual questions. This function lends itself to assessment efforts and the scholarship of teaching. This resource has been vital for our departmental goal of assessing pathology education at ISU.
75: WEDNESDAY SLIDE CONFERENCE ON THE WORLD WIDE WEB.
The Wednesday Slide Conference on the World Wide Web has been developed by the Armed Forces Institute of Pathology (AFIP), Department of Veterinary Pathology to allow maximum participation in this valuable educational forum by residents and veterinary pathologists around the world. Conferences are held on Wednesdays for 25 weeks each year, from September through May. Each week, four unknown cases of classic or recently documented diseases or entities are presented, encompassing modern concepts and the most recent literature references. Each final case manuscript has comments from the contributor, Department of Veterinary Pathology staff members, and AFIP pathologists and consultants. These comments are linked with images of gross and histopathologic lesions, special stains and immunohistochemical stains. In addition, journal references are linked to the appropriate PUBMED abstracts. Each conference focuses on morphologic and etiologic diagnoses, board quality descriptions, differential diagnosis, and classic gross and histologic lesions, with comparative pathology aspects among the common species affected.
76: CHARACTERIZATION OF EXPRESSION OF MMP 2, 3 AND 9 IN BREAST CANCER BRAIN METASTASIS.
In order to study the roles of MMP2, MMP3 and MMP9 in breast cancer brain metastasis, we used a rat model for distant brain metastasis developed in our lab. Evaluation of MMP2, MMP3 and MMP9 expression was carried out using semi-quantitative RT-PCR, Western blotting analysis, and immunohisto-chemistry. In addition, morphological assessment of the neoplastic process was also performed by histological evaluation. MMP2, 3 and 9 mRNA and protein are consistently up regulated in neoplastic brain tissue compared to control brain tissue. In situ zymography demonstrated that there are active forms of MMP2 and MMP9 in tumor foci in the brain. These results suggest that MMP2, 9 and MMP3 may be involved in the process of development of breast cancer brain metastasis that could be related to specific brain environmental characteristics. Additionally, these results show that our model can be used successfully as a model for in vivo study of MMPs in breast cancer brain metastasis.
77: MORPHOLOGY OF ANGIOINVASIVE T-CELL LYMPHOMA REPRODUCED IN RABBITS INOCULATED WITH HTLV-1 TRANSFORMED T-CELLS.
Pulmonary angioinvasive T-cell lymphoma can be a feature of HTLV-1 infection and adult T-cell leukemia and lymphoma (ATLL). Similar vascular manifestations occur in some non-Hodgkins lymphomas unassociated with virus. In these cases lesion occurrence is a poor prognostic sign and pathogenesis is not well established. To evaluate a model for studying malignant angioinvasive T-cell biology, 17 New Zealand White rabbits were injected with either RH/K34 HTLV-1-transformed rabbit T-cells, or cultured primary lymphocytes, and evaluated up to 4 days post inoculation for evidence of infection and disease. Transformed T-cells infiltrated pulmonary vascular walls and separated endothelium from the basement membrane zone. This change became more severe with time post inoculation and was accompanied by narrowed vessel lumens and perivascular cuffing by neoplastic T-cells, as well as multifocal consolidation of pulmonary parenchyma. Concomitant with increasing lesion severity post-inoculation, there was an increase in virus isolated from cultured spleen and thymus, as detected by HTLV-1 p19 gag ELISA. Additionally, HTLV-1 proviral load increased in lung, liver, kidney, spleen, thymus, heart, and skin by 4 days post inoculation. HTLV-1 was most concentrated in tissues with T-cell lymphoma-like infiltration including lung, liver and kidney. Controls lacked these changes. Rabbits given RH/K34 cells develop infection and lesions closely mimicking some ATLL patients. Morphogenesis duplicates the invasive biology of malignant T-cells trafficking into walls of blood vessels. Further analysis of the system is warranted to investigate the suitability for studying pathogenesis of malignant T-cell angioinvasive lesions.
78: REDUCED NUCLEAR TRANSLOCATION OF NFKB p65 IN CANINE DISTEMPER-INFECTED KERATINOCYTES IN VIVO AND IN VITRO.
The transition of keratinocyte proliferation to differentiation is tightly regulated and associated with a release of NFkB from its cytoplasmic inhibitor IkB. Subsequent translocation into the nucleus in suprabasal and subcorneal cells correlates with arrrest of proliferation. NFkB protein p65 was demonstrated in the nucleus of suprabasal and subcorneal keratinocytes in normal canine footpad epidermis. The localization of NFkB and its regulation in canine distemper virus (CDV)-associated keratinocyte proliferation was assessed in CDV-infected footpad epidermis and keratinocyte cultures using immunohistochemistry, immunofluorescence, and western blot analysis. In epidermis, which contained CDV nucleoprotein and mRNA, nuclear translocation of p65 was rarely observed. A similar lack of nuclear p65 translocation was demonstrated in keratinocytes of CDV-infected cultures compared to non-infected and mock-infected control cultures. Accordingly, nuclear extracts of CDV-infected keratinocyte cultures contained less p65 than control cultures. Higher levels of steady state IkBa were present in cell extracts of infected cultures. Intriguingly, CDV-infected cultures contained more phosphorylated IkBa compared to control cultures. IkB decreased markedly in control cultures 4 hours after cycloheximide treatment, reflecting IkB turnover. This decrease was far less pronounced in CDV-infected cultures. Treatment with okadaic acid, a strong NFkB activator, demonstrated that IkB degradation can be induced in CDV-infected cultures. These findings suggest that signals resulting in IkB phosphorylation occur in keratinocytes in CDV-infected cultures, but that degradation is not completed. Increased steady state concentrations of IkB could retain p65 in the cytoplasm resulting in decreased nuclear translocation. Reduced nuclear p65 activity may at least partly explain the increased proliferation of keratinocytes observed in CDV-infected canine footpad epidermis.
79: HUMAN T-LYMPHOTROPIC VIRUS TYPE 1 MITOCHONDRIAL LOCALIZING PROTEIN p13 SENSITIZES JURKAT T CELLS TO RAS-MEDIATED APOPTOSIS.
Human T Lymphotropic Virus-1 (HTLV-1), a deltaretrovirus similar to bovine leukemia virus (BLV), is the etiological agent of adult T-cell lymphoma/leukemia (ATL) and other lymphocyte-mediated inflammatory diseases. HTLV-1 is a complex retrovirus that encodes typical retroviral structural and enzymatic gene products, but also unique regulatory and accessory proteins. Using alternative splicing of its pX region, the virus encodes a series of viral regulatory and accessory proteins, including a singly-spliced open reading frame (ORF) II product p13. We have shown that selective mutations of infectious viral clones that ablate the expression of ORFII products (p30 and p13) resulted in reduced virus loads and host humoral responses in rabbits. p13 has also been shown to localize to mitochondria, and suppress cell growth and tumorgenicity in a mouse model, but its function in human lymphocytes remains undetermined. Herein, we analyzed functional properties of human lymphocytes expressing p13, using both transient expression plasmids, and lentiviral vectors stably expressing p13. Our data revealed that the p13-expressing Jurkat T cells were markedly sensitive to ceramide- and FasL-induced apoptosis in a dose-dependent manner, as measured by percentage of apoptotic cells by Annexin V staining assay. Furthermore, pre-incubation of the p13 expressing cell with farnesyl transferase inhibitor, a down-regulator of Ras, protected against cell death, providing evidence for the participation of Rasmediated signaling in the molecular alterations induced by p13 in human lymphocytes.
80: SCID/NOD MOUSE MODEL OF CANINE T-CELL LYMPHOMA: GENE EXPRESSION PROFILING AND INVIVO BIOLU-MINESCENT IMAGING.
Lymphoma is a malignant neoplasm arising from B or T cells. In dogs, one third are highly aggressive, multicentric T-cell lymphomas that are often associated with humoral hypercalcemia of malignancy (HHM). Parathyroid hormone-related protein (PTHrP) likely functions synergistically with other cytokines to induce hypercalcemia in canine T-cell lymphoma. There are no cell lines or animal models to investigate the pathogenesis of T-cell lymphoma in dogs. A xenograft model was established by injecting lymphoma cells isolated from a 4-year-old Irish wolf hound intraperitoneally in SCID/Nod mice. The mice developed multicentric lymphoma in the spleen, liver, thymus, and lymph nodes along with HHM and elevated plasma PTHrP as seen in the dog. Lymphoma cells from the mice were confirmed as canine T-cells by immunohistochemistry and canine-specific PCR for microsatellites. PTHrP mRNA expression was high in lymphomas compared to normal canine lymph nodes (NCLN) by quantitative real time RT-PCR. PTHrP protein was localized predominantly to the cytoplasm as detected by immunofluorescence. Cytokine gene expression profiles of NCLN and lymphoma xenografts were analyzed using cDNA arrays comprising 847 cytokine and cytokine receptors. We identified 27 genes that were up-regulated and 101 genes down-regulated two-fold or more in lymphoma compared to NCLN. Western blotting confirmed that cyclin A, PCNA, and P53 were deregulated in lymphoma. Finally, we monitored in vivo tumor progression and metastases in this lymphoma animal model using bioluminescerit imaging after transducing the lymphoma cells with a lentiviral vector expressing luciferase/yellow fluorescent protein reporter. Data from bioluminescent imaging revealed that the SCID/Nod mice exhibited trafficking patterns for lymphoma cells similar to that seen in dogs. This unique mouse model will be useful for investigating the pathogenesis of canine T-cell lymphoma.
81: AN INTRATIBIAL METASTASIS MODEL OF HUMAN LUNG CANCER: THE ROLE OF THE OSTEOCLAST, AND THE USE OF SERIAL IN VIVO BIOLUMINESCENT IMAGING IN DETERMINING RESPONSE TO THERAPY.
Certain tumor types have a propensity to metastasize to bone. The pathogenesis of bone metastases often involves, and may require, the activation of osteoclasts by tumor secreted factors, which promote important interactions with the bone microenvironment. Current methods of assessment of effective therapies to combat bone metastases are labor intensive and result in the euthanasia of large numbers of animals. We utilized an intratibial injection model of bone metastasis with serial bioluminescent imaging to monitor the effect of osteoclast inhibition on the interaction of the neoplastic cells with bone, and on overall growth of the tumor. Mice were injected with luciferase-transduced tumor cells (HARA, human pulmonary squamous carcinoma), and divided into three groups: (1) untreated, (2) biweekly treatment with a bisphosphonate, or (3) a proprietary anti-osteoclast agent. Mice were euthanized one month after tumor cell injection. Histomorphometry and serial imaging were used to evaluate tumor burden, and parameters of bone resorption and osteoclast activity. Mice in the treated groups demonstrated increased bone density and decreased osteoclast activity. In the bisphosphonate-treated group, tumor growth over time was decreased, as assessed by bioluminescent imaging. The use of serial in vivo bioluminescent imaging to monitor tumor growth over time resulted in the ability to quantify tumor growth and response to therapy using a relatively small number of animals, and was less labor intensive than evaluation by histomorphometry. Bisphosphonates may be useful as inhibitors of bone metastases both by decreasing osteoclast activity and slowing tumor growth.
82: PATHOLOGY OF SARS-CoV INFECTION OF MICE.
A mouse model of SARS-CoV infection was developed in female C57BL/6J mice. A human clinical isolate of SARS-CoV (Urbani strain) was introduced intranasally in 5–6 week old mice under anesthesia. Infection was detected by a cytopathic effect assay in Vero cell cultures as TCID50. Infected mice grew at a rate more slowly than normal to 40 days after infection but none died. Histologically, by day 3 after infection, there was a mild focal bronchitis and bronchiolitis with single cell and focal necrosis and degenerative epithelial changes and vasculitis. By day 5, bronchiole lesions were mostly resolved and only a few inflammatory foci composed of lymphocytes and macrophages were seen. The virus replicated in the lung to the highest levels by day 3 post-infection and the mice cleared lung virus by day 9. Expression of viral RNA was localized to bronchial and bron-chiolar epithelium but not within blood vessel endothelium or infiltrating inflammatory cells by in situ hybridization. Infected RAG1-/- mice developed similar pulmonary lesions and also cleared the virus quickly. These data indicate that C57B1/6 mice allow replication of SARS-CoV that produced mild pulmonary lesions which quickly resolved.
83: EXPERIMENTAL PATHOLOGY OF WEST NILE VIRUS INFECTION.
A mouse model of West Nile Virus infection is used at the Wadsworth Center to study the pathogenesis of the disease in mammals. Subcutaneous (footpad) inoculation is used to simulate a mosquito bite and is 100% effective in infecting mice, as are intracerebral and intraperitoneal routes, but not intranasal. C3H mice are most susceptible with 92% morbidity/58% mortality, followed by BALB/c (83%/25%), C57B6 (33%/25%), and ICR (25%/25%). Clinical signs include ruffled fur, weight loss, hunched posture, abnormal reflexes, and gait abnormalities; paralysis and circling were observed rarely. Virus spreads from the inoculated footpad to blood, spleen, and lymph nodes in approximately 2 days; by day 3, it's present in brain, spinal cord, heart, lungs, kidney, thymus, and contralateral footpad; and finally, virus is found in muscle and pancreas around days 4–5. Acute lesions in 13 mice of all 4 strains that died or were euthanatized early (average survival time ∼10 days) included lymphoid necrosis (12/12), gastritis, enteritis or colitis (12/13), hepatic lipidosis (3/13), necrotizing encephalomyelitis (2/13), and focal meningitis (1/13). The chronic lesions found in 12 largely asymptomatic survivors at 33–34 days post infection were lymphoid depletion (11/12), encephalitis (7/12) or myelitis (4/12) with gliosis, malacia and mineralization, and perivascular lym-phoplasmacytic cuffs, as well as mild, focal lymphocytic meningitis (1/12). These studies indicate that the mouse is an excellent model of West Nile virus infection, and that lesions are widespread and occur in both CNS and extraneural tissues.
84: MORPHOLOGIC AND HOST GENE EXPRESSION ANALYSIS OF EXPERIMENTALLY MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS INFECTED PEYER'S PATCHES IN LIGATED BOVINE ILEAL LOOPS.
Paratuberculosis is a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAPTB). During incubation periods that may last for years, extensive granulomatous inflammation occurs in the intestinal mucosa. The role of host genes in the establishment and regulation of MAPTB infection is complicated and marginally characterized. We evaluated the temporal morphology and gene expression profiles of bovine ligated ileal tissues infected with MAPTB. The bovine ligated ileal loop model was used to identify the host response during the early period of infection. Ileal loops were inoculated with PBS or MAPTB. Ileal loops were excised at 30 min, 1 hr, 2 hr, 4 hr. 8 hr and 12 hr post infection. Invasion and infection was characterized by electron microscopy. We compared the gene expression profiles of ileal tissues from MAPTB infected and PBS control using human cDNA microarrays, and confirmed selected microarray results with RT-PCR. Electron microscopy revealed the establishment of infection in bovine Peyer's patch as early as 1 hr post infection while light microscopy documented early inflammation of the mucosa. cDNA microarray analysis detected over 300 genes (18% of total) differentially regulated at a cut off value of 2 fold. At 95% confidence level, of the differentially regulated genes, 50% genes were up-regulated and 50% genes were down-regulated. Data mining disclosed the differentially regulated genes to function in various cellular processes including intracellular signaling, cytoskeletal rearrangement, oxidative stress, transcriptional regulation, cell surface receptors, cell-mediated immunity as well as a variety of cellular metabolic pathways, and may play a key role in establishment of infection. Heterologous cDNA microarrays successfully displayed a global profile of gene expression in control and MAPTB infected bovine Peyer's patches. The differentially regulated genes are predicted to be important in understanding the early interaction of MAPTB with Peyer's patches in intestine in bovine Johnes disease. Further time-course analysis of the differentially regulated genes will allow us to explore complex interactions between multiple genes that orchestrate the survival of pathogen within the host.
85: LESION QUANTITATION BY MAGNETIC RESONANCE IMAGING AND STEREOLOGY IN A GUINEA PIG MODEL OF CHILDHOOD TUBERCULOSIS.
Aerosol infection of the guinea pig with Mycobacterium tuberculosis (Mtb) is an important animal model of human tuberculosis. This model is used routinely to evaluate new anti-Mtb drugs and vaccine candidates however improved methods to evaluate efficacy is needed. Magnetic resonance imaging (MRI) and stereology were used to determine whole lung lesion burden following low dose aerosol infection of guinea pigs with the H37Rv strain of Mtb. MRI images were generated from lungs perfusion fixed with formalin, followed by serial sectioning for stereological analysis and routine histopathology. Infection resulted in progressive granulomatous inflammation and necrosis of the mediastinal lymph nodes and the pulmonary parenchyma that were visible on MRI images. Lung lesions were uniformly distributed throughout all lung lobes based on gross necropsy and MRI. Immunization of guinea pigs with BCG, six weeks prior to inoculation substantially reduced the lung and lymph node lesion burden and slowed lesion progression. Lymph nodes in non-immunized animals had extensive granulomatous lesions with extensive caseous necrosis. Lymph node inflammation in BCG immunized animals was reduced and subsequently replaced by fibrous connective tissue while lesions from non-immunized animals continued to progress. The rapid and progressive development of lesions of the mediastinal lymph nodes in advance of uniformly distributed lesions of the lungs is similar to pattern in Mtb infection in young children. In addition BCG immunization of guinea pigs conferred partial protection similar to children by reducing the lesion burden and slowing the progression of pulmonary and extrapulomary lesions. Quantitation of lesion burden by MRI on formalin fixed lung may prove to be an excellent method to evaluate drug and vaccine efficacy in experimental tuberculosis.
86: EFFECT OF LOW DOSES DEXAMETHASONE ON THYMUS MORPHOLOGY AND IMMUNE RESPONSES IN CALVES.
Glucocorticoids are illegally used in association with anabolic steroids as growth promoters in veal calves. An experimental administration of dexamethasone was carried out in order to assess the role of low doses of exogenous glucocorticoids on thymus atrophy induction and on the immune response. Three groups of five veal calves each were included in this study: group D was administered 0.4 mg/day of dexamethasone-21-phosphate per os for 25 days; group V was administered 2 mg of dexamethasone-21-isonicotinate im at days 14 and 21, and group K served as control. At slaughter the weight of the thymus was severely reduced in group D and in group V, compared to control animals. Lesions included severe lymphoid depletion and hyperplasia of adipose tissue. In situ evaluation of apoptosis in thymus, showed a reduction of the percentage of positive nuclear areas of animals belonging to group V in comparison with control animals. An overall decrease of lymphocyte proliferative response was detected after treatment with short acting dexamethasone, while humoral immune response wasn't affected by treatments. The results from this study suggest that the potential of corticosteroids to reduce the immunological responsiveness in beef cattle should be considered. Moreover, thymus weight and histology may be good indicators of illegal corticosteroids administration in veal calves. The improvement of control strategies to prevent the illegal use of growth promoters should also include the application of screening methods based on the measurement of body parameters and on histological investigations.
87: EARLY AND PERSISTENT LOSS OF MUCOSAL CD8+ T-CELLS IN FIV INFECTION.
We examined the yield and phenotype of cells from the mesenteric (MLN) and medial iliac (ILN) lymph nodes and intestinal intraepithelial lymphocytes (IEL) of cats infected with FIV-NCSU1 for greater than one year (chronic infection), cats infected for one day (acute), and uninfected controls. Cellularity was markedly decreased in the MLN and IEL and markedly increased in ILN of chronically infected cats. There was an overall loss in IEL, but not in LN, of CD5+ cells and a shift in the phenotype of CD5+ cells characterized by a progressive loss of CD5 + bright and increase in CD5+ dim cells. The CD5+ phenotype shift was observed as early as one day post FIV-exposure. A significant loss of CD8+ T cells was detected in IEL from both acute and chronically FIV-infected cats and this loss was comprised of both CD8+ and CD8+ T-cells. At one day post virus-infection, a decreased percentage of CD8+ T-cells (MLN and ILN) and CD8-+ T-cells (ILN) was seen in the lymph nodes, however this was not detected in lymph nodes from chronically infected cats suggesting a transient change. The loss of cellularity and decreased percentage of CD8+ T-cells indicates an absolute loss of IEL CD8+ T-cells that occurs as early as one day post FIV exposure and is persistent. As the majority of CD8+ T-cells in the mucosa are activated effector or memory cells, this represents a profound loss of the mucosal immune effector population.
NIH/NIAID RO1 AI46220, KO2 AI50432, Fort Dodge Animal Health Fellowship in Immunology.
88: ESTROGEN-DEPENDENT INDUCTION OF CYCLOOXYGEN-ASE-2 IN THE CANINE PROSTATE IN VIVO.
Cyclooxygenase-2 (COX-2) is involved in several physiological and pathological processes such as ovulation, parturition, inflammation and cancer. COX-2 expression can be induced by various stimuli including gonadotropins, cytokines, growth factors and tumor promoters. However, the effect of steroid hormones on COX-2 expression in the prostate has never been documented. In the present study, COX-2 expression was evaluated by immunohistochemistry in intact and castrated dogs treated with exogenous androgen or estrogen, as well as in control animals not receiving hormonal treatment. Results showed that prostatic tissues of untreated intact animals had a well-developed secretory epithelium, but none expressed COX-2. Castration led to the disappearance of the secretory epithelium, with only the basal cell layer remaining, whereas treatment of castrated dogs with the androgen 5alpha-androstane-3alpha, 17beta-diol (3, 17beta-diol) restored the secretory epithelium. No COX-2 staining was observed in untreated castrated animals, and in 3, 17beta-diol treated castrated dogs. In contrast, a clear pattern of COX-2 induction was observed in prostatic tissues of intact and castrated dogs treated with the estrogen 17beta-estradiol 17-cyclo-pentyl proprionate (ECP). Treatment of castrated dogs with estrogen resulted in the metaplasia of basal cells into a squamous epithelium, and intense COX-2 expression was observed in both the squamous epithelial cells, and in the basal cells of acini without metaplasia. Similar results were observed in prostates of intact ECP-treated dogs. Thus, this report demonstrates for the first time the induction of COX-2 by estrogen in prostatic tissues in vivo.
89: REGULATION OF SP-A AND SP-D mRNA EXPRESSION BY RETINOIC ACID IN HUMAN ALVEOLAR TYPE II CELLS.
The pathogenesis of a large number of infectious, inflammatory, and toxin-mediated lung diseases is tightly bound to deficiency and dysfunction of the surfactant component expression. Surfactant proteins A (SP-A) and D (SP-D), antimicrobial components of the pulmonary surfactant, are constitutively expressed by the alveolar type II and Clara cells in the normal lung. Since the expression of surfactant proteins is developmentally regulated, prematurely born infants are at higher risk of developing lung infections. A potential therapeutic agent all-trans-retinoic acid (RA) has been shown to enhance cell differentiation and have an effect on surfactant protein gene regulation. In H441 human pulmonary adenocarcinoma cells, RA markedly increases surfactant protein B (SP-B) mRNA, but does not have an effect on SP-A mRNA levels. Recently, it has been found that two genes encode SP-A in primates, namely SP-A1 and SP-A2. To assess the effect of RA on SP-A1/A2 and SP-D mRNA expression, H441 cells were incubated for 48h in a serum-free medium that contained no additions (negative control) or 500 nM RA. This was followed by cell collection, RNA isolation, cDNA synthesis, and fluorogenic real-time RT-PCR. The results revealed increased expression of SP-D mRNA and no significant alteration in SP-A1/A2 mRNA expression after the treatment compared to the negative control. These findings suggest that besides having different secretory pathways, SP-A and SP-D might also have different transcription regulation pathways. In addition, RA and its non-toxic derivatives such as all-trans-retinoyl -glucuronide should be further explored as the treatment of choice for enhanced lung development in prematurely born infants.
90: THE ROLE OF APOPTOSIS DURING THE RECOVERY PROCESS OF THE GLOMERULAR STRUCTURE IN RAT THY-1 NEPHRITIS.
Apoptosis might play an important role in the glomerulonephritis. The purpose of this study was to investigate whether the number of apoptotic cells and the levels of gene expression of apoptosis-relating proteins, Bax and Bcl-2, alter in the renal glomeruli during the course of rat Thy-1 nephritis. We dissected kidneys at 2, 3, 5, 7, 14, 28 and 56 days post-injection of rabbit anti-Thy-1 antibody. Severe mesangiolysis and influx of monocytes into the glomeruli were seen at day 2. The total cell number and PCNA-positive cells in the glomeruli significantly increased and peaked at day 7. Thereafter glomerular cells decreased and the glomerular structure returned to normal by day 56. Both Bcl-2 and Bax were localized in the mesangial area of glomeruli by immunostaining, moreover bcl-2 and bax mRNA was localized in the same area as their proteins by in situ hybridization at day 7. TUNEL-positive cells appeared at day 7 and their number increased at day 14. For quantitative analysis of the gene expression of bax and bcl-2, 200 glomeruli were dissected from the frozen sections of each kidney with a laser microdissection system. The level of gene expression of bax in the glomeruli was highest at day 14, whereas that of bcl-2 did not significantly change during the course of the nephritis. These results suggest that apoptosis might have an important role in excluding glomerular cells excessively proliferated in the glomeruli during the course of rat Thy-1 nephritis. Also, regulated expression of bax in the glomeruli might contribute to the reconstruction of the damaged glomeruli in Thy-1 nephritis.
91: 14-3-3 PROTEIN IN THE CNS OF SIV-INFECTED MACAQUES.
The presence of 14-3-3 protein in cerebrospinal fluid (CSF) has been reported to be a marker of neuronal damage in several CNS diseases including Creutzfeldt Jakob disease (CJD) and multiple sclerosis (MS). Our studies using the simian immunodeficiency virus (SIV)/macaque model of HIV CNS disease have demonstrated that 14-3-3 protein is present in the CSF of a subset of SIV-infected macaques during asymptomatic infection where it persists until terminal disease in animals that develop high CSF and CNS viral burdens, thus serving as an early predictive marker of the level of CNS viral replication. Furthermore, the zeta isoform of 14-3-3 appears to predominate in CSF as a marker of neuronal damage induced by SIV. In addition to serving as a CSF marker of neuronal damage, 14-3-3 proteins likely play key regulatory roles in neurodegenerative diseases by modulating neuronal and/or glial apoptosis. For example, by binding to phosphorylated Bcl-2-associated death protein (BAD), 14-3-3 protein can sequester BAD thus preventing BAD-triggered pro-apoptotic pathways. To test the hypothesis that 14-3-3 protein serves this neuroprotective role in the SIV model, 14-3-3-BAD interactions were evaluated by co-immunoprecipitating 14-3-3 and BAD from brain homogenates. The results suggest that 14-3-3 and BAD are dissociated in animals with SIV encephalitis, thereby promoting apoptosis.
92: THE EFFECTS OF NONCYTOPATHIC TYPE 2 BOVINE VIRAL DIARRHEA VIRUS ON BONE MARROW STEM CELL PROLIFERATION.
Bovine viral diarrhea virus (BVDV) causes significant economic loss in the North American cattle industry. Severe acute BVD, caused by highly virulent noncytopathic type 2 BVDV (ncpBVDV-2), results in unusually high morbidity and mortality. Clinical signs include pyrexia, anorexia, diarrhea, hemorrhage, severe leukopenia and thrombocytopenia. The purpose of this study was to investigate the effects of high and low virulent ncpBVDV-2 isolates on bone marrow stem cell proliferation and correlate the proliferative capacity with the presence of BVDV in bone marrow. We hypothesized that the inability of bone marrow stem cells to proliferate during high virulent ncpBVDV-2 infection contributes to prolonged neutropenia. Bone marrow samples were collected from 6–7 month old BVDV-naïve Holstein steers. Bone marrow mononuclear cells (BMMCs) were isolated and cultured for 5 days and the mean number of colonies (20 or more cells/colony) before and after virus inoculation was determined. Bone marrow proliferation was also evaluated by 3H-thymidine incorporation in BMMCs. The presence of BVDV in BMMCs and peripheral blood buffy coat cells was demonstrated by virus isolation. The preliminary results appear to support the hypothesis. BMMCs from calves inoculated with low virulent ncpBVDV-2 proliferated earlier and to a greater degree post-inoculation compared to calves inoculated with high virulent ncpBVDV-2. A surprising result was that BVDV was isolated from BMMCs in both groups as early as day 2–3 post inoculation. Overall, bone marrow proliferation was delayed in calves inoculated with the high virulent ncpBVDV-2 and bone marrow cells appeared to proliferate in a periodicity of approximately 5–7 days.
93: EXPERIMENTAL INFECTION WITH CHEOLWON/INCHEON ISOLATES OF CLASSICAL SWINE FEVER VIRUS IN PIG IN KOREA: CLINICAL SIGNS AND PATHOLOGY.
Several cases of classical swine fever (CSF) reoccurred in Cheolwon province and Incheon city in 2002. Two isolates (Cheolwon and Incheon) of classical swine fever viruses (CSFV) were successfully isolated from pigs in these areas and classified into genotype-2 with a phylogenetic analysis based on the sequence of 243 bp of 5 non-coding region and 190 bp of E2 envelope glycoprotein gene. We performed experimental infection in two groups of CSFV antibody free 50-day-week-old pigs with the Cheolwon isolate and the Incheon isolate of CSFV. Ten pigs were inoculated intramuscularly with each isolate of CSFV. Five or six additional pigs were housed in the same room but caged separately. Negative control pigs were kept in a completely isolated house. The infected pigs developed clinical signs such as pyrexia, anorexia, lethargy from 4–5 days post-inoculation (DPI) in both Cheolwon and Incheon groups, and diarrhea, staggering gait, and convulsion from 6–7 DPI in Cheolwon group and 9 DPI in Incheon group, respectively. Leukopenia could be detected from 3–5 DPI. All of the infected pigs were dead or euthanized 4–23 DPI. In contact pigs, clinical signs were observed from 10 DPI in Cheolwon group and 8 DPI in Incheon group. Leukopenia and pyrexia were detected from 9–14 DPI. Most of the contact pigs died from 13–18 DPI. Because of their moribund state, the rest of the pigs were euthanized within 23 DPI. Grossly rough hair coats and cyanosis were observed in most of the infected and contact pigs. Pathologic changes were frequently detected in lymph nodes and large intestine. The lymph nodes became swollen and peripherally hemorrhagic. Large intestine had severe diffuse necrosis or ulceration. Histopathologically nonsuppurative encephalitis, reticular cell hyperplasia in lymphoid tissue, and endothelial swelling of capillaries were observed in most pigs. The results suggested that CSFV isolates from Cheolwon and Incheon were highly virulent strains and caused acute forms of pathogenicity. We could not find any evidence of different pathogenicity between two strains of CSFV.
94: REDUCED EXPRESSION OF INTERFERON-ALPHA IS ASSOCIATED WITH ENHANCED PATHOGENICITY OF FIV INFECTION IN THE THYMUS.
Thymopoiesis requires a specific cytokine and physical microen-vironment for the appropriate generation of T cells, and this delicate system is influenced by direct infection with feline immunodeficiency virus (FIV) and the resulting influx of inflammatory cells. RNA and DNA were isolated from thymuses of control and neonatally infected, FIV-positive cats at 6–8 week, 12 week and 16–20 week time points. Real time PCR or RT-PCR was performed to measure FIV-gag DNA (proviral) load and mRNA levels for interleukin (IL)-7, IL-4, IL-15, interferon (IFN)-alpha, IFN-gamma, glyceraldehyde-3-phosphate dehydrogenase (G3PDH) and FIV-gag. Thymic IFN-alpha levels were significantly decreased in chronically infected animals and tended to decrease over the course of infection. Lower IFN-alpha levels correlated with higher thymocyte proviral loads, loss of double-positive (DP) thymocytes, increased numbers of intrathymic single-positive (SP) CD4+ and CD8+ cells and fewer peripheral CD4+ T cells. IFN-gamma levels were inversely correlated with IFN-alpha levels, and higher values were associated with increased proviral loads, greater viral transcription and increased loss of DP thymocytes. IL-7 levels were inversely correlated with those of IFN-alpha. IL-15 levels did not change significantly with infection, but did negatively correlate with the percentage of intrathymic SP CD8high cells. IL-4 levels in infected cats were similar to those in control cats, but were negatively correlated with viral load in PBMC and the percentage of double-negative thymocytes. These results show alterations in the transcription of thymic cytokines. Specifically, there is a reduction in thymic production of IFN-alpha in the course of FIV infection and this loss is associated with enhanced pathogenicity of FIV within the thymus. This decline in IFN-alpha may represent a loss of thymic lymphoid dendritic cells and may suggest an underlying loss of innate immune function that contributes to thymic pathogenesis.
95: PERIPHERAL NEUROPATHY IN SIV/HIV INFECTION.
Peripheral neuropathy is the most frequent neurological complication associated with HIV-1 infection, yet its pathogenesis remains undefined. Distal Symmetrical Polyneuropathy (DSP) is the most common form of peripheral neuropathy in HIV infection, affecting approximately 30–35% of HIV-infected individuals. We have developed an accelerated, consistent SIV/macaque model of HIV CNS disease with strong parallels to HIV infection in which SIV-infected macaques also develop moderate to marked multifocal ganglionitis characterized by focal and diffuse mononuclear infiltrates, neuronophagia and neuronal loss. To determine whether this SIV/macaque model paralleled HIV associated DSP, hematoxylin and eosin sections of dorsal root ganglia (DRG) and trigeminal ganglia (TG) from SIV-infected macaques euthanized during terminal infection were evaluated to identify infiltrating inflammatory cells and to assess the level of neurodegen-eration or neuronal loss. To further characterize the inflammatory cell population, tissue microarrays containing DRG and TG from SIV-infected and control uninfected macaques were immunostained for SIV gp41 protein (as a measure of viral burden), and a macrophage activation marker (CD68) followed by quantitative digital image analysis. Neuronal loss was measured by unbiased stereological methods (fractional area). There was a marked increase in the number of CD68-positive macrophages in perineuronal regions in the DRG and TG in SIV-infected animals compared to controls. Further there was marked loss of neurons associated with the presence of infiltrating/activated macrophages. Macrophages are hosts for virus replication and secrete neurotoxic viral gene products as well as pro-inflammatory cytokines and chemokines that promote neurodegeneration. These studies confirm that alterations in the PNS develop in SIV-infected macaques that closely parallel those observed in HIV-infected individuals.
96: VEGF INDUCES AIRWAY CELL PROLIFERATION IN PRETERM LAMBS.
Premature birth is one of the leading causes of pulmonary diseases in human neonates. Immature respiratory epithelia diminish the innate immune function and predispose the lung to many infectious agents such as respiratory syncytial virus (RSV). Current therapies include enhancement of lung maturation with corticosteroids (e.g. betamethasone); however this is not optimal. Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen that induces epithelial cell proliferation and differentiation and therefore has therapeutic potential in prematurely born infants. The purpose of this study was to evaluate the effect of VEGF on proliferation of respiratory epithelial cells and to compare it to the effect of betamethasone. Caesarean section derived lambs (139 ± 1 day of gestation) were divided into 3 groups. The first group (n = 5) received 20 ml (0.005mg/ml) of VEGF intra tracheally (IT). The second group (n = 4) received intra muscular (IM) betamethasone injections (0.5mg/kg). The third group (n = 5, control) received 20 ml of saline IT. After the inoculations (24 hr) lambs were euthanized and respiratory epithelial cell proliferation was assessed by Ki-67 immunohisto-chemistry. The VEGF group had significantly more bronchioles with Ki-67 staining than bethamethasone (p < 0.01) and saline (p < 0.01) groups and also significantly increased numbers of Ki-67 stained bronchiolar epithelial cells compared to the bethamethasone (p < 0.01) and saline (p < 0.03) groups. VEGF induced bronchiolar cell proliferation and may be useful as atherapeutic agent in premature infants for respiratory epithelial repair, growth and innate immunity.
97: TOLL-LIKE RECEPTOR GENE EXPRESSION IN WOUNDED PORCINE SKIN.
Large animal models of human skin innate immunity are lacking. The objective of this study was to assess mRNA expression for the innate pathogen recognition receptors, toll-like receptors (TLR) 2, 4, 6, 9 and CD14, in normal and wounded porcine skin. Full-thickness surgical skin wounds were created in the ventral abdomen of out bred, female Yorkshire-Landrace cross pigs, age 6–8 weeks (n = 5). Forty-eight hours after wounding, total cellular RNA was extracted from samples of the wound margin and normal adjacent skin utilizing RNeasy Mini Kits. Porcine gene specific primer-pair sequences were based on published sequences and designed with the Primer-3 and Mfold software programs. Utilizing the Platinum Quantitative RT-PCR Thermoscript One-Step System, standard RT-PCR confirmed constitutive expression of porcine TLR2, 4, 6, and 9 as well as CD14 mRNA in normal skin from all five pigs. Quantitative RT-PCR was performed on cDNA using SYBR Green Supermix on an i-Cycler. When wounded skin was compared to normal control skin, there was a relative fold increase in mRNA expression for TLR2 (1.52, ±0.45; mean, ±standard error, p = 0.3284), TLR4 (1.99, ±0.82, p = 0.2506), and CD14 (2.02, ±0.29, p = 0.0136). In contrast, mRNA expression in wounded skin appeared to decrease for TLR6 (0.78, ±0.20, p = 0.2949) and TLR9 (0.48, ±0.09, p = 0.0029). Prominent increases in IL-8 mRNA expression in wounded skin confirmed the delivery of a proinflammatory stimulus to extracted samples. At the whole organ level, porcine TLR and CD14 mRNA expression are differentially altered in wounded skin compared to control skin. This is the first report of TLR expression in porcine skin.
98: DELETION OF THE CRMA GENE FROM COWPOX VIRUS CAUSES VIRUS ATTENUATION IN C57BL/6 MICE.
Cowpox virus (CPV) encodes the serine protease inhibitor, CrmA, which inhibits interleukin-1 (IL-1) converting enzyme (ICE, caspase-1) and granzyme B in vitro. CrmA also prevents inflammation during CPV infection of chicken chorioallantoic membranes. The role CrmA plays during respiratory infection of mice has been debated. CPV lacking the CrmA gene (CPVCrmA) appears attenuated compared to CPV infection after intranasal inoculation of Balb/c mice. However, disease caused by intranasal inoculation of vaccinia virus (VV) lacking the CrmA homolog (B13R) is indistinguishable from VV infection. We show that intratracheal inoculation of C57BL/6 mice with 104 pfu of virus caused severe respiratory distress, weight loss and hypothermia which necessitated euthanasia in 100% of mice infected with CPV but only 50% of mice infected with CPVCrmA. Viral pneumonia with vascular necrosis and bronchiolar epithelialcell hyperplasia was observed in all mice euthanized due to respiratory disease. Disease signs were first observed on day 5 post inoculation (pi) in mice infected with CPV and day 6 pi in mice infected with CPVCrmA. The amount of virus recovered from the lung and brain was similar for both CPV and CPVCrmA from day 1 through day 7 pi. No significant difference was seen in bronchoalveolar lavage (BAL) fluid cytology or virus titer. Virus could not be recovered from serum of any mice. Serum cytokine levels were similar during infection with either virus with the exception of IL-10, which was increased on day 7 pi in mice given CPV. The differences between intratracheal infection with CPV and CPVCrmA are subtle but significant. Half of the mice infected with 104 pfu of CPVCrmA survive infection even though virus replication in the lungs and brain is as effective as CPV. Examination of cytokine levels in BAL fluid and cytotoxic lymphocyte function during virus infection is currently ongoing.
99: THE VAGUS NERVE IS ONE ROUTE OF TRANSNEURAL INVASION FOR INTRANASALLY INOCULATED INFLUENZA A VIRUS IN MICE.
Intranasally inoculated neurotropic influenza viruses in mice infect not only the respiratory tract but also the central nervous system (CNS), mainly the brain stem. Previous studies suggested that the route of invasion of virus into the CNS was via the peripheral nervous system, especially the vagus nerve. In order to evaluate the transvagal transmission of the virus, we intranasally inoculated unilaterally vagectomized mice with a virulent influenza virus (strain 24a5b), and examined the distribution of the viral protein and genome by immunohistochemistry and in situ hybridization over time. All the experiments were conducted according to A Guideline for Animal Experiments at Graduate School of Veterinary Medicine, Hokkaido University. An asymmetrical distribution of viral antigens was observed between vagal (nodose) ganglia: viral antigen was detected in the vagal ganglion of the vagectomized side two days later than in the vagal ganglion of the intact side. The virus was apparently transported from the respiratory mucosa to the CNS directly and decussately via the vagus nerve and centrifugally to the vagal ganglion of the vagectomized side. The present results, thus, demonstrate that neurotropic influenza virus travels to the CNS mainly via the vagus nerve.
100: IN VIVO LOCALIZATION, PENETRATION, AND EGFR SATURATION BY PANITUMUMAB (ABX-EGF) IN AN A431 XENOGRAFT MODEL SYSTEM.
Panitumumab, a fully human antibody, binds to the epidermal growth factor receptor (EGFR) with high affinity (5 × 10−11 M), preventing ligand-induced activation and resulting in apoptosis and arrest of tumor cell proliferation in some preclinical models. Human A431 epidermoid carcinoma cells were injected subcutaneously into the left flank of female CD1 nude mice and subsequent tumors were allowed to grow to a size of 300 cubic mm. Mice were then treated twice per week with intraperitoneal injections of 0, 20, 200, or 500 μg/mouse of panitumumab or 500 μg/mouse of isogenic IgG2. Panitumumab inhibited EGFR activation in a dose dependent manner in A431 xenograft tumors as measured by autophosphorylation of the receptor and downstream signaling activity. Immunohistochemical detection of panitumumab in A431 tumors using both an antibody directed against the Fc portion of human IgG and an antiidiotype antibody confirmed that administered panitumumab localized directly to the xenograft tumor. Staining was evident at 24 hours surrounding afferent blood vessels and demonstrated a dose-dependent tumor penetration. Double labeling for EGFR and panitumumab demonstrated a progressive replacement of EGFR staining with panitumumab tissue staining. Receptor saturation was further investigated at the single cell level using a 2-color flow cytometric-based ratiometric competition assay on cells obtained from concurrent frozen A431 tumors. Panitumumab at the 500 μg/mouse dose saturated <20% of the total EGFR at 24 hours, 22.5% at 96 hours, and 78% at 7 days. These experiments demonstrate that tumor penetration of an antibody therapeutic can be measured in preclinical models.
101: HUMAN T-LYMPHOTROPIC VIRUS TYPE-1 p30II INTERACTS WITH p300 AND MODULATES VIRAL GENE EXPRESSION.
Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes adult T-cell leukemia/lymphoma, and is implicated in a variety of lymphocyte-mediated inflammatory disorders. HTLV-1 provirus has regulatory and accessory genes in four pX open reading frames. HTLV-1 pX ORF-II encodes two proteins, p13II and p30II, which are incompletely defined in virus replication or pathogenesis. We have demonstrated that pX ORF-II mutations diminish the ability of the virus to maintain high viral loads in vivo and that p30II, a nuclear-localizing protein interacts with CREB-binding protein (CBP)/p300, disrupts CREB-Tax-CBP/p300 complexes bound to the viral Tax-Responsive Element repeats (TRE) and differentially modulates CREB and TRE-mediated transcription. Herein, we further characterized the role of p30II in regulation of viral gene expression, by identifying its motifs critical in binding p300 and regulating TRE-mediated transcription. Additionally, we demonstrate that p30II-mediated LTR repression can be rescued by p300 and that lysine 106 (K3) of p30II is critical in LTR repression. We have further characterized the interaction between p30H and p300 and demonstrate that p300 histone acetyl transferase (HAT) mutants do not rescue p30II-mediated LTR repression, unlike wildtype p300. In addition, we also show that p30II is acetylated and that deacetylation by HDAC-1 enhances while inhibition of deacetylation by trichostatin A decreases p30II-mediated HTLV-1 LTR repression. We are the first to demonstrate that HAT activity of p300 is crucial in modulating viral gene expression from the LTR by p30II. Collectively, our data indicates that HTLV-1, a complex retrovirus associated with lymphoproliferative disorders, uses accessory genes to promote cell-to-cell transmission of the virus, clonal expansion of infected cells and maintenance of proviral loads in vivo.
102: DENDRITIC CELL TOLERANCE DURING MALARIA INFECTION REGULATES THE PRODUCTION OF PROINFLAMMATORY CYTOKINES AND T-CELL RESPONSES.
An effective immune response to any infection is characterized by controlling pathogen growth while minimizing inflammation-associated pathology. Malaria infection presents a particularly narrow margin of error in this balance. Strong cellular immune responses can lead to immunopathology from excessive inflammation. However, less robust responses lead to poor control of parasite growth resulting in high parasitemia and anemia. Ideally, the host will activate a strong inflammatory response early to reduce parasite growth, then down regulate these pathways once adaptive responses have been established. We hypothesized that dendritic cells (DCs) control the tempo of pro- and anti-inflammatory cytokine production through their ability to influence T cell activation and cytokine production. To test this hypothesis we isolated DCs from different stages of infection then cultured these cells with naive CD4 T-cells. In an extensive series of experiments, we found that; while DCs from 0, 3 and 15 days post infection are equally efficient at activating naive T-cells, the cytokines these T-cells produce differ profoundly as a function of when the DCs were isolated. Naïve T-cells stimulated by DCs isolated early in infection (3 days post infection) produce high levels of IFNg, whereas those activated from DCs isolated late in infection (15 days post infection) produce high levels of IL-10 with little accompanying IFNg. These observations lead us to question whether DCs become tolerant to microbial stimulation as infection progresses, a phenomenon similar to endotoxin tolerance. Using LPS, CpGs or zymozan to directly stimulate DCs isolated from 0, 3, or 15 days post infection, we found that DCs late in infection become progressively less able to produce IL-12 in response to subsequent microbial stimuli. We hypothesize that this observed DC tolerance is an important mechanism in controlling the balance between pathogen mediated disease and inflammation-associated pathology.
103: PATHOGENESIS OF NEWCASTLE DISEASE IN TURKEYS EXPERIMENTALLY INFECTED WITH DIFFERENT ISOLATES.
Newcastle disease is a limiting disease of poultry worldwide and the cause of significant economic impact when outbreaks occur. The etiology, Newcastle disease virus (NDV), infects a wide range of avian species and has been well studied in chickens. However, the pathogenesis of NDV in turkeys is poorly understood. Control of an outbreak in a turkey-producing area will require more thorough appreciation of the action of the virus in this species. In this study, groups of four week old commercial turkeys were inoculated intraconjunctivally with 5 viral isolates (LaSota, Roakin, Turkey North Dakota, Iowa 1519 and CA 1083) representing all pathotypes of NDV. Clinical signs were monitored daily and 2 birds from each group were euthanatized, with collection of tissues into formalin on days 2, 5, 10 and 14 postinoculation. The tissues were examined by histopathology, immunohistochemistry for the presence of NDV nucleoprotein, and in situ hybridization for the presence of viral mRNA. No abnormal clinical signs were noted in birds infected with lentogenic strains; varying degrees of depression and nervous signs were observed in some birds of the mesogenic and velogenic groups.
104: ANTHRAX TOXIN RECEPTOR EXPRESSION AND CELLULAR TRAFFICKING PATTERNS OF BACILLUS ANTHRACIS.
Background: Mononuclear phagocytes are key players in the germination of B. anthracis spores and are targets of anthrax toxins. Two anthrax toxin receptors (ATRs) have been identified to date, tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2). Both ATRs have multiple splice variants and are type 1 transmembrane proteins with an extracellular domain that is highly related to von Willebrand factor type A and integrin inserted domains (VWA/I domains). The function of TEM8 or CMG2 has not been identified, however there are at least two potential phosphorylation sites located in the cytoplasmic portion of TEM8, which strongly suggests that TEM8 can transduce signals from the cell surface to the cytoplasm. Although expression data exist for these receptors in multiple tissue types, expression patterns in mononuclear phagocytes have not been evaluated. Methods: RT-PCR was used to evaluate TEM-8 expression in cell lines, primary mononuclear phagocytes, and tissues. Tissue samples, primary peritoneal and alveolar macrophages were collected from C57/BL6 mice. Human monocyte derived macrophages (MDM) were derived from PBMC samples. Results: TEM8 mRNA was detected in the HeLa, J774A.1, RAW 264.7, and THP-1 cell lines. TEM8 mRNA could also be found in mouse tissues. However, TEM8 mRNA could not be detected in primary human MDM, human PBMC, mouse alveolar macrophages, or mouse peritoneal macrophages. Furthermore, PMA-induced differentiation of THP-1 cells altered TEM8 mRNA expression. Conclusion: These data suggest that additional receptors could serve as an anthrax toxin receptor for primary mononuclear phagocytes. However, it remains to be determined if TEM8 expression is up-regulated or down-regulated under certain circumstances as is the case for THP-1 cells.
105: STUDIES FOR THE PATHOGENESIS AND PATHOGENICITY FOR THE PORCINE CIRCOVIRUS TYPE 2 KOREAN ISOLATES IN WEANED PIGS.
Post-weaning multisystemic wasting syndrome (PMWS) is a condition of piglets characterized by weight loss and dyspnea combined with pathological findings of interstitial pneumonia and generalized enlarged lymph nodes. Typical histological lesions include multifocal granulomatous pneumonia, and lymphocyte depletion and multinucleated giant cell formation in lymph nodes. A number of studies have indicated that infection with PCV2 alone is not sufficient to induce clinical PMWS. Several inoculation studies using PCV2 alone have resulted in asymptomatic infection with mild to moderate histopathologic lesions. In contrast, dual infection with PCV2 and PPV or PRRSV potentiates the replication and distribution of PCV2 and induces clinical disease in addition to more severe hitopathological lesions. The objective of the present study was to investigate the significance of immunostimulator/or immunosuppressor, and dual/or triple infection(PPV, PRRSV) on the development of PMWS in 3-week-old conventional piglets infected with PCV2 immediately after weaning. The virus inoculum was prepared from Korean isolates of PCV2, PRRSV and PPV. Thirty-six, 3-week-old PRRSV- and PCV2-seronegative piglets were randomly divided into six groups, comprising 6 piglets each. Group A: Negative control, Group B: PCV2 alone, Group C: PCV2+Prednisolone, Group D: PCV2+Ultra corn, Group E: PCV2+PRRSV, Group F: PCV2+PPV+PRRSV, respectively. Clinical signs were recorded daily. The piglets were euthanized on PIDs 21 and 42 for each group. Tissue samples were collected at necropsy for PCR, histological examination and immunohistochemistry, respectively. Pigs of dual/or triple infected groups had clinical signs such as growth retardation, rough hair-coat, cough and diarrhea. Group E and F were observed to have lungs that faiedl to collapse, were consolidated and rubbery, and generalized enlarged lymph nodes. Histologic lisions were interstitial pneumonia, lymphohistiocytic cuffing and peribronchiolar fibroplasia of lung, and lymphoid depletion of lymphoid organs and specific PCV2 IC/IB. These lesions were characteristic of pigs inoculated with PCV2/PPV/PRRSV.
106: HORMONE CHANGES ASSOCIATED WITH EXPERIMENTALLY INDUCED MARE REPRODUCTIVE LOSS SYNDROME (MRLS).
During late gestation in mares, uterine quiescence is maintained by metabolites of pregnenolone, collectively known as progestagens. Maternal serum progestagen concentration decreased significantly in two experiments, in which mares were induced to abort by administration of Eastern Tent Caterpillars (ETC). These included 50 g/d of ETC given by stomach tube to 6 late term pregnant mares in 2002 and 100 g/d of non-irradiated and 100 g/d of irradiated ETC given by stomach tube for 10 days to two groups, each of 6 mares, in 2003. Both experiments had control groups of 5 and 6 mares respectively. Daily serum progestagen concentrations were measured from day zero (pre-experiment) to the day of abortion. In the ETC treated mares that aborted, (6/6 mares in 2002 and 9/12 in 2003) serum concentration level dropped to < 7ng/ml on the day of abortion. All control mares (none aborted) in both experiments maintained progestagen serum concentration of > 25 ng/ml throughout the experiment. Since placenta is the source of progestagen during the second half of pregnancy in the mare, the findings indicate that ETC may induce abortion in mares by releasing one or more factors that are specifically toxic to placenta. Further work is ongoing to identify this factor(s) in ETC.
107: CHARACTERIZATION OF ADAMTS4 KNOCK OUT MICE.
The purpose of this work was to evaluate mice lacking the enzymatic activity of ADAMTS4 that may be involved in the progression of osteoarthritis. Exon 4 of ADAMTS4, which contains the catalytic domain of the enzyme Aggrecanase 1, was deleted in 129SvEv-Brd mice. At 14–18 weeks and 1 year of age mice were weighed and blood was collected for hematology and serum chemistry evaluations. Mice were then necropsied and body and organ weights were collected. A complete set of tissues was fixed and examined microscopically. ADAMTS4 knockout (KO) mice demonstrated no significant differences in gross findings, body and organ weights, serum chemistry or hematology values, or in microscopic findings at either age compared to age-matched control mice. Although expression of the ADAMTS4 gene has been found in many tissues throughout the body, deletion of enzymatic activity did not appear to have any effect on normal growth and physiology.
108: ROLE OF HUMAN T-LYMPHOTROPIC VIRUS 1 OPEN READING FRAME II-ENCODED P30II IN MODULATION OF LYMPHOCYTE APOPTOSIS.
Human T-lymphotropic Virus Type 1 (HTLV-1) is a member of the deltaretrovirus group, which also includes Bovine Leukemia Virus and Simian T-cell Leukemia Virus. It is the causative agent of adult T-cell leukemia/lymphoma. HTLV-1 p30II is encoded in open reading frame II of the viral pX region. Work from our laboratory has demonstrated that p30II is required for viral infectivity and maintenance in vivo. Gene array analysis indicates a role for p30II in modulating cellular expression of 19 apoptosis-related genes. We now seek to determine if the modulation of apoptosis-related gene expression by p30II translates into a functional role for p30II in modulating cellular apoptosis. Human lymphocytes immortalized with a wild-type (ACH) or a p30II-mutant (ACH.30) HTLV-1 molecular clone, 293T cells transiently expressing p30II, and Jurkat T cells stably expressing p30II were induced into apoptosis with camptothecin (specificity for cells in the S phase of the cell cycle), TNF-Related Apoptosis Inducing Ligand (TRAIL) (extrinsic apoptotic pathway), or etoposide (intrinsic apoptotic pathway). Apoptosis was assayed by annexin V flow cytometry (lymphocytes), or Western blot for cleaved PARP and sub-Gl flow cytometric peak analysis (293T cells). Compared to ACH cells, ACH.30 cells showed increased apoptotic induction following treatment with camptothecin, but no difference in apoptotic induction following treatment with etoposide or TRAIL. Transiently p30II transfected 293T cells and stable p30II expressing Jurkat cells showed no differential susceptibility to apoptotic inducing agents compared to empty vector transfected/transduced controls, and stable expression of p30II did not alter Jurkat cell proliferation rate. We conclude that HTLV-1 p30II does not modulate lymphocyte apoptotic susceptibility in activated transformed T lymphocytes but may suppress proliferation in quiescent or immortalized, non-transformed T lymphocytes.
109: CD4+ T CELL FUNCTION UPON EXPOSURE TO MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS INFECTED MACROPHAGES.
An experimental system to study the roles of T cell subsets in Mycobacterium avium subsp. paratuberculosis (Mptb) infection in calves was developed. Immediately prior to inoculation, the left superficial cervical lymph node (LN) was removed so that preinoculation tissues were available for comparison. Calves were inoculated subcutaneously in the right cervical region with live Mptb and the draining superficial cervical LN was removed at post-inoculation day (PID) 60–67. LN cells were cultured for 7 days in the presence or absence of Mptb-purified protein derivative (PPD) and sorted to obtain purified CD4+ and gamma-delta T cells. Macrophages were obtained from peripheral blood by selecting for plastic-adherent monocytes and grown for 7 days. Macrophages were infected in vitro with live Mptb, and autologous CD4+ or gamma-delta T cells were co-cultured with infected macrophages for 48 hours, at which time bacterial survival as well as production of nitrites and IFN-gamma were evaluated. Incubation of autologous Mptb-infected macrophages with unstimulated T cells from preinoculation LNs failed to significantly reduce bacterial viability compared to cultures without added T cells. There were no significant differences in IFN-gamma production or nitrite production between cultures incubated with or without T cells. In contrast, at PID 60–67, co-cultures containing PPD-stimulated CD4+ T cells produced significantly greater IFN-gamma compared to cultures containing unstimulated or antigen-stimulated gamma-delta T cells or unstimulated CD4+ T cells. Despite the production of significant IFN-gamma, bacterial viability was not reduced nor was production of nitrites increased in co-cultures containing PPD-stimulated CD4+ T cells. The data suggest that production of IFN-gamma by PPD-stimulated CD4+ T cells from Mptb-inoculated calves is insufficient to promote mycobacterial killing.
110: DISTRIBUTION OF BABESIA MICROTI IN EXPERIMENTALLY INFECTED HAMSTERS, AS DETERMINED BY IN SITU HYBRIDIZATION.
Babesia microti is an intraerythrocytic parasite that is increasingly incriminated as a cause of disease in humans. The natural reservoir is Peromyscus leucopus, the white-footed mouse, and the parasite is transmitted by Ixodes sp. ticks. Hamsters have previously been used as an experimental model of the disease. In this experiment, we developed a method of in situ hybridization to determine location of replicating parasites. Portions of the cysteine protease gene (557 bp) and of the 16S-like small subunit rRNA (598 bp) were amplified from blood collected from hamsters experimentally infected with B. microti. Resulting PCR product was cloned into a transcription plasmid and both positive and negative sense digoxigenin-labeled riboprobes were generated. In situ hybridization on formalin-fixed paraffin-embedded samples of liver, lung, kidney, and spleen from both infected and noninfected hamsters enabled the visualization of replicating parasites. Positive staining was most prominent in erythrocytes within blood vessels and spleen.
111: IMPAIRED SPLENIC CD8 T CELL RESPONSE TO INFLUENZA INFECTION IN THE TSSC6 TETRASPANIN DEFICIENT MOUSE.
The tetraspanins are a superfamily of transmembrane proteins with roles in cell motility and lymphocyte function. Tssc6 is a novel member of this family, expressed exclusively by cells of the haemopoietic system. The T lymphocytes of Tssc6 deficient mice hyperproliferate in response to agonists in vitro. To investigate the consequences of this in vitro phenotype to the T cell-mediated immune response in vivo, Tssc6 null mice were infected with the influenza virus. Cohorts of 4, age and sex-matched Tssc6 null and Tssc6+/+ mice were infected intranasally with 104 pfu of HKx31 influenza virus and analysed at day 8 and 10 post-infection. Cells were recovered from bronchoal-veolar lavage, mediastinal lymph node, and spleen. The number of B cells, and CD8 and CD4 T cells in each organ were demonstrated by flow cytometry. CD8 T cells specific for the virus were identified by 1) stimulating harvested cells with each of three immunodominant influenza peptides then staining for intracellular IFN-gamma, and 2) staining cells with tetrameric complexes of MHC class I (H-2Db) and the NP366 influenza peptide. Both assays were analysed by flow cytometry. The results of four experiments showed that primary, T cell-mediated immune response to influenza virus infection was impaired (P < 0.05) in the spleen of Tssc6 null mice. In contrast, the magnitude of the T cell response at both the site of pathology, in the lung, and the regional lymph node, where T cells are primed, was normal. These data implicate Tssc6 in the splenic immune response, and/or the trafficking of immune cells to the spleen.
112: PATHOGENESIS OF NEWCASTLE DISEASE VIRUS, USING INFECTIOUS CLONES TO EXAMINE CONTRIBUTIONS OF VARIOUS GENES TO VIRULENCE.
Infectious clones of Newcastle disease virus (NDV) were utilized to evaluate relative contributions of the fusion (F) protein, hemagglutinin-neuraminidase (HN) protein, and phosphoprotein (P) to virulence. Specifically, two different clones of the La Sota virus backbone with a F cleavage site mutated to virulent motif, virulent Beaudette C backbone with avirulent La Sota HN, and a virulent Beaudette C backbone with mutations in P were examined. Four-week-old White Leghorn chickens were inoculated with viruses, and tissues collected at intervals were examined by histology, immunohistochemistry, and/or in situ hybridization. Birds inoculated with recombinant La Sota with virulent F had histologic lesions and viral distribution that were greater than the lentogenic parent yet markedly less than the wild type virulent virus. Birds infected with an infectious clone of virulent Beaudette C exhibited marked histological lesions, and virus was distributed in numerous organs. Birds inoculated with recombinant Beaudette C chimera containing a La Sota HN displayed minimal histological lesions and virus distribution was limited. Birds inoculated with the Beaudette C clones with mutated P displayed reduced virulence with virus primarily restricted to inoculation sites. These preliminary findings indicate that presence of a virulent F within a lentogenic background moderately increases histological damage and viral distribution. However, the change is still reduced relative to virulent NDV. Conversely, substituting an avirulent HN into a virulent backbone greatly decreases virulence. Additionally, modifying the P within a virulent virus causes a decrease in severity of histological lesions and viral distribution.
113: PATHOLOGY OF EQUINE WEST NILE FLAVIVIRUS (WNV) INFECTION IN FLORIDA.
West Nile flavivirus, an arthropod-borne RNA virus introduced to the US in 1999, causes CNS disease in birds, horses, humans and a variety of other animal species. The national WNV outbreak reached Florida in 2001 affecting a naive population of susceptible hosts including horses. Through December of 2001, 550 Florida horses were confirmed to have WNV encephalomyelitis. A total of 46 WNV horses were treated at the University of Florida. Eleven of the 46 horses (24%) were submitted for necropsy. Three more horses were examined at necropsy as outside cases. Seven were diagnosed with WNV infection at necropsy the following year; in 2003 two more cases were added to the necropsy accessions totaling 23 horses with the confirmed diagnosis of WNV infection over three years. Of the 23 horses, five had gross changes of hemorrhage at the level of the caudal brain and spinal cord segments. Fifteen (65%) of the horses had microscopic changes of mild to moderate perivascular lymphocytic rhomboencephalitis. The spinal cord of 22 horses was affected by mild to marked lymphocytic infiltration with a few macrophages and glial nodules in all 22 horses (100%) mainly affecting the grey matter of the thoraco-lumbar segments. In 3 of the 22 horses, marked red blood cell extravasation into the neuropil had occurred. PCR or viral cultures were positive in 6 (27%) horses; indirect immunohistochemistry was positive in 10 of 20 (50%) horses. The morphologic findings observed prompted the conclusion that there is gross and microscopic overlap between WNV infection and other equine CNS disorders and must be considered as differential etiologic diagnoses. Glial nodules are suggestive of WNV infection and small quantities of WNV virions, antigen and RNA are present within the CNS making the viral detection and diagnosis difficult.
114: PATHOLOGY OF NATURAL WEST NILE VIRAL INFECTION OF RAPTORS IN GEORGIA.
Since its introduction to the US in 1999, West Nile virus (WNV) has caused significant mortality in wild birds of many species. Despite significant emphasis on surveillance and transmission, there has been little focus on basic pathology in wild birds. As part of the WNV surveillance effort, 300 raptors from Georgia were necropsied, and virus isolation was performed on heart, brain, and cloacal swabs. Histopathology and immunohistochemistry (IHC) for WNV were also performed. Of 300 birds examined, 26 were positive for WNV by virus isolation and 20 were also positive by IHC. Of these 26, 23 had histologic lesions consistent with WNV infection. The most common histologic lesions associated with WNV infection were lymphoplasmacytic to histiocytic myocarditis, myocardial necrosis and fibrosis, and lymphoplasmacytic encephalitis. Gross lesions included calvarial hemorrhage, myocardial necrosis, and splenomegaly. Red-tailed hawks were most commonly affected. Also affected were sharp-shinned, Coopers, and red-shouldered hawks, an osprey, a barred owl, and a great horned owl. Zero of 41 screech owls, seven barn owls, four broad winged hawks, and an American kestrel were positive for WNV. Positive cases were detected only during summer to late fall. WNV infection accounted for 7% of mortality in 2001, 10% in 2002, and 8% in 2003. Therefore, West Nile virus infection appears to be a stable and moderately low source of mortality in raptors in Georgia.
115: WEST NILE VIRUS INFECTION IN THE AMERICAN ALLIGATORS (ALLIGATOR MISSISSIPPIENSIS) IN LOUISIANA.
Since it first emerged in 1999, West Nile virus (WNV) infection has been established as a seasonal epidemic in North America. WNV generally circulates between mosquitoes and birds. The infected birds commonly have a high level viremia and serve as reservoir hosts. WNV infection has been reported in various species but primarily in warm-blooded animals. Recently, epizootic WNV infections in American alligators (Alligator mississippiensis) have been described from the states of Florida (2002) and Georgia (2003). Although the role of alligators in the transmission cycle of WNV infection is still largely unknown, there are concerns that these reptiles may be important regionally because they can develop high viremia and shed the virus in their feces. Between September and December of 2003, American alligators from several alligator farms in Louisiana died suddenly or showed various neurological signs including circling, ataxia, head and muscle tremors, and head tilt. Seven alligators were submitted for postmortem examination. With the exception of gas-filled gastrointestinal tracts with no digesta and a diffuse thin pseudo-membrane on the colonic mucosa, no significant gross findings were noted. Microscopically, common lesions were diffuse severe heterophilic, histiocytic, and necrotizing enterocolitis, heterophilic meningoencephalitis, necrotizing and heterophilic hepatitis, heterophilic and histiocytic splenitis, generalized heterophilic and histiocytic lymphadenitis, and necrotizing and heterophilic pancreatitis. Less common findings included mild multifocal heterophilic and lymphohistiocytic interstitial nephritis, gastritis, and mild pulmonary congestion and edema. Immunohistochemistry for WNV revealed abundant antigen within the intralesional macrophages, antigen presenting cell-like cells in the gut-associated lymphoid tissue, intravascular monocytes, hepatocytes, neurons, glial cells, ependymal cells, as well as occasionally in intestinal epithelial cells, endothelial cells, pancreatic exocrine cells, and renal tubular epithelial cells. Immunohistochemical studies on WNV infection in the alligators have not been previously described. The diagnosis was further confirmed by positive RT-PCR and virus isolation.
116: ACUTE RESPIRATORY DISEASE IN GREYHOUND DOGS INFECTED WITH H3N8 EQUINE INFLUENZA VIRUS.
An emerging fatal respiratory disease has been identified in greyhound dogs infected with H3N8 equine influenza virus. Recurrent episodes of fatal respiratory disease characterized by coughing, fever, and pneumonia terminating in severe pulmonary hemorrhage have been observed in greyhound dogs at racing tracks and kennels in Florida in recent years. Investigation of a respiratory disease outbreak in two kennels in Jacksonville Florida in January 2004 focused on 22 dogs showing signs of respiratory disease including cough, fever, hemorrhage from the nose and mouth and death. Eight dogs died or were euthanatized in moribund condition, and five of the dogs were necropsied and subjected to virus isolation studies and serology. Pathologic findings included: 1) severe pulmonary and pleural hemorrhage; 2) acute to subacute, erosive to hyperplastic tracheitis, bronchitis, and bronchiolitis; and 3) bronchopneumonia. An influenza virus was isolated from one of the dogs, and three dogs tested had antibody titers against equine influenza (eqny99). Hemagglutinin gene sequence analysis demonstrated that the isolated virus was closely related to A/Eq/Wisconsin/2003 (H3N8) isolated from a horse in Wisconsin. Subsequent immunohistochemistry with a monoclonal antibody against human H3 demonstrated influenza antigen in bronchial gland epithelial cells, bronchial and bronchiolar epithelial cells and in alveolar macrophages of 4 of the 5 dogs that were necropsied. The results indicate that an equine influenza infection has been associated with acute fatal respiratory disease in greyhound dogs.
117: T-CELL IMMUNE RESPONSES TO CANINE EHRLICHIA CANIS INFECTION.
Chronic Ehrlichia canis infection has been shown to cause a marked expansion of lymphocytes that share large granular lymphocytic morphology. We are interested in further characterizing these cells and therefore better understand the canine immune response to E. canis infection. The Clinical Immunopathology Service at the Colorado State University Diagnostic Laboratory recently evaluated cells from 27 dogs that were E. canis seropositive. Of the dogs, 12 exhibited a T cell receptor (TCR) clonal rearrangement, 4 exhibited an immunoglobulin (Ig) clonal rearrangement, and 2 expressed both TCR and Ig clonal rearrangements. Flow cytometry was performed on cells from 13 dogs; 10 had a lymphocytosis of CD8 positive cells. Based on these results, we hypothesized that E. canis infection is stimulating a clonal expansion of CD8 positive cells and began an epidemiological study. To date, we have evaluated 2 dogs with a CD21 lymphocytosis; neither had E. canis antibodies. In contrast, of the 8 dogs seen with CD8 lymphocytosis since beginning the study, 2 dogs were E. canis seropositive. Important questions that remain to be answered include the phenotype of clonally expanded cells and whether they are specific to E. canis antigens. Investigative laboratory work is still underway, and thus far includes PCR for clonality, phenotyping by flow cytometry, and real time PCR cytokine analysis of infected dogs.
118: ROLE OF IMMUNOHISTOCHEMICAL MARKERS IN DETERMINING VACCINE EFFICACY AND DISEASE SEVERITY IN MYCOBACTERIUM BOVIS INFECTED CATTLE.
Development of fibrotic granulomas in response to Mycobacterium bovis infection in cattle is pathognomonic for bovine tuberculosis. Cytochemical markers applied to bovine lymph node granulomas enhance assessment of the dynamics of infection and improve correlation between features of protection and pathology. Lymph nodes from cattle vaccinated with M. bovis bacillus Calmette-Guerin (BCG) and subsequently challenged with virulent M. bovis were compared immunohistochemically to lymph nodes from unvaccinated, challenged cattle. Expression of TGF-Beta and procollagen 1 mRNA, and cell marker identification of T cells, B cells, macrophages and Gamma Delta T cells were compared within lymph nodes and granulomas. Granulomas in vaccinated cattle were greatly reduced in number and area in comparison to controls and did not develop extensive caseous necrosis or mineralization. Macrophage marker CD68 provided an intense signal within granulomas of vaccinated cattle in contrast to the less robust pattern observed in unvaccinated controls. Non-vaccinated cattle developed large, coalescing granulomas with characteristic central necrosis and mineralization. Control cattle had more advanced fibrotic granulomas with increased procollagen mRNA, procollagen 1 expression, and expanded Gamma Delta T cell populations around and within granulomas. TGF-Beta signal was more evident in non-vaccinated cattle granulomas. B cells were rare within granulomas of either group and T cells were scattered along the granuloma margins. Lymph node evaluation employing immunohistochemical markers can better assess the immune response and discriminate lesion composition in relation to vaccine efficacy and disease severity.
119: PATHOLOGIC FINDINGS IN HARBOR PORPOISES (PHO-COENA PHOCOENA) STRANDED IN WASHINGTON STATE 2 MAY TO 2 JUNE 2003 COINCIDENT WITH THE MID-FREQUENCY SONAR EXCERCISES BY THE USS SHOUP.
Deployment of military sonar has recently been associated with abrupt mass strandings of multiple marine mammal species in the Bahamas, Canary Islands and possibly in Greece and Ireland. While transiting Haro Strait, WA, mid range tactical naval sonar was deployed by the USS Shoup on 5 May 2003. Between 2 May to 2 June, 2003, 14 harbor porpoises stranded. Due to the relatively small and biased sample size, it is difficult to resolve if there was a statistically significant increase in the number of associated stranded harbor porpoises (Phocoena phocoena) during (his time interval relative to prior stranding data. Comprehensive necropsies were conducted on ten animals. In addition to morphometric and computerized tomography (CT), representative tissues were harvested and evaluated by histopathology, microbiology, molecular studies and heavy metal analysis. Samples were also screened for chemical contaminants, domoic acid and fatty acid composition. Post mortem changes and freeze artifacts hindered definitive assessments. All examined animals had extravasated blood and minor pooling of serosanguinous fluid in the peribullar areas. These changes were distinct to previously reponed acute hemorrhage or hematoma formation and were most likely due to post mortem artifact. Scan images were examined from 7 intact animals and a single head. Intracochlear hemorrhage was detected in 1 of 8 porpoises. This animal presented with comminuted cranial fractures and the intracochlear hemorrhage was attributed to physical trauma. In this case series, there was no evidence of cochlear fenestral disruption, fresh intracochlear lesions, ossicular subluxation, nor were there visceral microcavitations, that are indicative of acoustic trauma or decompressionlike processes. Trauma and infectious disease resulted in the loss of 5 of the 10 porpoises and a cause of death could not be determined in the remaining 5 animals. There was insufficient evidence to confirm or discount the possibility that the porpoise strandings were related to acoustic trauma; however, the animals recovered in the northern Pacific Ocean did not feature any of the lesions as described in beaked whales stranded in the Bahamas and Canary Islands.
120: MICROSCOPIC AND SPECTROSCOPIC EVIDENCE THAT FELINE IDIOPATHIC PULMONARY FIBROSIS IS ASSOCIATED WITH BENTONITE PNEUMOCONIOSIS.
Chronic respiratory disease in domestic cats with clinical signs and pathology similar to idiopathic pulmonary fibrosis (IPF) in humans has recently been reported. Exposure to dusty environments is a risk factor for the development of IPF in humans. Because of this, we hypothesized that elemental and visual analysis of tissues in feline IPF will detect the presence of material similar to a respirable fraction of the aluminum-silicon clay bentonite, the primary ingredient in scoopable cat litters. To test this hypothesis we analyzed pulmonary tissues from cats with feline IPF and normal feline lung using light microscopy under polarized light, to detect crystalline material, and elemental analysis of whole lung using inductively coupled plasma-mass spectroscopy (ICP-MS) to detect increases in elements found in bentonite particles in the lungs of IPF cats. A respirable particle fraction with a mass mean aerodynamic diameter of 3.4μm was identified in a commercial brand of cat litter. Scanning electron microscopy with energy dispersive X-ray analysis and ICP-MS of the particles confirmed that the bentonite is an aluminum-silicon silicate with numerous trace elements. Levels of aluminum and trace elements (scandium, thorium, barium) were increased in IPF cats and the ratio of tissue trace elements was similar to the ratio in the bentonite. Small numbers of colorless acicular crystals similar in size and shape to the bentonite were found in the lungs and bronchial lymph nodes of the IPF cats but not in normal feline lung or lymph node. From this study we conclude that feline IPF is associated with inhalation of bentonite (cat litter) particles.
121: ARTERIOLAR ENDOTHELIAL PROLIFERATION AND MI-CROTHROMBOSIS ATTRIBUTED TO THROMBOTIC THROMBOCYTOPENIC PURPURA IN TWO CATS.
Two cats, a 9-year-old male domestic short hair and a 1-year-old female spayed Maine coon, were presented for necropsy. The DSH cat was found weak, cold, and dyspneic. The Maine coon cat had a 10-day history of weakness and poor appetite. Prior to this development, the cat was healthy and energetic, although thin. The DSH cat had mild pulmonary edema. Gross findings in the Maine coon cat were moderate pallor of tissues, watery red thoracic and pericardial effusions, and widespread petechial hemorrhages. The heart was moderately enlarged, weighed 23.5 g, and had moderate left ventricular hypertrophy. The urine was red. A 2 cm diameter hematoma was in the cerebrum. Histopathologic lesions were identical in both cats. Multiple organs had arterioles that were expanded and often obliterated by complex glomeruloid structures, occasionally with associated microthrombi. The proliferative intravascular cells emanated from the intima, variably formed vascular channels, and were positive for factor VIII-related antigen. Antemortem hematologic studies of the Maine coon cat revealed severe anemia, thrombocytopenia, neutropenia, platelet clumping, and poikilocytosis. Bone marrow examination indicated erythroid and megakaryocyte hyperplasia. No evidence of DIC was found (normal D-dimer, fibrinogen, and coagulation studies). In retrospect, criteria for diagnosis of the disease thrombotic thrombocytopenic purpura (TTP) were clearly met. This rare human disease is due to acquired or congenital deficiency of von Willebrand Factor cleaving protease. This subsequently results in large circulating complexes of von Willebrand factor leading to platelet clumping and microthrombi. Anemia is due to passage of erythrocytes through partially thrombosed or recanalized vessels. Chronic forms of TTP have been proposed in humans, consistent with the advanced lesions seen in the cats with this disease.
122: MITOCHONDRIAL EXPRESSION OF PROTEINS INVOLVED IN OXIDATIVE PHOSPHORYLATION IN NATURAL AND INDUCED FORMS OF CANINE DILATED CARDIOMYOPATHY.
Canine dilated cardiomyopathy (DCM) is characterized by dilation and impaired contraction of the ventricles leading to cardiac failure and sudden death. Among the numerous primary and secondary mechanisms implicated in the progression of disease, decreased mitochondrial energy production is commonly cited as a major functional defect of DCM. In order to better understand the pathophysiologic processes in DCM, we investigated changes in protein expression in mitochondria isolated from the left ventricles of 3 dogs (1 Boxer and 2 Doberman Pinschers) with spontaneously occurring DCM, and 3 dogs with DCM induced by rapid ventricular pacing. These results were compared with mitochondrial protein expression in heart tissue from the left ventricles of 7 healthy dogs. Fresh and frozen mitochondrial fractions were prepared and analyzed by 2-dimensional electrophoresis. No qualitative differences were observed comparing fresh and frozen heart mitochondrial samples. Out of greater than 1010 proteins, 96 were isolated that either increased or decreased by at least 2-fold between the groups. These proteins were subsequently identified using MALDI-TOF mass spectrometry and the Swiss-Prot database. The analysis revealed that the majority of altered mitochondrial proteins were involved in the oxidative phosphorylation pathway with complexes I and V most affected in both natural and induced forms of DCM. Most notably, chain 1 from complex I was universally down-regulated in both DCM groups, a finding that has precedent in other heart failure models and in man. Decreased chain I protein expression may be important to diminished Complex I function and reduced mitochondrial energy production in pacing-induced and naturally occurring canine DCM.
123: MICROSCOPIC GRADING OF CANINE CUTANEOUS MAST CELL TUMORS: A MULTI-INSTITUTIONAL REVIEW.
Currently, prognostic and therapeutic determinations for canine cutaneous mast cell tumors (MCTs) are primarily based on the histologic grade of the tumor. However, the use of numerous grading systems by different pathologists and especially institutional modifications in the absence of a validation of their prognostic significance make the prognostic value of histologic grading highly questionable. To evaluate the consistency of microscopic grading between multiple institutions and the prognostic significance of standardized grading as well as institutional modifications, 95 cutaneous MCTs from 95 dogs that had been treated with surgery only were graded in a blinded study by 31 pathologists from 16 institutions. The total survival time and the time to local or distant recurrence of MCTs were recorded for all dogs. There was an agreement of 74.6% for the grading of grade 3 MCTs, but only a 63.0% and 63.1% agreement for grade 2 and grade 1 MCTs, respectively. Features consistently characteristic of grade 3 identified MCTs have been reviewed and compiled. Based on these data and their prognostic association, a simple classification of canine cutaneous MCTs into high and low grade may be more consistent and prognostically significant than the currently used grading systems.
124: CORRELATION BETWEEN KIT EXPRESSION PATTERNS AND PRESENCE OF MUTATIONS IN THE C-KIT PROTO-ON-COGENE.
Cutaneous mast cell tumors (MCTs) are a common neoplasm in dogs. Currently, prognostic and therapeutic determinations for canine MCTs are primarily based on histologic grading. However, the majority of MCTs are of intermediate grade and therefore the prognostic value of histologic grading is questionable. Considering the prevalence of canine MCTs, the costs for therapy and the emotional stress for owners, a new prognostic classification is urgently needed. Recently, we demonstrated that KIT expression patterns, using immunohistochemistry, are prognostically significant for surgically removed canine MCTs. The goals of this study were: 1. to determine the relationship between KIT staining patterns and the presence of mutations in the c-kit proto-oncogene; and, 2. to determine the prognostic significance of KIT protein levels in MCTs. Fifty MCTs from 50 dogs that had been treated with surgical excision only were studied. Complete follow-up data were obtained for each case and sections were evaluated for KIT staining patterns. Neoplastic mast cells from each tumor were isolated using laser capture microdissection and DNA was extracted for mutation analysis of the c-kit proto-oncogene using PCR and sequence analysis. Punches from each neoplasm were incorporated into a tissue microarray. Sections of the tissue microarray were stained with an anti-KIT antibody followed by fluorescent-labeled secondary antibodies. Fluorescence was quantitated using a microarray scanner. Preliminary data indicate an association between KIT staining patterns and the presence of duplication mutations in the c-kit proto-oncogene. KIT immunostaining may be used as a high throughput screening procedure in order to identify MCTs with potential activating c-kit mutations.
125: THE EFFECT OF BOVINE VIRAL DIARRHEA VIRUS ON EXPRESSION OF DEFENSINS IN BOVINE TRACHEAL EPITHELIAL CELLS.
Co-infections of viruses and bacteria are important in the pathogenesis of respiratory disease in cattle. Bovine viral diarrhea virus (BVDV) infection is a risk factor for development of shipping fever pneumonia in feedlot cattle, but the mechanisms are not well defined. We hypothesize that BVDV predisposes to bacterial pneumonia by downregulation of innate immune responses in airway epithelial cells. The aim of this investigation was to compare the effect of noncytopathic BVDV infection on the LPS-stimulated expression of tracheal antimicrobial peptide (TAP) in bovine tracheal epithelial cells. Bovine tracheal epithelial cells were isolated from healthy calves and cultured on collagen-coated wells. The primary epithelial cell line was divided into four groups in triplicate. Groups 1 and 2 were infected with BVDV after 90% confluence, and groups 3 and 4 did not receive virus. After 48 hours, groups 1 and 3 were treated with LPS. Sixteen hours later, total RNA was extracted from cells in all four groups, and TAP mRNA expression was determined by real-time PCR, relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Treatment of tracheal epithelial cells with LPS alone resulted in a 50-fold increase in TAP mRNA relative to GAPDH, whereas TAP mRNA was increased only 2-fold in BVD-infected cells exposed to LPS. These preliminary data suggest that BVDV infection inhibits the LPS-induced upregulation of TAP mRNA in bovine tracheal epithelium, and this may be a mechanism by which this virus abrogates innate immune responses and predisposes to bacterial pneumonia in cattle.
126: A SURVEY OF TESTICULAR LESIONS IN HORSES.
Because of its invasiveness, testicular tissue collection for microscopic evaluation is not routinely performed in the stallion as a common diagnostic procedure. Very little is known or has been published about the incidence of testicular lesions in the general equine population. The aim of this survey was to assess the incidence of microscopic lesions in stallions slaughtered for food consumption at a facility in Fort Worth, Texas. From March to June 2003, testicular tissue from either the left or right testis was collected from 65 adult stallions. Tissue samples were fixed in Modified Davidson's solution for 24–72 hours, processed routinely, stained with HE and evaluated microscopically. A board-certified pathologist (UBM) evaluated the distribution and graded the severity of testicular lesions present, if any, from minimal to severe. Tissue alterations were grouped as occurring in the seminiferous tubules, testicular interstitium, rete testis, epididymis, or capsule. In the seminiferous tubules, 89% of the sampled stallions had evidence of tubular degeneration and 31% had evidence of tubular atrophy. Tubular dilation, intraepithelial cysts, and intratubular giant syncytial cells characterized the degenerated seminiferous tubules. Intratubular granulomatous inflammation was present in 38%. A malignant seminoma was identified in only one stallion. Of the 65 stallions, 92% had significant tissue alterations in the testicular interstitium; these consisted of interstitial edema, Ley-dig cell hypocellularity, perivascular lymphocytic inflammation, and interstitial fibrosis. The majority of the lesions graded as minimal to mild; some were moderate and occasionally severe. No significant tissue alterations existed in the rete testis, epididymis, or capsule. The survey indicates that the incidence of testicular lesions in the general equine population is high but that their severity is minimal to mild.
127: IDIOPATHIC ULCERATIVE SKIN DISEASE IN GREEN MORAY EELS (GYMNOTHORAX FUNEBRIS) TWO CASES.
Documented cases of skin disease in moray eels (Gymnothorax funebris) are very sporadic. Two male green moray eels with a history of progressive skin ulceration were euthanized. In a bilaterally symmetrical distribution pattern, multiple to coalescing erosions and ulcers extended from the head and face to the ventral aspects of the mandible and along the ventrum of the body to just proximal to the anal sphincter. Microscopically, the epidermis was irregularly thickened, with scattered foci of ulceration and erosion. Within the stratum spinosum and basale, there was moderate, multifocal swelling and vacuolation of epithelial cells, with apoptotic necrosis, goblet cell hyperplasia and foci of sub-basilar cleft formation. In some areas, focal fibroplasia and accumulation of proteinaceous eosinophilic material expanded the dermo-epidermal foci of separation. Ultra-structurally, the point of separation was identified below the basement membrane. Multifocally, the underlying dermis was edematous, with loosely arranged fibrous connective tissues, dilated blood vessels, and discrete perivascular collections of plasma cells, lymphocytes, histiocytes, and scattered eosinophilic granular cells. No pathogens were identified. There were no additional significant tissue alterations. After consideration of the history, the distribution of the lesions, and the absence of other remarkable signs or lesions generally attributed to infectious or toxic injurious agents along with the lack of known environmental stressors such as hypoxia, we propose that perhaps dysfunctional modulation of Cortisol levels in blood may have played a major role in the development of the ulcerative dermatosis described in these green moray eels.
128: MINOCYCLINE INHIBITS SIV REPLICATION IN SIV-INFECTED MACROPHAGES AND LYMPHOCYTES OF MACAQUES.
World Health Organization figures indicate that in 2003 over 40 million adults and children were infected with HIV globally. Current anti-AIDS therapies are expensive, require complex dosing regimens and have significant side effects and toxicity. In previous studies, we demonstrated that minocycline, a safe, readily available, inexpensive, semisynthetic tetracycline derivative suppresses replication of HIV and SIV in macrophages and lymphocytes, the major hosts for productive replication of the viruses in the CNS, in a dose-dependent manner. The purpose of this study was to define the mechanisms by which minocycline inhibits virus replication in these cells. Because HIV replication in primary human lymphocytes has been shown to be dependant on expression of the mitogen-activated protein kinase p38, we examined activation of p38 in SIV-infected primary macrophages and lymphocytes treated, or not, with minocycline. Cells were treated with 5, 10, 20, or 40 μg/ml of minocycline 24 hours prior to and concurrent with SIV infection. At 9 days post-inoculation, cells were lysed and western blots were performed to detect both activated and constitutively expressed p38. Levels of both p-p38 and p38 were significantly lower in minocycline-treated SIV-infected primary lymphocytes, but remained at pre-inoculation levels in minocycline-treated SIV-infected primary macaque macrophages. Thus, suppression of virus replication in lymphocytes likely occurs via a p38-dependant pathway, while in macrophages it is p38-independent. Future studies will examine the stage of virus replication at which minocycline acts. Minocycline is a safe, readily available antibiotic that should be investigated as an anti-HIV therapeutic.
129: PRESENCE OF ADIPONECTIN ISOFORMS IN CANINE SERUM.
Canine obesity is a common clinical entity believed to be caused primarily by long-term overeating and lack of exercise. Obese dogs tend to develop diabetes mellitus and total insulin secretion and fasting plasma insulin concentration are highly significant linear functions of obesity in dogs. Adiponectin is a recently discovered adipocytokine secreted exclusively by adipocytes and thought to link obesity to the development of type 2 diabetes in humans. Serum adiponectin concentrations are decreased in obese or type 2 diabetic humans and animals. Serum adiponectin concentrations are increased in type 1 diabetic humans. In humans, adiponectin circulates in serum as a hexamer of relatively low molecular weight (LMW), a larger multimeric peptide of high molecular weight (HMW), and as a trimer. A new index, SA, which represents the ratio, and not the absolute amounts, between the two oligomeric forms (HMW to LMW) is critical in determining insulin sensitivity in humans. In this study, serum from normal dogs was analyzed by SDS-PAGE and Western immunoblotting using mouse anti-Acrp30, a polyclonal antibody known to react with canine peptide sequences 18–32 and 187–200. Multiple adiponectin isoforms were identified under various reducing and denaturing conditions. Under normal and denaturating conditions, a 180 kD band analogous to the human LMW form was recognized. Under reducing conditions, two bands were recognized (120 kD and 90 kD) analogous to the human tetrameric and trimeric forms. Under reducing and denaturing conditions, bands were located at 90 kD, 60 kD, and 30 kD, analogous to the trimeric, dimeric, and monomelic forms, respectively. This study represents the first report of adiponectin isoforms in canine serum.
130: MENINGOENCEPHALITIS IN JUVENILE SALMON SHARKS ASSOCIATED WITH CARNOBACTERIUM SP.
The Salmon Shark (Lamna ditropis) is an apex predator which ranges from the north Pacific to central Baja California with adults reported primarily in the north part of the range and juveniles in the south. Documentation of juveniles comprises almost exclusively of reports of stranded sharks along the coast of California. Although these strandings have been recorded by local biologists for over 20 years, a cause for this phenomenon remains unidentified. We examined 10 juvenile salmon sharks found on central California beaches between June 2000 and August 2003. Tissues available for examination included formalin-fixed heads (5), frozen heads (3), formalin-fixed head and viscera with frozen liver (1), and an intact fresh carcass (1). Moderate to severe, suppurative, meningoencephalitis was diagnosed in 6 sharks and of these, 3 had fresh or frozen tissue available from which a Carnobacterium sp. was isolated. Multifocal necrotizing hepatitis was diagnosed in 2 cases of meningitis for which liver was available. One frozen brain not suitable for histological examination also yielded growth of Carnobacterium sp. Two sharks had mild to moderate, nonspecific nonsuppurative meningitis; bacteria were not identified histologically and tissue was not available for culture. One brain was normal. Molecular studies comparing 16S ribosomal DNA sequences of our 4 isolates demonstrated 100% homology with each other. A Genbank BLAST comparison placed the putative organism as a Carnobacterium species closely related to several Carnobacterium species in the Genbank. This is the first report of Carnobacterium sp. infection in sharks.
131: IMMUNOPHENOTYPING OF INFLAMMATORY CELLS IN HEPATIC ALVEOLAR ECHINOCOCCOSIS IN CYNOMOLGUS MONKEYS (MACACA FASCICULARIS).
Alveolar echinococcosis due to the metacestode larvae of the fox tapeworm Echinococcus multilocularis, has been described in cynomolgus monkeys. Usually the liver is affected with variable involvement of other organs, namely lung, mesenteric lymph nodes and pancreas. In these organs the metacestodes form multiloculated cysts compressing adjacent parenchyma. Histologically, lesions are characterized by a granulomatous and lymphocytic host response, which varies in character. Intact metacestodes are usually surrounded by macrophages, which are often multinucleated. In contrast, abundant, commonly degenerate granulocytes, in addition to macrophages surround collapsed laminated structures. In both these patterns, fibrosis is not a prominent feature, which is in contrast to a third, rare pattern of dense collagen deposition around cysts. In all patterns, various numbers of lymphocytes are present. Pro-toscolices can be demonstrated in the first and third pattern, but not within collapsed cysts. All patterns can be observed simultaneously in the same animal. To further characterize the inflammatory infiltrate, liver sections (35) from 13 animals with natural alveolar echinococcosis were stained with antibodies against CD3, CD20, CD79, lysozyme, and MAC387. Moreover, expression of iNOS was evaluated immunohisto-chemically. The anti-CD3 antibody stained mainly cell clusters near collapsed cysts. CD20-positive cells were present in the same locations but usually in lower numbers; however, individual cells present in the compressed hepatic parenchyma were also positive. Many cells randomly distributed within the hepatic parenchyma were positive for lysozyme and MAC387. Additionally, MAC387-positive cells and debris were often observed adjacent to laminated parasitic structures, whereas lysozyme was not common in these areas suggesting a specific response to metacestodal tissue. The anti-iNOS antibody stained occasional multinucleated cells adjacent to cysts.
132: SURVIVIN EXPRESSION IN CANINE PAPILLOMAVIRUS-INDUCED PAPILLOMAS.
Survivin, a member of the inhibitor of apoptosis (IAP) protein family, has a dual function of regulating normal development and inhibiting apoptosis. Although expressed during fetal development and in a wide range of tumors, it is rarely present in terminally differentiated tissues. Numerous studies have addressed the expression and role of survivin in various cancers, but survivin regulation of viral-induced apoptosis is poorly studied. Recurrent respiratory papillomatosis, caused by human papillomavirus, is characterized by unregulated papilloma growth on the laryngeal mucosa. Survivin expression is abundant in these papillomas, but so is the expression of apoptotic factors suggesting that dysregulation of apoptosis favors papilloma growth and survival. Papillomas caused by canine papillomavirus are common in young dogs and usually regress following the development of an immune response. We wanted to know if these tumors express survivin and if changes in expression are related to tumor regression. We examined 7 papillomavirus-induced papillomas from young dogs for survivin expression by immuno-histochemistry. There was survivin expression in all 7 tumors. In those with little or no inflammation, there was diffuse staining for survivin in epithelium above the basal layer. Staining was cytoplasmic and nuclear, but the cytoplasmic staining was much more intense. Stromal fibroblasts and infiltrating inflammatory cells were also survivin positive. As inflammation intensified, signifying regression, staining of the epithelium was patchy, had less intense cytoplasmic staining, and was transepithelial. In addition, there was intense nuclear staining of cells in the basal layer. Inflammatory cells in the stroma and infiltrating the epithelium stained intensely for survivin. Changes in the staining pattern of survivin during regression suggest that survivin may play a role in papilloma regression.
133: PROTEOMIC ANALYSIS OF NORMAL AND NEOPLASTIC CANINE PROSTATE AND BLADDER TISSUES.
Prostatic carcinoma is a significant health problem in men and dogs. The dog is a commonly-used animal model for investigating various aspects of the growth and metastasis of prostate cancer. The cellular origin of canine prostatic carcinomas is unclear to investigators, as the tumors may exhibit characteristics of glandular, transitional, and/or ductular differentiation. In order to further investigate the origin of these neoplasms in the dog prostate gland, we used proteomic analysis to compare the protein expression profile of normal dog prostate gland and urinary bladder with that of a canine prostatic carcinoma. Following protein extraction and 2-dimensional electrophoresis, a total of 1,018 proteins were compared between normal and neoplastic prostate tissue. Of these, 54 proteins (5.3%) were either over-expressed (19 proteins, 1.9%) or under-expressed (35 proteins, 3.4%) by a greater than two-fold difference in the carcinoma compared to normal prostate tissue. Comparison between normal bladder and prostatic carcinoma demonstrated a greater than two-fold difference in expression for 38 out of 874 proteins (4.3%). Fourteen of the proteins (1.6%) were over-expressed by the carcinoma, while 24 (2.7%) were under-expressed. On an individual basis, differences in protein expression between the normal and neoplastic prostate gland ranged from proteins that were not expressed differently between the two tissues to a protein that was under-expressed approximately 148-fold by the carcinoma. The greatest disparity in expression between normal bladder and prostatic carcinoma was a protein that exhibited a greater than 45-fold overexpression in carcinoma. Analysis of proteins expressed by normal and neoplastic prostate gland and bladder may be useful for identifying the origin of prostatic carcinomas in dogs.
134: SARCOIDS IN CAPTIVE ZEBRAS (EQUUS BURCHELLII): ASSOCIATION WITH BOVINE PAPILLOMAVIRUS INFECTION.
Sarcoids were diagnosed in four captive zebras from three facilities in the US and one facility in Mexico. Zebra No. 1 presented with a 9 × 7 × 5 cm inguinal mass. Seven months after surgical excision of the mass, the zebra showed a similar lesion in the right upper eyelid. Zebra No. 2 had a 10 cm diameter mass on the penile sheath. Zebras No. 3 and No. 4 each presented with a single raised, ulcerated mass on the nose, 2 cm to 5 cm in diameter. Histologically, all masses were dermal, compact, non-encapsulated, poorly demarcated, composed of well-differentiated spindle cells arranged in interlacing streams and whorls, and accompanied by moderate epidermal hyperplasia with long rete ridges. Based on the morphologic resemblance to the unique equine cutaneous neoplasm, a diagnosis of sarcoid was made. Association with bovine papillomavirus (BPV) was demonstrated by PCR, restriction enzyme digestion, nucleic acid sequencing, and in situ hybridization on paraffin-embedded tissues from the inguinal mass of zebra No. 1, and the penile and facial mass of zebras No. 2 and No. 3, respectively. The 244 bp amplicon had 98% identity with bovine papillomavirus type 1 (zebra No. 1). In situ hybridization for BPV DNA intensely stained the nuclei of neoplastic mesenchymal spindle cells. This is the first description of sarcoids in captive zebras. The topography and gross and histological presentation of sarcoids in zebras are similar to those in the horse. Our molecular findings further substantiate the role of BPV in the etiopathogenesis of sarcoids in equids.
135: HEPATIC AMYLOIDOSIS IN A CAPTIVE TRUMPETER SWAN (CYGNUS BUCCINATOR) WITH CHRONIC SCHISTOSOMIASIS.
A captive, 2-yr-old trumpeter swan (Cygnus buccinator) was referred to Iowa State University's Wildlife Care Clinic because of lethargy and severe weight loss. The bird died shortly following presentation and was submitted for post-mortem examination. Necropsy examination revealed diffuse muscle atrophy with yellow, gelatinous fluid and fibrin found on the surface of the liver and in the coelomic cavity. Bacterial and fungal cultures were negative. Microscopic examination of the liver revealed marked effacement of hepatic cords by an eosinophilic, amorphous to fibrillar material localized predominantly in Disse's space. In Congo red-stained sections the material was deep red-orange and turned apple-green under polarized light (consistent with amyloid). In the veins of the tunica muscularis and mucosa of the small intestine were numerous intravascular metazoan organisms. Also in the small veins and adjacent tissues of the lamina propria were 50 to 100 micron eggs with a translucent to golden-brown shell and internal larva (miracidium) consistent with schistosomiasis. Surrounding the schistosome eggs were variable numbers of macrophages, multinucleated giant cells, lymphocytes, plasma cells and a few heterophils. Captive waterfowl are prone to amyloidosis often secondary to social stressors or chronic inflammation. The combination of chronic intestinal inflammation due to the schistosome eggs, along with the captive status of this bird, likely instigated this tissue-specific, hepatic amyloidosis. This previously unrecognized association between schistosomiasis and amyloidosis in trumpeter swans is important from a diagnostic perspective and also may provide a foundation for future waterfowl models of human schistosomiasis-induced amyloidosis.
136: A NOVEL LEUKODYSTROPHY IN TWO BULL MASTIFF DOGS.
Leukodystrophies are inherited neurological disorders involving central nervous system white matter. They are uncommon in animals but a few, breed specific entities have been described. In 2002, two young-adult, purebred Bull Mastiff dogs from central New York state presented to their referring veterinarians displaying moderate to severe ataxia of all limbs, spastic tetraparesis that was worse in the hindlimbs, and a diffuse, action related, whole body tremor. Clinical signs were insidious in onset and slowly progressive. Anatomic diagnoses considered were a C1–C6 lesion or, based on the whole body tremor, a diffuse CNS disorder. No gross lesions were apparent in the brain or spinal cord. Histopathologically, numerous, multifocal, sharply demarcated, ovoid to angular areas of myelin pallor (plaques), occasionally surrounded by fine peripheral vacuolation, were present throughout white matter of the brainstem, principally involving the crus cerebri, cerebellar peduncles, transverse fibers of the pons, pyramids and the spinal tract of cranial nerve V. Numerous plaques were also present in the lateral and ventral funiculi of the spinal cord with smaller numbers throughout the dorsal funiculi. These plaques, which often were traversed by axons, did not stain with, luxol fast blue and were not associated with astroctytosis (GFAP negative). Preliminary ultrastructural findings include occasional hypertrophic glia in white matter, rare unmyelinated segments of axons and in one dog, focal proliferation of tubule-containing cytoplasmic glial cell processes (possibly oligodendroglial origin). The described clinical and histopathological findings and age of onset are similar to the well characterized, presumably hereditary, bovine syndrome known as Charolais ataxia or oligodendroglial dysplasia. This report presents the first description of a leukodystrophy similar to Charolais ataxia in another species.
137: CALCIUM-DEPENDENT ENHANCED TRANSCRIPTION OF p300 BY HUMAN T LYMPHOTROPIC VIRUS-1 p12I.
Transcriptional co-activator p300 mediates transcriptional control of cellular and viral DNA binding transcription factors, however transcriptional regulation of p300 is not known. We have recently reported that human T lymphotropic virus type-1 (HTLV-1) accessory protein p12I enhances p300 expression and p300-dependent transcription in a dose-dependent manner. 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. Earlier reports from our laboratory have demonstrated the critical nature of pl2I 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 indicates 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.
138: HISTOLOGIC AND IMMUNOHISTOCHEMICAL CHARACTERIZATION OF SPONTANEOUS PITUITARY ADENOMAS IN 13 CYNOMOLGUS MACAQUES {MACACA FASCICULARIS).
Pituitary adenomas were identified in 13 cynomolgus macaques necropsied at the Wake Forest University School of Medicine from 1994 to 2004. The affected monkeys included both males (8) and females (5) and ranged from 18 to 32 years of age. The pituitary adenomas were either focal, causing gross enlargement of the pituitary visible on postmortem examination, or multifocal microadenomas identified on histologic examination. A total of 34 adenomas were identified in the 13 macaques. Immunohistochemical stains for follicle stimulating hormone, luteinizing hormone, prolactin, human growth hormone, thyroid stimulating hormone, and adrenocorticotropic hormone were applied to pituitary tissue from all cases. Immunostaining results revealed 22/34 (64.7%) lactotroph cell adenomas, 5/34 (14.7%) plurihormonal cell adenomas, 2/34 (5.9%) corticotroph cell adenomas, 2/34 (5.9%) null cell adenomas, 1/34 (2.9%) somatotroph cell adenoma, 1/34 (2.9%) mixed corticotroph-somatotroph cell adenoma, 1/34 (2.9%) mixed lactotroph-corticotroph cell adenoma, 0/34 (0%) gonadotroph cell adenomas, and 0/34 (0%) thyrotroph cell adenomas. This study represents the first extensive retrospective case series performed to evaluate the histologic and immunohistochemical characteristics of pituitary adenomas in cynomolgus macaques. Our findings demonstrate that macaque pituitary adenomas frequently have mixed histological appearance and hormone expression. Similar to human pituitary adenomas, prolactin-secreting neoplasms were the most prevalent type.
139: TUMORS, TEARS, FUSION, DECALCIFICATION, FRACTURES AND INFECTION: TOUGH TIMES FOR A TYRANNOSAUR.
Preparation of a recently discovered skeleton of Gorgosaurus sp. (Theropoda: Tyrannosauridae) from the Two Medicine Formation of Montana (Campanian) has revealed a multitude of pathologic findings and yields insight into behavior, sexual dimorphism, and disease. These findings include: calloused gastralia (fracture and calcified hematoma); bifurcated pes ungual I (split claw sheath); remodelled scapula-coracoid (consistent with a proliferative process and fracture); calloused and deformed right fibula (fracture and tendon retraction); detached greater trochanter of the left femur, with bone remodeling and fistulous tracts (trauma, reattachment and osteomyelitis); and calloused, right and left dentaries with fistulous tracts and subsequent loss of tooth positions (trauma and tooth root abscess with osteomyelitis). Two pathologic findings appear to relate to sexual dimorphism in this robust (female) morphotype. The first is fusion of the 4th and 5th caudal centra (and their shared chevron), presumably in response to stress caused by the “overload” of a mounting male, as seen elsewhere in robust morphotype tyrannosaurids. The second is the collapse of the articular surfaces of at least eleven caudal centra, possibly the result of decalcification due to skeletal stripping of calcium for egg production. A heterogenous, spherical mass occupying the caudal aspect of the braincase is consistent with the presence of a large tumor which could have caused the incapacitation and death of this individual.
140: RENAL AND HEPATIC CYSTS IN A GROUP OF RHESUS MACAQUES {MACACA MULATTA) RESEMBLING AUTOSOMAL DOMINANT POLYCYSTIC RENAL DISEASE (ADPKD) IN HUMANS.
Renal and hepatic cysts were diagnosed in 4 male rhesus macaques, ranging from 20–29 years of age. Cystic lesions were found during routine necropsies, with none of the monkeys having a clinical history of renal or hepatic abnormalities. Three of the animals had the same sire, which was the grandsire of the remaining affected animal. No female offspring of the same sire had renal or hepatic cysts at necropsy. Cystic structures within the kidney were usually present in multiple discrete foci. The histological appearance varied from dilated, round structures lined by simple cuboidal epithelium to arborized stromal structures supporting pseudostratified columnar epithelium. In the liver, the cysts were also found in discrete foci, and typically consisted of irregularly shaped structures with diameters varying from 20–100 μm that were lined by simple cuboidal epithelium. Moderate amounts of fibrous connective tissue surrounded the hepatic cystic foci. In humans, renal cystic disease occurs in several hereditary syndromes, including autosomal recessive polycystic kidney disease, ADPKD and tuberous sclerosis. Given the appearance of the renal cysts, the presence of hepatic cysts, the maturity of the affected monkeys and the common ancestor, we submit that this condition has the greatest resemblance to human ADPKD. In our cases there is a clear Mendelian inheritance pattern; however, it is not typical of autosomal dominance as only males were affected. We cannot exclude that this condition may still occur in female macaques, due to the small sample size. We further suggest that immunologic and genetic investigation into this disorder in this group of macaques could result in the first viable primate model of ADPKD.
141: PATHOLOGIC FINDINGS IN CHICKENS, TURKEYS, AND PIGEONS EXPERIMENTALLY INFECTED WITH EXOTIC NEWCASTLE DISEASE VIRUS FROM AN OUTBREAK IN CALIFORNIA DURING 2002–2003.
Exotic Newcastle disease virus (NDV) isolated from chickens during the 2002–2003 California outbreak was inoculated into 4-week-old SPF White Leghorn chickens, 3-week-old SPF Beltsville White turkeys, 6-week-old commercial Broad Breast White turkeys, and 10 to 20-week-old racing pigeons. Birds were monitored clinically and euthanized sequentially, with oral and cloacal swabs, blood, and tissues collected. Tissues were examined by histopathology and by immunohistochemistry and/or in situ hybridization to detect viral replication and distribution. Clinically, infected chickens and SPF turkeys showed severe depression and all died or were euthanized due to severe clinical signs by 5 days post-inoculation. In these birds, histologic lesions were widespread and virus was detected in multiple organs. In commercial turkeys and in pigeons, however, less than 15% of birds showed overt clinical signs, histologic lesions were minimal, and viral distribution was limited. In this study susceptibility to highly virulent NDV did vary among chickens, SPF turkeys, commercial turkeys, and pigeons.
142: THE PROGNOSTIC CLASSIFICATION OF CANINE CUTANEOUS MAST CELL TUMORS BASED ON KIT EXPRESSION PATTERNS.
Cutaneous mast cell tumors (MCTs) are one of the most common tumors in dogs and have an extremely variable biologic behavior. Currently, prognostic and therapeutic determinations for MCTs are primarily based on the histologic grade of the tumor. However, the majority of MCTs are of intermediate grade and therefore the prognostic value of histologic grading is highly questionable. Considering the prevalence of canine MCTs, the costs for therapy and the emotional stress for owners, a new prognostic classification is urgently needed. To evaluate the prognostic significance of KIT expression patterns in canine cutaneous MCTs, we studied 98 cutaneous MCTs from 98 dogs that had been treated with surgery only. The total survival time and the time to local or distant recurrence of MCTs were recorded for all dogs. Using immunohistochemistry, MCTs were stained with anti-KIT antibodies. Three KIT staining patterns were identified. The KIT staining patterns were identified as: 1. membrane-associated staining alone; 2. focal to stippled cytoplasmic staining; and 3. diffuse cytoplasmic staining. Based on univariate and multivariate survival analysis, increased cytoplasmic KIT staining was significantly associated with an increased rate of local recurrence, and decreased survival duration. Based on these results, we propose a new prognostic classification of canine cutaneous MCTs according to their KIT staining pattern that can be used for the routine prognostic evaluation of canine cutaneous MCTs.
143: AN OUTBREAK OF CLOSTRIDIUM PERFRINGENS TYPE D ENTEROTOXEMIA IN MILKING DOES.
Three recently purchased milking does (two Saanen and one Alpine) were submitted for postmortem examination with a history of sudden death affecting 9 of 325 does (3%) in a milking flock. At postmortem, all does were obese. Significant lesions included mild to marked pulmonary edema and congestion in all 3 does, and severe colonic mucosal necrosis in 1 doe. Histologically, marked pulmonary edema was the only significant lesion present in 2 does, and the third doe had marked multifocal necrohemorrhagic colitis with intralesional colonization of clostridial-like bacteria. Bacterial culture of small intestine and/or colon yielded a heavy pure growth of Clostridium peifringens. A diagnosis of C. perfringens type D enterotoxemia was confirmed by the detection of epsilon toxin typical of C. perfringens type D on genotyping. Almost 90% of the affected does in this outbreak were recently purchased, and they had not been vaccinated for C. perfringens type D at their home farm. C. perfringens type D enterotoxemia should be considered in the differential diagnosis of sudden death in small ruminants. The most striking postmortem findings in goats naturally infected with C. perfringens type D consist of pulmonary edema and necrotizing pseudomembranous colitis. Colitis can be missed easily in autolysed cases, and pulmonary edema may be the only lesion observed. Cerebral vasogenic edema or bilaterally symmetrical encepha-lomalacia, the pathognomonic lesion of C. perfringens type D in sheep, is not a common lesion in goats naturally infected with C. perfringens type D. The gold standard for diagnosis of C. perfringens type D is the identification of epsilon toxin by genotyping of the isolated bacteria.
144: AN OVERVIEW OF MARINE MAMMAL DIAGNOSES IN THE NORTHEASTERN PACIFIC FROM 1999 TO 2003.
Over the last 5 years, there has been an intensive effort to recover and examine stranded marine mammals in order to identify and monitor enzootic, exotic and emerging disease concerns in the northeastern Pacific Ocean. To date, 322 animals, consisting of 4 sea otters, 14 otariids, 198 pinnipeds and 106 cetaceans have been evaluated. Although only small numbers of sea otters have been examined, there has been no serologic or molecular evidence of Toxoplasma gondii, Neospora caninum or Sarcocystis neurona. Most pinnipeds submissions are neonatal and juvenile harbor seals from rehabilitation facilities that present with in utero or post partum malnutrition. Infectious diseases due to bacteria likely represent opportunistic infections secondary to generalized debilitation or immunosuppression. Primary pathogens recovered from stranded pups include Salmonella spp and Clostridium difficile. Exposure to phocid herpesvirus was initially detected by antibodies in 1998 and the first epizootic was recorded in August, 2003. The majority of cetacean submissions consist of Dall's and harbor porpoises with fewer Pacific white sided dolphins and common dolphins. Bacterial infections and parasitism are commonly recognized with fewer diagnoses of trauma. The first multispecies outbreak of cryptococcosis due to Cryptococcus neoformans gatti (type B) includes 13 Dall's and harbor porpoises in British Columbia. Localized infections or generalized septicemias due to Pseudomonas spp, Edwardsiella tarda. Vibrio vulnificus. Salmonella spp and Erysipelothrix rhusiopathiae have been identified. Antibodies to Brucella spp have been detected in post mortem heart blood in 6 of 15 harbor porpoises, 1 of 2 common dolphins, 2 of 6 Dall's porpoises, 1 of 1 grey whale and 4 of 4 killer whales. The pathogenesis of infection with these bacteria is unknown and intensive efforts are currently underway to isolate and characterize this organism and further resolve its pathogenicity. Between 1972 and 2002, 17 vessel strikes were documented along the British Columbia coastline with 5 animals reported over a 7 month time span in 2003. The results of these studies provide an invaluable indication of marine ecosystem health and help to document the relative importance of anthropogenic impacts on marine mammals.
145: CUTANEOUS MAST CELL TUMORS IN POTBELLIED PIGS (SUS SCROFA).
Mast cell tumors have been rarely reported in swine. This study characterized the pathology of cutaneous mast cell tumors in potbellied pigs. Medical records available through a local spay/neuter program and the Duchess Fund database were reviewed for clini-copathological features. Mast cell tumors were identified in 10 animals and tissues were available from 8 animals for histological review. Pigs (7 male and 1 female) were 6 to 11 years of age and presented with 1 to 15, sessile, raised, sometimes ulcerated, skin masses (1.0 to 4.0 cm) on the head, trunk, and/or limbs. Masses developed over a few weeks to 2–3 years and recurred after surgery in some pigs. Hematoxylin and eosin, toluidine blue (pH-3.5), and Giemsa stained sections were evaluated from formalin-fixed, paraffin-embedded tissues for each case. Additionally, for 2 cases, samples were collected at surgery into Trumps fixative (4F-1G) for transmission electron microscopy and into 10% formalin, alcoholic formalin, Carnoys, Clarks, formalin-acetic-alcohol fixatives for comparison of fixation methods on mast cell staining quality. For these two cases, giemsa, toluidine blue (pH-1.0 and pH-3.5), alcian blue (pH-1.0 and pH-2.5), and chloroacetate esterase were performed. Histologically, unencapsulated mast cell tumors developed in the superficial dermis and extended to the panniculus in larger masses; all were accompanied by eosinophils. Neoplastic mast cells had faintly granular cytoplasm, mild anisokaryosis and anisocytosis, and rare mitotic figures. Electron microscopy revealed characteristic cytoplasmic granules and surface microvillar projections. Neoplastic mast cells stained poorly with formalin fixation and stained best with Clarks and Carnoys fixatives utilizing toluidine blue (pH-3.5) and Giemsa stains. This is the first report of cutaneous mast cell tumors in potbellied pigs.
146: SPONTANEOUS FOCAL RENAL DYSPLASIA IN A NEW ZEALAND WHITE RABBIT (ORYCTOLAGUS CUNICULUS).
Spontaneous, focal renal dysplasia was diagnosed in a four-month-old, male New Zealand White Rabbit (Oryctolagus cuniculus) that was part of a group sacrifice on day nine of a toxicology study. This animal had exhibited no abnormal clinical signs prior to its euthanasia. At necropsy, the principal findings were present in the right kidney, and included multiple renal cortical cysts (0.2–0.5 cm in diameter), and diffuse tan discoloration of the kidney. Microscopically, a locally extensive focus of renal dysplasia was present abutting the cortical cysts. The dysplastic focus was characterized by disorganized, dense nests of primitive cortical tubules surrounded by a loose mesenchymatous stroma (persistent mesenchyme). Primitive cortical tubules were variably dilated, and occasionally contained finely granular, pale eosinophilic casts. Large segments of both kidneys were within normal histological limits. Rare immature glomeruli, arborizing glomeruli, and hyperplastic tubular epithelium (epithelial mats) were seen in the right kidney. The spontaneous renal dysplasia was not test article related, and likely resulted from a failure of the metanephric blastema to achieve complete differentiation. To our knowledge, spontaneous renal dysplasia has not been previously characterized in the New Zealand White Rabbit, but has been seen in Himalayan Rabbits following in utero treatment with thalidomide.
147: RETICULAR AND ABOMASAL EROSIONS, HEPATIC ABSCESSES, AND BACTERIAL EMBOLI IN A BLUE DUIKER (CEPHALOPHUS MONTICOLA).
A mature, male blue duiker (Cephalophus monticola) died after a four-day history of anorexia, tachypnea, and tenesmus. The animal, on exhibit with other ruminants and non-human primates, had been fed alfalfa hay, alfalfa pellets, orange slices and canned primate chow prior to its death. At necropsy, the Duiker was in thin body condition. The serosa of the forestomachs were adherent to the peritoneal wall, and were covered with fibrinonecrotic debris. The liver contained numerous, 0.1–1.0 cm diameter abscesses. Microscopically, erosions were found in the reticulum and abomasum. Fibrinonecrotic serositis and capsulitis of the forestomachs, the liver, and the mesenteric lymph node were present. Abscesses containing bacterial colonies (Fusobacterium necrophorum and Arcanobacterium pyogenes) were randomly distributed in the hepatic parenchyma. Hepatic venous thrombi were found. Marked lymphoid depletion, necrotizing fibrinopurulent lymphadenitis, and sinus histiocytosis were seen in the mesenteric lymph nodes. Bacterial emboli were seen in pulmonary vessels. An incidental finding included the presence of Besnoitia spp. cysts in the male accessory sex glands, and the tongue. The cause of the gastric erosions was unapparent. As with domestic ruminants, the hepatic, mesenteric lymph node and pulmonary lesions likely developed as sequelae to the erosions in the reticulum and abomasum. To our knowledge, neither the gastric lesions and their sequelae, nor the hepatic bacterial culture results have been previously reported in the blue duiker.
148: OVARIAN PLACENTAL SITE TROPHOBLASTIC TUMOR IN A CYNOMOLGUS MONKEY (MACACA FASCICULARIS).
Placental site trophoblastic tumor (PSTT) is a neoplastic proliferation of intermediate trophoblasts. This rare neoplasm typically occurs in the postpartum uterus, and less commonly in the ovary and oviduct. An ovarian mass was removed from a two-and-a-half-year-old cynomolgus monkey. There was no evidence of recurrence or metastasis when the animal was euthanized nine months later. Histologically, sheets and cords of polygonal cells separated by fibrinoid matrix effaced the ovarian cortex. Neoplastic cells had a moderate to abundant amount of eosinophilic, occasionally vacuolated cytoplasm. Nuclei were hyperchromatic to irregularly shaped with moderately stippled chromatin and prominent nucleoli. Anisocytosis and anisokaryosis were prominent. Occasional binucleated cells that resembled syncytiotrophoblasts were present. The mitotic rate averaged 1 per HPF. Vascular invasion with replacement of the blood vessel wall by trophoblastic cells was identified. The tumor cells were diffusely and strongly positive for cytokeratin and inhibin. Approximately 50% of the cells were positive for human placental lactogen (hPL), and less than 2% of the cells were positive for human chorionic gonadotropin (hCG). PSTTs consist primarily of intermediate trophoblasts with a few widely scattered syncytiotrophoblasts. By immunohistochemistry, intermediate trophoblasts and syncytiotrophoblasts are positive for hPL and hCG, respectively. Therefore, placental site trophoblastic tumors have predominant staining for hPL, with rare cells positive for hCG, as in this case. The differential diagnosis includes epithelioid trophoblastic tumor, choriocarcinoma, anaplastic carcinoma and juvenile granulosa cell tumor. The histomorphology and immunohistochemical staining pattern are consistent with a tumor of intermediate trophoblasts. We are unaware of any other reports of PSTT in cynomolgus monkeys.
149: PYOMETRA CAUSED BY BACTEROIDES FRAGILIS IN A RHESUS MONKEY (MACACA MULATTA).
Anaerobic bacteria are recognized to be important pathogens in human female genital tract infections. Patients who contract genital tract infections are predominantly young, are otherwise healthy and generally respond well to treatment. With treatment, prognosis for cure is excellent; however, sequelae such as recurrent infections, infertility, or ectopic pregnancy can be serious. Among the anaerobic gram-negative bacteria, Bacteroides spp. are the most frequently encountered. Bacteroides fragilis is the most common anaerobic organism found in human clinical specimens. Although it is the anaerobe most frequently associated with bacteremia, a common isolate in intraabdominal infections of the female genital tract, and associated with wounds and abscesses in humans, B. fragilis is rarely the cause of infections in domestic, wild or laboratory animals. Here we report a case of pyometra due to Bacteroides fragilis in a rhesus monkey (Macaca mulatto). This animal was presented for clinical evaluation due to generalized pallor and heavy menstruation. On examination, the uterus was mildly enlarged and contracted with a smooth surface. Endometriosis was suspected and the monkey was euthanized and submitted for necropsy. Grossly, the uterus was markedly dilated with pus. Pure culture of Bacteroides fragilis was isolated from the uterus.
150: CRYPTOCOCCOSIS IN A MONGOOSE LEMUR (EULEMUR MONGOZ).
A 27 year-old female mongoose lemur (Eulemur mongoz) was euthanized after a three day history of rapidly progressive neurologic signs with polyuria, polydypsia and polyphagia. Blood-work and urinalysis revealed marked hyperglycemia and glucosuria. Histopathologic examination of tissues revealed numerous 4 to 10 micron yeasts surrounded by a clear halo (Cryptococcus sp.) throughout the meninges and brain parenchyma. Also observed were numerous acute and chronic thromboses within the parenchymal vessels, not associated with fungal organisms. There was evidence of systemic atherosclerosis and glomerulosclerosis. The constellation of lesions in this animal are considered to be consistent with chronic diabetes mellitus and opportunistic fungal infection in a compromised host. To our knowledge, this is the first report of cryptococcosis in a mongoose lemur.
151: DISSEMINATED TOXOPLASMOSIS IN A CAPTIVE RING-TAILED LEMUR (LEMUR CATTA).
Ring-tailed lemurs (Lemur catta) are diurnal, herbivorous primates indigenous to southern Madagascar. They are currently listed as vulnerable to extinction due to the loss of dry brush, scrub and closed canopy forest habitats. Substantial numbers of captive ring-tailed lemurs exist and despite efforts to maintain populations, these animals are often predisposed to a variety of captivity related infectious diseases. A 3-year-old, secundiparous, captive female ring-tailed lemur died following a brief history of dyspnea, lethargy and anorexia. The predominant histomorphologic features included diffuse lymphohistiocytic interstitial pneumonia and massive hepatocellular and splenic necrosis with parenchymal collapse. Numerous, crescent-shaped, 2 × 6 micrometer long, free and intrahistiocytic tachyzoites, and several 20 to 50 micrometer diameter, thin walled tissue cysts were associated with the histologic lesions. The tachyzoites reacted strongly with polyclonal anti-Toxoplasma gondii antibody in an avidin-biotin immunohistochemical procedure. Ultrastructurally, T. gondii tachyzoites had distinct electron lucent rhoptries, conoids and parasitophorous vacuoles. PCR with RH strain specific primers (TOX4 and TOX5) amplified a 529 bp internal transcribed spacer (ITS) rDNA fragment and the gene sequence was consistent with genotype II, avirulent murine T. gondii strains. These pathologic and molecular observations suggest that ring-tailed lemurs and other captive primates may be uniquely susceptible to relatively avirulent strains, hence posing a risk for captive, zoological and nature reserve collections.
152: TUBERCULOSIS DUE TO MYCOBACTERIUM AVIUM IN A CAPTIVE BENGAL TIGER (PANTHERA TIGRIS).
A captive adult female Bengal Tiger (Panthera tigris) died after a prolonged anorexia in the zoo in South Korea. Necropsy examination showed multiple nodules in the lungs, liver and spleen. Histopathologically there were typical granulomas composed of necrotic areas surrounded by lymphocytes and foamy macrophages. Serology for Toxoplasma gondii was negative. PAS stain did not revealed mycotic agents. Ziehl-Neelsen stain revealed a few acid-fast organisms within liver and lung. Multiplex polymerase chain reaction (PCR) analysis of both the liver and lung showed a perfect match for Mycobacterium avium. This is a rare case of infection of a wild mammal with the avian type of tuberculosis and strongly suspect that the infection originated from culled chickens being fed regularly to the captive tigers in the zoo.
153: DEMODEX SPP. IN THE PERINEAL HAIR FOLLICLES OF RHESUS MACAQUES (MACACA MULATTA).
The perineal skin was evaluated from 69 rhesus macaques as part of a necropsy protocol. Microscopic evaluation of hematoxylin and eosin stained skin sections revealed 20 animals positive for the mite Demodex spp. Reaction to the mites ranged from minimal follicular epidermal hyperplasia to furunculosis. To our knowledge this is the first report of Demodex spp. in rhesus macaques.
154: MALIGNANT MENINGIOMA WITH VISCERAL METASTASES IN A GOLDEN POISON DART FROG (PHYLLOBATES TERRIBILIS).
A 6-year-old female Golden poison dart frog (Phyllobates terribilis) developed progressive ataxia and coelomic effusion. Necropsy revealed numerous, coalescing, 1 to 3 mm diameter, off white, moderately circumscribed, firm infiltrates throughout the liver and kidneys. Serial sections of the head revealed an approximately 3 mm diameter, poorly circumscribed and infiltrative neoplasm arising within the meninges along the dorsolateral aspect of the brain. The neoplasm was composed of dense, plump, spindle to polygonal cells arranged in sheets within sparse, vascular stroma. The cells had sparse, lightly eosinophilic cytoplasm, oval or indented nuclei, and coarsely stippled and hyperchromatic chromatin. The mitotic index was 4 per 10400x fields. The cells were invading the medullary space of the calvaria, and through the calvaria into the dorsal skeletal muscle of the head. The neoplasm was compressing the brain but not invading it. The liver and kidney lesions were infiltrative metastases of similar cells replacing most of the parenchyma. Smaller and fewer metastases were also detected in lung and thyroid gland. Ultrastructurally, the neoplastic cells had numerous cell processes forming interdigitations with frequent connecting desmosomes between cells. The cells stained strongly positive for vimentin by immunohistochemistry. These features supported a diagnosis of primary malignant meningioma with visceral metastases. To the authors’ knowledge this is the first report of a meningioma in an amphibian.
155: MULTIPLE TRICHOEPITHELIOMAS IN AN ALPACA (LLAMA PACOS).
The incidence of development and clinical behavior of camelid neoplasias are rarely characterized in current veterinary literature. A spontaneously developing trichoepithelioma in an aged adult alpaca is examined. Trichoepitheliomas are benign follicular tumors most commonly reported in domestic canines, less so in domestic felines, and rare or unrecognized in other species. These benign tumors of follicular origin have a predilection for the back, thorax, and tail, with infrequent multicentric presentation. Treatment is via surgical excision, without local recurrence or metastasis reported in domestic species. Trichoepitheliomas are unreported in camelids. A thirteen year old, male alpaca (Llama pacos) presented in early May 2004 with disseminated, well circumscribed, non-ulcerated intradermal masses of unknown duration. Individual masses were variably alopecic and approximately 1–4 cm in diameter. Several of the masses were excised and submitted to the Veterinary Medical Diagnostic Laboratory in Columbia, MO for evaluation. Histologically, all specimens were consistent with well circumscribed, dermal cysts lined by a mixed population mainly composed of multiple layers of polygonal, basilar-type cells, with euchromatic nuclei and having variable amounts of pale, eosinophilic cytoplasm. These cells undergo abrupt keratinization, without the development of a granular cell layer. The cyst lumens are filled with concentric layers of lamellar keratin and clumps of melanin pigment. Currently, the alpaca is doing well with no further clinical concerns.
156: NF-kappaB EXPRESSION IN SPONTANEOUS CUTANEOUS SQUAMOUS CELL CARCINOMAS OF DOGS, CATS, HORSES AND CATTLE.
The transcription factor NF-kappaB has been implicated in the oncogenesis of a variety of tumors; however, its role in the development of squamous cell carcinoma (SCC) has not been fully characterized. Some human and rodent studies indicate activation of NF-kappaB promotes SCC, while, in others, inhibition of NF-kappaB was associated with the spontaneous development of SCC. The goal of this project was to characterize the NF-kappaB expression in spontaneous SCC of domestic animals. Immunohistochemistry (IH) for the NF-kappaB subunit p65 was performed on biopsies from naturally occurring canine (n = 46), feline (n = 47), equine (n = 53) and bovine (n = 29) SCC. Both cytoplasmic and nuclear staining was assessed and compared to that present in normal and hyperplastic epidermis. All results reported were significant (p < 0.05). Canine SCC had increased nuclear staining for NF-kappaB p65 over both that observed in normal and in hyperplastic epidermis, and increased cytoplasmic staining over that observed in normal epidermis. Increased nuclear staining over that in normal epidermis was observed in both SCC and hyperplastic epidermis in feline skin biopsies. Equine SSC exhibited increased nuclear staining compared to both normal and hyperplastic epidermis, and increased cytoplasmic staining compared to normal epidermis. Bovine SCC and hyperplastic epidermis had increased cytoplasmic staining compared to that in normal epidermis. IH for IkappaBalpha and Hras was also performed to further characterize potential tumor pathogenesis. In addition to little nuclear staining for p65, bovine SCC had increased staining for both Hras and IkappaBalpha. These results imply there may be species differences in the molecular oncogenesis of SCC.
157: HEMORRHAGIC BOWEL SYNDROME (HBS) IN CATTLE.
Previous observations of segmental jejunal impaction and jejunal coagulated blood cast accumulations in dairy cows in the US and Europe have indicated an association with Clostridium perfringens type A. Isolation of clostridial organsims in pure culture and of clostridial toxins from affected cows has given support to the hypothesis that the bacilli may be involved in the pathogenesis of this syndrome. Typically, the syndrome is sporadic and seasonal in Florida (November–January), involves adult cows and has a low mortality rate. Affected animals exhibit clinical signs of inappetence, depression and abdominal distension, pass jelly-like feces intermingled with blood coagula, and have a marked drop in milk production. Animals at surgery or necropsy have jejunal loops focally distended by doughy reddened fecal masses and bleeding ulcers beneath. The common microscopic diagnosis is that of an erosive-ulcerative, necrohe-morrhagic jejunitis with intraluminal hemorrhage or blood clots. Gram stains demonstrate clusters of gram-positive, medium-sized rods in areas of the intestinal necrotic debris. A recently observed hemorrhagic intestinal impaction scenario at the University of Florida involved 2 adult Angus cows. Unlike in dairy cows where the jejunum is involved, the 2 animals had lesions of HBS in the spiral colon. Fecal cultures were positive for Clostridium perfringens genotype A in both animals. HBS needs differentiaton from winter dysentery, cecal torsion, intussusception, intestinal coccidiosis and environmental and feed toxins. Tests for Salmonella sp. and bovine mucosal disease virus are commonly negative.
158: PROTEASOME INHIBITORS DO NOT INDUCE INCREASED ENDOGENOUS PRION PROTEIN (PRP) OR THE FORMATION OF PROTEINASE K-RESISTANT PRP IN MURINE AND HUMAN NEURONAL CELL LINES IN VITRO.
Prion diseases are a group of closely-related neurodegenerative conditions that are associated with the conversion of cellular prion protein (PrPC) to a protease-resistant isoform (PrPSc). Studies in PrP transfected cells caused the accumulation of a PrPSc-like form of PrP suggesting a potential theoretical link between proteasome inhibition and the pathogenesis of prion disease. However, these findings were not duplicated in nontransfected neuronal cell lines and primary neurons. This discrepancy has been attributed to the potential of proteasome inhibitors to selectively induce transcription from expression constructs carrying a heterologous viral promoter. We determined if pharmacologically relevant concentrations of various proteasome inhibitors resulted in the accumulation of normal or proteinase K resistant PrP in nontransfected mouse (N2A, GT-1) and human (NT-2) neuronal cells. Cells were exposed to lactacystin, epoxomicin, and MG 132 for 16 hours and then examined by Western analysis. Verification of proteasome inhibition was demonstrated by accumulation of c-jun after exposure of each cell line to the proteasome inhibitors. All cell lines had abundant normal PrPC expression and no indication of an increase in the amount of endogenous PrPC or the conversion of it into a PrPSc-like form upon treatment with the proteasome inhibitors. In conclusion, this study indicates that in vitro studies with nontransfected murine and human neuronal cell lines expressing wild-type PrP exposed to proteasome inhibitors do not show increased levels of PrP or the formation of proteinase-resistant forms. Thus, the experimental data indicates that there is negligible risk associated with proteasome inhibition and prion-related disease.
159: STRATEGY FOR HAZARD ASSESSMENT AND RISK MANAGEMENT OF DRUG-INDUCED PHOSPHOLIPIDOSIS.
Drug-induced phospholipidosis refers to an excessive reversible accumulation of phospholipids and drug in lysosomes. It is usually manifested in tissue as cytoplasmic vacuolation or in the case of lung, foamy alveolar macrophages. Ultrastructurally, the change is manifested by multi-layered lamellar lysosomal inclusions. The presence of phospholipidosis signals a change in cell membrane integrity and is predictive of drug or metabolite accumulation in affected tissues. Phospholipidosis presents a formidable challenge for hazard assessment and risk management in humans because it varies greatly with species and strains of animal model, organs affected, therapeutic agent and dose, duration and route of treatment, etc. Although a reliable biomarker has not been fully defined, the characteristic cationic amphiphilic chemical structure of the agent and in vitro models may aid in early detection. In vivo assessments of organ involvement in addition to the therapeutic index and class, dosage regimen (short or long-term use), and potential for reversibility are factors that should be considered in risk management of phospholipidosis. This presentation will provide guidelines for hazard assessment and risk management of phospholipidosis in drug development.
160: SCREENING STRATEGIES IN HIT-TO-LEAD AND EARLY LEAD OPTIMIZATION WITH GOOD PREDICTIVITY FOR DEVELOPMENT LIMITING SIDE EFFECTS AND TOXICITY.
The early identification of development limiting toxicity via small scale non-GLP pharmacology and toxicology studies has reduced pharmaceutical attrition from the contemporary benchmark 35-to-45% to <15%. Further improvement in efficiency in R&D lies in leveraging “best in class” technology and early integration with medicinal chemistry and pharmacology activities during hit-to-lead (H2L) and early lead optimization (LO). Liver toxicity represented the predominant reason for drug withdrawals and “black box” warnings from 1975 to 1999, now supplanted by CNS and CV functional side effects. H2L and early LO toxicity and safety pharmacology screening, to be practical, must be relatively high throughput with low test agent consumption (<10 mg/assay). To meet this challenge we have adopted a discovery assay by stage (DABS) paradigm that clearly delineates the timing and type of assay required for selection of the lead series and for representatives from the lead series to progress. The DABS paradigm utilizes target expression profiling and knockout phenotyping to predict pharmacology; leverages in vitro toxicology assays (mutagen and clastogen assessment, hERG binding and transcription profiling of primary rat hepatocytes) as selection criteria for in vivo efficacy testing; integrates chemical and on/off target toxicity assessments into in vivo efficacy studies; and facilitates early identification of serum and tissue biomarkers of efficacy and toxicity. Our early LO in silico and in vitro testing paradigm for toxicity and side effects has well defined predictivity for human response (data we will share) and for development limiting barriers focusing on QT interval prolongation, liver toxicity, and genetic toxicity. Our primary rat hepatocyte TP screening has 75% concordance with liver toxicity with no false positives and is now in progress to “roll out” as a routine screen.
161: IDENTIFICATION AND CHARACTERIZATION OF TESTICULAR TOXICANTS IN RATS WITH DNA MICROARRAYS.
Testicular toxicity is not uncommon following exposure to environmental toxicants and to pharmaceutical compounds. However, little is known of the mechanisms by which these agents cause testicular toxicity, and no reliable biomarker exists to detect early testicular changes. We evaluated whether transcription profiling data generated with microarrays (Affymetrix RAE230A) could provide a mechanistic understanding of testicular toxicity and predict toxicity before morphologic changes are observed. Testes were collected from male CD rats (n = 4), treated orally for 1 (Day 1) and 4 (Day 4) days with one of 3 Sertoli cell toxicants [boric acid at 1000 mg/ kg,dibromoacetic acid (DBAA) at 250 mg/kg,mono-(2-ethylhexyl) phthalate at 1000 mg/kg,single dose] or a germ cell toxicant [ethylene glycol monomethyl ether (EGME) at 200 mg/kg]. Testes were collected for transcription profiling and histology. Histological changes were observed only in rats treated with DBAA and EGME. All compounds regulated the expression of a relatively low number of genes at both timepoints. At Day 1, there were no consistent gene expression changes that distinguished compounds or the two mechanistic classes of testicular toxicants. However, at Day 4, gene expression changes allowed the differentiation of the Sertoli cell toxicants from EGME. Analysis of these gene expression changes provided some potential mechanisms for the toxicity induced by the Sertoli cell toxicants, including the disruption of Sertoli and germ cell intercellular junctions. Moreover, we identified several genes with a consistently altered expression pattern, providing a basis for the identification of potential biomarkers. Collectively, these results indicate that transcription profiling can be used to predict and gain a molecular understanding of testicular toxicity in the rat.
162: INVESTIGATION OF HEMATOTOXICITY IN DOGS AND RATS WITH PSEUDO PELGER-HUËT ANOMALY.
Pelger-Huët anomaly is characterized by a reduced number of nuclear segments of granulocytes and can be hereditary or acquired (pseudo Pelger-Huët (PPH)). PPH has been associated, in man and animals, with a variety of primary hematological disorders, infections and drugs. Oral administration of an experimental compound in dogs and rats during 4-week toxicity studies revealed the presence of PPH cells in the peripheral blood, associated in dogs with decreased erythron and increased platelet counts and in rats with increased reticulocyte and leukocyte counts, suggestive of dysfunctional hematopoiesis. Examination of bone marrow smears in both species did not reveal abnormalities. To elucidate potential compound effect on multipotential and progenitor cells, hematotoxicity testing via colony forming assay procedure was performed, using human, canine and rat bone marrow cells cultured in vitro. Colony-forming cell-granulocyte, erythroid, macrophage, megakaryocyte (CFC-GEMM), burst-forming unit-erythroid (BFU-E) and granulocyte-macrophage colony-forming cell (GM-CFC) were cultured in presence of growth factors and rising doses of experimental compound. For all species and cell populations tested, the compound demonstrated hematotoxicity by inhibition of the CFC-GEMM, BFU-E and GM-CFC, indicating an action at the multipotential stem cell level, affecting erythroid, myelomonocytic and megakaryocytic cell lineages. Test set up did not permit the determination of experimental compounds’ mechanism of action nor PPH cell formation, and could not predict if stem cell toxicity would be partial or permanent but confirmed toxic effect on bone marrow. This case illustrates how in vitro stem cell testing can be used, in addition to conventional testing, to evaluate potential hematotoxicity of xenobiotics and can play a key role in early detection of adverse toxicity and improve safety assessment.
163: CANCER PHENOTYPE BIOMARKERS IN HUMAN XENOGRAFT BEARING MICE BY SERUM PROTEOMIC PATTERN RECOGNITION USING SURFACE ENHANCED LASER DISORPTION AND IONIZATION MASS SPECTROSCOPY ANALYSIS.
Low molecular weight proteins detected in serum of cancer patients analyzed by mass spectroscopy can reliably predict specific cancer disease states. Employing a similar approach to pattern recognition analysis of peptide ions discovered by SELDI time of flight MS, we sought to determine if serum from mice bearing human cancer xenografts could be predictive for cancers in analogous preclinical studies. Nude mice given threshold doses of human ovarian and colon cells developed cancer xenografts. Study protocols, serum collection and processing on weak cation exchange resin surfaces for SELDI-TOF assay were harmonized with methods used in clinical studies. All mass spectra generated met quality assurance criteria. Computer algorithms were used to establish training and testing models in n-dimensional space representing foci of cancer and non-cancer using sera from known treatment groups. When 86 longitudinally collected sera were used to establish models for comparing known cancer with prebleed sera, disease states representing benign and cancer conditions were predicted with 94.4% sensitivity and 95.8% specificity. Using models created from 95 longitudinally collected sera representing both mice with cancer and disease-free sham control mice inoculated with saline vehicle only, cancer was predicted with 100% sensitivity and specificity. Results to date indicate that preclinical studies of serum proteomics can be aligned to clinical studies. Preliminary findings suggest differences between cancer types and among different cancer burdens are detectable. Additional analyses are being conducted to elucidate these differences.
164: IMMUNOSUPPRESSIVE-RELATED LYMPHOPROLIFERATIVE DISORDER (IRLD) OF MACAQUES AS A MODEL OF POST-TRANSPLANT LYMPHOPROLIFERATIVE DISORDER (PTLD).
Following treatment with an immunomodulator drug, a spectrum of lymphoproliferative lesions was diagnosed in cynomolgus monkeys (Macaca fascicularis), based on a modification of the diagnostic criteria for PTLD. In this study, lesions were associated with infection by a lymphocryptovirus (LCV) similar to those described in immunosuppressed humans and monkeys following transplantation. In human patients PTLD is potentially fatal even in the early lesion stage, but may go into remission in most stages. Only individual lymph nodes and a spleen were diagnosed with early lesions, characterized by some architectural preservation, but containing lymphoblasts along with plasma cells and lymphocytes. Polymorphic IRLD with destruction of the underlying architecture showing a full range of B cell maturation was the most common lesion type in monkeys. Monomorphic IRLD, containing confluent sheets of transformed cells was present in a few animals. An additional animal had a Hodgkin disease-like lesion with Reed-Sternberg cells which were CD30 +. One lesion was CD3+, but most were CD20+, and with the exception of two cases that were polytypic, they were monotypic for light chains. All lesions hybridized with an EBER-1 probe and most had EBNA-2 immunoreactivity. Other changes included epithelial lesions due to LCV, candidiasis and protozoiasis of the GI tract. Following a recovery period, no IRLD occurred, suggesting reversibility of lesions in macaques
165: ANALYSIS OF MICRO-DISSECTED MESENTERIC VASCULAR ELEMENTS FROM RATS GIVEN SKF-82526 (FENOL-DOPAM): TEMPORAL GENE EXPRESSION USING AFFYME-TRIX GENECHIP®.
Numerous vasoactive drugs are known to produce vascular damage in species used for pre-clinical drug safety assessment. To further evaluate drug-induced vascular injury, Fenoldopam (DA1 agonist) was administered to rats at a dose known to induce mesenteric arterial damage. Laser capture microdissection of selected regions of the mesenteric arteries followed by RNA linear amplification, and GeneChip® microarray analysis were used to evaluate differential gene expression in vehicle control rats and those terminated 1 or 4 hours following dosing. Comparative analysis of endothelial or smooth muscle enriched samples between control and treated rats revealed a number of dramatically regulated transcripts. Selected Fenoldopam regulated genes were confirmed by Real-Time RT-PCR and/or in situ hybridization and immunohistochemistry. Fenoldopam regulated transcripts belong in many key biological pathways including, cell adhesion, cell growth, metabolism, signal transduction, stress response and inflammation/immunoregulation. In depth bioinformatic analysis of the regulated genes identified signaling pathways involved in vasoregulation (dilation/resistance) and hemorrhage. Many of these pathway alterations make intuitive sense relating to development of morphologic lesions; such as protease upregulation/extracellular matrix remodeling, and down-regulation of adherence molecules which might lead to intramural hemorrhage and medial damage seen after 12 hr post-dose. Laser capture microdissection of tissue elements followed by transcriptomics has allowed rapid determination of a large number of previously unknown pathogenic pathways which can now be experimentally tested for their involvement in evolution of this drug-induced vascular lesion.
166: CARDIAC GENE PROFILING IN SPRAGUE-DAWLEY RATS ASSOCIATED WITH MINIMALLY INVASIVE SURGERY.
Surgery is a stressful event associated with increased serum catecholamines in humans and animal models, despite attempts at mitigation with various anesthetic/analgesic regimens and psychological counseling in humans. The purposes of this study were to profile cardiac gene expression in a rat model, and to explore the underlying mechanism of perioperative cardiac ischemia, a common surgical complication in cardiac and non-cardiac surgery in human patients. A group of 10 male Sprague-Dawley rats 2–3 months of age received open-chest surgery under general endotracheal anesthesia with isofiurane. At 3, 6, 9, 24 and 48 hours after surgery, 2 rats were euthanized with CO2 and the heart was collected for gene profiling with RG-U34A GeneChips. Another 6 male rats, without any surgical manipulation, served as controls. In surgically manipulated rats, approximately 80 genes or gene fragments were down-regulated (< 2 fold over the controls) and 134 were up-regulated (> 2-fold over the controls) in the heart at least at one time point. The affected genes included those associated with cardiomyocyte structure components, ion channels, signaling pathways, cell cycles, nuclear factors and metabolic enzymes. The most down-regulated genes were associated with cardiomyocyte structural components and enzymes involved in carbohydrate, protein and lipid metabolism. The most up-regulated genes were those associated with stress responses, inflammation, immunity and tissue remodeling. Many genes associated with ion channels, signaling pathways, cell cycles or nuclear factors were either down- or up-regulated. Results indicated that the cardiac gene expression in response to surgery was multifaceted; the stress responses play important roles in the perioperative cardiac injury.
167: LASER MICRODISSECTION (LMD) FOR TOXICOGENOMIC ELUCIDATION OF MECHANISMS OF RENAL TOXICITY.
Laser microdissection (LMD) is a powerful technology for isolation of small areas or cell populations from heterogeneous histologic sections allowing analysis of DNA, RNA or proteins while effectively avoiding cross contamination. The goal of this study was to optimize and validate a LMD technique for RNA gene expression studies. The Leica AS Laser Microdissection system was used to isolate and excise specific regions of the kidney. Qiagen RNeasy Micro kit was found to be the optimal RNA extraction technique based upon RNA integrity and yield. LMD processing times up to 4 minutes had no negative impact on RNA integrity and yield. Similar RNA quality was obtained from all sample sizes (500 to 40,000 cells) and increasing amounts of RNA were obtained with increasing cell number. RNA extractions from whole kidney homogenates and cortex, outer medulla and inner medulla zones obtained by LMD were evaluated. Fourteen differentially regulated genes identified from the literature were evaluated by RT-PCR to validate uniformity and/or differential expression of cellular and region specific genes in the various LMD and whole tissue samples. A pilot toxicogenomic study utilizing LMD was initiated. Administration of AVE XY to Sprague Dawley rats resulted in histopathological changes of dilated tubules localized specifically to the outer medulla of the kidney. RNA expression profiling of outer medulla using the Affymetrix RGU34A chip identified 205 differentially regulated genes in controls versus treated animals. Previous profiling of whole kidney homogenates had identified 195 differentially regulated genes. Comparison of data sets identified 88 similarly regulated genes with unique sets of genes identified from whole kidney homogenates and LMD acquired outer medulla. Further analysis of the potential role of these genes in toxicologic mechanisms is in progress.
168: TCDD-INDUCED PROLIFERATION OF LUNG EPITHELIAL CELLS POST-INFLUENZA VIRUS INFECTION IS NOT DUE TO INCREASED TISSUE DAMAGE.
Examination of histopathologic changes in lungs of mice nine days after influenza virus infection revealed aberrant epithelial proliferation in the mice pretreated with the pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent aryl hydrocarbon receptor agonist. Since TCDD has been associated with an increased incidence of lung tumors, we wanted to explore the mechanism of increased epithelial proliferation. Increased epithelial proliferation can be a response to lung injury. Therefore, we wanted to assess whether there is increased lung damage in TCDD treated mice. To accomplish this, 8-week-old female C57B1/6 mice were gavaged with 10 fig/kg TCDD or vehicle one day prior to a sublethal infection with influenza A virus (H3N2). Mice were sacrificed and bronchoalveolar lavage fluid (BALF) was collected 0.5, 1, 3, 5, 7, and 9 days post influenza infection. Lactate dehydrogenase (LDH), a marker of cellular injury, and protein, a marker of vascular leakage, were measured. A separate group of mice was sacrificed 7 days after infection for lung wet/dry weight ratios to further assess edema. There were no differences in BALF LDH or protein concentrations or in wet/dry weight ratios. These findings suggest that the increased proliferation seen in the TCDD-exposed animals is not the result of increased injury to the lung, but instead is due to some other effect of TCDD. Thus, this model may be useful in assessing the carcinogenicity of TCDD.
169: AN OVINE MODEL OF ACUTE RESPIRATORY DISTRESS SYNDROME (ARDS) SECONDARY TO INHALATION OF CHLORINE GAS.
Chlorine gas inhalation leads to rapid and severe lung injury. A widely available industrial chemical, it could be used as a weapon of opportunity by terrorists. We developed a severe, survivable, large animal model of acute chlorine gas inhalation injury. Sixteen tracheostomized, anesthetized sheep were exposed to variable doses of chlorine, ranging from 56–500 ppm × 300–600 liters, delivered by closed circuit over 30–60 minutes (10 liters/minute). Animals were maintained on a ventilator for 96 h post injury. A bimodal pattern in the hypoxia was seen, featuring early (most likely reflex-mediated) hypoxia, followed by partial recovery, then ARDS. Pneumothorax was seen in some animals, occasionally resulting in cardiovascular collapse. Computed tomography (CT) scans demonstrated both ground-glass opacification, suggesting an alveolar lesion; as well as peribronchiolar edema, suggesting airway injury. With disease progression, there was often complete consolidation of large portions of the lung. On histopathology, the extent and severity of lesions varied both between animals and within lung regions. In severely affected animals, there was widespread necrosis involving all pulmonary tissues. The lungs of less severely affected animals were characterized by multifocal to coalescing, variably severe combinations of necrosis, acute to subacute inflammation, bronchiolar epithelial attenuation, type 2 pneumocyte hyperplasia, congestion, hemorrhage, and intra-alveolar fibrin and edema. The rapid, severe hypoxia induced in this model make it highly suitable for the study of ARDS. The model will now be used to test new technologies for lung support.
170: PHYSEAL HYPERTROPHY IS A PHARMACOLOGIC BIOMARKER FOR TRANSFORMING GROWTH FACTOR-β RECEPTOR INHIBITORY ACTIVITY IN RATS.
Transforming growth factor-β is critical in the pathogenesis of fibrosis and its signalling pathway, including the activin-like kinase 5 (ALK5) receptor domain, is a therapeutic target for drug discovery. In toxicologic investigational studies of 4–10 days in 10-wk old Sprague-Dawley rats, using 10 different proprietary ALK5 inhibitors, expansion of the hypertrophic and proliferation zones of the physes of the femur and tibia were noted. Chondrocytes were increased with increased deposition of chondroid matrix. Subphyseal hyperostosis was also present. The degree of hypertrophy was dose and time dependent. Aged rats were refractory, presumably due to decreased physeal growth. Zones of physes were laser microdis-sected from ALK5 inhibitor treated and control rats after 3 and 5 days and transcripts for TGF-β1 and 2, ALK5, IHH, VEGF, OP-1, IGF-1, βFGF and PTHrp were amplified by real-time PCR. TGF-β1 transcripts were increased in all zones after 3 days, while TGF-β 2 expression decreased in all zones. IHH expression was decreased in resting/proliferative zones after treatment, but increased in the pre-hypertrophic zone and perichondrium. IGF-1 and βFGF were down-regulated in perichondrium while BMP-7 expression was increased. PTHrP expression was elevated in perichondrium and proliferative zones. VEGF expression decreased in the hypertrophic zone. Since physeal hypertrophy coincided with areas of TGF-β activity, and expression patterns of cytokines associated with TGF-β and known to affect physeal maturation were altered after treatment, we propose that physeal hypertrophy could be used as a pharmacologic bio-marker of ALK5 inhibitory activity in the rat.
171: EXPERIMENTAL TRANSMISSION OF CHRONIC WASTING DISEASE AGENT TO CATTLE BY INTRACEREBRAL ROUTE: FINAL OUTCOME OF THE STUDY.
Thirteen calves were inoculated intracerebrally with brain suspension from mule deer naturally affected with CWD. Three other calves were kept as uninoculated controls. The experiment was terminated at 6 years post inoculation (PI). During that time, prion protein (PrPres) was found in the central nervous system (CNS) of 5 cattle. Microscopic lesions suggestive of spongiform encephalopathy in the brains of these PrPres positive animals were subtle in the first 3 and absent in the latter 2 cases. However, all 5 animals were positive for PrPres by both immunohistochemistry and Western blot. The 3 uninoculated control cattle and 8 other inoculated animals euthanized during this time did not have PrPres in their CNS. Degenerative changes indicative of neuroaxonal dystrophy (NAD) were seen in dorsal medulla oblongata and appeared to be related to advancing age in both inoculated and control cattle. Analysis of the gene encoding bovine PRNP revealed similar findings, i.e., homozygosity for alleles encoding 6 octapeptide repeats, serine (S) at codon 46 and S at codon 146 in all samples. Findings of this study show that although PrPres amplification occurred following direct inoculation into the brain, none of the affected animals had classical histopathological lesions of spongiform encephalopathy. Furthermore, only 38% of the inoculated cattle demonstrated amplification of PrPres. Although intracerebral inoculation is an unnatural route of exposure, and is the most severe challenge possible, this experiment shows that CWD transmission in cattle can have long incubation periods (up to 5 years). This finding suggests that oral inoculation of cattle with CWD would require not only a much larger dose of inoculum, but also, may not result in amplification of PrPres within CNS tissues during the normal lifespan of cattle. It is possible that a second bovine passage of material (brain infected with CWDmd) from this study may result in a larger proportion of affected cattle with a shortened incubation time, and may produce different clinical and pathological findings. Such a study is now in progress. Also, experimental inoculations of cattle with CWD isolates from white-tailed deer and elk are needed to compare clinicopathological findings with the present study and these studies will be initiated in the near future.
172: USE OF CALBINDIN D-28 AND MICROTUBULE ASSOCIATED PROTEIN-2 IMMUNOHISTOCHEMISTRY TO CHARACTERISE NEUROTOXICITY AS PART OF A REGULATORY TOXICITY STUDY.
Background: Cerebellar toxicity can be difficult to detect using conventional histological staining procedures, such as haematoxylin and eosin (H&E). The use of immunohistochemistry (IHC) in the CNS can provide benefits in terms of sensitivity and specificity. However one of the problems with their use in detecting toxicity in regulatory studies is their application to routinely prepared material, e.g. immersion formalin-fixed, paraffin embedded (FFPE) sections. Aim: To develop an IHC method and evaluate expression of calbindin D-28 and microtubule associated protein-2 (MAP-2) as markers in the detection, characterisation and grading of unexpected CNS toxicity in the rat. Results: High power examination of H&E stained brain sections of treated rats 2 days following a single oral dose of a novel compound revealed irregular vacuolation of the molecular layer and Purkinje cell degeneration. Animals killed following 14 days recovery showed Purkinje cell degeneration but vacuolation of the molecular layer was absent. In treated animals, low power examination revealed loss of calbindin D-28K expression in degenerating neurons arranged in parasagittal stripes within the vermis. In control animals, MAP-2 expression was high in Purkinje cell dendrites and cell bodies in the molecular layer, basket and stellate perikarya. Following treatment, MAP-2 expression was reduced in Purkinje cell bodies and molecular layer of the vermis forming pale parasagittal stripes. Conclusions: This is the first description of successful use of these two markers in a regulatory toxicity study using FFPE brain. These markers provide a sensitive method for characterising CNS toxicity which can be detected at low power enabling easier detection, screening and grading of neurotoxicity.
173: DIFFERENTIAL EXPRESSION OF COX-1 AND COX-2 IN THE GASTROINTESTINAL TRACT OF THE RAT.
Background: Sustained inhibition of cyclooxygenase (COX) enzymes by nonsteroidal anti-inflammatory drugs (NSAIDS) can cause intestinal ulceration in humans and laboratory animals. The central dogma that COX-1 inhibition causes the gastrointestinal (GI) side effects has recently been challenged by the observation that COX-2-/- mice can spontaneously develop ileocaecal perforation. Aim: To use immunohistochemistry with morphometry to investigate COX-1 and COX-2 expression in the normal rat GI tract and examine if sites of ulceration previously observed with long term COX-2 inhibitor administration in mice correlate with differential COX-1/COX-2 expression. Results: COX-2 positive cells were observed predominantly in the apical lamina propria of intestinal villi with fewer cells in the mucosal epithelium. The highest level of COX-2 expression was observed at the ileocaecal junction (ICJ). COX-2 expression was present in parasympathetic ganglia of the submucosa and muscularis. In the stomach, the highest grade of COX-2 expression was observed in the apical lamina propria of the fundus adjacent to the junctional ridge. In contrast, COX-1 positive cells within the lamina propria were evenly distributed along the GI tract but were present in higher numbers than COX-2 positive cells. At the ICJ, COX-1 positive cells were observed in the lamina propria but the mean level of expression of COX-1 was not significantly different from the ileum and caecum. COX-1 expression was also observed in the mucosal epithelium, submucosal vascular endothelium, muscularis and muscularis mucosae. Conclusions: In contrast to COX-1 which is evenly expressed along the GI tract, COX-2 expression in the rat is highest at the ICJ and this may relate to the mechanism of spontaneous lesions observed in COX-2-/- mice.
174: HISTOPATHOLOGICAL EFFECTS OF CIPROFLOXACIN ON RAT FETAL HEART DEVELOPMENT.
Ciprofloxacin is a broad spectrum fluoroquinolone antibiotic, widely used in the treatment of various infectious diseases. Since there is little information about ciprofloxacin-induced side effects on fetal hearts, this preliminary study was planned to evaluate potential ciprofloxacin-induced changes on rat fetal heart histopathology. Thirty Wistar rats were selected and randomly divided into two groups; control (n = 15) and test (n = 15). The test group received 14 mg/kg (PO) ciprofloxacin daily during pregnancy and the control group received vehicle. After parturition, heart tissue of neonates of treatment and control groups were taken and prepared for light microscopy. Tissues were stained by H&E. Microscopic study of heart tissue slices showed that myocardial cells were smaller and nuclei were denser in neonates from ciprofloxacin-dosed dams compared to controls. Cardiac weight was reduced in neonates from ciprofloxacin-dosed dams compared to controls (P < 0.05). Since Ciprofloxacin induced changes in the myocardium of rat feti, caution should be used in dosing of pregnant women with Ciprofloxacin.
Key words: Ciprofloxacin, fetal heart development, histopathological effects, Rat.
175: GENETIC ALTERATIONS IN BRAIN TUMORS FROM 1,3 BUTADIENE-TREATED B6C3F1 MICE.
The nervous system of the B6C3F1 mouse has rarely been a target for chemical carcinogenesis in the National Toxicology Program (NTP) bio-assays. However, six malignant gliomas and 2 neuroblastomas were observed in B6C3F1 mice exposed to 1,3-butadiene (NTP technical reports 288 and 433). These mouse brain tumors were evaluated with regard to morphology and profile of the genetic alterations that are observed in human brain tumors. The histologic characteristics of the brain tumors were similar to those of human counterparts. Most of the tumors examined had genetic alterations in the p53 tumor suppressor gene characterized by mis-sense mutations in 3/6 malignant gliomas and 2/2 neuroblastomas, loss of heterozygosity in the vicinity of p53 gene locus in 4/5 malignant gliomas and 2/2 neuroblastomas, and nuclear staining of p53 protein in 3/5 malignant gliomas and 2/2 neuroblastomas. Loss of heterozygosity in the vicinity of Ink4a/Arf gene locus was observed in 5/5 malignant gliomas and 1/1 neuroblastomas. One of 3 malignant gliomas had loss of heterozygosity in the vicinity of epidermal growth factor receptor (EGFR) gene locus. None of the tumors examined exhibited loss of heterozygosity near the phosphatase and tensin homologue (PTEN) gene locus. One of 2 neuroblastomas had a mutation in codon 61 of H-ras, while no H-ras mutation was observed in the malignant gliomas examined. Most of the mutations in the exons 5–8 of p53 gene were G to A transitions. Only one brain tumor (malignant glioma) was reported in control mice in over 500 NCI/NTP studies and it did not show evidence of p53 overexpression, p53 mutation, or K- and H-ras mutation. In conclusion, the specific genetic alterations observed was most likely induced by 1,3-butadiene treatment and was contributed to the development of mouse brain tumors. The findings were consistent with the major genetic alterations reported in human brain tumors, and suggest a causative role of environmental chemicals in human neurocarcinogenesis.
176: ENHANCED IMAGE ANALYSIS OF A TRANSPARENT FISH MODEL.
Small fish models are increasingly being used in the laboratory to determine basic mechanisms of development/gene expression, toxicity, and carcinogenicity which are often broadly applicable across phyletic levels. Along with the availability of complete genomic sequences from such models as zebrafish and Japanese medaka comes the need for determination of structural/functional relationships to phenotypic expression and high resolution methods to image such expression. We are developing methods for in vivo imaging of hepatic development and response to classical toxicants in a see-through Japanese medaka fish model. Specific Aims: 1) To define the liver of the medaka emphasizing relationships of hepatic tubules to microvascular system and quantitatively determine and characterize specific cells types during late embryogenesis and after adult spatial relationships and major vascular afferent route have been achieved; 2) to image hepatocyte and biliary epithelial cell interactions; and 3) to follow in the same individuals the tissue- and cell-specific responses to classical toxicants. High resolution light microscopy was performed on livers at each of the stages of development. We have observed rapid elongation of the gut and hepatic growth after the establishment of the hepatic portal vein and the initiation of exogenous feeding. Using fluorescence imaging, we performed in vivo analysis of bile formation in the embryolarval medaka liver from 4 to 17 days after fertilization. Embryo exposures to 7-benzyloxyresorufin indicated that embryonic hepatic cyp 3A cleaved the benzyloxy and that resorufin was conjugated and transported into bile passageways. In addition, 7-ethoxyresorufin, a substrate for hepatic CYP 1A, was used following induction by β-naphthoflavone. Resorufin fluorescence in biliary passageways of intact organisms indicated that the CYP 1A was inducible, and that conjugate was transported into bile passageways. With in vivo imaging, we are able to monitor molecular events in control and exposed organisms and to determine adult consequences of early life stage toxicity.
177: BRONCHOSCOPIC ADMINISTRATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR INDUCES A DOSE-DEPENDENT MONOCYTE RECRUITMENT IN NEONATAL LAMBS.
Preterm birth is an increasing statistic and premature infants are more prone to respiratory distress syndrome due to lack of adequate surfactant synthesis. Vascular endothelial growth factor (VEGF) is a growth factor that stimulates vascular development. In recent studies of preterm mice, intratracheal instillation of recombinant human VEGF (rhVEGF) in mice was shown to improve clinical scores and survivability with lack of described side-effects. To further evaluate this mechanism, a preliminary study was designed in neonatal lambs to assess various rhVEGF dosages for extrapolation to a perinatal lamb model. Lambs were given 20cc of rhVEGF (dosages—0.05 mg/ml, 0.005 mg/ml, or 0.0005 mg/ml) or sterile bovine serum albumin (BSA, 0.5 mg/ml) by sterile bronchoscope to the right pulmonary bronchus. Lung tissues were collected at 16, 24, and 32 hours post inoculation. Control lungs (with BSA) had no lesions at any time point. However, the rhVEGF treated groups had a dose-dependent deep red, well-defined consolidation in the right lung that was consistent in location with bronchoscopic deposition of rhVEGF. Microscopically, alveolar septa and alveolar spaces of lesions contained monocytes (>95%) with a small number of neutrophils. The lambs given the lowest rhVEGF dose had relatively sparse monocytic infiltration. This preliminary study demonstrates a dose-dependent and rapid recruitment of monocytes following rhVEGF administration. This acute monocytic infiltration following pulmonary rhVEGF deposition is a novel finding that warrants caution for the prophylactic or therapeutic intervention with rhVEGF in preterm infants.
178: LESIONS OF SKELETAL MUSCLE INDUCED BY CLOFIBRATE IN RATS.
Hypolipidemic drugs such as peroxisome proliferators activated receptor alpha agonists (fibrates) and HMG-CoA reductase inhibitors (statins) are known to cause rhabdomyolysis in human skeletal muscles. However, very little has been reported on the muscular toxicity induced by hypolipidemic drugs in animals. In this study, we investigated the muscular lesions induced by clofibrate, a well-known fibrate, in soleus muscle (type-I predominant skeletal muscle), tensor fasciae latae muscle (type-II predominant skeletal muscle) and biceps femoris muscle (type-I and II mixed skeletal muscle) in rats. Six-week-old female CD(SD)IGS rats were treated with clofibrate (750 mg/kg/day) or vehicle by oral gavage for 28 days. Samples of soleus, tensor fasciae latae and biceps femoris muscles, and blood were collected on Day 29 for histopathological examination and clinical chemistry, respectively. In addition, soleus and tensor fasciae latae muscles were examined electron-microscopically. Microscopically, degeneration of muscle fibers, vacuolation of muscle fibers and cellular infiltration were noted in the soleus muscle in rats treated with clofibrate. Ultrastructurally, increase of lipid droplet and hypertrophy of mitochondria were seen in the soleus muscle. The microscopic change of muscular vacuolation corresponded with increase of lipid droplet. No change was seen in either tensor fasciae latae or biceps femoris muscles. The value of aspartate aminotransferase, alanine aminotransferase, creatine phosphokinase, and lactate dehydrogenase were not changed. These data suggest that the sensitivity to clofibrate-induced muscular lesions is different among the muscles. It is also suggested that soleus muscle, type-I predominant muscle, is one of the susceptible muscles to muscular toxicity caused by clofibrate.
179: EFFECTS OF NONYLPHENOL ON THE EXPERIMENTAL HEPATOCARCINOGENESIS IN MALE RATS.
In this study, nonylphenol (NP) was selected as a representative endocrine disruptor. NP is used in the preparation of lubricating oil additives, plasticizers and surface active agents. It has also been found in polyvinyl chloride (PVC) used in the food processing and packaging industries. The objective of our study was to evaluate the effects of NP on DEN-induced hepa-tocarcinogenesis. F344 male rats were randomly divided into DEN+NP groups, DEN and NP alone groups. These groups were separated into 4 subgroups (control, NP2.8, NP14, NP70 group) for total 8 groups. For induction of liver tumors, mini-osmotic pumps providing a continuous infusion of DEN were implanted into the abdominal cavity in DEN+NP groups at 6 weeks of age. Pellets containing 2.8, 14, 70 of NP were implanted subcutaneously after 2 weeks of DEN treatment. Rats were sacrificed after 14, 20 and 26 weeks DEN treatment. NP did not effect reproductive organ weights during whole period. In 26 weeks, liver weight in DEN+NP groups significantly decreased compared to DEN alone. Histopathology showed a reduced incidence of foci multiplicity and hepatocellular carcinoma in DEN-treated groups given NP. BrdU-labeling indices and area of GST-P positive foci were decreased significantly in NP low-dose group compared to the control group at 26 weeks. These data indicate that NP reduces DEN-induced hepatic tumors and their precursors. The mechanism of this response was considered related to suppression of hepatocullar proliferation.
180: ENHANCED NEUTROPHIL TRANSMIGRATION IS THE POSSIBLE MECHANISM BEHIND INCREASED SUSCEPTIBILITY OF FEMALES TO ALCOHOLIC STEATOHEPATITIS.
Alcoholic steatohepatitis (ASH) is a rate-limiting step and an important pathologic finding in the progression of alcoholic liver disease (ALD). It is well known that females are at significantly higher risk of developing alcoholic liver injury than males. However, the mechanism behind the increased susceptibility of females to ALD is not completely understood. The objective of this study was to investigate the gender differences in the temporal pattern of hepatic neutrophil infiltration in an ASH model produced by chronic ethanol (EtOH) ingestion. ASH was induced in the male and female SD rats (weight matched) by feeding EtOH-containing, Lieber-DeCarli diet (36% calories by EtOH) for 6 weeks followed by a single injection of lipo-polysaccharide (LPS, 10 mg/kg, ip). The control rats received an iso-caloric diet where the calories were adjusted with maltose-dextrin. Liver injury as measured by plasma transaminase elevations (ALT and AST), and assessed by H&E stained liver sections, revealed a significantly higher liver injury (>20-fold) in the female ASH model compared to the males. Steatosis, neutrophilic infiltration, and multifocal coagulative necrosis was evident both in the male and female rats. However, hepatic neutrophilic necrotic foci were noted as early as 2 hr with a peak at 12 hr post LPS administration in the females. In the males, significant neutrophilic necrotic foci was delayed till 12 hr after LPS challenge. Furthermore the number of neutrophils in the female liver was nearly 2-fold greater than the males. CYP2E1, an enzyme involved in the metabolism of EtOH resulting in oxidative stress, was not significantly different between EtOH treated males and females. In conclusion, these data indicate that neutrophil transmigration is earlier and much higher in the females following EtOH +LPS treatment, which may explain the possible reason for higher susceptibility of female rats to ALD.
181: EFFECT OF COMBINED ABAMECTIN AND BIFENTHRIN ON MACRO AND MICROMINERAL TISSUE CONCENTRATIONS OF BLACK ROCK PIGEONS (COLUMBA LIVIA).
Lavish use of pesticide in agriculture industry is posing a great threat to the population of free living birds. This study was designed to investigate the toxic effects of abamectin and bifenthrin on the macro and microminerals tissue concentrations of black rock pigeons. Abamectin and bifenthrin at a purity of 93.5% was orally administered to twenty birds irrespective to gender to investigate the acute effect of these pesticides on the macro and microminerals of visceral organs. Sampling of liver, lungs, heart, kidneys, brain, breast and thigh meat was carried out at 12 hours and 48 hours and at 7 days after the administration of these pesticides. Mean concentration of sodium showed a significant increase at 12 hours in liver and significant decrease in thigh muscle at 48 hours and at 7 days but potassium remained unchanged. Chloride increased significantly in liver, lungs and kidney at 7 days. Mean magnesium concentration increased but iron and manganese concentration decreased significantly in all the organs. It can be concluded that abamectin and bifenthrin together alter the concentration of several macro and microminerals in different parts of the body at the doses given.
182: EFFECT OF COMBINED ABAMECTIN AND BIFENTHRIN ADMINISTRATION ON HEMATOLOGICAL AND PLASMA BIOCHEMICAL PROFILE OF BLACK ROCK PIGEONS {COLUMBA LIVIA).
The population of free flying birds is in danger due to massive use of pesticides in agriculture industry. This study was designed to investigate the hazardous effects of Abamectin and bifenthrin. These compounds were dosed at 70mg/kg body weight and purity of 93.5% by oral gavage to twenty black rock pigeons (Columba livia) irrespective of gender to investigate the acute effects of pesticides on hematological parameters. Blood was collected by cardiac puncture at 12 hours and 48 hours after the compound administration. Mean body weights of pigeons significantly decreased after 48 hours but visceral organ weights revealed nonsignificant differences as compared to control. Mean erythrocyte count significantly decreased after 48 hours of drug administration while hemoglobin and leukocyte count remained unchanged and stayed within normal range. Lymphocyte and neutrophil concentrations were non-significantly different among different time intervals, but monocyte concentration was significantly decreased. Plasma glucose, cholesterol, alkaline phosphatase and glutamic-pyruvate transaminase (GPT) also revealed non-significant differences but total proteins as well as albumin concentration was significantly decreased. A significant increase in plasma glutamic-oxaloacetic transaminase (GOT) concentration was observed after 48 hours of drug administration. It can be concluded that abamectin and bifenthrin together alter several hematology and clinical chemistry parameters at the doses given.
183: CHARACTERIZATION OF RAT ENDOMETRIAL STROMAL SARCOMA-DERIVED TUMOR LINES.
Endometrial stromal sarcomas (ESSs) have been reported in the uterus of humans and rats, and the incidence is very low. ESSs are characterized by proliferation composed of exclusively of cells differentiated as endometorial stroma, but the histogenesis remains poorly understood. To clarify the cellular nature, we established a transplantable tumor (RY) and cell lines (RY-PB and cloned RY-B-E3) from a spontaneously occurring ESS encountered in a 24-month-old female F344 rat. The primary tumor and RY tumors, which had been serially passaged in syngeneic female rats up to the 10th generation, consisted of spindle or round cells arranged in sheets or ill-defined bundles. Neoplastic cells of the primary and RY tumors, as well as cultured cells of RY-PB and RY-B-E3, showed positive reactions to vimentin, ED1/ED2 (macrophages/histiocytes), OX6 (for dendritic cells expressing rat MHC class II antigens), and lysosomal enzymes such as acid phosphatase and non-specific esterase in varying degrees. Ultrastructurally, neoplastic cells had tubulovesicular system-like structures and variously developed lysosomes in the cytoplasm. Neoplastic cells also exhibited immunoexpression to α-SMA. The addition of TGF-β1 to RY-PB and RY-B-E3 cultures increased the number of α-SMA-positive cells, whereas the positive cell number was decreased by anti-TGF-β antibody. The RT-PCR method revealed the expression of TGF-β1 mRNA in the cultured cells. The present study showed that rat ESS-derived cells exhibited dendritic cell-like and myofibroblastic cell-like phenotypes; the latter was induced by TGF-β1. These tumor lines may be useful for studies on the histogenesis of ESSs.
184: GENETIC ALTERATIONS IN RAS, P53, AND BETA-CATENIN GENES IN HEMANGIOSARCOMAS OF B6C3F1 MICE FOLLOWING EXPOSURE TO ENVIRONMENTAL CHEMICALS FOR TWO YEARS.
The most prominent neoplastic lesions in mice in the 2-year feed study of o-nitrotoluene were hemangiosarcomas. Fifteen o-nitrotoluene-induced and 13 spontaneous hemangiosarcomas were examined for genetic alterations in ras, p53 and beta-catenin genes. Mutations in at least one of these genes were identified in 13 of 15 (87%) of the o-nitrotoluene-induced hemangiosarcomas, and missense mutations in p53 exons 5–8 were detected in 11 of 15 (73%) of these lesions. Four hemangiosarcomas from the 5,000 ppm group exhibited double mutations in p53; one of these also had a beta-catenin mutation, while another had a CTA mutation at codon 61 of K-ras. Seven of 15 (47%) hemangiosarcomas from mice exposed to o-nitrotoluene had splice site mutations at the beginning of exon 2 or deletions in the beta-catenin gene. Spontaneous hemangiosarcomas from control mice lacked p53 and beta-catenin protein expression. Our data indicated that p53 and beta-catenin mutations in these hemangiosarcomas most likely occurred as a result of the genotoxic effects of o-nitrotoluene and that these mutations may play a role in the pathogenesis of o-nitrotoluene-induced hemangiosarcomas in B6C3F1 mice.
185: THE OVARY OF THE RAT AND YOLK SAC CARCINOMA.
Bilateral yolk sac carcinoma metastatic to the visceral organs was found on Day 561 of a carcinogenicity study in the ovaries of a Sprague-Dawley rat. In birds, the yolk sac, connected by its vasculature, provides a continual source of nutrients to the developing embryo. In mammals, the yolk sac, while not a major source of nutrients, is retained as part of the extra-embryonic membranes, where it surrounds and fuses with the outer surface of the amnion. The yolk sac plays a vital role in early embryonic development because it is the source of both the primitive nucleated red blood cells that migrate to the embryo and become the hemopoietic stem cells, and because it is the first site at which the primordial germ cells can be identified. The primordial germ cells migrate from the yolk sac wall to the median part of the urogenital ridge where they play a key role in gonadal differentiation. This poster reviews the anatomy, embryology and physiology of the yolk sac and ovary, and describes some of the congenital, degenerative, hyperplastic and neoplastic pathologic conditions that can derive from the vestigial yolk sac components of a female rat.
186: THE ACVP/STP COALITION FOR VETERINARY PATHOLOGY FELLOWS.
A 2002 employer and training program survey confirmed a critical shortage of existing veterinary anatomic and clinical pathologists, and predicted the situation would worsen with time due to continuing deficit in supply, increases in demand, and retirements in the current workforce (http://www.acvp.org/career/employsurv.pdf). In response to this shortage and to unite their efforts to increase the supply of veterinary pathologists to fill diverse set of employment positions, The American College of Veterinary Pathologists (ACVP) and the Society of Toxicologic Pathology (STP) have developed “The ACVP/STP Coalition for Veterinary Pathologists.” The initial goal of the Coalition is to create 15 new positions to train residents or PhD graduate candidates. These positions will be funded by industry and private foundations, and will provide support for stipend, tuition, health benefits, travel and educational supplies. Positions will be awarded to academic training institutions based on review of competitive responses to a Request for Applications issued by the Coalition. A close interaction is envisioned between training programs, students and funding sponsors to more effectively cross-communicate goals and objectives. Coalition Fellows will be expected to complete the ACVP Certification Examination or their PhD degree within two years after the end of funding and pursue careers in either veterinary anatomic or clinical pathology, but otherwise will have no pay back obligation. The Coalition has multiple advantages; it will (1) increase the supply of veterinary pathologists, (2) provide a unified mechanism for ACVP/STP-endorsed solicitation and distribution of funds to train veterinary pathologists, and (3) provide a conduit for enhanced interaction between academic and industrial veterinary pathologists.