Abstract
Combined 53rd and 37th Annual Meetings of the American College of Veterinary Pathologists and the American Society for Veterinary Clinical Pathology
New Orleans, LA
December 8-11, 2002
Specialty Group Coordinator Anne M. Ryan
Abstracts 1-21 are reprinted with permission from Veterinary Clinical Pathology, Vol 31(3) 2002.
1: Comparison of Systemic and Liver-Targeted Delivery of Adeno-Associated Virus Vectors (Aav) for Hepatic Gene Therapy
A canine model for glycogen storage disease Ia (GSD Ia), has been described by our group that is equivalent to severe GSD Ia in humans. We are investigating the use of hepatic gene therapy as a novel treatment modality for this disease. An important limitation for hepatic gene therapy is the inadequate delivery of the therapeutic gene. The objective of this study was to determine in a murine model the most efficient route and dose of gene delivery. We administered an AAV vector encoding human placental alkaline phosphatase to C57/B6 mice via three routes (tail vein, intraparenchymal and hepatic portal vein injection), using high and low numbers of vector particles. To assess gene transfer into mouse livers, genomic DNA was extracted and quantitative PCR analysis for vector PNA was performed. All mice (100%) that received high dose vector by portal vein injection demonstrated the presence of the provirus at > 10 copies/cell. Of the mice receiving high dose vector by tail vein, 40% demonstrated between 1-10 vector copies/cell, compared to 50% of those receiving intraparenchymal injections. These preliminary findings confirm that direct administration of vector to the liver via the hepatic portal vein produce high levels of gene transfer, and that intraparenchymal administration of vector achieves levels similar to tail vein injection. Ongoing experiments will determine whether differences in anatomical distribution of the vector exist. The results of this investigation will aid in the development of our unique canine model of hepatic gene therapy for GSP Ia.
2: Cerebrospinal Fluip Finpings in Florida Horses with Confirmed West Nile Virus Infection: 30 Cases (2001)
The purpose of this study was to summarize CSF analyses from 30 horses with acute neurologic signs due to confirmed West Nile Virus (WNV) infection. We hypothesized that CSF results in these horses would be influenced by the site of CSF collection (AO vs. LS). CSF analyses from horses that presented between July and November 2001 with acute WNV infection that was confirmed by immunoglobulin M antibody capture ELISA were reviewed. Of 30 CSF results, 8 (27%) were normal; 22 (73%) were abnormal. Among the abnormal CSF results, a mononuclear pleocytosis was found in 16 (73%) and an elevated protein concentration with normal nucleated cell count was found in 6 (27%). A lymphocytic predominance was found in 11 of 16 (69%) samples with a mononuclear pleocytosis. Of 30 CSF results, 12 (40%) were collected from the AO site; 18 (60%) were from the LS site. Six of 12 (50%) AO samples and 16 of 18 (89%) LS samples were abnormal. A 1-sided Fisher exact test revealed that this difference was significant (P = 0.027). These results suggest that in horses with acute onset of neurologic signs due to WNV encephalomyelitis, the CSF findings will likely be abnormal, that a mononuclear pleocytosis with lymphocytic predominance is most commonly observed, and that collection from the LS site is more sensitive than collection from the AO site.
3: Evaluation of Serum Ferritin as a Marker for Malignant Histiocytic Tumors
Differentiating malignant histiocytic tumors (MHTs) from inflammation and other tumors by cytology or biopsy can be problematic. A serum marker that identifies MHTs would enhance diagnosis of these neoplasms and aid in staging disease, assessing treatment response, and detecting recurrence. Ferritin is a soluble protein-iron complex synthesized by mononuclear phagocytic cells and hepatocytes. It represents the mobile storage form of iron and is an acute phase protein in people. Hyperferritinemia occurs with iron overload, hemolytic anemia, inflammation, hepatic disease, and neoplastic diseases. Using a canine-specific double monoclonal antibody capture ELISA technique, we measured serum ferritin in dogs with malignant histiocytosis, histiocytic sarcomas, benign histiocytomas, hemolymphatic neoplasia, acute and chronic inflammation, hepatic disease, and immune-mediated hemolytic anemia (IMHA) and in dogs that had received transfusions. Concentrations of serum iron, TIBC, and ceruloplasmin, an acute phase protein, also were measured. All dogs with MHTs had hyperferritinemia ranging from mild to marked (geometric mean 3,361 ng/ml, range 819-86,980 ng/ml, reference interval 80-800 ng/ml). Ferritin concentrations also were elevated in all dogs with IMHA and frequently were elevated in dogs with hepatic disease and hemolymphatic neoplasia. Ferritin and iron were correlated only in dogs with hemolymphatic neoplasms and inflammation (p < 0.05). Ferritin was independent of TIBC and ceruloplasmin in all dogs. Based on calculated likelihood ratios, moderate hyperferritinemia does not differentiate among dogs with MHTs, IMHA, hemolymphatic neoplasms, and hepatic disease. MHTs or IMHA are likely in dogs with marked hyperferritinemia. We conclude that serum ferritin is a useful marker for MHTs; however, for diagnostic purposes, other diseases must be excluded in dogs with hyperferritinemia.
4: Evaluation of a Rapid Agglutination Method for Detection of Equine Red-Cell Surface Antigens (Ca and Aa) as Part of Pre-Transfusion Testing
Blood typing prior to transfusion minimizes the risks of transfusion reactions and prevents immunization of the recipient against incompatible RBC antigens. The major RBC antigens that warrant identification prior to RBC transfusions in horses are Ca and Aa. Standard blood typing protocols are time consuming (2.5 to 3 hours) and impractical in emergency settings. The objective of this study was to determine whether equine RBC could be typed for Ca and Aa antigens within 15 minutes using sera from horses with RBC antibodies. Serum was obtained from a horse with anti-Ca antibody and from another horse with anti-Aa antibody. The presence of agglutinating antibody was confirmed with antibody screening. Venous blood samples, collected in citrate-phosphate-dextrose, were obtained from 24 horses of various breeds. Samples were blood-typed in the VMTH Hematology Laboratory using standard methodology. Washed RBCs from each of the 24 horses were incubated individually with anti-Ca and anti-Aa sera at dilutions of 1:4, 1:8 and 1:16 for 15 and 30 minutes at room temperature or 37°C. Seventeen Aa-positive horses and 7 Aa-negative horses were examined. All 17 Aa-positive horses had a positive agglutination reaction at all dilutions, times and temperatures, while all Aa-negative horses were negative. Eighteen Ca-positive and 6 Ca-negative horses were also examined. Each Ca-positive horse was positive for agglutination at all time points and temperature up to the 1:16 dilution. All Ca-negative horses were negative at all times, temperatures and dilutions. These results support the hypothesis that equine RBC can be rapidly typed in 15 minutes, at room temperature, for the presence of Ca and Aa antigens using equine derived sera. This technique may be beneficial for pre-transfusion testing of equine patients in an emergency setting.
5: Evaluation of a Flow Cytometric Method to Determine Lymphocyte Proliferation in the Dog
Measurement of lymphocyte proliferation has been an important part of assessing the immune response in experimental animal studies. The objective of this study was to evaluate a flow cytometric technique for measuring lymphocyte proliferation and compare results to those obtained through the use of 3H-thymidine incorporation. This flow cytometric technique utilizes a fluorochrome, car-boxyfluorescein diacetate succinimidyl ester (CFSE), which is equally distributed between daughter cells as they divide. Stimulated mononuclear cells from 15 dogs were stained with CFSE and incubated for 72 hours. Phycoerythrin-labeled IL-2 was then added to identify the activation marker, IL-2R. Flow cytometry results were reported as percent proliferation, number of divisional peaks, and percent of IL-2R staining. Separate stimulated mononuclear cell al-iquots were pulsed for 4 hours with 3H-thymidine with results expressed as counts per minute (CPM) and stimulation index (SI). The percent proliferation, number of divisional peaks, percent IL-2R staining, CPM, and SI were explored to evaluate any relationship between the methods of recording cell proliferation. Spearman's rank correlation indicated that only IL-2R staining showed a moderate relationship to percentage proliferation, indicating that most proliferating cells were also expressing the IL-2 receptor. The lack of correlation between data obtained by the CFSE method and the 3H-thymidine method was hypothesized to be due to shortcomings of the 3H-thymidine method. Analysis of individual dog results indicates that CFSE showed a comprehensive record of the proliferation activity of the cells whereas 3H-thymidine incorporation gave only limited information. The use of flow cytometry with CFSE appears to be a better indicator of lymphocyte proliferation and can be used with other fluorescent labeled markers.
6: Spirochetemia Due to Borrelia Turicatae Infection in Dogs from Texas
Over an approximately 2.5 year period, spirochetes were found on blood smears from two dogs from North Texas and one dog from West Texas. Two of the affected dogs were Siberian huskies and clinical and hematologic findings were variable. Spirochetes were isolated from all three dogs by first inoculating mice with infected dog blood and then culturing organisms from mouse blood inoculated into modified Kelly's medium. The spirochetes were identified as Borrelia turicatae, an agent of tick-borne (endemic) relapsing fever in humans, by reactivity with monoclonal antibodies and DNA analysis. Two dogs recovered with treatment. One died, leaving uncertainty as to whether the conditions leading to death were directly the result of spirochete infection. Borrelia turicatae is distinct from Borrelia burgdorferi, the agent of Lyme disease in humans, dogs, and other species. Serologic cross reactivity may confuse the diagnosis of these two organisms when using whole-cells as the test antigen. The incidence of B. turicatae infection in dogs and humans is unknown. Spirochetemia may be transient, and sample handling may affect ability to recognize it when present.
7: Specimen Collection Comparison for Clinical Pathology Analysis
Specimen collection methods can affect the results of various clinical pathology assays. This study compared blood anticoagulants and collection sites, and urine collection techniques, to determine the best way to collect samples for clinical pathology assays using Han-Wistar rats. Our study compared results from hematology and clinical chemistry analysis of blood collected from the orbital sinus and from the vena cava, and urine collected over a 6-hour (room temperature) and 18 hour (refrigerated) period. Blood for hematology was collected into EDTA tubes; blood for chemistry analysis was collected into tubes containing lithium heparin or a gel clot activator. In the male rats, hematocrits were greater from vena cava samples as compared to orbital sinus samples. White blood cell counts from females were lower from samples taken via orbital sinus than from the vena cava. All samples collected into tubes containing lithium heparin exhibited some degree of hemolysis. In both males and females, concentrations of phosphorus, glucose and potassium were greater in samples taken from the vena cava than from the orbital sinus. AST activity was greater in heparinized samples taken from the vena cava than in samples taken from orbital sinus. In both males and females, specific gravity and pH were greater in 6 hour, room temperature urines. The preferred collection techniques as determined by this study are 1) orbital sinus and serum for clinical chemistry, 2) either collection site for hematology and 3) 18 hour refrigerated urine.
8: Correlation of Canine Troponin I Levels with Size of Myocardial Infarct
Cardiac Troponin I (cTnI) is an important biomarker used in diagnosing acute myocardial infarction (AMI) in humans. Both cTnI and creatinine kinase isoenzyme MB (CK-MB) are elevated after AMI. Evidence-based literature indicates cTnI levels are more specific for myocardial necrosis, and increases in cTnI have been adopted by recent guidelines redefining AMI criteria. Less is known about the application of cTnI in animals. This study was conducted to validate the use of the human cTnI assay in the dog by assessing correlation of cTnI levels with size of myocardial infarcts in dogs. Seven dogs enrolled in 2 different studies were included. Infarcts were generated by manipulation of the left anterior descending (LAD) coronary artery: via LAD ligation (n = 4) or via intraarterial occlusion using coronary stents (n = 3). Serum samples were collected at: baseline (pre-infarct), 6, 20, 24 and 48 hours post-infarct. cTnI and CK-MB concentrations were measured using a Bayer Immuno 1 Chemistry analyzer. At 4 weeks post-infarct, gross and his-topathological evaluations were performed. Cardiac scar tissue or infarct volume was represented as percent of total left ventricular volume. There was a positive correlation between the infarct size (volume) and cTnI levels. Each animal had elevated cTnI and CK-MB levels following infarct (baseline less than 0.1 ng/ml). Peak levels (ranging from 0.5 to greater than 200 ng/ml) were observed between 6 and 24 hours post-infarct. cTnI and CK-MB levels in two dogs were markedly lower compared with other dogs. In both cases, gross anatomical observations revealed smaller infarct sizes than other subjects. Findings indicate that cTnI levels in the dog are predictive for degree of myocardial damage.
9: Evaluation of Normal Sprague-Dawley Rat (Titrated Plasma Using the Acl™ Advance Coagulation Analyzer
The objective of this work was to verify the use of the random access ACL™ Advance automated coagulation analyzer (Beckman Coulter, Inc.) on Sprague-Dawley rat citrated plasma for PT, APTT, and fibrinogen. Precision, accuracy, and preliminary reference intervals were determined for each test; linearity of dilution was also checked for fibrinogen. The results from the ACL® Advance instrument were also compared with the MLA Electra 900C. Accuracy and precision were determined for each test using five manufacturers’ controls (Assess® normal, low, high, calibration plasma, and IL Test® low fibrinogen) with the manufacturer's reagents; each control was tested three times a day for 10 days. The mean result obtained for each control was within the expected range; the %CV obtained for PT and APTT was less than 3.2% and fibrinogen less than 4.9%. Linearity of dilution for fibrinogen shows a correlation coefficient of 0.9956 for a concentration range of 123.4-222.0 mg/dL. Twenty citrated plasma samples, collected from 56-57 day old male and female Sprague-Dawley rats, were compared on the MLA Electra 900C and the ACL® Advance instrument. Preliminary reference intervals were calculated for each test as follows: PT (10.4-11.5 s.), APTT (13.65-21.53 s.), fibrinogen (222.8-263.8 mg/dL). In conclusion, the accuracy, precision, preliminary reference intervals obtained on the ACL® Advance shows this instrument is acceptable for testing rat plasma samples.
10: A Method for Detergent Extraction and Enzymatic Determination of Fecal Triglyceride in Sprague Dawley Rats
Conventional techniques for the extraction of fecal lipids for quantification require the use of organic solvents that have liabilities such as flammability and inaccuracies due to extraction of both dietary and endogenous lipids. A modified extraction method that uses the non-ionic detergents Triton X-100 and Brij 30 is described and validated which facilitates the precise and accurate determination of fecal triglycerides based on the enzymatic determination of the triglyceride concentration. An inhibitor of lipases, orlistat, was administered by oral gavage twice daily for 2 days to male Sprague-Dawley rats and the fecal pellets were collected over a 24-hour period. Aliquots from pooled fecal samples were initially subjected to varying ratios of detergent diluent until the optimum extraction ratio was identified for each pooled sample. Untreated rats served as controls. Extraction efficiency was determined following complete fecal triglyceride extraction (no measurable triglyceride) by spiking aliquots of feces with a triglyceride standard. Following complete mixing and extraction, the triglyceride recovery ranged between 97% and 114%. Precision, as assessed by replicate measurements from the same aliquot of feces, was determined to have a coefficient of variation of 8.7%. Inter-assay variation was approximately 8.8%. The results indicate that the use of non-ionic detergents for the extraction of fecal lipids and subsequent measurement of the triglyceride content is an accurate and precise method for assessing fecal triglycerides.
11: Changes in Plasma Protein Concentrations in Healthy Horses and in Horses with An Acute Abdomen Submitted to Laparotomy
The purpose of this study was to determine whether plasma protein concentrations were altered in horses with an acute abdomen before and after laparotomy. Ten horses with an acute abdomen— colon impaction (5), colon entrapment (5)—and ten healthy horses (control) were examined. Blood samples were collected before and daily during 10 days post-operative. Plasma protein concentrations were determined by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were identified by using reference markers and by comparison with electrophoretic mobility of purified proteins. To determine whether plasma protein concentrations were significantly different between groups throughout time, data were analysed by ANOVA. Plasma protein concentration was higher in horses with an acute abdomen than healthy horses, and the largest value occurred 24 hours after laparotomy. Twenty-four plasma proteins with molecular weights ranging from 24,000 to 340,000 daltons were identified in all 10 healthy horses. Horses with an acute abdomen showed three more proteins with molecular weights of 58,000, 63,000, and 77,000. Plasma concentration of proteins with molecular weights 43,000 (acid glycoprotein), 47,000 (haptoglobin), 61,000 (antitrypsin), 105,000 (C-reactive protein), 128,000 (ceruloplasmin), and 340,000 (fibrinogen) increased in both healthy and sick animals, but the values were significantly higher in horses with an acute abdomen. The protein with the largest percentage increase in concentration was ceruloplasmin at 24-48 hours after surgery (207.66% and 494.54% in healthy and sick horses, respectively). Plasma protein concentrations decreased 72 hours after laparotomy. In conclusion, changes in plasma protein detectable by SDS-PAGE may be useful in monitoring the progression of the postoperative period in healthy horses and in horses with an acute abdomen.
12: Hematologic and Biochemical Changes in Burmese Python (Python Molurus Bivittatus) Blood Caused by Commonly Used Anticoagulants
Current recommendations on which anticoagulant should be used in sampling reptilian blood are based on studies in avian or mammalian species. This study evaluated the preservation of Burmese python blood cells stored in lithium heparin, sodium citrate and potassium EDTA with and without the addition of albumin. Intracardiac blood samples were taken from ten mature Burmese pythons and processed at 0, 3, 12 and 24 hours post-collection to simulate the transit time to a laboratory. Time zero samples were processed within one hour of collection and all samples were refrigerated between processing times. Total protein concentrations and PCV were significantly different between those samples collected in lithium heparin and sodium citrate. There was no significant difference between those samples collected in heparin and EDTA in any value measured. The manual white blood cell counts and the differential counts for heterophils and lymphocytes were not significantly different in any anticoagulant; likely related to the inherent variability of the hemocytometer method. There was increased lysis noted in the sodium citrate samples as evidenced by a decreased PCV, increased protein concentration, and increased erythrocyte fragments on slides examined. There was no significant difference in the number of lysed cells with and without the albumin additive. Glucose, potassium, and chloride concentrations changed significantly over the 24-hour sampling period in the lithium heparin samples. Python blood cells may be adequately preserved in lithium heparin and potassium EDTA, but not sodium citrate, over a 24-hour period if refrigerated. The addition of albumin does not improve preservation of blood cells. Blood collected for biochemical analysis should be separated immediately to prevent artifactual changes in glucose and potassium concentration.
13: Hematologic and Biochemical Changes in Macaw (Anodorhynchus Spp.) Blood Caused by Commonly Used Anticoagulants
There is controversy over which anticoagulants to use for avian blood collection. A review of the few published studies in this area does not provide a clear recommendation for the most efficacious anticoagulant. Heparin is currently the predominant anticoagulant used in practice. Albumin has been added to heparinized nucleated blood samples as a cell stabilizer. EDTA and sodium citrate have also been advocated for use and are better suited for instrument analysis. We sought to determine the most effective anticoagulant for use in avian blood collection, using the three most common anticoagulants: lithium heparin, potassium EDTA, and sodium citrate, with and without the addition of albumin. Cell lysis and changes in chemistry values were evaluated in 10 macaws over a 24 hour time period at 0, 3, 12, and 24 hours. Manual WBC counts (using Natt and Herrick stain), estimated WBC counts, differential cell counts, packed cell volumes, total solids and plasma chemistries were evaluated. More than half of the samples had greater than 50% lysis at 24 hours in all anticoagulants. There was no difference in the ability of the different anticoagulants to preserve cells at 3 and 24 hours. There was a significant decrease in PCV in those samples collected in sodium citrate versus those samples collected in heparin (10% at 3 hours). Albumin did not improve cell preservation in any value examined. There was a high coefficient of variation in all the manual WBC counts making the determination of differences between anticoagulants impossible. Avian blood samples should be evaluated within 12 hours of collection for best results. Sodium citrate is not recommended for collection of avian blood due to significant changes in PCV and increased cell lysis.
14: Use of a Polymerase Chain Reaction Assay to Study the Carrier State in Infection with Came-Lid Mycoplasma Haemolama, Formerly Eperythro-Zoon Spp. Infecting Camelids
An Eperythrozoon species affecting camelids has recently been reclassified as Mycoplasma haemolama. This hemoparasite is associated with mild to marked anemia in stressed, immune-suppressed, and debilitated animals, but has also been identified in low numbers in apparently healthy camelids. The mode of transmission is not known. A polymerase chain reaction (PCR)-based assay to amplify the 16S ribosomal RNA gene of M. haemolama was developed using primers based on GenBank sequence accession AF306346 and blood from a naturally-infected alpaca. Specificity was shown by failure of these primers to amplify related Mycoplasma species. Parasitemia was detected in llamas and alpacas from a variety of geographic locations using this assay. The detection limit is estimated to be 1 organism in 3.8 × 109 to 7.7 × 109 RBCs. A splenectomized alpaca infected with M. haemolama provided blood for experimental infection of 8 other alpacas. All developed parasitemia detectable by PCR at least two days before organisms were seen on blood smears. Four of the infected alpacas were treated with a commonly-used tetracycline regime. The alpacas have been monitored for 16 weeks postinfection. Three of four tetracycline-treated alpacas have been intermittently PCR positive with one of these occasionally having detectable parasitemia on blood smear. Two of four untreated alpacas have been intermittently positive by PCR, with no detectable organism on blood smears. These results suggest that parasitemia is not cleared by the standard tetracycline regime used in camelids though organisms are no longer seen on blood smears.
15: Characterization of the Major Antigenic Protein 2 of Ehrlichia Canis and Ehrlichia Chaffeenses
Canine monocytic ehrlichiosis (CME) may be caused by either Ehrlichia canis or Ehrlichia chaffeensis. A serological assay that can distinguish between these two infections is currently not available and would be a valuable tool for epidemiological studies. Previously, we used the Major Antigenic Protein 2 (MAP2) of E. chaffeensis and E. canis to develop two indirect ELISAs that would serologically diagnosis infections with either agent. In this study, we examined the conservation of the MAP2 among various geographic isolates of E. canis and E. chaffeensis, and determined if a single copy gene encodes the protein. We also evaluated cross-reactivity by determining if there are conserved epitopes between E. canis and E. chaffeensis rMAP2. Southern blot analysis using digested genomic DNA from each species of organism was hybridized with species-specific DIG-labeled map2 probes. Results suggested that map2 is a single copy gene, indicating a lesser probability for antigenic variation. The map2 gene from different isolates of E. chaffeensis and E. canis was PCR amplified, cloned, sequenced, and compared. The map2 gene was found to be highly conserved among the various geographic isolates of each species. Cross-reactivity was evaluated using ELISA and Western blots by reacting the rMAP2 of E. chaffeensis and E. canis with sera from dogs experimentally infected with E. canis. The rMAP2 ELISA was not able to serologically distinguish between E. canis and E. chaffeensis infections. The amino acid sequence of the MAP2 of E. canis and E. chaffeensis had a 79% identity. Therefore, differences in the translated amino acid sequence were observed between the two species. If these differences result in distinct epitopes in each species, these epitopes could be targeted to develop a serologic assay that could rapidly distinguish between the two infections.
16: Leptin Opposes the Negative Inotropic Effect of Interleukin-1 Beta
Interleukin-1beta (IL-1β) is a potent negative inotrope, which contributes to the pathogenesis of myocardial dysfunction and heart failure. Leptin, an adipocyte-derived cytokine, regulates fat mass and may protect against some of the effects of IL-1β in endotoxic shock. With age and weight gain, individuals often acquire resistance to leptin. Studies have associated leptin resistance with development of cardiac hypertrophy. We hypothesize that this may be due, in part, to unopposed effects of IL-1β. This study examined whether leptin opposes the negative inotropic actions of IL-1β and if leptin resistance is associated with impaired protection against the cardio-suppressive effects of IL-1β. Myocytes were isolated from leptin sensitive (wildtype, +/+) and leptin resistant (heterozygous for a null mutation in the leptin receptor, +/cp) SHHF rats. Myocyte contractility and calcium transients were simultaneously determined by edge movement and by 340/380 nm fluorescence ratios following fura loading. Myocytes were incubated with IL-1β (10 ng/ml), leptin (25 ng/ml) or leptin followed by IL-1β. When given alone, IL-1β and leptin acutely decreased contractility by 20% and 40% respectively, in myocytes from +/+. The negative inotropic effect of leptin was associated with blunted calcium transients in +/+. Myocytes from +/cp showed a similar negative inotropic response to IL-1β but not to leptin, compatible with leptin resistance. In both +/+ and +/cp, preincubation with leptin, followed by IL-1β, abrogated the negative inotropic effects of either cytokine alone. After 3 hr, cardiosuppression by IL-1β or leptin was no longer seen and the combined cytokines actually resulted in increased contractility. In conclusion, leptin alone transiently inhibits myocyte contractility in leptin sensitive rats. Pre-incubation with leptin prevents the transient negative inotropic effects of IL-1β.
17: Immunohistochemistry of Atrial and Brain Natriuretic Peptides in Control Cats and Cats with Hypertrophic Cardiomyopathy
Atrial and brain natriuretic peptides (ANP and BNP) are hormones synthesized by cardiomyocytes that participate in electrolyte-fluid homeostasis by inducing diuresis, natriuresis, and peripheral vasodilation. Although ANP and BNP tissue and plasma levels are reportedly increased in human patients with hypertrophic cardiomyopathy (HCM), these peptide levels in HCM cats and the usefulness of cats as a human model for HCM are still unknown. Cardiac tissues from five control and three HCM cats were collected, fixed in 4% paraformaldehyde, routinely processed and embedded in paraffin. An indirect immunoperoxidase method was performed with polyclonal IgG fractions from rabbit antisera against synthetic peptides corresponding to feline ANP (1-28) and proBNP (43-56). In control cats, ANP/BNP immunoreactivity was restricted to the atria. Atrial cardiomyocyte staining for both was more intense and diffuse adjacent to the endocardial surface as compared to epicardial surface; auricles stained more diffusely than atria for both. The ventricular cardiomyocytes of control hearts did not stain for ANP or BNP. The interstitial capillaries and nerve fibers within the heart were positive for BNP. The HCM atrial immunoreactivity for ANP/BNP was more diffuse and the layered pattern less distinct than controls. The HCM ventricular cardiomyocytes were negative for ANP and lightly and diffusely stained for BNP. The capillaries and nerve fibers remained positive for BNP. We conclude that in control hearts, ANP/BNP are restricted to cardiomyocytes of the atria, more specifically those closer to the endocardial surface. In cats with HCM, ANP/BNP are more diffuse in the atria and BNP has novel expression in the ventricular cardiomyocytes.
18: Pcr for Antigen Receptor Rearrangements (Parr): An Objective and Specific Assay to Aid in the Diagnosis of Lymphoma
Although the diagnosis of canine leukemia and lymphoma in advanced stages is usually uncomplicated, some presentations of the disease can be a diagnostic challenge. In certain situations, lymphoma and leukemia can be difficult to distinguish from a benign reactive proliferation of lymphocytes. Because clonality is the hallmark of malignancy, we have developed an assay that uses the polymerase chain reaction to amplify the variable regions of immunoglobulin genes and T cell receptor genes in order to detect the presence of a clonal lymphocyte population. Samples from approximately 350 dogs were tested using the assay. Dogs were only included in the study if a definitive diagnosis of lymphoid malignancy was made using criteria other than PCR; if a condition other than lymphoid malignancy was definitively diagnosed; or if, in the absence of a definitive diagnosis, one year of follow-up was available. In this group of dogs the assay was approximately 82% sensitive and 98% specific. We then analyzed the performance of the assay on samples derived from patients with diagnostic dilemmas. These included: dogs with chronic, mild lymphocytosis consisting of lymphocytes with a uniform appearance; dogs with a cytologic or histologic diagnosis of lymphoid hyperplasia or reactive lymph node with no apparent cause; dogs with unexplained hypercalcemia. The sensitivity and specificity of the assay in each of these situations will be presented.
19: Polymerase Chain Reaction for Clonal Antigen Receptor Gene Rearrangement is a Sensitive Assay for Detecting Peripheral Blood Involvement in Canine Lymphoma
Uniquely rearranged immunoglobulin and T cell receptor gene sequences can be amplified and electrophoretically separated by size to detect a clonal population of lymphocytes. We determined the presence of neoplastic lymphocytes using both microscopic examination of blood smears and PCR amplification of blood-derived DNA, and compared the two methods for frequency of detection of lymphoma. For microscopic examination (200 leukocytes counted), the samples were categorized as negative (less than or equal to 1 % immature lymphocytes), equivocal (> 1% immature lymphocytes, no lymphoblasts), or positive (greater than or equal to 1 lymphoblast). A PCR amplifed sample was positive if a single band was seen on the gel, or negative if no bands, a smear or a faint ladder was seen. PCR diagnosed peripheral blood as positive approximately 3-fold more frequently than did microscopic diagnosis. Nearly three quarters of the microscopically negative samples were positive using PCR. Our conclusion is that, overall, PCR detects lymphoma in peripheral blood more frequently than microscopy, and is especially useful for microscopically negative samples. This study also revealed that neoplastic lymphocytes exist in the peripheral blood of dogs at the time of initial diagnosis of lymphoma at a higher frequency than previously reported. The high sensitivity of this assay may be useful for detecting lymphoma, monitoring response to therapy, identifying dogs out of remission prior to clinical signs, and screening breeds at risk.
20: Expression of Probability in Cytologic Diagnosis
Qualifying terms or modifiers are used by pathologists to express (un)certainty or probability in cytologic diagnosis. Words are imprecise in meaning, however, and may be interpreted differently by pathologists and clinicians. The goals of this study were to assess: 1) the frequency of use of 18 modifiers, 2) the percentage of (un)certainty implied by those modifiers, 3) preferred modifiers for defined probability levels, and 4) factors that impact diagnostic certainty. We surveyed 195 ACVP board-certified clinical pathologists in October 2001. Ninety-six (49%) pathologists responded, primarily from veterinary schools (59) and diagnostic laboratories (30) in the US (77) and Canada (11). All but 3 pathologists preferred the use of words to express probability. Eleven terms were used often or sometimes by at least 50% of respondents. Most terms (12/18) were used to imply a diagnostic probability between 70% and 90%. The adjective “highly” added about 10% probability, irrespective of modifier. The least variability in terminology occurred with probabilities of 95-100%; the most variability occurred with probabilities of <70%. Preferred terms for defined probability levels were: “diagnostic for” or no modifier (100%), “most/consistent with” or “highly suggestive of” (95%), “probable” (75-95%), “suggestive of” (50-75%), “possible” (25-50%), “can't rule out” (5-25%), “unlikely” (5%), and “no evidence for” (0-5%). The most important factors affecting (un)certainty were sample quality and cellularity. Compared with less experienced pathologists, experienced pathologists ranked other patient diagnostic information as less important in affecting diagnostic probability. The results of this study indicate wide discrepancy in the implied likelihood of a diagnosis using words, and suggest the need for defined terminology. Future studies are indicated to determine how clinicians interpret probability expression in cytologic reports.
21: Feline Pulmonary Hemosiderosis
A differential diagnosis for hemosiderosis in cats typically includes hemorrhage secondary to trauma, neoplasia or a coagulopathy, or pulmonary congestion secondary to heart failure. The observation of chronic inflammation accompanying hemosiderosis, without evidence of frank hemorrhage, in tracheal washes from several cats with chronic coughing and suspected allergic bronchitis prompted a 3-year retrospective study to determine the range of diseases which can result in this type of profile. We identified 14 cats with tracheal wash fluids characterized as having chronic inflammation (predominantly macrophages) and hemosiderosis (Prussian blue positive macrophages and/or coarse hemosiderin detected in macrophages). Two of these cats also had eosinophilic components to the inflammation. These two cats, plus one other cat, were clinically diagnosed as asthmatic. Four cats had congestive heart failure, with three of these diagnosed with obstructive hypertrophic cardiomyopathy and one diagnosed with congenital mitral valve insufficiency. Two cats had a combination of liver and pancreatic disease, with pulmonary edema detected in one of these cats at necropsy. One cat had ingested Draino with subsequent development of tongue ulceration. Definitive diagnoses were not determined in 4 of the 14 cats. Clinical signs and physical examination results for these cats were vague and included anorexia, chronic coughing, vomiting, dehydration, and lethargy. This study serves to add to the list of diagnoses potentially linked to feline pulmonary hemosiderosis. Furthermore, several of these cases are similar to the human disorder known as idiopathic pulmonary hemosiderosis.
22: Metastatic Urogenital Carcinomas in California Sea Lions (Zalophus Californianus) Are Highly Associated with Otariine Herpesvirus-1 (Othv-1) Infection
Preliminary studies suggested that DNA from a newly described Otariine herpesvirus 1 (OtHV-1) was often present in tumor tissue of California sea lions (CSL) dying of urogenital carcinomas. As part of part of a larger multidisciplinary study to investigate environmental, infectious, and genetic factors that contribute to the development of cancer, these initial studies have been expanded to include a systematic analysis of CSL with and without carcinomas. To date we have examined standard sets of fifteen tissues from 25 CSL including 9 adult tumor-free females, 11 adults with urogenital tumors (9 female, 2 male), 4 tumor-free juvenile males and a single juvenile female with retrobulbar sarcoma. Presence of virus was determined by polymerase chain reaction amplification of a 650 base pair, OtHV-1 specific fragment of the viral DNA polymerase gene (Pol). Pol amplicon was detected in at least one tissue of all 11 adult animals with urogenital tumors, most often in the lower genital tract (50-79% of examined tissues) and lumbar lymph nodes (60% of examined nodes). In contrast, Pol amplicon was detected in only the salivary gland from a single adult tumor-free female and was not detected in any juvenile animals, including the one with retrobulbar sarcoma. Thus, we conclude that genital infection with OtHV-1 is highly correlated with the presence of tumors in adult female CSL. Further studies to examine additional adult males and juveniles are underway.
23: Chronic Obliterative Arteritis in Axis Deer Due to Sheep-Associated Malignant Catarrhal Fever
Ten axis deer were examined at the North Carolina diagnostic laboratory as part of an investigation of deaths of animals in a mixed species game farm. From October 2001, to April 2002, the farm experienced deaths in Pere David's deer, axis deer, blackbuck antelope, white-tailed deer, and elk. In contrast to other species, clinical disease in axis deer was protracted. The axis deer developed depression, separated from the group, and progressively lost condition over an interval of at least 2 weeks before dying. Two animals were killed 3 months following initial recognition of any illness. Histologic examination of deer with prolonged clinical signs revealed numerous widely disseminated large proliferative arteries in multiple visceral organs. The arteries had prominent subendothelial intimal proliferation of spindle cells with intervening collagen and matrix. Moderate to heavy predominantly lymphocytic infiltrates were within the thickened vascular walls and extended transmurally to the surrounding adventitia. Laboratory findings revealed all clinically affected axis deer examined were positive for MCF viral antibody by competitive inhibition ELISA. PCR amplified DNA fragments from peripheral blood lymphocytes of affected deer. When sequenced, the amplicons were 100% identical to published Ovine Herpesvirus 2 sequences. A similar syndrome of proliferative arteriopathy has been reported in cattle. These cattle survived up to 150 days after clinical onset of MCF and had similar significant fibrointimal arterial plaques. A chronic disease form of MCF in cattle subsequent to partial clinical recovery was documented, and distinctive arterial lesions were the sequela. At this time, no axis deer have been proven to recover from clinical MCF, although chronic MCF is confirmed.
24: Spontaneous Idiopathic Pulmonary Fibrosis in Cats: Morphologic Comparison to the Human Disease
Idiopathic pulmonary fibrosis (IPF) is a chronic pulmonary disease of humans characterized by chronic-progressive remodeling of the alveolar parenchyma. Current animal models of IPF fail to develop the spectrum of morphologic features of the disease. Lungs from thirteen cats with chronic dyspnea had changes consistent with usual interstitial pneumonia (UIP), the pathology characteristic of IPF. The 6 male and 7 female cats, ages 2-13 years (mean = 8.4 yrs) were a variety of breeds (6 mixed, 2 Persians, 2 DLH, and 3 DSH). Histopathology revealed heterogeneous alveolar remodeling that appeared to originate subpleurally. The changes consisted of: 1) interstitial fibrosis with fibroblast/myofibroblast foci, 2) alveolar epithelial metaplasia, and 3) alveolar interstitial smooth muscle metaplasia. Interstitial lymphocytic inflammation was not a prominent feature in the disease, graded minimal to mild in 9/13 (69%) of cats and moderate in 4/13 (31%) of the cats. Mucous cell metaplasia (Alcian Blue/ Periodic Acid-Schiff +) was the predominant pheno-type in the areas of alveolar epithelial metaplasia in 8/13 (62%) of the cats; in the remaining 5 cats (38%) the metaplastic cells were squamous, columnar or type II pneumocytes. Mast cells (tryptase +) were increased in 10/13 (77%) of the cats, associated with foci of epithelial metaplasia and collagen. Myofibroblasts (smooth muscle actin +) were prevalent subjacent to epithelial cells in the areas of remodeling. We conclude: 1) remodeling of the alveolar parenchyma of the feline lung can mimic the changes of UIP, 2) cats may be a valuable model for the study of UIP/IPF in humans, and 3) the cause of UIP/IPF in cats is unknown.
25: Immunohistochemical Detection of Uroplakin Iii in Canine Urothelial Tumors
The transitional epithelium of the mammalian urinary bladder (urothelium) consists of six to ten layers including superficial (also called umbrella), intermediate, and basal cells. Umbrella cells are characterized by a highly specialized plasma membrane that forms plaques covering the apical surface of the urothelium, forming the asymmetric unit membrane. The major protein components of the asymmetric unit membrane are four transmembrane proteins named uroplakins Ia, Ib, II, and III. Uroplakins are highly conserved through a broad range of mammalian species. Immunohistochemical detection of uroplakins in urothelial tumors has been reported in human, bovine, and some laboratory animal species. Immunohistochemistry for uroplakin III (UP HI) using a commercially available antibody was performed in normal canine urinary bladder and 72 canine urinary bladder neoplasms that had been fixed in formalin and embedded in paraffin. Prolonged fixation (3 to 28 days) did not significantly alter the immunostaining for UP III. UP III was detected in superficial (umbrella) and some intermediate cells of the normal urinary bladder, 7 of 7 transitional cell papillomas (TCPs), 50 of 55 transitional cell carcinomas (TCCs), and 4 of 5 metastatic TCCs. The staining was typically in the cytoplasmic membrane but diffuse or focal cytoplasmic staining was also observed. Intracytoplasmic lumina were usually positive for UP III. One squamous cell carcinoma of the bladder, four nonepithelial bladder tumors, and 285 non-urothelial tumors from non-urinary sites were negative for UP III. UP III is a specific and sensitive marker for canine transitional epithelial (urothelial) neoplasms, detecting 91% of TCCs. Negative results may be observed with anaplastic tumors.
26: Exploring Functional Relationships within the Apoptotic Network
Despite identification of numerous mutations causing retinal degeneration in humans and animals, the molecular events which precede photoreceptor apoptosis are poorly understood. To identify genes which are critical for this cascade, the expression profile of 500 retinal genes was compared in wild-type and rd-1 mice using cDNA arrays. Array results were confirmed by RT-PCR. The initial retinal expression profile in rd-1 retina suggests that both pro-and anti-apoptotic signals interact concurrently. Altered gene expression falls into four classes: 1) Early response genes c-fos and EGR-1 are elevated. 2) APAF-1 and caspases-3 and -7 are elevated. These are components of the mitochondria-associated apoptotic pathway. 3) TNFR1 expression is increased, suggesting pro-apoptotic contribution via the cell surface death receptor pathway. 4) EGFR, PI3Kp85 and Akt are all reduced, suggesting that this pro-survival mechanism is downregulated. Elimination of caspase-3 imparts partial retinal protection to the rd-1 mouse. cDNA array results imply the existence of several interacting pathways during apoptosis, and suggest that these may need to be manipulated at several levels to prevent photoreceptor death.
27: Malignant Transformation of Lens Epithelium in the Cat
The lens epithelium is considered refractory to neoplastic transformation. Although lens tumors can be induced experimentally in mice by genetic manipulation, spontaneous lens epithelial tumors have not been reported in any species. To ascertain the cell of origin of post traumatic ocular sarcomas in cats, 10 ocular sarcomas from cats with a history of head trauma were examined and compared to normal cat eyes. In general, tumors occupied ciliary body and iris, and expanded intraocularly to fill the posterior and anterior chambers. Local extraocular spread typically occurred at the limbus. Neoplastic tissue consisted of bland spindeloid cells arranged in interlacing fascicles, alternating with sheets of plump epitheloid cells which produced PAS, Trichrome and collagen Type IV positive basement membrane material. 3/10 tumors stained positively for crystallin alphaA; one of these was also positive by ISH. All tumors stained strongly for vimentin, and variably for other markers. The majority of tumors examined were undifferentiated sarcomas, with a preponderance of fibrosarcoma- or leiomyosarcoma-like features. Spontaneous transformation of lens epithelial cells is a unique finding in any species. The phenomenon is reminiscent of postvaccinal sarcomas in the cat, and suggests that feline tissues may be more susceptible to neoplastic transformation after local insult than most other species.
28: Feline Post-Traumatic Ocular Sarcoma: A Review of 110 Cases
Records and selected slides from 110 archived cases of feline ocular post-traumatic sarcomas (FPTS) were reviewed and follow-up information was collected for 31 cases. Results: 79 cases were spindle cell tumors, 16 were osteosarcomas, and 15 were round cell tumors. All cases had lens capsule rupture. Advanced tumors filled the globe with extrascleral extension directly or within the optic nerve or peripheral nerves. The time between trauma and enucleation ranged from two months to over ten years. Spindle cell FPTS have thick basement membranes surrounding neoplastic cells in some areas. Round cell tumors exhibit abundant necrosis with neoplastic cells surviving around vessels. Of the 31 cases with follow-up information 14 have had life-ending reoccurrences. Five others died within a year after enucleation with reoccurrence complicating other issues. In these 19 cats the average survival time was 7 months. 7 cats with no reoccurrence have been alive for an average 2 years. 5 cats died one to seven years after enucleation but we were not able to establish a relationship between the cause of death and tumor recurrence. Extrascleral extension of the tumor was seen in 21 of the 31 cases with follow-up information; 5 of the 10 cases where the tumor was confined to within the sclera are either alive more than one year after enucleation or died without recurrence more than one year after enucleation. Conclusions: Cats with traumatic lens capsule rupture are at risk of developing FPTS. Extrascleral extension is a bad prognostic indicator. Spindle cell tumors may develop from released lens epithelial cells. Post-traumatic round cell tumors have a different morphology. Serious consideration should be given to prophylactic removal of blind traumatized eyes when lens capsule damage can be documented.
29: Expression of Survivin in Non-Neoplastic Proliferative and Neoplastic Canine Tissues
Survivin is a recently discovered inhibitor of apoptosis that, in humans, is expressed in embryonic tissues and a variety of cancers, yet is undetectable in most terminally differentiated tissues. Over-expression of survivin in human tumors is being used as an independent negative predictor of survival in cancer patients. Several experiments have shown that increasing levels of survivin in tumor cells is inversely correlated with the long term survival of cancer patients. The goal of this study was to determine if survivin expression could be used to differentiate neoplastic cells from their hyperplastic or non-neoplastic proliferative counterparts. Specifically, survivin expression was determined in canine nasal carcinomas as compared to hyperplastic nasal epithelium and survivin expression in canine fibrosarcomas was compared to granulation tissue. Survivin expression was detected by immmunohistochemical staining using anti-human survivin antibody. Both nasal carcinomas and hyperplastic nasal epithelium expressed survivin, however, the neoplastic cells stained as intensely or more intensely than the hyperplastic cells. Fibrosarcomas did not express survivin whereas nonneoplastic fibrovascular proliferation stained intensely. Although canine nasal carcinomas express survivin, the fact that hyperplastic nasal epithelium also expresses survivin excludes the use of this inhibitor of apoptosis as a specific marker of nasal carcinomas in dogs. While the absence of survivin expression in fibrosarcomas excludes its use as a specific marker of canine fibrosarcomas, its presence in fibrovascular granulation tissue suggests a possible role of survivin in wound healing.
30: The Niemann-Pick C1 Protein in Feline Fibroblasts
Niemann-Pick C1 (NPC1) disease is a rare inherited metabolic disorder characterized by hepatosplenomegaly, progressive neuro-degeneration, and storage of lipids such as cholesterol and glyco-sphingolipids within most tissues, including the central nervous system. The current study was conducted to partially characterize the Niemann-Pick C1 (NPC1) protein in normal feline fibroblasts. Rabbit polyclonal antibodies were generated against a peptide corresponding to amino acids 1256-1275 of the feline NPC1 protein. Using immunoblot analysis, two major proteins were identified that migrated at approximately 140 and 180 kDa. These two proteins were absent when immunoblots were incubated in the presence of NPC1 antibody and immunizing peptide, or preimmune serum. Fl-uoresence microscopy of feline fibroblasts incubated with the feline NPC1 antibody revealed granular staining within the perinuclear region. This staining was diminished when feline fibroblasts were incubated in the presence of feline NPC1 antibody and immunizing peptide, or completely absent when feline fibroblasts were incubated in the presence of preimmune serum. Additional studies using double-labeled fluorescence microscopy indicated that feline NPC1 partially co-localized with markers for late endosomes/lysosomes, endoplasmic reticulum, and microtubules, but not the trans-Golgi network. The NPC1 protein in feline fibroblasts has a similar subcellular distribution as that previously described in human and murine fibroblasts. These experiments are being repeated using NPC1 affected and NPC1 heterozygous feline fibroblasts to determine if the NPC1 mutation results in altered NPC1 protein localization.
31: Tyzzer'S Disease in a Dog Associated with Cyclosporin and Azathioprine Administration
An 18-month-old spayed female Standard Poodle was presented to the Veterinary Teaching Hospital (VTH) of the Ontario Veterinary College with a history of lethargy, anemia and severe weight loss. At admission, a complete blood count revealed a severe normocytic, hypochromic anemia with reticulocytopenia and low numbers of spherocytes. The hematocrit rose following two transfusions with packed red blood cells, but reticulocytes remained low despite the persistent anemia. A bone marrow aspirate indicated an increased prominence of the erythroid component with a predominance of rubriblasts and prorubricytes and little maturation beyond the mitotic pool, consistent with ineffective erythropoiesis. A diagnosis of immune-mediated hemolytic anemia (IMHA) was made and the dog was treated with cyclosporin (100mg orally Q 12h) and azathioprine (50 mg orally Q 24h). One week post-treatment, the serum hepatic function parameters were dramatically increased. The animal died twenty-two days post-admission after showing a progressive worsening of signs of lethargy and acute hepatic failure. The dog was icteric at necropsy and the liver was very friable. Histologically, numerous multifocal to coalescing areas of coagulative necrosis were present in the liver. Numerous argyrophilic filamentous bacilli consistent with Clostridium piliformis were present at the periphery of these necrotic areas. The presence of C. piliformis was confirmed by using immunohistochemistry and nested PCR. C. piliformis, the causative agent of Tyzzer's disease affects a wide variety of animals, including dogs. However, the disease in dogs must precipitated by various stressors. In this case the dog was treated with cyclosporin and azathioprine, both well-known immune suppressant drugs. To the best of our knowledge this is the first reported case of canine Tyzzer's in association with cyclosporin and azathioprine administration.
32: Genetic Diversity of Canadian Feline Immunodeficiency Virus Isolates
Lentiviruses have marked sequence variation rendering universal classification of primary isolates into subtypes by gene amplification difficult. We examined several Canadian feline immunodeficiency virus (FIV) primary isolates that could not be amplified by the polymerase chain reaction (PCR) with previously described primers. The reactions either did not yield a product or resulted in multiple amplicons. Therefore, an extensive search of available viral sequences was performed to identify more highly conserved regions. A nested PCR assay was optimized using primers designed to amplify approximately 1100 base pairs of a region encompassing the viral long terminal repeat (LTR) and part of the gag gene. These primers successfully produced a single amplicon from the FIV subtypes A, B, C, and D, and all Canadian isolates examined. PCR products were sequenced and aligned with other published sequences in order to construct a phylogenetic tree. Analysis of nucleotide homology indicated that the Canadian FIV isolates clustered with previously identified subtypes A, B and C, and that subtype A was highly prevalent. Thus, we have established a robust method for amplification of primary FIV isolates, and identified the presence of subtype A in Eastern Canada, which to our knowledge has not been previously reported.
33: Leishmaniasis and Neoplasia in American Foxhound Dogs
For several years, our group has been actively investigating a widespread outbreak of canine leishmaniasis across 21 states in the United States and 2 provinces in Canada. In an attempt to limit further disease spread, many owners of low-titer IFA-positive dogs elected either the dogs’ euthanasia or donation to scientific institutions for disease investigation. Forty foxhound dogs were donated to our group at Walter Reed Army Institute of Research. Within 9 months of arrival, 9 of these dogs (22%) were euthanized due to complications secondary to Leishmania infection, neoplasia or both. A complete necropsy was performed on each dog. Six dogs (67%) were positive for Leishmania by serology, culture and/or histology; 6 dogs (67%) had significant malignant neoplastic lesions. Fifty percent (3 of 6) of the Leishmania positive dogs had concurrent neoplastic disease. In these dogs Leishmania amastigotes were easily cultured from neoplastic tissue. Two of the three dual affected dogs were less than 3-years-old, and both likely were infected with Leishmania through vertical transmission. Two had hemangiosarcoma, which interestingly has previously been reported to occur with canine leishmaniasis. In addition, 5 non-institute dogs have been identified with concurrent leishmaniasis and neoplasia. Neoplasms in all dogs were of diverse origin. Based on these preliminary data, we propose that the foxhound dogs in our study group have an increased incidence of neoplasia and Leishmania infection compared to the general canine population. In addition, we hypothesize that even before clinical signs of leishmaniasis are evident, chronically infected dogs experience cell mediated immune depression, which may predispose them to manifest cancers at an earlier age.
34: Bartonella Associated Valvular Endocarditis in Dogs
The purpose of this study was to describe gross and histological lesions associated with valvular endocarditis by Bartonella spp. in the dog. Thirty-one cases of valvular endocarditis presenting to necropsy service at the University of California at Davis between 1997 and 2001 were examined. Twelve cases were associated with Bartonella spp. based on a combination of polymerase chain reaction (PCR) amplification, histology, and special staining methods. PCR and serological titers were performed on fresh valve tissue and with blood samples respectively when possible. In historic cases, DNA was extracted from paraffin embedded aortic valve tissue and tested by amplification of a 400 bp fragment of the citrate synthase gene. By histological analysis, valvular leaflets infected with Bartonella are chronic lesions based on the presence of fibrous tissue, mineral, and degree and character of inflammation. Endothelial immunohistochemical analysis of the valve surface revealed that the endothelium of the outflow and inflow tracts was often disrupted by regions of vascular clefts, neovascularization, immature matrix, and small numbers of inflammatory cells. Further analysis of the endothelium in this region by silver staining methods and electron microscopy revealed the presence of Bartonella organisms in both extracellular regions and within endothelial cells.
35: Lesions of Spontaneous Bovine Uterine Trichomoniasis
In a study of fifty spontaneous bovine pyometras, one uterus had unique lesions characterized by an exaggerated inflammation of the lamina propria of the endometrium. Tritrichomonas (Trichomonas) sp. was isolated from only this pyometra. Subsequently, during study of a beef herd with infertility problems, Tritrichomonas sp. was cultured from the vaginal discharge of three bred but non-pregnant cows. The open cows from this herd were culled and followed to slaughter. Two pyometras were observed, sampled for light microscopy, and cultured (aerobically, anaerobically and for Tritrichomonas sp.). Tritrichomonas sp. was cultured from these three spontaneous cases. No anaerobic bacteria were cultured from any of the uteri. Corynebacterium sp. was isolated from three uteri, Alcaligenes sp. from one uterus and Pseudomonas aeruginosa from one uterus. The histologic lesions were similar in all three cases and consisted of a severe, periglandular and submucosal, lymphoplasmacytic infiltration with minimal fibrosis. Lymphoid follicle formation was common. Some glandular lumens were distended by neutrophils and mucin containing trichomonads. The cervices of the uteri also had a mononuclear, mucosal infiltrate with lymphoid follicle formation. The histologic lesions are similar to those of experimental bovine trichomoniasis; however, pyometra is rare in experimental infections. It is presumed that the pyometra seen with spontaneous cases is related to the mixed infection with other bacterial pathogens. Despite characterization in textbooks that the histology of uterine trichomoniasis is “relatively mild and non-specific,” the spontaneous lesions are striking when compared to nontrichomonad-induced uterine infections. Unfortunately, the lesion pattern is not pathonomonic for trichomoniasis. The uterine lesions of Campylobacterium sp. infections are similar to those of trichomoniasis.
36: Enteric Helicobacter in Captive Cheetahs (Acinonyx Jubatus) in Japan
To confirm the presence and implication of Helicobacter in enteric lesions among captive cheetahs in Japan, pathological studies were carried out on intestinal samples from 36 cheetahs that died in captivity (7 duodenum, 28 jejunoileum, 17 colon, 15 cecum, 20 rectum). Techniques included immunostaining using anti H. pylori antibody and transmission (TEM) and scanning (SEM) electron microscopy. Immunostaining identified enteric Helicobacter in 21 of the 36 cheetahs (58%). The prevalence according to organ was duodenum 0, jejunoileum 6 (21%), cecum 9 (60%), colon 9 (53%) and rectum 8 (40%). In infected animals, Helicobacter were found in either the small intestine or the large intestine, but rarely in both, with the majority of animals (over 50%) infected in the large intestine. Animals in the latter group were mostly positive for Helicobacter throughout the cecum, colon and rectum. The Helicobacter were mostly present in the crypts, either scattered or in clumps, and were seen by SEM to be straight and rod-like, 3-3.5 × 0.3 microns in size, with regular, densely aligned pericytoplasmic fibers. Intestinal lesions in the cheetahs included lymphocytic enteritis, enlarged lamina propria, expanded crypts, amyloid deposition, and erosion and ulceration. In all animals examined, lymphocytic enteritis was present to some degree in at least part of the intestine, and was severest in the cecum. Amyloid deposition was also highly prevalent, appearing in 27/28 samples from the small intestine (96%) and 28/52 samples from the large intestine (54%), in tissue without autolysis, and the degree of amyloidosis was advanced. Gastric Helicobacter infection is well known in cheetahs. Our results indicated there is also a high rate of enteric Helicobacter infection in these animals. Ultrastructural observation suggested the bacteria may be H. bilis or Flexispira rappini. Lymphocytic enteritis and intestinal amyloidosis were also highly prevalent in the cheetahs observed, but correlation with the Helicobacter infection was unknown. Our results suggested that in addition to amyloidosis in other organs, amyloid deposition in the intestines is a major life-threatening factor in cheetahs.
37: Fatal Case of Acute Pleuritis by Bordetella Bronchiseptica in a Squirrel Monkey (Saimiri Sciureus)
Bordetella bronchiseptica is one of the major respiratory pathogens in animals. It is implicated in porcine atrophic rhinitis, canine kennel cough, and bronchopneumonia in many species. B. bronchiseptica was isolated from a fatal case of bronchopneumonia in an African green monkey. Also it induced pneumonia in Callicebus spp. primates. But the fatal case of acute pleuritis by B. bronchiseptica has been rarely reported in animals. This paper describes the gross and microscopic lesions of a 3 year-old female squirrel monkey (Saimiri sciureus) submitted to National Veterinary Research & Quarantine Service, South Korea. The dead monkey and other 7 squirrel monkeys were imported from Japan, November 6. 2002. The dead monkey has showed lethargy, recumbency and inappetence since November 14, 2002 and died in November 17. Grossly, fibrinous epicarditis was seen. Fibrin was deposited on the ventral and dorsal pleura of right lung and diaphragm, and lobular to lobar, reddish consolidation of the right lung was seen. No lesion was seen in other organs. Histopathologically, the lung had severe fibrinous pleuritis, edema, thrombosis and focal necrosis with fibrin and mononuclear cells deposited on the epicardium. There was also severe lymphoid depletion in the splenic white pulp. B. bronchiseptica was isolated from the lung pleura and parenchyma and epicardium. The isolate was nitrate reduction positive, urease production positive and was agglutinated with anti-rabbit B. bronchiseptica serum. We thought that death of that monkey was related with transport and other stresses. Considering gross and microscopic lesions, bacterial culture, and biochemical tests of the isolate, this case was confirmed as the fatal case of acute pleuritis by the infection of B. bronchiseptica in squirrel monkey.
38: Cerebral Parastrongyliasis in Four Virginia Opossums (Didelphis Virginiana)
Parastrongylus (= Angiostrongylus) cantonensis is the lung worm of rats and was first reported in the United States in 1987 in New Orleans. Cerebral parastrongyliasis was diagnosed in four Virginia opossums (Didelphis virginiana), all of which were captured wild in the vicinity of Baton Rouge, Louisiana, a distance of 124 km from New Orleans. The common clinical signs were weakness, ataxia, and neurological signs such as circling. Microscopically, multifocal areas of cerebral and cerebellar leptomeninges were thickened with infiltrates of a few to moderate numbers of eosinophils, macrophages, lymphocytes, a few multinucleated giant cells and occasional cross-sections of nematodes that were morphologically consistent with those known to be P. cantonensis. This is the first report of parastrongyliasis in the Virginia opossum, the only native North American marsupial. Recent increase in the number of parastrongyliasis in various species in Louisiana, radiating away from New Orleans, indicates that P. cantonensis is endemic in the Gulf Coast of North America.
39: Causes of Mortality in Confined White-Tailed Deer (Odocoileus Virg1Nianus) in Pennsylvania
To characterize naturally occurring diseases in confinement raised deer in Pennsylvania, the postmortem records of 66 white-tailed deer (Odocoileus virginianus) were reviewed to determine the primary cause of death of each animal. The deer, originating from 33 different confinement operations in Pennsylvania, had been submitted to the Pennsylvania Animal Diagnostic Laboratory System at the Pennsylvania State University (PADLS-PSU) for postmortem examination between August, 2000 and April, 2002. The most common causes of death were pneumonia (13 cases) and enteritis (13 cases) in this study. Arcanobacterium pyogenes, Fusobacterium necrophorum and Pasteurella sp. were the most commonly isolated bacteria from the pneumonic lungs in which a significant bacterial pathogen was obtained. Etiologic agents associated with enteritis included Clostridium perfringens, Mycobacterium paratuberculosis and nematode parasites. Other primary causes of deer mortality, in decreasing order of frequency, included systemic abscessation, trauma, malnutrition, abomasitis, hepatic necrosis, myocardial degeneration with mineralization and cellulitis with septicemia. One case of each of the following conditions was also found in this study: meningoencephalitis, systemic granulomas, cecal rupture with peritonitis, rumenitis/acidosis, acute asphyxia secondary to tranquilization, nasopharyngeal obstruction secondary to nasal bots, urolithiasis, tubular nephritis, pulmonary hemorrhage, polioencephalomalacia and nephrosis. The cause of death was undetermined in 7 cases. In conclusion, pneumonia and enteritis were considered the most common causes of death in confined white-tailed deer in this study.
40: Pathologic Findings in Domoic Acid Intoxicated California Sea Lions (Zalophus Californianus)
Over 100 adult free-ranging California sea lions (Zalophus californianus, CSL), predominantly adult females, were intoxicated by domoic acid during the course of three harmful algal blooms between 1998 and 2000 in central and northern California. The vector prey item was Northern anchovy (Engraulis mordax) and the primary domoic acid producing algal diatom was Psuedo-nitzschia australis. Post mortem examination revealed gross and histologic findings that were distinctive and aided in diagnosis. Animals examined died between one day and ten months after admission to a marine mammal rehabilitation center. Seizures and status epilepticus with obtundation were the main clinical findings. Frequent gross findings in animals dying acutely were seizure-related and consisted of myocardial pallor, bronchopneumonia, and uterine catastrophe (in gravid animals). Gross findings in animals dying months after intoxication included bilateral hippocampal atrophy. Histologic observations implicated limbic system seizure injury consistent with excitotoxin exposure. Peracutely, there was microvesicular vacuolation within the neuropil of the hippocampus, amygdala, pyriform lobe, and other limbic structures. Acutely, there was also ischemic neuronal necrosis. Necrotic neurons were particularly apparent in the granular cells of the dentate gyrus and the pyramidal cells within the CA4, CA3, and CA1 sectors of the hippocampus. Chronically, there was gliosis, mild nonsuppurative inflammation and loss of laminar organization in affected areas. These lesions were both similar and different from neural lesions in primates and rats with experimental domoic acid intoxication.
41: Concurrent Coccidian and Myxozoan Parasitism in Indian Flap-Shelled Turtles (Lissemys Punctata Andersoni)
Two adult Indian Flap-shelled turtles from the Baltimore Zoo were presented for necropsy. Both animals were found dead after a brief period of lethargy and inappetance. Numerous pale yellow caseous nodules were present in multiple organs and free within the coelom; microscopically these were granulomas. Coccidian parasites were found in the lung, liver, and GI tract associated with pneumonia, hepatitis, and enteritis. Sporulated spherical oocysts contained at least 6 club-shaped sporozoites arranged in opposed pairs and were tentatively identified as Eimeria sp. This genus has previously been reported in chelonian GI tract and less frequently in liver; however, the lung appears to be a newly reported and possibly aberrant location in this host. The coccidian parasite caused a marked host inflammatory response in all sites in which it was found. Coccidian parasites have previously been reported to cause visceral granulomas in avian species. Myxozoan plasmodia, containing fusiform spores each with two pyriform polar capsules opposite each other (Myxidium sp.), occluded 10-15 percent of the renal tubules. There was no notable inflammatory reaction, implying a long-established host-parasite relationship. TEM showed that the renal microvilli were compressed by the Plasmodium. If more of the kidney were infected with an increased percentage of occluded tubules, it would probably cause renal dysfunction. Although Myxidium mackeii has previously been reported for turtles in India, its ultrastructure is now newly described.
42: Spontaneous Cardiovascular Lesions in Exotic Mammals
In exotic mammals, cardiovascular lesions have been characterized as incidental or experimental. Previous reports describe cardiac failure caused by encephalomyocarditis virus in African Elephants; myocardial degeneration and endocarditis in dolphins; protozoal and filarial parasite lesions in hoofed stock, fox, otters, and wallabies (experimental), and atherosclerotic plaques in non-human primates. This report describes cardiomegaly and vascular mineralization in a one-year-old, male wallaby; atherosclerosis in a 20-year-old, female capuchin monkey; myocardial degeneration and intimal mineralization in a 36-year-old Asian Elephant; and a non-septic thrombus, and incomplete subaortic stenotic ring in a 13-year-old, female mountain lion. The wallaby died from necrotizing myositis (white muscle disease) and acute gastric ulceration. Macroscopically, the heart was enlarged. Microscopically, cardiac myofibers appeared normal, but intimal mineralization of myocardial arteries was found. No microscopic lesions suggestive of heart failure were noted. The capuchin monkey died from necrotizing cholangiohepatitis (bacterial and fungal cultures were unremarkable). An atherosclerotic plaque was present in the aorta. The Asian Elephant died of multiple organ failure secondary to necrotizing endometritis and peritonitis. Intimal mineralization was seen in multiple arteries. Portions of myocardium were degenerate and mineralized. The mountain lion was euthanized due to chronic weight loss induced by metastatic thyroid follicular adenocarcinoma. Grossly, the septal leaflet of the left aortic valve (AV) was thickened, and a subaortic white fibrous endocardial band was present. Histologically, the fibrous band was determined to be an incomplete subaortic stenotic ring. The left AV lesion was a non-septic thrombus. In all species, except the elephant, the cardiovascular lesions were not directly contributory to the animals’ deaths. These lesions have either not been described, or infrequently described in these exotic animals.
43: Seven Cases of Vaccination Site Fibrosarcomas in Ferrets
Between 1996 and 2002, 10 ferrets were diagnosed with cutaneous fibrosarcomas at the University of Georgia. Seven tumors were located in vaccination sites (interscapular, dorsal thorax, and dorsal neck) while 3 were not (ventral abdomen, base of tail, paw). Of the 7 ferrets with vaccination site fibrosarcomas (VSF), 5 had received canine distemper and rabies vaccinations in the preceding 12 months. The vaccination history of the other 2 ferrets was unknown. Histologically, all VSF were well demarcated and located within the hypodermis adjacent to the panniculus muscle. Two NVSF were located within subcutaneous adipose, but panniculus muscle was not present in the sections. One NVSF was located within the superficial dermis. Mild to moderate multifocal, predominantly lymphoplas-macytic, inflammation with discrete peripheral lymphoid aggregates was visible within 5 of 7 VSF. Four VSF also contained rare to numerous giant cells (>2 nuclei). Intracellular basophilic granular material was present within 2 neoplasms. None of the NVSF contained lymphoplasmacytic inflammation, giant cells or basophilic material. Necrosis of >20% of neoplastic cells was visible within 2/ 7 VSF and 1/3 NVSF. Immunohistochemistry demonstrated smooth muscle actin in 20-50% of cells within 3/7 VSF. Approximately 10% of cells in 1 VSF contained desmin. None of the NVSF contained muscle cytoskeletal proteins. The high proportion of fibrosarcomas at vaccination sites observed in this study suggests a relationship between vaccination and fibrosarcoma development in ferrets. Intratumoral lymphoplasmacytic inflammation, giant cells, intracellular basophilic material and myofibroblast differentiation are also reported features of feline vaccine-associated sarcomas. To the authors’ knowledge, vaccination-associated neoplasia has not been reported in non-feline species.
44: Molluscum Contagiosum in a Dog
A 2 mm circular raised, reddened papule of 4 months duration was removed from the cranial ventral thorax of a 5 year old neutered male Great Dane. The lesion had enlarged slightly prior to removal and was mildly pruritic. Histologically, the epidermis was moderately to severely hyperplastic with numerous, 12 to 24 micron diameter, round to oval eosinophilic inclusions expanding the cytoplasm of multifocal keratinocytes from the stratum spinosum to the stratum corneum. The inclusions often filled the cytoplasm and compressed the nucleus to the periphery of the keratinocyte. Ultrastructurally, the inclusions were composed of numerous, densely packed, poxvirus particles measuring approximately 148 to 184 nm in width and 237 to 265 nm in length. The virions had a flattened, biconcave central nucleoid. A mild, lymphoplasmacytic superficial dermatitis was also present histologically. The histologic and ultrastructural lesions are consistent with molluscum contagiosum, a human epidermotropic molluscipoxvirus infection that has been rarely reported in horses, chimpanzees, and a red kangaroo. To the authors’ knowledge, this is the first report of molluscum contagiosum in a dog. The dog had no known contact with an infected human, and the source of the infection was undetermined. The lesion has not recurred eight months after removal.
45: Oral Chondroid Melanoma in a Dog
Chondroid formation without concurrent osteoid differentiation is an extremely rare feature of malignant melanomas; only four have been described in humans. A 3 × 3 × 7 mm, ovoid, lightly pigmented mass surgically removed from the left commissure of the lip of an 11-year-old Cocker Spaniel dog was examined by light microscopy. The mass was composed of nests of polygonal cells with concomitant areas of chondroid differentiation that focally altered the architecture of the adjacent dermis and submucosa, as well as the basal layer of the overlying epidermal and mucosal epithelium. The cells throughout the mass, including those entrapped within the chondroid matrix, contained melanin and were immunoreactive for S-100, vimentin, and melan A, but exhibited no staining for cyto-keratin. These results are consistent with melanoma and suggested a melanocytic histiogenesis for the chondroid matrix. To the author's knowledge, this report describes the first example of an oral chondroid melanoma in an animal.
46: Nodule Or Crust Forming Diseases of Bovines in Korea
We have examined fluid and tissue samples from 74 animals with nodules or crust forming lesions in the oral or nasal mucosa, teat, udder or other skin occurring during the period January and December 2001. All 74 cases were confirmed negative for Foot and Mouth disease. The distribution of species was 68 cattle (91.9%), 4 pigs (5.3%), 1 goat (1.4%) and 1 deer (1.4%). Electron microscopy, PCR and serological tests were performed on 798 samples, including sera (543), crusts or nodules (85), saliva (114), vesicle fluids (11) or other samples (45). In cattle, BVD was detected in 33 cases (48.5%). Six papilloma (8.8%), 4 cases of pseudocowpox (5.8%) and 3 cases of IBR (4.4%) were also detected. Additionally, contagious ecthyma was detected in the goat and BVD was identified in the deer. Cattle affected by BVD showed erosion or ulceration of nasal and oral mucosa or diarrhea. Cattle affected by papilloma or pseudocowpox showed vesicles, crusts or nodules in teat or udder. We concluded that major diseases in nodules or crusts forming cases were BVD, papilloma and pseudocowpox in Korea.
47: Variability of Neutrophil and Pulmonary Alveolar Macrophage Function in Swine
Neutrophils and alveolar macrophages are important components of the innate defense of the lung against bacterial infection. This study measured neutrophil and alveolar macrophage function in swine, to identify the effect of age and production stage on leukocyte function, and to estimate the variability of these assays. Neutrophil function assays measured chemotaxis, phagocytosis, oxidative burst, and degranulation. Phagocytosis and oxidative burst were evaluated in alveolar macrophages isolated from bronchoalveolar lavage fluid. Neutrophil and alveolar macrophage functions varied greatly from day-to-day and between pigs. When the same pigs were analysed weekly, individual pigs did not have consistently high or low neutrophil and macrophage responses compared to their cohorts. Neutrophil oxidative burst was significantly lower in younger suckling and weaner pigs compared to older grower-finisher pigs. Similarly, oxidative burst responses were significantly lower in alveolar macrophages from suckling and early weaner pigs less than 40 days of age than those from older pigs. Phagocytosis, chemotaxis, and granule secretion did not vary with age. These results demonstrate that neutrophils and alveolar macrophages from suckling and weaner pigs have lower oxidative burst responses than those from older pigs, but that other measures of function of these leukocytes do not vary among various stages of swine production.
48: Equine Cns Lymphoma and Meningovascular Amyloidosis: Connection Or Coincidence?
Only two reports describe equine lymphoma with intralesional amyloid. We have observed vascular or meningovascular amyloid in horses with CNS lymphoid infiltrates (lymphoma or possible lymphoma). Amyloidomas of the CNS of humans has been associated with low grade monoclonal B lymphocyte neoplasms. We studied 24 horses with CNS lesions: (1)8 cases of apparent CNS amyloid with lymphocytic infiltrates, (2) 7 equine CNS lymphomas, and (3) 9 equine encephalitis cases. Representative tissue blocks were evaluated immunohistochemically with leukocyte markers and PCNA and histochemically with Congo red and Congo red/potassium permanganate pretreatment. CNS amyloid deposition was confirmed in 5/8 primary cases; 4/8 were classified as lymphoma from H&E morphology and 3/4 were Congo red positive. Of the 7 lymphomas, characterized immunohistochemically as B cell type, one also had vascular amyloid. Congophilic material was not detected in any of the encephalitis cases. The primary cases tended to be older, ranging from 6 to 20 years, mean 12; the lymphoma cases ranged from 0.3 to 18 years, mean 8; and the encephalitis cases ranged from 1 to 12 years, mean 5. Although associated with lymphoid infiltrates, none of the amyloid was AL type. All 3 groups showed angiocentric infiltrates but only the encephalitis group had parenchymal infiltration. Although lesion clonality in the primary cases was not established, we have demonstrated the presence of intralesional amyloid in 4 CNS lymphomas diagnosed via H&E morphology. One amyloid positive horse in the primary group with milder lymphoid infiltrates may be a low grade lymphoma. Further studies are indicated to establish whether CNS amyloid is a marker for cerebral lymphoma in the horse.
49: Detection of Borna Disease Virus in the Brain of Bovine Fetus in the Uterus
Borna disease virus (BDV) is the causative agent of Borna disease (BD), characterized by nonpurulent meningoencephalomyelitis. Recently, vertical transmission has been indicated in horse (Hagiwara et al., 2000). However, no fetal infection in cattle has been proved. The purpose of this study is to demonstrate BDV infection in the fetal bovine brain. Detection of BDV antigens and nucleic acid in the central nervous system (CNS) was done by polymerase chain reaction (PCR), in situ hybridization (ISH) and immunohistochemistry (IHC). A 5-month-old fetus was obtained from a 4-year-old Holstein-Friesian cow showing the clinical signs of lethargy, depression, and progressive paresis and was diagnosed as BD by PCR, ISH and IHC. In contrast, no morphologic and histopathologic changes were noted in any regions of the fetal brain. BDV p24 RNA positive signals were found in several regions of the fetal brain and peripheral blood by PCR. ISH using BDV p40 sense and antisense riboprobes disclosed the positive hybridization signals in the neurons of cerebrum and cerebellum. Immunoreactivities for BDV p40 protein diffusely appeared in nuclei of numerous neurons throughout the brain. The BDV-positive fetal brain homogenates were intracranially inoculated to neonatal gerbils. In the gerbil brains, positive BDV RNA and antigens could be confirmed by PCR, ISH and IHC at day 14 after inoculation. To our knowledge, this is the first report of the occurrence of intrauterine BDV infection in cattle.
50: Use of Prnp Alleles in Scrapie Resistant Prnp Heterozygous Sheep
In certain hereditary transmissible spongiform encephalopathies (TSE), disease in heterozygous individuals is modified by differential use of the prion protein (PrP) gene PrnP. Susceptibility to Scrapie in sheep homozygous for valine (V) at PrP codon 136 and arginine (R) at codon 154, is defined by glutamine (Q) at codon 171: the genotype 136VV/154RR/171QQ is susceptible, while genotypes 171QR and 171RR are resistant. Based on observations in hereditary Creutzfeldt Jakob disease E200K, 129M, where delayed onset and incomplete penetrance in 200EK heterozygous patients is associated with preferential transcription of the wild type PrnP E200 allele, the hypothesis is tested that 171R is preferentially transcribed in Scrapie resistant 171QR sheep. RNA was isolated from 3 171QR sheep, reverse transcribed and used to amplify by PCR a 490 bp fragment within the main PrnP ORF using primers spanning codons 136, 154 and 171. Amplicons were cloned and sequenced. Polymorphism was limited to a single base at position 365 of the amplicon, corresponding to the second base in codon 171. 28 of 45 clones were 365G resulting in CGG for R. The remaining 17 clones were 365A resulting in CAG for Q. Expression of PrP, which is necessary for susceptibility, was ubiquitous in 171QR mesenteric lymph nodes by indirect immunofluorescence with tyramide amplification and similar to that in a 171QQ lymph node. Importantly, PrP colocalized with a marker of follicular dendritic cells, which accumulate the disease-associated conformer PrPSc during preclinical Scrapie. These qualitative data suggest that the 171R allele may be preferentially used in 171QR sheep to produce PrP. Since 171R PrP is less readily converted to PrPSc than 171Q PrP, predominant use of the 171R allele may contribute to resistance.
51: Spongiform Neurodegenerative Disease in a Young Rottweiler in Italy
A spongiform neurodegenerative disease of unknown origin has been described in a few young Rottweiler dogs in the USA, Switzerland, Netherlands, Spain and Australia. The purpose of this report is to describe the lesions of the first case of Rottweiler spongiform encephalopathy in Italy. A 7-month-old male Rottweiler dog was referred with a 3-month history of dyspnea, cough, inspiratory stridor and progressive ataxia more severe in the pelvic limbs. Slowed proprioceptive placing reactions were detected in all limbs. Patellar reflexes were exaggerated and crossed-extensor reflexes were evident in all limbs. No muscle atrophy was detected. The dog showed swallowing difficulties and remaining cranial nerve function was normal. Thoracic radiographs and myelography showed no significant lesions. The dog was euthanatized and a complete necropsy was performed. Histologic lesions were restricted to the central nervous system and were characterized by bilateral symmetrical neuronal vacuolation especially of cerebellar nuclei (fastigial, interposital, lateral), nucleus of the spinal tract of the trigeminal nerve, vestibular and thalamic nuclei. Vacuolation of single neurons was observed in the caudal colliculus, pons and spinal cord. Moderate Wallerian degeneration was observed in the caudal brainstem and ventromedial funiculi of the spinal cord. Vacuolation and loss of Purkinje cells and proliferation of Bergmann's glia were also observed. The neuronal vacuoles were negative with histochemical methods (PAS, Luxol fast blue, Oil red O) and some Purkinje cells were positive with hyperphosphorylated neurofilaments. Immunohistochemical staining for protease-resistant prion protein was negative. Ultrastructurally, the neuronal vacuoles were empty or contained granular material.
52: Gliomatosis Cerebri in the Dog
Gliomatosis cerebri is a rare disorder characterized by unusually widespread infiltration of the neuraxis by neoplastic glial cells with general preservation of brain architecture. Although well recognized in humans, the disease has not been previously described in animals. This report describes the pathologic features of the disease in six dogs. The dogs ranged from 3 to 9 years of age (mean 6.1 years) and there was no evidence of breed predilection. 5 of the 6 dogs were neutered or intact males. The clinical findings were variable, including depression, postural reaction deficits, circling, and cranial nerve deficits. Histologically, there was remarkably diffuse infiltration of the white and gray matter of the brain by small numbers of elongated neoplastic glial cells. Areas of greater cellularity formed grossly visible lesions in four cases, and cells in densely cellular areas exhibited greater anisocytosis and pleomorphism. Other features of tumor growth included neuronal satellitosis, perivascular cuffing, tropism for cranial nerve and brain stem nuclei, and subpial accumulation in the cerebellum. The clinical and pathologic findings were comparable to those described in man. The histogenesis of the neoplastic cells in gliomatosis cerebri is uncertain. In the human form of the disease, an astrocytic origin is suspected, based on cellular morphology and in some cases, positive staining with glial fibrillary astrocytic protein (GFAP). In these canine cases, immunohistochemical staining for GFAP and leukocyte markers were negative. Gliomatosis cerebri should be considered in the differential diagnosis of widely disseminated canine intracranial neoplasms.
53: Two Cases of Feline Craniopharyngioma
Craniopharyngioma is a tumor in the sellar region and derived from remnant epithelial cells of Rathkes pouch. The tumor is occasionally encountered in human infants, but very rare in animal species. Though there are several case reports of canine and murine craniopharyngioma, no feline cases of the tumors have ever been reported. We report the first two cases of craniopharyngioma in cats. Case 1: A castrated male Chinchilla cat (9.5 years old) was presented to the Veterinary Hospital of the Univ. Tokyo (VH-UT) due to anorexia, nasal discharge and wheezing. MRI examination revealed an egg-shaped mass (3 × 3 × 1 cm) in the cranial base. The cat was euthanized because of neurological signs and cognitive impairment. A white-colored mass pressed the brain and involved the optic nerve. Although osteolysis at the cranial base and right ocular fundus was observed, the tumor did not invade to the brain and right eyeball. Histopathological examination revealed sheet-like, acinar and tubular proliferation of epithelial cells. Large cysts filled with eosinophilic fluid were occasionally observed. The tumor cells were positive for keratin and/or vimentin. Case 2: A castrated male American short haired cat (7 years old) was presented to the VH-UT due to open-mouth breathing and wheezing. MRI revealed a nasopharyngeal mass invading to the intracranial space and tympanic bulla. Histopathology of the tumor revealed sheet-like proliferation of epithelial cells intermingled with irregularly branched tubular structures. The tumor cells were positive for keratin and/or vimentin.
54: High Grade Malignant Astrocytoma with Granular Cell Change in a Sprague-Dawley Rat
We describe the histologic and ultrastructural features of a spontaneous, high grade malignant astrocytoma with unusual granular cell change in a 2 year old Sprague-Dawley rat that presented clinical signs of hind limb paralysis. This poorly demarcated neoplasm effaced the left cerebral hemisphere, and was primarily composed of sheets of neoplastic round to oval astrocytes with a pale eosinophilic, finely granular cytoplasm. There were multifocal areas of necrosis and hemorrhage occasionally lined by palisades of neoplastic astrocytes. A distinctive feature of this astrocytoma was the presence of randomly distributed, large binucleated cells that contained numerous hypereosinophilic, 1-2 microns granules and occasional clear, 2-3 microns cytoplasmic vacuoles. The neoplastic astrocytes, but not the binucleated granular cells, stained positively for vimentin (VIM), lysozyme and occasionally phosphotungstic acid hematoxylin (PTAH). Within the binucleated granular cells, cytoplasmic granules stained with PTAH, and Periodic Acid Schiff before and after diastase digestion, but not with VIM, glial fibrillary acidic protein or lysozyme. Staining for wide spectrum cytokeratin, toluidine blue, and pan-neurofilament were negative in both cell populations. Ultrastructurally, neoplastic astrocytes were characterized by cytoplasmic aggregates of electron dense, 8-12 micron intermediate filaments consistent with VIM and desmin. Within the cytoplasm of binucleated granular cells, there were low to moderate numbers of fat globules and autophagocytic vacuoles that contained myelin figures and multiple aggregates of 20-30 nanometer electron dense granules interpreted as glycoprotein or glycolipid. Intermediate filaments were not present. This is the first report in the rat of a neoplasm with features resembling the human granular cells astrocytoma. Our findings suggest that the binucleated granular cells in this neoplasm might be of astrocytic origin.
55: End-Stage Hypertrophic Cardiomyopathy in a Family of Cats
About fifteen percent of human hypertrophic cardiomyopathy (HCM) patients develop end-stage HCM characterized by relative thinning of the ventricular walls and septum with dilation of the ventricular lumen, decreased fractional shortening, and progression to heart failure. Baty et al., recently documented similar progressive changes to end-stage HCM in a family of four cats through serial echocardiograms. At the time of heart failure, these cats exhibited relative decreases in interventricular septal (IVS) and left ventricular free wall (IVFW) thickness with reductions in indices of systolic ventricular function and relative dilation of the left ventricular lumen. Our objectives were to describe the pathologic alterations associated with end-stage HCM and investigate the pathogenesis in three of the four cats. Grossly, there was mild to marked left atrial dilation with mild thinning of the IVS and LVFW relative to earlier stages of HCM. The left atrium contained large thrombi in two of the three cats and all three cats died following thromboembolization of the aortic bifurcation. Histologically, all three cats had subendocardial and myocardial fibrosis, predominantly of the IVS and LVFW, and one cat had acute, multifocal, myocardial infarcts with mononuclear inflammatory cell infiltrates. The pathogenesis of end-stage HCM is uncertain, but theories implicate occlusion of the coronary blood flow by thickening of the coronary vessels, apoptosis of myocytes, myocardial hypertrophy beyond the ability of the vasculature to supply blood, coronary vascular thromboembolism, and coronary vessel spasm. Apoptosis assays using a TUNEL kit did not reveal significant numbers of apoptotic myocytes. Considering the presence of atrial thrombi and/or arterial thromboembolism in these cats, coronary vascular thromboembolism seems to be the most likely major contributing factor.
56: Immunohistochemical Localization of Apolipoprotein B-100 and A-I in Canine Atherosclerotic Lesions
We attempt to determine and compare the localization of apo B-100 and apo A-I in normal and atherosclerotic lesions of canine aortas, coronary arteries, and peripheral arteries, using ABC immunohistochemical techniques. The tissues (aorta, coronary artery, spleen and kidney) in this study were collected from the 10 dogs over 10 years of age with systemic atherosclerosis as well as 3 normal dogs (3 years old). Histopathologically, the atherosclerotic lesions were characterized by the deposition of lipids and infiltration of lipid-laden foam cells in the tunica intima and tunica media, sometimes forming fibrofatty plaques containing abundant sudanophilic and mineralized materials. Immunohistochemically, canine apoB (CapoB)-100 and canine apoA (CapoA)-I immunopositive signals were simultaneously observed in mild and severe atherosclerotic lesions of the aorta, coronary arteries, splenic arteries, and renal arteries in the double immuno-labeled sections. Both CapoB-100 and CapoA-I positive signals were seen in the swollen cytoplasm of endothelial cells and smooth muscle cells and the foamy cytoplasm of macrophages. The subendothelial space and extracellular matrix in the tunica intima and media were also positive. Neither CapoB-100 nor CapoA-I positive signals were seen in normal arteries. These findings closely resemble those of the localization of apoB-100 and apoA-I in human atherosclerotic lesions.
57: The Leukocyte Immunophenotype in Clinically Healthy Dogs and Dogs with Lymphoma in Remission is Similar, but Differs from Dogs with Leukemia
The objective of this study was to compare the immunophenotype of circulating leukocytes in clinically healthy dogs, dogs with primary leukemia or leukemia secondary to lymphoma, and dogs in clinical remission from lymphoma. Blood samples were collected from 20 clinically healthy dogs, 20 dogs with leukemia with or without lymphoma, and 20 dogs in clinical remission from lymphoma. Leukocytes were stained with reagents specific for 23 canine cell surface markers: CD1a, b, and c, CD3, CD4, CD8a, CD8b, CD11a, b, c, and d, CD14, CD18, CD21, CD34, CD45, CD45RA, CD49d, CD54, CD90, MHC II, TCRαβ, and TCRγδ. Surface marker expression on lymphocytes or neoplastic cells was quantified by flow cytometry. Analysis of the frequency of cells expressing each of the markers was performed on the clinically healthy dogs to establish reference ranges. The dogs with lymphoma or leukemia were treated with a standard protocol of chemotherapy, and responses to therapy as well as survival were evaluated. Results indicated that leukemia in dogs arises from many different types and stages of hematopoietic cells. There were differences in the response to therapy predictable from the immunophenotype. Dogs that had achieved clinical remission while being treated for lymphoma showed no abnormalities in the immunophenotype of their circulating lymphocytes.
58: Coordinate Expression of Cytokeratin 7 and Cy-Tokeratin 20 in Canine Urothelial Tumors: An Immunohistochemical Study
The urinary bladder is the site of 0.5-1.0% of all canine tumors. Immunohistochemical characterization of epithelial neoplasms of the urinary bladder is done mainly with antibodies to cytokeratins (CKs). The urothelium contains simple-epithelium type cytokeratins (CKs 7, 8, 18, 19, 20) and stratified epithelium cytokeratins (CKs 13 and 17). The most common cytokeratins used in human pathology to distinguish urothelial tumors from other carcinomas are CKs 7 and 20; both cytokeratins are expressed in many urothelial carcinomas. Immunohistochemistry for cytokeratin 7 (CK 7) and cyto-keratin 20 (CK 20) using commercially available antibodies was done in normal canine urinary bladder and 72 neoplastic canine urinary bladder tumors that had been fixed in formalin and embedded in paraffin. There was moderate reduction in the intensity for CK 7 and CK 20 after 1 week's fixation. CK 7 was detected in 7 of 7 transitional cell papillomas (TCPs), 53 of 54 transitional cell carcinomas (TCCs), and 5 of 5 metastatic TCCs. The staining for CK 7 was diffuse cytoplasmic. CK 20 was detected in 1 of 7 TCPs, 36 of 54 TCCs, and 1 of 5 metastatic TCCs. Staining with CK 20 was cytoplasmic and weaker than that for CK 7. There was coordinate expression of CK 7 and CK 20 in 36 (67%) TCCs. Although more sensitive than uroplakin III to characterize urothelial tumors, CK 7 is less specific. We recommend use of CK 7 as a marker for suspected urothelial tumors that are negative for uroplakins.
59: Cyclooxygenase-2 is Induced in Canine Mammary Tumors
Mammary tumors are the most common neoplasms in female dogs. Induction of cyclooxygenase-2 (COX-2), a key rate-limiting enzyme in prostaglandin biosynthesis, has been implicated in the oncogenesis of various cancers in humans, including breast cancer. However, expression of COX-2 has not been investigated in canine mammary tumors, and the objective of this study was to determine whether COX-2 is expressed in mammary tumors in dogs. Normal mammary gland (n = 4), simple or complex adenomas (n = 63), and simple or complex adenocarcinomas (n = 84) were studied by immunohistochemistry using a selective anti-COX-2 antibody. Results showed that COX-2 was not expressed in the normal mammary gland but was detected in 24% of adenomas (15/63 cases) and 56% of adenocarcinomas (47/84 cases) (P < 0.001). The incidence of COX-2 expression and intensity of the COX-2 immunoreactive signal were higher in adenocarcinomas than in adenomas (P < 0.001). In both simple and complex adenocarcinomas, COX-2 expression was often localized to the cytoplasm of tumor cells. In 15 adenocarcinomas of the complex type, the cytoplasm of myoepithelial cells was COX-2 positive, and COX-2-expressing inflammatory cells were noted in the stroma of few adenocarcinomas. These results demonstrate for the first time that COX-2 is induced in a proportion of canine mammary gland tumors, and that COX-2 expression is more frequent and more intense in malignant than in benign tumors, suggesting a potential role for COX-2 in canine mammary tumorigenesis. (Supported by grants from the American Kennel Club/Canine Health Foundation and the Natural Sciences and Engineering Council of Canada)
60: Cyclooxygenase-2 and Inos Expression in the Cornea of Dogs with Keratitis
Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are not present in normal tissues but are over-expressed at inflammatory sites such as in arthritic conditions. These therapeutic targets have potential uses to treat inflammatory conditions including ophthalmic diseases in veterinary medicine. Keratitis is considered the most common inflammatory eye disease in dogs. In this study we evaluated the expression of COX-2 and iNOS in normal eyes (n = 3) and in eyes with keratitis (n = 12) by immunohistochemistry using isoform-specific antibodies. In the normal eyes, no COX-2 or iNOS immunoreactivity was observed. In keratitis, COX-2 and iNOS expression was observed in all corneal layers (epithelium, stromal cells, and endothelium). COX-2 immunoreactivity was also noted in the stromal and epithelial cells of the iris and stromal cells of the trabecular meshwork. iNOS immunoreactivity was present in new corneal vessels, infiltrating inflammatory cells, and in the stromal cells of the trabecular meshwork. These data suggest that COX-2 and iNOS may play a pathologic role in keratitis and suggest potential therapeutic implications of COX-2 and iNOS inhibition in inflammatory eye diseases.
61: Clinical, Ophthalmological, and Pathological Findings of Hypovitaminosis a in Korean Native Beef Cattle
Hypovitaminosis A has been suspected in Korea because most cattle are fed a ration composed of a little green grass, a lot of cereal and dry rice straw. Recently, an outbreak of blindness in cattle was reported in the Dangjin Area of Chungnam Province. In this report, ophthalmoscopic findings of fundus, serum vitamin A concentration and pathologic findings in the affected calves are described. Blindness occurred only on the farms where green pasture was not fed. Both pupils were totally dilated with the absence of pupillary light reflex in blind cattle. After administration of vitamin A as feed additives or by parenteral injection, no new cases of blindness were found. In the ophthalmoscopic examination, the fundus revealed multifocal linear white mottling which was more severe in nontapetal fundus than tapetal fundus. Optic disc atrophy was observed in all calves examined although there was some variation in severity. The average serum vitamin A concentration was 4.1 microgram/dl in the calves of the affected farms. Grossly, main lesions were confined to the optic canals. The optic canals were bilaterally narrowed dorsoventrally as compared to normal size. In the portion of optic nerves located within the optic canals, severe constriction was present. Histopathologically, the dura matter in optic canals was moderately thickened by fibrous tissues. There was multifocal retinal dysplasia also. Outer nuclear layer and photoreceptor layer were absent, and inner nuclear layer was slightly distorted. These findings were similar to other reports.
62: Proventriculitis in the Broiler Chickens: Chronic Lymphocyte Responses in the Glandular Interstitium
Proventriculitis occurs naturally in commercial broiler chickens. Its economic impact is due to proventricular thinning and rupture during evisceration with resultant carcass condemnation for contamination. Chronic proventricular glandular lumenal ectasia, fibroplasia, and inter-glandular interstitial lymphoid aggregates occur. Normal proventriculi have subepithelial lymphoid aggregates at the orifices of the deep proventricular glands. Chickens with proventriculitis have severe lymphoid infiltrates in the glands and the mucosa that are unique to this disease. To characterize these infiltrates, one-day-old commercial and SPF boilers were orally gavaged with a proventricular homogenate produced from broilers with naturally occurring proventriculitis. Lymphoid inter-glandular infiltrates occurred as the chronic response to this exposure. Lesions were initiated at 7 days post-exposure, and were more prominent at 14 days post-inoculation with well-developed nodular aggregates present. Lymphocytes were characterized in situ by immunohistochemistry using monoclonal antibodies against T and B cells. Results showed both cell types were increased in this chronic disease response. Reactions were more intense in commercial broilers, and suggest the presence of additional antigenic exposure in these chickens compared to that occurring in the SPF broilers.
63: Proventriculitis in Broiler Chickens: Experimental Reproduction and Characterization of Acute Phase Glandular Necrotic Lesions
Acute necrotic proventriculitis is a naturally-occurring disease in broiler chickens of undetermined etiology. It consists of glandular damage leading to proventricular rupture and economic losses due to carcass contamination. Lesions progress chronologically from acute glandular necrosis to chronic interstitial fibrosis. This progression occurs over an unknown time course. The objective of this work was to reproduce proventriculitis using a tissue homogenate, compare lesions produced to naturally-occurring disease, and describe the morphologic features of the acute phase lesions. Proventriculi from commercial broiler chickens with proventriculitis and those with grossly normal proventriculi were collected, homogenized in equal volumes of saline, and administered by gavage into the crops of 1-day-old broiler chickens. Initial morphologic responses occurred in randomly-oriented proventricular glands at 6 days postexposure. The first change was intra-glandular secondary and tertiary duct ectasia, oxynticopeptic epithelial cell swelling and hypertrophy, epithelial cell necrosis, and detachment from underlying intra-glandular connective tissue septal stroma. Early changes were preferentially present in the basal third of affected glands with subsequent spread within affected glands. Inter-glandular septa successfully limited lateral spread to adjacent normal glands Extracted RNA from all lesions was negative for Infectious Bursal Disease (IBD) genomic nucleic acid using PCR primer sets for VP2 sequences shared by all known IBD strains. No lesions were present in proventriculi of chickens gavaged with the homogenate of normal proventriculi. We conclude that acute lesions of broiler proventriculitis identical to natural disease: 1) Can be reproduced by gavage of an organ homogenate produced from affected chickens; 2) Appear as ductular ectasia and necrosis in basal glandular segments; and 3) Contain no evidence of previously known IBD strains.
64: Bone Lesions in the Hemochromatosis of Salers Cattle
Hemochromatosis in Salers cattle is a familial wasting disease characterized by accumulation of iron and death due to liver failure in young adults (Vet Pathol 38: 372-389, 2001). Soft bones and incisor loss have been reported but skeletal lesions have not previously been examined in detail. An affected heifer from a colony at Wyoming State Veterinary Laboratory was monitored from birth until euthanasia at 18 months. An unaffected, age matched steer was used for comparison. The heifer fell two weeks before euthanasia, developed non-weight bearing lameness of the right forelimb, then became recumbent and lost weight rapidly. Both animals received fluorescent bone markers 5 and 12 days before euthanasia. At necropsy, the heifer had fractures of the right humerus, left femur, and two ribs. The cortex was somewhat thicker in larger long bones of the affected heifer when compared to the control. Distinctive circumferential light-dark laminations in the outer third of the cortex were visible along the shafts of all long bones and the mandible. These were seen radiographically as layers of decreased density. Bone analysis revealed iron levels in the affected calf that were 30 to 50 times greater than the control and decreased % ash in the outer cortex. Histologically there were osteopenic outer circumferential lamellae and decreased mineralization where excess iron was found in the matrix. The fluorescent marker indicated irregular mineralization in the outer cortical layers of the affected heifer. Endochondral bone formation beneath the growth plate in the affected heifer was markedly decreased as compared to the control. Results indicate that in hemochromatosis increased iron levels in the bone matrix are associated with dysplastic periosteal bone formation in the outer cortex where there is periodic inhibition of osteoblastic bone formation and mineralization that led to pathologic fractures.
65: Enamel Sheath Protein Producing Ameloblastic Fibro-Dentinoma in Three Dogs
Enamel sheath protein, including ameloblastin (Sheathlin) and amelogenin are produced by ameloblasts of the tooth germ at the late bell stage. The epithelial component of odontogenic tumors may be ameloblasts only. However, the cellular differentiation of ameloblasts is variable for each odontogenic tumor. We obtained three ameloblastic fibro-dentinomas that have an immunopositive reaction of enamel sheath protein on the neoplastic cells, and analyzed cellular differentiation characteristics of these tumors. Tissue samples were fixed in 10% neutral buffered formalin and embedded in paraffin. Serial sections were stained with HE and immunohistochemically with primary antibodies for porcine sheathlin N-terminal, rat ameloblastin, porcine amelogenin, human cytokeratin AE1/AE3 and bovine vimentin. Histopathologically, the neoplastic cells of these tumors had a follicular-like pattern exhibiting palisading of the basaloid cells and stellate reticulum-like pattern of the spindle and stellate-shaped cells. There were squamous and ghost cells in all three tumors. Eosinophilic material was observed among neoplastic cells. In addition, there was mesenchymal material, dentinoid and fibrous tissue in all three tumors. There was no the differentiation of the odontoma towards mature enamel matrix and dentin. We diagnosed as these ameloblastic fibro-dentinoma. Eosinophilic material was immunohistochemically positive for sheathlin, ameloblastin and amelogenin. Also, the cytoplasm of the neoplastic cells was positive, especially spindle cells in the stellate reticulum. Basaloid, squamous, ghost cells and dentinoid materials were negative for these antibodies. In conclusion, these tumors showed cellular differentiation of functional secretory ameloblasts and enamel matrix protein. Immunodetection of enamel sheath protein is used to check functional secretory ameloblasts and distinguish essentially ameloblastic tumors from other odontogenic tumors.
66: Pathological Characteristics of the Calves Cloned from Somatic Cells
Many calves cloned from somatic cells have been produced by nuclear transfer since 1998. Sometimes they have problems such as large offspring, prolonged gestation, and increased rate of abortion/ stillbirth. We have examined these calves to understand the pathogenesis of the lesions. Tissues were collected from nuclear transferred (NT) calves that were stillborn or died as neonates. The placentas were sampled during Caesarian sections. Three calves produced by artificial insemination (AI) were sacrificed as controls. The tissues were evaluated by histopathology and electron microscopy. Immunohistochemical analysis was also performed for hormones related to parturition, such as ACTH. In several cases, severe bleeding from the cut end of the umbilical vessels was the cause of the death of the neonatal NT calves. Enlargement of the umbilical vessels was observed in most cases and the retraction of the umbilical vessels did not occur normally in the NT calves. The umbilicus was fragile and easily cut. Edema of the vessels was observed histopathologically, which might have contributed to the bleeding that resulted in neonatal death. In the placentomes of the NT calves, tight epitheliochorial attachments and proliferation of trophoblasts were present after the full term of gestation, whereas these lesions were not observed in AI calves. Ultrastructurally, irregular length and width of the microvilli were prominently observed between the trophoblasts and the uterine epithelial cells. Tight junctions were also detected at the microvillous connection. In the pituitary anterior lobe, the number of ACTH-positive cells in AI calves was increased 2-fold compared to NT calves. These findings may be related to the increased gestation observed in calves cloned from somatic cells.
67: Differential Diagnosis of Viral Encephalomyelitis in Horses
Naturally occurring cases of rabies, equine herpesvirus, West Nile virus and Eastern equine polioencephalomyelitis and Bornavirus encephalitis were studied with histologic, immunohistochemical, and others ancillary methods and compared with other viral infections (Louping ill, Japanese, Murray Valley and tickborne encephalitides). Rabies is a nonsuppurative polioencephalomyelitis and ganglionitis with Negri bodies and abundant intracytoplasmic virus. Equine herpesvirus 1 infects the cytoplasm and nucleus of endothelium and myocytes causing vascular necrosis, thrombosis, ischemic myeloencephalopathy with segmental malacia and perivascular lymphocytes. West Nile polioencephalomyelitis primarily involves ventral and lateral horns of thoracic and lumbar spinal cord and rhombencephalon, with flavivirus sparsely distributed within neurons and glial cells. Eastern equine encephalitis alphavirus causes polioencephalomyelitis with heart and smooth muscle wall necrosis, localization within neurons, leukocytes, cardiocytes and smooth muscle cell cytoplasm. Borna disease lesions are rather severe in the hippocampus, often sparing spinal cord, sometimes with intranuclear eosinophilic Joest-Degen inclusions. Louping ill, Japanese encephalitis and tickborne encephalitis are characterized by striking necrosis of Purkinje cells, whereas Murray Valley encephalitis virus can be associated with extensive cerebrocortical necrosis. The post mortem use of immunohistochemistry, serology, virus isolation and polymerase chain reaction, singularly or collectively, is influenced by the histological findings. (The two authors equally contributed to this work).
68: Encephalomyelitis Associated with Japanese B Encephalitis in Horses in Korea
An outbreak of natural Japanese B encephalitis occurred in a group of thoroughbred horses in Korea. Twenty one of 28 horses died or were euthanized following three to ten days history of malaise and progressive neurologic deficits. The diagnosis was made based on the results of histopathology and various laboratory procedures to identify the viral infection, including serology, fluorescent antibody (FA) test, polymerase chain reaction (PCR), electron microscopy (EM), and virus isolation. Histopathologically, the horses had nonsuppurative encephalomyelitis characterized by perivascular mononuclear cell infiltration, astrogliosis, and neuronal necrosis with neuronophagia in the brain and spinal cord. Japanese B encephalitis virus (JEV) antigens were detected in the brain by indirect FA test. The target band of expected 477 base pairs was produced by PCR. JEV was isolated from 2 horses after third passage in mice. Toga-virus-like particles were found in the brain tissue homogenates on EM. Hemagglutinating inhibition titers for JEV were sharply increased. This report describes the first epidemic outbreak of natural Japanese encephalitis in horses in Korea.
69: Natural West Nile Flavivirus Infection in Horses in the Usa and Italy
Ten horses from the American northern east coast and 6 horses from Tuscany (Italy) with natural West Nile flavivirus (WNV) infection were examined. Specific polyclonal and monoclonal antibodies were used to detect viral antigen in tissues. There was mild to severe polioencephalomyelitis with small T lymphocyte perivascular infiltrate, multifocal glial nodules, neutrophils, spheroids, and occasional neuronophagia in all animals. Perivascular hemorrhage, also macroscopically noted in 25% of the horses, was observed in 50% of the horses. Lesions extended with different severity from the basal nuclei through the brain stem and to the sacral spinal cord. WNV antigen was moderate in most cases and was identified within the cytoplasm of a few neurons, axons, glial cells and macrophages. In the horses from the USA, lesions were more prominent in the mid- and caudal brainstem, whereas in the horses from Italy severe inflammatory lesions were identified only in the ventral horns of the thoracic and lumbar spinal cord. We believe that differences in topographic distribution of neural lesion might also depend on WNV isolates. The most appropriate specimens for histological, immunohistochemical, molecular and virus culture diagnosis appear to be midbrain-caudal brainstem and thoracolumbar spinal cord of horses infected with USA and Italian WNV isolates, respectively. An examination limited to cerebrum and viscera, and failure to examine the thoracolumbar tract of the spinal cord may lead to inaccurate diagnosis of this zoonotic disease.
70: Encephalitis Due to Actinobacillus Species in Three Adult Horses
Encephalitis due to Actinobacillus sp. was identified in three female adult horses (Mares 1, 2, 3). Mare 1 and 2 had acute neurological disease and Mare 3 had valvular cardiac disease and pneumonia. In Mare 1 there was diffuse suppurative meningoencephalitis and meningomyelitis. Suppurative inflammation was observed in sections of cervical, thoracic and lumbar spinal cord. Actinobacillus sp. was isolated from the brain and meninges. The brain of Mare 2 had multifocal areas of necrosis with heavy infiltration of neutrophils, marked hemorrhage and several small bacterial colonies within neuropil of cerebrum and cerebellum. This mare also had embolic suppurative bacterial nephritis and myocarditis. Special staining of brain sections revealed gram-negative bacteria having morphological characteristics similar to Actinobacillus sp. In Mare 3, cerebrum and cerebellum had bacterial emboli surrounded by neutrophils and marked hemorrhage. A few spinal cord vessels contained bacterial emboli. The mare also had severe suppurative endocarditis of mitral valve, myocarditis, embolic bacterial nephritis and bronchopneumonia. Numerous Actinobacillus sp. were isolated from the kidney, lung and liver. The 16S rRNA gene of the bacterium isolated from Mare 1 was sequenced and a blast comparison of the sequence to GenBank indicated that the strain was 99% identical to two Actinobacillus capsulatus submissions (accession #s AF145255 and M75067). Although rarely reported to cause lesions in internal organs of adult horses, Actinobacillus sp. has not been reported to produce encephalitis in adult horses. These cases occurred within the 4 weeks of Mare Reproductive Loss Syndrome (MRLS) epidemic of 2001. The same bacteria {Actinobacillus sp.) have been isolated from many MRLS associated late term aborted fetus.
71: Polysaccharide Storage Myopathy: A Common Metabolic Disorder of Horses
Equine polysaccharide storage myopathy (EPSSM) is a metabolic myopathy thought to involve abnormal carbohydrate metabolism. This disorder has been recently recognized to be the most common cause of recurrent exertional rhabdomyolysis in horses of various breeds. Other manifestations include postanesthetic myopathy, progressive muscle wasting and weakness, abnormal pelvic limb gait, back soreness, and sudden onset of recumbency. Samples of semimembranosus or semitendinosus muscle samples from all horses presented for necropsy were fixed in formalin, stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) stain for glycogen, and examined for evidence of abnormal glycogen and amylase resistant complex polysaccharide. Samples from 39 draft-related horses and 72 light horses were obtained. Multiple (3-7) transverse sections and 1 longitudinal section from each sample were examined. Two-thirds of all draft-related horses and one-third of all light horses examined were found to have pathologic changes characteristic of EPSSM. Only the most severe cases were detected on HE stained sections. In most cases, detection of EPSSM relied on examination of PAS stained sections. Chronic myopathic changes, including excessive fiber size variation and increase in internal nuclei, were found to be age-related changes and were not always present in EPSSM horses. The clinical significance of underlying EPSSM in horses presented for necropsy with various disorders is not known, but on-going myofiber necrosis detected in some affected horses suggests that EPSSM may contribute to clinical signs of pain or weakness. Given the high incidence of this metabolic myopathy in horses, evaluation of muscle samples with PAS stain for glycogen is indicated, especially in cases with clinical histories or clinico-pathologic findings that could reflect underlying myopathy.
72: Mare Reproductive Loss Syndrome: Pathologic Observations
An abortion storm that affected the equine population in central Kentucky and surrounding regions commenced in the last week of April 2001 and extended through the month of May. The syndrome recurred in the spring of 2002 spanning the same time frame as in 2001. Since there was no immediate explanation for the abortion outbreak, the condition has been referred to as Mare Reproductive Loss Syndrome (MRLS). At least 17 breeds of horses were affected. Mares in early gestation (bred to foal in 2002) and late gestation aborted. Veterinary practitioners identified increased incidences of epicarditis and eye problems that now are considered conditions associated with the syndrome. A heavy eruption of the eastern tent caterpillar (ETC), Malacosoma americanum has been associated epidemiologically with MRLS. During the outbreak, aborted equine fetuses from 32 counties were delivered to the Livestock Disease Diagnostic Center for diagnostic testing. Significant laboratory findings include lesions observed in late term aborted fetuses and placentas and results of cultures of late term aborted fetuses for bacteria. Most late term aborted fetuses have a prenatal pneumonia, which is characterized by infiltrates of inflammatory cells in bronchioles and alveoli, colonies of bacteria in bronchioles and alveoli, engorged pulmonary vasculature and presence of abundant squames. Placental lesions are characterized by umbilical amnionitis and interstitial placentitis. Cultures of 400 + fetuses and placentas have yielded Streptococcus spp. (51%) and Actinobacillus spp. (17%). At this time, the cause of MRLS has not been identified.
73: Gene Expression by Bovine Macrophages in Response to Mycobacterium Avium Subsp Paratuber-Culosis
The biochemical processes within macrophages that determine whether mycobacterial organisms are killed or establish a chronic infection are poorly defined. To better understand the interaction of bovine macrophages with Mycobacterium avium subspecies para-tuberculosis (M. a. ptb), we determined gene expression by bovine monocyte-derived macrophages 24 hours after addition of M. a. ptb organisms using a human noncompetitive oligonucleotide microarray. To identify genes most likely to be of importance to resistance to infection, we compared genes expressed by macrophages incubated with M. a. ptb to macrophages incubated with the less pathogenic organism Mycobacterium avium subspecies avium (M. a. a). Of 42 genes that were differentially expressed, the majority were involved in cells signaling (n = 11), apoptosis (n = 11), transcription/translation (n = 8), and inflammation (n = 5). When compared to M. a. a-infected macrophages, M. a. ptb-infected macrophages had greater expression of protein tyrosine phosphatases (n = 4) and lesser expression of protein kinases (n = 3). Expression of STAT-induced STAT inhibitor 3, a potent suppresser of cytokine signaling, was also greater in M. a. ptb-infected macrophages. Over expression of protein phosphatases and suppressers of cytokine signaling and under expression of protein kinases may inhibit organism- and cytokine-induced activation of macrophages and inhibit their capacity to kill organisms and induce inflammatory and immune responses.
74: Identification of Conserved Calcineurin Binding Motif in Human T Lymphotropic Virus-1 P121: Role in Modulating Nfat-Mediated T Cell Activation
Human T-lymphotropic virus type 1 (HTLV-1) infection is associated with a diverse range of lymphoproliferative and neurodegenerative diseases, yet pathogenic mechanisms induced by the virus remain obscure. This complex retrovirus, like bovine leukemia virus, contains typical structural and enzymatic genes, but also unique regulatory and accessory genes in the pX region of the viral genome (pX ORF I-IV). Emerging evidence indicates that the HTLV-1 accessory proteins are critical for establishment of viral infectivity. We have reported that p121 (encoded in p × ORF I) is required for resting T cell infectivity and activates the Nuclear Factor of Activated T cells (NFAT) by releasing calcium from the endoplasmic reticulum (ER). In this study, we identified a highly conserved motif in p12I (PxIxIT), which is highly homologous to a calcineurin-binding motif of NFAT. Wild type p12I and a C-terminal mutant (aa 48-99) containing the PxIxIT motif expressed from transfected plasmids bound endogenous calcineurin in both 293T and Jurkat T cells using immunoprecipitation and calmodulin agarose bead assays. However, a N-terminal mutant (1-47) lacking the motif failed to bind calcineurin and an alanine mutant (AxAxAA) had decreased binding affinity. p12I competed with NFAT for calcineurin binding in calmodulin bead pull down experiments. Interestingly, the AxAxAA mutant had increased NFAT transcriptional activity (∼ 2 fold) in the Jurkat T cells compare to wild type p12I. Ours is the first report to identify a retroviral protein that binds calcineurin. We propose that p12I modulates NFAT activation and thereby T cell activation by both causing calcium release from ER stores, as well as by binding calcineurin.
75: Cns Immune Activation in Acute Siv Infection.
Neurological disease ranging from subtle cognitive deficits to overt dementia is a common manifestation of HIV infection but the pathogenesis of HIV CNS disease remains poorly defined. Systemic immune responses accompanying initial plasma viremia, such as production of pro-inflammatory cytokines, induce innate CNS immune responses including activation of the endothelial cells forming the blood-brain barrier and brain macrophages. This study examined the evolution of innate CNS immune responses during acute infection in a well-characterized SIV/macaque model of HIV CNS disease to determine whether activation of CNS immune responses may promote initial viral entry and replication in the brain. The levels of endothelial cell ICAM-1 expression and macrophage/microglial expression of CD68 and antigen detected by HAM56 in basal ganglia were measured by immunohistochemical staining followed by computerized image analysis. To specifically evaluate activation of the perivascular macrophage subset of brain macrophages, an anti-CD 163 antibody specific for macaque perivascular macrophages was characterized by confocal laser scanning microscopy. Subsequently, perivascular macrophage activation was quantitated by image analysis. In parallel, CNS viral load was quantified by real-time RT-PCR. Statistically significant increases in both endothelial and brain macrophage activation (p < 0.05, Mann-Whitney test) were found 10 days post-inoculation with SIV coincident with initial SIV replication in the CNS. Although overt encephalitis does not develop during acute infection, dramatic CNS immune activation that develops during acute HIV infection may paradoxically contribute to development of HIV CNS disease by providing a fertile field for initial HIV replication in the brain.
76: A Mouse Model of Hepatocellular Carcinoma: Ectopic Expression of Fgf 19 in Skeletal Muscle of Transgenic Mice
Most mouse models of hepatocellular carcinoma have expressed growth factors and oncogenes under control of a liver-specific promoter. In contrast, we describe here the formation of liver tumors in transgenic mice overexpressing human fibroblast growth factor 19 (FGF19) in skeletal muscle. FGF19 transgenic mice had elevated hepatic alpha-fetoprotein mRNA as early as 2 months of age, and hepatocellular carcinomas were evident by 10 months of age. Increased proliferation of pericentral hepatocytes was demonstrated by BrdU incorporation in the FGF19 transgenic mice prior to tumor formation and in non-transgenic mice injected with recombinant FGF19 protein. Areas of small cell dysplasia were initially evident pericentrally, and dysplastic/neoplastic foci throughout the hepatic lobule were glutamine synthetase positive, suggestive of a pericentral origin. Consistent with chronic activation of the Wingless/Wnt pathway, 44% of the hepatocellular tumors from FGF19 transgenic mice had nuclear staining for beta-catenin. Sequencing of the tumor DNA encoding beta-catenin revealed point mutations that resulted in amino acid substitutions. These findings suggest a previously unknown role for FGF19 in hepatocellular carcinomas.
77: Pthrp Expression and Bone Metastases of Human Breast Cancer (Lcc15) and Lung Cancer (Hara) Cells in Nude Mice
Breast and lung cancer exhibit a propensity to metastasize to bone, however the pathogenesis of this is not well understood. Parathyroid hormone-related protein (PTHrP), which is involved in the activation of osteoclasts, is secreted from several tumor types that metastasize to bone. We investigated the development of bone metastases using the cell lines LCC15 (human breast carcinoma) and HARA (human pulmonary squamous carcinoma) after intracardiac injection in nude mice. Both cell lines aggressively metastasize to bone, however LCC15 produces minimal PTHrP and HARA secretes high levels of PTHrP. Plasma total calcium and PTHrP concentrations were measured. TRAP-positive osteoclast numbers were measured in long bones. PTHrP mRNA was measured by quantitative RT-PCR. LCC15 injected mice developed extensive bone metastases (evaluated histologically and radiographically) that were highly infiltrative, but demonstrated minimal osteoclast activation and bone destruction. HARA bone metastases were much fewer in number, however they demonstrated marked osteoclast activation and pronounced bone destruction. Plasma total calcium levels were increased in two mice from each group; plasma PTHrP levels were elevated in 12 of 17 mice from the HARA group, and were undetectable in the LCC15 group. There was a 17-fold increase in PTHrP mRNA in HARA bone metastases compared to muscle metastases. Although both the LCC15 and HARA cell lines metastasize to bone, the nature of the metastases are different. HARA cells produce and secrete PTHrP, which likely plays an important role in the osteolytic nature of the bone metastases. LCC15 cells produce little PTHrP, and must have other features that allow them to target bone.
78: Mouse Models of Experimental Breast and Prostate Bone Metastasis: Non-Invasive Visible Light Imaging of Luciferase-Labeled Tumor Cells Vs. Digital X-Ray Examination
Breast and prostate models of experimental bone metastasis were developed using non-invasive visible light imaging of luciferase-labeled human prostate (PC-3M) and breast (MDA-MB-231) cancer cell lines (in male and female mice, respectively). After intracardiac injection into the left ventricle through the diaphragm, tumor cells were present in the metaphyses of the tibia and femur, the roots of the teeth, and sometimes in the humerus, ribs and other sites. Growth of these tumors was often associated with significant bone lysis, which was more pronounced in prostate metastases. Bone damage was evaluated ex vivo by digital x-ray examination (Faxitron). Tumor burden was assessed in vivo by quantifying visible light emission from luciferase-labeled tumor cells (IVIS CCD camera system), and was confirmed by histopathological examination of the bones. Animals were imaged starting 10 days after tumor cell injection, and those with characteristic tumor burdens were sacrificed, then radiographed 34 days after implantation. Bones were examined by routine histology. While the x-ray examination provided accurate assessment of the extent of bone damage, bone lysis was not always a reflection of the tumor burden. Metastases of PC3M caused significantly more destruction of the bone in the tibia than MDA-MB-231, despite the presence of comparable numbers of tumor cells. Thus, the two analytical approaches, digital x-ray, and visible light imaging are complementary, and should be used together in evaluation of progression of bone metastases in animal models. We will present and discuss effects of a novel bis-phos-phonate, zoledronate, and a novel COX-2 inhibitor, lumiracoxib, on inhibition of growth of tumor cells and preservation of bones in the experimental model of prostate-to-bone metastasis.
79: Differential Response of Bovine Macrophages to Mycobacterium Avium Subsp. Paratuberculosis and Mycobacterium Avium Subsp. Avium
Mycobacterium avium subspecies paratuberculosis (M. a. ptb) and Mycobacterium avium subspecies avium (M. a. a) are genetically and antigenically very similar but differ in virulence for cattle. M. a. ptb causes a severe chronic intestinal infection (i.e. Johne's disease), whereas, M. a. a causes a self-limiting infection. We compared the response of bovine monocyte-derived macrophages to ingestion of M. a. ptb and M. a. a by determining organism survival, superoxide and nitric oxide production, and expression of the cytokines tumor necrosis factor-alpha (TNF), interferon-gamma (IFN), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-12 (IL-12), and granulocyte-monocyte colony-stimulating factor (GM-CSF) at 6, 24, 72, and 96 hour of incubation. Macrophages infected with M. a. a were able to kill approximately half of the organisms within 96 hours, whereas M. a. ptb were not killed. No differences were detected in superoxide or nitric oxide production between macrophages infected with M. a. ptb or M. a. a. M. a. ptb-infected macrophages had greater expression of IL-10 and GM-CSF (all time periods) and IL-8 (72 hr) and had lesser expression of TNF (6 hr), INF (6 hr), and IL-12 (72 hr) when compared to macrophages activated by addition of lypopolysaccharide and interferon. M. a. ptb-infected macrophages had greater expression of IL-10 (24 hr) and lesser expression of TNF (6 hr) when compared to M. a. a-infected macrophages. These data indicate that the virulence of M. a. ptb organisms may be due to inherent resistance of the organism to intracellular degradation and suppression of macrophage activation by over secretion of IL-10.
80: Lamb Age Affects Susceptibility and Lesion Severity in Bovine Respiratory Syncytial Virus Infection
Bovine respiratory syncytial virus (BRSV) infection is a significant cause of respiratory disease in cattle and sheep. The purpose of this study was to evaluate the effect of age on the severity of BRSV infection in lambs. In this study, two groups of commercial lambs (3-5 days old or 12-weeks old) were infected intranasally and intratracheally with BRSV inoculum. Each group of lambs was randomly placed in time points (3, 6-7, and 14 days post inoculation [PI]) for evaluation. Evaluation of disease severity was assessed by gross and microscopic examination, and BRSV antigen was detected by immunohistochemistry. At 6-7 days PI, the neonatal lambs grossly had multifocal consolidation (3-5 mm) in all lung lobes. Microscopically, infected bronchiolar epithelium had segmental necrosis with intraluminal neutrophils and cell debris. Remaining areas of bronchiolar epithelium were proliferative with frequent mitotic figures. Older lambs had minimal to no lesions. Immunohistochemistry for BRSV antigen intensity and distribution showed increased staining in the neonatal tissue compared to the older lambs. Our results suggest neonatal lambs are more susceptible to BRSV infection and exhibit more severe gross and microscopic lesions.
81: Experimental Infection by Leptospira Interrogans Serovar Pomona in Dogs
An established experimental model for studying leptospirosis in dogs was used to study infection by Leptospira interrogans serovar pomona. Seronegative 9-week-old female beagle dogs were inoculated with serovar pomona (principals, n = 12) or leptospire-free media (controls, n = 5) via the conjunctiva. Clinical signs were recorded and clinical pathology assays and necropsies (3/timepoint) were done at 7, 10, 14, and 20 days post-inoculation (PI). Control dogs remained healthy and also were necropsied (1/timepoint). Clinical signs in serovar pomona inoculated dogs began 7 days PI and included lethargy, fever, inappetence, dehydration, and diarrhea. On day 10, gross lesions included multifocal renal hemorrhage, perirenal edema, and pulmonary hemorrhage. Pulmonary hemorrhage also was present in dogs necropsied on day 14 and 20 PI. Inoculated dogs had histopathologic lesions at 7, 10, 14, and 20 days PI. Lesions at day 7 were subtle and confined to liver (perivascular inflammation) and kidney (multifocal interstitial nephritis). On day 10, interstitial nephritis was diffuse and infiltrates around hepatic portal veins were more severe. Interstitial nephritis was multifocal and more intense in dogs necropsied on days 14 and 20. However, the periportal inflammation was less severe by day 20. Dilated proximal convoluted tubules and cortical mineral deposits were present at day 20 only. Multifocal alveolar pneumonia was present in dogs on days 10, 14, and 20 PI. Elevations in BUN, anion gap, and bilirubin were sporadic in infected dogs. Platelet counts from culture positive dogs were decreased from baseline values on post inoculation days 10, 12, and 14. This work demonstrates that dogs are susceptible to serovar pomona and develop clinicopathologic changes, some of which progress in severity with time.
82: The Role of Black Flies in the Transmission of Vesicular Stomatitis Virus New Jersey Serotype Infection in Pigs: A Pathologist's Viewpoint
Vesicular stomatitis (VS) is a disease of economic importance to the livestock industry not only because of economic loss associated with decreased production but because the clinical signs in cattle and swine mimic those of foot-and-mouth disease. The causative agents are a group of antigenically related but distinct viruses of the genus Vesiculovirus, family Rhabdoviridae. Despite the repeated isolation of vesicular stomatitis virus New Jersey serotype (VSV-NJ) from insects during epizootics, their role in virus transmission and the pathogenesis of the disease remains controversial. We present visual evidence that black flies can be infected with VSV-NJ and transmit the virus to pigs. Following intrathoracic inoculation, VSV-NJ antigen was detected throughout the body, including the salivary gland, of black flies (Simulium vittatum) by immunohistochemistry. Similarly infected black flies were able to transmit the virus to pigs when fed on the snout, resulting in lesions and virus antigen distribution similar to that seen in natural disease and with other experimental models of disease. When infected flies fed on the snout, vesicular lesions developed on the snout and secondary vesicles occurred on the lower lip and tongue. Microscopically, the snout skin had vesicle formation, intense epidermal and perivascular infiltrations of inflammatory cells, and viral antigen in snout epidermis. Viral antigen was also detected in tonsillar crypt epithelium and in epithelium of secondary vesicles, but not in lymph nodes. These findings support the hypothesis that insect transmission plays a role in the epidemiology of VSV-NJ.
83: Physical Map and Genome Sequencing Survey of Haemobartonella Felis (Mycoplasma Haemofelis)
Haemobartonella felis (Mycoplasma haemofelis) is a member of a newly defined group of mycoplasmas that parasitize the red blood cells of mammals and cause a hemolytic anemia. A renewable source of H. felis DNA, a large insert genomic library, was created by cloning partially digested H. felis DNA into a bacterial artificial chromosome (BAC) vector. BAC clones were placed in order and a physical map of restriction enzyme sites was made using a combination of hybridization and fingerprinting techniques. A set of 24 BAC clones was identified that represented the complete genome with minimal overlap. The total genome size of H. felis was approximately 1200 kilobases and contains 28 Nru I sites, 21 Sal I sites and 9 Not I restriction sites. To identify genes that may be involved in pathogenicity and survival, BAC clones dispersed throughout the genomic encyclopedia of H. felis were sub-cloned into a plasmid vector and used to generate genomic sequencing surveys (GSSs). Among the 624 GSSs obtained, 165 (26.4 %) had significant BLASTn or BLASTx homology scores. This analysis revealed the two previously identified H. felis genes (16S rRNA and ribonuclease P RNA) and 75 distinct genes that had not been previously reported in H. felis. The majority of significant scores were to genes and proteins in the Mycoplasma and Ureaplasma genera. The remaining GSSs with significant homology scores were split evenly between members of the Bacillus/Clostridium group and other miscellaneous bacteria. The average guanidine and cytosine percentage of the GSSs was 38.5%. This manuscript represents the first in depth genomic study of any of the haemotrophic mycoplasmas and is the first physical map and genomic encyclopedia of an uncultured organism.
84: In Vivo Analysis of Human T-Lymphotropic Virus Type 1 Surface Envelope Mutants in a Rabbit Model of Infection
Human T-cell leukemia virus type 1 (HTLV-1) infection causes adult T-cell leukemia/lymphoma. The HTLV-1 envelope consists of surface glycoprotein gp46 (SU) and transmembrane glycoprotein gp21 (TM). HTLV-1 SU regions between aa 75-101 and 181-208 are important for syncytia formation in vitro. To identify motifs important for in vivo viral spread, Env point mutations at aa 95, 105, and 195 were generated within an infectious HTLV-1 clone, ACH. All mutated viral clones were capable of immortalizing CD4+ lymphocytes. Lymphocyte cell lines immortalized by wild type ACH, and ACH-env point mutant clones (ACH-95, ACH-105, ACH-195), were intravenously injected into rabbits (10 million cells per rabbit). Serum and peripheral blood lymphocytes (PBL) were harvested at 2, 4, and 8 weeks (wks) post inoculation (p.i.), and rabbits were necropsied at 8 wks p.i. Rabbits inoculated with wild type ACH, ACH-95 or ACH-105 produced strong antibody responses to both Env and Gag proteins by 4 wks p.i. Rabbits inoculated with ACH-195 had no anti-HTLV-1 antibody at wk 2, and weak or indeterminate responses at 4 and 8 wks pi. Ex-vivo cultures of PBL tested for viral antigen (p19 Matrix) production correlated with serologic data. We conclude that 1) in vitro assays for HTLV-1 infectivity do not always correlate to in vivo behavior, and 2) aa 195, previously identified to be an important immunodominant region of HTLV-1 SU, may be also important for mediating viral infectivity in vivo. Future studies will be directed at delineating additional critical motifs in the HTLV-1 SU that allow viral spread in the rabbit model of infection.
85: Protozoal Gastritis in Rhesus Macaques Infected with Simian Immunodeficiency Virus
Many of the opportunistic infectious agents recognized in AIDS patients are also seen in rhesus macaques infected with simian immunodeficiency virus (SIV). We performed a retrospective study examining gastritis in SIV-infected rhesus macaques. Of 467 complete necropsies performed from 1993 to 2002 on SIV-infected rhesus macaques at the New England Regional Primate Research Center, we identified 107 (23%) with gastritis. Protozoa histologically compatible with Trichomonas sp. were found in the gastric lesions of 18 of these animals (17%). Because of the unusual features of the trichomonads (variation in shape and size), unusual location (gastric mucosa) and severity of the lesions with protozoa, transmission electron microscopy (TEM) and polymerase chain reaction (PCR) were used to further identify these protozoa. Using TEM we were able to confirm the histologic finding of trichomonads, with flagella and an undulating membrane. Preliminary results of PCR confirmed that trichomonads are present. In order to determine the role of the trichomonads and the impact of various factors contributing to the development of SIV-associated gastritis, additional histologic stains (acid fast, silver stain) and immunohistochemistry for cytomegalovirus (CMV) were performed to rule out other microorganisms. In all 18 cases of gastritis with intralesional protozoa, Trichomonas sp. were identified. The number of intralesional trichomonads correlated positively with the severity and type of gastritis (necrosuppurative). In all of the animals with Trichomonas gastritis, AIDS confirming opportunistic infections, including CMV, adenovirus, SV40 and microsporidia were identified in different tissues. We conclude that Trichomonas is a significant co-factor in the development of necrosuppurative gastritis in SIV-infected rhesus macaques.
86: Myxoma Virus Serp2 Alterations Have Unexpected Effects on Viral Pathogenesis
Wild type myxoma virus (MYX) encodes a variety of virulence factors including the serine protease inhibitor (serpin) SERP2. Like CrmA of cowpox virus, SERP2 has been shown to inhibit caspases 1, 8, 10 and granzyme B in vitro. However, CrmA is a more effective inhibitor than SERP2. MYX viruses, in which serp2 was mutated or replaced by crmA, were constructed to evaluate the role of SERP2 during infection. MYXserp2::IacZ is a serp2 deletion mutant. MY-Xserp2::crmA expresses CrmA instead of SERP2. MY-Xserp2D294A replaces the P1 aspartate of SERP2 with alanine, which inactivates the serpin. MYXserp2D294E contains glutamate at the P1 site and was examined because the serpin PI-9 has glutamate at P1 and inhibits granzyme B but not caspases. We show that SERP2D294E inhibits caspase-1 efficiently but activity against caspases-8, 10 and granzyme B is significantly reduced. Consistent with these findings, MYX and MYXserp2::crmA block apoptosis during infection of RK-13 cells but MYXserp2::lacZ, MYXserp2D294A and MYXserp2D294E do not. During infection of NZW rabbits, MYXserp2::lacZ was attenuated and did not cause myxomatous lesions and only a few secondary lesions formed. Infections with MY-Xserp2D294A were far more attenuated than MYXserp2::lacZ with few signs of illness. MYXserp2D294E infections caused viremia, myxomatous lesions and signs of illness indistinguishable from MYX infection. Infections with MYXserp2::crmA were similar to those seen with MYX in terms of illness and viremia but somewhat less mortality was observed. Skin lesions caused by MYXserp2::crmA were non-myxomatous and resembled those of MYXserp2::lacZ. These results suggest that CrmA does not fully substitute for SERP2 in dermal lesions and that the ability to control apoptosis in infected cells does not correlate with virulence.
87: Gastric Cytokine, Growth Factor, and Receptor Transcripts Are Elevated in Mice with Severe, Rapidly Progressive Gastritis Due to Helicobacter Pylori Infection
CD4+ T-cells are necessary for development of gastritis, a DTH-response, and gastric epithelial proliferation, and CD4+ splenocytes secrete interferon-gamma in response to H. pylori antigen. The goal of this study was to further test the hypothesis that interferon-gamma indirectly elicits proliferation of gastric epithelium via stromal or epithelial growth factors. Five mice from the following groups were used: H. pylori strain SS1 infected C57BL/6 severe combined immunodeficient (SCID) mice recipients of splenocytes from congenic wild-type (WT) mice, uninfected non-recipient SCID, and infected and uninfected WT mice. Mice were killed 8 weeks after inoculation. RNA was isolated from gastric mucosa. Quantification of transcripts using real-time RT-PCR was performed with primers for interferon-gamma, interferon-gamma receptor, IL-12, IL-10, KGF, KGF receptor, TGF-alpha, and GAPDH. Transcript levels were normalized to GAPDH and reported as fold increase over negative controls (uninfected, non-recipient SCID mice). Interferon-gamma, interferon-gamma receptor, and IL-12 transcript levels in infected, recipient SCID mice were significantly elevated (3-4 log-fold) over negative controls. There were no significant differences in IL-10 transcript levels. KGF and KGF-receptor transcript levels in infected mice were significantly elevated over negative controls (6-7 log-fold). TGF-alpha transcript levels in infected mice were elevated (3 log-fold) over negative controls. The transcript levels support a strong association between interferon-gamma and stromal and epithelial growth factors. There was an apparent up-regulation in interferon-gamma receptor in infected, recipient SCID compared to infected WT mice. This might relate to the rapid progression of this model. Experiments in progress (ELISA, IHC, in situ hybridization) will help further define this model.
88: Upregulation of Il-1 and Il-6 in Bluetongue Virus Infected Blood Mononuclear Cells of White-Tailed Deer (Odocoileus Virg1Nianus)
Hemorrhagic disease, caused by closely related Orbiviruses in the bluetongue and epizootic hemorrhagic disease virus serogroups, is the most important viral disease affecting white-tailed deer (WTD) throughout their range in North America. Clinically, the disease caused by these viruses is highly variable with effects ranging from subclinical to death. The reasons for this clinical variation are unclear and poorly studied, but this variation is possibly related to variable degrees of acquired and innate immunity. We hypothesize that the clinical outcome of BTV infection in WTD is due to the response of the microvasculature to inflammatory mediators released from infected cells, specifically mononuclear cells and endothelium. The objective of this study was to evaluate the upregulation of inflammatory mediators, specifically IL-1 and IL-6, in BTV infected WTD blood mononuclear cells. WTD blood mononuclear cells were infected with two doses of partially purified BTV serotype 10 and harvested at various time points post infection to study infection kinetics and upregulation of the inflammatory mediators IL-1 and IL-6. BTV infection of WTD blood mononuclear cells resulted in the upregulation of both IL-1 and IL-6. These data suggest that the pathogenesis of BTV infection in WTD may involve these inflammatory mediators, and future studies to evaluate their effect on the microvasculature is warranted. Such studies would help in understanding the pathogenesis of BTV infection and its management in the free ranging and captive deer population.
89: The Role of Macrophages in Pathogenesis of West Nile Virus in Birds
West Nile Virus has been a significant cause of mortality in wild birds in the United States since it was first recognized in New York in 1999. Marked differences in susceptibility between species have consistently been demonstrated, although the cause for these differences is not well understood. Immunohistochemistry on 269 birds with WNV has consistently demonstrated WNV antigen in large macrophages in the heart, reticuloendothelial cells in the spleen, and Kupffer cells in the liver. Therefore, we hypothesize that macrophages are a major target for WNV and that differences in species susceptibility are due to differences in macrophage/virus interaction. We attempted to assess the macrophage/virus relationship by measuring viral titers and production of nitric oxide by infected macrophages. Macrophages were isolated from the spleens of Northern cardinals and rock doves and infected with WNV. Viral titers and a standard nitrite assay to assess levels of nitric oxide were performed on samples from multiple time points. Viral titers demonstrated an increase in viral titer over time consistent with replication of virus in the cultured macrophages. The nitrite assay demonstrated an increase in nitric oxide post-infection. These findings support the hypothesis that macrophages play a major role in the pathogenesis of WNV in birds and may help to explain differences in species susceptibility.
90: The Effects of Immunosuppression on the Pathogenesis of Avian Leukosis Virus Subgroup J in Broiler Chickens Exposed to Marek'S Disease Virus
Avian leukosis virus subgroup J (ALV-J) infection causes significant economic loss because of increased mortality, tumor production, decreased production, and high costs for eradication. This research was performed to investigate the effects of immunosuppression on ALV-J pathogenesis. The effects of the immunosuppression on ALV-J pathogenesis were determined in chickens with natural exposure to Marek's disease virus. Suppression of B-cell and T-cell was induced by treatment of cyclophosphamide (CY) and cyclosporin A (CSP), respectively. Chickens with immunosuppression of T- and B-cell function were more frequently ALV-J viremic with a higher virus titer compared to non-immunosuppressed groups. In immunohistochemical staining using monoclonal antibodies against ALV-J envelope glycoprotein, expression of viral antigen was present in more chickens in CY treated group compared to the control group at 3 weeks post-infection. With CSP treatment, the overall mean score of viral antigen expression was significantly higher compared to the control group at 10 weeks post-infection. With CY and CSP treatment, expression of viral antigen was significantly higher at 9 and 10 weeks post-infection at the end of the experiment compared to that present in 3 and 4 weeks post-infection, respectively.
91: Imaging and Pathogenesis of Bone Metastases of Human Breast and Prostate Cancer in Nude Mice
Breast and prostate cancer commonly metastasize to bone. The overall goals of this project were to investigate (1) the role of parathyroid hormone-related protein (PTHrP) in metastasis to bone, (2) use luciferase to image bone metastases in vivo, and (3) develop gene therapies to prevent or treat bone metastases in vivo. PTHrP expression by cancer cells is increased in bone due to the release of TGF-beta from bone at sites of resorption. Injection of human breast cancer cells (MDA-231) into the left ventricle of the heart were used to induce bone metastases. The MDA cells induced lytic bone metastases that were demonstrated by radiography at 5 weeks. PTHrP mRNA expression in the MDA cells were measured using quantitative real-time PCR (Roche LightCycler). TGF-beta induced a 2.3-fold increase in PTHrP mRNA. The MDA and human prostate cancer (PC-3) cells were transfected with a cDNA that encodes a combined luciferase/yellow fluorescent fusion protein. The PC-3 cells were injected into the left ventricle and the mice were imaged for bioluminescence (using a Xenogen IVIS Imaging CCD Camera System) after injection of luciferin (3 μg). PC-3 cells were distributed in all areas of the body at 3 min; in lungs, bones, and kidneys at 15 min; no viable tumor cells were present at 24 hr; and bone metastases were detectable at 20 days. This demonstrated the feasibility and increased sensitivity of detection of bone metastases compared to radiography. The MDA cells were stably transfected with an expression vector containing luciferase, cytosine deaminase and tyrosine kinase cDNAs. The cytosine deaminase and the tyrosine kinase will allow treatment of breast and prostate cancer bone metastases with 5-fluorouracil and gancyclovir. These data demonstrated that the MDA cells induced lytic bone metastases in vivo that may have been enhanced by TGF-beta stimulation of PTHrP expression and imaging of bone metastases in vivo using light emitted from luciferase was more sensitive compared to conventional radiography.
92: Chondrocyte Apoptosis Directly Induced by Cyclic Hydrostatic Pressure
Recent studies have indicated that chondrocyte apoptosis is responsible for hypocellularity in osteoarthritic cartilage and could directly contribute to matrix degradation. We investigated whether mechanical stress, in the form of hydrostatic pressure, can directly induce chondrocyte apoptosis by using a novel pressure-loading system. Equine chondrocytes were isolated separately from superficial and deep layers of articular cartilages, embedded in alginate constructs, and intermittently pressurized at 1,000 psi (approximately 6.8 MPa) for 12 hours. Chondrocyte apoptosis was identified with immunocytochemical TUNEL assay. The cyclic hydrostatic pressure-treated chondrocytes had a higher percentage of apoptosis compared with the controls (p < 0.001). The present study demonstrated that articular chondrocyte apoptosis could be directly induced by cyclic hydrostatic pressure. Chondrocyte apoptosis may be an early change of osteoarthritis and may cause matrix degradation.
93: T-Cell Activation Upregulates Parathyroid Hormone-Related Protein Expression in Transformed Human T Lymphocytes
Parathyroid hormone-related protein (PTHrP) is the causative factor of humoral hypercalcemia of malignancy (HHM) observed in most patients with adult T-cell leukemia/lymphoma (ATLL), an aggressive and fatal malignancy of activated T-lymphocytes resulting from HTLV-1 infection. It has been previously shown that HTLV-1 oncoprotein Tax can upregulate PTHrP expression in vitro. However, we showed that PTHrP is highly expressed in ATLL in a Tax-independent manner using a SCID/beige mouse model of HHM. Moreover, very low levels of tax mRNA expression are present in ATLL cells from patients. The objective of the present study was to determine how the signaling transduction in T-cell activation can regulate PTHrP gene transcription independently of tax expression. We observed a strong inverse relationship between PTHrP and tax mRNA expression by quantitative RT-PCR in five ATLL cell lines. Transcription of the PTHrP gene is under the control of three promoter regions (P1, P2, and P3). Examination of alternative PTHrP promoter usage in ATLL cell lines by quantitative RT-PCR demonstrated that P3-initiated transcripts were the most abundant in cell lines with low tax levels, while P2-initiated transcripts were present at greater levels in cells with high tax expression. P1-initiated transcripts were negligible. In ATLL cells tested, we observed constitutive binding of NF-kappa B transcription factors to P2 region, and nuclear factors to ETS/STAT binding sites in the P3 region. In addition, we were able to induce PTHrP mRNA expression in Jurkat cells, a HTLV-1 negative transformed T-cell line, by treatment with PMA, ionomycin, and IL-2, altogether mimicking T-cell activation. Finally, transfection experiments in Jurkat cells resulted in strong transcriptional activity of P2 and P3-luciferase constructs in response to PMA and ionomycin. NF-KB, AP-1, MAPK (upstream of ETS), and STAT signaling pathways are typically induced during T-cell activation. In conclusion, our data suggest that PTHrP mRNA expression in ATLL is transcriptionally upregulated during T-cell activation in a Tax-independent manner via NF-KB, ETS, and STAT pathways.
94: Retinoic Acid Receptor Beta 2 Inhibition of Metastasis in Mouse Mammary Gland Xenografts
The retinoic acid receptor beta 2 protein is a putative tumor suppressor that inhibits proliferation and can induce apoptosis when introduced into breast, cervical, lung, and pancreatic cancer cell lines. To determine if RAR beta 2 suppresses proliferation of mammary-derived cancer cells in vivo, we transduced MDA-MB-435 breast cancer cells with the LXSN retroviral vector containing RAR beta 2 and implanted LXSN vector- or RAR beta 2-transduced cells into the mammary fat pads of nude and severe combined immune deficiency (SCID) mice. We analyzed the xenografts for several tumor parameters, including tumor size, inflammation, vascularity, mitoses, tumor recurrence at the primary site following resection, and metastases. We found that 19 of 52 mice inoculated with vector-transduced cells developed metastases in multiple organs while only 1 of 55 mice receiving RAR beta 2-transduced cells displayed evidence of metastases (p < 0.000001, combined experiments two-tailed Fisher's exact test). Moreover, RAR beta 2-tumor cell recipient mice had a lower incidence of post-resection tumor recurrence (8/55 vs. 25/52, p = 0.0004), 34% less necrosis (in three of four experiments, p = 0.001), and 39% fewer mitoses in tumor tissue (p < 0.000001). Our findings suggest that RAR beta 2 may play a role in inhibiting the metastatic cascade in a mouse mammary gland xenograft tumor model and is a potential candidate for therapeutic intervention in human breast cancer.
95: Canine Prostate Stimulates Osteoblast Activation via An Endothelin-Dependent Mechanism. B
Bone metastases are common in humans and dogs with late-stage prostate cancer. A unique feature of prostate cancer bone metastases is new bone formation at metastatic sites (“osteoblastic metastases”). Most tumors that metastasize to bone cause bone destruction, not new bone formation. The mechanisms of how prostate cancer induces bone formation at sites of bone metastasis are not known. We hypothesized that stimulation of osteoblasts by prostate tissue at metastatic sites is due to the paracrine actions of growth factors produced by prostate epithelial cells. We have previously shown that normal canine prostate tissue induced new bone formation when implanted adjacent to the calvarium of nude mice. To complement this in vivo model, we developed an in vitro system of prostate-stimulated osteoblast activation to investigate mechanisms of prostate-induced new bone formation. We found that treatment of cultured rat calvarium for 24 hours with 100 micrograms of protein from normal dog prostate homogenates stimulated alkaline phosphatase activity 4-6-fold compared to controls. Stimulation began approximately 8 hours after treatment, and was diminished after 72 hours. ALP activity also increased in a dose-dependent manner, with 100 micrograms prostate protein providing maximal stimulation. Calvaria treated with homogenates of normal dog salivary gland, kidney, bladder, and muscle did not increase ALP activity. Pretreatment of the calvaria for 1 hr with endothelin antagonists, but not anti-PTHrP antibody or indomethacin, abrogated the stimulation of ALP. Our results indicated that osteoblast activation by canine prostate occurs via an endothelin-dependent mechanism, and that PTHrP or prostaglandin synthase-mediated pathways are likely not involved. In conclusion, this is a reliable, reproducible assay for determining the roles of molecules important in the activation of osteoblasts by prostate.
96: Pharmacologic Inhibition of Mucus Secretion in Mice Exposed to An Aerosolized Muscarinic Agonist, Methacholine
Asthma and chronic obstructive pulmonary disease (COPD) are disabling diseases that affect over 30 million people in the United States. One of the hallmarks of both diseases is airflow obstruction caused by a combination of bronchoconstriction and excessive mucus plugging, the latter due to increased mucus production and secretion by submucosal glands. Parasympathetic stimulation of airway smooth muscle and submucosal glands via muscarinic receptors promote both bronchoconstriction and mucus secretion. To assess whether inhaled antimuscarinic compounds had potential to block mucus secretion in human patients, an animal model of mucus secretion was developed in mice. Mice were challenged with saline or methacholine, a potent muscarinic agonist. Larynx and trachea were removed from challenged mice and step-sectioned to evaluate laryngeal submucosal glands for mucus using alcian blue/periodic acid Schiff (AB/PAS) staining. Methacholine dramatically reduced AB/PAS staining in laryngeal submucosal glands, consistent with para-sympathetic-mediated mucus secretion. To determine whether aerosolized antimuscarinics could block the reduction in AB/PAS staining, mice were pretreated with the antimuscarinic ipratroprium (Atrovent®) before methacholine challenge. Ipratroprium partially blocked the methacholine-induced decrease in AB/PAS staining suggesting that antimuscarinics may have potential for reducing mucus secretion in COPD and asthma patients.
97: Parainfluenza Virus-3 Pulmonary Lesions Are Not Enhanced by Bovine Viral Diarrhea Virus
Parainfluenza virus-3 (PI-3) is a common respiratory pathogen of cattle and sheep. Bovine viral diarrhea virus (BVDV) is a common bovine pathogen that may enhance respiratory disease. Two groups of neonatal lambs were inoculated intranasally and intratracheally with PI-3/BVDV or PI-3 alone. Both infected lambs and controls (receiving only media) were bled and euthanized 3, 6, and 17 days post-inoculation (p.i.), and the respiratory tract tissues were subsequently collected. The purposes of the study were to: 1) compare acute, subacute and chronic pulmonary lesions in PI-3/BVDV versus PI-3 inoculated lambs, and 2) determine cellular distribution of PI-3 and BVDV antigens within the lung by immunohistochemistry. The histopathological lesions were similar in both groups of animals. In acutely infected animals, they consisted of multifocal to coalescing histiocytic and nec-rosuppurative bronchointerstitial pneumonia. During subacute stages of the infection, histiocytic and suppurative interstitial pneumonia was present accompanied by type II pneumocyte hypertrophy and hyperplasia, and bronchiolitis with epithelial cell hyperplasia. Finally, in chronically infected animals, there was mild fibrous interstitial pneumonia with lympho-histiocytic peribronchitis, peribronchiolitis, and perivasculitis. According to immunohistochemistry, PI-3 viral antigen was abundant 3 days p.i., and present within the cytoplasm of the bronchiolar epithelial cells, to a lesser extent in the macrophages and type II pneumocytes, and very rarely in the bronchial epithelial cells. On day 6 p.i., the amount of viral antigen varied among the animals from small amounts in the most severely affected areas to complete absence, while complete viral clearance was noted on day 17 p.i. In conclusion, the simultaneous presence of PI-3 and BVDV viruses in the lung of neonatal lambs does not enhance lesion severity, or persistence and distribution of PI-3 antigen.
98: Restoration of Cftr Chloride Channel Activity in Ib3 Cells with Aav-Minigene Constructs
rAAV vectors have been shown to stably transduce respiratory epithelium, but the AAV packaging capacity limits the size of the expression cassette to ∼4.5kb, preventing the inclusion of a strong promoter with full-length CFTR. Previous experiments have demonstrated restoration of CFTR activity with a N-terminally truncated minigene (D264CFTR) in Xenopus oocytes and IB3 cells (a CF bronchial cell line), and we have recently shown that the CMV-enhanced beta actin promoter (CBA) is 100-fold stronger than CMV in the lung. We hypothesize CFTR minigenes driven with the CBA will enhance overall vector-mediated CFTR expression. IB3 cells were stably transfected or transduced with pCBA-D264CFTR or pRSV-D264CFTR, and cell cultures were expanded. A 36C1-efflux assay was used to screen for CFTR function, comparing the rate of efflux before and after stimulation with an agonist mixture. Patch clamp analysis was used to determine CFTR-specific bioelectric activity before and after stimulation with an agonist mixture. pCBA-D264CFTR-transfected IB3 cells demonstrated a significant increase in cAMP-activated chloride efflux (1.31 ± 0.71) as compared to the non-transfected negative control IB3 cells (0.80 ± 0.10) (p < 0.05). Patch clamp analysis verified CFTR specific bioelectric activity for the pCBA-D264-CFTR transfected IB3 cells. Preliminary results on IB3 cells stably transfected with pRSV-D264CFTR also demonstrate bioelectric patterns characteristic of CFTR, and 36C1-efflux upon stimulation. Functional restoration of CFTR activity was demonstrated in IB3 cells with rAAV constructs containing a 264 amino acid N-terminally deleted CFTR gene driven by CBA and RSV promoters. These constructs are currently being developed for in vivo application. Funded by grants from the NIH (HL51811) and the CF Foundation.
99: Effect of Bovine Granulocyte Colony Stimulating Factor on the Development of Pneumonia Caused by Mannheimia Haemolytica
The role of recruited neutrophils in Mannheimia haemolytica infection is controversial. We hypothesized that elevated neutrophil count in blood prior to infection with M. haemolytica would lead to rapid bacterial clearance and less severe lesions. This experiment investigated the effect of recombinant bovine granulocyte colony stimulating factor (G-CSF) on blood neutrophil counts, and the response to intrabronchial challenge with M. haemolytica. Two replicate experiments (A and B) were conducted, in which 4 calves per experiment were treated daily with 5 micrograms/kg G-CSF, and 4 calves per experiment were treated with saline. At day 10 of treatment, all 16 calves were challenged with 5 × 109 CFU/ml (experiment A) or 4.5 × 108 CFU/ml (experiment B) M. haemolytica bacteria, into the right bronchus by bronchoscope-placed catheter. The average blood neutrophil counts in G-CSF-treated and untreated calves after four days of treatment were 60.8 and 8.4 × 109/L, respectively. Two untreated calves became neutropenic and were euthanized two days after infection because of severe respiratory distress. Clinical signs in the other calves were similar in G-CSF-treated and non-treated calves, and these calves were euthanized five days after infection. G-CSF-treated calves had 37% lower lung lesion scores than non-treated calves, and this difference was significant (p = 0.04) when the effect of previous antibody titer to leukotoxin was considered. Lung weights were similar in G-CSF-treated and non-treated calves (1.93 and 2.03% of body weight, respectively). Histologically, G-CSF-treated calves had less severe pulmonary parenchymal necrosis and infarction. In conclusion, G-CSF induced neutrophilia, and G-CSF-treated calves had milder gross pulmonary lesions following experimental infection with M. haemolytica.
100: Dextran Sulfate Sodium Causes Suppurative and Ulcerative Inflammation in the Cecum and Large Intestine in Piglets
Dextran Sulfate Sodium (DSS) induces suppurative and ulcerative colitis in rodents and primates when administered in the drinking water. We previously developed a 3-day-old pig model of E. coli O157:H7 infection in which colostrum-fed pigs infected with E. coli O157:H7 at 72 hours of age develop classic attaching and effacing lesions, but lack a histologically detectable host inflammatory response in the enteric mucosa and evidence of systemic Shiga toxin (Stx) absorption. In order to assess the effect of inflammation on mucosal permeability to Stx, a reliable method to induce a host inflammatory reaction in the 3-day-old pig model is needed. Colostrum fed York/Duroc cross newborn pigs were housed in climate controlled stainless steel cages and fed Esbilac milk replacer. Treatment pigs received 1.0, 1.5, 3.0, 5.0, 7.5, or 10.0 gram% DSS in 20 ml milk replacer 2 times daily, starting 1 day after arrival. Control pigs received a sham treatment of 20 ml milk replacer. Pigs were monitored for appetite, activity level, bloody diarrhea, and dehydration. Pigs were euthanized by barbiturate overdose at 4, 5, or 6 days of age or when clinical signs dictated. Samples of jejunum, ileum, cecum, spiral colon, and rectum were collected in 10% neutral buffered formalin. Treatment pigs, but not controls, developed severe bloody diarrhea by 4 days post-treatment and had hemorrhagic typhlocolitis and spiral colon edema at necropsy. Histologically, by 3 days post-treatment, treatment pigs had dose-dependent neutrophil infiltration in the cecum, spiral colon, and rectum. By 4 days post-treatment, treatment pigs had histologic hemorrhagic, ulcerative, and suppurative typhlocolitis. In conclusion, DSS induces suppurative, ulcerative, and hemorrhagic typhlocolitis in piglets in a dose dependent manner.
101: Does Long Polar Fimbriae Facilitate Colonization by Escherichia Coli O157:H7 in Pigs?
The contribution of long polar fimbriae (1pf) has been evaluated for the development of attachment and effacement (A/E) lesions in the intestines of germ-free piglets. Loci have been identified in E. coli O157:H7 that bear homology to 1pf genes in Salmonella enterica, serovar typhimurium that are important colonization and virulence factors for Salmonella. Two isogenic mutants of Escherichia coli O157:H7 strain 86-24 were evaluated, a single mutant, 1pfAl-, and a double mutant, 1pfA1-1pfA2-. Germ-free pigs were inoculated at 24 hours old with 105 cfu of bacteria. Eight pigs were infected with the single 1pfA1- mutant strain, four with the double mutant, nine with the wild type, and three were inoculated with a nonpathogenic strain of E. coli. Pigs were necropsied at 24 hours post-inoculation. To evaluate colonization, tissues were collected for histopathology. Multiple sections of intestine were scored for the extensiveness of A/E lesions. The results showed a minimal reduction in colonization in the intestine by the single 1pfA1- mutant when compared to the wild type strain, with the average colonization scores in the same category (moderate, 10%-50% affected). However, the tissues from the double mutant had a greater reduction in colonization with the A/E scores in the minimal category (0%-10% affected). The data suggests 1pf facilitates colonization. Colonization and lesion development occur to a lesser extent in the absence of Lpf. Additionally, these results support the in vitro work that demonstrates a reduction of adherence to tissue culture cells by the mutant strain.
102: Characterization of Gene Expression in Mouse Hepatoblastoma-Derived Cell Line
Characterization of MHB-2, a cell line derived from mouse hepatoblastoma, was performed by RT-PCR and immunocytochemical methods to cast light on the histogenesis of mouse hepatoblastoma. RT-PCR for c-kit, CD34 and Thy-1, all of which are expressed in oval cells or hematopoietic stem cells, were carried out on MHB-2 cells. Expression of alpha-fetoprotein (AFP) and albumin (ALB) mRNA was also examined as markers of fetal or adult hepatocytes. In addition, the cell line was investigated immunocytochemically for c-kit, AFP and ALB. Analysis of gene expression revealed MHB-2 cells expressed c-kit, CD34 and Thy-1, whereas no expression of AFP and ALB gene was detected. Immunocytochemically, MHB-2 cells were positive for c-kit but negative for AFP and ALB. MHB-2 cells expressed c-kit as well as CD34 and Thy-1 genes, suggesting MHB-2 cells possessed an immature character similar to oval cells or hematopoietic stem cells. On the other hand, absence of AFP and ALB gene expression suggested that MHB-2 cells were on a different lineage from hepatocyte or originated from less-differentiated cells.
103: Histologic, Angiographic and Tissue Perfusion Changes Associated with Progressive Vascular Occlusion by Ameroid Constrictors
Ameroid constrictors are rigid-jacketed devices used to model chronic ischemia that cause progressive constriction when implanted around vascular structures. We compared the histologic, angiographic and tissue perfusion changes in the normally perfused distribution of the left anterior descending (LAD) coronary artery with the ameroid constricted distribution of the left circumflex (LCX) coronary artery. The proximal LCX of 12 female Yorkshire swine (age: 3.9 ± 0.9 mo) were implanted with a 2.25 mm titanium-jacketed ameroid constrictor. Coronary blood flow was assessed by selective coronary angiography prior to and at 3-4 and 6 weeks post implant. Myocardial perfusion was assessed by neutron activated microsphere techniques at baseline and during adenosine-induced hyperemia (140 microgram/kg/min IV). Angiography revealed progressive LCX constriction with 95-100% occlusion at 6 weeks. Patchy, mild to moderate fibroplasia and myocardial cell degeneration and loss were seen over time (p < 0.001) in the LCX distribution. At 6 weeks, baseline blood flows in the LAD and LCX distributions were 1.26 ± 0.34 and 1.02 ± 0.34 ml/g/min, respectively. Hyperemic flow was significantly reduced (p = 0.025) in the LCX distribution (LAD = 2.67 ± 1.29 versus LCX = 1.19 ± 0.56 ml/g/min), a 48% decrease in perfusion reserve. This study demonstrates patchy, mild to moderate fibroplasia and myocardial cell degeneration and loss associated with diminished hyperemic blood flows in the distribution of chronic coronary artery constriction by ameroid occlusion.
104: Morphologic Features of a Clinically Relevant Piglet Model for Lethal Staphylococcal Enterotoxin B (Seb) Intoxication
Staphylococcal enterotoxins cause serious gastrointestinal illness, and intoxication with these powerful superantigens can lead to lethal shock. In order to overcome significant shortcomings of current rodent and non-human primate models of SEB intoxication, we developed a piglet model of lethal SEB intoxication that exhibits a clinical syndrome of vomiting, diarrhea and anorexia with progression to prostration and death. These sequelae are similar to that observed in humans exposed to SEB. In this pathogenesis study, piglets were euthanized at 24, 48, 72 and 96 hours after intravenous SEB administration. At 24 hours, there was subtle diffuse edema suggesting vascular leakage; and lymphoid tissues, excluding the thymus, were enlarged and congested. By 72 hours there was severe widespread edema, which was most prominent in the mesentery between loops of spiral colon and in the retroperitoneal connective tissue. The severity of lymphadenopathy was also progressive and by 72 hours post-treatment, lymph nodes were markedly enlarged, dark red and firm. Early histologic changes in these lymph nodes included diffuse lymphoid hyperplasia and congestion, and by 72 hours there was severe hemorrhage and edema with perivascular fibrin deposition and widespread lymphocytolysis. These changes were first detected and were most severe in the mandibular and mesenteric nodes. Electron microscopy was used to further define the vascular and lymph node changes. Additional histologic changes included perivascular aggregates of large lymphocytes variably present in the lung and lymphocytic portal hepatitis. Study of this piglet model will further elucidate the pathogenesis of SEB intoxication and enable us to test new therapeutic regimes.
105: Histopathologic Features in the Congenitally Carnitine-Deficient Juvenile Visceral Steatosis (Jvs) Mouse
Carnitine is an essential cofactor to transport the long-chain fatty acids for beta-oxidation in the mitochondria. The morphological features of carnitine deficiency were studied in a mutant strain of juvenile visceral steatosis (JVS) mice with congenital carnitine deficiency. Untreated young mice showed marked growth retardation associated with severe hepatomegaly and fatty liver. In contrast, cardiac hypertrophy and systemic circulatory impairment were the main findings in mice at adult and old age when maintained under L-carnitine treatment. The principal lesions common to homozygous mutant mice consisted of the following changes: 1) accumulation of fat droplets in various kinds of cells (hepatocytes, renal tubular epithelial cells, salivary gland ductal cells, Brunner's gland epithelial cells, brown adipose cells, cardiac muscle fibers and striated muscle fibers in the thigh muscles, diaphragm and extrinsic muscle), 2) enlargement of hepatocytes with infrequent hyaline globules in untreated young mice, 3) fine cytoplasmic granules and hypertrophy of cardiac muscle fibers with bizarre nuclei, 4) atrophy of adrenocortical cells, 5) necrosis of lymphocytes in the thymic cortex and spleen, and 6) erosion or ulceration of the gastric mucosa. A wide variety of abnormalities in JVS mice, as reported in human with carnitine deficiency, strongly suggest the variable and complicated physiological role of carnitine in individual cells or even in the same type of cell at different ages.
106: Effect of Feline Immunodeficiency Virus and Methamphetamine on the Expression of the Astrocytic Glutamate Transporter Glt-1
Feline immunodeficiency virus (FIV) infection of cats results in alterations in neurophysiological and behavioral function during periods of low brain proviral load. Excess of the major excitatory neurotransmitter glutamate is suspected to lead to neuronal injury through excitotoxic mechanisms during lentiviral infection. Extracellular glutamate levels could become elevated through decreased scavenging by astrocytic glutamate transporters. Methamphetamine has been suggested to increase glutamate transporter expression in rats due to hyperglutamatergic activity, and therefore might act synergistically with lentiviral infection to enhance neurotoxicity. Our hypothesis is that FIV infection leads to suppression of glutamate transporter expression and that co-administration of methamphetamine will exaggerate excitotoxic effects. In our first study group, six specific pathogen free cats were infected with 1000 TCID50 of the Maryland isolate of FIV intravenously at 8 weeks of age and necropsied 20 months later. In our second study group, two groups of six 6 month old cats were administered methamphetamine intravenously on a cyclical dosing schedule prior to necropsy at 12 months of age. One of these two groups was infected with 1000 TCID50 of FIV-Maryland 1 month into dosing. GLT-1 levels were analyzed by immunoblotting total protein homogenates with a commercially available polyclonal antibody recognizing the C-terminal peptide sequence of feline GLT-1 and ratioed against beta-tubulin immunoreactivity. Ratios in treated cat groups were compared against uninfected age-matched control cats. Preliminary semi-quantitative immunoblotting for GLT-1 expression in the frontal cortex suggests a non-significant trend toward decreased transporter expression in the dual methamphetamine-FIV treatment group. Analysis of GLT-1 mRNA expression using real-time PCR will be performed to assess whether the GLT-1 expression is downregulated at the transcriptional level.
107: Sensory Ataxia in a Mouse with Targeted Deletion of a Transcription Factor Specific to Trkc Neurons
Runx 3 belongs to a small family of transcription factors, which play key roles in hematopoiesis and osteogenesis. During development, it is detected in several tissues including a subset of neurons in cranial and dorsal root ganglia (DRG). Immunohistochemical examination of DRG shows that Runx3 is predominantly expressed in large diameter neurons and co-localizes with trkC and parvalbumin, markers of proprioceptive neurons. It is undetectable in trkA and trkB neurons. Homozygous disruption of the Runx3 gene results in severe ataxia, limb hyperextension, small stature, and reduced survival. In KO embryos, trkC neurons are generated but undergo progressive loss in DRG as monitored by X-Gal staining. At P0, L5 ganglia reveal 26% reduction in neuronal numbers. In contrast to DRG, neuronal loss is not observed in cranial ganglia. Commensurate with this discrepant effect, muscle spindles are absent from limb muscles but survive in masticatory muscles. A marked reduction in the number of large diameter fibers is observed in dorsal roots and in the dorsal column of the spinal cord, where collaterals of Ia afferents ascend to the cerebellum. Tracing studies employing anterograde DiI labeling and parvalbumin immunostaining demonstrate absence of Ia afferents terminating in the ventral gray matter of the spinal cord. Electrophysiological examination reveals that short latency monosynaptic potential is virtually abolished, indicating loss of connectivity between Ia neurons and motor neurons. Taken together, the data shows that Runx3 is a transcription factor specific to trkC neurons and that its disruption causes congenital-onset ataxia. Emphasis in this presentation will be placed on the various modalities utilized to generate the morphological data.
108: Stress Conditioning Promotes Viral Clearance from Brain in a Mouse Model of Persistent Infection
Elevated levels of the major inducible 70 kDa heat shock protein (hsp72) can mediate cellular stress conditioning, a state of heightened tolerance to noxious stimuli. Stress conditioning may also enhance priming of cell-mediated immune responses through hsp72-dependent cross-presentation of antigen. The present work tested the hypothesis that heat-induced stress conditioning can enhance clearance of persistent viral infection of brain, using a mouse model of measles virus (MV) encephalitis. Balb/c mice 36 h of age were exposed to a 41°C hyperthermic treatment for 30 min. Western blot analysis showed elevation of hsp72 in brain at 6 h post-treatment, the time of intracranial viral inoculation. Brain viral burden was monitored using real time RT-PCR of total RNA. Viral burden was equivalent between heated and non-heated treatment groups at 2, 4, and 7 d post-infection (PI). At 14 d PI, evidence of clearance was present in 2 of 8 heated mice and 1 of 8 non-heated mice. By 21 d PI, significant differences were apparent between treatment groups, with 19 of 20 heated mice having cleared virus in contrast to only 10 of 20 of the non-heated animals. Viral burden in the latter group was associated with lymphocytic meningoencephalitis and selective neuronal necrosis. Cytopathic effect was not observed in the non-heated group. The temporal onset of clearance was correlated to the detection of splenocyte blastogenic responsiveness to purified MV antigen. Collectively, these results show that stress conditioning promotes clearance of MV persistent infection in mouse brain, and that this clearance event is correlated to the onset of MV-specific cell-mediated immune responses.
109: Mechanisms of Anemia in Protein-Tyrosine Phophatase Deficient “Viable Motheaten” Mice
The autosomal recessive viable motheaten (mev) mutation disrupts the structural gene for hematopoietic cell phosphatase. This gene, located on mouse Chromosome 6, encodes a protein-tyrosine pho-phatase that is commonly termed Src-homology 2-domain phosphatase-1 (SHP-1). This signaling molecule is expressed primarily in hematopoietic cells and functions as a critical negative regulator of signal transduction in a number of signaling pathways in the immune and hematopoietic systems. Viable motheaten mice are deficient in SHP-1 retaining approximately 20% of wild type activity. These mice have a regenerative anemia, severe immune dysfunction and develop inflammatory lesions in the lung and skin and die of acidophilic macrophage pneumonia. The majority of human patients with polycythemia vera, a myeloproliferative disorder, have reduced expression of SHP-1 in their erythroid progenitor cells. These progenitors are hypersensitive to erythropoietin. Erythroid progenitor cells in mev/mev mice are also hypersensitive to erythropoietin but paradoxically these mice develop anemia. In this study we investigated the mechanisms by which erythrocytes are destroyed in viable motheaten mice. We found that this anemia was associated with erythrocyte membrane changes that were independent of the presence of anti-erythrocyte antibodies and that erythrocytes from mev/ mev mice showed increased fragility and susceptibility to oxidant damage. Measurements of oxidant production by macrophages from mev/mev mice demonstrate a higher basal level and enhanced oxidant production after stimulation. Additionally we found a significant elevation of lipid peroxidation and diminished levels of total glutathione, a critical antioxidant, in erythrocytes from mev/mev mice. We hypothesize that as a consequence of severe inflammatory disease, mev/mev erythrocytes are subject to exceptionally high oxidative stress resulting in oxidation of phospholipids in the erythrocyte membrane with subsequent hemolysis.
110: Expression and Regulation of Cyclooxygenase-2 in Canine Normal and Neoplastic Keratinocytes
Squamous cell carcinoma is one of the most common cancers in dogs, yet relatively little is known about the molecular events involved in its development. Rapidly mounting evidence implicates COX-2 in the oncogenesis of various types of cancer. We have previously reported that COX-2 is strongly expressed by keratinocytes in canine squamous cell carcinomas while normal canine skin does not express COX-2. The objective of this study was to characterize the regulation of COX-2 in canine keratinocytes using an in vitro model. Cell lines derived from normal (CK) and neoplastic canine keratinocytes (SCC) were cultured in the absence or presence of agonists, and immunoblots, immunohistochemistry, radioimmunoassays and a cell proliferation assay were used to study COX-2 regulation and effect. Results showed that SCC had a higher baseline expression of COX-2 protein than CK, and that COX-2 expression in both cell lines decreased in a time-dependent manner with serum starvation. In both cell lines, stimulation with 5 ng/ml PMA or 50 ng/ml TNF-alpha induced a time-dependent increase in COX-2 protein, with COX-2 induction being stronger in SCC than in CK. Measurement of PGE2 (one of the most common prostanoid with potent biological activities) revealed that SCC produce more PGE2 than CK, under both baseline and stimulated conditions. Additionally, cell growth of both CK and SCC could be inhibited in a concentration-dependent manner by the specific COX-2 inhibitor NS398. These results indicate that canine neoplastic keratinocytes expressed more COX-2 than normal keratinocytes, suggesting that COX-2 could be involved in skin carcinogenesis in dogs. (Supported by a grant from the Morris Animal Foundation).
111: What Does the Canine Darier-Like Disease Teach Us about Epidermal Renewal?
The molecular mechanisms of an inherited hyperproliferative and acantholytic canine skin disease resembling human Darier Disease were investigated. Using confocal microscopy we determined altered calcium homeostasis and defective function of a SERCA-type calcium pump in long-term keratinocyte cultures established from le-sional and non-lesional skin of an affected dog. Interestingly, comprehensive in vitro and in vivo analysis revealed that non-lesional cells, which contain the defect, had no alterations in differentiation marker and adhesion structure expression and formed a normal stratified squamous epithelium in vitro and in vivo. These cells, however, exhibited impaired regulation of p21CIP1, a protein involved in checkpoint control in the cell cycle. Our results demonstrate for the first time that SERCA function has no direct influence on keratinocyte differentiation as was suggested in the literature, but is associated with altered cell cycle progression. Interestingly, this defect can be compensated in normal epidermal turnover but may explain the higher susceptibility of the skin of patients to mechanical stress, heat and UV radiation resulting in focal lesions. In conclusion, the analysis of this acantholytic and hyperproliferative canine disease provided insight into the regulation of epidermal proliferation and differentiation.
112: Diosgenin-Associated Hepatotoxicosis and Ne-Phrotoxicosis in Goats Grazing Switchgrass
The purpose of this study was to elucidate the cause of clinical photosensitization, elevated liver and kidney serum enzymes, and histologically evident hepatopathy and nephropathy observed in a herd of Boer cross goats grazing Alamo Switchgrass (Panicum virgatum L.) during the summer of 2001. This herd consisted of 81 goats, ranging in age from 3-6 months. Twenty-four goats (30%) subsequently developed clinical signs of lethargy, poor body condition, and skin ulcerations with crusting on the dorsal midline, left and right flank, lateral aspect of the limbs, dorsal pinnae, and face. Necropsies revealed hepatocellular necrosis, Kupffer cell hypertrophy and hyperplasia, biliary hyperplasia and fibrosis, and mild cholangitis. Birefringent crystals were present in the bile ducts and Kupffer cells. Hepatic lesion severity appeared to correlate with increased Switchgrass exposure time. Renal lesions included proximal tubule dilation with evidence of necrosis admixed with various stages of degeneration and regeneration, primarily in the medullary region. Erosive to ulcerative skin lesions, suggestive of photosensitization, were also observed. Serum chemistry abnormalities include elevated BUN, creatinine, total bilirubin, AST, GGT, and decreased values for albumin and total protein. Diosgenin and other steroidal sapogenins are believed to be the principle toxic agents in Panicum spp. Ingestion of Panicum spp., primarily Kleingrass, has been reported to be hepatoxic in sheep and horses but not cattle. Using gas chro-matography-mass spectrometry, diosgenin was determined to be the major steroidal sapinogen in the Switchgrass grazed by the affected goats. To date, there have been no published reports of Switchgrass-associated toxicity in goats. This study suggests that the diosgenin present in the Switchgrass sample is both hepatoxic and nephrotoxic to goats.
113: Biomarkers of Fumonisin B1 Toxicity (Equine Leukoencephalomalacia, Elem) in Horses
Fumonisin B1 (FB1) alters sphingolipid biosynthesis by inhibiting ceramide synthase and induces hepatotoxicity in all species but neurotoxicity only in horses (ELEM). We evaluated FB1 induced changes in serum, urine and cerebrospinal fluid (CSF) in horses and correlated changes with morphologic effects. FB1 was administered IV at 0.20, 0.10, 0.05, 0.01 or 0 mg FB1/kg BW/day (n = 3 or 4). Blood samples were obtained every second day; urine and CSF just prior to euthanasia. Horses were euthanized when neurologic signs became severe (≥7 d, ≥0.05 mg FB1/kg) or on day 28. FB1 induced 1) dose- and time-dependent increases in serum: sphinganine, sphingosine, total bilirubin, bile acids and total protein (all doses); SDH, AST, ALP, GGT and cholesterol (≥0.05 mg FB1/kg); and creatinine (≤0.10 mg FB1/kg); 2) dose-dependent increases in urine: sphinganine (all doses), as well as GGT, protein and creatinine (NOT all doses); and 3) dose-dependent increases in CSF: sphinganine (0.01 mg FB1/kg) and protein (≥0.05 mg FB1/kg). In the nervous system, a dose-dependent vasculopathy with severe proteinaceous perivascular edema, leukocytosis and hemorrhage was present in meninges, brain and spinal cord at all doses. Icterus was present at >/ = 0.05 mg FB1/kg, with hepatocellular apoptosis (± necrosis) at all doses but especially at 0.05 mg FB1/kg. Renal lesions were not observed. Treated horses had biochemical evidence of hepatic, renal (without histologic lesions) and neurologic (CSF) toxicity. Increases in sphin-gosine and sphinganine concentrations appear to be specific for FB1 exposure and occurred in serum at all doses examined. Based on this study and the literature, the most sensitive indicator of FB1 exposure in horses is serum sphingolipid concentration.
114: Interactions of Pcb and Arsenic in Hepatic Promotion and Progression
Polychlorinated biphenyls (PCBs) and arsenic are common pollutants and are likely to be found as mixtures in the environment. The coplanar PCB126 (3,3’,4,4’,5-pentachlorobiphenyl) exhibits dioxin-like promotional effects on hepatocarcinogenesis. Arsenic is a clastogen that has been suggested to act at the progression stage of carcinogenesis or as a co-carcinogen. This study evaluated mixtures of these two chemicals to determine potential interactions at the promotion and progression stages of carcinogenesis. Staining for glutathione-S-transferase (placental form) (GST-P) identifies promoted foci, while staining for transforming growth factor alpha (TGF alpha) is presumed to represent progression. Rats were injected with an initiating dose of diethylnitrosamine (DEN) and received a partial hepatectomy three weeks after DEN injection. Gavage with corn oil or 10 ug/kg PCB126 in corn oil was started at two weeks after DEN administration. Arsenic (75 ppm) in drinking water was started in two groups at two weeks and in two groups at 8 weeks. Ethylnitrosurea was injected i.p. at 8 weeks as a control for progression. Rats were sacrificed at 8, 16, and 24 weeks. Our data indicate an increase in GST-P preneoplastic foci in livers of rats exposed to PCB126 alone and in combination with arsenic. Approximately ten percent of foci in the PCB-treated groups, particularly those foci largest in size, also stained for TGF alpha. Arsenic as a single agent did not increase GST-P or TGF alpha positive foci over DEN controls. Foci number and size appeared greater in rats treated with PCB126 and arsenic compared with PCB126 alone. This suggests that while arsenic alone may not act in promotion and progression, it may do so in combination with PCB126. This research is supported by NIEHS Grant #5 K08 ES00380.
115: Organophosphorus Neurotoxicant Tri-Orthotolyl Phosphate: Serum Analyte and Histologic Liver Alterations in Rats
The interactions of stress and exposure to neurotoxic chemicals are a matter of considerable interest in toxicology. Young adult male Long-Evans rats were administered the following (alone or in various combinations): 400 ug/ml corticosterone in the drinking water (the stress model) from days 1-63; 60 mg/kg sc chlorpyrifos on days 7 and 42; tri-otho-tolyl phosphate (TOTP, 75, 150 or 300 mg/kg) by gavage every other day from days 14-26 and 49-61. This study evaluated clinical chemistry, hematologic, and liver histologic findings at sacrifice. Corticosterone exposure alone or in combination with chlorpyrifos produced no significant variation of analytes. However, a significant alteration in cholesterol (mean 114 mg/dL compared to 85 mg/dL in controls, p = 0.017) occurred with TOTP exposure at 150 mg/kg. Exposure to TOTP at 300 mg/kg produced alterations in cholesterol (mean 125 mg/dL, p = 0.003) and alanine aminotransferase (ALT) (mean 142 U/L compared to 52 U/L in controls, p = 0.041). TOTP at 75 and 150 mg/kg combined with corticosterone and chlorpyrifos individually or in combination produced significant alterations in cholesterol (mean range 127-171 mg/dL, p = 0.028-0.056). In addition, TOTP at 300 mg/kg, combined as described above, resulted in alterations in albumin (mean 2.8 g/dL; p = 0.055), albumin:globulin ratio (mean 0.073, p = 0.055), urea nitrogen (mean 13.3 mg/dL, p = 0.001), cholesterol (mean 131 mg/dL, p = 0.030), ALT (mean 153 U/I, p = 0.032), and liver histologic features (necrosis). Therefore, multiple administration of TOTP to Long Evans male rats at doses of 75 mg/kg and greater appears to alter hepatocellular membrane permeability and cholesterol metabolism, and TOTP at 300 mg/kg results suggest the possibility of reduced hepatic function along with necrosis.
116: Expression Levels of Hepatic Enzymes and Nuclear Receptors following Treatment of Rats with a Bile Acid Sequestrant, Cholestyramine
Most lipid-lowering agents, despite acting by different mechanisms, have been associated with mild, asymptomatic elevations of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The mechanisms for these elevations of serum transaminases are unclear. In this study, using the cholestyramine-treated rat model, we investigated whether interruption of the enterohepatic circulation of bile acids would affect serum and hepatic ALT and AST levels. In addition, we investigated the effect of inhibition of bile acid reabsorption on two nuclear receptors (LXR and FXR) involved in cholesterol metabolism in the liver. There were transient, reversible increases of both serum ALT and AST following treatment with cholestyramine (4% in the diet) for 2, 4, and 13 weeks in both genders. ALT and AST levels returned to control levels after treatment for 26 weeks. These changes did not correlate with any histologic change. These elevations of serum ALT and AST were not due to increases in gene transcription, since hepatic ALT and AST mRNA levels were similar to those of control animals. As anticipated, the activity and mRNA levels of cholesterol 7□-hydroxylase and HMG-CoA reductase increased dramatically at all time points following cholestyramine treatment. However, there were no significant changes in hepatic LXR and FXR mRNAs. In conclusion, our data suggest that transient, reversible elevations of serum ALT and AST observed with lipid-lowering agents, are a pharmacologic, rather than a toxic, response. The increase in 7□-hydroxylase and HMG-CoA reductase activities in response to cholestyramine treatment is likely the result of bile acid depletion and removal of the FXR inhibitory effect on these enzymes.
117: Amiodarone Hepatotoxicity
Amiodarone is a potent antiarrhythmic drug with severe multi-organ toxicity. To assess hepatotoxicity by gene expression analysis using Affymetrix microarrays, 5 male Wistar rats were treated once with vehicle, 100 mg/kg or 600 mg/kg amiodarone intraperitoneally and samples collected after 6 hours. In a second experiment, rats were treated with vehicle, 50 mg/kg or 100 mg/kg amiodarone intraperitoneally daily for 4 days. Livers were sampled for histopathology and measurement of lipids and triglycerides. Serum was analyzed for standard clinical parameters. Liver RNA was collected for gene expression analysis using Affymetrix Genechips (RG-U34A). Histopathological examination did not reveal treatment-related findings in rats after a single dose of amiodarone. Rats treated for 4 days revealed a dose-related increase in incidence and severity of microvesicular steatosis and a mild increase in triglycerides in the liver. Expression of carnitine palmitoyltransferase I and NADPH-cytochrome P450 oxidoreductase were slightly upregulated 6 hours after amiodarone treatment. Betaine homocysteine methyltransferase was significantly decreased in rats treated with amiodarone for 4 days compared with rats treated for 6 hours. Expression of CYP450 lauric acid omega hydroxylase, CYP452, and squalene synthetase were increased in rats treated with 50-100 mg/kg/day amiodarone for 4 days. These results are in agreement with reports suggesting that amiodarone-induced hepatotoxicity, characterized by microvesicular steatosis, is secondary to drug-induced inhibition of mitochondrial beta-oxidation of fatty acids and the respiratory chain. Interestingly, expression of phospholipase A2 was markedly increased in rats treated for 4 days compared with rats 6 hours after a single dose. Amiodarone is a cationic amphiphilic drug known to produce phospholipidosis by inhibition of phospholipase activity, allowing lysosomal accumulation of phospholipids and formation of typical lamellar inclusions. In conclusion, analysis using Affymetrix Genechips reveals alterations in gene expression related to toxicity of compounds.
118: Liposome Formulation to Decrease Systemic Toxicity of High Dose Drug Delivery
Liposomes are phospholipid vesicles used to improve solubility of compounds that need to be administered at high doses. They are cleared from the circulation by the mononuclear phagocyte system in the liver and spleen. Liposome composition, size, charge, dose, and host factors in the plasma influence clearance. In the first study, the drug of interest was incorporated within the phospholipid bilayers of multilamellar liposomes composed of soybean lecithin with a mean diameter of 200 nm. Wistar rats were given liposomes at a dose of 400 mg/kg/h in a continuous intravenous infusion for 3-4 days (total daily dose 9.6 g/kg). The spleen was enlarged and clay-colored. Hepatic sinusoids were markedly dilated and contained numerous, huge, foamy macrophages and Kupffer cells. Moderate periportal edema, infiltration, and periacinar necrosis were seen in high dose rats. The spleen showed atrophy of the red pulp, lymphoid depletion, dilated sinuses with numerous foamy macrophages, and hemosiderosis. Congestion with or without vascular dilation was noted in several organs in high dose rats. Intravenous administration of high doses of liposomes resulted in systemic toxicity due to clearance by the MPS and to severe blood stasis from a large amount of particulate material in the circulation. In the second study the drug of interest was adsorbed onto the negatively charged surface of 200 nm diameter liposomes, thus markedly reducing the dose of liposome required while allowing delivery of the same dose of compound. Wistar rats were given liposomes in a continuous intravenous infusion for 4 days at 60 mg/kg/h (total daily dose 1.44 g/kg). Systemic toxicity was eliminated by reducing the amount of phospholipids administered by a factor 6.7 in comparison to the previous liposomal formulation.
119: Thiazolyl Peptide Antibiotic-Induced Macrophage Hypertrophy/Hyperplasia in the Dog and Mouse
BMS-411886, a poorly soluble (at pH 7.4) thiazolyl peptide antibiotic that fluoresces at 490 nm, induces hypertrophy/hyperplasia of cells of the monocyte-macrophage system with cytoplasmic pigment accumulation. Studies were conducted to characterize the microscopic lesions induced by BMS-411886 and to evaluate their reversibility. BMS-411886 was administered intravenously to dogs at 25 mg/kg for 14 days, followed by a 14-day dose-free period, and to mice at 30 mg/kg for 7 days, followed by a 14-day dose-free period. At necropsy, tissues were collected and processed for light (liver, kidney, spleen, lung, heart, thymus, and injection site), fluorescence (liver, kidney, spleen, and lung), and electron (liver and kidney from dogs) microscopy. Light microscopic findings were similar in both species and included the accumulation of enlarged, pigmented macrophages in hepatic and splenic sinusoids and within or lining alveolar septa of the lung, and lymphoid depletion in the spleen. By fluorescence microscopy, BMS-411886 was localized in the cytoplasm of macrophages of the liver, spleen, and lung, and of renal tubular epithelium. By electron microscopy, electron-dense residual bodies were present within phagolysosomes of Kupffer cells and renal tubular epithelium. In summary, BMS-411886 induced a persistent macrophage hypertrophy/hyperplasia in the liver, spleen, and lung, tubular degeneration and necrosis with regeneration in the kidney, and cytoplasmic pigment accumulation in affected cells. Fluorescence microscopy demonstrated BMS-411886-related material within the cytoplasm of macrophages and renal tubular epithelium, which by electron microscopy was localized to phagolysosomes. Persistence of BMS-411886-related material in phagolysosomes was attributed to insolubility of drug, and cellular uptake and accumulation in excess of lysosomal degradation.
120: Expression of Transforming Growth Factor Beta (Tgf-β) and Tgf-β Ii Receptor in Preneoplastic Hepatic Lesions in Rats following Treatment with Polychlorinated Biphenyls
Control of cell proliferation is vital to understanding the mechanisms of action of promoters. Growth fact-ors can stimulate or inhibit progression through the cell cycle. TGF-β1 normally inhibits cell proliferation and promotes differentiation in hepatocytes. Once activated, TGF-β1 binds to the heterodimeric TGF-β receptor, inhibits cell proliferation, and induces apoptosis at the G1/S stage of the cell cycle. TGF-β is also important in carcinogenesis with a loss of TGF-β1 responsiveness and a decrease in TGF-β type II receptors (TGR2) being reported in hepatocellular carcinoma cells. However, expression of TGF-β1 and TGR2 expression in PCB-induced preneoplastic lesions has not been established. This study was designed to investigate the mechanistic role of TGF-β1 and its receptor TGR2 as a modulator of hepatic tumor promotion following administration of two environmentally relevant PCBs, the planar PCB 126 (3,3’,4,4’,5-pentachlo-robiphenyl) and the non-planar PCB 153 (2,2’,4,4’,5,5′-hexachloro-biphenyl). Promotion of altered hepatic foci was evaluated utilizing a medium-term eight-week bioassay for promoters of hepatocarcinogenesis in the rat. Both PCBs induced significant increases in preneoplastic, glutathione-S-transferase positive foci. An immunohistochemical study showed an increase in TGF-β1-positive staining in larger preneoplastic hepatic foci, and this correlated with a decrease of TGR2 expression in preneoplastic lesions. Our data suggest that the expressions of TGF-β1 and TGR2 are significantly altered during the promotion stage of hepatocarcinogenesis and that these changes might contribute to the development and progression of preneoplastic lesions. NIEHS Superfund Basic Research Program Project, P42-ES05949 and NIEHS K08ES00314 supported this work.
121: Ultrastructure of Mast Cells Observed in the Ear Skin of Iqi/Jic Mice with Picryl Chloride-Induced Contact Dermatitis
In this study, we examined ultrastructural changes of mast cells in the skin of IQI/Jic female mice following topical application (left ear) of PCL at 4, 11, 18 and 25 days after the sensitization of the abdominal skin with PCL. In the dermis at 4 to 9 hr after the 1st application, a small number of mast cells had several swollen granules with low electron density and showed extrusion of a few membrane-free granules through openings in the cell membrane. In a few mast cells, membrane-bound granules were protruded from the cytoplasm and dropped into the dermis. Neutrophils sometimes attached to the mast cells and phagocytized extruded membrane-bound mast cell granules. In the dermis at 4 to 9 hr after the 4th application, many immature mast cells characterized by well-developed Golgi apparatus, many free ribosomes and a few small electron-dense secretory granules in the peripheral cytoplasm were observed. In addition, prominent formation of intracytoplasmic degranulation channels and extrusion of multiple swollen membrane-free granules through the cell membrane were observed in several mast cells. Thus, piecemeal degranulation of mast cells was mainly observed after the 1st application, and anaphylactic degranulation of mast cells was commonly found after the 4th application. In addition, a close spatial relationship between mast cells and neutrophils was common after the 1st application, and immature mast cells were frequently observed after the 4th application. Studies on mast cell-related cytokine profiles are now in progress.
122: Evaluation of Cell Proliferation and Apoptosis in the Liver Using Immunohistochemistry and Transcription Profiling: A Comparative Study
Increased hepatocyte proliferation and apoptosis are common responses to various hepatic toxicants, especially non-genotoxic carcinogens. Cell proliferation and apoptosis are traditionally evaluated by histologic examination, but can also be quantified using iramunohistochemistry. Transcription profiling of a wide array of genes using DNA microarrays offers the opportunity to evaluate various cell signaling pathways, including cell proliferation and apoptosis, but it is unclear how these data correlate with morphologic responses. In this study, we evaluated whether transcription profiling data could predict changes in hepatocyte proliferation and apoptosis. Male CD rats were treated orally by gavage for 5 days with either methapyrilene at 35, 70 or 140 mg/kg/day, or clofibrate at 200, 400 or 800 mg/kg/day. Livers were collected for transcription profiling and histology. After RNA isolation and labeling, labeled cDNA probes were applied to Incyte RatGEM 1, which contained approximately 7,800 arrayed cDNA elements. Cell proliferation and apoptosis were quantified using computer-assisted image analysis following immunohistochemical staining for PCNA and activated caspase 3. Methapyrilene treatment induced a dose-dependent increase in hepatocellular proliferation and apoptosis, which was correlated with increased expression of numerous genes (>50) regulating these processes. Clofibrate treatment caused significant increases in hepatocellular proliferation at all dose levels and regulated a number of genes involved in cell proliferation. However, following clofibrate treatment, hepatocellular apoptosis was not significantly increased and comparatively few apoptosis pathways were regulated. Collectively, these results indicate that transcription profiling can be used to assess hepatocellular proliferation and apoptosis and to identify specific cellular pathways that may be utilized in predictive toxicology studies.
123: A Global Intranet-Based Robotic Telepathology Platform for Toxicologic Pathology Consultation
Many pharmaceutical companies now have multiple global discovery and lead optimization research centers due to the advent of ongoing mergers and acquisitions. To capitalize on institutional knowledge and experiences, it is important to facilitate collaborations and consultations between research sites in an efficient and time-sensitive manner. We have established an Aventis Telepathology Platform (ATP) for rapid, multi-site histopathology consultation and live, on-line, image sharing. To create the ATP, various digital pathology imaging and telepathology technologies were first evaluated as were robotic remote controlled digital micro/macro imaging systems. Strategies for the ATP were then designed utilizing the Aventis intranet-based web-interface and the robotic system chosen in collaboration with Trestle Corporation (Newport, CA). Results of internal testing indicated that the ATP was a secured intranet platform with adequate speed transfer times. The ATP is simultaneously accessible from multiple sites from pathologist's desktop computers, resulting in a global ‘multi-head microscope’. Near instantaneous responses occur between global sites following a variety of desktop computer commands, such as slide navigation, fine-focus, objective lens magnification changes, and live-image notations. A multi-slide robotic tele-microscopy system upgrade is currently in beta testing as is a high-resolution large screen monitor system with near optic quality display capabilities. Creation of the ATP has resulted in a global network of internal pathology expertise that facilitates rapid consultation between DSE sites.
124: Comparison of Bouin'S and Modified Davidson'S Fixative for Testicular Morphology and Immunostaining of Markers for Sertoli Cells and Spermatogonia
Recent revisions of regulatory guidelines for testing effects of chemicals on reproduction recommend Bouin's fluid (BF) or a ‘comparable’ fixative instead of formalin to preserve morphology of the testis. BF has also been the fixative of choice for immunohistochemical staining (IHC) of testicular antigens. However, picric acid in BF is a health and safety hazard, as well as a waste disposal problem. Furthermore, its use is labor intensive, requiring multiple alcohol rinses to remove picric acid that would otherwise compromise histological and IHC staining. Recently a modified Davidson's fluid (mDF) has been reported as an alternative to BF to fix testes for routine histopathologic examination. This study compared the two fixatives for their overall histomorphologic clarity and their IHC properties using antigenic markers to differentially stain Sertoli and spermatogonial nuclei. Additionally, dual staining using IHC to detect androgen receptor (AR) and periodic acid-Schiff-hematoxylin for the acrosome assessed the utility of this method for detecting important functional proteins in different stages of the spermatogenic cycle. The mDF resulted in noticeably less shrinkage of the seminiferous tubules and superior overall morphologic clarity compared to BF, and also allowed good PAS-staining of the acrosome. IHC detection of AR and PCNA (to respectively identify Sertoli cells directly and indirectly) as well as protein gene product 9.5 (to label spermatogonia) was superior in mDF compared with BF-fixed specimens. These techniques could be useful when quantification of these cells is required. Dual staining to detect both AR and the acrosome demonstrated differential staining for AR in Sertoli cells in a stage-specific pattern. This procedure could be useful for mechanistic studies in reproductive toxicology where cell- and stage-specific effects are hypothesized.
125: A New Quail Model, F1(Awe X We), to Evaluate Sex Reversal Effects of Endocrine Disrupters in Early Development
F1(AWE X WE) Japanese quail (Coturnix coturnix japonica) have been produced by mating between male AWE (albino plumage color) and female WE (wild plumage color) strains of Japanese quail. The male and female F1 quail exhibit exactly wild and albino phenotypes, respectively, ruled by a criss-cross inheritance. We investigated usefulness of the F1(AWE X WE) quail in sex reversal effects of sex hormones in early development. F1(AWE X WE) eggs received 17 beta-estradiol (E2) or methytestosterone (MT) at 0, 20, 200, 2,000, and 20,000 ng/20 micro-L corn oil just before the incubation. At 16-day of incubation, embryos were phenotypically observed and necropsied. Testes and ovaries were examined histopathologically. Survival rates of the embryos were not significantly different between the control and all E2 groups, and the control and the 20, 200, and 2,000 ng MT groups. Grossly, the genetic sex (plumage colors) completely coincided with genital organs (testes and ovaries) in all F1 embryos of the control group. Histopathologically, ovotestes were detected in the left gonad of F1 male embryos in the E2 groups at doses of 200 ng and above. E2 exposure resulted in a dose-dependent increase in the feminization of the left testis. No ovotestes were detected in all F1 male embryos of all MT groups. E2 and MT exposure induced no noticeable changes in ovaries of any F1 female embryos. The present study suggests that F1(AWE x WE) quail are useful to evaluate feminization effects of the estrogenic disrupters.
126: Retrospective Histologic and Molecular Study of Helicobacter Pylori in Cynomolgus Monkeys (Ma-Caca Fascicularis)
Because Helicobacter pylori-related gastric alterations may confound toxicologic studies, 38 cynomolgus monkeys (Macaca fasci-cularis, 3 to 8 years of age) were evaluated retrospectively for H. pylori infection. Sections of archival, formalin-fixed, paraffin-embedded gastric antrum were stained with hematoxylin and eosin (HE), Warthin-Starry (WS) and Alcian yellow-toluidine blue (AY) stains for histologic evaluation and detection of bacteria. In addition, oligonucleotide primers that recognized regions of the 16S rRNA gene were used in a nested polymerase chain reaction (PCR) to specifically detect H. pylori in 21 of these animals. Nine of the 38 animals had histologic lesions characteristic of H. pylori infection. Typical H. pylori lesions in the gastric antrum included diffuse thickening of the mucosa with hyperplasia of glandular epithelium, lymphoplasmacytic infiltrate within the lamina propria, and prominent lymphoid follicles. In 6 of these 9 animals, organisms compatible with H. pylori were detected with both WS and AY stains. Both special stains were negative in two animals that had characteristic H. pylori lesions. With PCR, H. pylori was detected in 16 of 21 animals tested, 8 of which had histologic alterations. In conclusion, H. pylori infection was detected in cynomolgus monkeys using a combination of histology, special stains, and PCR amplification. PCR results alone were considered too sensitive to use as a screen for H. pylori lesions since bacterial DNA was detected in the absence of histologic alterations. In contrast, characteristic microscopic lesions in gastric tissues that yielded H. pylori product with PCR did not consistently correlate to identifiable organisms with special stains, even though H. pylori specific amplimers were detected after PCR. Therefore, a combination of PCR and histologic techniques may be used to identify H. pylori infection, where PCR would be used primarily to confirm the presence of H. pylori in suspect tissues.
127: Effect of Storage Conditions on the Stability of Rna in Paraffin Histologic Sections
In a previous study, we have demonstrated that the prolonged storage of paraffin sections affects the successful detection of mRNAs by radioactive in situ hybridization. This phenomenon is thought to be related to RNA degradation in non-deparaffinized histologic sections that are stored at room temperature before use. In the present study, we expanded this work by evaluating whether different storage conditions may prevent the loss of signals following storage. Paraffin blocks of mouse experimental skin wounds and rat myocardial infarcts were serially sectioned at different times, so that the resulting sections could be stored for different periods of time (up to 60 days). Paraffin sections were stored under one of the following conditions: room temperature, 4°C, or under vacuum in sealed plastic bags kept at room temperature. All sections were then hybridized with 33P-labeled riboprobes specific for rat Type I collagen, rat Type III collagen and rat matrix metalloproteinase-2.
Signal intensities were analyzed using a phosphorimager and by blinded microscopic evaluation. For all 3 storage conditions, stronger signals were consistently observed in freshly sectioned slides compared to slides sectioned 2 weeks or more before hybridization as previously reported. No significant differences were observed among the 3 storage conditions. In conclusion, these results indicate that the prolonged storage of paraffin sections negatively affects the successful detection of mRNAs by in situ hybridization and that the loss of signals occurring during storage is independent of the storage conditions evaluated in this study.
128: The Expression and Activity of Selected Matrix Metalloproteinases during Skin Wound Healing in the Mouse
Cutaneous wound healing is a complex series of events that ultimately leads to restoration of the injured tissue. The hallmarks of wound healing are hemostasis, re-epithelialization, granulation tissue formation, angiogenesis, and stromal remodeling. All of these events require the controlled degradation of various parts of the extracellular matrix (ECM). Through the proteolytic degradation of various ECM components, matrix metalloproteinases (MMPs) contribute to the dynamic process of cutaneous wound healing. Many MMPs are up-regulated and show distinct spatial and temporal mRNA expression patterns in cutaneous wound healing. This suggests that each MMP may provide a distinct function during wound repair. However, whether these patterns match MMP activities is still poorly understood. By using RT-PCR, in situ hybridization, in situ zym-ography, and laser capture microdissection, we have studied the expression and activities of selected MMPs in a mouse skin wound healing model and found that, at the early stages of the wound healing process, most MMP activities were detected in migrating epithelial cells. In contrast, MMP activities were more commonly located in stromal cells in the later wound healing stages. The MMP transcripts which were up-regulated rapidly following wounding were MMP-1, MMP-3 and MMP-9, while MMP-2 and MMP-14 mRNA expression levels were mostly up-regulated in the later wound healing stages. These data suggest that MMP-1, MMP-3 and MMP-9 provide the most important function in the early stages of the wound healing process, while MMP-2 and MMP-14 have a more important role during the remodeling phases of the healing process.
129: Expression and Localization of Cox-1 and Cox-2 in the Eye
The cyclooxygenases (COX) are enzymes that catalyze the fist two steps in the biosynthesis of eicosanoids from arachidonic acid. The COX orchestrate a variety of homeostatic processes and participate in various pathophysiological conditions. Two COX isoforms have been identified, COX-1 and COX-2. COX-1 is constitutively expressed in almost all tissues and displays the characteristics of a “housekeeping” enzyme. By contrast, COX-2 is the product of an immediate early gene that is rapidly inducible and tightly regulated. Under normal conditions, COX-2 expression is highly restricted to certain tissues, but it can be dramatically increased in various tissues by pathological stimuli, such as inflammation and neoplasia. Prostaglandins (PGs), mostly PGE2 and prostacyclin, are important mediators of inflammation, pain, and fever. Studies have shown that PGs have a pivotal role in the regulation of physiological and pathological conditions of the eyes in humans and animals, suggesting that the COX might be involved in these disease conditions. Little is known about the distribution of COX-1 and COX-2 in the normal eye. To investigate the distribution and the potential participation of COX-1 and COX-2 in ocular pathology and physiology, we evaluated their expression in the lens, iris, optic nerve, sclera, retina, and cornea from different species (rat, rabbit, dog, monkey and human) using real-time RT-PCR and Western blot. Both COX-1 and COX-2 were constitutively expressed at the mRNA and protein levels in all different parts of the normal eye, except in the lens.
130: Hematein Inhibits Atherosclerosis by Inhibition of Nf-Kb-Dependent Gene Expression and Reactive Oxygen Generation
Hematein, a natural flavonoid, is known as an analgesic and antiinflammatory agent. Here we investigated the effects of this compound on atherosclerosis in low density lipoprotein receptor deficient (Ldlr−/-) mice fed high cholesterol diet alone or in combination with hematein for 8 weeks. Mice fed high cholesterol diet alone develop the fatty streak lesion in the aortic sinus, whereas this lesion was significantly reduced by hematein treatment. Infiltrated macrophages, iNOS protein, TNF-alpha, interleukin-6, interleukin-1beta and nitrotyrosine expression were localized in the atherosclerotic lesion. Hematein treatment reduced serum levels of nitrite and nitrate, TNF-alpha, IL-1beta, and lipid peroxide, but did not changed the plasma cholesterol level, compared with the levels of control mice. In addition, hematein inhibited NF-□B activation and the activity of transient tranfected NF-□B and iNOS promoters in peritoneal macrophages from Ldlr−/-mice. This compound also suppressed NO production and iNOS expression in LPS+INF□-stimulated peritoneal macrophages from Ldlr−/-mice. Hematein inhibited the production of NF-□B-dependent cytokines TNF-alpha, IL-1beta, IL-6 in LPS+INF□-stimulated peritoneal macrophages. Hematein also inhibited superoxide generation in LPS-stimulated peritoneal macrophages. These results suggest that hematein inhibited atheroscleotic lesion formation, possibly by inhibiting NO production, lipid peroxidation, and pro-inflammatory cytokine expression through the inhibition of NF-□B activation and reactive oxygen production.
131: Soluble Lymphotoxin-Beta Receptor Alters Antigen Responsive Lymph Node Microarchitecture.
A murine lymphotoxin-beta receptonimmunoglobulin G1 fusion protein (mLTBR:mIgG1) has been shown to affect follicular dendritic cell (FDC) function, thereby altering formation and maintenance of normal lymphoid microarchitecture. A four week repeat dose study was performed in mice using this murine construct. Mice were treated with mLTBR:mIgG1 once per week for four weeks by subcutaneous injection at doses of 0, 0.2, 1, 10 and 40 mg/kg and necropsied on either day 29 or day 85. To assess the effects of administration of mLTBR:mIgG1 on cellular immunity, an oxazolone based delayed type hypersensitivity (DTH) test was performed in the left ear four days prior to necropsy on Day 29. Pharmacologic effect of treatment with mLTBR:mIgG1 was evidenced by: 1) a dose-dependent increase in peripheral blood lymphocytes and neutrophils on Day 29, 2) an increase in spleen weights which correlated histologically with a reversible splenic lymphoid hyperplasia, and 3) a treatment-related decrease in follicular dendritic cell immunohistochemical staining. mLTBR:mIgG1 produced no treatment-related effects on non-lymphoid tissues or on clinical chemistry parameters and all effects of mLTBR:mIgG1 showed significant signs of reversibility by two-months after termination of treatment. T-cell mediated immunity was not impaired as measured by the DTH response. Lymph node architecture was essentially unchanged in the mesenteric, right mandibular and right axillary lymph nodes of treated animals. However, in the nodes providing lymphatic drainage for the DTH test site, the left mandibular and left axillary nodes, there was reactive paracortical hyperplasia and diminution of germinal center activity. Thus it appears that while mLTBR:mIgG1-mediated alteration of FDC function does not prevent localized antigen-driven lymphoproliferation, such diminution may result in a loss of the normal microarchitecture seen in a lymph node undergoing reactive hyperplasia. These findings are cited as examples of unexpected microscopic changes noted in the lymphoid tissue of animals exposed to an immunomodulatory drug.
132: Evaluation of 1,3-Butadiene Induced Lung Tumors for K-Ras and P53 Mutations and Allelic Losses in the Region of the K-Ras Gene following Inhalation for up to 20 Weeks
Over the past 20 years the general assumption is that the ras gene family represents dominant oncogenes. Of the three ras genes, Hras1, Nras and Kras2, Kras2 is the most frequently mutated in human neoplasms and in chemical induced rodent neoplasms. Recently, however, it was shown that the Kras gene has two major functions where it promotes the development of lung tumors as a gain of function oncogene and inhibits cancer as a loss of function tumor suppressor gene. These new data are interesting as they suggest that the two major genes in cancer (ras and p53) may have more in common than previously recognized; the p53 gene was first described as an oncogene and later it was discovered that the wild type gene functions as a tumor suppressor gene. In this study, a high frequency of the signature Kras codon 13 G to C transversion mutation was detected in 1,3-butadiene induced lung neoplasms and accompanying these mutations were allelic losses. Furthermore, the mutant p53 protein was detected in a subset of the 1,3-butadiene induced lung neoplasms.
133: Mucous Cell Metaplasia Induced by Inhaled Fine Airborne Particles and Allergen in the Pulmonary Airways of Rats
The purpose of our study was to test the hypothesis that inhalation of concentrated airborne particles (CAPs) during allergen (ovalbumin; OVA) sensitization will exacerbate allergen-induced mucous cell metaplasia in the pulmonary airways of rats following allergen challenge. A mobile air research laboratory equipped with inhalation chambers, air pollution monitors, and an air particle concentrator was parked in a southwestern Detroit community when concentrations of ambient fine particulate matter (0.1-2.5 microns in mean aerodynamic diameter; PM2.5) were estimated to be high. Brown Norway rats, with and without OVA sensitization, were exposed to filtered air (controls) or PM2.5 CAPs generated by a Harvard air particle concentrator (26x ambient PM2.5 concentration). Rats were exposed to CAPs at an average chamber concentration of 1 milligram per meter cubed, 10 hours per day, for five consecutive days. Immediately following the daily exposure, rats were intranasally sensitized with OVA. Two weeks later, all rats were intranasally challenged with OVA for 3 consecutive days. Forty-eight hours following allergen challenge, rats were sacrificed and pulmonary airway tissues were processed for light microscopy. The amounts of intraepithelial mucosubstances (IM) within the airway were estimated using standard morphometric techniques. CAPs alone caused an 88% increase in IM, and OVA challenge alone induced a 94% increase in IM compared to controls. CAPs-exposure did not enhance OVA-induced IM. These results suggest that inhaled PM2.5 during sensitization may not exacerbate allergen-induced mucous cell metaplasia. However, PM 2.5 exposure alone may cause mucous cell metaplasia. Research supported by the Michigan Life Sciences Corridor Fund and the ACVP/Pfizer Minority Fellowship in Veterinary Pathology.
134: Effects of Anti-Connective Tissue Growth Factor Monoclonal Antibody on Development of Fibrosis in the Rat Remnant Kidney (5/6 Nephrectomy) Model
Transforming growth factor beta (TGF-beta)-mediated tubuloin-terstitial fibrosis and glomerulosclerosis are common sequelae in progressive renal disease. Recent therapeutic strategies for inhibiting detrimental effects of fibrosis and glomerular collagen deposition include blocking TGF-beta downstream mediators, particularly Connective Tissue Growth Factor (CTGF). This study evaluated effects of a proprietary anti-CTGF monoclonal antibody on progressive renal failure in the rat remnant kidney (5/6 nephrectomy) model. Forty-three Sprague Dawley rats underwent subtotal nephrectomy and were assigned to five groups. Group 1 controls were euthanized 14 days postnephrectomy. Groups 2 and 3 received intraperitoneal (IP) saline or anti-CTGF Ab, respectively, every 72 hours for 15 days starting 14 days post-nephrectomy and were euthanized three days post-treatment. Groups 4 and 5 received saline/antibody protocols as for Groups 2 and 3, but were euthanized 14 days post-treatment. Weekly CBC, serum chemistries and urinalyses monitored renal disease and correlated renal function with histologic lesions. Remnant kidneys were stained with H&E, trichrome and sirius red stains and subjectively evaluated for fibrosis in blinded fashion. Immunohis-tochemical stains were performed on sections for collagen types I, III, IV, and hydroxyproline/proline content was quantified. Saline-treated 18- and 28-day rats had greater fibrosis scores (12.6% and 16.9%, respectively) and greater cortical and medullary fibrosis compared to anti-CTGF Ab treated rats. Saline-treated rats had increased glomerular fibrosis, predominantly type IV collagen and 11% greater serum urea nitrogen (SUN) and urine protein levels. Our results suggest intraperitoneal anti-CTGF antibody mildly inhibits collagen I, II, HI deposition and tubulointerstitial fibrosis, markedly inhibits glomerular collagen IV deposition and reduces SUN and protein levels in the rat remnant kidney model of renal failure.
135: Cgrp and Substance P Are Important in Epithelial Repair, but Not Neutrophil Dependent Removal of Injured Airway Epithelial Cells, after Ozone Exposure in Conscious Rats
We investigated the role of neutrophils in removing injured airway epithelial cells and the importance of neuropeptides, CGRP and substance P, in neutrophil function and epithelial repair in conscious rats exposed to ozone. Wistar rats were exposed to 1-ppm ozone or filtered air for 8 hours followed by an 8-hour post-exposure period. For some experiments, rats were neutrophil depleted by an intraperitoneal injection of anti-rat neutrophil serum or remained neutrophil sufficient. In other experiments, rats were systemically administered a CGRP- and/or a neurokinin-1-receptor antagonist or administered a vehicle control. Microdissected lung lobes were stained with Ethidium Homodimer-1, a marker of cellular injury, and BrdU, a marker of epithelial repair. Positive epithelial cells were quantified in specific airway generations. We found neutrophils were critical in removing ozone-injured airway epithelial cells. Rats treated with either the CGRP- or NK-1- receptor antagonist had delayed epithelial repair; while no significant differences were found in rats treated with combined receptor antagonists or vehicle. Neither CGRP nor substance P appears critical in neutrophil aided removal of injured epithelial cells in vivo. However, in vitro, substance P primed rat neutrophils increasing superoxide production after PMA stimulation. Finally, we found no significant correlation between the presence of neutrophils and epithelial repair at specific airway generations. We conclude that neutrophils are important in the removal of airway epithelial cells in conscious rats after acute ozone exposure. Additionally, while CGRP and substance P are not critical mediators involved in neutrophil aided removal of injured cells, they are important in subsequent epithelial repair.
136: Pathogenesis of Microcystin-Lr Toxicosis: Sublethal Histologic and Metabonomic Changes.
The cyanobacterial (blue-green algae) toxin microcystin-LR is known to cause necrosis of centrilobular hepatocytes. Although there are few in vivo sublethal dose studies in the literature, there has been extensive in vitro work and many in vivo studies with lethal doses. In a recent sublethal study, mice were treated with a single dose of 45 ug/kg and euthanized at times up to 24 hours post-dosing. Here, we followed the effects of sublethal doses for a longer time in a rat model. Rats were treated with a single sublethal dose, either 80 or 20 ug/kg i.p. and sacrificed at 2 or 7 days post-dosing. Changes in the high-dose, 2-day sacrifice group and in one rat in the high-dose group that was found moribund at 3 days included centrilobular hepatic necrosis and congestion, accompanied in some animals by centrilobular regeneration, as evidenced by stem cell proliferation and neovascularization. By 7 days post-dosing, the animals had recovered. The necrotizing process had ended and the centrilobular areas had been replaced by regenerative, usually hypertrophic hepatocytes. This study is part of the Consortium for Metabonomic Toxicology (COMET) project to develop and use metabonomics as a predictor of tissue- and mechanism-specific toxicities. Urinary changes in small molecular endogenous metabolites are determined by 1H-NMR and pattern recognition analysis. A large reference database and predictive chemometric models are currently being developed using studies with toxins with specific topologic sites of injury and mechanisms of action.
137: A Tough Bioelastomer for Implantation Delivery of Drugs, Cells and Genetic Material
Biodegradable polymers are limited by their inferior mechanical properties and unsatisfactory compatibility with cells and tissues. BioRubber or poly(glycerol-sebacate) (PGS) is a newly designed tough biodegradable elastomer composed of biocompatible monomers. This elastomer forms a covalently crosslinked, three dimensional network of random coils with hydroxyl groups attached to the backbone. Design features allowing flexibility, strength and gradual surface degradation include limited crosslinking and hydrogen-bonding interactions between the hydroxyl groups. Subcutaneous autoclaved PGS slabs were implanted in 15 seven-week-old female Sprague-Dawley rats and examined with histopathology at 7, 14, 21, 28 and 36 days postimplantation. The transient tissue reaction to the implants included PMN and mononuclear cells, edema and neovascularization peaking at 14-21 days with complete resolution by 6 weeks postimplantation. These findings are in marked contrast to the most commonly implanted material, polylactide polyglycolide copolymers (PLGA), which becomes chronically walled off by giant cells and a thick collagen capsule and undergoes rapid full thickness degeneration with concurrent loss of structural integrity. PGS shows promise as a delivery vehicle or implantable structural support in a variety of potential applications.
138: Using Vadds as a Teaching Tool in Senior Clinical Pathology Rotations
Senior students at the College of Veterinary Medicine, Iowa State University, must take a one-week rotation in clinical pathology, taught in groups of 12 students. Traditionally, this consisted of examining glass slides, cases, and didactic sections. Negative reviews of the didactic course section were addressed by integration of active clinical learning using a diagnostic reporting system, VetStar Animal Disease Diagnostic System (VADDS) and medical records system (VetStar). Groups of 2 or 3 students were assigned to find a “Case of the Day” consisting of a currently hospitalized patient with unique abnormalities in cytology, urinalysis, clinical chemistry, or hematology. During each afternoon of the rotation, students reported their case using VADDS linked to a computer projection system. Cytology, hematology, and urinalysis cases were augmented by using a microscope projection system during the presentations. The link between VetStar and VADDS allowed easy tracking of cases and demonstration of cases in the classroom. Students followed one case during the week or chose a new case each day. This approach promoted generation of differential diagnoses for active, “unsolved” cases rather than recitation of classic disease entities. Course review responses have been positive compared to the previous year on a scale from 10 (poor) to 1 (excellent). Ratings for instructional aids went from 2.45 to 1.72, stimulation of thought from 2.46 to 1.62; efficiency of lab time from 2.77 to 1.64; efficiency of class time from 2.95 to 1.68; and overall course evaluation from 2.58 to 1.62. In conclusion, requesting students to chose and display clinical pathology data from currently hospitalized cases stimulated engaged discussion, encouraged recall abilities, and reinforced incorporation of clinical pathology abnormalities in differential diagnoses.
139: Enhancing Problem Solving Skills for Clinical Pathology Students
The Problem List Generator (PLG) is a case-based software tool designed to help clinical pathology students improve diagnostic problem solving skills. The PLG presents patient data that includes history, signalment, physical exam and laboratory data, and then requires students to a) identify all abnormal laboratory data, and b) communicate their diagnostic reasoning by organizing that data into a problem list, which is a hierarchical outline of relationships between data and pathophysiologic mechanisms of disease. Students then compare their problem lists to one created by a faculty clinical pathologist in the same outline format. Previously presented data from the first two years of PLG implementation, and data collected during the Spring semester of 2002 suggest that using the PLG improves student diagnostic problem solving ability. All three years of data (student survey results, faculty member interviews, and final exam scores) were combined to assess the PLG's effectiveness. One treatment group of 180 students used the PLG for both case-discussion sessions and homework. Another treatment group of 84 students participated in PLG-based case-discussion sessions without using the PLG for homework. A comparison group of 334 students did not use the PLG for case-discussion sessions or homework. Final exam scores suggest that the PLG helps students learn clinical pathology more effectively. Both treatment groups scored significantly higher on the final exam than the comparison group (p = .000 and .001 respectively). Faculty interviews and student surveys mirrored final exam results. These findings suggest that providing an expert problem solving process to students, combined with a system that requires both students and teachers to document their diagnostic reasoning in a consistent and explicit format, improves student diagnostic problem solving ability.