Abstract

A number of viral infections have been reported in tortoises. These include reports of herpesviruses associated with necrotizing stomatitis, which appears to be responsible for significant morbidity and mortality among captive land tortoises in Europe and has also been reported in the United States.^{2,5-7,9,11-15,17} Iridoviruses have been identified by electron microscopy in lesions of tortoises suffering from hepatitis and respiratory disease.^8,16,23 Togaviruses have been isolated from the blood of Texas tortoises, but have not been reported in association with any disease.^4,21 Reoviruses have not been reported in chelonians, but have been identified and isolated in other reptiles.^1,3,10,19,22 The only viruses isolated from and associated with a distinct disease process in land tortoises are herpesviruses.^2,14 Koch’s postulates have not been fulfilled for any viral infections of tortoises. This study reports the isolation of herpes-like, irido-like, and reo-like viruses, as well as an unidentified, nonenveloped virus from organ samples of land tortoises.

In 1996, 315 organ samples from 27 land tortoises necropsied at the Department of Zoo Animal Pathology, Institute of Animal Pathology, Bern were tested in cell culture for the presence of cytopathic agents. The tortoises tested consisted of nine Hermann’s tortoises (Testudo hermanni), seven spur-thighed tortoises (T. graeca), four red-foot tortoises (Geochelone carbonaria), four leopard tortoises (G. pardalis), one yellow-foot tortoise (G. denticulata), one radiated tortoise (G. radiata) and one parrot-beaked tortoise (Homopus aereolatus). The animals tested were chosen on the basis of pathologic and histopathologic findings indicative of a virus infection, including inclusion bodies and unclear diagnoses.

Organ samples were collected in cell culture medium (basal medium Eagle) (BME) with Earle’s salt solution (Seromed, Biochrom KG, Berlin, Germany) and frozen until testing. Before testing, the samples were thawed at room temperature, sonified and centrifuged at low speed (1000xg, 5 min) for the removal of cell debris. Supernatant was inoculated onto Terrapene heart cells (TH-1, CCL (50), ATCC, Rockville, Maryland USA) and chicken embryo fibroblasts (CEF) prepared according to Schat and Purchase.²⁰ All cultures were incubated at 28°C and observed daily for cytopathic effects (CPE). If no CPE was visible after 14 days, a second passage was performed. Cytopathic agents were tested for their sensitivity to chloroform, inhibition of replication in cell culture by 5-iodo-2’-desoxyuridine (IUDR) and cell culture supernates were examined by electron microscopy.

Herpes-like viruses were isolated in TH-1 cell cultures from the esophagus of two, and the small intestine of one, spur-thighed tortoises and the tongue, trachea and brain of one yellow-foot tortoise. The isolates caused round cell formation in TH-1 cells typical of tortoise herpesviruses^2,14 and no CPE in CEF. They were further identified as herpesviruses on the basis of their sensitivity to chloroform, inhibition of replication by IUDR and by electron microscopy. Histopathologic examination also revealed eosinophilic intranuclear inclusion bodies in cells of affected organs.

Irido-like viruses were isolated from the tongue, trachea, lung, liver, esophagus, small and large intestine and cloaca of one and the tongue of another Hermann’s tortoise. These isolates caused a round cell CPE and vacuolization in both TH-1 cells and CEF. They were also found to be sensitive to chloroform and IUDR, and electron microscopy revealed viral particles morphologically consistent with iridoviruses. Histopathologic examination of tissues of these animals revealed basophilic intracytoplasmatic inclusion bodies in several organs.

A reo-like virus was isolated from the tongue, esophagus, lung, liver and kidney of one spur-thighed tortoise. This isolate caused syncytium formation in CEF and no CPE in TH-1 cells. The isolate was not sensitive to chloroform or IUDR, showing it to be non-enveloped and possibly an RNA-virus. Electron microscopic examination revealed reo-like virus particles in the cell culture supernatant.

An agent that caused lysis in TH-1 cells, but no CPE in CEF was isolated from a number of organs from two leopard tortoises, three Hermann’s tortoises, and four spur-thighed tortoises (Table 1). This agent was not sensitive to chloroform or IUDR, could be serially passaged in TH-1 cell culture and was able to pass through a 100 nm filter without a reduction in titer. Despite repeated efforts, we have not yet been able to visualize it in an electron microscope, despite the fact that it reaches titers of 7–8 log₁₀ TCID₅₀/ml (mean tissue culture infectious dose) in TH-1 cell cultures. Herpesviruses were also isolated from three of the spur-thighed tortoises. All of the animals from which these unidentified viruses were isolated suffered from disease symptoms typically described for herpesvirus stomatitis. Histologic examination demonstrated intranuclear inclusion bodies in various tissues in all but one of the Hermann’s tortoises. The significance of these viruses in the disease process is unclear. Since they replicate faster in cell culture than herpesviruses, it is possible that all of the animals from which these viruses were isolated were also infected with herpesviruses which could not be isolated because the cell monolayer was destroyed by this agent first.

Koch’s postulates have not yet been fulfilled for any viral infections of tortoises. Although histologic findings have so far strongly suggested that herpesviruses are responsible for viral stomatitis in tortoises,¹⁸ the fact that another virus was isolated from so many animals also diagnosed with herpesvirus infection suggests that it could also be a factor in this disease. It is also possible that this virus is a secondary infection in animals whose immune systems have been weakened by other disease processes. The irido-like virus infection in the two Hermann’s tortoises was also clinically similar to herpesvirus stomatitis. These data indicate that a number of viruses are potentially virulent for tortoises and are therefore of concern for owners, breeders, zoos, the pet trade and conservation programs. It is important for veterinarians to consider various viral diseases as differential diagnoses in tortoises. Studies to identify and characterize the isolated viruses are ongoing.

Table 1. Animals and organs from which an unidentified, nonenveloped virus was isolated

Acknowledgments

The authors would like to thank Dr. Werner Herbst, Institut für Hygiene und Infektionskrankheiten der Tiere, Justutus Liebig University, Giessen, Germany and Dr. Peter Wild, Institute of Veterinary Anatomy, Laboratory for Electron Microscopy, University of Zurich, Switzerland for electron microscopy.

Literature Cited

1.  Ahne, W., I. Thomsen, and J. Winton. 1987. Isolation of a reovirus from the snake Python regius. Arch. Virol. 94: 135–139.

2.  Biermann, R. 1995. Isolierung und Charakterisierung von Herpesviren bei Landschildkröten. Veterinary Medical Dissertation, Justus Liebig University, Giessen, Germany.

3.  Blahak, S., I. Ott, and E. Vieler. 1995. Comparison of six different reoviruses of various reptiles. Vet. Res. 26: 470–476.

4.  Bowen, G.S. 1977. Prolonged western equine encephalitis viremia in the Texas tortoise (Gopherus berlandieri). J. Trop. Med. Hyg. 26: 171–175.

5.  Braune, S, V. Geiss, and W. Thiel. 1989. Eine neue durch Herpesviren verursachte Erkrankung bei Landschildkröten. Tierärztl. Prax. 17: 416–419.

6.  Cooper, J.E., S. Geschmeissner, and R.D. Bone. 1988. Herpes-like virus particles in necrotic stomatitis of tortoises. Vet. Rec. 123: 554.

7.  Harper, P.A.W., D.C. Hammond, and W.P. Heuschele. 1982. A herpesvirus-like agent associated with a pharyngeal abscess in a desert tortoise. J. Wildl. Dis. 18: 491–494.

8.  Heldstab, A., and G. Bestetti. 1982. Spontaneous viral hepatitis in a spur-tailed Mediterranean land tortoise (Testudo hermanni). J. Zoo Anim. Med. 13: 113–120.

9.  Heldstab, A., and G. Bestetti. 1984. Herpesviridae causing glossitis and meningoencephalitis in land tortoises (Testudo hermanni). Proc. Sec. Int. Col. Path. Reptiles and Amphibians.

10.  Jacobson, E.R. 1986. Viruses and viral associated diseases of reptiles. Acta Zool. et Path. Antverpensia 79: 73–90.

11.  Jacobson, E.R., S. Clubb, J.M. Gaskin, and C. Gardiner. 1985. Herpesvirus-like infection in Argentine tortoises. J. Am. Vet. Med. Assoc. 187: 1227–1229.

12.  Kabisch, D., and J.W. Frost. 1994. Isolation of herpesvirus from Testudo hermanni and Agrionemys horsfieldii. Verh. Ber. Erkrg. Zootiere. 36: 241–245.

13.  Lange, H., W. Herbst, J.M. Wiechert, and T. Schliesser. 1989. Electron microscopical evidence of herpesvirus and high mortality in Hermann’s tortoises (Testudo hermanni) and four-toed tortoises (Agrionemys horsfieldii). Tierärztl. Prax. 17: 319–321.

14.  Marschang, R.E., M. Gravendyck, and E.F. Kaleta. 1997. Herpesviruses in tortoises: investigations into virus isolation and the treatment of viral stomatitis in Testudo hermanni and T. graeca. J. Vet. Med. B. 44: 385–394.

15.  Mueller, M., W. Sachsse, and N. Zangger. 1990. Epidemic herpesvirus infection in spur-tailed (Testudo hermanni) and spur-thighed Mediterranean land tortoises (Testudo graeca) in Switzerland. Schweiz. Arch. Tierheilk. 132: 199–203.

16.  Mueller, M., N. Zangger, and T. Denzler. 1988. Iridovirus-Epidemie bei der griechischen Landschildkröte (Testudo hermanni hermanni). Verhandl. Ber. 30. Intern. Symp. Erkrg. Zoo und Wildt., Sofia: 271–274.

17.  Pettan-Brewer, K.C.B., M.E. Drew, E. Ramsey, F.C. Mohr, and L.J. Lowenstine. 1996. Herpesvirus particles associated with oral and respiratory lesions in a California desert tortoise (Gopherus agassizii). J. Wildl. Dis. 32: 521–526.

18.  Posthaus, H., R.E. Marschang, M. Gravendyck, and L.N. Bacciarini. 1997. Study on herpesvirus infections in land tortoises in Switzerland. Proc. Am. Assoc. Zoo Vets., Houston, TX; 17–18.

19.  Raynaud, A., and M. Adrian. 1976. Lesions cutanees a structure papillomateuse associees a des virus chez le Lezard vert (Lacerta viridis). Comptes Rendues Academie des Sciences Paris 283: 845–847.

20.  Schat, K.A., and H.G. Purchase. 1989. Cell culture methods. In: H.G. Purchase, L.H. Arp, C.H. Domermuth, and J.E. Pearson (eds.). A Laboratory Manual for the Isolation and Identification of Avian Pathogens. Kendall/Hunt Publ. Co., Dubuque, IA. Pp. 171–172.

21.  Sudia, W.D., V.F. Newhouse, L.D. Beadle, D.L. Miller, G.J. Johnson, Jr., R. Young, C.H. Calisher, and K. Maness. 1975. Epidemic Venezuelan equine encephalitis in North America in 1971: vector studies. Am. J. Epidemiol. 101: 17–35.

22.  Vieler, E., W. Baumgärtner, W. Herbst, and G. Köhler. 1994. Characterization of a reovirus from a rattlesnake, Crotalus viridis, with neurological dysfunction. Arch. Virol. 138: 341–344.

23.  Westhouse, R.A., E.R. Jacobson, R.K. Harris, K.R. Winter, and B.L. Homer. 1996. Respiratory and pharyngo-esophageal iridovirus infection in a gopher tortoise (Gopherus polyphemus). J. Wildl. Dis. 32: 682–686.