Greek tortoise male 6 years Testudo graeca.This was a pet tortoise that awoke from hibernation with nasal and ocular discharge. At clinical examination the tortoise was dehydrated and had a severe intestinal parasitic infection (ascarids and oxyurids) diagnosed by the referring veterinarian. Despite antibiotic, antiparasite and rehydrating therapy, clinical signs including open mouthed breathing worsened and the tortoise died spontaneously.
A full necropsy was performed by the referring veterinarian who reported the presence of ulcerative glossitis and stomatitis with pannus formation and severe hyperaemia of lungs.Â Esophageal and tracheal mucosal linings were reported to be diffusely ulcerated.Â Heart, liver, kidney, spleen, testes, stomach and intestine were grossly normal.
Several organs, including the lungs, were formalin-fixed and sent for histopathology.
Lung: Severe and diffuse inflammatory changes involving 80% of the pulmonary parenchyma are present in all sections.Â Upper and lower airways (ediculae) contain variably abundant luminal accumulation of mucus and fibrin admixed with numerous sloughed necrotic epithelial cells, moderate numbers of heterophils, viable or occasionally degenerated (karyolysis and karryorrhexis), and rare reactive macrophages.Â The epithelium lining larger airways and ediculae is multifocally eroded and ulcerated.Â In the epithelial cells (sloughed and viable), nuclei are multifocally characterized by chromatin margination and occasionally contain amphophilic, homogeneous variably sized and shaped inclusions bodies often filling the nucleus (consistent with herpesviral inclusions).Â
The pulmonary interstitium is moderately to severely and diffusely expanded by hyperaemia and edema, numerous heterophils and lesser numbers of lymphocytes and plasma cells.Â Pleura is severely, diffusely edematous and contains a small number of heterophils.
Severe, diffuse, acute, necrotizing pneumonia with amphophilic intranuclear inclusions consistent with herpesvirus, Greek tortoise, Testudo graeca.
Etiology: Chelonid herpesvirus (most likely type 3)
Herpesviruses are enveloped viruses with a double stranded DNA surrounded by icosahedrally arranged caspomeres.Â Herpesvirus infections are widespread and occur in most classes of vertebrates including fish, amphibians, and reptiles.Â Infection with herpesvirus has been reported in chelonians, lizards, snakes and in crocodilians.Â In chelonians, herpesviruses have been associated with several disease complexes, which are characterized by diphtheritic-necrotizing stomatitis, hepatitis, rhinitis, tracheitis, and pneumonia in tortoises.(8) Tortoises in the genus Testudo including Greek tortoise (T.Â graeca), Hermanns tortoise (T.Â hermanni), and Russian tortoise (Agrionemys horsfieldi) are particularly prone to infection with Chelonid herpesvirus (ChHV).Â ChHV infections have been associated with glossitis, stomatitis, enteritis, and meningoencephalitis in Hermanns tortoises (Testudo hermanni); with stomatitis and enteritis in Afghan tortoises (Testudo horsfieldii); with stomatitis, tracheitis, and pneumonia in desert tortoises (Gopherus agasizii)(11); and with stomatitis and encephalitis in spur-thighed tortoises (Testudo graeca).(8) Green turtles older than one year of age have also been susceptible to pneumonia, tracheitis and conjunctivitis ascribed to herpesvirus.(8)
Herpesviruses have also been associated with oral, respiratory, cutaneous, and genital lesions in Atlantic loggerhead sea turtles (Caretta caret)(13) and skin diseases in sea turtles, such as gray patch disease and fibropapillomatosis in green turtles (Chelonia mydas).(1,7,12)
The exact route of transmission of herpesvirus in wild chelonids is still unknown.Â In captive animals, a major means of transmission is the exchange of pet tortoises between private collections.(8) It is very likely that direct contact between affected animals and unaffected tortoises represents the primary route of transmission.Â The finding of viral particles in testicular epithelium of Greek and Hermanns tortoises suggests the possibility of vertical transmission.(8) Herpesviruses identified in various species of turtles and tortoises have been preliminarily named chelonid herpesviruses (ChHVs), but represent an up-to-now unassigned species in the herpesvirus family.Â Classification of ChHVs is mainly based on putative differences in the host spectrum and 4 variants have been recognized.Â ChHV-1 was first described in association with gray patch disease in captive green sea turtles (Chelonia mydas) in the West Indies.(12) This disease is characterized by patchy gray areas of hyperkeratotic and necrotic papules that occur over the head, neck, and flippers.Â ChHV-2 was seen in two Pacific pond turtles (Clemmys marmorata) with fatal hepatic necrosis.Â A similar disease has been seen in painted turtles (Chrysemys pict a) and in map turtles (Graptemys pseudogeographica) in association with herpesvirus-like particles.
These viruses have been preliminarily named ChHV-3.6 ChHV-4 was seen in tissues of Argentinian tortoises (Geochelone chilensis) with necrotizing stomatitis or mouth rot.Â Interestingly, red-footed tortoises (Geochelone carbon aria) kept together with the diseased Argentinian tortoises remained clinically healthy.(4) Epizootics of chronic seromucous rhinitis (running nose syndrome) were described in large populations of captive T.Â graeca.Â This outbreak was part of a series of epidemic ChHV infections that have occurred in Europe during the last decade.Â In most cases, outbreaks follow shared housing of different tortoise species after addition of new animals.Â In all of these cases, presumed carrier species, especially T.Â graeca, remained healthy, whereas other, presumably less resistant species, became sick or died.Â
Clinical signs in tortoises include nasal serous to mucopurulent discharge, open mouth breathing, wheezing, dyspnea, lethargy, anorexia, weight loss and ataxia.Â Radiographs, magnetic resonance, CT scans, bronchoscopy, and cytology have been used for clinical diagnosis.(8)
Pathological findings in most chelonids with respiratory disease include necrotizing caseous stomatitis that extends in the oral cavity and nares and necrotizing glossitis with presence of diphtheritic plaques.Â Lower airways can be involved resulting in necrotizing pneumonia and emphysema.Â Enteritis and hepatomegaly have been also reported.(8) In tortoises, eosinophilic intranuclear inclusions are commonly seen in epithelial cells of affected tissues stained with haematoxylin and eosin and are associated with syncytial cells.(8) Secondary bacterial complications are associated with development of multiple bacterial granulomas.Â Intranuclear inclusions have also been reported in lung and trachea of green turtles (Chelonia mydas) with respiratory disease(5) and in cutaneous fibropapillomas.(7) Using TEM, virions can be detected in the nucleus and cytoplasm of infected cells of the tongue, trachea, bronchi and alveoli, endothelial cells of glomerular capillaries and within neurons and glial cells of the medulla oblongata and diencephalon.(8)
Tests that have been developed to diagnose chelonid herpesvirus include enzyme-linked immunosorbent assay (ELISA) for the detection of herpesvirus antibodies in plasma samples of Mediterranean tortoises.(9) Indirect and direct immunoperoxidase assay have been used either for assessing the presence of anti-herpesvirus antibody in tortoise plasma or for detecting herpesvirus antigen in tissues.(10)
ChHV DNA has been demonstrated in a broad range of formalin fixed and paraffin embedded tissues in tortoises suffering from stomatitisrhinitis complex by in situ hybridization and PCR.Â The ISH signal colocalizes to the same areas and cell types that contain intranuclear inclusions in haematoxylin and eosin stained tissue sections from tortoises of different geographic provenances.Â Nuclear hybridization signals have been detected in epithelial cells of the lingual mucosa and glands, in tracheal epithelium, pneumocytes, hepatocytes, the renal tubular epithelium, cerebral glial cells and neurons, intramural intestinal ganglia and in endothelial cells of many organs.(14)
Lung: Pneumonia, bronchointerstitial, fibrosing, heterophilic and histiocytic, subacute, diffuse, severe, with type II pneumocyte hypertrophy and exfoliation with numerous epithelial intranuclear viral inclusions.
The differential diagnosis discussed by conference participants included adenovirus, ranavirus, and fibropapillomavirus.Â Previous reports of chelonian adenoviruses include an atAdenovirus in a leopard tortoise (Geocehlone pardalis) and a Siadenovirus in Sulawesi tortoises (Indotestudo forsteni).(15) Reported gross lesions include hepatosplenomegaly, fibrinonecrotic membranes in the lumen of the colon, oronasal fistulae, and ulceration of the tongue and oral mucosa.Â The histologic appearance of adenovirus is very similar to herpesvirus, including epithelial, endothelial and myeloid necrosis with basophilic to amphophilic intranuclear viral inclusions in many tissues.(3,14)
Ranavirus is in the family Iridovirus, with gross lesions including hepatic necrosis, ulcerative tracheitis, pneumonia, and ulcerative pharyngitis and esophagitis.Â Histopathologic findings include basophilic intracytoplasmic viral inclusions in hepatocytes and epithelial cell, and fibrinoid vasculitis in multiple organs.Â Reptiles are thought to aquire ranavirus from amphibians.(3)
Fibropapillomatosis is found in all species of sea turtles except for the leatherback, and is caused by a herpesvirus.Â Fibropapilloma-associated turtle herpesvirus causes a debilitating disease characterized by large numbers fibropapillomas which result in decreased mobility and occasional blindness when located near the eyes.Â These are often accompanied by anemia and immunosuppression.Â Histology of the masses is that of a typical fibropapilloma, and intranuclear viral inclusions are rarely seen.Â Fibropapillomas can also be seen on the viscera, with the kidney and lung being primary target tissues(16).Â
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9.Â Origgi FC, Klein PA, Mathes K, et al.Â Enzyme-linked immunosorbent assay for detecting herpesvirus exposure in Mediterranean tortoises (spur-thighed tortoise [Testudo graeca] and Hermann's tortoise [Testudo hermanni]).Â J Clin Microbiol. 2001;39:3156-3163.Â
10.Â Origgi FC, Klein PA, Tucker SJ, et al.Â Application of immunoperoxidase-based techniques to detect herpesvirus infection in tortoises. J Vet Diagn Invest. 2003;15:133-140.
11.Â Peitan-Brewer KCB, Drew ML, Ramsay E, et al.Â Herpesvirus Particles Associated With Oral and Respiratory Lesions in a California Desert Tortoise (Gopherus agassizi).Â J Wildlife Dis. 1996;32:521-526.Â
12.Â Rebell G, Rywlin A, Haines H.Â A herpesvirus-type agent associated with skin lesions of green sea turtles in aquaculture.Â Am J Vet Res. 1975;36:12211224.
13.Â Stacy BA, Wellehan JF, Foley AM, et al.Â Two herpesviruses associated with disease in wild Atlantic loggerhead sea turtles (Caretta caretta).Â Vet Microbiol. 2008;126:63-73.
14.Â Teifke JP, Lo HR CV, Marschang RE, et al.Â Detection of Chelonid Herpesvirus DNA by Nonradioactive In Situ Hybridization in Tissues from Tortoises Suffering from StomatitisRhinitis Complex in Europe and North America.Â Vet Pathol. 2000;37:377385.
15.Â Rivera S, et al.Â Systemic adenovirus infection in Sulawesi tortoises (Indotestudo forsteni) caused by a novel siadenovirus.Â J Vet Diagn Invest. 2009;21(4):415-26.
16.Â Wyneken J, Mader DR, Weber III ES, et al.Â Medical care of seaturtles.Â In: Mader DR, ed.Â Reptile Medicine and Surgery. 2nd ed.Â St.Â Louis, MO: Saunders Elsevier; 2006:986-91.