Abstract
Combined 52nd and 36th Annual Meetings of the American College of Veterinary Pathologists and the American Society for Veterinary Clinical Pathology
Salt Lake City, Utah
December 1–5, 2001
Specialty Group Coordinator
Anne M. Ryan
1: Defective Platelet Collagen Signaling in a Thoroughbred Filly
The objective of this study was to characterize a bleeding diathesis in a 2 year-old Thoroughbred filly. Initial testing included a CBC, chemistry and electrolyte panel, PT, PTT, and assays for ATIII, vWF, fibrinogen, and coagulation factors VIII, IX, XI, and XII. Platelet function was evaluated by aggregometry. A platelet adhesion assay was performed using microtiter plates pre-coated with collagen. SDS-PAGE and Western blots were used to characterize protein fractions in various platelet preparations. The platelet count was normal (138,000/μL). Laboratory abnormalities included slight increases in PTT (53.4 seconds, reference interval 31.5–48.5 seconds) and fibrinogen concentration (187 mg/dL, reference interval 65–180 mg/dL), and mildly decreased activities of factor VIII (40%, reference interval 50–200%) and factor XI (56%, reference interval 60–150%). Platelet aggregation in response to ADP and ristocetin was normal. Platelets had a mildly diminished response to thrombin, and virtually no response to collagen. Immunoassays for the major collagen receptor GPVI were inconclusive, but the horse's platelets bound collagen normally in the adhesion assay. SDS-PAGE indicated a missing band in the range of 70–79 kD. Immunoblots for phosphotyrosine showed a missing band of approximately the same mass. Platelets expressed the membrane glycoproteins GPIIb-IIIa and GPIb as well as the intracellular signaling molecules Syk, fyn and lyn. Immunoblots for another tyrosine-phosphorylated signal transduction protein, SLP-76, were inconclusive. On the basis of these results, the bleeding diathesis is not likely the result of thrombocytopenia or deficient clotting factor(s). Aggregometry and adhesion assays suggest a lesion downstream of the collagen receptor. Collagen activation occurs via a tyrosine kinase-dependent pathway. SLP-76 is a key adapter protein in this signaling pathway, and a similar bleeding phenotype has been described in SLP-76 knockout mice. Further investigation is underway to characterize the platelet function defect in this horse.
2; Laboratory Findings in Mycobacterium Tuberculosis Culture-Positive Asian Elephants Compared to Clinically Normal Elephants
Mycobacterium tuberculosis currently infects 3% of elephants housed in captivity in North America. An evaluation of CBC and clinical chemistry results was undertaken to determine if values could be identified as consistently different between culture positive and clinically normal elephants. Approximately 50 Asian elephants were routinely evaluated over a 2-year period. Animals with signs of clinical disease or significant changes from baseline blood values were excluded from the data set. Results from 20 healthy elephants were compared to values from 5 culture-positive elephants. Values that were significantly lower (P < .05) in positive animals included A:G ratio, mean cell hemoglobin concentration (MCHC), and glucose concentration. Values that were significantly higher (P < .05) in positive animals included platelet, band neutrophil, eosinophil, calcium, and bicarbonate (TCO2) concentrations. The A:G ratio as a measure of general inflammation was of particular interest in these animals. All animals that were initially negative had a decreased A:G ratio at the time of positive trunk wash, compared to baseline A:G ratios in these animals. When the A:G ratio, determined using the Hitachi 911, was analyzed with a ROC curve, the value of 0.7 was determined to be the most effective decision threshold below which tuberculosis should be considered. This value had 100% sensitivity and 77% specificity in the population examined. Cytochemical staining of a bilobed leukocyte unique to elephants was chloroacetate esterase and peroxidase positive. This cell was classified as a monocyte for this study. Misidentification of these cells may result in underreporting of monocytes and falsely increased lymphocyte counts.
3: Defective Carboxylation of Vitamin K Dependent Clotting Factors in Rambouillet Sheep
Increased periparturient lamb mortality, due to a heritable coagulopathy, is being investigated in an inbred flock of Rambouillet sheep. Affected offspring have prolonged activated clotting times (7–8 minutes), prothrombin times (>200 seconds), and activated partial thromboplastin times (>300 seconds). Lambs have decreased factor II (45–50%), factor VII (8–20%), factor IX (1–4%), and factor × (20–25%) activities in comparison to normal pooled sheep plasma controls. Vitamin K antagonists cannot be attributed to the clinical signs. Postmortem examination of affected offspring is unremarkable, and biochemical profiles of affected offspring are within normal limits. These findings suggest a heritable defect of vitamin K cycling as the cause of the coagulopathy. Further investigation required that affected animals be transfused with plasma every 7–10 days to correct the bleeding diathesis. Daily administration of vitamin K1 prolonged these transfusion intervals to 60–80 days; however, vitamin K-dependent factor activities remained unchanged in vitamin K-supplemented animals. Inappropriate response to vitamin K administration suggested an abnormality in the enzyme gamma-glutamyl carboxylase. Enzyme activities were determined from liver microsomal preparations of a homozygous affected lamb and a normal 1-day-old lamb. Gamma-glutamyl carboxylase activities were 5 to 10 fold lower compared with enzyme activity in the control lamb. These results support a heritable defect of gamma-glutamyl carboxylase as the cause of the clinical signs.
4: Adjuvant Induced Arthritis Alters Expression of Inducible Nitric Oxide Synthase (Inos) in Foreign Body Granulomas
Literature from fields as diverse as leprosy and hip replacement indicates that cytokine balance, especially that between INOS and transforming growth factor-B1, is important for regulation of the granulomatous inflammatory response. Aberrant regulation is associated with uncontrolled disease, excessive tissue destruction and scarring, and rejection of implants. Systemic inflammatory diseases have aberrant regulation of the inflammatory response and are associated with abnormal healing. Many of these diseases have systemically elevated nitric oxide (NO) levels from INOS. Effects of NO are concentration dependent. At low levels produced by endothelial NO synthase and neuronal NO synthase, NO is a physiologic messenger. At high levels produced by INOS, NO can have pathologic effects. While investigating the hypothesis that elevated levels of INOS in an adjuvant induced arthritis (AIA) model of inflammation may be associated with abnormal wound healing, we also found that expression of INOS in foreign body granulomas was altered. In AIA, a model of rheumatoid arthritis, there is systemic elevation of inflammatory cytokines including INOS. Wound healing is abnormal in this model associated with elevated INOS in hyperproliferative epidermis. INOS is expressed around infection and foreign bodies in control and AIA wound beds. Expression in foreign body granulomas was found to decrease with time and resolution of inflammation. In AIA, expression of INOS in foreign body granulomas was increased and prolonged. Our results indicate that cytokine balance within foreign body granulomas can be altered by systemic inflammatory disease.
5: Cardiac Expression of Atrial Natriuretic Peptide Gene in Feline Cardiomyopathy
Atrial natriuretic peptide (ANP) is a hormone produced by the heart that when released produces diuresis, natriuresis, and peripheral vasodilation. We have sequenced feline ANP and shown that it is normally produced only in the cardiac atria. In this study, we report on ANP gene expression in cats with cardiomyopathy. Seven feline hearts with cardiomyopathy were used: 5 with hypertrophic cardiomyopathy (HCM), 1 with dilated cardiomyopathy (PCM) and 1 with restrictive cardiomyopathy (RCM). Tissue samples were collected from the left and right atria (LA and RA), left and right ventricle (LV and RV), and interventricular septum (IS), and stored in RNA stabilization solution at -20°C. Total RNA was extracted from each sample and used for Northern blot analysis. As in normal cats, hearts from the cats with all forms of cardiomyopathy expressed ANP mRNA in both LA and RA. In addition, cardiac tissue from cats with HCM expressed ANP mRNA in up to 5 heart regions with the strongest expression in the atria. Cardiac tissues from the cat with RCM showed ANP expression only in the atria; cardiac tissues from the cat with DCM showed expression in the atria and very slight expression in the IS. It is concluded that cats with cardiomyopathy maintain mRNA ANP expression primarily in the atria. In addition, cats with HCM may express ANP mRNA in the ventricles as well. Increased expression of ANP mRNA during cardiomyopathy may result in elevated cardiac and blood ANP concentrations, which may allow the use of ANP as a plasma marker of cardiac disease in cats.
Other Oral Platform Presentations
6: A Formalized Process for Developing Successful User Interfaces for the Problem List Generator
The Problem List Generator (PLG) is an Internet-based learning tool designed, developed and tested by the Biomedical Informatics Research Group (BIRG), a 16-member multidisciplinary team of faculty, graduate students, and veterinary students. The BIRG is composed of experts in veterinary clinical pathology, instructional design, educational technology, educational psychology, computer science, interface design, informatics, and assessment techniques. The PLG uses a systematic approach to guide students through the diagnostic reasoning process as they analyze laboratory data from clinical cases. Students use the PLG to produce a problem list, which is an outlined causal hierarchy of data grouped under explanatory headings. The problem list provides a method for visualizing and comparing student and expert solutions for each case. The PLG interfaces were designed using a systematic method of usability testing including paper prototypes, one-to-one evaluations, small group evaluations, field trials, and a priority table. Additionally, whole-group usability questionnaires were administered formatively and summatively. PLG interface changes were made iteratively in response to student feedback. Formative evaluations of the prototype indicated that the PLG was only slightly more usable than the paper alternative; however, once technical problems were resolved, summative evaluations were overwhelmingly positive. The PLG was used extensively at VMRCVM during the Spring Semester 2001 Clinical Pathology course, with 83 of 89 students choosing it to do their course homework of analyzing 93 cases. Students enthusiastically expressed that the PLG was highly intuitive and helped develop their diagnostic skills better than the alternative paper and pen method of completing their homework.
7: Mouse Serum Cytokine Analysis by Multiplexed Microsphere-Based Flow Cytometry Using the Luminex 100
Quantitation of proinflammatory cytokines in serum is desirable for understanding the pharmaco-toxicity of novel compounds. Mouse serum sample volume limits cytokine analysis by current ELISA methods. We investigated microsphere-based multiplexed analysis on the Luminex 100 flow cytometer as a sample-sparing alternative for cytokine quantitation. Proinflammatory cytokine production can be induced in mice by injection of lipopolysaccaride (LPS). Saline or LPS (1 mg/kg) was administered (i.p.) to 60 male mice (Charles River Laboratories, CD-1). Five mice per group were killed and blood was collected at 0.5, 1, 2, 4, 24, and 72 hours. Anti-mouse cytokine antibodies were covalently coupled to uniquely dyed 5.5 μm microspheres (Luminex Corp) and used to develop sandwich type immunoassays. The assay procedure measured 9 cytokines simultaneously from a 50 μL serum sample. Microsphere classification and fluorescence measurement was performed using the Luminex 100 flow cytometer. Excellent positive responses with clear peak times were measured for TNFα (1 hour), IL6, KC (murine IL-8 homolog), GM-CSF (2 hour) and G-CSF (4 hour) compared with minimal response for saline controls. G-CSF remained increased at 24 hours, while other cytokines were similar to control values by or before 24 hours. Minimally increased values for IL-1β, INF-γ and IL-12 were noted, but similar less pronounced increases were also noted for saline-treated mice. IL-3 was not affected by either treatment. Multiplex analysis provided an excellent tool to quantitatively screen serum samples for cytokine response; 5 cytokines were identified as potential markers of immune responsiveness after LPS challenge of mice.
8: Urinary Albumin as a Sensitive Marker for Nephropathy in Aged Male Rats
Chronic progressive nephropathy is a common age-related finding in most strains of ad libitum fed rats. Proteinuria is generally considered a hallmark of end-stage nephropathy in rats, and increased urinary albumin excretion is used as an early marker of renal microvascular disease in human diabetic nephropathy. The purpose of this study was to evaluate the predictive value of urinary albumin excretion in spontaneous rat nephropathy. Urine volume and protein and albumin concentrations were determined on three 16-hour urine collections from 15 male rats (Sprague-Dawley, Charles River Laboratories, >6 months old) at 2 week intervals. Urine albumin and protein were determined by immunoturbidimetric and colorimetric analysis on the Hitachi 717 automated clinical analyzer using commercially available kits (Roche Diagnostics, Tina-quant Albumin; Biotrol, pyrogallol red reagent for protein). H&E and PAS-stained sections of kidney were evaluated independently by 2 pathologists. Minimal to mild nephropathy was identified in 10/15 rats. Urinary albumin excretion was consistently increased in rats with nephropathy while protein excretion was less predictive. Urinary albumin excretion in rats with no histopathologic lesions was 0.25–1.68 mg/day. In comparison, mean urinary albumin excretion in rats with minimal and mild nephropathy was 2.9–14.2 mg/day and 40.6–62.5 mg/day, respectively. Urinary protein excretion ranged from 6.8–12.7 mg/day in rats with no histopathologic evidence of nephropathy, while protein excretion for rats with minimal and mild nephropathy was 12.3–26.0 and 52.0–86.4 mg/day, respectively. Protein excretion of 10–20 mg/day was less consistently associated with histologic changes. Nephropathy was only identified by increased urinary albumin excretion in these rats. Urinary protein excretion is a sensitive indicator of nephropathy in male rats, but urinary albumin excretion provides superior correlation with microscopic observations.
9: Increased Salt Sensitivity Secondary to Leptin Resistance in Shhf Rats is Mediated by Endothelin
Obesity is associated with an increased incidence of hypertension and cardiovascular disease. Studies have suggested that there may be a link between leptin resistance in obese individuals and increased salt sensitivity. SHHF/Mcc-facp (spontaneous hypertension heart failure) rats were used to study the effect of gene dosage of a null mutation of the leptin receptor (cp) on salt sensitivity and renal endothelin production. Obese (cp/cp), heterozygous (+/cp), and lean (+/+) male SHHF rats were fed a low salt diet (0.3% NaCl) for 7 days, followed by a high salt diet (8.0% NaCl) for 7 days. No significant differences in blood pressure or 24-hour urinary endothelin excretion were observed between groups on the low salt diet. On the high salt diet, systolic blood pressure significantly increased in cp/cp rats, compatible with increased salt sensitivity. The high salt diet resulted in a significant increase in renal endothelin excretion by all groups, and cp/cp rats had significantly greater endothelin production compared to either +/+ or +/cp rats. Compared to the low salt diet, the high salt diet was also associated with a suppression of plasma nitric oxide, a potent vasodilator. Treatment with an endothelin antagonist (bosentan, 100 mg/kg/day) prevented the increase in blood pressure associated with the high salt diet in cp/cp rats. These studies suggest that leptin resistance (cp/cp) is associated with increased salt sensitivity that is mediated, in part, by increased renal endothelin production.
10: Flow Cytometric Murine Bone Marrow Toxicity Assessment in Pre-Clinical Drug Development
Microscopic evaluation of bone marrow for the delineation of erythroid and myeloid composition is time consuming, labor intensive, and subject to human error or bias. Limited numbers of cells are evaluated and only semiquantitative morphologic information is obtained. In contrast, flow cytometry can assess phenotype and function at the single cell level in a rapid, reproducible, and objective manner. We exploited this potential of flow cytometry to evaluate murine bone marrow toxicity in preclinical drug development. A panel of monoclonal antibodies (CD45, CD3, CD11b, TER119, CD117, Ly6E-A, CD34) was used to phenotype mouse bone marrow progenitors of the lymphoid, myeloid, and erythroid series, and stem cells. Consistent with flow cytometric evaluations of human bone marrow, murine bone marrow cells could be uniquely identified on a cytogram of CD45 fluorescence plotted against forward angle scatter. The maturity and lineage characteristics of these discrete areas were then determined by electronic gating and evaluation for the presence or absence of CD34 with and without the lineage markers. Microscopic evaluation of Wright's stained cytocentrifuged preparations of sorted cells was used to confirm flow cytometric classification of proliferating and non-proliferating lymphoid, myeloid, and erythroid populations. This methodology facilitated identification of selective modulation of specific bone marrow populations by novel xenobiotics in drug development in a rapid and cost-effective fashion.
11: Bone Marrow and Hematologic Findings in Cats with Lymphosarcoma
Recent publications have helped to elucidate the distribution of feline lymphosarcoma and its response to treatment. However, it is difficult to determine from these reports the extent of bone marrow involvement and the presence of hematologic abnormalities at the time of diagnosis as well as their clinical significance. A retrospective study was undertaken to characterize the bone marrow, hematologic, and clinicopathologic changes in cats with lymphosarcoma. A search of the medical records of the Veterinary Hospital of the University of Pennsylvania revealed 118 cats with histologically or cytologically confirmed diagnoses of lymphosarcoma that underwent staging evaluation between July 1995 and April 2001. Full staging procedures included complete blood count, serum biochemical screen, thyroid hormone levels, urinalysis, thoracic radiographs, abdominal ultrasound, bone marrow aspiration, FeLV/FIV testing, and other diagnostics as indicated. Evidence of lymphoid neoplasia within the bone marrow was detected in approximately one-quarter of cats on initial bone marrow aspirate cytology while other nonspecific abnormalities were more common. All cats that had neoplastic cells circulating in the peripheral blood had infiltration of the bone marrow. However, less than one-half of the cats with bone marrow involvement had malignant cells detected on a complete blood count. Nevertheless, preliminary review of the data revealed that these cats were more likely to be anemic, thrombocytopenic and to have abnormal neutrophil counts. In addition, cats in which the kidneys were identified as the primary site of disease were most likely to have lymphosarcoma detected within the bone marrow.
12: Artifactual Macrocytosis in Dogs and Cats
Macrocytosis without polychromasia was observed in mailed-in blood samples. A small pilot study was performed to determine the effects of room temperature sample storage on mean cell volume (MCV) and hematocrit (HCT) by automated methods and manual packed cell volume (PCV). Samples from 7 dogs and 6 cats submitted in EDTA for routine CBC analysis were stored at room temperature and analyzed at 0, 8, 24, 48, and 72 hours for PCV and automated HCT and MCV. Automated results were obtained from a Bayer Advia 120 and a Coulter S+IV. Manual, PCV, Advia HCT and Coulter S+IV HCT results changed over the 72-hr time period from a mean of 45% to 52%, 45% to 53%, and 44% to 51%, respectively in dogs and from 35% to 38% for PCV and from 35% to 37% for HCT in cats. Advia MCV and Coulter S+IV MCV results changed from a mean of 69 to 83 fL and 67 to 78 fL, respectively, in dogs and from 42 to 45 fL and 41 to 45 fL, respectively, in cats. Manual PCV and HCT results by both instruments increased 7 to 8% and 2 to 3% in dog and cat samples, respectively, over 72 hours of sample storage at room temperature, but values stayed within reference intervals. Mean cell volume increased by 14 fL and exceeded the reference interval in dog samples stored over 72 hours. Mean cell volume increased by 3 to 4 fL in cat samples over 72 hours but stayed within reference intervals. Manual PCV, HCT and MCV determinations may be artifactually increased due to storage of samples at room temperature. Microcytosis associated with iron deficiency anemia may be masked under these conditions.
13: Plasmodium Yoelii Uses the Murine Duffy Glycoprotein for Invasion of Normocytes: Evidence for An Alternate Receptor on Reticulocytes
Plasmodium vivax, a human malaria parasite, and P. knowlesi, a monkey malaria parasite, require the Duffy glycoprotein to enter human erythrocytes. Whereas P. vivax has an absolute requirement for Duffy to enter human erythrocytes and is restricted to reticulocytes, P. knowlesi and P. falciparum (the most virulent form of human malaria) have alternate pathways to invade their host erythrocytes and are capable of invading both reticulocytes and normocytes. These pathways are likely mediated by different erythrocyte binding proteins expressed by the parasite, and by different erythrocyte receptors. The parasite proteins are considered candidates for a multivalent vaccine. We show that the murine Duffy glycoprotein is used by P. yoelii for invasion of normocytes. Mice with their Duffy gene knocked out (DKO mice) had a 2.3 to 5 fold lower parasitemia than wild type (WT) mice. In these same experiments, the infection frequency of normocytes was markedly lower for DKO mice, but the infection of reticulocytes was identical between the 2 strains. This indicated that the Duffy glycoprotein is used for invasion of normocytes but not reticulocytes. Indeed, short term invasion assays revealed that DKO normocytes were invaded 7.5 to 8.5 fold less frequently than WT normocytes, but invasion of reticulocytes was almost identical between the 2 strains. Our conclusion is that the Duffy glycoprotein is used by P. yoelii for invasion of normocytes, but that there is an alternate receptor for the invasion of reticulocytes. This murine model is useful for dissecting the molecular basis of invasion and host cell preference, and for evaluating strategies for prevention or amelioration of disease. Poster presentations
14: Analysis of Methods for Feline Urine Ph Measurement
Monitoring of urine pH, often done at the patient's home, is essential for proper clinical treatment and management of conditions such as urolith formation. The purpose of this experiment was to assess the agreement of pH methods readily available for home monitoring. Urine samples were obtained by cystocentesis from 40 clinically healthy cats and pH was measured within 2 hours of collection (0-hr). Each sample was evaluated using pH paper, urinalysis reagent strip (Multistix), 2 brands of portable meters (Chek-Mite and Checker), and a standard laboratory bench-top meter. Urine samples were refrigerated and a second pH reading was taken with the laboratory bench-top meter after 24 hours. Using statistical software we evaluated the degree of agreement between the 2 clinical methods using Deming regression and NCCLS bias plot tests with the reference method as the laboratory bench-top meter at time zero. A paired t-test was used to compare the mean differences between 0-hr and 24-hr pH. Results indicated closest agreement using one portable meter (Chek-Mite) and worst agreement using the other portable meter (Checker) related to a constant negative bias of 0.305 units. As expected, pH paper and reagent strips had poor and intermediate agreement, respectively. The reagent strip method when compared with the bench-top analyzer had a negative bias of 0.121 units and a wide disagreement at the low pH level. Similar to a previous study, the reagent strip did not strongly agree with the reference method in that only 50% of values were within 0.25 pH units of each other. Statistical significance between 0-hr and 24-hrs post-refrigeration samples was not considered clinically significant. In conclusion, some portable meters are excellent for home monitoring. When collected at home and kept refrigerated, urine may be measured within 24 hours at institutions using the reference method or portable meter.
15: Wright Staining of Urine Sediment: An Improved Method for Detection of Bacteriuria in Dogs
Bacterial urinary tract infections are important causes of urinary tract disease in dogs. Two screening methods for detecting bacteriuria in dogs were compared to quantitative urine culture. Urine was collected via cystocentesis from 441 dogs. A portion of the specimen was aliquoted for quantitative aerobic bacterial culture. In addition, unstained wet mount and air-dried Wright-stained (Diff Quik) urine sediment preparations were examined by light microscopy for bacteria. Of a total of 459 samples submitted, 74 were culture positive (>1000 CFU/mL). Receiver operating characteristic (ROC) curve analysis was performed to determine optimum cut-off levels for significant bacteriuria in the 2 urine sediment examinations. In unstained urine sediment, significant bacteriuria was defined as 10 or more bacteria per high power field. In Wright-stained sediment examination, significant bacteriuria was defined as 10 or more bacteria per 20 oil immersion fields. Compared to urine culture, unstained wet mount sediment examination had 66% sensitivity and 97% specificity, with positive and negative predictive values of 81% and 94%, respectively. In contrast, the Wright-stained sediment examination had 93% sensitivity and 99% specificity, with positive and negative predictive values of 95% and 99%, respectively. The diagnostic performance of the Wright-stained examination of urine sediment was an improvement over routine examination of unstained urine sediment for detection of bacteriuria. False positive results with unstained sediment preparations were attributed to the presence of amorphous particles (lipids, cytosol, etc.) that resemble bacteria in size and shape but cannot easily be distinguished from true bacteria without staining.
16: Retinal Degeneration in Domestic Ferret
Eyes from several unrelated clinically blind adult domestic ferrets were submitted for examination. In all examined globes, there was complete atrophy and loss of the photoreceptor layer, outer nuclear layer and outer plexiform layer of the retina, affecting both tapetal and nontapetal areas. The inner nuclear layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, and optic nerves were largely unaffected. There were occasional retinal pigmented epithelial cells at various levels throughout the remaining retinal layers. Several globes also contained focal areas of full thickness retinal atrophy and chorioretinal adhesions, or a thin glial scar with multifocal mild hyperplasia of the underlying retinal pigmented epithelium. This retinopathy is histopathologically similar to that seen in the group of canine heritable retinopathies (progressive retinal atrophy) and human degenerative retinopathy (retinitis pigmentosa). We believe this condition may be underdiagnosed because of the normally poor vision of ferrets and their ability to accommodate for loss of visual acuity.
17: Towards Understanding Retinal Degeneration usingg a Functional Genomics Approach
Numerous initiating mutations for retinitis pigmentosa (RP) have been identified; however, subsequent cellular events that result in photoreceptor death are complex and poorly understood. Using cDNA microarrays, we identified groups of coordinately expressed genes during retinal degeneration in a murine model of retinitis pigmentosa, the rd mouse. Retinas were collected from age-matched rd and control mice at post-natal (PN) days 5, 8, 12 and 15, and used to prepare RT-PCR-derived probes that were hybridized to 4.6K mouse cDNA microarrays. Over the four time points, 262 genes had significant differences in expression between rd and control retina. The temporal expression pattern of both over- and under-expressed genes in rd coincides with the sequence of photoreceptor death. Differentially expressed genes were divided into the categories of cellcycle control, apoptosis, signal transduction, mitochondrial metabolism, vesicular transport, cytoskeletal function and proteosome pathways. Of these pathways, cellcycle control in rd was chosen for further analysis. Expression alterations suggested that aberrant reactivation of cell cycle occurs in rd. This was supported by BrdU staining of rd retinas and by immunohistochemical evaluation of selected cell cycle control proteins. Further evaluation of cell cycle mechanisms using virtual Northern blots and crossbreeding studies are underway.
18: A Phenotypic Variant of Hereditary Alpha Mannosidase Deficiency in Two Inbred Calves
Two related, six-month-old, female, inbred mixed-breed beef calves, displayed posterior paresis that progressed to tetraparesis and eventual cerebellar signs over a four week period. A complete blood count, serum chemistry, and cerebral spinal fluid analysis were all within normal limits. Blood lead levels and cholinesterase activity were within normal limits. Because of the progression of clinical signs, the animals were euthanized and complete necropsies were performed. Microscopic evaluation revealed moderate to marked cytoplasmic vacuolation of numerous neurons within the brain, especially prominent in the cerebellum and caudal brainstem, spinal cord ventral horns and multiple ganglia. Ultrastructural examination of the neurons of the caudal brainstem, thoracic spinal cord and the cranial mesenteric ganglion revealed finely flocculent material accumulating within membrane-bound cytoplasmic vacuoles compressing organelles to the periphery of the cell. Light microscopic and ultrastructural changes of hepatocytes and renal tubular epithelial cells were not observed. Frozen brain tissue from one affected calf and a normal control calf were analyzed for deficiencies of ten separate lysosomal enzymes using 4-methyIumbelliferyl substrates and measured by fluoro-spectroscopy. Activity was expressed as nmol/hour/mg of protein. The results demonstrated a severe deficiency in alpha mannosidase in the affected calf (affected = 1.8 nmol/hr/mg protein; control = 32.0 nmol/hr/mg protein). A diagnosis of alpha mannosidase deficiency was made based on light microscopy, transmission electron microscopy, and enzyme analysis. This is the first case of presumptive hereditary alpha mannosidase deficiency in calves without involvement of the liver and kidney.
19: The Diagnosis and Prognosis of Synovial Tumors in Dogs: 35 Cases
Although synovial cell sarcoma is reported to be the most common neoplasm of the canine synovium, this retrospective study of 35 canine synovial tumors found that the majority were of histiocytic origin. Five (14.3%) synovial cell sarcomas were identified by positive immunohistochemical staining with antibodies to cytokeratin. Eighteen (51.4%) histiocytic sarcomas were identified by cell morphology and immunohistochemical staining with antibodies to CD18. Six (17.1%) synovial myxomas were identified by histologic pattern. The remaining six (17.1%) synovial tumors represented two malignant fibrous histiocytomas, one fibrosarcoma, one chondrosarcoma, and two undifferentiated sarcomas. Rottweilers were overrepresented in the histiocytic sarcoma category, and Doberman Pinschers were overrepresented in the synovial myxoma category. The average survival time was 31.8 months for dogs with synovial cell sarcoma, 5.3 months for dogs with histiocytic sarcoma, 30.7 months for dogs with synovial myxoma, and 3.5 months for dogs with other sarcomas. Among the dogs with follow-up information available, metastatic disease was detected in 25% of dogs with synovial cell sarcoma, 91% of dogs with histiocytic sarcoma, none of the dogs with synovial myxoma, and 100% of dogs with other sarcomas. Immunohistochemical staining for cytokeratin, CD18, and actin is recommended to make the diagnosis and thereby predict the behavior of synovial tumors in dogs.
20: Characteristics of Primary Spindle Cell Neoplasms of the Anterior Uveal Tract in Eleven Dogs
Eleven primary spindle cell neoplasms of the anterior uveal tract were identified from 4634 canine ocular submissions during 1978–2001. The present study evaluated the age, sex, breed, laterality, histologic features and immunohistochemical (IHC) staining profiles of these cases. The median age was 8.3 years (range 4.5–12). Females and males were equally represented. There was a notable breed predilection for Siberian Huskies (8/ 11); Australian Shepherds (2) and a Border Collie were also represented. Right and left eyes were equally affected. Iris color was determined to be blue (8/11), brown (1/11), or indeterminate (2/11). Histologically, all tumors involved the iris (4/11) or iris and ciliary body (7/11). Tumors were composed of spindle cells arranged in fascicles and whorls with areas of Antoni A (5/11) and Antoni B (2/11) patterns. Four tumors had a cellular organization resembling peripheral nerves with fine periodic acid-Schiff positive outlines of cellular processes. Variable amounts of fibrillar eosinophilic extracellular matrix were a prominent feature in all tumors. Pre-iridal fibrovascular membranes were present in 10 cases, and glaucoma was diagnosed in 8 cases. All tumors were positive for vimentin (7/7) and of 9 tumors, 7 were positive for glial fibrillary acid protein. All tumors were negative for smooth muscle actin (7/7), desmin (7/7), and melan A (9/9). We believe these dogs have a distinct anterior uveal spindle cell tumor, presenting in blue irides with morphologic features of schwannoma.
21: Gastrointestinal Stromal Tumors and Leiomyomas in the Dog—A Clinicopathologic, Immunohistochemistry and Molecular Genetic Study of 50 Cases
We reviewed diagnostic material and records from 50 canine gastrointestinal mesenchymal tumors from the archives of the AFIP. Twenty-one (42%) were histologically reclassified as gastrointestinal stromal tumors (GISTS) and 29 (58%) as leiomyomas. Both tumors occurred in multiple breeds, with the German Shepherd predominating. The GISTS occurred in 10 males and 10 females, with a mean age of 10.5 years (range 5–14 years). Five (24%) produced clinical signs and 6 (29%) had metastasis to the liver or abdominal cavity. The GISTS occurred in the colon (10, 48%), small bowel (6, 29%), stomach (4, 19%), and mesentery of small bowel (1, 5%), and were composed of densely packed spindle cells with an overall basophilic appearance. Mitoses ranged from 0–19/40X field. Eleven (52%) were positive for CD 117 (kit); 7 (33%) were positive for smooth muscle actin, and all were negative for desmin and S-100 protein. No ultrastructural features of smooth muscle or neural differentiation were identified. Exon 11 c-kit mutations were documented in 2 of 5 cases. The leiomyomas occurred in 23 (82%) males and 5 (18%) females with a mean age of 10.8 years (range 8–17 years). Nine (31%) tumors produced clinical signs. The leiomyomas occurred in the stomach (22, 76%), esophagus (4, 14%) and 1(3%) each in small bowel, colon and rectum; they were composed of mature smooth muscle cells with eosinophilic cytoplasm. No mitoses were observed. Twenty-eight (97%) were positive for smooth muscle actin and 18(62%) for desmin. All were CD117 negative. Electron microscopy verified smooth muscle differentiation. We conclude that although both GISTS and true leiomyomas occur in the GI-tract of dogs, these tumors have distinctive pathologic features.
22: Camelid Cutaneous Fibropapillomas: Clinicopathologic Findings and Association with Papillomavirus
Four camelid cutaneous fibropapillomas with histologic features similar to equine sarcoids were diagnosed. They were characterized by a dermal fibroblastic proliferation and overlying, often ulcerated, hyperplastic epidermis with thin rete pegs extending down into the dermis. Two of the tumors came from llamas and two from alpacas. All four animals were 6-year-old females. The fibropapillomas from the two llamas and one alpaca were located on the nose. The location of the other alpaca's tumor was not given. One of the llama tumors underwent partial regression before surgery but recurred and spread following surgery. All four of the tumors were positive for papillomavirus DNA by polymerase chain reaction testing. This report provides the microscopic and clinical features of fibropapillomas in camelids as well as evidence for a papillomavirus etiology.
23: Cervical and Vaginal Epithelial Neoplasms in Cynomolgus Macaques
Cancer of the cervix is the second leading cause of cancer death in women worldwide. In this report, naturally occurring cervical and vaginal intraepithelial neoplasms are described in histologic specimens obtained from 6 of 148 cynomolgus macaques (Macaca fascicularis) being used in long-term studies. Lesions were observed in both treated and control animals without respect to treatment. The lesions were located in the vaginal epithelium, ectocervix, and at the cervical os. Morphologic changes included papillomas; intraepithelial lesions ranging from focal dysplasia to carcinoma in situ; and one invasive squamous cell carcinoma. Lesions demonstrated proliferative expansion of the basal epithelium, anisokaryosis, and koilocytic atypia. Because papillomavirus infection is a major risk factor for cervical carcinoma in women, tissues from two of the six cases were examined with immunohistochemistry using a rabbit polyclonal antibody for human papillomavirus (NCL-PVp). Lesion staining was positive for papillomavirus antigen. Additional lesions have subsequently been identified clinically by colposcopic examination and acetowhite staining. These findings represent a previously unreported range of cervical and vaginal intraepithelial neoplasms in non human primates and suggest that papillomavirus infection may be a factor in lesion pathogenesis. The unique similarities between the observed lesions and those seen in women suggest that macaques may provide a suitable animal model for studies of the pathogenesis and prevention of papillomavirus-associated cervical cancers.
24: Argyrophilic Nucleolus Organizer Regions (Ag-Nors)-A Potential Prognostic Parameter in Veterinary Tumor Pathology: A Review and Proposal for a Standardized Approach
Interphase AgNORs contain all necessary components for ribo-somal RNA synthesis, including two non-histone argyrophilic proteins, C23 (nucleolin) and B23 (nucleophosmin), which allow visualization of AgNORs by light microscopy. More than 1000 papers in human medicine, but fewer than 50 papers in veterinary medicine reported on the evaluation of numerous qualitative and quantitative AgNOR parameters as diagnostic and prognostic tools for various neoplasms. The number of AgNORs has been demonstrated to be directly correlated with rRNA transcriptional activity. In contrast to methods that evaluate the percentage of proliferating cells (growth fraction) such as Ki-67, PCNA or BrdU, in vitro studies have confirmed AgNORs as one measure of proliferation rate. Standardization of silver staining methods, AgNOR parameters measured, (such as mean and total AgNOR area), AgNOR number and methods to count AgNORs are necessary to generate valid data. To achieve standardization of the AgNOR method and to establish AgNORs as a routine prognostic tumor marker, the “International Committee on AgNOR Quantitation” has proposed guidelines for AgNOR staining and evaluation in human tumor pathology. This presentation reviews the current knowledge of the structure and function of AgNORs as well as the experimental and theoretical justifications for AgNORs as a prognostic marker in human tumor pathology. In addition, the potential use of AgNORs as a prognostic tool in veterinary tumor pathology and an example of how AgNORs can be used as a prognostic marker for canine malignant lymphomas are presented. Furthermore, a method to evaluate the proliferation activity of neoplastic cells by combining analysis of the growth fraction (Ki-67) and the proliferation rate (AgNORs) is proposed.
25: Peripheral PrpSC Accumulation in Preclinical Sheep Scrapie
To determine the temporal and cellular accumulation pattern of PrPSc in natural preclinical disease, two flocks (80 sheep), were analyzed for Prnp genotype and examined for PrPSc accumulation at various potential accumulation sites (CNS and non-CNS) by immunohistochemistry using monoclonal antibodies. None of 19 sheep younger than 12 months had PrPSc deposits in any tissue. Of 61 sheep older than one year, 13 had PrPSc deposits. Nine had deposits within and outside of the CNS. Four had deposits restricted to non-CNS sites. All 13 animals with PrPSc deposits had involvement in gut associated lymphoid tissue (GALT) and the intestinal nervous supply. Other accumulation sites included lymphoid tissues unrelated to the gastrointestinal tract and placenta. PrPSc was not detected in bone marrow or mammary gland. PrPSc occurred only in sheep of Prnp genotype 136AA 171QQ. Cellular specificity of PrPSc deposition was demonstrated by double immunolabelling in lymphoid and intestinal tissues. PrPSc colocalized predominantly with follicular dendritic cells, rare macrophages and neurons. Consistent with experimental models of scrapie, these results demonstrate early involvement of the intestinal tract with amplification of PrPSc in specific cells of the GALT, regional lymph nodes and the PNS during preclinical disease. The data provide a rationale to study specific immune cells and their interactions with the peripheral nervous system in disease pathogenesis.
26: Phenotypic Characterization of Lymphoid Cells Accumulating Chronic Wasting Disease Prion Protein in Mule Deer
The abnormal isoform of the prion protein, PrPCWD, accumulates in lymphoid tissues in several of the transmissible spongiform encephalopathies, or prion diseases, including variant Creutzfeldt-Jakob disease, scrapie, and chronic wasting disease of deer and elk (CWD). Using multi-immunofluorescent labeling and confocal microscopy to phenotype PrP-containing lymphoid cells, we have found that PrPCWD accumulates on the membrane surface of S100 and vimentin-expressing follicular dendritic cells (FDC) within the follicular germinal centers, potentially in association with complement or Fcγ receptors. PrPCWD colocalized with immunoglobulin and cc21 (CD21), indicating that PrPCWD was primarily extracellular in the germinal center. Cells morphologically consistent with tingible body macrophages (TBM) also contained PrPCWD. These findings suggest that FDC and TBM within lymphoid follicles harbor PrPCWD in deer with CWD.
27: Binding of Shiga Toxin 2E (Edema Disease Toxin) to Porcine Red Blood Cells
Edema disease is caused by swine-adapted Escherichia coli (STEC) strains that produce Shiga toxin 2e (Stx2e). Previous studies have shown that Stx2e receptor globotetraosylceramide is expressed on porcine red blood cells (RBC), that Stx2e binds to porcine RBC in vitro and that Stx2e can be detected in the blood of some STEC-inoculated pigs. The first objective of this study was to determine if the levels of Stx2e in blood of STEC-inoculated pigs correlate with the incidence, the severity of disease, or both. Blood samples from 50 STEC-inoculated pigs were collected every other day 2 weeks postinoculation and tested in a Vero cell assay. Eleven of 15 (73%) clinically affected pigs and 5/35 (14%) subclinically affected pigs had Stx2e in the RBC fraction of their blood but not in serum or plasma. Sub-clinically affected pigs with Stx2e in their blood developed more extensive vascular lesions in the brain and ileum than pigs without Stx2e in blood (average number of necrotic arterioles 63% and 27.5%, respectively, p = 0.001). The second objective was to determine if pigs vary in the capacity of their RBC to bind Stx2e in vitro. FACS analysis of blood from 34 naive pigs demonstrated two RBC phenotypes based on their capacity to bind Stx2e. Although RBC from most pigs consistently bound high amounts of Stx2e (high-binding phenotype, n = 29), consistently low Stx2e binding was detected in RBC from few pigs (low-binding phenotype, n = 5). We concluded that the incidence and severity of edema disease correlated directly with detectable levels of Stx2e in the blood of STEC infected pigs and that pigs vary in the capacity of their RBC to bind Stx2e in vitro.
28: Halicephalobiasis with Mandibular Destruction in a Young Mixed Breed Horse
Halicephalobiasis is an uncommon, yet important, parasitic disease of horses and humans that is caused by Halicephalobus gin-givalis. A two year old, mixed-breed horse with a chief complaint of mandibular swelling was referred to an equine practice for further examination and biopsy. The mandible was markedly enlarged and radiographs revealed extensive osteolysis with bony proliferation of the rostral 40% of the mandible. Small amounts of white mandibular tissue were evaluated histopathologically. There was widespread loss of normal bone architecture with replacement by a marked granulomatous infiltrate composed of macrophages, fewer multinucleated giant cells, and rare lymphocytes, plasma cells, and eosinophils. Within this infiltrate, numerous nematode larvae exhibited the following morphology: a cross-sectional diameter of 18–25 microns; longitudinal lengths of >30 microns; rhabditiform esophagus; thin cuticle; pseudocoelom; platymyarian-meromyarian musculature; digestive tract and tapered ends. Rare larvae were seen in the margins of bone or within osteoid. Based on the morphology and host response, a diagnosis of Halicephalobiasis was reported. Treatment was not attempted, and the horse was euthanized. The pathogenesis and life cycle of Halicephalobus gingivalis is poorly understood and only females, reproducing parthenogenetically, have ever been identified; attempts at hatching eggs from this fresh tissue were initially unsuccessful. Halicephalobus gingivalis is found in areas of decaying organic matter and is thought to infest horses through oral, nasal, or skin wounds. Reported sites of localization include skin, the urogenital tract, the central nervous system, oral and nasal cavities, and bone and joints.
29: Chronic Leptospirosis in An Asiatic Water Buffalo (Bub-Alus Arnee) and Preliminary Survey for Leptospira Infection in Captive Nondomestic Ruminants at a Suburban Zoo
A 14 year-old, captive-born, male Asiatic water buffalo (Bubalus arnee) died following progressive weight loss. Laboratory findings included azotemia, hyperphosphatemia, hypocalcemia, hypoglycemia, and elevated LDH and AST. It had been housed in a large outdoor, mixed-species enclosure; llamas, guanacos, mouflons, addax, Barbary sheep and capybaras were housed in the same enclosure. Necropsy revealed the absence of fat stores, severe serous atrophy of pericardial and renal pelvic fat, moderate to severe generalized muscle atrophy, moderate generalized lymphadenomegaly and moderate hydropericardium. The kidneys were firm and had numerous whitish coalescent streaks throughout the cortex and medulla. There were two tan nodules in the liver. Microscopically, there was severe chronic lymphoplasmacytic and fibrosing tubulointerstitial nephritis with Warthin Starry-positive spirochetes within the lumen of renal tubules. Other relevant findings included hepatocellular carcinoma in the liver, moderate to severe renal tubular hemosiderosis, arteriosclerosis of medium-sized renal arteries, and moderate to severe mes-angioproliferative glomerulopathy. A preliminary serologic survey using the microscopic agglutination test for 12 Leptospira interrogans serovars in 53 captive ruminants belonging to 10 species revealed positivity in 17 animals (32%) with titers up to 1:800 and the 12 serovars represented (mostly L. i. icterohaemorrhagiae, L. i. autumnalis and L. i. pomona). L. i. icterohaemorrhagiae, -autumnalis and/or pomona antibody titers up to 1:200 were found in three weak newborn calves (two nilgai antelope and a common waterbuck). Serologic testing in the Asiatic water buffalo index case was performed with banked sera from 1996, 1997 and the time of deat, and was negative. The clinicopathologic findings in the Asiatic water buffalo are consistent with chronic leptospirosis, and the results of the preliminary serologic survey suggest that exposure to multiple L. interrogans serovars occurs frequently in captive ruminants at Africam Safari. Leptospirosis may be a significant disease and mortality factor in captive ruminants at Africam Safari.
30: Malignant Catarrhal Fever in Bison
In recent years, malignant catarrhal fever (MCF) has become an important disease in the bison industry. Herds have experienced up to 100% mortality. Bison have a rapid clinical disease, usually dying within 48 hours of onset of illness. Bison usually have hemorrhagic cystitis and colitis, but lymphadenomegaly and corneal opacity occur in fewer than half the cases in bison. Vasculitis is often subtle and fibrinoid vascular necrosis is rare. Direct contact with sheep has not been demonstrated in all bison herds experiencing MCE PCR testing consistently demonstrates ovine herpesvirus-2 (OHV-2) in tissues of bison and cattle with MCF. OHV-2 has been demonstrated in peripheral blood leukocytes of animals that later die of MCF. Other herpes viruses and retroviruses have not been found consistently. OHV-2 was not found in tissues from culled bison in a herd with no history of MCF. OHV-2 has been found in peripheral blood of almost all adult domestic sheep tested but has not been found in two herds of Rocky Mountain bighorn sheep that have not had contact with domestic sheep. The means of transmission of this disease is not known.
31: Encephalomyelitis Associated with Akabane Virus Infection in Adult Cows and Calves in Korea
Five cows, 2 to 7 years old, exhibiting neurologic signs in Korea were diagnosed as Akabane virus infection based on the results of histopathology, immunohistochemistry, serology, and RT-PCR analysis. Immunohistochemistry and RT-PCR were equally effective and sensitive for diagnosing Akabane virus infection during the early stage of infection. Histologically, typical lymphohistiocytic inflammation characterized by perivascular mononuclear cell infiltration, gliosis, neuronophagia, and neuronal loss were noted in the brain and the spinal cord ventral horn gray matter. The lesions in the brain were most prominent in the pons and medulla oblongata. Akabane virus antigens were detected in the brain, spinal cord or both, mainly in the degenerating neurons and glial cells. RT-PCR generated the expected band in four cases. This is the first report on the outbreak of natural Akabane virus infection in adult cattle. An outbreak of Akabane virus infection was also diagnosed in 15 newborn calves. Further study is underway to characterize the virus isolated from 2 adult cases.
32: Lesions in Green Tree Pythons (Morelia Viridis) Infected with a Novel Iridovirus
Three of 10 juvenile green tree pythons (Morelia viridis) died shortly after being confiscated by Australian customs authorities following illegal importation from Indonesia. The snakes that died were emaciated and had large ulcers of the buccal mucosa. There was moderate chronic ulcerative necrosis of the nasal mucosa and severe diffuse, periacinar vacuolar change of the liver with extensive areas of hepatocyte necrosis in 2 snakes. One of them also had severe necrotizing pharygitis. Immunohistochemistry with a rabbit polyclonal antibody against epizootic hematopoietic necrosis virus (EHNV) detected viral antigen in the liver, heart, lung, kidney, stomach, small intestine, skin, bone, and nasal and oral epithelium. Immunoreactivity was observed in a variety of cell types including vascular endothelium, fibroblasts and osteoblasts, as well as in nasal exudate. No abnormalities were detected on gross or histopathologic examination of the other seven snakes. A virus isolated from two of three snakes that died was characterized by electron microscopy, enzyme linked immunosorbent assay (ELISA), polyacrylamide electrophoresis, PCR and sequence analysis, restriction endo-nuclease digestion, and DNA hybridization. The virus differs from other reported Ranaviruses and has been provisionally named Wamena virus (WV) after the geographical location in Irian Jaya where the snakes were collected. This also is the first report of a systemic Ranavirus in any species of snake.
33: Bovine Herpesvirus-4 Associated Endometritis: An Emerging Syndrome in Dairy Cattle
More than 35 cases of BHV-4 associated metritis have now been confirmed in Georgia dairies. All cases have initially occurred in the 3 to 28 day postpartum period and have followed a similar course. The endometrial lining epithelium becomes necrotic and ulcerates 3 to 7 days postpartum with only mild inflammation in the lamina propria and submucosa. Over the ensuing 21 days, the ulcers become confluent and diffuse. Epithelium is replaced by fibrinonecrotic, suppurative mats and severe transmural suppurative inflammation, eventually resulting in severe bacterial pyometra. Arcanobacterium pyogenes, Escherichia coli, and Clostridium perfringens have been isolated from the uterus and are consistently refractory to antibiotic and uterine infusion therapy. Seroprevalence to BHV-4 in one Georgia dairy with 15 clinical cases approaches 50% and a few cats from the premises are also seropositive. Heminested PCR, fluorescent antibody assays and viral isolation from the uterus are preferred diagnostic assays. Venereal transmission may be important, and infected bulls could be latent carriers for life. The clinical signs, post-parturient timing and histopathologic lesions are very similar to those reported in Belgium with BHV-4. In addition, RFLP and sequencing analysis of PCR products suggests the strain of virus may be more similar to Movar 33 than to DN599 (more commonly isolated in the U.S.). Lipidosis and hypocalcemia often accompany the metritis and may be requisite cofactors in disease expression following parturition. Because death is often due to other factors, the diagnosis of BHV-4 could be easily overlooked. BHV-4 associated endometritis appears to be an emerging syndrome in southern U.S. dairies.
34: A Feline Panleukopenia Outbreak in New Mexico Cats—Use of a Canine Parvovirus Elisa Test for Diagnosis
A major outbreak of panleukopenia (feline parvovirus infection) occurred in cats in Albuquerque, New Mexico, in 1999 and 2000. A total of 22 cats were necropsied and confirmed to have died from panleukopenia over a 1.5-year period. In the three years before this outbreak, panleukopenia had been diganosed only once in a cat from the Albuquerque area. The disease had been rarely diagnosed in cats from other parts of the state. Cats that died of panleukopenia were primarily kittens less than four months of age that were from animal shelters. Four adult cats were found to have died from panleukopenia. Severe enteritis with marked epithelial crypt necrosis was found in all cats diagnosed with panleukopenia. Additional lesions seen in some cats included lymphoid depletion in Peyer's patches and lymph nodes and bone marrow hypoplasia. Basophilic intranuclear inclusion bodies were observed in crypt epithelial cells in a few cats. Electron microscopy on intestinal content revealed parvovirus-like viral particles in one cat examined. Intestinal content from five cats with intestinal lesions typical of panleukopenia were tested for parvovirus antigen using a commercial canine ELISA test. All five tested positive for parvovirus with this ELISA test. Four cats with diarrhea, but without histologic lesions typical of panleukopenia, tested negative. It appears that the canine parvovirus ELISA test may be a reliable antemortem test for panleukopenia in cats.
35: A New Form of Amyloid in So-Called Canine Calcifying Epithelial Odontgenic Tumor
The precursor protein for amyloid deposits contained in the so-called canine calcifying epithelial odontogenic tumor (c-CEOT) has not been investigated. We attempted to definitively establish the specific type of amyloid protein and/or precursor amyloid protein in a cCEOT. Specimens from a cCEOT were obtained by surgery and crude amyloid substance was extracted with subsequent purification by SDS-PAGE, electroblotting onto PVDF membranes, excision and elution of amyloid protein related bands, and reversed-phase HPLC. The 15 N-terminal residues of the amyloid protein band were compared with the published N-terminal sequence of the rat ameloblastin. Eleven of the 15 amino acids sequenced were identical. Amelogenin and ameloblastin (sheathlin) are produced by ameloblasts in different stages and considered to play various roles in the modulation of mineral deposition and crystals during tooth morphogenesis. Immunohistological analyses were completed on the cCEOT and tooth germ from canine aborted fetus, using anti-porcine sheathlin N-terminal (pS), anti-rat ameloblastin (rAB) and anti-porcine amelogenin (pAG) antibodies. Immunopositive reaction against pAG, rAB, and pS was found in the amyloid of the cCEOT and the immature enamel of tooth germ. No immunopositive reaction against pAG, rAB, and pS was detected in the neoplastic epithelial cells of the cCEOT. In conclusion, amyoid may be derived from amelogenin and ameloblastin (sheathlin) components produced by ameloblasts in the cCEOT.
36: Histopathologic Examination of Hepatic Microvascular Dysplasia without Portsystemic Shunt in Dogs: 10 Cases
Hepatic microvascular dysplasias (HMD) were compared with portsystemic shunt (PSS). Ten dogs with HMD and 10 dogs with PSS were studied. Both groups of dogs had various clinical signs including seizures, ataxia, lethargy, vomiting and diarrhea. Both groups had elevated levels of total bile acid and ammonium. All dogs underwent portography. Histopathologically, there were increased arteriolar profiles in the portal triads of both groups. In PSS, basic hepatic architecture was normal. HMD showed abnormal lobular architecture associated with considerable variation in the size of hepatic lobules. Portal triads were increased in HMD and less developed compared to PSS. HMD is a congenital disorganization of the liver's microscopic architecture. In 3 of the HMD cases, the prognosis was poor and the dogs died within a few months.
37: Does Infectious Bursal Disease Virus Cause Proventriculitis in Broiler Chickens?
Acute necrotic proventriculitis is a naturally occurring disease of broiler chickens in the United States. It decreases proventricular wall tensile strength leading to proventricular rupture during evisceration and economic losses caused by carcass contamination. Morphologically, lesions progress from acute glandular epithelial necrosis to interstitial edema and fibroplasia with severe multifocal lymphocytic infiltrates in the inter-glandular stroma. Infectious Bursal Disease Virus (IBDV) has been implicated as the cause of this disease, and IBDV vaccination protocols are being recommended to prevent its development. We collected proventriculi from naturally affected broiler chickens during acute and chronic phases of this disease. Samples were formalin fixed and paraffin embedded. Total RNA was extracted from samples and examined by real time RT-PCR using primer sets specific for IBDV. No sequences matching known classical or variant IBDV strains were detected in these extracts. These results show typical IBDV strains are not present in situ. We conclude acute proventriculitis in broiler chickens is caused by alternate strains of IBDV not detected by these primer sets or is caused by another agent whose effects are exacerbated by previous IBDV-induced immunosuppression.
38: Differences in Mycobacterial and Parasitic Infections Observed among Three Groups of Co-Housed Doves (Streptopelia Risoria, Geopelia Cuneata)
The clinical diagnosis of mycobacteriosis in an aviary resulted in the euthanasia of a flock of 23 doves, comprised of 10 ringneck doves (Streptopelia risoria) with wild-type coloration (RN), 6 white color mutants of the ringneck doves (WRN), and 7 diamond doves (DD; Geopelia cuneata). All birds had been maintained in the same outdoor facility for several years, some having been hatched there. Significant differences among the 3 groups of birds were observed with respect to several clinical and histologic parameters. Serology for anti-mycobacterial antibodies was positive in 10/10 RN, 7/7 DD, and 1/6 WRN. The severity of granulomatous disease was evaluated grossly and histologically by number, severity, and organ distribution of lesions. Multiple organ involvement was observed in 10/10 RN, 1/6 WRN, and 1/7 DD doves. Severe to effacing lesions were observed in 7/10 RN, 1/6 WRN (same bird as above), and 3/7 DD doves. Amyloidosis (primarily hepatic, but sporadically renal, splenic, pancreatic, and/or adrenal) wass present in 4/10 RN, 2/6 WRN, and 0/7 DD doves. In addition to lesions attributable to mycobacteriosis, variations in parasitism exist among the three groups. The proventricular mucosae of the majority of the RN doves (7/10) contained Tetrameres spp. organisms, which were not observed in any birds of the other two groups. The intestinal mucosae of 1/10 RN, 2/6 WRN, and 0/7 DD doves contained moderate numbers of Cap-Maria spp. organisms. Given the common environmental influences, immunogenetic variations among the three groups of birds are believed to play a significant role in the observed differences in mycobacterial disease and endoparasitism.
39: Morphogenesis of Subchondral Bone Failure: An Sem Study in Horses
Subchondral bone failure occurs focally in racehorses with traumatic osteochondrosis of the fetlock joint. It is often symptomless, but more severe forms are associated with lameness. Scanning electron microscopy (SEM) was performed on horses with various stages of disease to study the morphogenesis. Palmar fetlock condyles from 23 racehorses with mild, moderate or severe subchondral bone sclerosis; subgross cracks beneath the calcified cartilage; and flattening or indentation of the cartilage were studied. Parasagittal slices 2 mm thick were photographed, radiographed and prepared for SEM. Fine intralamellar cracks between collagen fibers and short radiating fine cracks in the matrix were seen in most samples. This change was a possible drying artifact but was more prominent in the layer of bone immediately beneath the calcified cartilage and in the more sclerotic bone at the failure site. The earliest subgross cracks developed within 1–2 mm of the calcified cartilage layer and extended parallel to it for 4–5 mm in irregular branching lines. Fragmentation of crack margins developed as a gap developed in the crack. In one fortuitous sample, smoothly ground fragments were seen within the gap along with an entrapped vessel. In other samples, loss or fragmentation of bone at the crack margins had led to the collapse and compaction of the subchondral bone beneath sites where there was flattening or indentation of the intact overlying cartilage layers. There was little evidence of reactive change at the site, but evidence of osteoclastic enlargement of vascular canals was often seen a few millimeters deep to the lesion site and could be recognized radiographically. Interestingly, none of these obviously chronic lesions were known to be responsible for clinical lameness.
40: Progesterone Accelerates the Development of Hypertension in a Rat Model of Genetic Hypertension and Congestive Heart Failure (Shhf-Facp)
This study examines the effect of estrogen and progesterone on blood pressure in lean female SHHF-facp rats (SHHF). Female SHHF rats develop hypertension later than males with accelerated development of cardiovascular disease after reproductive senescence. In contrast to people and other strains of hypertensive rats like the spontaneously hypertensive rat that respond to pregnancy with lower blood pressure, SHHF show dramatic increases in blood pressure during pregnancy which return to baseline values after delivery. These studies investigated the effect of pseudopregnancy and subcutaneous implants containing physiologic doses of estrogen, estrogen plus progesterone, progesterone, and saline on conscious systolic tail cuff blood pressure. The ability of the type II progesterone antagonist RU468 to block the effects of progesterone was also studied. The progesterone treated group showed significantly higher mean blood pressure than controls (P<0.05). Differences in blood pressure among the estrogen group, estrogen/progesterone group, and control groups were not statistically significant. The blood pressure for the pseudopregnant group (baseline = 133, day 15 = 174) was almost identical to blood pressure for the progesterone group (baseline = 132, day 15 = 172). Progesterone-treated rats had significantly higher systolic blood pressure compared with control and RU486+progesterone-treated rats, indicating the ability of RU486 treatment to prevent the progesterone-mediated increase in blood pressure. After discontinuation of the RU486 treatment, the blood pressure of the rats in this group had increased to be similar to the progesterone-treated group and significantly greater than the control group. These data indicate that changes in progesterone are an important mechanism for hormone dependent modulation of hypertension in SHHF females and may contribute to exacerbation of high blood pressure during pregnancy and after reproductive senescence.
41: Vascular Neoplasia in the Domestic Ferret (Mustela Putorius Furo): A Retrospective Study
Vascular tumors are occasionally seen in pet ferrets. Over a nine-year period, 14 cases were received at the AFIP. Hemangiosarcoma was identified in 13/2825 cases (<0.5%), versus 1 case of hemangioma, all being confirmed via positive immunohistochemical staining of neoplastic cells with a polyclonal anti-Factor VIII antigen stain. All the affected animals were adults with a mean age of 5.1 years (range 1–7 years). Of the 14 ferrets, 8 were male, 5 were female, and 1 of unknown gender. Six of the 14 tumors (43%) occurred in the haired skin, with no fur color predilection. Five of 6 cutaneous vascular neoplasms showed evidence of malignancy, with aggressive infiltration along deep and/or lateral margins and the presence of mitotic figures within the neoplastic population. There were no reports of local recurrence or metastasis after complete excision. Although the sample is small, these cutaneous neoplasms appear to have similar biological behavior to their counterpart in dogs. An interesting finding in this study is that all the cutaneous hemangiosarcomas were seen in males. Eight of the 14 tumors (57%) were of visceral origin, with these organs involved: spleen (4 cases, 50%), peritoneum (2 cases, 25%), abdominal lymph nodes (1 case, 14%), and liver (1 case, 14%). In two cases, primarily involving the spleen and peritoneum respectively, the neoplasm appeared to be related to the cause of death. We believe that the spleen is a likely site of origin for hemangiosarcomas of the abdominal cavity in ferrets and that their pattern mirrors that seen in the dog with a potential for widespread abdominal metastasis. The long-term prognosis for ferrets with visceral hemangiosarcoma, as would be expected, is poor.
42: Neoplasms of the Liver in the Domestic Ferret (Mustela Putorius Furo)
Neoplasms involving the liver are occasionally seen in ferrets. In a review of 2825 cases archived at the AFIP from 1992 to 2000, hepatic neoplasia was identified in 60 cases (2.1%). Secondary neoplasms of the liver (those involving the liver by extension or metastasis) accounted for 43/60 cases (72%), whereas primary neoplasms account for 17 (28%). The distribution of secondary neoplasms is as follows: malignant lymphoma (16/ 37%), adrenocortical carcinoma (14/32%), poorly differentiated carcinoma/adenocarcinoma (10/23%), pancreatic exocrine adenocarcinoma, (1/2.3%), mast cell tumor (1/2.3%), poorly differentiated sarcoma (1/2.3%). Cholangioma was the most common primary hepatic neoplasm in this study (6/35%), followed by hepatocellular carcinoma (3/18%), hepatoma (3/18%), hemangiosarcoma (3/18%), and cholangiocarcinoma (2/12%). All three forms of malignant lymphoma seen in ferrets (lymphocytic, lymphoblastic, and immunoblastic-polymorphous) were represented in this study, with both leukemic and solid tumor forms in evidence. Although the majority of the neoplasms seen in this study are morphologically distinct, the differentiation of metastatic adrenocortical carcinoma from hepatocellular carcinoma may be difficult and often requires immunohistochemical confirmation. Diagnostic and prognostic features of the neoplasms listed above will be discussed.
43: Systemic Cytomegalovirus Infection in a Xenograft Transplant Recipient Cynomolgus Macaque
An adult male cynomolgus macaque (Macaca fascicularis) developed a fever of unknown origin 28 days post surgery for heterotopic transplantation of a transgenic piglet heart. The monkey was depressed, anorexic, and developed diarrhea at day 31. Immunosuppressive drugs used here previously in xenograft transplant monkeys consisted of cyclosporine, methylprednisolone, and ERL, but in this monkey, antithymocyte globulin (ATG) was also added. Therapies initiated at the onset of clinical signs included ticarcillin/clavulonic acid, gentamicin, fluconazole, and metronidazole. All macaques used by this transplantation group are Herpes B, SIV, SRV, STLV and TB negative, but at euthanasia (day 36) serology from this animal was positive for Simian Cytomegalovirus. Gross necropsy lesions consisted of rare petechial hemorrhages in the lung and kidney, and swollen, pale kidneys. Histologically, disease was most severe in the lung and consisted of mild, nonsuppurative interstitial pneumonia, alveolar, perivascular, and intramural edema, and numerous markedly swollen endothelial cells that often contained large intranuclear viral inclusions. Cytomegalic, inclusion-bearing cells of mesenchymal origin (i.e., endothelial cells, fibroblasts and monocytes) were also noted in liver, kidney, spleen, salivary gland, adrenals, testis, heart, intestines and brain. Other lesions included disassociation of hepatic cords and acute renal tubular necrosis. Cytomegalovirus is an opportunistic pathogen frequently encountered in immunosuppressed human transplant or HIV/ AIDS patients. Because CMV had not previously been diagnosed in other transplant macaques here, the addition of ATG to the immunosuppressive drug regime was presumed responsible for disease manifestation. To our knowledge, CMV has not been reported in cynomolgus macaques chemically immunosuppressed for transplantation purposes, and this case offers a useful model for CMV pathogenesis in human transplant patients.
44: Distribution and Expression in Disease of a New Family of Molecules Based on Homology to Jam: Tissue Macrophage-Like Cell Specific Stigma, Endothelial Cell Specific Jamit and Its Receptor on Nk and Dendritic Cells, Shatr
JAM is a junctional adhesion molecule expressed on the lateral surface of epithelial cells as well as on neutrophils and may have a role in transmigration of cells across mucosal barriers. Here we describe two novel membrane molecules related to JAM by sequence homology: JAMIT (or VE-JAM) and STIGMA and describe the receptor for JAMIT, SHATr. By in situ hybridization, JAMIT is expressed on high endothelial venules in lymphoid tissues and has inducible expression in vascular endothelium during inflammation or neoplasia. SHATr binds to JAMIT based on protein-protein and cell-based assays and is specifically expressed on CD56+ NK cells, CD56+/CD3+ NK-T cells, CD8+ cytotoxic cells and dendritic cells as determined by FACS, in situ hybridization and immunohisto-chemistry. The expression pattern of SHATr and JAMIT suggests their interaction may function during homing or trafficking of these cell types. STIGMA, an additional novel family member, is an ITAM-motif bearing trans-membrane protein specifically expressed in an exclusive population of tissue macrophage-like cells including placental Hofbauer cells, resident macrophages in the intestinal lamina propria, microglial cells, synovial cells, Kupffer cells, and alveolar macrophages. STIGMA expression is increased in these cells when activated by the presence of neoplasia or inflammatory disease including rheumatoid arthritis, hepatitis, pneumonia, asthma and bronchitis. Together these molecules comprise a new family of molecules whose biological relevance in disease is inferred by their expression pattern and ligand-receptor interactions.
45: The Anatomic and Immunologic Function of the Piscine Heart and Its Role in the Diagnosis of Infectious Disease
Many diagnosticians do not appreciate the anatomic and functional differences within the piscine heart and the potential impact that examination of these sites may have on one's ability to make a diagnosis of infectious disease. The are four distinct anatomical components to the fish heart: the sinus venosus, the atrium, the ventricle, and the bulbus arteriosus. The ventricle, the largest component, is anatomically and functionally similar to the ventricle in other species and may be a target for degenerative, toxic or infectious diseases. The atrium and bulbus arteriosus are less commonly sampled, but their unique anatomic and functional characteristics make examination of these sites essential. The atrial lumen is lined by endothelial cells that function as a component of the reticuloendothelial system. These cells function in the innate immune response and are capable of phagocytosis of particulate foreign material and, more importantly, infectious agents. Bacterial phagocytosis by these endothelial cells has been identified and associated with systemic infections of Vibrio sp. and Mycobacterium sp. Viral infection of these cells has been identified in numerous fish infected with iridovirus. The bulbus arteriosus, a structure unique to fish, is composed of a complex meshwork of elastic fibers and smooth muscle. This complex meshwork may serve to trap circulating infectious agents. Histopathologic examination of the bulbus arteriosus has proved useful in the diagnosis of systemic fungal infections in multiple cases. As a diagnostician or prosector, it is important to understand the unique anatomical and functional differences among components of the heart so that those components may be sampled appropriately to aid in the diagnosis of infectious disease in fish.
46: Prevalence and Pathological Characteristics of Helicobacter Spp. in Gastric Mucosa of Domestic Pet Dogs
This study was performed to survey the prevalence of Helicobacter infection in pet dogs and examine the pathological changes. Twenty-one animals dying of various causes were obtained from several animal hospitals and necropsied. Gastric samples were fixed in 10% neutral formalin and frozen at -20°C for PCR assay. With Steiner's silver stain, Helicobacter-like organisms (HLO) were observed in the gastric mucosa of 5 (23.8%) of 21 stomachs. The bacteria colonized mainly the mucus, gastric pits, cytoplasm of parietal cells, and the lumen of gastric glands. PCR with Helicobacter genus specific primers amplified a 374 bp 16S rDNA fragment in the 5 gastric samples. Immunohistochemistry with a rabbit polyclonal antibody against H. pylori demonstrated positively stained bacteria in gastric pits, the lumen of gastric glands, and the cytoplasm of parietal cells. HLO infection in dogs seemed unrelated to gastritis.
47: Isolation and Identification of Porcine Circovirus and Postweaning Multi-Systemic Wasting Syndrome in China
Postweaning multisystemic wasting syndrome (PMWS) has become an emerging disease in swine worldwide. Two outbreaks of PMWS in swine had been recently observed in two separate geographic locations (Beijing City and Hebei Province) in China. Affected pigs showed similar clinical signs, for example, dysnea, tremor, anorexia, jaundice, debility and death. Necropsy revealed enlarged liver and mesenteric and inguinal lymph nodes, hemorrhage in the large and small intestines and renal capsule, consolidation of the lung, and atrophy of the spleen and pancreas. Histopathological examination revealed lymphoid depletion in the spleen and lymph nodes with infiltration of lymphohistiocytic cells and hemosiderinladen macrophages in the sinusoidal spaces, mucoid bronchiolitis and bronchointerstitial pneumonia, erosive to ulcerative enteritis with atrophy or collapse of intestinal villi and atrophy of pancreatic acinar cells. Congestion and/or infiltration of lymphohistiocytic cells were observed variably in the liver, kidney, heart and pancreas. Electron microscopy revealed numerous small non-enveloped porcine circovirus (PCV) -like viral particles in selected sample from the lymph node and spleen. Polymerase chain reaction and indirect immunofluorescent stain revealed that both isolates were positive for porcine circovirus type 2 (PCV-2). PCV was successfully isolated from both geographic locations, and were cultured in Dulac cells. Electron microscopy on the cultured virus showed numerous small, nonenveloped PCV-like viral particles that were approximately 17 nm in diameter.
48: Intracranial Ganglioneuroma in a Dog
Ganglioneuroma is a benign tumor of neural crest origin most often arising in the peripheral autonomic nervous system. Intracranial ganglioneuroma is extremely rare in all species, but has been previously reported associated with the trigeminal nerve in a dog and a child. A 10 year old West Highland White terrier was euthanized after a progressive 7 week course of neurologic deficits involving the cranial nerves IV and X. Other clinical sign included circling to the right, hypermetria and left head tilt. Gross examination revealed marked atrophy of the left temporal and masseter muscles and a necrotic ulcer on the left cornea. Examination of the brained showed a discreet, firm mass occupying the cerebello-pontine angle. On histologic examination the mass was composed of well differentiated neurons surrounded by whorls of unmyelinated axons with associated Schwann cells and perineurial fibroblasts. Most neurons were positive for neuron-specific enolase (NSE) but were negative for S-100 antigen. Schwann cells were weakly positive for NSE but strongly positive for S-100. Perineurial cells were negative for both. This is only the second case of intracranial ganglioneuroma reported in the dog. Clinical and gross findings were compatible with an origin in the trigeminal nerve.
49: Diagnosis of Infectious Canine Hepatitis Virus (Cav-1) Infection in Puppies with Encephalopathy
Infectious canine hepatitis (ICH) is an adenoviral infection with worldwide distribution that affects primarily young Canidae and Uridae. We report an outbreak of fetal ICH virus-induced encephalopathy in a litter of 11 puppies, 9 of which died with acute CNS disease. The puppies were less than one week old and had signs of lethargy, vocalization, head pressing, ataxia, circling, blindness, and vomiting. Formalin-fixed sections of brain, liver, kidney, and lymph nodes were submitted from one puppy, whereas frozen tissues were submitted from another. Immunohistochemistry (IHC), virus isolation, and PCR methods were used to detect virus. The brains from the two puppies were slightly swollen and on cut surface had multifocal petechial hemorrhages and gray discoloration in the brain stem. Sections of brain had multifocal areas of congested blood vessels that contained swollen endothelium. Adjacent neuropil contained coalescing areas of hemorrhage, mild spongiosis and individual neurons that were swollen to pyknotic. Although not obvious on H&E stained sections, large intranuclear inclusion bodies were seen in endothelium stained stained for virus by IHC. The liver had minimal foci of periacinar to peripheral hepatocellular coagulation necrosis. Large basophilic, IHC-positive intranuclear inclusion bodies were occasionally seen within randomly scattered viable and degenerate hepatocytes. ICH virus was isolated from the liver in inoculated cell cultures. Lymph nodes were diffusely enlarged and edematous with ecchymotic hemorrhages. PCR detected canine adenovirus DNA in supernatant fluids from viral infected cell cultures. CAV-1 is an endotheliotropic and hepatotropic virus that rarely causes acute, severe, hemorrhages in the brain.
50: Borna Disease in a Heifer in Japan
Borna disease (BD) in cattle has been reported in Switzerland and Germany. This study investigated a Japanese heifer suffering from a severe and acute progressive CNS disorder using histopathology. Detection of BD virus (BDV) antigens and nucleic acid in the CNS by immunohistochemistry, PCR and in situ hybridization (ISH). A 2-year-old Holstein-Friesian heifer was suspected of having bovine herpes encephalitis (BHV) or listeriosis because of sudden hyperesthesia and tremor. Neuropathological lesions consisted of a nonsuppurative encephalomyelitis characterized by thick perivascular cuffs of mononuclear cells, the presence of inflammatory cell infiltrates in the neural parenchyma, neuronophagia and focal gliosis. Although these lesions were widespread in the cerebrum, cerebellum, and spinal cord, inflammatory changes were predominantly located in the grey matter. Strong immunopositivity against the BDV p40 protein was diffusely distributed in the cytoplasm of small and large neurons in the areas with inflammatory changes. Immunopositivity against BHV-1, C. psittaci and Listeria spp. antibodies was not found in any regions in the CNS. Positive hybridization signals with antisense riboprobes were present in the nuclei and cytoplasm of the neurons. BDV p24 RNA-positive signals in the paraffin-embedded brain tissue were found in the frontal lobe of heifer by RT-nested PCR. The histologic and immunohistochemical features of CNS lesions in the heifer differed from those of CNS lesions caused by BHV-1, C. psittaci and Listeria spp. In summary, a heifer was diagnosed as having Borna disease. This is the first report describing an evidence of spontaneous Borna disease in cattle in Japan.
51: Immunohistochemical Differentiation of Canine Central Nervous System Diseases Producing Mononuclear Pleocytosis
A total of 39 dogs with histopathologically confirmed central nervous system CNS mononuclear pleocytosis were characterized immunohisto-chemically using antibodies against CD3, lysozyme, canine immunoglobulin (IgG, IgM, IgA), canine distemper virus, Toxoplasma gondii, and Neospora caninum. Of 39 dogs, 26 demonstrated lesions compatible with granulomatous meningoencephalomyelitis (GME), 6 canine distemper, 1 toxoplasmosis, 1 neosporosis, 2 CNS lesions of unknown cause, and 3 had CNS lymphoma involving the spinal cord. All dogs were evaluated immunohistochemically for distemper, toxoplasmosis and neosporosis. Dogs ranged in age from 2 months to 14 years. The ratio of females to males was 2:1. There was no breed predilection. CNS lesions consisted primarily of macrophages and lymphocytes in perivascular cuffs and granulomatous parenchymal and meningeal infiltrates. Dogs with GME had primarily CD 3 antigen positive T cells, as well as lysozyme positive cells. The distemper subgroup demonstrated less numerous cellular infiltrates overall, with lysozyme expression barely predominating over anti-canine Ig expression. CD3+ expression was minimal. The two dogs with toxoplasmosis and neosporosis had marked lymphocytic perivascular infiltrates that were predominantly B-cell origin; lysozyme staining was less common, but predominated in the neosporosis case. CD 3 antigen positive T cells were rare in this subgroup. The two unclassified cases had variable outcomes, with lysozyme, CD 3 and B cell staining occurring in equal numbers. The three spinal lymphoma cases were B-cell origin. These results demonstrate the potential use of immunochemical staining of T and B cell lymphocytes and monocyte/macrophages in CNS tissues to better correlate postmortem disease diagnosis.
52: Enterotoxemia Caused by Clostridium Perfringens Type a in Formosan Deer
The case reports for Clostridium perfringens Type A entero-toxemia in formosam deer have rarely been recorded. This paper describes a natural case of Type A enterotoxemia in farmed formosam deer in Korea. A dead 10-month-old male formosam deer had been fed with assorted grain feed, oak leaves, acorn and bean curd. Grossly there was no visible external change. Despite the carcass being examined within 12 hours of death, there was significant post-mortem decomposition. There was severe hemorrhage in the serosa of abomasum and small intestine and much blood-tinged and watery contents were present in the lumen. In addition, there were severe swelling of spleen and some red foci in hepatic parenchyma. Microscopically there were severe congestion and hemorrhage in the mucosa, submucosa, muscular layer, serosa of abomasum and small intestine. The spleen and pancreas also showed severe congestion and hemorrhage. There were multifocal hemorrhage with hepatic necrosis in the periportal area and focal mononuclear cells deposition in sinusoid. Clostridium perfringens was isolated from the small intestine. Toxin typing demonstrated alphatoxin and that the strain belonged to Type A. In electron microscopy of feces, no virus particles were detected. Considering clinical signs, gross lesions, microscopic lesions, bacterial culture, and toxin typing of the isolate, this case was diagnosed as a enterotoxemia by Clostridium perfringens Type A.
53: Multiple Cell Types Contain Virus in Feline Leukemia Virus-Associated Myelopathy
A myelopathy associated with chronic feline leukemia virus (FeLV) infection was recently described in domestic cats. Affected cats have gradually progressive lower motor neuron dysfunction manifesting with paresis and eventual paralysis. Histopathologic changes predominate in the thoracic and lumbar spinal cord and consist of diffuse white matter degeneration characterized by dilated myelin sheaths. Neuronal loss affecting the ventral horn and occasional gliosis are observed. Neither tumors nor immunodeficiency nor hematological changes typically observed with FeLV infection are present. Laser microdissection of endothelial cells, neurons, astrocytes, oligodendroglial and microglial cells with subsequent quantititative PCR amplification indicate that provirus is consistently present in neurons and small blood vessels, but inconsistently and at fewer copy number in glial cells. These findings suggest that myelopathy is a novel disease syndrome associated with FeLV infection of multiple cell types in the central nervous system.
54: Bovine Ovarian Epidermoid Cysts
Ovaries from 1971 cows were examined at an abattoir. One ovary each from three animals was noted to have unusual morphology. Case 1 was from a heifer having a 5.5 × 4.0 × 3.0 cm left ovary containing over two dozen, 0.5 to 3.0 cm smooth-walled cysts containing a liquid to semi-solid white material. The opposite ovary was normal and inactive. Case 2 was observed in the right ovary from a mature cow. The abnormal ovary had three (1.5, 2.0 and 0.5 cm), smooth-walled cysts containing milky-white fluid. The left ovary appeared normal and had developing follicles and a mature corpus luteum. Case 3 was in the left ovary of a mature cow. This ovary contained a 5 cm, thin-walled cyst containing white, turbid fluid and a 3.0 × 1.5 cm cyst containing pasty, pale-tan material. Multiple sections of the ovaries demonstrated that the cysts were epidermoid cysts (EC) lined only by stratified squamous epithelium and containing multiple layers of noncompacted, keratin debris. In affected ovaries, no follicles or oocytes were observed. Ovarian ECs have not been reported in the veterinary literature, and human ovarian ECs are rare. The relatively high incidence of ovarian ECs in the cows of this study is unusual given the lack of published cases of this condition in the veterinary literature. The macroscopic similarity between ECs and abscesses would suggest they could be misdiagnosed as abscesses, or it may be that ECs in domestic species have been classified as dermoids or teratomas. Simple ECs do not contain epidermal adnexal structures such as hair and sebaceous glands, and they do not contain structures from the other embryonic germ layers. ECs are probably congenital malformations and should not be considered neoplasms.
55: Uterine Necrosis, Peritonitis, Toxemia, and Vascular Thrombosis in An Asian Elephant (Elephas Maximus) following Dystocia and Caesarian Section
A 36-year-old, female, Asian Elephant (Elephas maximus) died 19 days following dystocia, and a resultant caesarian section delivery of a stillborn bull calf. Despite intensive therapy, the elephant developed multiple complications, postoperatively, that included: azotemia, proteinuria, elevated liver (LDH) and skeletal muscle (CPK) enzymes, metabolic acidosis, increased thrombin time and fibrinogen degradation products, and a leukogram with a degenerative left shift. A complete necropsy was performed. Externally, a well-healed, 54 cm long, vertical incision was visible over the left lateral abdominal wall. The significant macroscopic observations were found in the reproductive tract. The left uterine wall was darkly discolored, friable, and contained a 36 cm long sutured surgical site. The omentum was multifocally adherent to the necrotic uterine wall. Numerous fibrin tags were present. Microscopically, severe diffuse transmural necrosis and vascular thrombosis of the uterine wall was detected. Additional histological observations included fibrinous peritonitis, renal necrosis and thrombosis, myocardial hemorrhage and necrosis, epicardial thrombosis, hepatocellular dissociation and necrosis, lymphoid depletion and necrosis, and myodegeneration of the skeletal muscle. The death of this elephant was attributable to multiple organ failure caused by sepsis and thrombosis.
56: Localization of Swine Influenza Virus in Naturally Infected Pigs by in Situ Hybridization and Immunohistochemistry
Swine influenza virus (SIV) RNA and antigen were detected in 15 naturally infected pigs by in situ hybridization using a nonradioactive digoxigenin-labeled cDNA probe and by immunohistochemistry using a influenza virus H1N1 specific monoclonal antibody. A 582 base-pair cDNA probe for viral RNA encoding the nucleocapsid protein of SIV type A H1N1 strain was generated by the reverse transcription polymerase chain reaction. In situ hybridization and immunohistochemistry gave similar results for serial sections from each of 15 lung samples. Positive cells typically exhibited a dark brown (in situ hybridization) or red (immunohistochemistry) reaction product in the nucleus and cytoplasm without background staining. A strong positive signal for both in situ hybridization and immunohistochemistry was detected mainly in the bronchial and bronchiolar epithelial cells. A less intense signal was detected in the interstitial and alveolar macrophages. Simultaneous detection of hybridization and immunohistochemical signals on serial sections provided evidence that SIV had replicated in positive cells. The in situ hybridization technique developed in this study was useful for the detection of SIV RNA in tissues taken from naturally infected pigs and may be a valuable technique for studying the pathogenesis of SIV infection.
57: Concurrent Pulmonary Paragonimus Kellicotti Infection and Squamous Cell Carcinoma in a Cat
An adult, spayed domestic long-haired cat presented to the Watson Road Veterinary Clinic in St. Louis in mid-January with a history of vomiting, mild weight loss and anorexia of three days duration. The cat had been found at a state park in southestern Missouri five years previously and was thereafter kept strictly indoors. The owner also reported a chronic nonproductive cough that occurred once or twice daily. A tentative diagnosis of feline asthma was rendered, and the cat was treated with prednisone. The cough resolved, but the anorexia persisted. Radiographs revealed numerous miliary nodules in the right and left caudal lung fields. Faced with a differential of pulmonary mycotic infection or neoplasia, the client elected to euthanize the cat. Necropsy was performed by the referring veterinarian who described a 2 × 3 cm mediastinal mass and a renal infarct. Samples of lung, mediastinal mass and kidney were submitted to the Veterinary Medical Diagnostic Laboratory. Histologically, the pulmonary parenchyma contained well-demarcated cysts lined by stratified squamous epithelium and surrounded by thick fibrous connective tissue. One cyst contained two trematodes in cross-section. Paired testes with developing and mature spermatozoa and uteri with operculated ova and golden, refractile shells were present within one of the trematodes. Contiguous with metaplastic bronchial epithelium were distinct nests and cords of neoplastic squamous cells. The cells were polygonal with moderately aniskaryotic, round to oval, vesicular nuclei, one to two, prominent nucleoli and moderate amounts of eosinophilic glassy cytoplasm. The final diagnoses included squamous cell carcinoma and Paragonimus kellicotti infection.
58: Microscopic Lesions of the Dermis in Quarter Horses with Hyperelastosis Cutis
Hyperelastosis cutis is an inherited connective tissue disease of Quarter horses. Affected horses have hyperextensible, easily torn skin. Gross skin lesions can be evident at birth or may not be noticed until the horse begins training and skin trauma is caused by the saddle. There is no treatment, and affected horses are usually euthanized. To date, the histopathological, ultrastructural and biochemical traits of equine hyperelastosis cutis are not well characterized. Electron microscopy may reveal variation in dermal collagen fiber diameter and loose packing of fiber bundles. Light microscopy of routinely processed and stained sections of affected skin may reveal loosely arranged bundles of dermal collagen, but in many cases, no microscopic lesions are evident. A 1-yr-old Quarter horse filly with hyperelastosis cutis was euthanized and necropsied. Full-thickness sections of skin were of normal thickness; microscopically, the deep dermis contained a linear horizontal zone of loose collagen with partial to complete separation of the upper part of the skin from the lower deep dermis. Severely affected skin contained an empty horizontal cleft through the deep dermis. We suggest the term “zonal dermal separation” for this lesion. Mild acute and chronic dermal microhemorrhage were also present. Similar microscopic lesions were in deep incisional skin biopsies from a 2-yr-old Quarter horse gelding. Based on these two cases, zonal dermal separation appears to be a distinctive histopathologic lesion associated with hyperelastosis cutis in Quarter horses. Skin biopsies should be full-thickness (approximately 5 mm in depth in the lumbar and sacral regions) to achieve adequate representation of deep dermis for histopathologic examination.
59: Histologic Characterization of Canine Subepidermal Vesicular Diseases: Bullous Pemphigoid, Epidermolysis Bullosa Acquisita, and Mucous Membrane Pemphigoid
In the dog, subepidermal autoimmune vesicular diseases include bullous pemphigoid (BP) and recently described epidermolysis bullosa acquisita (EBA) and mucous membrane pemphigoid (MMP). The study objectives were to better characterize histologic features of the diseases and to determine if they could be distinguished histologically, as the diseases differ prognostically. Twenty-eight dogs with subepidermal vesicles evident histologically and clinical and immunologic characteristics of these diseases were included (11 BP, 7 EBA, 10 MMP). Twenty-four parameters were assessed with H&E stained sections. In 24 cases, six parameters were assessed using the Luna stain for eosinophils. Two pathologists independently reviewed the slides blindly and subsequently reconciled differences. The binomial proportion test was used to assess probability trends of the parameters with specific diseases. The Fisher's exact test was done to determine if an association exists when comparing individual diseases. Although several parameters had probability trends with both stains, most had no significant association using the Fisher's exact test. There was a significant association between five or more single file or aggregated eosinophils at the basement membrane (BM) using the Luna stain for BP compared with EBA but not with MMP. Using the Luna stain, combining the parameters of more than five eosinophils at the BM and eosinophilic spongiosis in the epidermis, 55% of BP cases would be identified (5/9) with one erroneous inclusion of MMP (1/9). Therefore, the Luna stain combined with H&E staining aids in the distinction of BP from EBA and MMP and should be used to evaluate canine subepidermal vesicular diseases.
60: Immunohistochemical and Spectrophotometric Differentiation of Canine Prostate Neoplasms Using Tissue-Specific Markers
Prostate carcinoma and transitional cell carcinoma (TCC) often affect the canine prostate gland. These tumors have morphologic similarities using light microscopy and conventional hematoxylin and eosin staining. The dog is the only large mammal other than humans to develop prostate carcinoma with any regularity and is a commonly used animal model for studying human prostate carcinoma. To assure the relevancy of biomedical research involving canine prostate neoplasia to human prostate carcinoma, it is important to accurately differentiate tumors arising from the prostate epithelial cells (prostate carcinoma) from those arising from transitional epithelial cells of the prostatic urethra (TCC). We used the tissue-specific markers cytokeratin 7 and arginine esterase (AE) to more accurately differentiate prostate carcinoma from TCC. Tissue sections of normal and neoplastic canine prostate and bladder samples were deparaffinized. Endogenous peroxidase activity was blocked with hydrogen peroxide. Nonspecific protein binding was blocked by incubation in normal horse serum. Tissue sections were incubated for 18 hrs at 4°C with a mouse anti-human antibody against cytokeratin 7 (DAKO) known to cross-react with canine cytokeratin 7. An avidin-coupled horse anti-mouse secondary antibody system (ABC System, Pierce Chemical Co.) was used to detect cytokeratin 7 binding. Immunohistochemical positivity was detected in normal canine bladder but not normal canine prostate tissue. Most canine TCCs (12/15) showed marked positivity. Occasional canine prostate carcinomas (2/4) exhibited mild or focal positivity. AE activity was measured spectrophotometrically by the change in absorbance at 253 nm following hydrolysis of a substrate (benzoyl arginine ethyl ester, BAEE, Sigma). Canine prostate tissue homogenates contained high AE activity, whereas canine bladder homogenates contained no AE activity. These markers will be useful tools to more accurately diagnose canine prostate carcinoma.
61: Neoplasia in Captive African Hedgehogs (Atelerix Al-Biventris) with Emphasis on Two Adrenocortical Carcinomas
A retrospective review of the pathology records for African hedgehogs (Atelerix albiventris) kept at Africam Safari (Puebla, México) revealed that 6 of 18 (33.3%) animals had one or two neoplasms at necropsy. The age range of affected animals was 3 months to 4.5 years; the sex ratio was 1:1. Four hedgehogs had been born at Africam Safari. Two hedgehogs had two different neoplastic disorders. Neoplasms diagnosed in these 6 cases included adrenocortical carcinoma (2), mammary carcinoma (1), cutaneous mast cell tumor (1), oral squamous cell carcinoma (1), uterine adenocarcinoma (1), malignant round cell tumor (1) and spindle cell sarcoma (1). Some of these hedgehogs had concurrent (infectious or noninfectious) disease processes at necropsy. Grossly, the adrenocortical carcinomas presented as a single large soft mass arising from one of the adrenal glands with locally extensive hemorrhage. In one of them, there were multiple metastases seeding the peritoneal serosa and the liver, accompanied by a moderate to severe serosanguineous effusion; whereas the other case did not have metastases but presented with a concurrent mammary carcinoma. Microscopically, both tumors were multilobular and consisted of packed cords and perivascular rosettes of round to polyhedral cells in a delicate fibrovascular stroma. The cells had large round nuclei and a moderate amount of slightly basophilic or eosinophilic cytoplasm containing few vacuoles. The mitotic index was low. The unaffected adrenal gland was histologically within normal limits. Neither animal had clinical evidence suggesting that the adrenal tumors were functional. Although most neoplasms described in this study are common in captive African hedgehogs (especially mammary neoplasia, oral squamous cell carcinoma, and malignant round cell tumors), adrenal neoplasia is uncommonly reported in this species. However, adrenocortical carcinomas accounted for 25% neoplasms in this series.
62: Relation of Ulcers, Viral Hemorrhagic Septicemia Virus, and Ichthyophonus Hoferi to Pacific Herring Population Biomass in Prince William Sound, Alaska, 1994–2001
After the 1989 Exxon Valdez oil spill in Prince William Sound, Alaska, the estimated biomass of adult Pacific herring in Prince William Sound declined 80% from the fall of 1992 to April 1994. Comprehensive disease study was initiated with the primary objective to determine the relationship between disease and population biomass. Each year from 1994–2001, adult herring were subjected to complete necropsy (n = 233–400 per year). Samples were collected in the spring (spawning time) and fall (time of peak condition). Analysis of each fish included determination of age (from scales), histopathology of 10 organs, a plasma chemistry panel, and tissue culture for virus isolation. Disease prevalence data were used to modify a model that estimates population biomass: natural survival was linearly and negatively related to disease prevalence. Pacific herring in Prince William Sound had 12 potential pathogens that affected at least 10% of adult fish. However, only two abnormalities were significantly correlated with population change: (1) the North American strain of viral hemorrhagic septicemia virus (VHSV), and (2) ulcers covered by filamentous bacteria. Ichthyophonus hoferi caused disseminated granulomatous disease, but multiyear prevalence of I. hoferi was related more to fish age than to changes in population biomass. We conclude that disease-related mortality is most likely in early spring when fish are in poor condition and that epizootic mortality is related to increased prevalence of VHSV and ulcers.
63: Pathological Study of Cheetahs (Acinonyx Jubatus) Dying in Captivity in Japan
AIM: To clarify the common lesions and cause of death among captive cheetahs in Japan. METHODS: Pathological studies were carried out on 58 cheetahs, including, in 18 cases, transmission and scanning electron microscopy. PCR analysis was also carried out on frozen fresh gastric tissue from 24 of the cheetahs, using primers respectively against Helicobacter spp. (HC), H. heilmannii (Hh) and H. felis (Hf). In addition, the feces of 48 living cheetahs were tested by PCR for infection using the HC primer. RESULTS: A high percentage (69%, 40/58) of the cheetahs evaluated had renal failure. All 49 for which histopathological examination was possible showed atrophic and /or lymphocytic and plasmacytic gastritis. Helicobacter infection was visible by Warthin-Starry stain in 21/36 cases (58%). Electron microscopy confirmed the presence of Helicobacter morphologically similar to Hh, Hf, H. acinonyx and Flexispira rappini in 15 of the 18 animals. By PCR analysis of fresh gastric tissue, 20/ 24 cheetahs (83%) were positive for HC, 18/20 were positive for Hh, and 3 of these 18 were also positive for Hf, indicating mixed infection. In PCR analysis of fecal samples from 48 cheetahs, 42 (87%) were positive for Helicobacter spp. Systemic amyloidosis was observed in 51/56 cheetahs (91%), frequently affecting the kidneys, often accompanied by papillary necrosis. The amyloid deposits were immunohistochemically identified as protein AA type. Amyloidosis-induced renal and/or multiple organ failure was a significant cause of morbidity and mortality. Accidents, feline infectious peritonitis, feline parvovirus infection and pseudotuberculosis were also identified as causes of death. CONCLUSION: Severe amyloidosis, and gastritis accompanied by Helicobacter infection are highly prevalent in cheetah populations in Japan. Amyloidosis was identified as a major cause of death.
64: Adrenal Cortical Carcinomas with a Myxoid Component in Domestic Ferrets (Mustela Putorius Furo)
A retrospective study of 11 adrenal cortical carcinomas revealed an unusual histologic variant in the ferret. The cases were from surgical biopsy and necropsy cases from the archives of The Ohio State University Department of Veterinary Biosciences from 1992 to 2000. Of 29 cases of adrenal gland lesions in ferrets, 15 (52%) were adrenocortical carcinomas of which 11/15 (73%) had a myxoid component. The myxoid component was a variable part (5–95%) of the adrenal cortical neoplasm and consisted of sheets and cords of small, polygonal neoplastic cells that form lumen-like spaces. Spaces contained a variable amount of acellular alcian blue (pH 2.5)-positive mucinous matrix (i.e. acidic mucopolysaccharides). Neoplastic cells were negative for the argentaffin reaction. Immunohistochemically, this population of neoplastic cells and more typical areas of the adrenal masses were strongly positive for vimentin and alpha-inhibin and variably positive for synaptophysin. PCNA-labeling indices (LI) indicated that adrenal cortical neoplastic cells within the myxoid component of the neoplasm had a significantly elevated LI (p<0.05) compared with more typical neoplastic adrenal cortical cells or non-neoplastic zona reticularis cells. Ultrastructurally, these cells were similar to cells with a more typical adrenocortical phenotype (i.e., cytoplasmic lipid vacuoles, prominent smooth and rough endoplasmic reticulum profiles, and zonula adherens). This lesion was interpreted as a myxoid adrenal cortical carcinoma of the ferret. This variant appeared to be more aggressive based upon the LI and the rate of invasion of adjacent tissue. Morphologically this lesion resembles the myxoid variant of adrenocortical carcinoma, a rare entity described in humans.
65: Comparative Pathology of Baboon and African Green Monkey Alpha Herpesviruses
The comparative pathology of Herpesvirus papio 2 (HVP2) of baboons (Papio spp.) and Simian Adenovirus 8 (SA8) of African green monkeys (Cercopithecus aethiops) relative to human Herpes simplex virus 1 (HSV1) was investigated using a mouse model system. Young adult mice were inoculated with virus intramuscularly and followed for 21 days. The ability of each virus to infect mice was determined by the virus dose needed to induce an anti-viral IgG antibody response. The 50% infectious dose (ID50) for HVP2 was 101.75 PFU while the ID50 for HSV1 and SA8 was 102.5 and 103.8, respectively. There were marked differences in the ability of these three viruses to invade the CNS and cause clinically evident neurological disease. While HSV1 produced neurological signs in a few animals with a 50% CNS disease dose (CNSD50) of >106, SA8 did not produce clinical signs of CNS disease using doses as high as 106 PFU. In contrast, HVP2 readily invaded the CNS and produced fatal disease with doses as low as 102 PFU. Histopathological examination of tissue from HVP2-infected mice revealed severe inflammation and necrosis in the central, peripheral, and autonomic nervous systems. Viral antigens were detected in lesions by immunohis-tochemistry. This study shows that, based upon the development of a humoral immune response, HVP2 and SA8 can infect mice; however, there are marked differences in the ability of each virus to cause clinical disease and CNS lesions. Therefore, this murine model can be further utilized for the investigation of either the viral or host determinants responsible for the varying neurovirulence of these simian alpha-herpesviruses.
66: Human T-Lymphotropic Virus Type 1 P30Ii Regulates Gene Transcription by Binding Creb Binding Protein/P300
Human T-cell lymphotrophic virus type 1 (HTLV-1), a complex retrovirus, causes adult T-cell lymphoma/leukemia (ATL) and is linked to a variety of immune-mediated disorders. HTLV-1 encodes typical gag, pol, and env gene products, as well as unique regulatory and accessory genes encoded in four open reading frames (pX ORF I-IV). We have reported that the nuclear localizing protein p30II, encoded in pX ORF II, functions as a transcription factor, differentially modulates CREB-responsive promoters and is critical for maintenance of proviral loads in rabbits. To further evaluate the function of p30II we tested if Ga14(BD)-p30II-mediated transactivation was enhanced following expression of CREB Binding Protein (CBP) and p300, conserved co-adapters of transcription. p30II-mediated transactivation was enhanced following p300 expression and was competitively repressed by the p300 binding protein, adenovirus E1A and the E1A-CR2 (mutated for Rb binding, but retaining p300 binding). Importantly, we demonstrate that p30II colocalizes with p300 in cell nuclei and directly binds to CBP/p300 in cells. Deletion mutants of CBP/p300 were used to localize the site critical for binding p30II to a highly conserved KIX region. DNA binding assays confirmed the interference of p30II with the assembly of CREB-Tax-p300/CBP multiprotein complexes on 21 bp repeat oligonucleotides in vitro. Ongoing studies seek to test the role of acetylation in p30II-mediated transcription using site-directed mutants. Collectively, our data suggests that p30II, via an interaction CBP/p300, modulates viral transcription to allow the virus to maintain proviral loads in vivo.
67: Pems-Associated Astrovirus Infection in Adult Turkeys
Poult enteritis mortality syndrome (PEMS) is an incompletely understood multifactorial syndrome of young turkeys defined by mortality rates, immunosuppression, and enteritis. No studies to date have addressed the effect of PEMS on adult turkeys. This study investigated the response of 6-week old commercial turkeys to an inoculation of filtered PEMS homogenate and the humoral response to the PEMS-associated astrovirus. Innoculated birds developed watery diarrhea and weight loss. Enzyme linked immunosorbent assays (ELISAs) revealed a mild humoral response to the astrovirus peaking 21 days after initial infection, but no evidence of an anamnestic response was found following reinfection. Total immunoglobulin measurements failed to demonstrate evidence of humoral immune suppression. Reverse transcriptase-polymerase chain reaction (RT-PCR) for astrovirus performed on fecal samples indicated some animals were passing viral genome in feces. In situ hybridization detected viral replication in the distal ileac, cecal, and proximal colonic epithelial cells, with positive staining concentrated at the basolateral surface of the villus. No other sites of replication were found. These findings contradict the current thinking that PEMS agents do not affect adult turkeys. In our study, turkeys were both clinically affected and served as sources of viral amplification and environmental contamination. Consequently, control efforts should include consideration of adult turkeys in the transmission and maintenance of the disease in a flock.
68: Parathyroid Hormone-Related Protein Regulation and Alternative Promoter Usage in Adult T-Cell Lymphoma in a Sod/Beige Mouse Model of Humoral Hypercalcemia of Malignancy
The majority of patients with adult T-cell lymphoma (ATL) resulting from human T-cell lymphoma virus type 1 (HTLV-1) infection develop humoral hypercalcemia of malignancy (HHM). We utilized SCW/beige mice to study the pathogenesis of HHM. SCID/beige mice were inoculated intraperitoneally with a primary human ATL line (RV-ATL) and developed lymphomas in the mesentery, liver, thymus, lungs, and spleen. The lymphomas stained positively for human CD45RO surface receptor and normal mouse lymphocytes stained negatively confirming the human origin of the tumors. The ATL cells were immunohistochemically positive for parathyroid hormone-related protein (PTHrP). In addition, PTHrP mRNA was highly expressed in lymphomas when compared to other immortilized HTLV-1-positive cell lines. Mice with lymphoma developed severe hypercalcemia, and their plasma PTHrP concentrations were markedly increased in comparison to controls. Bone densitometry and histomorphometry in lymphoma-bearing mice revealed significant bone loss due to a marked increase in osteoclastic bone resorption. RV-ATL cells contained an intact proviral copy of the tax gene. However, tax expression was not detected by Western blot or RT-PCR in RV-ATL cells, which suggests that factors other than tax are modulators of PTHrP gene expression. Transcription of the PTHrP gene is under the control of three promoter regions (P1, P2, and P3). Examination of alternative PTHrP promoter usage in xenografted RV-ATL and other HTLV-1-positive cell lines by RT-PCR analysis demonstrated that P3-initiated transcripts were the most abundant, while P2-initiated transcripts were present at low levels, and P1-initiated transcripts were not detectable. Interestingly, P2 and P3 promoters contain three different putative transcription factor binding sites potentially involved in PTHrP transactivation in ATL cells.
69: Pathology of Multiple Endocrine Neoplasia in Induced Mutant Mice
Multiple endocrine neoplasia 1 (MEN1) is an autosomal dominant human syndrome with multiple endocrine tumors in parathyroids, pancreatic islets, pituitary, adrenal cortex and other tumors. We have generated a mouse model of the human disorder, the Menl± mouse by homologous recombination. The null mutant was embryic lethal, while the Menl± mouse is viable and developed multiple endocrine and other tumors after 6 months of age. In some tissues, such as pancreatic islets, adrenal cortex, parathyroid and pituitary, focal hyperplasia or dysplasia first developed, followed by atypical hyperplasias and adenomas. Islet cell hyperplasias and adenomas highly expressed insulin. Islet cell adenomas evaluated showed loss of the wild-type Menl allele, providing evidence of the role of Menl as a tumor suppressor gene. The mice also developed tumors in the ovary, testis, adrenal medulla, prostate gland, and stomach. Most of these tumors were slow growing and developed after 12 months of age. The human and mouse disorders have many similarities which will allow us to study the mechanisms of carcinogenesis in mice and their possible relevance to humans. Rare multiple endocrine neoplasias in domestic animals have been reported but their genetic etiology is unknown. The findings in the human and mouse studies could provide an incentive for determining the genetic changes in these domestic animal cases.
70: Abnormal Glandular Growth, Myocardial and Skeletal Atrophy and Ascites in Zebrafish of Mixed Genetic Background
Zebrafish within multiple tanks in a modern research facility displayed a pattern of ventral enlargement and general physical decline. The fish represented a variety of commonly available stocks including wild type lines and mutants on variable genetic backgrounds. The tanks used a reverse osmosis system supplemented with Instant Ocean and sodium bicarbonate. Common findings at gross necropsy were small overall size, an enlarged fluid filled coelomic cavity and minimal gonadal tissue. Histopathology revealed a 100–200 micron reniform glandular growth partially enclosing the sinus venosus adjacent to the pericardium and esophagus. The tissue contained nests and acinar structures lacking lumens composed of tall cuboidal to columnar cells with a basophilic staining pattern. Unaffected individuals had a smaller focus of similar tissue. Additional changes included a dilated thin-walled myocardium with atrophied myocytes, pectoral fin muscular atrophy, pancreatic atrophy and occasionally peritonitis. The location and character of the glandular tissue is strongly suggestive of the ultimobranchial gland, the equivalent of the mammalian thyrocalcitonin-producing parafollicular “C” cells of the thyroid gland. This tissue may control a variety of ionic balances within fish similar to its control of calcium stores in mammals. Low calcium concentrations ranging from 34.2 to 51.3 ppm may have contributed to abnormal glandular growth in a subset of the population. Further work will characterize the glandular tissues and serum ion balance parameters in an attempt to understand the mechanisms and genes driving this phenomenon resembling parafollicular abnormalities observed in bulls.
71: Morphologic and Molecular Characterization of Salmonella Typhimurium Infection in Neonatal Calves
The host response to Salmonella spp. plays a major role in the outcome of the infection. The present study was aimed to further characterize Salmonella typhimurium infection of neonatal calves both at the morphologic and molecular level using the ligated ileal loop model. Eight 4–5 week-old male Holstein calves underwent laparotomy and loops were prepared in the ileum. The loops were either inoculated with a S. typhimurium strain pathogenic for cattle or injected with sterile LB broth as control. Samples for histology, transmission and scanning electron microscopy, and RNA extraction were collected at time points between 5 minutes and 12 hours postinfection. Invasion of both M cells and enterocytes began at 15 minutes post-infection. Intracellular bacteria were observed in the lamina propria after 1 hour post-infection. A severe acute neutrophilic response was associated with invasion of the Peyer's patches. Upregulated expression of CXC chemokines (IL-8, GRO-alpha, GRO-gamma, and GCP2) was detected by RT-PCR beginning at 1 hour post-infection. Expression of pro- (IL-1beta, IL-18, and TNF-alpha) and anti-inflammatory (IL-10, IL-1Ra, and IL-4) cytokines were also assessed. A marked increase in expression of IL-1beta was observed whereas IL-18 and TNF-alpha did not change their profile of expression after infection. Up-regulation of IL-1Ra and IL-4, but not IL-10 was observed. Our findings indicate that infection of bovine ligated ileal loops with S. typhimurium results in an acute neutrophilic inflammatory response that is associated with the upregulation of CXC chemokines (IL-8, GRO-alpha, GRO-gamma, and GCP2), IL-1beta, IL-1Ra, and IL-4.
72: Use of a Mouse Model to Evaluate Helicobacter Pylori Virulence Determinants
Adoptive transfer of unfractionated CD4+, or CD4+ CD45RBhigh splenocytes results in severe, rapidly developing gastritis in H. pylori-infected immunodeficient (SCID) mice. The purpose of this study was to use the recipient SCID mouse model to compare the role of two putative bacterial virulence factors in severity of disease due to H. pylori. Wild-type H. pylori strains were SS1 and M6. Mutant strains were SS1:0826, deficient in beta-1,4-galactosyltransferase and expressing truncated LPS O-antigen, and M6-delta-ureB (cag1:ureB), a urease transformant that expressed weak urease activity. SCID mice were inoculated with mutant or wild-type bacteria, given fractionated or unfractionated splenocytes 2–4 weeks later, and killed 8 weeks after transfer. C57BL/6 wild-type mice were killed 8 weeks after bacterial inoculation. Gastritis was quantified histologically by determining percent affected gastric mucosa. Bacterial colonization was quantified by plate count. In wild-type mice, gastritis was mild, and there were no differences between colonization groups. In contrast, recipient SCID mice developed severe gastritis, but only in response to wild-type H. pylori or the urease mutant, not in response to the O-antigen deficient mutant. Absence of gastritis in mice colonized by O-antigen deficient H. pylori resulted in higher colonization rates by the mutant compared to wild-type. These results demonstrate that 1) the adoptive transfer SCID model can be used to quantify differences in virulence attributable to bacterial virulence factors, and 2) LPS O-antigen but not urease contributes to gastritis due to H. pylori in mice.
73: Pathogenicity of a Hong Kong-Origin H5N1 Avian Influenza Virus for Four Passerine Species
The first zoonotic transmission of a highly pathogenic hemagglutin 5, neuraminidase 1 (H5N1) avian influenza virus occurred in 1997, with the virus causing significant human disease and six fatalities. This investigation assesses the ability of the A/chicken/Hong Kong/220/97 (H5N1) highly pathogenic avian influenza virus (chicken/Hong Kong) to infect and cause disease in zebra finches (Taeniopygia guttata), house finches (Carpodacus mexicanus), house sparrows (Passer domesticus), and European starlings (Sternus vulgaris) following intranasal administration. The morbidity, mortality, gross and histological lesions, and distribution of viral antigen, as determined with immunohistochemistry, were evaluated. Zebra finches developed severe anorexia and depression, and there was 100% mortality of this species. Along with severe anorexia and depression, house finches also developed neurological signs, including tremors and incoordination. Within 2 days of the onset of neurological signs, affected house finches were moribund or found dead. Conversely, sparrows exhibited transient mild depression but no mortality, and starlings demonstrated neither clinical disease nor mortality. Grossly, both species of finches had splenomegaly. In addition, pancreatic mottling was observed in the house. finches. Gross lesions were not observed in the sparrows or starlings. Histological lesions in the zebra finches were widely distributed through multiple organs and corresponded to the presence of viral antigen. In house finches, histological lesions and viral antigen were observed primarily in the brain and pancreas. Viral antigen and corresponding lesions were observed only in the testes and heart of few sparrows. Tissues from the starlings contained neither histological lesions nor viral antigen. These results indicate that there is significant variation in the pathogenicity of the chicken/Hong Kong virus for different species of passerine birds.
74: Role of Arf-P53 Pathway in An in Vivo Murine Azox-Ymethane-Induced Colon Tumor Model
A differential susceptibility (susceptible and resistant) phenotype to the organotropic colon carcinogen, azoxymethane (AOM), has been characterized in A/J and AKR/J mice. We have reported activating Ki-ras mutations at a frequency of approximately 20% within A/J colon tumors and about 30% in preneoplastic aberrant crypt foci of both strains. Several oncogenic stimuli including Ki-ras are known to induce p53-mediated growth arrest/apoptosis via ARE We hypothesize that failure of growth arrest within A/J colon tumors is related to deregulation of ARF-p53 pathway. In this study, 5-week old male A/J and AKR/J mice were injected i.p. with AOM weekly for 6 weeks and sacrificed 24 weeks after the last injection. PCR-based mutational analysis of ARF and p53 cDNAs in AOM-induced colon tumors failed to detect mutations in either of these two critical tumor suppressor genes. In addition, laser capture microdissection of tumors followed by PCR-based sequencing of exons 5–8 of genomic p53 was performed as a means of determining whether sub-populations of tumor cells may have acquired p53 mutations. However, no carcinogen-induced alterations were found even in the most pleimorphic cells. By ribonuclease protection assay, ARF overex-pression was observed at both mRNA (10-fold, p<0.05) and protein levels within colon tumors. Despite induction of ARF and sequence normal p53, upregulation of p53 transcriptional target, p21 was not observed. On the contrary, there was a decrease in p21 mRNA levels (30% of controls, p<0.05) within the tumors indicating that the susceptible A/J strain have a deregulated ARF-p53 pathway. Additional studies are underway with a goal toward further defining potential alterations in this pathway in our murine colon cancer model.
75: Interpreting Transcriptomics
Gene microarray technology has provided a powerful new tool for pathologic interpretation of lesions. The ability to view the molecular summation of thousands of gene transcripts at a single moment in time provides a unique opportunity to connect morphology with molecular events and perhaps to predict outcomes based on early transciptional responses. Involvment of pathologists in differential gene expression experiments is essential for gene expression interpretation. The failure to heed caveats of faulty study design, absence of histopathologic or clinical pathologic correlates, poor characterization of samples, and poor clinical knowledge of the test animals is creating a morass of conflicting information in the literature. To correct this a standard approach for involvement of pathologists must be adapted. A simple approach to interpretation begins with appropriate statistical analysis of the data set followed by a pathophysiologic sorting of significant gene changes by pathways. Integration of the resulting data in view of clinical, light and ultrastructural morphology and in situ hybridization and in association with appropriate biochemical confirmations is best achieved by a cooperative association of pathologists and molecular biologists.
76: Distribution of Substance P Receptor (Neurokinin-1 Receptor) in Normal Ovine Lung and during the Progression of Bronchopneumonia in Sheep
Substance P contributes to the physiologic homeostasis of pulmonary airways and vasculature. During pneumonia, alterations in substance P production and receptor expression can influence bronchoconstriction and vascular perfusion. We determined the distribution of substance P receptor (neurokinin-1 receptor [NK-1R]) in lungs of normal sheep and sheep with acute (1 day), subacute (15 days) and chronic (45 days) bronchopneumonia caused by Mannheimia haemolytica. We compared the immunohistochemical distribution of three rabbit polyclonal antibodies generated to the cytosolic C-terminal portion of NK-1R (393–407). Control sections lacked staining and included no primary antibody, normal rabbit serum, and NK-1R antibody pre-incubated with a synthesized 15 aa peptide of NK1-R residues 393–407. NK-1R immunoreactivity was traced in digital images (Adobe Photoshop 6.0) and quantified with IPLAB software (Scanalytics). There were significant differences among antibodies in distribution of NK-1R immunoreactivity but not between treatments. Antibody 1 (#653) had the broadest distribution and stained alveolar septae, smooth muscle cells of airways and vessels, epithelial cells of airways and alveoli, and submucosal glands. Antibody 2 (#652) had the highest affinity for smooth muscle cells, and antibody 3 (#654) had the highest affinity for submucosal glands. This work suggests that: 1) NK-1R is widely distributed in the lung, 2) antibodies to the same NK-1R cytosolic region can vary in affinity, 3) possible alterations of NK-1R during the progression of bacterial bronchopneumonia may not be detected by immunohistochemistry.
77: Pathologic Changes in Late Term Fetuses and Neonates Cloned from An Adult Jersey Cow
Ovarian granulosa cells were aspirated from an adult Jersey and cultured in the presence of 10% fetal bovine serum. MII oocytes were enucleated between 17 and 21 h post maturation and fused with individual granulosa cells. Resulting cloned embryos were activated, allowed to develop for 6 days and were then transferred to surrogate recipients. Pregnancy was established in 20/38 recipients as determined by the presence of embryonic heartbeat between days 29–35 using ultrasound. One calf was delivered with a normal placenta and has remained healthy for 8 months. Hydramnios and hydrallantois accompanied by marked placental edema occurred in 6 of the remaining pregnancies that progressed to late term. Necropsy examinations were performed on three fetuses that died between 174 to 255 days of gestation and on two cesarean derived neonates that survived for one hour and 6 days. Renal lesions consisting of increased mesenchyme, primitive tubules, vacuolation of tubular epithelium and small to moderate numbers of oxalate crystals in lumens of tubules were observed in both late term fetuses and neonates. Hepatic lesions consisting of perivenular fibrosis and biliary duct hyperplasia were also observed. Meconium and amniotic squamous cells were present in the lungs of all late term fetuses and the two neonatal calves. Some of the findings may be due to placental insufficiency, however, hydrallantois and placental edema are also features of fetal nephrogenic syndromes.
78: Epithelial-Mesenchymal Transformation in Uvr-Induced Murine Skin Tumors
Epithelial-mesenchymal transformation (EMT) takes place during normal embryonic development and tissue repair. During EMT, closely associated immobile epithelial cells expressing cytokeratin are transformed into dissociated and motile fibroblast-like cells that express vimentin. Similar events occur during carcinoma progression and generally herald more aggressive tumor behavior. We are investigating the occurrence of EMT in ultraviolet radiation (UVR)-induced squamous cell carcinomas of the skin of mice and the factors that control this process. Our studies show that UVR-induced squamous cell carcinomas in mice undergo EMT during transition to spindle cell carcinomas. During this process, epithelial cells lose cytokeratin expression and begin to express vimentin. This change is accompanied by loss of E-cadherin expression. Our studies also indicate that UVR exposure of keratinocytes transiently enhances expression of the Slug transcription factor, a regulatory molecule that is an important modulator of EMT during embryonic development. UVR induction of Slug appears to involve activation of the epidermal growth factor receptor and mitogen-activated protein kinase signaling pathways downstream from this receptor. We are currently examining expression of Slug and the related transcription factor Snail in UVR-induced murine epidermal tumors to determine if expression of these factors is increased relative to normal skin. If so, persistently elevated Slug expression may account for EMT occurring late in the development of UVR-induced skin tumors.
79: Taci, a Tnfr Family Member, is Important in the Regulation of B Lymphocyte Growth and Function and Inhibition of Its Ligands via Taci-Fc Ameliorates Arthritis in Mice
TACI and BCMA are TNFR superfamily members that are expressed on B lymphocytes and activated T lymphocytes (TACI) or B lymphocytes (BMCA) and interact with both BlyS and APRIL. We generated TACI KO mice and evaluated their histological and immunological phenotype. We also used a TACI-Fc immunoadhesin in vivo in murine collagen-induced-arthritis (CIA) to evaluate the effect of blockade of BlyS and APRIL in the pathogenesis of this disease. We found TACI KO mice to have dysregulated B lymphocyte production with increased numbers of follicular B lymphocytes and resultant enlargement of the spleen and lymph nodes. In addition, resting IgM levels were elevated and there was an enhanced humoral response to antigen in KO mice. This phenotype is similar to that described for BlyS transgenic mice and suggests that TACI has a negative immuno-regulatory role in its interaction with BlyS or APRIL. In murine CIA treatment with TACI-Fc, capable of inhibiting both BlyS and APRIL, had profound ameliorative effects on disease. TACI-Fc treated mice had a significant reduction in disease incidence and severity as determined by clinical scores, radiographic analysis and histology. TACI-Fc treated mice had reduced levels of anti-collagen IgG formation and a reduced T lymphocyte response to collagen in vitro. Taken in context with the immuno-proliferative phenotype of TACI KO mice, TACI-Fc inhibits the interaction of BlyS and APRIL with BCMA or another undiscovered receptor to ameliorate and prevent disease in this model.
80: Expression of Anaplasma Marginale Major Surface Protein 2 in Transmitting Ticks
Ehrlichia spp. infection develops when infected ticks are allowed to feed on a mammalian host. In contrast to multiple polymorphic forms of Major Surface Protein 2 (MSP 2) in bovine hosts, MSP2 in South Idaho strains of Anaplasma marginale is restricted to just 2 variants that are expressed in the salivary gland of feeding Dermacentor andersoni ticks. The aims of the study were to determine when and where MSP2 restriction occurs in the tick, how MSP2 expression is regulated in the salivary gland of feeding ticks and if the number of A. marginale per salivary gland is significantly increased in feeding ticks. Western blot, real time PCR and DNA sequencing demonstrated that expression and restriction of MSP2 takes place in the midgut during the first 48h after the blood meal through which ticks acquire infection. The MSP2 mRNA and protein levels increase only minimally and transiently in salivary glands of transmission-feeding ticks, when compared to unfed ticks. A. marginale numbers per tick increase gradually in salivary glands both of fed and unfed ticks. It is concluded that MSP2 restriction is an early event in the tick and that restricted MSP2 variants are expressed in the midgut and salivary glands. While MSP2 may be required for infectivity, there is no strict correlation between MSP2 expression and the development of infectivity.
81: Murine Cmv Chemokine Homolog Mck-2 Recruitment of Leukocytes to Sites of Infection
Murine cytomegalovirus (MCMV) chemokine (MCK-2), a viral beta chemokine homolog expressed following MCMV infection, exhibits pro-inflammatory properties. The function of this protein is being studied by comparing behavior of MCK+ and MCK- viruses in the mouse host as well as following injection with recombinant MCK-2 protein into mouse footpads (FP). A higher level of inflammation, including macrophages, dendritic cells, and neutrophils was induced at sites of infection by viruses expressing MCK-2 compared to mutants. Infection with MCK+ virus induced a greater swelling response compared to mice infected with MCK- virus. Histological analysis revealed more intense inflammatory infiltrates and edema in MCK+ virus inoculated mice that peaked at 72 hours. Twice the number of inflammatory cells were recovered from MCK+ compared to MCK- virus infected FP even though virus replication was equivalent. Cytologic analysis of inflammatory cells revealed a greater number of virus positive neutrophils and monocytes in MCK+ virus and more virus positive macrophages in MCK-virus inoculated feet. Flow cytometry was used to show that greater numbers of macrophages and dendritic cells were present in feet during infection by MCK+ virus compared to MCK-virus. Inoculation with recombinant MCK-2 induced considerable FP swelling and complemented the poor swelling with MCK- virus. MCK-2 induced a predominantly mononuclear cell response. Together, these findings suggest that MCK-2 may function as a viral chemokine by recruiting a particular subset of inflammatory cells during MCMV infection.
82: Bovine Leukemia Virus Transmembrane (Tm) Protein is Not Phosphorylated
Bovine leukemia virus (BLV) causes persistent lymphocytosis, a pre-neoplastic polyclonal expansion of B lymphocytes. This expansion increases the probability of disease transmission to new hosts. The mechanisms of this expansion have not been completely elucidated. Viral mimicry of host cell signaling proteins is one potential mechanism to increase viral propagation or shield virus from the host immune system. The cytoplasmic portion of BLV TM (gp30) has multiple motifs homologous to those of host signaling proteins, including two immunoreceptor tyrosine-based activation motifs (ITAMs) which have significant homology to B cell receptor (BCR) ITAMs. In normal B cells, binding of the BCR results in phosphorylation of the tyrosine residue of the BCR ITAMs, initiating signal cascades that are important in cell proliferation and survival. Because of the sequence homology, we hypothesized that the TM ITAMs function as BCR mimics and are phosphorylated in infected B cells. To examine TM phosphorylation status we used both peripheral blood mononuclear cells isolated from naturally infected, persistently lymphocytotic cattle, and two other BLV-expressing cell lines. Various stimulation techniques were used. Methods of phosphorylation detection included immunoprecipitation of the TM followed by immunoblotting for tyrosine phosphorylated proteins, and 32-P incorporation into cells followed by detection of TM by immunoprecipitation. Both detection methods in all three cell types demonstrated that TM is not phosphorylated, at least under these conditions. These results suggest that TM is unlikely to mimic the BCR by direct interaction with B cell tyrosine kinases or phosphatases through phosphorylated ITAMs.
83: Correlation of Gastric Interferon-Gamma and Growth Factor Transcript Levels in a Mouse Model of Helicobacter Pylori Infection
Gastric epithelial proliferation in experimental Helicobacter spp. infection of mice is associated with CD4+ cell infiltration, and CD4+ spleno-cytes secrete interferon-gamma (IFNG) in response to H. pylori antigen. These experiments tested the hypothesis that IFNG indirectly elicits proliferation of gastric epithelium via stromal or epithelial growth factor secretion. Four to six mice from the following groups were used: H. pylori strain SS1 infected and uninfected C57BL/6 severe combined immunodeficient (SCID) mice transferred with splenocytes from congenic wild-type (WT) mice, and infected and uninfected C57BL/6 WT mice. Mice were killed 8 weeks after inoculation. RNA was isolated from gastric mucosa. Relative quantification of transcripts using one-step real time RT-PCR was performed using primers for the following cDNAs: IFNG, IL-4, IL-10, TNF-alpha, IL-1, TGF-alpha, EGF, HGF, KGF, and GAPDH. Areas under melting curves of amplified products were determined, and reported as fraction of GAPDH. Transcripts shown to be elevated by relative quantification (IFNG, TGF-alpha, and KGF) were quantified by comparison to serial dilutions of control cDNA using two-step real time RT-PCR. Infected, transferred SCID mice had significantly elevated levels of IFNG, TGF-alpha, and KGF cDNAs standardized to GAPDH compared to the other groups. IFNG, TGF-alpha, and KGF transcript levels (ng) determined by absolute quantification in infected, transferred SCID mice were significantly elevated over the other groups. The data support an association between transcript levels of IFNG, TGF-alpha and KGF. Gastric epithelium and stroma might respond to high levels of IFNG with TGF-alpha and KGF secretion respectively. In conclusion, elevated IFNG, TGF-alpha and KGF transcripts in infected, transferred SCID mice support an indirect effect of IFNG on gastric epithelial proliferation in H. pylori infection.
84: Histomorphologic and Immunohistochemical Charcterization of Experimental Marburg Virus Infection in Cynomolgus Macaques (Macaca Fascicularis)
Marburg virus is responsible for lethal outbreaks of hemorrhagic fever in both humans and nonhuman primates. Previous studies showed a predilection of Marburg virus for macrophages, hepatocytes, adrenal cells, pancreatic islet cells, and fibroblast-like cells. The objective of this study was to determine the distribution of lesions and Marburg viral antigen in the tissues of eight adult cynomolgus macaques (Macaca fascicularis) that died 7 to 10 days after intramuscular inoculation of Marburg subtype Musoke (MBG-M). Macaques were infection controls or placebo controls in experimental vaccine studies. Tissues obtained at necropsy were formalin-fixed and prepared for routine histopathology. Tissues were also immunostained with monoclonal antibodies specific for Marburg viral antigen by the indirect immunoperoxidase technique. A novel finding was the immunohistochemical localization of virus in endocrine and neuroendocrine cells in the pituitary gland. Other endocrine cells immunoreactive for virus included adrenal cortical cells, pancreatic islet cells, thyroid C-cells, and parathyroid chief cells. Viral antigen was also immunolocalized in cells of the mononuclear phagocytic system and mesenchymal cells within many organs. Epidermal cells and mucosal epithelial cells had widespread viral antigen localization. We conclude that the the endocrine, gastrointestinal, integumentary, lymphoid, and hematopoietic systems are sites of predilection for Marburg viral antigen. The mechanistic role of Marburg virus-infected cells in the functional context of the hypothalamic-pituitary-adrenal axis warrants further investigation.
85: Experimental Vesicular Stomatitis in Horses
Vesicular stomatitis, a disease of cattle, horses, and swine, is caused by related viruses in the genus Vesiculovirus, family Rhabdoviridae. Although recognized for at least 160 years, the epidemiology and pathogenesis of this disease remains undefined. In this study we infected horses with Vesicular Stomatitis Virus New Jersey serotype (VSV-NJ) and Indiana serotype (VSV-I) by routes compatible with contact (intraepithelial inoculation of tongue, oral inoculation via scarification of lip, and per os) and vector transmission (intradermal inoculation of chin skin). Horses inoculated with VSV-NJ in tongue, lip, or chin skin developed lesions at the site of inoculation as early as post inoculation day (PID) 1 or 2. Secondary lesions occurred in some animals inoculated in the lip and chin skin. Only one of three horses inoculated per os developed lesions. Viral shedding from the oral cavity occurred in all horses except one infected per os. Shedding occurred as early as PID 1 and lasted from 3 to 8 days in horses inoculated in tongue, lip, or chin skin. Shedding occasionally occurred from the nasal cavity, but virus was never isolated from feces or blood. Serum neutralizing antibodies were first detected on PID 6 or 7. Results were similar in horses infected with VSV-I. Horses can be infected with VSV-NJ and VSV-I by routes that simulate contact or vector transmission. Viral shedding is predominantly from the oral cavity; viremia cannot be detected. Serum neutralizing antibodies develop quickly resulting in the rapid termination of viral shedding.
86: Mouse Model of Aerosolized Glanders (Burk-Holderia Mallei)
Twenty-five specific pathogen-free male BALB/c mice were exposed by aerosol to a lethal dose of Burkholderia mallei (China 7 strain), while 10 served as uninfected controls. Five infected and two uninfected control mice were killed beginning on days 0 (at 6 hr) through 4 post exposure. Following aerosol exposure to B. mallei the most consistent lesion was acute, progressing to pyogranulomatous, inflammation. Histopathologic changes were first seen at 6 hr (day 0) post exposure in the nasal cavities of all mice. On day 1, lesions were present in lung, trachea, spleen, liver, submandibular lymph node, and one mouse brain. On day 2, changes were also present in the bone marrow and in mediastinal lymph nodes. Prominent lesions in the nasal cavity included an acute inflammatory cell infiltrate, erosion and ulceration of both respiratory and olfactory epithelium and a sero-cellular exudate. Involvement of the olfactory tract and olfactory lobe of the brain was present in all mice by day 4. We believe the infection extended into the brain from the nasal olfactory mucosa through the olfactory nerves foramina. Because of this direct extension of inflammation following aerosol exposure, postexposure therapy may be problematic. We have shown that the mouse is a susceptible animal model for B. mallei infection by the aerosol route.
87: Disease in Domestic Chickens after Inoculation with Newcastle Disease Viruses from Wild and Exotic Birds
Three Newcastle disease virus (NDV) isolates recovered from wild (anhinga) and exotic birds (pheasant and dove) were passaged four times in domestic chickens prior to this pathogenesis study. Groups of ten, four-week-old SPF White Leghorn chickens, inoculated intraconjunctivally with each of the passaged isolates, were observed for clinical disease and were sampled at 2, 5, 10, and 14 days post-inoculation (dpi) or at the occurrence of mortality. Tissues were examined by histopathology, by immunohistochemistry (IHC) for presence of NDV nucleoprotein, and by in situ hybridization (ISH) for mRNA using a digoxigenin-labeled riboprobe to the NDV matrix gene. Birds inoculated with pheasant and dove isolates had severe disease, characterized by marked depression, and died at 4 and 5 dpi, respectively. Although birds inoculated with the anhinga isolate did not show clinical signs, mild to severe lymphoplasmacytic encephalitis was detected histologically at 5, 10 (more prominent), and 14 dpi. Severe diffuse necrosis of the lymphoid organs was the main histologic finding in birds inoculated with the pheasant (at 4 dpi) and dove (at 4 and 5 dpi) isolates. Viral nucleoprotein and viral mRNA were detected by IHC and ISH, respectively, among all the affected organs. The results of this study demonstrate that moderate to highly virulent NDV isolates from wild and exotic birds represent a serious threat for commercial poultry flocks.
88: Immunohistochemical Findings in Brucella Melitensis-Induced Hepatitis in Rhesus Monkeys
M.
Brucella melitensis is a significant bacterial pathogen that may be considered a threat for illegitimate use. Definition of the adaptive immune and inflammatory responses to this pathogen may provide information for novel immunotherapeutic strategies. Our initial characterization involved four Rhesus macaques aerosol exposed to 1x107 Brucella melitensis 16M organisms. Tissues from each animal were collected at necropsy nine weeks post exposure. H&E stained sections of liver revealed hepatitis characterized by multifocal collections of lymphocytic and histiocytic cells. To further characterize the inflammatory cell lineage, sections of liver were evaluated using antibodies directed against T cells (CD3), B cells (CD20), dendritic cells (p55, CD83) and the proliferation antigen Ki67. The predominant cells were CD3+ T cells with fewer dendritic cells. Immunohistochemical assays were negative for B cells and cell proliferation. These results indicate that inflammatory foci observed in livers from Rhesus macaques infected with Brucella melitensis nine weeks following aerosol exposure appear to be predominantly of T cell lineage admixed with fewer dendritic cells. B-cells were not observed associated with the inflammation. Furthermore, the negative staining using Ki67 antibody suggests inflammatory cells are recruited to the region without evidence of local proliferation at the timepoint examined. Further work is needed to ascertain the role of antigen presenting cells in the pathogenesis of brucellosis in Rhesus macaques.
89: In Vitro Epithelial Cell Invasion Models for the Study of Bacterium-Host Cell Interactions of Edwardsiella Ictaluri
Enteric septicemia of catfish, caused by the bacterium Edwardsiella ictaluri, is the most significant disease of farmed channel catfish (Ictalurus punctatus). Invasion of Edwardsiella ictaluri into cultured mammalian and fish enteric epithelial cells is described. Gentamicin survival assays were used to demonstrate the ability of multiple isolates of this catfish pathogen to invade IEC-6 (rat small intestinal epithelium origin), Fat Head Minnow (FHM) and channel catfish enteric epithelial cells. Invasion of all cell types occurred within 2 hours of contact at 26° C, in contrast to E. coli DH5α, which did not invade cells tested. Preincubation of IEC-6 cells with chemical blocking agents including cytochalasin D (microfilament depolymerizer) and mon-odansylcadaverine (blocks receptor mediated endocytosis) significantly reduced invasion, whereas colchicine (microtubule depolymerizer) had no effect on bacterial internalization. Results indicate that actin polymerization and receptor-mediated endocytosis are involved in uptake of E. ictaluri by IEC-6 epithelial cells. This study provides the first documentation of invasion of cultured mammalian and fish cell lines by this pathogen, and identifies possible mechanisms used for intracellular access. Additionally, the study describes several functional in vitro invasion models. Disease control is dependent on understanding the mechanisms used by this bacterium to access host tissues. These models provide working systems for the detailed study of E. ictaluri pathogenesis at the cellular level.
90: Suppression of P38 Reduces T Cell-Induced Inflammation in the Skin but Not the Colon in a Murine Adoptive Transfer Model of Inflammatory Bowel Disease and Psoriasis
Transfer of naive T cells into minor MHC mismatched, severe combined immunocompromised (SCID) murine recipients induces dermatitis and colitis. This model is useful for investigating pathogenesis and pharmacologic intervention of inflammatory bowel disease and psoriasis. This study investigated whether p38 suppression altered development or progression of inflammatory lesions. C.B-17 SCID (H2d) mice were transplanted with naïve splenic T cells (CD45RBhi CD4+) sorted by flow cytometry and CD4-enriched to >95% purity. Vehicle-treated, control mice had a 100% incidence of gross and microscopic bowel and skin lesions. Colonic lesions consisted of thickening of the colon wall with mixed or granulomatous inflammatory infiltrates expanding the lamina propria and effacement of the mucosal crypt epithelium, often associated with adjacent crypt mucosal hyperplasia and occasional ulceration. There were dermal and epidermal mixed inflammatory infiltrates with occasional deep dermal granulomatous foci. The epidermis was multifocally thickened by edema, hyper/parakeratosis, serocellular crust formation and occasional ulceration. Mice given a p38-suppressive agent had colonic lesions similar to those of vehicle-treated mice, but skin inflammation and epidermal changes were markedly reduced. These results indicate subtle differences in T cell-mediated inflammatory pathways between skin and the colon.
91: Overexpression of Murine Il-17E Induces a Th2-Like Response and Multi-Organ Inflammation in Transgenic Mice
IL-17E is a recently described member of an emerging family of IL-17 related cytokines. IL-17E has been shown to bind IL-17Rh1, a protein distantly related to the receptor for IL-17, suggesting that IL-17E likely possesses unique biological functions. Here we have identified the murine ortholog of IL-17E and developed transgenic mice in order to characterize the actions of this new cytokine. Overexpression of IL-17E resulted in eosinophilia, and increased serum IgE and IgG1, but not IgG2a, suggesting a Th2-like immunologic response. Consistent with this, serum levels of IL-13 and IL-5 were also elevated in the transgenic mice. Elevated gene expression of several Th2 cytokines, including IL-4, IL-5, IL-10 and IL-13 were observed in multiple tissues. Overexpression of IL-17E induced mixed inflammatory cell infiltration and epithelial hyperplasia and hypertrophy in multiple tissues including heart, liver and lungs. These findings suggest that IL-17E is a unique pleiotrophic cytokine that engages a systemic Th2-like response with tissue-specific immunological and pathological changes.
92: Tissue Distribution of Dc-Sign, a Dendritic Cell-Specific Hiv/Siv Receptor, in Siv-Infected and Uninfected Macaques
DC-SIGN, an external C-type lecti, is expressed on the surface of immature and mature dendritic cells (DCs). It has been proposed that DC-SIGN plays a pivotal role in HIV/SIV infection by allowing peripheral DCs to capture and specifically bind HIV-1 gp120, transport the virus to lymph nodes and present it to T-cells. Although the location of DC-SIGN expression has been established in a limited number of human tissues, its distribution in the Rhesus macaque, a key HIV/SIV animal model, has not been shown. This study characterized DC-SIGN expression in SIV infected and uninfected macaque tissue by immunohistochemistry and confocal microscopy. Immunohistochemistry using monoclonal and polyclonal antibodies against DC-SIGN (Gift of Robert Doms, U Penn) was performed on a variety of tissues from SIV infected (n = 3) and uninfected (n = 3) macaques. In both infected and uninfected macaques, DC-SIGN positive cells were present within the submucosa and lamina propria of tongue, vagina, rectum and tonsil. No positive cells were present within the epithelium of any tissue. In liver, Kupffer cells were strongly positive. Within lymphoid tissues, numerous positive cells were present within sinusoids. In addition, two distinct populations of dendritic cells were labeled: those cells consistent with interdigitating reticular cells in the paracortex and scattered follicular dendritic cells within germinal centers. DC-SIGN expressing dendritic cells have been proposed as a “Trojan-horse” involved in trafficking HIV/SIV from the peripheral mucosal surfaces to lymphoid tissues. In our studies, the absence of intraepithelial DC-SIGN positive cells suggests that DC-SIGN does not play a significant role in transmucosal passage.
93: Effect of Na+/I− Symporter Expression on Growth of Matlylu Cells in Vitro and in Vivo
The human sodium iodide symporter (hNIS) mediates iodide uptake in thyroid follicular cells and provides a mechanism for effective radioiodide treatment of residual, recurrent, and metastatic thyroid cancers. Several studies have investigated the therapeutic applications of hNIS gene transfer for various types of cancer in vivo, primarily through the use of immunodeficient mice bearing subcutaneous xenografts. In this study, we used a biologically relevant rat model of prostate cancer, which consistently develops metastases in distant lymph nodes and lungs to explore the clinical applications of hNIS gene transfer for advanced prostate cancer. The objective of this study was to determine the effect of hNIS expression on growth of MATLyLu cells in vitro and in vivo in the absence of radioiodide therapy. MATLyLu cells were transduced with hNIS or empty vector (LXSN) retrovirus, and tumor growth and progression were monitored in Copenhagen rats subcutaneously injected with parental or transduced MATLyLu cells. Tumors were examined histologically and immunohistochemically to evaluate proliferation (PCNA, Ki-67) and apoptosis (Caspase-3). Although metastases consistently developed in all rats, tumor growth and metastatic progression were significantly delayed in rats injected with MATLyLu-hNIS cells compared to rats injected with MATLyLu-LXSN or parental MATLyLu cells. Mixed and clonal populations of MATLyLu-hNIS cells also grew significantly slower than MATLyLu-LXSN or parental MATLyLu cells in vitro. These results suggest that hNIS inhibits MATLyLu cell growth. Such growth rate differences between tumors with and without hNIS expression prior to radioiodide therapy make interpretation of tumor size, metastatic progression, and animal survival after radioiodide treatment difficult unless appropriate controls are included.
94: Expression of Monocyte Chemoattractant Protein-1 (Mcp-1) in Pigtailed Macaques (Macaca Nemestrina) with Siv Encephalitis
AIDS Dementia Complex (ADC) is a constellation of signs including motor and cognitive deficits affecting a large proportion of HIV-infected individuals. Studies of autopsy specimens reveal a broad array of pathological changes including focal accumulations of macrophages with or without associated giant cells in both white and gray matter, cerebral cortical atrophy and neuronal loss. We have developed an animal model of ADC using SIV-infected macaques that recapitulates the neurological events in HIV encephalitis. Co-inoculation of pigtailed macaques (Macaca nemestrina) with a neurovirulent viral clone (SIV/17E-Fr) and an immunosuppressive viral swarm (SIV/DeltaB670) produces neurologic disease in over 90% of inoculated animals within 3 months post-inoculation (p.i.). SIV encephalitis is characterized by infiltration and activation of macrophages and formation of microglial nodules. To investigate the role of the macrophage chemoattractant MCP-1, we measured its expression in 30 infected and 3 uninfected macaques in CSF and plasma by ELISA and in paraffin-embedded brain tissue by immunohistochemistry (IHC) and in situ hybridization (ISH) using quantitative image analysis. There was a statistically significant increase in CSF:plasma ratios of MCP-1 detected by ELISA at 28 days p.i. in animals with moderate or severe encephalitis as compared to animals with mild or no encephalitis, and elevated expression of MCP-1 in tissue detected by IHC and ISH at 8 weeks and 3 months p.i. We conclude that elevated CSF:plasma MCP-1 ratios may be a useful early predictor of HIV-induced encephalitis. Understanding the role of MCP-1 in SIV encephalitis will help us to understand the role of this important chemokine in HIV encephalitis.
95: High Salt Diet Induces Fundic Gland Atrophy in Mongolian Gerbils
Outbred Mongolian gerbils obtained from a supplier within the USA were fed a high salt (2.5%) Purina test diet from 8 to 60 weeks of age. Control animals received a basal Purina diet containing 0.25% salt. The five animals receiving the high salt diet experienced a moderate to marked loss of parietal and chief cells of the fundic glands. Some areas of the glands were replaced by a large mucoid phenotype glandular cell population. In addition, gastric pits were significantly elongated and lined by proliferating foveolar cells. Inflammation was not a key characteristic. The varying degree of fundic gland atrophy, mucoid metaplasia and gastric pit elongation between individuals is consistent with the genetic variation present in an outbred population in contrast to what might be observed for inbred strains. These changes are similar to the preneoplastic changes observed in Helicobacter spp. infected mice and gerbils. Similar infection-related gastric changes may preceed the development of human gastric adenocarcinoma. Previous Helicobacter infection of mice on high salt diets has suggested a synergism during the development of preneoplastic gastric changes. In humans, the combination of a high salt diet and Helicobacter infection may act synergistically to increase the incidence of gastric adenocarcinoma particularly within some cultural groups. Further work will focus on the characterization of this possible synergism.
96: Influence of Gender on Hepatocarcinogenesis in Japanese Medaka, Oryzias Latipes
Japanese medaka (Oryzias latipes) are used widely in hepatocar-cinogenesis research. Because livers of female fish are responsive to estradiol, and because of proliferative effects of estradiol, female medaka may be at higher risk of developing hepatic tumors. This study had three objectives: 1. identify any gender difference in total DNA adducts shortly after diethylnitrosamine exposure, 2. determine if female sexual maturity is associated with diethylnitrosamine-induced lesions, and 3. semi-quantify the expression of two estrogen-responsive gene transcripts in diethylnitrosamine-induced hepatic lesions in medaka. Total DNA adducts were quantified by accelerator mass spectrometry at times ranging from 0 to 672 hours after 14C-diethylnitrosamine exposure. Sexual maturation was delayed in a subset of animals through photoperiod manipulations, allowing lesion incidence comparisons between sexually mature and immature females 4, 6 and 8 months after diethylnitrosamine exposure. Iso-topic in situ hybridization was used to semi-quantify estrogen receptor (ER) and choriogenin transcripts in a variety of lesions. Females had fewer DNA adducts than males at 10 of 11 time-points after diethylnitrosamine exposure. At 4, 6 and 8 months after exposure, lesion incidence was highest in mature females followed by immature females, then males. ER mRNA expression in hepatic lesions of females increased as a function of histological grade. Altered foci and hepatocellular carcinomas in females expressed more ER and choriogenin mRNA than those in males. In conclusion, estradiol promotes hepatocarcinogenesis in female medaka, possibly through a proliferative effect on hepatocytes, particularly in mature females who are under the most estradiol influence.
97: A Null Mutation in the Homeobox Gene, Hex, Severely Retards Hepatic Development in the Mouse Embryo
The homeobox gene, Hex, is expressed in the definitive en-doderm of the developing mouse embryo, where it directs normal forebrain development. In addition, other domains of Hex expression are found in the liver, thyroid gland, lungs, endothelial cells and multipotent hematopoeitic cells. In order to study extracranial mechanisms controlled by this gene, a null mutation of the Hex gene was created by homologous recombination in embryonic stem (ES) cells. Null mutants die in utero between embryonic days 12–1 (E12–15). The mutant liver fails to develop trabeculae, and the liver resembles a blood-filled sac. The capsule is lined by a layer of spindloid and cuboidal cells, and similar cells are seen in the residual trabeculae. Hematopoeitic elements appear comparable to those of the wild type (WT) animal. At E12.5, the liver has enlarged markedly in the WT animal, due to development of hepatic cords and continued hematopoeisis. In Hex mutants, the liver undergoes minimal further development and assumes a sac-like appearance. Hematopoeitic elements are present in normal proportions, but are admixed with necrotic debris and appear less robust than in the WT littermates. In addition, there is failure of complete cardiac septation in a variable proportion of Hex null animals. The serosal surfaces of heart and liver are smooth and avascular in the WT animals, but are prominently vascularized in Hex mutants. Contrary to previous reports, no abnormalities of pancreatic or thyroid development were noted.
98: Progressive Glomerulonephritis and Tumors in Null Mice Deficient in a Single P450 Enzyme, Cyp1B1
The cytochrome P450 CYP1B1 metabolically activates poly-cyclic aromatic hydrocarbons and is a major P450 isoenzyme in human monocytes and macrophages. We have previously shown that mice deficient in CYP1B1 were resistant to induced tumors after 7,12-dimethylbenz[a]anthracene exposure. The pathology of aging CYP1B1 mice on a B6;129 background was studied in groups of 30 males and 30 females. By 12 months, 50% of the female mice had become ill or died due to a progressive glomerulonephritis while males developed similar renal lesions later in life. This disease followed a sequence of proliferative, mem-branoproliferative and sclerotic glomerulonephritis. Anti-DNA antibodies were found in the blood of the mice along with dense deposits in glomerular basement membranes. The lesions were unlike those found in aging wild type B6;129 mice. Male mice also had histiocytic sarcomas and other tumors later in life. The spectrum of tumor incidences were different than those in wild type B6;129 mice. Our study provides evidence that deficiency of CYP1B1 can play a role in the development of glomerular disease and tumors of the mononuclear phagocyte system.
99: Quantitative Image Analysis of Cerebral Beta-Amyloidosis in the Appv717F Transgenic Mouse Model of Alzheimer'S Disease
Cerebral beta-amyloidosis is a major feature of the neuropathology of Alzheimer's disease (AD). The APPV717F transgenic mouse is a model of cerebral beta-amyloidosis for studies on the pathogenesis of AD and the efficacy of potential treatments. Histopathologic lesions of cerebral beta-amyloidosis that occur in the APPV717F mouse can be measured using quantitative image analysis. We developed image analysis methods that included novel sampling strategies at the microscopic slide and field levels. Analyses of cerebral beta-amyloidosis in a population of homozygous APPV717F control mice produced results with high variability (standard error). The model and analysis methods were examined for sources of variability. The image analysis methods were shown to be adequate to accurately capture the severity of the pathology in a given mouse and, within the limits of the analyses, were free from systematic error. However, the largest contribution to the standard error of the analyses was phenotypic variability inherent in the mouse model population. Measures of inter-animal variability in cerebral beta-amyloidosis were used to perform power analyses for use in the design of future studies using the APPV717F mouse. Furthermore, we demonstrated that background genes of this outbred mouse strain influence phenotypic variability.
100: Quantitative Trait Loci (Qtls) for Virus-Induced and Allergen- Accentuated Chronic Airway Inflammation and Remodeling Map to Chromosomes 7 and 20 in Rats
Brown Norway (BN) rats infected with Sendai virus were used as a model to study the genetics of virus-induced asthma. Genome-wide scan and linkage analysis was used in BN × F344 F1 rats backcrossed to BN rats to identify quantitative trait loci (QTL) controlling susceptibility to chronic airway inflammation and remodeling induced by Sendai virus infection during early life and subsequent recurrent ovalbumin exposure. The linkage analysis study was conducted in 176 backcross rats using 187 PCR-based genetic markers following innoculation with Sendai virus and sensitization and repeated aerosol exposure to ovalbumin. Asthmatic phenotypes studied included chronic bronchiolar inflammation, bronchiolar fibrosis, bronchiolar mucous cell hyperplasia and high serum IgE. Genetic analysis identified the strongest linkages of chronic bronchiolar inflammation to be on chromosome 7 in the interval between D7Rat35 and D7Rat103. Candidate genes identified on rat chromosome 7 include MadCAM1, Igf1, and Tbxa2r. Bronchiolar mucous cell hyperplasia and elevated serum IgE were linked to the interval containing genes for rat MHC (RT1) and TNF-alpha on chromosome 20. Four polymorphic loci were identified in the promoter region of TNF-alpha between BN and F344 rats. Funded by NIH HL61018.
101: Increased Expression of Tracheal Antimicrobial Peptide (Tap) Mrna in the Lungs of Neonatal Calves with Bacterial Pneumonia
Innate immunity of the respiratory mucosa is particularly vital shortly after birth. We determined whether an inducible beta-defensin, tracheal antimicrobial peptide (TAP), is expressed in neonatal calves, and the extent of changes in expression during bacterial pneumonia. Eighteen 1- to 3-day-old, colostrum-deprived calves were inoculated with either Mannheimia haemolytica or pyrogen-free saline by fiberoptic bronchoscopy into the main bronchus of the left cranial lung lobe, and necropsied 2 or 6 hours post-inoculation (PI). Expression of TAP mRNA, as well as two molecules vital to leukocyte infiltration, interleukin 8 (IL-8) and intercellular adhesion molecule 1(ICAM-l), were determined by real-time relative quantitative reverse transcriptase-polymerase chain reaction (TaqMan). TAP mRNA was expressed at a basal level in control (saline-inoculated) neonates. Both TAP (P = 0.1) and ICAM-1 (P = 0.01) were significantly increased in calves that received M. haemolytica versus calves that received saline; TAP expression varied among animals. Also, individual relative mRNA levels between TAP and IL-8 and between IL-8 and ICAM-1 had positive correlations (r2 = 0.89, p<0.0001; r2 = 0.41, p = 0.09, respectively). These results suggest that in neonates: (i) expression of TAP mRNA is present at a basal level; (ii) both TAP and ICAM-1 are rapidly upregulated during pneumonia and TAP expression varies between animals; and (iii) within individuals, correlations between TAP, IL-8 and ICAM-1 mRNA suggest common pro-inflammatory stimuli for upregulation.
102: Differentiation of North American and European Genotypes of Porcine Reproductive and Respiratory Syndrome Virus in Formalin-Fixed Paraffin-Embedded Tissues by Multiplex Rt-Pcr
North American and European genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) were distinguished in formalin-fixed, paraffin-embedded tissues from PRRSV-infected pigs by multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR). A method based on xylene deparaffinization followed by proteinase K digestion yielded quality RNA for reliable and consistent RT-nPCR analyses. PRRSV nucleic acid was detected in lung, mediastinal lymph node, tonsil, and liver samples from pigs inoculated with North American strain virus, European strain virus, or both North American and European strains. All 30 archival formalin-fixed, paraffin-embedded lung tissues from pigs naturally infected with PRRSV were positive for PRRSV by RT-nPCR amplication and gave a pattern that corresponded to the North American genotype. Multiplex RT-nPCR could be applied successfully to formalin-fixed, paraffin-embedded tissues for the detection and differentiation between North American and European genotypes of PRRSV.
103: Xenotransplantation of Sheep Lung into Nude Mice as a Model for Ovine Pulmonary Carcinoma
This study investigated the survival, differentiation, and proliferation rate of epithelial cells in sheep lung that was xeno-transplanted subcutaneously in nude mice. Four fragments taken from macroscopically normal lungs of 6-month-old lambs were transplanted into the subcutis of weanling athymic (nude) mice. The implants were removed and examined by light microscopy, electron microscopy, and immunohistochemistry at 1, 2, 4, 6, and 8 weeks post transplantation. At 1 week there was necrosis of 25–50% of the tissue but by 2 weeks, the tissue present was 80–100% viable. Grafts retrieved at 2–8 weeks exhibited good differentiation into alveoli, bronchioles, bronchi, cartilage, capillaries, and an additional fibrous component in some sections. The bronchiolar and bronchial epithelial cells secreted mucus as was evidenced by periodic acid-Schiff staining. Cilia also were present on bronchial epithelium. In addition, the tissue was stained with antibody to Ki-67 and approximately 50% of the cells in sections taken at 2 weeks stained, showing prominent proliferation of all epithelial cell types in these sections. By 8 weeks, only 5% of the cells showed evidence of proliferation by immunostaining for Ki-67. Sections of lung containing microscopic tumor nodules from lambs infected with Jaagsiekte sheep retrovirus (JSRV) also proliferated when transplanted. Tumor nodules were unencapsulated, papilliferous proliferations composed of a single layer of well-differentiated cuboidal epithelial cells. Xenografted lung may prove to be a good model for the study of the pathogenesis of JSRV based on the survival, growth and differentiation of the pulmonary epithelial cells in this environment.
104: A/J Mice Are More Susceptible to Cigarette Smoke-Induced Pulmonary Emphysema than B6C3F1 Mice
Many animal models of emphysema have been developed that are useful in physiology and pathology, but few have been produced by exposing animals to cigarette smoke (CS), the main cause of emphysema in humans. Previous work demonstrated that female B6C3F1 mice develop emphysema after 7–13 months of exposure to mainstream CS. Here, we demonstrate murine gender and strain differences in the susceptibility to CS-induced emphysema after relatively short exposure periods. Male and female mice of the B6C3F1 hybrid strain and the A/J inbred strain were exposed whole-body for 6 hours /day, 5 days /week for 15 weeks to CS at 250 mg total particulate material/m3. At necropsy lungs were inflated with formalin at a constant pressure, and H&E-stained sections from paraffin-embedded tissues were assessed stereologically for alveolar air space expansion (mean linear intercept [Lm] of alveolar septa, volume density of alveolar air space [VVair]) and tissue loss (volume density of alveolar septa [VVspt], total alveolar surface area [Sa]). Based on significant changes in several stereologic parameters, the A/J mice were more susceptible to CS-induced emphysema than the B6C3F1 animals. Female mice were particularly affected. These results open avenues of exploration for strain- and gender-specific emphysema susceptibility determinants, such as differences in types of inflammatory reactions, imbalances between matrix proteinases and anti-proteinases, and defenses against oxidative stress. Better understanding of emphysema models produced by CS exposure is needed, because other models usually recapitulate only a few aspects of this complex disease, whereas CS produces pulmonary changes in mice that likely mimic those in the pathogenesis of the human disease. Funded by Tobacco Settlement Cooperative Research Agreement UNM PO 881757.
105: Antiatherogenic Effect of Agastache Rugosa in Ldl Receptor Deficient Mice
The anti-atherogenic effect of extract from aerial part of Agastache rugosa was investigated in low density lipoprotein receptor (LDLR) deficient mice. We investigated whether the extract can modulate endothelial activation involved in leukocyte recruitment. The diet of all animals contained 1.25% cholesterol, 15% fat and 0.5% sodium cholate. We divided the 20 female mice into two groups. After 8 weeks, total plasma cholesterol level was not significantly reduced compared to the control group. The mean body weight was not different between extract-treated group and control group. Histological examination showed mature atherosclerotic lesions composed of foam cells under the endothelium, numerous proliferative smooth muscle cells, and necrotic cores in control animals. In contrast, the extract-treated group showed only early lesions containing mainly superficial cells. Immunohistochemical stains demonstrated that macrophage infiltration was reduced in the extract-treated group. As assayed by morphometry, the lesion area in aortic sinus was reduced by 53% in extract-treated group compared with control group (p<0.01). In conclusion, the aerial part of Agastache rugosa has an anti-atherogenic effect through anti-inflammatory activity.
106: Mice Lacking Platelet Derived Growth Factor (Pdgf) B-Chain from Leukocytes and Platelets Have Increased Angiogenesis
Platelet derived growth factor (PDGF) B-chain stimulates connective tissue and endothelial cell proliferation in vitro and in vivo. Important sources of PDGF B-chain are cells of hematopoietic origin, particularly platelets and macrophages; it is believed that PDGF B-chain augments wound repair and may be involved in vascular diseases such as atherosclerosis. To test the hypothesis that hematopoietic-derived PDGF B-chain is necessary for wound repair, we created hematopoietic PDGF B-chain knockout mice in which the hematopoietic system of an irradiated recipient wild type mouse is replaced with bone marrow from a PDGF B-chain−/- or +/+ donor. We examined two different lesions in these mice: 1) granulation tissue formation induced by implanting subcutaneous sponges, and 2) an organized thrombus was created by ligating the left common carotid artery. We show that 75–85% of PDGF B-chain transcripts within the granulation tissue are of hematopoietic origin. Unexpectedly, we found that the absence of hematopoietic-derived PDGF B-chain is necessary for wound repair, we created hematopoietic PDGF B-chain did not affect the extent of granulation tissue formation, nor did it affect the size of the organized thrombus. However, the vascularity of the sponge-induced granulation tissue, as well as the vascularity of the organized thrombus, were increased in the mice lacking hematopoietic PDGF B-chain. We conclude that hematopoietic-derived PDGF B-chain is not necessary for granulation tissue formation or for the formation of an organized thrombus but may inhibit vascularization in both of these models, probably through disabling the short range chemotactic gradients recruiting vascular support cells.
107: www.vetpathresidents.homestead.com: An Online Study Resource for Veterinary Pathology Board Preparation
The culmination of residency training in veterinary pathology is board certification by the American College of Veterinary Pathologists. Certification is essential for career advancement and future employment. As evidenced by current board pass rates, this task is daunting, given the breadth of material that must be reviewed. Group study with motivated individuals improves preparation and eliminates duplication of effort. Even in large pathology training programs, often only a small number of trainees are studying to sit the board examination in any given year. By allowing access to the thoughts and strategies of multiple study groups, all can benefit from the synergism of a large, diverse network. Furthermore, isolated individuals in non-traditional or smaller training programs, as well as board-eligible candidates in the workplace, are able to access to and benefit from this interaction. A large portion of a candidate's time is spent acquiring material before they actually begin studying for boards. Upon completion of the examination, their study material is often discarded and the benefits of their countless hours of preparation are lost. The purpose of this web site is to serve as a repository of veterinary pathology board preparation material for distribution among veterinary pathology residents and board-eligible candidates regardless of their location. If you are currently a resident, a board-eligible candidate, or, an ACVP diplomate, we invite you to join us in our academic pursuits at: http://vetpathresidents.homestead.com. Password access to this free website can be obtained by contacting the authors’ e-mail links on the homepage.
108: Cyclooxygenase-2 Inhibition Does Not Affect Mononuclear Phagocyte Function
Two isoforms of cyclooxygenase (COX), COX-1 and COX-2, catalyze the production of prostaglandins (PGs) that are involved in various physiologic and pathophysiologic processes. COX-2 is the only isoform induced at the site of inflammation and is responsible for the generation of pro-inflammatory PGs. It is not known whether inhibition of COX-2 at the site of inflammation will affect primary inflammatory cell functions. Therefore, a series of in vitro and in vivo studies were conducted to evaluate the functional capacity of mononuclear phagocytes following treatment with a selective COX-2 inhibitor (SC-58236) and a non-selective COX-1/COX-2 inhibitor (naproxen sodium). The in vitro studies evaluated the effects of these drugs (1, 10, or 100 μM) on the capacity of rat peritoneal macrophages to phagocytose opsonized S. aureus-coated bioparticles. Cytochalasin B, a microfilament disruptor, was used as a positive control and gave the expected inhibitory response. The in vivo studies evaluated the functional activity of tissue-specific macrophages by monitoring the clearance of 51Cr-labeled sheep erythrocytes (sRBC). Female B6C3F1 mice were treated orally for 3 days with vehicle or 3, 10, or 30 mg/kg SC-58236 or naproxen sodium. Maleic vinyl ether was used as positive control and gave the expected inhibitory response. Peripheral blood clearance of labeled sRBC was monitored for 60 minutes, then the animals were sacrificed, exsanguinated, and liver, spleen, lungs, thymus, skin, intestine and kidneys were removed and analyzed for radioactivity. The in vitro data showed no effect with SC-58236 at any concentration and a statistically significant inhibition of phagocytosis at all concentrations tested with naproxen sodium. The degree of inhibition noted with naproxen sodium was slight (8.5% decrease). The in vivo studies showed no effect of SC-58236 or naproxen sodium on phagocytic indices (organ weights, % uptake or total counts/ mg tissue). Collectively, these data indicate that COX-2 is not involved in the phagocytic function of macrophages and that its inhibition at the site of inflammation does not affect the primary phagocytic activity of inflammatory cells.
109: Alterations in Local Lymph Node Cytokine Expression Associated with Suppression of Contact Hypersensitivity (Chs) in Adult Rats Perinatally Exposed to Low-Level 2,3,7,8-Tetrachloro-Dibenzo-Dioxin (Tcdd)
The developing immune system is one of the most sensitive targets of tetrachloro-dibenzo-dioxin (TCDD) toxicity in rodents; low-level perinatal exposure has been shown to induce alterations primarily of acquired cell-mediated immunity. Work from this laboratory indicated that F344 rats exposed to TCDD in utero and lactationally have reduced contact hypersensitivity (CHS) to 2, 4-dinitrofluorobenzene (DNFB) for up to 6 months of age. In this study, the relationship between CHS suppression and cytokine profiles of the draining lymph node in these offspring were investigated. Lymph nodes draining CHS reaction (DNFB-LN) were evaluated by real-time RT-PCR for gene expression of T-helper type-1- (Th-1-), T-cytotoxic type-1- (Tc-1-), and T-helper type-2- (Th-2-) associated cytokines in 2-month-old offspring of dams exposed to 1 or 3 mg TCDD/kg on gestational day 14. In a duplicate experiment with 3-month-old offspring, cells of the DNFB-LN were evaluated by fluorocytometry for phenotype and intracellular cytokine content. Significantly greater IL-12p40 and IFN-γ, and lower IL-10 mRNA occurred in DNFB-LN of 2-month-old offspring perinatally exposed to 3 mg TCDD/kg with suppressed CHS relative to controls. In 3-month-old offspring exposed to 3 ml kg TCDD with suppressed CHS, intracellular cytokine analysis for IL-2, IL-4 and IFN-γ indicated significantly greater frequency of IFN-γ+CD8b+ T-cells in DNFB-LN. Collectively, results suggest that the suppressed CHS in perinatally TCDD-exposed offspring is linked with enhanced IL-12p40 and IFN-γ, and decreased IL-10 expression within DNFB-LN, with CD8+ (Tc-1) cells being primarily responsible for the IFN-γ production. The enhanced cytokine expression may represent a compensatory, or unregulated response of CD8+ T-cells and antigen-presenting cells in these rats. The reduced expression of IL-10, suggests a reduction in activity of a CD4+ (Th-2-like) subset in draining nodes. These results indicate disruption of the cytokine cascade during the efferent phase of CHS in draining nodes of adult rats perinatally exposed to TCDD. (This abstract does not reflect EPA policy.)
110: Effects of Rapamycin Administration on Renal Lesions of Nzbxnzw F1 Female Mice
The purpose of this study was to evaluate the effect of rapa-mycin therapy on glomerulopathies that develop in NZBxNZW F1 female mice. Rapamycin, an immunosuppressive agent, was dosed 3 times per week over a 2-week period in 29-week-old mice. Kidneys were evaluated from 12-, 25-, 36-, 42-, and 55-week-old mice. The 12- and 25-week-old mice had no to slight glomerular changes prior to initation of therapy. Renal lesions seen in untreated 36- to 55-week-old NZWxNZB mice included thickened glomerular basement membranes, decreased numbers of glomeruli, decreased size of glomeruli, and increased cellularity of glomeruli. Thickened basement membranes were observed as increased width of capillary walls with homogeneous eosinophilic material; this is characteristic of the antigen/antibody deposition anticipated with the development of SLE in this strain of mouse. Small glomeruli with few cells and prominent homogeneous eosinophilic material were considered to be sclerotic (glomerulosclerosis). Mice administered rapamycin had kidneys that were microscopically within normal limits. Mice that received no therapy had slight to moderate membranopro-liferative glomerulopathy and some tubule changes including dilatation and basophilia. Amelioration of anticipated lesions in mice administered rapamycin was considered to be directly related to the immunosppressive effects of rapamycin. Overall, under the conditions of these studies, treatment of NZWxNZB mice with rapamycin is associated with decreased prevalence and severity of renal lesions.
111: Transitional Cell Proliferative Lesions of the Ureter with Hydronephrosis in B6D2F1/Cr Mice
Minimal to moderate unilateral abdominal swelling was detected in untreated, 8 to 11 week old female B6D2F1/Cr mice. Otherwise, the mice were clinically heathy. Approximately 10% of animals received in a number of shipments of 200 hybrid female mice from a commerical supplier were affected. Grossly, one kidney, usually on the right side, was enlarged and fluid filled; the proximal 4 mm of its ureter was enlarged (2 mm) and thickened. The contralateral kidney and ureter and the urinary bladder usually were normal grossly and microscopically. Histologically, transitional cells of the thickened proximal ureter had a spectrum of proliferative lesions ranging from marked hyperplasia to papillary adenoma and carcinoma. The larger lesions protruded into the renal pelvis. Hyalinosis, which was immunohistochemically positive for Ym1/Ym2 protein, was present in the proliferative transitional cells. The affected ureter frequently had severe transmural chronic inflammation with extensive infiltrates of eosinophils. Affected kidneys had moderate to severe hydronephrosis with associated fibrosis. Pelvic transitional epithelium tended to be flattened. Hyalinosis was absent in pelvic transitional epithelium in the affected or contralateral kidney and the urinary bladder. Minimal hyalinosis was present in nasal epithelium but not in the stomach, gall bladder or lung. Other organs were normal or had no consistently related lesions The pathogenesis of proliferative ureteral lesions and their incidence in male B6D2F1/Cr mice are not known at this time.
112: Immunolocalization of Cox-2 in the Equine Kidney
The cyclooxygenase (COX) enzyme catalyzes the production of prostaglandins and exists as two distinct isoforms, COX-1 and COX-2. COX-1 is constitutively expressed and, in the kidney, is considered important in the regulation of basal renal functions such as renal blood flow and salt and water homeostasis. Conversely, the inducible isoform, COX-2, functions in normal physiologic processes as well as in stressed conditions (e.g. volume-depletion) in some species (such as dogs, but not monkeys). These species differences may explain the species-specific nephrotoxicity associated with non-steroidal anti-inflammatory drugs (NSAIDs). NSAID use in the horse for the treatment of pain and inflammation is widespread; however, to our knowledge, no information exists as to the renal localization of COX-2 in this species. In this study we used immunohistochemistry to evaluate COX-2 expression in the kidneys of 3 adult male horses. Each animal had been treated with an NSAID and developed NSAID-related renal medullary crest necrosis and, in one case, right dorsal ulcerative colitis. COX-2 was localized to the papillary interstitial cells, parietal cells of Bowman's capsule, as well as the collecting duct. However, minimal COX-2 staining was observed in the macula densa. This pattern of COX-2 staining differs from that observed in the rat and dog in which prominent COX-2 staining is observed in the macula densa and thick ascending limbs of loop of Henle. The COX-2 expression pattern observed in the glomerulus and collecting ducts of the equine kidney has not been reported in other species. These observations suggest that COX-2 may be important in renal function of the horse under pathophysiologic conditions and may have functions distinct from those reported in other species.
113: Pharyngeal Aspiration for Exposing the Mouse Respiratory Tract to Respirable Particles
The most common techniques for experimental exposure to re-spirable particles are inhalation and intratracheal instillation. Inhalation exposures are expensive, technically difficult and potentially hazardous. Intratracheal instillation in the mouse is technically difficult and associated with bolus effects, uneven pulmonary distribution, invasiveness and technical difficulties. Therefore, we have evaluated a pharyngeal aspiration technique previously used for exposure to protein antigens as a method for exposing the mouse respiratory tract to particles. Four female, 15-16-week-old mice were lightly anaesthetized with isoflurane, the tongue was extended, and 50 μL of a suspension of 1 μM red-fluorescent polystyrene amine-modified latex beads was placed at the base of the tongue. Tongue restraint was continued until at least two deep breaths were completed. The mice were sacrificed 1-hour post exposure. Lungs were inflated with O.C.T. (Tissue-Tek) TBS mixture and frozen in liquid nitrogen. To measure the area of the lung occupied by the fluorescent particles, 5 random 0.13 mm2 sections of the terminal bronchiole and adjacent alveoli and 5 random 0.13 mm2 sections of the alveoli without the terminal bronchioles were recorded for the left and right lungs of each mouse. Large numbers of fluorescent beads were located in alveolar spaces without preferential distribution in peribronchiolar alveoli. In all exposed mice, both the left and right side of the lung were exposed. These findings suggest that the aspiration technique deposits particles in the deeper lung and because it is less invasive as well as technically easier than the intratracheal instillation technique, may be more suitable for repeated exposures to re-spirable particles.
114: Optimization of An Automated Non-Isotopic in Situ Hybridization Protocol
We have implemented an automated non-isotopic in situ hybridization (ISH) protocol with signal amplification to detect relatively low abundance mRNA using the Ventana Discovery System® and the Dako Autostainer®. Protocol parameters for ISH were optimized on murine tissues using 1 kb digoxigenin-labeled PCR-generated riboprobes to cathepsin S, biotinyl-tyramide, and alkaline phospha-tase-based visualization derived from manual methods. Both cry-osections and paraffin sections were evaluated. Parameters optimized included fixation, pretreatments (proteinase K digestion, avidin-biotin blocking), probe concentration, stringency wash conditions with/without formamide, RNase treatment, and post-stringency fixation. Pretreatments, hybridization, and stringency washes were performed on the Discovery System® and immunodetection was performed on the Autostainer®. We determined that optimal signal was achieved in most tissues from cryosections fixed for 1 hr. in 4% paraformaldehyde followed by successive treatments with 0.1% active DEPC, proteinase K, and 0.2M HC1. Background was minimized with probe concentrations of ∼30-50 ng/mL (0.5μL/mL). Signal was optimal when hybridization at 55°C was for a minimum of 9 hrs. RNase A digestion followed by successive stringency washes at 60°C containing 1XSSC, 0.5XSSC, and 0.1XSSC was effective in eliminating non-specific probe binding. Post-stringency fixation in 4% paraformaldehyde reduced non-specific staining during immunodetection. For some tissues, such as brain, optimal signal was achieved in paraffin sections. The features of the Discovery System® which include individual slide heating control and reagent agitation under liquid coverslip greatly contribute to uniformity of signal across the section and the reduced signal-to-noise ratio. The combination of the instrumentation offers the opportunity to rapidly assess simultaneously numerous variables within a protocol.
115: The Prolonged Storage of Paraffin Sections Affects the Successful Detection of Mrnas by in Situ Hybridization
Whereas RNA degradation in fresh tissues is a well-recognized and well-understood phenomenon, little is known about the stability of RNA in non-deparaffinized histological sections that are stored before use. In this study, we evaluated whether prolonged storage of paraffin sections affects the detection of specific mRNAs by radioactive in situ hybridization (ISH). Paraffin blocks of rat experimental skin wounds, myocardial infarcts and fibrotic lungs were serially sectioned at different times, so that the resulting serial sections could be stored at room temperature for different periods of time (up to 35 days). All sections were then hybridized with 33P-labeled riboprobes specific for rat Type III collagen and rat matrix metalloproteinase-2 (MMP-2). Signal intensities were analyzed using a phosphorimager and by blinded microscopic evaluation. Stronger signals were consistently observed in freshly sectioned slides compared to slides sectioned 2 weeks or more before hybridization. Signal intensities were negatively correlated with the storage period of the sections. In conclusion, our results indicate that it is preferable to use freshly prepared sections (i.e., stored for 4 days or less) when trying to locate specific mRNAs in formalin-fixed, paraffin-embedded tissues by radioactive ISH.
116: A Mouse Model to Evaluate the Effect of Compounds on Incisional Skin Wound Healing
In the skin, wound healing is a dynamic, complex, and highly regulated process associated with the release of large numbers of growth factors and cytokines, the recruitment of inflammatory cells, and the proliferation and migration of keratinocytes, fibroblasts, and endothelial cells. Because of the large number of signals and biologic modifiers involved, many compounds affecting cell proliferation, cell migration, inflammation, angio-genesis or protein synthesis could potentially impair the healing of skin wounds. To evaluate the potential impact of investigational compounds on the healing of skin wounds, we have developed an incisional wound model in mice. Hairless, but immunocompetent SKH1 mice were treated orally with vehicle or dexamethasone at 0.2 and 1 mg/kg/day BID 6 days before wounding and during the healing process. A 1.5 cm transverse incisional wound was created on the dorsum of mice and was sealed by 3 equidistant sutures. The healing process was monitored at different time points macroscopically, histologically, by immunohistochemistry and in situ hybridization and by evaluating the tensile strength of the wounds. Dexamethasone at 1 mg/kg/day significantly altered the healing and morphology of skin wounds and greatly affected the tensile strength of the wounds. In conclusion, this mouse model is useful to evaluate the potential impact of various experimental compounds on the healing process in the skin. Dexamethasone at 1 mg/kg/day is recommended as a positive control for such studies.
117: Comparison of Caspase-3 Immunohistochemistry and the Tunel Method for the Quantification of Apoptosis in Histologic Sections of Pc-3 Subcutaneous Xenografts
The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method has been the gold standard for the detection and quantification of apoptosis in histologic tissue sections for many years. However, this method is associated with a number of technical problems and has been shown to be non-specific, especially in neoplasms. Recently, antibodies against several caspases have become commercially available. Caspases are cysteine proteases that are known as the “executioners of apoptosis”. The purpose of this study was to evaluate whether an immunohistochemical stain for caspase-3 would improve our ability to detect and quantify apoptosis in tissue sections. We used PC-3 tumor xenografts as an example, since these tissues contain large numbers of cells undergoing apoptosis. Our results indicated that caspase-3 immunohisto-chemistry was a more convenient and easier method to detect and quantify apoptosis in this model. In addition, we found a good correlation (r2 = 0.75) between the apoptotic indices measured by computer-assisted image analysis following identification of apoptotic cells by either the TUNEL method or caspase-3 immunohistochemistry. Therefore, we recommend the use of caspase-3 immunohistochemistry for the detection and quantification of apoptosis in tissue sections.
118: Preferred Sites and Measurements for Rapid Osteoclast Quantification in Rat Adjuvant Arthritis
Bone erosion is pronounced in rats with mycobacterial-induced adjuvant arthritis (AdA). Significant bone preservation in association with reduced osteoclast numbers occurs in AdA following administration of osteoprotegerin (OPG), a soluble RANK ligand receptor that thwarts osteoclastogenesis (Nature 402: 304, 1999), or the anti-cytokines interleukin-1 receptor antagonist or PEG-conjugated soluble tumor necrosis factor receptor type I (Arth. Rheum. 43 [9 Suppl]: S233, 2000). This study was performed to define sites for rapid osteoclast enumeration in AdA as an aid to providing quantitative comparisons between bone-sparing anti-arthritic therapies. Male Lewis rats (n = 6/group) were necropsied 7 days after the onset of arthritis (day 16 after adjuvant inoculation). Osteoclasts were detected by an indirect immunohistochemical method for cathepsin K. Erosions and osteoclast numbers were assessed in the “ankle” (tibia and tarsals) using a five-tiered, semi-quantitative histopathologic scale as well as by 8 quantitative measurements in the calcaneus, navicular tarsal, and distal tibia acquired using a commercial image analysis system (Osteometrics, Decatur, GA, USA). The simplest yet most robust parameters for quantifying skeletal damage were “bone area as a percentage of total area” (an index of erosion) and “osteoclast numbers per square millimeter of total area” (a measure of cells associated with disease). Osteoclast counts in the navicular tarsal, the most sensitive site in arthritic joints, were approximately 200-fold higher than controls. Comparable ratios were much reduced in calcaneus (7-fold elevation) and tibia (3-fold increase). Quantitative data were correlated to the semi-quantitative histopathology grades. These findings indicate that osteoclasts may be quantified reliably in the navicular tarsal bone using two simple and robust endpoints of relevance to the AdA disease process.
119: Effect of Ileal Resection on Hepatocyte Proliferation and Apoptosis
Bile acids have recently been demonstrated to be ligands for the nuclear receptor FXR and to regulate the expression of various molecules involved in cholesterol and lipid metabolism. In addition, evidence suggests that bile acids may regulate the proliferation of hepatocytes. To investigate the role of bile acids on hepatocyte proliferation and apoptosis, we used a rat model of ileal resection to reduce the enterohepatic recirculation of bile acids. Rats were sacrificed 2, 4, and 13 weeks following ileal resection. Cholesterol 7α-hydroxylase activity and fecal bile acid excretion were determined to verify the decrease in ileal bile acid reabsorption. Proliferating and apoptotic hepatocytes were detected by immunohistochemistry using PCNA and caspase-3, respectively. The proliferation rate was quantified using computer-assisted image analysis, while the apoptotic rate was evaluated by semi-quantitative analysis. Our results demonstrated a slight decrease in liver weights and significant increases in 7α-hydroxylase activity and fecal bile acids following ileal resection. In addition, a moderate, but significant decrease in hepatocyte proliferation was observed 2 and 4 weeks after surgery, while there was no difference in the apoptotic rate among groups at any time points. These results suggest that physiologic concentrations of bile acids can regulate hepatocyte proliferation and that inhibition of bile acid reabsorption are associated with slight changes in hepatocyte proliferation, which may result in slight decreases of liver weight.
120: Comparison of Regional Hepatic Cxcr2 Gene Expression in Dog and Rabbit Using Laser Capture Microdissection Anp in Situ Hybridization
Pifferences in regional expression of pharmacologically targeted receptors may, in some cases, explain differences in pharmacologic responsiveness and/or toxicity profiles of different species. Expression of an IL-8 chemokine receptor (CXCR2) was characterized in four anatomically distinct regions of rabbit and dog liver. Tissues were isolated by laser capture microdissection (LCM) and expression of CXCR2 was analyzed by TaqMan using cDNA generated with either gene-specific primers or random hexamers. Neutrophils, known to highly express CXCR2, were included as positive controls. Relative abundance of CXCR2 was expressed as average number of copies/106 copies of 18S rRNA. Validity of LCM data was confirmed by comparing slopes of standard curves generated for both conventional and LCM derived cPNAs. Non-isotopic in situ hybridization with biotinylated riboprobes was employed to confirm localization of these low abundance transcripts in rabbit and dog. CXCR2 expression was not detected in any of the LCM isolated liver regions using random hexamers or gene-specific primers. However, comparable expression of CXCR2 transcript was detected in neutrophils of both species when gene-specific primers were employed. In additon, robust expression of CXCR2 in neutrophils was evident in the dog and rabbit by in situ hybridization. CXCR2 expression was not evident in liver by in situ hybridization. Although no differences in relative CXCR2 transcript abundance between dog and rabbit was observed, a sensitive and correlative method for evaluating differences in tissue specific gene expression was established.
121: Identification of Cocaine Hepatic Protein Targets in Vivo Using 2D Gel Electrophoresis and Mass Spectrometry
Previous studies have shown that cocaine undergoes oxidative metabolism to one or more reactive metabolites capable of binding to proteins. Protein binding of cocaine metabolite(s) correlates closely with the location and extent of cocaine hepatic injury. Experiments using an anti-cocaine antibody and Western blots have found cocaine adduction of several specific proteins following an hepatotoxic cocaine dose in mice. With gel electrophoresis and sequence analysis, we have recently identified two of the protein targets as hsp60 and transferrin. We report here the identification of three additional protein targets using mass spectrometry. Male ICR mice were treated with cocaine (50 mg/kg, IP). Livers were harvested after 3 hr and homogenized. Proteins in whole liver homogenate were separated using two-dimensional gel electrophoresis. Proteins from saline-treated mice served as controls. Adducted proteins were detected by Western blot using anti-cocaine antibody. Identified spots on the gel were isolated, trypsin digested and then analyzed using a matrix-assisted laser desorption ionization (MALDI) mass spectrometry (Per-Septive Systems Voyager-DE Pro). The Swiss Prot database was used to determine the identity of the target proteins. Large MOWSE score differentials ensured a high level of confidence in protein identification. Three proteins, Liver arginase-1, 1-Cys peroxyredoxin, a non-selenium dependent glutathione peroxidase (GP), and mercaptopyruvo-sulfotrans-ferase protein were identified as cocaine protein targets. Arginase-1 (A-1, liver-type arginase; EC 3.5.3.1) is known to play an important role in liver metabolism as a component of the urea cycle, and GP is an antioxidant enzyme for the detoxification of reactive peroxides. If impaired by adduction, either protein target could play an important role in cocaine hepatotoxicity. (Supported by DA-06601 from the National Institute on Drug Abuse).
122: Loss of Hepatic Glutathione S-Transferase Expression in Largemouth Bass by Estradiol as Demonstrated by Real-Time Polymerase Chain Reaction
Despite the growing body of research targeting the effects of natural and synthetic estrogens in aquatic species, little is known regarding the ability of these agents to affect xenobiotic detoxification pathways. In the present study, a real-time quantitative PCR method was optimized to analyze the effect of 17β-estradiol exposure (E2, 2mg/kg, IP) on hepatic glutathione S-transferase (GST) steady state gene expression in largemouth bass (Micropterus salmoides). The quantity of steady state GST-A mRNA transcripts were indirectly determined relative to the expression of β-actin using real time PCR and the results were compared to those using Northern blotting. Exposure to E2 elicited a significant time-dependent loss of steady-state GST-A mRNA expression, with the normalized level of GST-A mRNA expression assessed by real-time PCR being decreased to 61%, 37%, and 18% of time zero GST mRNA levels at 12, 24, and 48 hr after injection, respectively. The results from the real-time PCR were highly positively correlated to analysis of mRNA expression using Northern blotting (R>0.8, p<0.01). These results indicate that a single injection of E2 can markedly decrease the expression of hepatic steady state GST-A mRNA in largemouth bass, and raises the possibility that exposure to environmental estrogens may affect the ability of bass to conjugate xenobiotics via GST-A. We have established a highly specific, high-throughput technique using real-time PCR, that will allow us to quantitatively measure GST-A gene expression and, therefore, to further study the effects of estradiol as well as other GST-A inducers and regulators in largemouth bass.
123: Pcb and Arsenic Interactions in Hepatocarcino-Genesis
Polychlorinated biphenyls (PCBs) and arsenic are common pollutants and are likely to be found in mixtures in the environment. The coplanar PCB126 (3,3’,4,4’,5-pentachlorobiphenyl) exhibits dioxin-like promotional effects on hepatocarcinogenesis and is considered the most toxic of the PCB congeners. Arsenic is a clastogen which is thought to act during the progression stage of carcinogenesis. This in vivo study was designed to evaluate mixtures of these two chemicals to determine potential interactions at the promotion and progression stages of carcinogenesis. Female 12-week-old Fischer 344 rats were injected with an initiating dose of diethylnitro-samine (DEN). Gavage with corn oil alone or 10ug/kg PCB126 in corn oil was begun two weeks later (three times weekly for duration of study). All rats received a partial hepatectomy three weeks after DEN injection to stimulate liver regeneration. Arsenic at 75 ppm was added to the drinking water in two groups starting at two weeks: one that received corn oil and one that received PCB gavage. In two other groups arsenic treatment was started at 8 weeks; again, one group received corn oil and one received PCB gavage. As a control for progression, ethylnitrosurea (positive control) or saline (negative control) were injected i.p. at 8 weeks into two additional groups that received corn oil gavage. Rats were sacrificed at 8, 16, and 24 weeks. Preneoplastic foci were seen in the livers of all rats exposed to PCB126, alone and in combination with arsenic. The largest foci were seen in rats exposed to the combination of chemicals. Arsenic as a single agent did not produce grossly visible foci. These data suggest that arsenic acts during progression to increase preneoplastic foci in the liver after exposure to PCB126.
124: Hepatic Tumor Promoting Activity and Cell Cycle Kinetics following Oral Administration of Planar and Non-Planar Pcb Mixtures in Rats
This study evaluated the interactive hepatic tumor promoting effects and alterations in cell proliferation of two polychlorinated biphenyls (PCBs), the planar PCB 126 (3,3’,4,4’,5-pentachlorobiphenyl) and the non-planar PCB 153 (2,2’,4,4’,5,5′-hexachlorobiphenyl) which are common environmental pollutants. Promotion of altered hepatic foci and cell proliferation was evaluated utilizing a medium-term bioassay for promoters of hepatocarcinogenesis. The assay employs placental glutathione-S-transferase positive (GST-P+) liver cell foci as markers of preneoplasia in rats after treatment with the initiator diethylnitrosamine (DEN) followed by exposure to test chemicals and partial hepatectomy. Area and number of GST-P+ foci were analyzed by histomorphometry. Hepatocyte proliferation was evaluated utilizing implantation of an osmotic pump to deliver bromo-deoxy uridine (BrDU). Cell proliferation was quantified following immunohistochemical staining for BrDU and was reported as percent labeled hepatic nuclei per 1,000 hepatocytes. For PCB 126, the doses were 0.1, 1, and 10 μg/kg body weight. For PCB 153, the doses were 10, 100, 1,000, 5,000, and 10,000 μg/kg. Combined PCB 126 and 153 exposures were; 0.1 + 10, 1 + 100, 10+1,000, 10+5,000, and 10+10,000 μg/kg, respectively. Treatment with PCB 126 or 153 alone resulted in increased number and area of preneoplastic foci; however, treatment with the mixture of PCB 126 and 153 resulted in less than additive GST-P+ focus formation. Cell proliferation results with PCB 126 or PCB 153 alone or in combination did not differ from controls. Since the antagonism appears to exist at environmentally relevant levels of both PCBs these results could have major implications for risk assessment.
125: Chemopreventive Effect of Conjugated Linoleic Acid in the N-Methyl-N-Nitrosourea-Induced Mammary Tumor Model in Rats
Conjugated linoleic acid (CLA) is a term that refers to a collection of positional and geometric isomers of octadecadienoic acid with conjugated double bonds. Milk and other dairy products are good sources of CLA because of the bihydrogenation of dietary unsaturated fatty acids by rumen bacteria. We examined the pre-initiation effects of CLA in the N-methyl-N-nitrosourea (MNU)-induced mammary tumor model in rats. Female SD rats were randomly divided into five groups. The animals of groups 1, 2 and 3 were treated with MNU and the animals of groups 4 and 5 were injected with saline only at the age of 50 days. Animals of groups 1 and 2 were given diet containing 0.5% or 1% CLA from week 1 before MNU treatment. The animals of group 4 were given basal diet containing 1% CLA without MNU treatment. All animals were killed at week 25. The incidences of mammary tumors in the groups 1, 2 and 3 were 65% (13/20), 45% (9/20) and 68% (19/28), respectively. Histopathological examination of tumors demonstrated most of them to be adenocarcinomas. In the MNU alone group (group 3), benign tumors constituted 10.8% and malignant tumors 89.2%. In the MNU+CLA 0.5% and MNU+CLA 1% groups, benign tumors were 18.8 and 27.8% and malignant tumors 81.2 and 72,2% respectively. Conclusion: CLA was effective against MNU-induced mammary carcinogenesis in rats. CLA is a unique anticarcinogen because it is a naturally occurring substance primarily found in food products derived from animal sources.
126: Lack of Effect of 94-Ghz Radio Frequency Radiation Exposure in An Animal Model of Skin Carcinogenesis
Although there is no evidence that electromagnetic energy in the radio frequency radiation (RFR) band is mutagenic, there have been suggestions that exposure might serve as either a promoter or co-promoter in some animal models of carcinogenesis. Recent developments in electromagnetic technology have resulted in the manufacture of RFR sources capable of generating frequencies in the millimeter wave (MMW) length range (30–300 GHz). Because absorption of MMW energy occurs in the skin, it is to be expected that long-term detrimental health effects, if any, would most likely be manifest in the skin. In this study, we investigated whether a single (1.0 W/cm2 for 10 sec) or repeated (two exposures per week for 12 weeks, 333 mW/cm2 for 10 sec) exposure to 94-GHz RFR serves as a promoter or co-promoter in the 7,12-dimethyl-benz[a]anthracene (DMBA)-induced SENCAR mouse model of skin carcinogenesis. Neither pattern of MMW exposure significantly affected papilloma development, as evidenced by a lack of effect on tumor incidence and multiplicity. There was also no evidence that MMW exposure served as a co-promoter in DMBA-induced animals repeatedly treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). We therefore conclude that exposure to 94-GHz RFR under these conditions does not promote or co-promote papilloma development in this animal model of skin carcinogenesis.
127: Purification of a 7000 Dalton Metallothionein-Like Protein from Juvenile Desert Tortoises (Go-Pherus Agassizii)
Metallothionein (MT), a metal-binding protein with a molecular weight of approximately 7 kD, is inducible by a number of metals including cadmium. Fourteen juvenile desert tortoises were injected intracoelomically daily with 1 mg/kg body weight cadmium chloride for 5 days to induce synthesis of MT. At necropsy, the mean dry weight cadmium (Cd) concentration in livers from treated tortoises was 45 ppm. In control tortoise livers, the mean Cd concentration was 0.26 ppm. Microscopic changes in kidney and liver included mild renal tubular dilatation and proteinosis, and mild dilatation of bile canaliculi. Low molecular weight Cd-binding proteins were isolated from pooled hepatic and renal cytosol by molecular weight gel-filtration column chromatography, and one cadmium peak was identified by atomic absorption spectrophotometry (AAS). Proteins within Cd peak fractions were purified further by anion exchange column chromatography, and two distinct Cd peaks were detected by AAS. PAGE-SDS electrophoresis, amino acid sequencing and amino acid composition analysis of these Cd peak fractions revealed a 6.5 kD ubiquitin protein in peak one, a 7 kD MT-like protein in peak two and a 14 kD Cd-induced protein in peaks one and two. Ubiquitin is a highly conserved protein involved in nonlysosomal degradation of damaged intracellular proteins. Reversed-phase high performance liquid chromatography (RP-HPLC) of anion exchange peak two revealed an elution peak similar to that of rabbit MT standard. Results suggest the presence of only one isoform of MT in liver and kidney of juvenile desert tortoises. Antibodies to the purified 7 kD MT-like protein will be used to develop ELISA and immunohistochemical tests for MT in tissues and body fluids of the desert tortoise.
128: Lesions Induced by Cyclophosphamide Treatment in Broiler Chickens
Cyclophosphamide (CY) is an immunosuppressant and inhibits cell division by alkylating nucleic acids. This study was performed to determine the toxic effects of CY in broiler chickens. One-day-old White Plymouth Rock broiler chickens were treated with either 4 mg/day CY or phosphate buffered saline intraperitoneally for 4 consecutive days. Mortality of the CY-treated chickens was 65.4% (34/52), while that of the PBS-treated chickens was 10% (5/50). CY-treated chickens were significantly smaller, had delayed feathering, and had a lower relative bursal weight. Histologically, the lymphocytes were markedly depleted in the spleen and bursa, and the bone marrow was severely hypocellular. There was multifocal hepatocellular necrosis and vacuolation. Intestinal crypts were occasionally dilated and contained necrotic cell detritus in their lumens.
129: Fibrinoid Vascular Necrosis Associated with Sir-Olimus Treatment in Rhesus Monkeys
The rhesus monkey {Macaca mulatto) was utilized as a renal allograft transplant model to test a new steroid-sparing immunosuppressive regimen. In humans, allogeneic islet transplantation has been treated with a combination of sirolimus, tacrolimus, and daclizumab to provide adequate immunosuppression and allograft survival. This study was designed to determine if this regimen adequately prevents renal allograft rejection. Renal allotransplantation was performed in rhesus macaques with immunosuppressive treatment (n = 5) or without treatment (control n = 6). Sirolimus and tacrolimus were administered (with targeted trough levels) via gastric gavage and/or orally; daclizumab was given intravenously. All controls developed acute cellular rejection (mean survival = 7 days) characterized by lymphohistiocytic interstitial nephritis with tubulitis. Treated monkeys had prolonged allograft survival (mean survival = 36 days). Most of these treated monkeys (4/5) had prominent fibrinoid vascular necrosis in the small intestinal submucosa. Two treated monkeys had to be euthanized prior to renal rejection due to gastrointestinal (GI) toxicity and wasting. Grossly, both had a severe hemorrhagic enteropathy of the small intestine; histologically, there was marked submucosal edema, hemorrhage, and prominent fibrinoid vascular necrosis of the small arteries and arterioles in the submucosa. None of the control animals had these lesions. A review of GI tract histopathology from multiple other immunosuppressive protocols suggested that the GI lesions were an adverse effect of sirolimus. A regimen utilizing sirolimus, tacrolimus, and daclizumab prolongs the renal allograft survival in this rhesus model. However, this combination therapy is significantly limited by profound GI toxicity in this model.
130: Non-Steroidal Anti-Inflammatory Drugs Do Not Affect Erythropoietin Synthesis
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of pain and arthritis. Depletion of red blood cell (RBC) mass is frequently associated with NSAID use in humans and animals, for which a potential mechanism is not well understood. The kidney plays an important role in erythro-poeisis, as it is the major source of erythropoietin (EPO), but it is also a target organ of NSAID toxicity, associated with the marked inhibition of renal prostaglandin synthesis. Therefore, it is possible that the observed hematologic changes in response to NSAID use may be related to the NSAID effects on renal EPO production. We evaluated the hematologic effects and EPO levels in dogs (6/group) given a NSAID, loxoprofen sodium, for 2 weeks. Loxoprofen was administered orally at dosages of 2, 6 or 24 mg/kg/day that produced systemic exposures at 1-, 2-, and 8-fold, respectively, above the efficacious exposures. At dosages ≥6 mg/kg/day, red blood cell number, hematocrit and hemoglobin were decreased while an increase in white blood cells (26%) was observed at 24 mg/kg/day. These changes were most prominent in male dogs. No changes in EPO levels were observed in any animal. Upon pathological examination, we observed typical NSAID-related lesions including severe gastrointestinal erosions and ulcerations at dosages ≥6 mg/kg/day as well as renal papillary necrosis at 24 mg/kg/day. Collectively, these data demonstrate that NSAIDs do not affect EPO levels at dosages associated with both erythroid changes and renal toxicity, and suggest that the potential mechanism of erythroid changes may be related to blood loss from the injured gastrointestinal mucosa.
131: Immunolocalization of Pde4 Isoenzymes in Rat Mesenteric Arteries Using Confocal Microscopy
Phosphodiesterase type-4 (PDE4) inhibitors are among several drug classes that induce segmental medial necrosis of mesenteric arteries when administered to rats. The nature and distribution of the lesion suggest a consequence of extreme localized disturbance of smooth muscle tone. We hypothesized that regional distribution of PDE4 isozymes predisposes the rat mesenteric artery to damage; therefore, we explored the distribution of PDE4 isozymes within and along the rat mesenteric arterial wall. Mesenteric arteries were collected from male Sprague-Dawley rats. Frozen transverse sections were prepared and fixed in ethanohacetone, blocked with bovine serum albumin, incubated with primary antibody (rabbit anti-rat PDE4A, B, or C, or mouse anti-rat PDE4D), followed by secondary antibodies conjugated to the fluorochrome Cy5, and counterstained with DAPI (fluorescent nuclear stain). Whole mounts of mesenteric arteries, briefly fixed with formaldehyde, were stained as above and enrobed in agar. All tissues were examined with a Zeiss LSM-510 confocal laser scanning microscope. In sections, all PDE4 isozymes were localized, to varying degrees, throughout the medial smooth muscle layer and within the endothelium. In whole mounts, all 4 isozymes were localized diffusely throughout the medial smooth muscle; there was no evidence of segmental distribution along the vascular wall. Results indicate that the principal PDE4 isozymes (A, B, C and D) are expressed in medial smooth muscle and endothelium of rat mesenteric arteries and therefore could be involved in mediation of altered tone, either dependent upon or independent of endothelial activity. Diffuse expression of PDE4 isozymes along the vascular wall suggests that the segmental lesion induced by PDE4 inhibitors is not a consequence of local differences in PDE4 enzyme distribution.
132: Evaluation of Biochemical Markers of Myocardial Necrosis in Rats. Diagnostic Value of Cardiac Troponin I
The limited information available on biochemical markers of myocardial injury in laboratory animals prompted us to investigate, in rats with isoproterenol-induced myocardial injury, the relevance of markers used in human medicine. Six to seven week-old Sprague Dawley male rats were divided into 7 groups of 5 animals each. Six groups were treated once with isoproterenol (4 mg/kg IV) and one control group received 0.9% NaCl (IV). Blood samples were obtained in treated animals at 6, 12, 18, 24, 48, and 96h after injection and at 6 and 96h in controls. Five treated animals were sacrificed at each blood sampling time point and five controls at 96h. Hearts were sampled for histological examination. Serum cardiac troponin I (cTnI), creatine kinase (CK) and its isoenzymes, and CK-MB mass were evaluated. Levels of cTnI, although variable, were most elevated at 6h, had decreased by 12h and were nearly undetectable by 48h. cTnI was not detected in controls. Changes in CK and its isoenzymes were attributed to individual variations and exogenous factors rather than treatment with isoproterenol. CK-MB mass values were consistently under the test detection limits suggesting that the human-based immunoassay was not appropriate for rats. Isoproterenol-induced microscopic cardiac changes varied with the time of examination and were characterized by multifocal areas of myofiber degeneration/necrosis, interstitial edema, inflammatory cell infiltration and ensuing fibroplasia. Lesions involved primarily the left ventricular wall and septum near the apex, and extended into the papillary muscles. cTnI peak values noted at 6h correlated with acute myocardial changes. In conclusion, among the biochemical markers evaluated, only cTnI appeared to be a relevant marker of early myocardial damage in rats.
133: Are Mice Good Models for the Preclinical Safety Assessment of Adenoviral Vectors?
Background: Hepatic fibrogenesis has recently been identified in macaques treated with an adenoviral vector. Our aim was to determine if mice would develop similar changes due to E1/E2a/E3-deflcient vectors. We also wanted to know if a null vector would produce similar changes to those of a factor VIII-encoding vector. Design: Four groups of CD1 mice were treated with either 0 or 1.5x1013 particles/kg of AV3null2c or 3x1012 or 1.5x1013 particles/kg of Av3H8201. Ten animals per group were necropsied on SD (Study Day) 8, 29 and 57. Results: Smooth muscle-specific, actin-positive stellate cells (myofibroblasts) were present by SD 8 in all mice receiving either doses of the vectors. Increased laminin immunoreactivity and collagen staining were most prominent on SD 57. Fibrillar nuclear inclusions were recognized in hepatocytes and sinusoidal lining cells following treatment with either vector. Conclusion: Liver fibrosis in CD1 mice was similar to that observed in monkeys following a single intravenous administration of an adenoviral vector. Inclusion bodies noted in monkeys were present in mice as well. The null vector and the vector that encoded for Factor VIII caused essentially similar changes. Mice could serve as good animal models for the safety asessment of adenoviral vectors.
134: Genetics of Chlordane-Associated Liver Tumor Regression in B6C3F1 Mice
Gene microarray technology was applied in the study of gene expression and discovery of new genes involved in the regression of mouse liver tumors induced by chlordane, a non-genotoxic murine hepatocarcinogen. Upon cessation of chronic exposure to chlordane in B6C3F1 mice at least 30% of hepatocellular adenomas and carcinomas regress. Chlordane was administered in the feed at 55 ppm to 210 male mice and interim sacrifices were performed between 408 and 570 days of age for mice treated continuously with chlordane or after cessation of treatment (63 mice) at 491 days. Total RNA was extracted from ∼0.4 gm each of 12 archived snap-frozen hepatocellular carcinomas, 6 from the continuously treated and 6 from the stop group. mRNA [1 μg/μL] was used as the template for generation of fluorescent-labelled cDNA and subsequent hybridizations on the NIEHS “Mouse Chip” which contains ∼9,000 mouse gene sequences. Quadruplicate direct comparisons were made between mRNAs from the continuous and stop group tumors. Results have identified 66 gene sequences that were statistically determined to have a range of 1.5-5 fold difference in the level of expression. Among them were some proposed or known to be involved in: 1) carcinogenesis (cathepsin S, superoxide dismutase, and c-fos), 2) immediate early and stress responses (cyr61, hsp70, and pip92), 3) immune function (MHC antigens and polymeric Ig receptor), and 4) lipid metabolism (Cyp4a14, Cyp4a10, VLDL receptor, and Apoli-poprotein C-2). Differential expression for 3 out of 3 genes tested was confirmed by non-isotopic RNase protection assay (c-fos, Cyp4a14, and cyr 61). c-fos was highly and differentially expressed in liver tumors and not in normal mouse liver, suggesting it may be involved in chlordane hepatocarcinogenesis and regression.
135: Pathology of 129S4/Svjae and B6;129 Mice Used in the Generation of Genetically Engineered Mice
Use of genetically engineered mice (GEM) has dramatically expanded in medical research and shows potential for similar increase in toxicologic testing. Embryonic stem (ES) cells used for targeted gene disruption or “knockout” are commonly derived from 129 strain sublines. Microinjection of ES cells into C57BL/6 blastocysts result in chimeras, which are then backcrossed to C57BL/6 mice to produce B6;129 mice. In order to differentiate between induced and background lesions, the pathologist should be familiar with the common pathology of these strains. In 40 male and 48 female 129S4/SvJae mice maintained for up to 27 months, the most common neoplasms were Harderian gland adenomas, pulmonary alveolar adenomas, uterine and ovarian tumors, and hemangiomas/sarcomas in various organs. Only a few lymphomas and teratomas developed. No mammary tumors were present. Common non-neoplastic lesions included hyalinosis in respiratory tract and stomach, acidophilic macrophage pneumonia, lymphocytic infiltrates in several tissues, karyomegaly of epididymal epithelium, megaesophagus, hyperostosis (females), pancreatic islet cell hyperplasia, dental dysplasia, uterine angiectasis, and widespread arteritis. Cause of death included vascular lesions, megaesophagus, acidophilic macrophage pneumonia, lung tumors, and Harderian gland tumors. In 50 male and 49 female B6;129 mice maintained for up to 24 months, the most common neoplasms were lymphomas, pulmonary alveolar tumors, ovarian cystadenoma, and hepatocellular tumors. Common non-neoplastic lesions included otitis media, karyomegaly of the epididymal epithelium, membranoproliferative glomerulonephritis, pancreatic islet cell hyperplasia, skin ulceration, megaesophagus, and lymphocytic infiltrate in several organs. Cause of death included lymphoma, skin ulceration, megaesophagus, histiocytic sarcoma, duodenal adenoma, and hemangiosarcoma. These two studies should help investigators to characterize these lines and their GEM.
136: Effects of Cytokine Suppression on Differentiation and Maturation of Murine Bone Marrow Erythroid Progenitors
Recent studies indicate that p38α MAP kinase plays a direct role in regulating erythropoiesis. Cytokine suppressive drugs (CSD) that inhibit p38α mitogen activated protein (MAP) kinase induce bone marrow hypocellularity and decrease red cell mass in rodents. Therefore a short-term mouse bone marrow liquid culture system was initiated to further identify the target population and mechanisms leading to red cell depletion. Mouse bone marrow cells were collected and subsequently enriched for Sca-1+ stem cells using anti-Sca-1 magnetic beads. Isolated Sca-1+ cells (cultured in IMDM supplemented with recombinant mouse (rm) stem cell factor (SCF), rm interleukin-3 (IL-3), recombinant human (rh) IL-6, and rh erythropoietin) were cultured in the continuous presence or absence of 0.1–50 μM CSD for up to 14 days. Multiparameter flow cytometry and colony forming unit (CFU) assays were used to evaluate effects on erythroid differentiation of Sca-1 cells. Differentiation and maturation of hematopoietic progenitors (Sca-1highTer 119lowCD 44high) into committed erythroid (Sca-1lowTerhighCD44low) cells and DNA cell cycle analysis (7-amino actinomycin D) was used to identify modulation of cell cycle patterns in the target population. Flow cytometric analyses revealed a delay in progression of Sca-1highTer 119lowCD 44high to Sca-1 low-TerhighCD44low with significant effects on cell cycle progression. Burst-forming units-erythroid (BFU-E) and colony-forming units-granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM) colonies were counted. The data demonstrates significant CSD-induced decreases in both BFU-E and CFU-GEMM colonies initially. However, colony formation recovered to control levels at later time points. These observations, therefore, lead us to conclude that inhibition of p38 kinase results in a transient delay in erythroid maturation by targeting differentiation of Sca-1+ stem cells.
137: A New Perspective on Thymic Hematoma in Young Dogs
A review of the literature revealed that thymic hematoma of undetermined cause usually occurs in dogs <1 year old. Many cases, including our cases, occurred at 3 to 4 months of age. Most dogs die suddenly and thymic hematoma is usually fatal unless they receive supportive treatment. Gross examination reveals hemomediastinum. In addition to hemorrhage, reported histopathological descriptions of the thymus range from normal to complete lymphoid depletion with necrosis and fibrosis and were interpreted as thymic involution. The premise that spontaneous thymic hematoma in young dogs is simply a potential complication of thymic involution triggered by excitement, exertion, or trauma, even mild, has been proposed in several articles. Microscopic examination of our case material revealed significant lesions that are not consistent with normal physiologic involution of the thymus. Hemorrhage often resulted in loss of normal lobule architecture and formation of cystic spaces. Acute inflammation was present in and around the cystic lobule walls. Adipose tissue was not present in the lobules and was sparse in the interlobular septa. These findings suggest that thymic hemorrhage is the cause of the thymic injury rather than a sequel to normal physiologic involution. The thymus should be increasing in size rather than involuting in 3- and 4-month-old dogs. Since our laboratory developed the ability to test for anticoagulants, 3 of 5 cases of thymic hematoma were positive for anticoagulant toxicosis. Our findings indicate that hemorrhage associated with rodenticide toxicosis can be confined to the mediastinum and that testing for rodenticides is indicated in cases of thymic hemorrhage.
138: Locoweed (Oxytropis Sericea) Poisoning in Mule Deer (Odocoileius Hemionus)
Locoweeds (Astragalus spp. and Oxytropis spp.), which contain swain-sonine, poison both livestock and wildlife. Swainsonine inhibits cellular mannosidases resulting in lysosomal dysfunction and altered glycoprotein metabolism. Though poisoning is reported in elk and there are anecdotal reports of poisoning antelope, it is unknown if mule deer are susceptible. Recently in locoweed endemic areas in Wyoming and Colorado, deer and elk have developed an infectious spongiform disease, chronic wasting disease (CWD). Though spongiform diseases and locoweed poisoning produce distinct clinical and histologic lesions, no comparative studies documenting these differences are available. The purpose of this study is to induce and describe chronic locoweed poisoning in deer. Subsequent studies will compare locoweed-induced lesions with those of CWD. Two groups of four mule deer were fed alfalfa pellet or pellet containing 15% Oxytropis sericea. Within 12 weeks, locoweed treated deer, ingesting about 1.5 mg swainsonine/kg bw/day, developed lymphocyte vacuolation and increased serum enzyme activities. Though similar dose and duration causes severe clinical disease in other livestock, treated deer were nearly normal. Mild clinical changes included poor body condition, weight loss, and subtle intention tremors. After 16 weeks, two treated and two control deer were euthanized and necropsied. Poisoned deer had neuronal swelling and cytoplasmic vacuolation that was especially severe in cerebellar Purkinje cells. Epithelial cells of the kidney, thyroid, and exocrine pancreas were also swollen and vacuolated. Macrophages in the spleen, lymph nodes and liver were also vacuolated. Though neurons were vacuolated, few were degenerative or pyknotic as is seen in chronic locoism in other species. These findings indicate that deer are susceptible to locoweed poisoning, but they are affected less than other livestock or wildlife species.
139: Morphologic and Gene Expression Alterations Induced by Ppar Alpha Agonists in Liver and Muscle
Synthetic PPAR alpha agonists cause slight increases in serum ALT in multiple species, and in addition, cause liver cell proliferation and hypertrophy, liver tumors and skeletal myopathy at high doses in rodents. While it is known that PPAR alpha itself is necessary for most of these responses, the downstream effectors have yet to be elucidated. We hypothesized that modulators of key endpoints may behave in a dose-responsive manner relative to therapeutic effects and toxicity/carcinogenicity. Gene expression analyses were done in conjunction with morphologic and clinicopathologic evaluation at multiple doses of PPAR alpha agonists in rat. Significant increases in levels of hepatic ALT mRNA were not observed. Prototypical PPAR alpha-induced alterations in gene expression were observed, such as those involved in beta-oxidation, oxidative stress, cell proliferation and cell communication. The relative degree of expression generally reflected dose and severity of morphologic effects. Myodegeneration was observed in the soleus but not gastrocnemius muscle of animals exposed to high doses of PPAR alpha agonists and corresponded to enhanced expression of oxidative genes in the soleus compared to the gastrocnemius. These data may support the potential for threshold determination of PPAR alpha gene alterations relative to key morphologic endpoints.
140: Pathogenomics: Pathology Screening and Tissue Archiving for Enu Mutagenesis
Objectives: The goals of the Centre for Modeling Human Disease (CMHD) are to use genome-wide N-Ethyl-N-nitros urea (ENU) mutagenesis in mice to functionally annotate the mouse genome, to discover new models of human disease, and to create useful new models for testing disease therapies. The Canadian Mouse Mutant Repository (CMMR) works in partnership with the CMHD to recover and archive the expansive germ cell and tissue resources generated by the CMHD. Methods: The Pathology Core (PC) of the CMHD provides detailed gross- and histopathological, immunohistochemical, morphometric, and slide-based analyses as essential components of the CMHD's gene and model discovery program. We have developed a high throughput facility for morphology and tissue-slide based assays of all organ systems of mutant mice. Important considerations in high throughput screening of our ENU mutants include sample identification and tracking, expert interpretation of bona fide phenotype in mouse tissue, and reproducibility and consistency of specimen collection. The pathology and phenotype of several heritable mutants discovered in the program will be presented and discussed. The Pathology Core also collects and maintains the CMMR's physical and digital archive that consists of germ cells, frozen tissue, formalin-fixed and paraffin-embedded tissue, and glass-slide sections. To integrate, store, and make available the phenotype information from pathology and physical resources from the CMMR, the PC is developing a comprehensive networked database that will be made available as a searchable database on the world-wide-web (www). Conclusions: Comprehensive and targeted pathology screens, multiple tissue archiving, and pathogenomic data management and distribution are essential components of the Pathology Core's contribution to the CMHD/CMMR's functional genomics initiative.
141: The Effects of Inhaled Octamethylcyclote-Trasiloxane (D4) on Ovulation and Ovarian Morphology in a Study of the Preovulatory Lh Surge and other Gonadotropin and Ovarian Sex Hormones
In previous reproductive studies in the Sprague Dawley (SD) rat, high octamethylcyclotetrasiloxane (D4) exposures decreased fetal implantation sites and litter size. We report here the most recent of several studies used to illuminate the mode of action. SD rats with jugular cannulas were exposed to 0, 700, or 900 ppm D4 by whole body vapor inhalation for 3 days (6 hr/day) from diestrus day 1 through the day of proestrus. Blood for hormone levels was collected on the day of proestrus at 2, 4, 6, 8, and 10 PM, and at necropsy at 8 AM on the day of estrus. On the day of proestrus, following separation of plasma, red cells were resuspended in heparinized sterile saline and returned to the animals at subsequent blood collections. Plasma levels of LH, FSH, prolactin, estradiol, and estrone were determined. At necropsy, wet mounts of oviducts were examined and ova were counted. The ovaries were routinely fixed and processed and step-sectioned. Six sections from each ovary were examined. Sections through basophilic corpora lutea, mature follicles, and atretic follicles were counted. D4 exposure increased the percentage of rats with ablation or delay of the LH surge and failure to ovulate. Rats that failed to ovulate had numerous mature follicles and lacked basophilic CLs. There was no treatment effect on follicular atresia. Control and exposed rats that failed to ovulate had similar hormonal and ovarian morphology profiles; conversely, control and exposed rats that ovulated normally were also similar.
142: Cutaneous Vasculitis and Necrotizing Dermatitis after Implantation of a Penrose Drain Containing Zinc Diethyldithiocarbamate
We previously described lethality in mice 14 hours after implantation of a sterile commercial latex Penrose drain containing the vulcanization accelerator, zinc diethyldithiocarbamate (ZDEC). Local and systemic morphologic alterations after sublethal exposure to this drain have now been investigated. In an experiment terminated 12 hours after surgery, mice received a sham operation, a 200 mg subcutaneous control latex Penrose drain, or a 200 mg subcutaneous ZDEC drain. In an experiment terminated 36 hours after surgery, mice received a sham operation, a 150 mg subcutaneous control latex Penrose drain, a 100 mg subcutaneous ZDEC drain, or a 150 mg subcutaneous ZDEC drain. In all mice 12 hours after implantation, the ZDEC drain was associated with cutaneous vasculitis and vacuolation of basal and sebaceous gland epithelial cells. In all mice 36 hours after surgery, cutaneous necrosis was present above ZDEC drains. While mice with larger ZDEC drains were depressed, mice receiving 100 mg ZDEC drain implants had pruritus and self-trauma in this group potentially exacerbated cutaneous alterations. Tissue damage extended into epaxial muscles beneath ZDEC drains, with myositis in 4 of 5 mice 12 hours after implantation of 200 mg drains and myofiber degeneration or necrosis in all mice 36 hours after implantation of 150 mg drains. ZDEC drain implantation was also associated with apoptotic necrosis of the thymus and apoptotic necrosis of the adrenal “X-zone”. These findings suggest that local and systemic tissue damage can be produced by at least one brand of Penrose drain containing ZDEC. More investigation is needed to establish the potential for ZDEC contaminated latex implants to exacerbate local tissue injury and produce systemic toxicity.
143: Enterotyphlocolitis in Dogs Given the Anti-Muscarinic Pnu-171990
Drugs that block muscarinic receptors have potential utility in the treatment of several diseases including asthma and urinary incontinence. The antimuscarinic PNU-171990 was evaluated in a 2-week (30, 60 and 120 mg/kg/day) oral toxicity study in the dog. Dogs demonstrated dose-dependent sedation that was most severe at the approximate time of highest plasma drug levels. In the second week of testing, several high-dose (120 mg/kg/day) dogs had decreased food consumption that progressed to anorexia. The anorectic dogs developed profuse and bloody diarrhea and fecal cultures from two dogs were positive for Clostridium perfringens or bacilli consistent with C. perfringens. Histologically, high-dose dogs had necrotizing inflammmation that involved predominantly the ileum, cecum, and colon. In dogs with the most severe clinical signs, lesions were florid, diffuse, and involved the full thickness of the mucosa. The histologic characteristics of the intestinal lesions were not consistent with direct irritation and may have been secondary to alterations in intestinal motility and flora. Antimuscarinics decrease intestinal motility and PNU-171990 was demonstrated to increase charcoal transit time, a measure of intestinal motility, in the mouse. Findings in dogs that were likely secondary to the anorexia and necrotizing enterotyphlocolitis included ketonuria and hepatic lipid accumulation (altered metabolism secondary to anorexia), thymic atrophy (stress/cortisol), portal inflammation in the liver (ascending inflammation from intestine), and prolonged PT and APTT (decreased vitamin K absorption).
144: Renal Pars Recta Degeneration and Necrosis in Rats Given the Antimuscarinic Pnu-171990.
Drugs that block muscarinic receptors have potential utility in the treatment of several diseases including asthma and urinary incontinence. The antimuscarinic PNU-171990 was evaluated in a 4-week (75, 225, 450, and 675 mg/kg/day) oral toxicity study followed by a 4-week recovery period in the rat. Rats given high doses had several clinical signs that were likely secondary to the exaggerated pharmacology associated with high doses of an antimuscarinic. These included squinting (dilated pupils), distended abdomens (decreased intestinal motility), choking on food (decreased salivation), and decreased activity/unsteady gait. Histologically, rats sacrificed at 30 days from all dose groups had degeneration or necrosis of the tubular epithelium in the renal pars recta and, in males, an increased incidence or severity of proximal tubular hyaline droplets. Serum creatinine, blood urea nitrogen, and urinalyses were unremarkable. Development of the pars recta lesions required several days of dosing; the first change, macrovesicular vacuolation, was observed at Day 7 in high-dose males and at Day 12 in high-dose females. Epithelial necrosis was not observed until Day 19 of dosing and the pars recta lesions were completely reversible after a 30 day recovery period. Pars recta lesions were observed at a dose that produced plasma drug levels approximating the target drug levels for humans, and the development of the drug was terminated. The possible pathogenesis of the pars recta lesions and the evaluation of a potential biomarker (alpha-GST) for the lesions will be discussed.