Charles A. Baldwin

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.

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.

Efficacy of select disinfectants at inactivating Ranavirus

Ranavirus can cause disease in reptiles and amphibians. Because survival time outside of a host remains uncertain, equipment must be disinfected to prevent transmission of ranaviruses. However, disinfectant efficacy against amphibian ranaviruses has not been investigated for chlorhexidine (Nolvasan), sodium hypochlorite (bleach), or potassium compounds. Our goal was to determine the efficacy of Nolvasan (0.25, 0.75 and 2.0%), bleach (0.2, 1.0, 3.0 and 5.0%), and Virkon S (1.0%) at inactivating Ranavirus at 1 and 5 min contact durations. Potassium permanganate (KMnO4) (2.0 and 5.0 ppm) was also tested with a 60 min contact time. Nolvasan at 0.75 and 2.0% and bleach at 3.0 and 5.0% concentration were effective for both contact durations. Virkon S was effective for both durations, but KMnO4 was not effective at either concentration. Concentrations of Nolvasan, bleach and Virkon S that are at least 0.75, 3.0 and 1.0%, respectively, are effective at inactivating Ranavirus after 1 min exposure time.

Concurrent Infection with Ranavirus, Batrachochytrium dendrobatidis, and Aeromonas in a Captive Anuran Colony

Abstract Four species (Dendrobates auratus, Phyllobates terribilis, Pyxicephalus adspersus, and Rhacophorus dennysi) of captive anurans with a clinical history of lethargy and inappetence were found dead and were submitted for necropsy. Gross lesions included irregular patches of sloughed skin and rare dermal ulcerations. Histologic findings included epidermal proliferation that was most pronounced on the digits and that included intracytoplasmic chytrid organisms. Bacteria were often associated with the epidermal lesions. Intracytoplasmic inclusion bodies were observed in hepatocytes. Real-time polymerase chain reaction yielded positive results for both Ranavirus and Batrachochytrium dendrobatidis (Bd). Bacterial culture of internal organs yielded Aeromonas hydrophila. This is the first report of concurrent infections in anurans by Ranavirus and Bd and A. hydrophila.

Diagnosis of Encephalitic Bovine Herpesvirus Type 5 (BHV-5) Infection in Cattle: Virus Isolation and Immunohistochemical Detection of Antigen in Formalin-Fixed Bovine Brain Tissues

1. Barry R, Vincent M, Kent S, et al.: 1988, Characterization of prion proteins with monospecific antisera to synthetic peptides. J Immunol 140:1188-1193. 2. Bendheim PE, Barry RA, DeArmond SJ, et al.: 1984, Antibodies to a scrapie prion protein. Nature 310:418-421. 3. Berg LJ: 1994, Insights into the role of the immune system in prion diseases. Proc Natl Acad Sci USA 91:429432. 4. Caughey B, Race RE, Vogel M, et al.: 1988, In vitro expression in eukaryotic cells of a prion protein gene cloned from scrapieinfected mouse brain. Proc Natl Acad Sci USA 85:4657-4661. 5. Chesebro B, Race R, Wehrly K, et al.: 1985, Identification of scrapie prion protein-specific mRNA in scrapie-infected and uninfected brain. Nature 315:331-333. 6. Hadlow W, Race R, Kennedy R, Eklund C: 1979, Natural infection of sheep with the scrapie virus. In: Slow transmissible diseases of the nervous system, ed. Pruisner S, Hadlow W, vol. 2, pp. 3-12. Academic Press, New York, NY. 7. Katz JB, Pedersen JC, Jenny AL, Taylor WD: 1992, Assessment of western immunoblotting for the confirmatory diagnosis of ovine scrapie and bovine spongiform encephalopathy (BSE). J Vet Diagn Invest 4:447449. 8. Maciulis A, Hunter N, Wang S, et al.: 1992, Polymorphisms of a scrapie-associated fibril protein (PrP) gene and their association with susceptibility to experimentally induced scrapie in Cheviot sheep in the United States. Am J Vet Res 53: 19571960. 9. Miller JM, Jenny AL, Taylor WD, et al.: 1993, Immunohistochemical detection of prion protein in sheep with scrapie. J Vet Diagn Invest 5:309-316. 10. Oesch B, Westaway D, Walchli M, et al.: 1985, A cellular gene encodes scrapie PrP27-30 protein. Cell 40:735-746. 11. Pruisner SB, Fuzi M, Scott M, et al.: 1993, Immunologic and molecular biologic studies of prion proteins in bovine spongiform encephalopathy. J Infect Dis 167:602-613. 12. Rogers M, Serban D, Gyuris T, et al.: 1991, Epitope mapping of the Syrian hamster prion protein utilizing chimeric and mutant genes in a vaccinia virus expression system. J Immunol 147:3568-3574. 13. Scott MR, Butler DA, Bredesen DE, et al.: 1988, Prion protein gene expression in cultured cells. Protein Eng 2:69-76. 14. Summers MD, Smith GE: 1988, A manual of methods for baculovirus vectors and insect cell culture procedures. Tex Agric Exp Stn Bull 1555: 18-32. 15. Wells GA, Scott AC, Johnson CT, et al.: 1987, A novel progressive spongiform encephalopathy in cattle. Vet Rec 121:419420.

PATHOLOGIC FINDINGS IN LARVAL AND JUVENILE ANURANS INHABITING FARM PONDS IN TENNESSEE, USA

Amphibian populations are declining globally, yet general pathologic surveys for free-ranging amphibians are uncommon. Pathologic surveys are necessary to provide insight into the impacts of humans on emergence of pathogens in amphibian populations. During 2005, 104 American bullfrog (Rana catesbeiana) and 80 green frog (Rana clamitans) larvae and 40 green frog juveniles were collected from farm ponds in Tennessee, and complete necropsies were performed. Diagnostic testing included bacterial culture, virus testing, fecal parasite analysis, and histologic examination. Gross and histologic examination revealed that all individuals, except one bullfrog tadpole, could be classified as clinically normal. The clinically abnormal tadpole had swollen erythemic legs, and was positive for Aeromonas hydrophila but negative for Ranavirus. Parasites were common (43%) among specimens, with myxosporidium and trematodes most often noted. Commensal and opportunistic microorganisms were cultured from the tissues. Ranavirus was detected in 29% of individuals but generally not associated with significant histopathologic changes. Myxosporidia and Ranavirus coinfections occurred in 7 and 26% of green and bullfrog tadpoles, respectively, with the highest coinfection rate (83%) in bullfrog tadpoles during winter. Protozoans were most common in fecal examination. These data can serve as a baseline to evaluate the presence of clinical disease in larval and juvenile amphibians.

The Seroprevalence of Johne's Disease in Georgia Beef and Dairy Cull Cattle

Beef and dairy cattle serum samples, collected during 2000 at sale barns throughout Georgia, were obtained from the Georgia State Brucellosis Laboratory and were used to conduct a retrospective epidemiological study. Statistical samplings of 5,307 sera, from over 200,000 sera, were tested for antibodies to Mycobacterium avium ssp. paratuberculosis, (Johnes disease) using a commercial enzyme-linked immunosorbent assay test kit. An overall period seroprevalence in all classes of cattle tested was 4.73%. The period seroprevalence in dairy cattle was 9.58%, in beef cattle it was 3.95%, and in cattle of unknown breed it was 4.72%. It was concluded that the seroprevalence of Johnes disease in cull beef and dairy cattle in Georgia is economically significant.

Equine Herpesvirus 2-Associated Granulomatous Dermatitis in a Horse

Granulomatous dermatitis in horses has been linked to many etiologies, including various parasites, fungi, and bacteria. Idiopathic forms of granulomatous inflammation-producing diseases, some of which are localized to the skin, also have been reported in horses. Herein we describe a case of recurrent equine granulomatous skin disease characterized by intranuclear viral inclusions within macrophages and giant cells. The histologic changes were primarily noted in the deep dermis and included multifocal to coalescing areas of necrosis marked by histiocytic cell infiltration and presence of giant cells. Electron microscopic examination revealed intranuclear and intracytoplasmic viral particles consistent with herpesvirus. Sequence results of the polymerase chain reaction product were consistent with equine herpesvirus 2, adding another possible etiology to the list of differentials in cases of equine granulomatous skin disease.

Eastern equine encephalitis in dogs

Eastern equine encephalitis virus (EEEV) is an Alphavirus that is endemic in the Southeastern United States. From 1993 to January 2005, the Veterinary Diagnostic and Investigational Laboratory in Tifton, Georgia, performed postmortem examinations on over 101 domestic canines exhibiting clinical neurological disturbances. In 12 of these dogs, brains were histologically suggestive of infection with EEEV. All dogs were less than 6 months of age, with no breed predilection. Clinical signs included pyrexia, depression, nystagmus, and lateral recumbency. Microscopically, brains from all 12 puppies contained infiltrates of lymphocytes, plasma cells, and histiocytes, with occasional neutrophils and random foci of astrocytosis and gliosis. There were mild to moderate perivascular infiltrates of neutrophils along with scattered lymphocytes, plasma cells, and macrophages in the meninges. Viruses isolated from brain homogenates of all 12 puppies were confirmed by indirect fluorescent antibody testing to be EEEV. Additionally, RNA extracted from the brains and viral cultures of 2 dogs were determined by a specific reverse-transcriptase polymerase chain reaction (RT-PCR) to contain EEEV. The single available serum sample exhibited a 1:8 serum neutralization titer to EEEV.