Tortoise Herpesvirus Infection

History:

The first report of a herpes-like virus infection in a tortoise was that of a captive-hatched and reared 6-yr old cachectic desert tortoise (Gopherus agassizi), that had a pharyngeal abscess (Harper et al, 1982). 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 in the respiratory tract, along with syncytial giant cells, and bacterial granulomas (Pettan-Brewer et al, 1996). By electron microscopy, herpesvirus-like particles were found within the inclusions.

Of 2200 recently imported Argentine tortoises (Geochelone chilensis), 1200 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, 1996). 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 carriers 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 1995).

Etiologic Agent:

Members of the family Herpetoviridae are double stranded DNA viruses that are approximately 100-150 nm in diameter. The viral nucleocapsid has a cubic symmetry with 162 capsomers, surrounded by an envelope. Replication occurs within the nuclei of cells with envelopment from the nuclear membrane. Sites of intranuclear replication are often seen by light microscopy as intranuclear inclusions with hematoxylin and eosin staining. Many herpesviruses may exist in a silent form in the natural host, resulting in active infection and disease when a susceptible species is exposed.

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. horsfieldiii; 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).

Host:

A virus compatible with herpesvirus has been seen in several species of tortoises including desert tortoises, Argentine tortoise, leopard tortoise, four-toed (Russian) tortoise, Hermann's tortoise, Greek tortoise, and yellow foot tortoise Until proven otherwise, all tortoises should be considered susceptible.

Distribution:

Herpesvirus has been detected in captive tortoises worldwide. In Europe, tortoises in the genus Testudo appear to be particularly susceptible to infection with herpesvirus. Ill and infected tortoises also have been seen in Germany, France, Spain, Switzerland, United Kingdom, and Japan. It seems to be highly prevalent among private pet collections. No information is available on prevalence of infection in wild populations.

Clinical Signs:

General clinical signs include rhinitis with a serous to mucous nasal discharge. These signs overlap with those seen in chelonian mycoplasmosis. In typical cases, necrotizing stomatitis, glossitis, and pharyngitis are seen (Figure 1; Figure 2; Figure 3). Dyspnea may occur. Conjunctivitis has been observed in association with the respiratory signs. Other more general clinical signs includes anorexia and cachexia. Involvement of the central nervous system may lead to incoordination and ataxia.

Pathology:

Necrotic caseous stomatitis (involving the palate and internal nares) and glossitis with presence of diphtheritic plaques are common findings in tortoises with herpesvirus infection (Figure 4; Figure 5). The involvement of the upper respiratory tract is generally associated with rhinitis and pharyngitis. The lower respiratory tract also may be affected as seen by tracheitis, pneumonia and eventually emphysema. By light microscopy, intranuclear inclusions (Figure 6; Figure 7; Figure 8) may be observed within epithelial cells at multiple tissues, along with syncytial giant cells and bacterial granulomas. Desquamated degenerating epithelial cells may contain eosinophilic intranuclear inclusions. Using electron microscopy, viral particles at various stages of development (Figure 9; Figure 10) can be found within nuclei and cytoplasm in epithelial cells in the tongue, trachea, bronchi, alveolae, hepatocytes, intestinal epithelial cells, endothelial cells of capillaries of the glomeruli, and within neurons and glial cells in the medulla oblongata and diencephalon.

Transmission:

The exact route of infection is unknown. However, based upon what we know about transmission in other species, direct tortoise to tortoise contact probably results in transmission between animals. No one knows how long the virus may persist in the environment, continuing to remain infectious. Vertical transmission through the egg may also occur.

Diagnosis:

Herpesvirus infection should always be considered in the differential diagnosis when gross lesions such as stomatitis/pharyngitis are observed. While finding intranuclear inclusions is also supportive of a diagnosis, it is necessary to examine tissue using transmission electron microscopy in order to determine their composition. Negative staining electron microscopy can be used to identify virions in exudates, scrapings, and fecal specimens. Viral isolation is currently the most definitive method for determining infection. The virus can be isolated in commercially available Terrepene heart cells. 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 for determining exposure to this virus(es).

Control and Treatment:

Suspect and ill tortoises should be isolated from clinically healthy tortoises. All new tortoises should be quarantined minimally for 90 days before being introduced into the main collection. Treatment with anti-viral drugs such as acyclovir has been recommended. Antimicrobials may be useful in controlling secondary infections. Disinfection of enclosures depends upon the type of enclosure used for housing pet tortoises. Outdoor pens probably should be rotated since the virus may persist in the soil or environment for several weeks. If tortoises are being kept in metal livestock tanks or rubber-made containers, the containers can be disinfected with 3% sodium hypochlorite.

Current and Future Research:
  • Development of an ELISA and Immunoperoxidase based serological tests to determine the exposure of tortoises to herpesvirus. Testudo immunoglobulin has been isolated and purified for both monoclonal and polyclonal antibody production.
  • Development of a Polymerase Chain Reaction (PCR) method to determine the presence of specific nucleotide sequences for herpesvirus in viral DNA for rapid detection in tissues and exudates.
  • Cloning of a viral glycoprotein to serve as the antigen in a serologic test.
References:
  • Harper PAW, Hammond DC, Heuschele WP. 1982. A herpesvirus-like agent associated with a pharyngeal abscess in a desert tortoise. J Wildl Dis 18:491-494.
  • Pettan-Brewer KCB, Drew ML, Ramsay E, Mohr RC, Lowenstine LJ. 1996. Herpesvirus particles associated with oral and respiratory lesions in a California desert tortoise (Gopherus agassizii). J Wildl Dis 32:521-526.
  • Jacobson ER, Clubb S, Gaskin M, Gardiner C. 1985. Herpesvirus like infection in Argentine tortoises. J Am Vet Med Assoc 187:1227-1229.
  • Cooper JE, Gscheimeissner S, Bone DR. 1988. Herpesvirus like particles in necrotic stomatitis in tortoises. Vet Rec 123: 554.
  • Muller M, Sachsse W, Zangger N. 1990. Herpesvirus-epidemie bei der Griechischen (Testudo hermanni) und der Maurischen Landschildkrote (Testudo graeca) in der Schweiz. Schweiz Arch Tierheilk 132:199-203.
  • Biermann RH. 1995. Isolierung und Charakterisierung von Herpesviren bei Landschildkroten. Med. Vet. Diss. Giessen.
  • Biermann RH, and Blahak S. 1994. First isolation of a herpesvirus from tortoises with diphtheroid-necrotizing stomatitis. Second World Congress of Herpetology. Abstracts. Jan 6: 27.
  • Kabisch D, Frost JW. 1994. Isolation of herpesvirus from Testudo hermanni and Agrionemys horsfieldii. Verh ber Erkrg Zootiere 36:241-245.
  • Frost JW, Schmidt A. 1997. Serological evidence for susceptibility of various species of tortoises to infections by herpesvirus. Verh ber Erkrg Zootiere 38:25-28.
For More Information Contact:

Dr. Francesco Origgi
Box 100126
Department of Small Animal Clinical Sciences
College of Veterinary Medicine
University of Florida
Gainesville, Florida 32610-0126
E-mail: origgif@vetmed.ufl.edu

Dr. Elliott Jacobson
Box 100126
Department of Small Animal Clinical Sciences
College of Veterinary Medicine
University of Florida
Gainesville, Florida 32610-0126 USA
E-Mail: jacobsone@vetmed.ufl.edu

Dr. Paul Klein
Box 100275
Department of Pathology and Laboratory Medicine
College of Medicine
University of Florida
Gainesville, Florida 32610-0275 USA
E-Mail: paklein@college.med.ufl.ed