Murray E. Hines

West Nile Virus in Farmed Alligators

Seven alligators were submitted to the Tifton Veterinary Diagnostic and Investigational Laboratory for necropsy during two epizootics in the fall of 2001 and 2002. The alligators were raised in temperature-controlled buildings and fed a diet of horsemeat supplemented with vitamins and minerals. Histologic findings in the juvenile alligators were multiorgan necrosis, heterophilic granulomas, and heterophilic perivasculitis and were most indicative of septicemia or bacteremia. Histologic findings in a hatchling alligator were random foci of necrosis in multiple organs and mononuclear perivascular encephalitis, indicative of a viral cause. West Nile virus was isolated from submissions in 2002. Reverse transcription-polymerase chain reaction (RT-PCR) results on all submitted case samples were positive for West Nile virus for one of four cases associated with the 2001 epizootic and three of three cases associated with the 2002 epizootic. RT-PCR analysis was positive for West Nile virus in the horsemeat collected during the 2002 outbreak but negative in the horsemeat collected after the outbreak.

Bacterial Pathogens Isolated from Cultured Bullfrogs (Rana Castesbeiana)

A commercial bullfrog (Rana castesbeiana) operation in south Georgia had multiple epizootics of systemic bacterial infections over a 3-year period, 1998–2000. A number of potential pathogens (Aeromonas hydrophila, Chryseobacterium (Flavobacterium) meningosepticum, Chryseobacterium (Flavobacterium) indolgenes, Edwardsiella tarda, Citrobacter freundii, Pseudomonas spp., and (Streptococcus iniae) were isolated from various tissues. Clinically, frogs demonstrated acute onset of torticolis, stupor, and indifference to stimuli. Cutaneous hyperemia, subcutaneous and muscular hemorrhage, and peripheral edema were consistent gross findings. Histologically, clusters of lymphocytes, monocytes, and occasional acidophiles with scattered granulomas occurred in liver, kidney, and spleen. This is the first report of S. inae and C. meningosepticum as potential disease agents in R. castesbeiana. These findings suggest that a variety of bacteria may be associated with redleg and that culture results must be obtained for accurate diagnosis.

Endometritis in Postparturient Cattle Associated with Bovine Herpesvirus-4 Infection: 15 Cases

Suppurative, ulcerative endometritis associated with bovine herpesvirus-4 (BHV-4) infection was identified in 15 postparturient dairy cows from 5 separate dairies. Characteristic eosinophilic to amphophilic intranuclear viral inclusion bodies were identified within degenerate endometrial lining epithelium and endothelial cells. Bovine herpesvirus-4 was confirmed as the etiology by a combination of fluorescent antibody assays, viral isolation, heminested PCR, ultrastructural examination of the uterus and inoculated tissue culture cells, and negative-stain electron microscopy of tissue culture supernatant. Viral particles measuring 70–95 nm were demonstrated in uterine epithelial and endothelial cells by electron microscopy. Bacteria including Arcanobacterium pyogenes, Escherichia coli, and an α-Streptococcus isolate were isolated from all uteri. Bovine herpesvirus-4-associated endometritis has been previously reported in sporadic cases in Europe but has not been previously reported in the United States. Endometritis associated with BHV-4 appears to be an emerging syndrome in Georgia dairy herds.

Seroprevalence and Comparison of Isolates of Endometriotropic Bovine Herpesvirus-4

Sixty-eight cases of suppurative, ulcerative endometritis associated with Bovine Herpesvirus-4 (BHV-4) in postparturient dairy cows (62 Holsteins and 6 Jerseys, mean age 4.2 years) were confirmed by a combination of histopathology, fluorescent antibody assays, electron microscopic evaluation of uterus, and polymerase chain reaction (PCR). All cases occurred in the 3- to 28-day postpartum period, and histologic lesions among various cows were consistent when compared with postpartum interval. The endometrial lining epithelium was necrotic and ulcerated from 3 to 7 days postpartum, with only mild inflammation in the lamina propria and submucosa. From 1 to 4 weeks postpartum, the ulcers were confluent to diffuse. Epithelium was replaced by fibrinonecrotic, suppurative mats, resulting in severe bacterial pyometra by day 24. Seroprevalence to BHV-4 in one dairy with a history of 18 mortality cases was 36% (107 of 296). In a random sample of 8 cows from this herd, none had serologic titers in blood sampled 2 weeks prepartum, but 3 of 8 seroconverted with significant titers of 1:8 to 1:16 at 2 weeks postpartum. By 10 weeks postpartum, all 8 cows returned to negative serologic status. Two of 6 cats from the premises also had positive titers. Random serum samples taken from 480 dairy cattle at sale barns indicated 76 (16%) were positive by serum neutralization. Clinical signs, postparturient timing, and histologic lesions were very similar to those previously reported in Belgium with BHV-4. But sequence analysis of PCR products of the glycoprotein B region of 4 separate field isolates of endometriotropic BHV-4 suggests these field isolates were more closely related to the North American nonvirulent strain DN-599 than to the endometriotropic European strain V.

Diagnosis of Enteritis and Enterotoxemia due to Clostridium Difficile in Captive Ostriches (Struthio Camelus)

petted normal ranges. Seven of the expired birds were necropsied immediately after death. Two live ostriches with clinical signs were euthanized, and tissues were obtained for histopathology, bacterial culture, and viral isolation. At necropsy, all of the ostriches examined had similar gross lesions that differed only in severity. The colon and cecum were markedly dilated and diffusely hemorrhagic and contained no formed feces. The lungs were variably congested, and the liver was frequently pale yellow in color. Two of the birds had hemorrhagic foci in the proventriculus. No other significant gross lesions were observed in any of the birds. Microscopic examination of the tissues revealed an acute, severe, necrotizing typhlitis and colitis in all birds, with massive numbers of large gram-positive bacilli present within glands and in the intestinal lumen (Figs. 1, 2). There was moderate, diffuse hepatocellular vacuolar change in 6 birds and multifocal lymphoid necrosis in the bursa of Fabricius of 2 of the ostriches. No viral inclusions were noted. Moderate to severe congestion was present in the lungs of 7 of 9 birds. Two of the birds had a mild necrotizing proventriculitis with mild hemorrhage. Sections of the intestine were submitted for aerobic and anaerobic culture. Enrichment for Salmonella in selenite broth for 12 hours yielded negative results, and no clinically significant aerobic pathogenic bacteria were found. Two different species of Clostridium were anaerobically cultured from the intestine. They were tentatively identified as C. hastiforme and C. clostridiiforme by API 20A biochemical assay. b After isolation and subculture of the original cultures in cycloserine-cefoxitin-fructose agar and additional biochemical analysis by An-IDENT biochemical assay, b the C. hastiforme culture was shown to be C. difficile. The API-20A biochemical assay was then repeated on the pure culture, and it too indicated the presence of C. difficile. The intestine was submitted for clostridial enterotoxin assay c by enzyme-linked immunosorbent assay (ELISA). Extracts of multiple sections

Evaluation of novel oral vaccine candidates and validation of a caprine model of Johne's disease

Johnes disease (JD) caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a major threat to the dairy industry and possibly some cases of Crohns disease in humans. A MAP vaccine that reduced of clinical disease and/or reduced fecal shedding would aid in the control of JD. The objectives of this study were (1) to evaluate the efficacy of 5 attenuated strains of MAP as vaccine candidates compared to a commercial control vaccine using the protocol proposed by the Johnes Disease Integrated Program (JDIP) Animal Model Standardization Committee (AMSC), and (2) to validate the AMSC Johnes disease goat challenge model. Eighty goat kids were vaccinated orally twice at 8 and 10 weeks of age with an experimental vaccine or once subcutaneously at 8 weeks with Silirum® (Zoetis), or a sham control oral vaccine at 8 and 10 weeks. Kids were challenged orally with a total of approximately 1.44 × 10(9) CFU divided in two consecutive daily doses using MAP ATCC-700535 (K10-like bovine isolate). All kids were necropsied at 13 months post-challenge. Results indicated that the AMSC goat challenge model is a highly efficient and valid model for JD challenge studies. None of the experimental or control vaccines evaluated prevented MAP infection or eliminated fecal shedding, although the 329 vaccine lowered the incidence of infection, fecal shedding, tissue colonization and reduced lesion scores, but less than the control vaccine. Based on our results the relative performance ranking of the experimental live-attenuated vaccines evaluated, the 329 vaccine was the best performer, followed by the 318 vaccine, then 316 vaccine, 315 vaccine and finally the 319 vaccine was the worst performer. The subcutaneously injected control vaccine outperformed the orally-delivered mutant vaccine candidates. Two vaccines (329 and 318) do reduce presence of JD gross and microscopic lesions, slow progression of disease, and one vaccine (329) reduced fecal shedding and tissue colonization.

Preliminary characterization of chemically generated Mycobacterium avium subsp. paratuberculosis cell wall deficient forms (spheroplasts).

Cell wall deficient forms (CWD, spheroplasts) genetically indistinguishable from M. avium subsp. paratuberculosis (MAP) have been isolated from patients with Crohns disease and sarcoidosis. These MAP CWD may be important in the pathogenesis of these diseases and in Johnes Disease in other animal species. CWD forms are extremely difficult to isolate and generally revert to cell wall competent forms (CWC) when cultured in vitro. Cultured MAP strain 19698 were chemically treated to generate sufficient CWD to compare to CWC organisms by electron microscopy, chemotype profile (matrix solid-phase dispersion and thin layer chromatography), silver-stained SDS-PAGE gels with and without periodic acid treatment and Western blots with antigen recognition by sera from confirmed Johnes positive and Johnes negative cattle. On electron microscopy, CWD organisms were larger and rounder than cell wall competent forms and had lost the majority of their cell walls, being bounded only by a plasma membrane. Chemotype profiles of CWD lacked bands generally associated with cell wall glycolipids. Silver-stained SDS-PAGE gels of CWD demonstrated loss of bands that migrate in the same region as lipoarabinomannan (LAM) and some bands likely representing proteins and weakening of bands that migrate similarly to phosphatidylinositol mannosides (PIM). Western blots of CWD demonstrated bands with loss or attenuation of signal that migrate similarly to LAM and other constituents. In summary, CWD and CWC forms of MAP 19698 had marked differences in morphology, chemotype profile, cell wall constituents, and antigens recognized by Johnes disease positive and negative bovine sera.

A rational framework for evaluating the next generation of vaccines against Mycobacterium avium subspecies paratuberculosis

Since the early 1980s, several investigations have focused on developing a vaccine against Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johnes disease in cattle and sheep. These studies used whole-cell inactivated vaccines that have proven useful in limiting disease progression, but have not prevented infection. In contrast, modified live vaccines that invoke a Th1 type immune response, may improve protection against infection. Spurred by recent advances in the ability to create defined knockouts in MAP, several independent laboratories have developed modified live vaccine candidates by transpositional mutation of virulence and metabolic genes in MAP. In order to accelerate the process of identification and comparative evaluation of the most promising modified live MAP vaccine candidates, members of a multi-institutional USDA-funded research consortium, the Johnes disease integrated program (JDIP), met to establish a standardized testing platform using agreed upon protocols. A total of 22 candidates vaccine strains developed in five independent laboratories in the United States and New Zealand voluntarily entered into a double blind stage gated trial pipeline. In Phase I, the survival characteristics of each candidate were determined in bovine macrophages. Attenuated strains moved to Phase II, where tissue colonization of C57/BL6 mice were evaluated in a challenge model. In Phase III, five promising candidates from Phase I and II were evaluated for their ability to reduce fecal shedding, tissue colonization and pathology in a baby goat challenge model. Formation of a multi-institutional consortium for vaccine strain evaluation has revealed insights for the implementation of vaccine trials for Johnes disease and other animal pathogens. We conclude by suggesting the best way forward based on this 3-phase trial experience and challenge the rationale for use of a macrophage-to-mouse-to native host pipeline for MAP vaccine development.

Analysis of DNA Aneuploidy and c-myc Oncoprotein Content of Canine Plasma Cell Tumors Using Flow Cytometry

To derive a method for determining malignant potential of plasma cell tumors, a retrospective analysis of the DNA ploidy and relative p62c-myc oncoprotein content using bivariate flow cytometry was performed on 23 formalin-fixed, paraffin-embedded tissues from 23 dogs. The samples included one tissue each from 17 males and six females 2 to 16 years of age (mean = 7.5 years). Twelve breeds were represented, including three Cocker Spaniels, three Golden Retrievers, and five of mixed breed. Ten of the samples were histologically classified as malignant-plasma cell tumors, and ten specimens were classified as benign. Three samples of plasmacytic inflammation, from two Cocker Spaniels and one Shih Tsu, were included as controls. The ploidy and relative c-myc content data obtained were compared with the histologic grade. A significant difference in ploidy was found between benign and malignant tumors (P ≤ 0.05). Five of nine malignant plasma cell tumors were aneuploid; the remainder were diploid (4/9) or tetraploid (1/9). Only one of the benign plasmacytomas was aneuploid (1/10), whereas six were diploid (6/10), and three were tetraploid (3/10). All of the controls were diploid (3/32). When relative amounts of p62c-myc from malignant and benign tumors were compared by flow cytometry, a greater significant difference was established (P ≤ 0.01) than by using aneuploidy alone. Relative values of p62c-myc content ranged from 219 to 553 units in 8/10 malignant plasma cell tumors and from 86 to 392 units in 3/10 benign plasmacytomas. The remainder of the neoplasms (2/10 malignant and 7/10 benign) lacked measurable values of p62c-myc above background fluorescence concentrations. Two atypical cutaneous plasmacytomas with later metastasis were included in the study. The results indicate that simultaneous analysis of ploidy and relative p62c-myc concentration can be used as an aid in assessment of malignant potential in canine plasma cell tumors.

Rete Testis Mucinous Adenocarcinoma in a Dog

A bilateral testicular neoplasm from an 11-year-old mixed-breed male dog was removed surgically and examined histologically. The neoplasm was nonencapsulated and composed of acinar and tubular structures lined by one or more layers of neoplastic polyhedral epithelial cells with an abundant mucinous secretion. On histochemistry, all neoplastic cells and associated secretions were periodic acid-Schiff positive. Some neoplastic cells and all associated secretions were positive on mucicarmine stain, and some neoplastic cells, all the stroma, and associated secretions were positive on alcian blue stain. On immunohistochemistry, the neoplastic cells had strong diffuse cytoplasmic immunoreactivity for cytokeratin and vimentin, weak scattered cytoplasmic immunoreactivity for carcinoembryonic antigen and neuron-specific enolase, and no immunoreactivity for S-100. On the basis of histopathology, histochemistry, and immunohistochemical findings, a diagnosis of mucinous adenocarcinoma of rete testis was made. Rete testis adenocarcinoma is a well known but very rare neoplasm in humans. To our knowledge, this is the first report of the mucinous variant of adenocarcinoma of the rete testis in a dog.