Twenty-Fourth Annual Meeting and Symposium of the Desert Tortoise Council, March 5-8, 1999
Abstracts

Stomatitis/pharyngitis, with or without rhinitis, is a significant health problem of tortoises. The first report involved a 6 year old cachectic desert tortoise, Gopherus agassizii, which was in captivity since hatching. The tortoise was found to have a pharyngeal abscess which upon histologic examination had intranuclear inclusions in the superficial epithelial cells of the palatine mucosa (Harper et al. 1982). Electron microscopy demonstrated various developmental stages of a virus morphologically compatible with members of the family Herpetoviridae. In a second report, a 60-year-old captive desert tortoise with caseous necrosis of the oral cavity, choanae, trachea, and lungs, had intranuclear inclusions within epithelial cells at those sites, syncytial giant cells, and bacterial granulomas (Pettan-Brewer et al. 1996). By electron microscopy, herpesvirus-like particles were found within the inclusions.

Of 2,200 recently imported Argentine tortoises (Geochelone chilensis), 1,200 died over a 3 month period; red-footed tortoises (Geochelone carbonaria) imported with the Argentine tortoises and housed together remained clinically healthy (Jacobson et al. 1985). At necropsy, necrosis of the oral mucosa with accumulations of necrotic cellular debris around the glottis, the roof of the oral cavity, and internal nares was seen. By light microscopy, desquamated degenerating epithelial cells contained eosinophilic intranuclear inclusions. Electron microscopy, demonstrated inclusions to consist of viral particles containing an electron-dense core. Particles consistent with herpesvirus were seen enveloping from cell membranes and mature enveloped particles measuring approximately 125 nm were seen in the cytoplasm.

There are several reports of herpesvirus infection in Mediterranean tortoises (Testudo graeca and T. hermanni). Of 13 Greek tortoises (T. graeca) from two private colonies, herpes like particles were detected by electron microscopy in two animals with stomatitis (Cooper et al. 1988). Initially, while swabs taken from the oral lesions resulted in the isolation of a variety of microorganisms, treatment with a number of systemic and local antibiotics had no effect on the course of the disease. Eventually, viral particles consistent with herpesvirus were demonstrated by electron microscopy within bronchial and palatine mucosal epithelium. In 16 Hermann’s tortoises and 8 Greek tortoises with necrotizing glossitis/stomatitis, intranuclear inclusions were found in epithelial cells in the tongue, trachea, bronchi, alveolae, endothelial cells of capillaries of the glomeruli and within neurons and glial cells in the medulla oblongata and diencephalon (Muller et al. 1990). Electron microscopic examination of the liver and trachea demonstrated hexagonal nucleocapsids in the nuclei of hepatocytes and epithelial cells of the trachea. Enveloped virions in the cytoplasm were 110-120 nm and were morphologically consistent with herpesvirus. The authors considered imported tortoises to be latent carrier of this virus. Stress and parasitism may have contributed to the clinical manifestation of the virus in the imported tortoises. By electron microscopy, herpes-like particles have also been seen in the intestinal contents of a Hermann’s tortoise, several of which had caseous material in the upper digestive tract, hepatomegaly, and enteritis (Biermann et al. 1995).

There are multiple isolates of herpesvirus from Mediterranean tortoises with stomatitis/pharyngitis. Herpesvirus has been isolated in cell culture from brain, lung/trachea, and- liver from two Hermann’s tortoises and a Russian tortoise (T. horsfieldii) (Biermann and Blahak 1994) and from spleen, liver and brain of seven Hermann’s tortoise and one Russian tortoise (Kabisch and Frost 1994). A serum neutralization test was used to determine exposure of tortoises to herpesvirus and in one study, 42.5% of Greek tortoises and 18.5 % of Hermann’s tortoises were seropositive (Frost and Schmidt 1997).

While serum neutralization is often considered the gold standard when measuring an animal’s antibody response to a viral pathogen, it has limited utility because of certain practical problems since 9 to 10 days are required to determine the titer of a potentially exposed tortoise. Therefore we initiated studies designed to develop a more rapid and practical assay that would have wide application in private, zoological, rehabilitation, and breeding programs designed for releasing captive tortoises to the wild. Mediterranean tortoise immunoglobulin has been purified in the Core Hybridoma Laboratory, ICBR, University of Florida and mouse monoclonal and polyclonal antibodies have been produced against this immunoglobulin. With this reagent we will develop an immunoperoxidase and ELISA based approach to determining exposure to this virus(es).

References

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Pettan-Brewer, K. C. B., M. L. Drew, E. Ramsay, R. C. Mohr, L. J. Lowensine. 1996. Herpesvirus particles associated with oral and respiratory lesions in a California desert tortoise (Gopherus agassizii). J. Wild. Dis. 32:521-526.