25th Annual Meeting and Symposium of the
Desert Tortoise Council, April 21-24, 2000
Abstracts

Investigation of Herpesvirus Infection in Chelonians

Francesco Origgi¹, Elliott Jacobson², Paul Klein^3
1Departments of Small Animal Clinical Sciences and Pathobiology,
College of Veterinary Medicine, University of Florida
²Department of Small Animal Clinical Sciences,
College of Veterinary Medicine, University of Florida
³Department of Pathology, College of Medicine, University of Florida

Beginning in the mid 1970s, herpesviruses have been reported as pathogens associated with different diseases in several species of chelonians (Cox et al. 1980, Drury et al. 1998/1999, Frye et al. 1977, Harper et al. 1982, Heldstab and Bestetti 1989, Jacobson et al. 1982/1985/1986/1991, Kabish and Frost 1994, Marschang et al. 1997/1998/1999, Muro et al. 1998, Rebel et al. 1975). More recently herpesvirus infection has been identified as a significant health problem in Mediterranean tortoises (Testudo graeca and hermanni) (Drury et al. 1998/1999, Heldstab and Bestetti 1989, Kabish and Frost 1994, Marschang et al. 1997/1998/1999, Muro et al. 1998). Several herpesvirus strains have been isolated from pet Mediterranean tortoise in Europe and in the US, showing a worldwide distribution of these viruses. Two isolates have been recently purified in our laboratory. A variety of clinical signs, including stomatitis rhinitis, conjunctivitis, pneumonia and signs of central nervous system disease can be observed in ill tortoises (Heldstab and Bestetti 1989). Glossitis can be severe and diphteritic plaques on the tongue as well on the hard palate of the tortoises in the advanced stages of the disease are a common finding. The disease seems to target more aggressively the upper respiratory tract of the tortoise, but involvement of the lower respiratory tract has also been reported. Using light microscopy, intranuclear eosinophilic inclusions have been detected in multiple epithelial tissues and in the brain. Electron microscopy has shown inclusions to consist of virions that are morphologically compatible with herpesvirus.

The transmission route is totally unknown in the wild. Captive tortoises are commonly exchanged between different collectors, resulting in spreading of the disease. A six month quarantine is necessary to reduce the risk of transmission of the pathogen from infectious to naive tortoises. In naive collections the mortality can be as high as the 100% (Drury et al. 1998).

No specific therapy is currently available. An in vitro study with acyclovir and gancyclovir has shown the ability of these chemotherapeutics in reducing viral replication (Marschang et al. 1997).

An early diagnosis is the most effective preventive measure for reducing the risk of infection. The most definitive diagnostic test for herpesvirus infection in Mediterranean tortoises is virus isolation. Unfortunately virus isolation is not always achievable. In tortoises in early stages of the disease, relatively little or no virus at all is shed.

Of serological tests serum-neutralization is considered the "gold standard" for detection of exposure to herpesvirus in tortoises. This test detects the presence of serum-neutralizing antibodies and 11-14 days are needed for a diagnosis to be made (Frost and Shmidt 1997).

A new enzyme-linked immunosorbent assay (ELISA) has been recently developed at the University of Florida (Origgi and Jacobson 1999). Preliminary unpublished data shows a high sensitivity and specificity of the test compared to SNT. In a survey conducted on 175 plasma samples obtained from Mediterranean tortoises in a rehabilitation facility in France, 35 samples were positive for the presence of SN antibodies while 38 were positive using the ELISA test. All the samples positive by SN were positive also by ELISA. This assay is currently been validated through a transmission study conducted on Mediterranean tortoises. Furthermore we are currently screening a total of 31 recombinant DNA fragments obtained from the cloning of an American herpesvirus isolate (HV1976). Partial sequencing of several recombinants has already been accomplished. Sequencing information could allow the development of several new diagnostic tests that could complement the serological assays described above. These tests will allow rapid screening of tortoises in wild, private, breeding and zoo populations.

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