LYMPHOPROLIFERATIVE DISEASE IN TESTUDO HERMANNI AND GEOCHELONE PARDALIS TORTOISES ASSOCIATED WITH HERPESVIRUS-LIKE INFECTION.

S D J McARTHUR BVet. Med., M.R.C.V.S. Holly House Veterinary Surgery, 468 Street Lane, Moortown, Leeds, LS17 6HA.

Presented at the Symposium at the University of Bristol, 28 March 1998

The following report describes an outbreak of disease, in a mixed species breeding colony, owned by a member of the BCG. In order to accurately describe some of the events that occurred, it has been necessary to use some medical and scientific terminology. 1 have therefore tried to include short explanations of some terms (in brackets) that may confuse those readers without a medical background .

I have also drawn up the following points to help readers worried that a similar disease could infect their colony or tortoise.

• The disease described later was able to affect at least two species of chelonia (Leopard tortoises and Herman tortoises). It is feared that this disease was precipitated by the mixing of tortoises of different species. It is wise to try and maintain chelonia in isolated species groups and to resist the temptation to mix species together.
• The incubation period of this disease was seen to be greater than 8 months in at least one isolated individual. 1 currently suggest isolating any new chelonia before introducing them into an existing colony for 12-18 months, or alternatively accept any disease outbreaks that occasionally result otherwise.
• When introducing new chelonia to a stable colony, it is wise to mix them first with a small sample group (sentinels), and then to wait and see if any disease becomes apparent over the next 12-18 months. If the sentinels remain disease free one could then cautiously increase the exposure of new individuals to a larger group. It appears wise to maintain captive tortoises and turtles subdivided in small isolated groups in order to limit possible spread of serious disease through larger colonies.
• A second episode of this disease has been found in Essex, a year after the initial outbreak. Fortunately this only appeared to affect a single Leopard tortoise. It is not known how many tortoises this animal contacted whilst in the earlier possession of an animal dealer. It is unwise to rescue and nurse sick chelonia in the presence of other healthy chelonia without practising a very high standard of hygiene and disinfection.
• It is wise to make enquiries as to the disease history of any colony from which you are considering accepting new stock. Most tortoise breeders understand disease outbreaks. 1 would always advise a strict quarantine period from tortoises that are obtained with unknown history.
• It is wise to remove any ill tortoises from a colony as quickly as possible. This is very important if the disease signs are consistent with infectious disease. (e.g. breathing difficulties, unexplained jaundice, unexplained lethargy, anorexia and inactivity, increased oral and nasal secretions or even sudden death).
• It is wise ensure that sick chelonia are nursed away from healthy specimens, possibly at another site such as a veterinary surgery familiar with chelonian treatment methods. Alternatively sick chelonia should be barrier nursed with a high level of hygiene and disinfection in order to limit possible transfer of disease to healthy animals.
• It is wise to ensure that your tortoise is kept isolated from others if it is taken to a site where other tortoises are gathered. Examples include pre and post hibernation check ups.

In 1996 1 was asked to help with the investigation and management of a disease epizootic (epidemic) in two species of tortoise Testudo hermanni (Herman 's tortoise) and Geochelone pardalis (Leopard tortoise) associated with profound systemic disease, wasting and high mortality. After extensive investigations it is proposed that this epidemic was the result of virus infection. It appeared that any causative agent was able to affect more than one species of chelonia .

Disease was associated with dyspnoea (respiratory distress) due to upper and lower respiratory tract disease, hepatitis (inflammation of the liver), pancreatitis (inflammation of the pancreas), nephritis (inflammation of the kidney) intermittent paralysis and disseminated lymphoproliferative disease. The term lymphoproliferative disease describes a condition where white blood cells infiltrate and divide within organs inappropriately, eventually causing pathology that impairs their normal function. It may also impair the function of the immune system. (Leukaemia and lymphoma are some more common names of human lymphoproliferative diseases. It is not yet clear if this chelonian lyrnphoproliferation should be classified as benign or malignant.)

Microbiological investigations isolated bacterial, chlamydial, fungal and parasitic agents. In addition a herpesvirus was observed in hepatic and splenic tissue from which histopathological diagnosis of lymphoproliferative disease had already been made. It is proposed that the primary cause of the outbreak was a herpesvirus infection and that this was associated with a variety of secondary and/or concurrent infections. A histopathological diagnosis of lymphoproliferative disease was made from tissue from six individuals. Negative contrast electronmicroscopy of tissue samples from six individuals (Five T hermanni one C pardalis) demonstrated herpesvirus particles. This episode resulted in 100% morbidity and 100% mortality in this group of 29 tortoises.

It is not yet known whether the presence of this chelonian herpesvirus could be secondary to another agent such as a retrovirus. In birds a similar disease caused by a herpesvirus is called Mareks Disease. A second avian lymphoproliferative disease called Avian Leukosis is caused by a retrovirus, so both agents currently seem plausible. Horizontal transmission (two animals in contact) between species suggests herpesvirus to be a more likely cause than a retrovirus as this would be expected to be mainly vertically transmitted (between parents and offspring). It is possible that both types of virus are necessary and and similar outbreaks should be investigated for the presence of antigens associated with retrovirus. It is hoped that samples stored from this outbreak can be screened at a future date.

This affected colony contained several units, but only one was affected. This was made up of twenty five T hermanni tortoises and four Leopard tortoises (Geochelone pardalis) which had temporarily shared the same enclosure. Unaffected tortoises consisted of one Graeca ibera, four Testudo hosfeldii one Kinyxis erosa, two Geochelone sulcata and a further sixteen juvenile T hermanni These were kept in individual species groups, isolated from each other, and from the main affected group. Some of these had temporarily shared a common airspace with the affected group but not the same ground enclosures.

G. pardalis were the first to show the disease, the main group of T hermanni demonstrated significant clinical signs after a time lag of 3 or 4 months. This disease affected 29 individuals, 17 of which died from the infection. Twelve individuals were euthanased. The ultimate morbidity in this group was 100%. The ultimate mortality was also 100% (59% dying directly from the disease and 41% were moribund individuals euthanased to prevent suffering)

The presenting signs in the G. pardalis included excessive secretion Of ocular and nasal fluids, prolonged anorexia, muscle wastage and fore and hind limb paralysis. Later what appeared to be a neck muscle paralysis permanently retained the neck of two tortoises in flexion. The disease in the G. pardalis tended to be chronic and animals were anorexic and wasted over a period of around 6 months.

T hermanni generally showed a more acute onset of signs which included profound debility and collapse. This acute phase generally lasted three to four days. These animals had pale greyish mucus membranes, dyspnoea (troubled breathing) and whitening of the corneal surface of the eyes that cleared over a period of a week or so.

Some of the T hermanni tortoises died within 12 hours of displaying these signs, other animals were nursed through this initial crisis and then progressed into the chronic phase of the disease. Those that survived seemed to recover for 3 or 4 weeks more and were then either overwhelmed by bacterial, fungal and helminth (parasitic worm and fluke) infections, or they went into acute hepatic (liver), pancreatic or renal (kidney) failure. This was demonstrated from blood biochemistry and haematology and later post-mortem examinations and histopathology.

Blood samples from two hospitalised T hermanni were monitored to try and assess viability and response to treatment. Most of the changes observed were considered to be non-specific, but it did appear that renal function was significantly compromised despite intensive fluid therapy and supportive treatment. Haematology during both acute and chronic phases showed panleucopaenia (low number of total white blood cells) with a profound lymphopaenia (low number of a specific type of white blood cell).

Various bacterial fungal and helminth agents were isolated from affected animals but none could account for the morbidity observed.

Tissue samples were sent to histopathologists at two separate laboratories, both of whom reached a similar diagnosis of an infiltrative lymphoproliferative disease. Lyrnphoproliferation was observed in spleen, liver, heart and intestine.

Liver and spleen samples from 6 affected tortoises were processed for negative contrast electron microscopy (EM). Herpesvirus- like particles were seen in samples submitted from all six cases. The average size of the particles was 10nm in diameter and the typical surface structure and icosohedral symmetry of herpesviruses was clearly visible. No enveloped viral particles were seen.

Conclusion

Tortoises in this colony suffered an outbreak of infectious lymphoproliferative disease associated with the presence of a herpesvirus infection. It is possible that this herpesvirus is the causative agent. Some individuals also suffered from concurrent chlamydial, bacterial, fungal and helminth infections, probably as a result of impaired immunocompetence. The outbreak was notable for its ultimate 100% morbidity and 100% mortality in a group of twenty five Testudo hermanni and four Geochelone pardalis.

Suggested Reading

Biermann, R. 1995. Isolierung und Characterisierung von Herpesviren bei Landschildkroten. Veterinary Medical Dissertation pp34-43. Justus-Lieburg-Gniversitat, Giessen, Germany.

Braune, S_ Geiss, V and Theil, W. 1989. A new herpesvirus -caused disease in tortoises. Tierarztl Prax.; 17(4): pp416-419. German.

Calneck, B.W and Witter, R.L. 1984. Mareks Disease. Disease of Poultry (8th Ed) pp325-354. Editor Hofstad, M.S., Iowa State University Press.

Cooper, J.E., Gschmeissner, S. and Bone, R.D. 1988. Herpesvirus- like Particles in Necrotic Stomatitis of Tortoises. Veterinary Record 123, (21), p544.

Cooper, J.E., Lawton, M.P.C., Jacobson, E.R. and Zwart, P. 1991. Deaths in Tortoises. Veterinary Record 128, (15), p364.

Cooper, J.E. 1994. Viral Diseases of Reptiles. British Herpetological Society Bulletin 47 pp9 1.

Cox, W.R., Rapley, W.A. and Barker, I.K. 1980. Herpesvirus- like infection in a painted turtle (Chryemys picta). J. Wildl Dis. Jul; 16 (3): pp445-449.

Frost, J.W. and Schmidt, A. 1997. serological evidence for susceptibility of various tortoise species to herpesvirus infection. 38 Intemationalen Symposiums uber Erkrankungen der Zoo und Wildtiere Vom 7-11 Mai. 1997. Zurich / Scweiz. pp25-27.

Frye, F.L., Oshiro, L.S., Dutra, F.R. and Carney, J.D. 1997. Herpesviruslike infection in two Pacific Pond Turtles. Journal of the American Veterinary Medical Association Vol 171, (9) pp883-883.

Haines, H. and Klees, W. 1977. The Effect of Water Temperature on a Herpesvirus Infection of Sea Turtles. Infection and Immunity, Vol 15, (3), pp756-759.

Harper, P.A. Hammond, D.C. and Heuschele, W.P. 1982. A herpesviruslike agent associated with a pharyngeal abscess in a desert tortoise. J. Wildl Dis. Oct; 18(4); pp491-494.

Heldstab, A. and Besetti, G. 1982. Spontaneous Viral Hepatitis in a Spur-Tailed Mediterranean Land Tortoise. Journal of Zoo Animal Medicine. 13, pp113-120.

Jacobson, E.R., Gaskin, J.M. and Walkquist, H. 1982. Herpesvirus -like infection in map turtles. J Am Vet Med Assoc. Dec 1; 181 (11); pp 13221324.

Jacobson, E.R. Gaskin, JX, Clubb, S. et al. 1982. Papilloma like virus infection in Bolivian side necked turtles. J Am Vet Med Assc 181 (11) p1325.

Jacobson, E.R., Club, S., Gaskin, J.M. and Gardiner, C. 1985. Herpesvirus- like infection in Argentine tortoises. J Am Vet Assoc. Dec 1; 187(11; pp1227-1229

Jacobson, E.R., Gaskin, J.M., Roeike, M., Greiner, E.C. and Allen J. 1986. Conjunctivitis, tracheitis and pneumonia associated with herpesvirus infection in green sea turtles. J Am Vet Assoc. Nov 1: 189(9); pp1020-1023.

Jacobson, E.R., Burgelt, C., Williams, B et al. 1991. Herpesvirus in cutanous fibropapilomatosis of the green sea turtle. Dis Aqua Organ 12A.

Jacobson, E.R. 1994. Causes of mortality and diseases in tortoises: a review. Journal of Zoo and Wildlife Medicine vol 25(1) pp2A7.

Kabish, D. and Frost, J.W. 1994. Isolation of Herpesvirus from Testudo hermanni and Agryonemys horsfieldi Verhandlungsbericht uber Erkrankungen der Zootiere 36 pp241-245.

Lange, H., Herbst, W., Wiechert, J.M. and Schliesser, Th. 1989. Electron microscopical Evidence of Herpes Virus and high mortality in Hermann's tortoises and four toed tortoises. Tierarzd. Prax 17, pp319321.

Marschang, R.E., Gravendyke, M. and Kaleta, E.F. 1 a. Herpesviruses in tortoises: Investigations into Virus Isolation and the Treatment of Viral Stomatitis in Testudo hermanni and T graeca. J Vet Med B 44, pp385-394.

Marschang, R.E., Gravendyke, M. and Kaleta, E.F. 1997b. New Investigations on herpesviruses in tortoises. 38 Internationalen Symposiums uber die Erkrankungen der Zoo und Wildtiere Vom 7-11 Mai 1997. Zurich / Scweiz. pp29-34.

Muller, M., Sachsse, W. and Zangger, N. 1990. Herpesvirus- Epidemie bei der griechischen (Testudo hermanni) und der maurischen Landschildkrote (Testudo graeca) in der Schweiz. Scheiz. Arch. Tierhilkd. 132: pp199-203.

Oettle, E.E., Steytier, Y.G.M. and Williams, M.C. 1990. High Mortality in a Tortoise Colony. South African Journal of Wildlife Research 20, (1), pp21-25.

Randal, C.J. 1991. Marek's Disease (including transient paralysis). A Clor Atlas of Diseases and Disorders of the Domestic Fowl and Turkey. (70-75) Wolf Publishing Ltd.

Rebell, G., RywIin, A. and Haines, H. 1975. A Herpesvirus-type agent Associated with Skin Lesions of Green Sea Turtles in Aquaculture. Am. J. Vet. Res. Vol 36, (8), pp 1221-1224.

Richie. 1995. Avian Viruses (Function and Control) Lake Worth, Florida Wingers. Chapter 14, Retroviridae pp 363-377.

The investigation of this disease outbreak was limited by availability of funds. The costs were shared by the BCG member Glynn Gould and myself in order to provide as much information as we could to limit similar disease outbreaks in the future. Glynn deserves all our thanks for this.

Facilities to investigate potential chelonian viral diseases are currently limited in the UK and in order to try and improve matters in the future a project has been started by Stephen Divers MRCVS (BCG Liason Officer), Mike Jessop MRcvs and myself (Both BCG members) to try and enable virus culture of chelonian viral diseases in the UK. The project known as CVI (Chelonian Virology Investigation) is currently in its infancy, but we are hopeful it can be developed in time.

Testudo Volume Four Number Five 1998

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