Signalment:  

Adult age unknown male eastern box turtle, (Terrapene carolina carolina).A colony of 27 box turtles has been maintained at the Maryland Zoo in Baltimore for many years without major disease issues. In 2011, 2 of the turtles were found dead and considered to be too autolyzed for necropsy submission. At that time, clinical findings in many of the other turtles included lethargy, inappetence, plaques on the tongue, soft palate, and cloaca. Remaining turtles were triaged, separated based on severity of clinical signs, and treated with antimicrobials, gavage feeding, and additional supportive care. Some turtles were euthanized due to lethargy, severe oral and cloacal plaques, and overall clinical decline. Over six weeks a total of 13 box turtles died or were euthanized. This turtle was placed in the severely affected group when triaged and did not respond to supportive therapy. It was found dead three days after triage and was submitted to the Johns Hopkins Department of Molecular and Comparative Pathobiology for evaluation.

Gross Description:  

Gross findings included severe multifocal to coalescing fibrinonecrotic plaques within the oral cavity including the tongue and soft palate and the hard palate. Mucosal plaques into the proximal half of the esophagus and mild multifocal mucosal plaques were present in the cloaca. The stomach had intralumenal non-adherent fibrinonecrotic debris, and 1-6mm ulcerated mucosal nodules with adherent superficial fibrinonecrotic material. The turtle was in poor to fair body condition with scant perivisceral fat.

Histopathologic Description:

Decalcified transverse section of the head, including oral cavity. The hard palate has multifocal to coalescing regions of mucosal loss with replacement by abundant fibrin, necrotic debris, and mixed heterophilic and lymphocytic infiltrates (including degenerate heterophils), with an overlying pseudomembranous crust consisting of sloughed epithelial cells, necrotic heterophils, fibrin, and bacterial colonies. At some edges of ulcerated areas, remnant squamous mucosa has ballooning degeneration, scattered intraepithelial heterophils and lymphocytes, intraepithelial edema, and occasional intracorneal foci of heterophilic and lymphocytic inflammatory cells. The submucosa contains multifocal perivascular lymphocytic and histiocytic infiltrates. 

The lacrimal gland has multifocal mild interstitial lymphocytic inflammation with necrosis. 

Morphologic Diagnosis:  

1. Oral cavity (hard palate): Stomatitis, heterophilic and lymphohistiocytic, ulcerative, fibrinonecrotic, multifocal to coalescing, subacute, severe, with moderate submucosal lymphohistiocytic perivasculitis, superficial pseudomembrane formation, and superficial bacteria.
2. Lacrimal gland: Adenitis, lymphocytic, necrotizing, multifocal to coalescing, moderate.

Lab Results:  

PCR positive for ranavirus; sequencing result in other individuals: frog virus 3, PCR negative for herpesvirus.

Condition:  

Ranavirus; frog virus

Contributor Comment:  

The submitted case was one of 13 adult captive eastern box turtles from a zoological exhibit that all died over a span of several weeks. In addition to the severe stomatitis, microscopic findings included ulcerative and fibrinonecrotic glossitis, esophagitis, gastritis, cloacitis with pseudomembrane formation, and fibrinoid degeneration of vessel walls in the spleen, subacute interstitial nephritis, subacute periportal hepatitis, lymphocytic enterocolitis, and lymphocytic perivasculitis in several organs. Inclusion bodies were not definitively identified in most cases and were rare in the lung and liver of one case. Incidental findings in several turtles included nodular gastritis with mixed necrotizing inflammation and intralesional nematode larvae. Primary differentials were ranavirus, herpesvirus, and septicemia.

Antemortem oropharyngeal samples were collected from many turtles and submitted for PCR detection. Ranavirus was confirmed in 8 of the 10 tested turtles submitted for necropsy, including all of the turtles with oral plaques similar to the submitted case. In two turtles, PCR was followed by DNA sequencing, identifying the ranavirus frog virus 3 in both cases. Herpesvirus was confirmed in 4 of the 10 tested turtles. Tissue from this turtle was negative for herpesvirus. Several bacterial agents were detected in oropharyngeal and blood samples from other ranavirus-positive turtles in this population, highlighting the potential role of secondary bacterial pathogens as factors contributing to inflammation, sepsis, and death of ranavirus-infected turtles. 

Ranavirus currently is classified as a genus in the Iridoviridae family. Iridoviruses are large (120-200nm), icosahedral, double stranded DNA viruses that replicate in the cytoplasm. Ranavirus infections are important causes of disease in fish(9) and amphibians.(4) The ranavirus frog virus 3 has been reported with increasing frequency as a significant cause of mortality in several reptile species.(5) Environmental stressors, na+�-�ve or suppressed immunity, or introduction of novel strains may play a role in outbreaks that emerge in wild and captive reptiles. Amphibians and reptiles have been suggested as important reservoirs for ranaviruses that may cause economically and ecologically important disease in finfish.

Typical presentations of ranavirus infection in turtles includes cervical edema, palpebral edema, rhinitis, and stomatitis-glossitis.(5) A series of cases of ranavirus in captive eastern box turtles in North Carolina(1) describes clinical signs that also included cutaneous abscesses, oral erosions and abscesses, and respiratory distress. Other studies that include several species of turtles and tortoises describe similar signs as well as yellow-white oral plaques.(6,7) In these studies, histopathology revealed fibrinoid vasculitis of skin, mucous membranes, lungs, and liver, multifocal hepatic necrosis, multicentric fibrin thrombi, fibrinous and necrotizing splenitis, and necrotizing stomatitis and esophagitis. While basophilic intracytoplasmic inclusion bodies have been reported in ranavirus infections,(5) often they are not observed, even with ranavirus infection confirmed by PCR, electron microscopy, or virus isolation.(3,6)

JPC Diagnosis:  

1. Oral cavity (hard palate): Stomatitis, necrotizing, focally extensive, severe.
2. Lacrimal gland: Dacryoadenitis, necrotizing, multifocal, moderate.

Conference Comment:  

Due to mild slide variation, the degree of lacrimal gland necrosis and inflammation within submitted sections varies; however, most conference participants appreciated some degree of necrotizing dacryoadenitis. The moderator concurred with this observation, but points out that reptiles and birds tend to have relatively high numbers of plasma cells within the normal lacrimal gland, so dacryoadenitis must be diagnosed with caution in these species. In addition to the differential diagnosis addressed by the contributor, including herpesvirus, bacterial septicemia and ranavirus, participants briefly discussed fungal infection (Candida spp.) and poxvirus as rule-outs for fibrinonecrotic stomatitis with pseudomembrane formation. These conditions can generally be differentiated histologically. Herpesvirus results in characteristic intranuclear viral inclusions, and poxvirus, while rarely reported in turtles, produces large intracytoplasmic inclusions.(1) Candidiasis can be distinguished microscopically by the presence of budding yeast, pseudohyphae and true hyphae.(2) In this case, viral inclusions were not identified and ranavirus was confirmed by PCR.

The contributor does an outstanding job of covering all the salient features of ranavirus infection in reptiles. Ranavirus, specifically frog virus 3, was initially associated with widespread disease epizootics in amphibians. Affected tadpoles (who are particularly vulnerable to infection) and frogs typically present with cutaneous hemorrhage/ulceration or disseminated disease with multiorgan necrosis. Subclinical infections are common in frogs; the kidneys and macrophage populations are considered the primary sites of virus persistence.(7) Both adult and larval salamanders are susceptible to a ranavirus known as Ambystoma tigrinum virus, which results in splenic, hepatic, renal and gastrointestinal necrosis, sloughing of the skin, and discharge of inflammatory exudate from the vent. Interestingly, ambient temperature appears to play a significant role in disease pathogenesis, as high mortality is observed in those salamanders infected at 18^oC, while those infected at 26^oC tend to survive.(8) Ranavirus infection in fish populations was first reported in Australian redfin perch and rainbow trout in the 1980s; it has since been implicated in multiple disease episodes in both farmed and wild freshwater fish worldwide. Fingerlings and juveniles are most susceptible, and disease is characterized by severe necrosis in the liver, pancreas and renal/splenic hematopoietic cells. In addition to these tissues, Santee-Cooper virus, a ranavirus in wild largemouth bass, also causes enlargement and inflammation of the swim bladder, resulting in moribund fish that tend to float to the surface. As in amphibians, ranavirus infections in fish can be subclinical.(8) Furthermore, inter-species transmission between amphibians and fish has been demonstrated, implicating both species as potential reservoirs for the virus.(8) Ranavirus is such a significant problem in both fish and amphibians that it meets the criteria for listing by the World Organization for Animal Health (OIE).(1)

References:

1. Ariel E. Viruses in reptiles. Vet Res. 2011;42(1):100-112.

2. Brown CC, Baker DC, Barker IK. Alimentary system. In: Maxie MG, ed. Jubb, Kennedy and Palmers Pathology of Domestic Animals. Vol 2. 5th ed. Philadelphia, PA: Elsevier Limited; 2007:230.

3. De Voe R, Geissler K, Elmore S, Rotstein D, Lewbart G, Guy J. Ranavirus-associated morbidity and mortality in a group of captive eastern box turtles (Terrapene carolina carolina). J Zoo Wildl Med: official publication of the American Association of Zoo Veterinarians. 2004;35:534-543.

4. Gray MJ, Miller DL, Hoverman JT. Ecology and pathology of amphibian ranaviruses. Dis Aquat Org. 2009;87:243-266.

5. Jacobson ER. Infectious Diseases and Pathology of Reptiles: Color Atlas and Text. Boca Raton, FL: CRC/Taylor & Francis; 2007:288, 404-406, 440.

6. Johnson AJ, Pessier AP, Jacobson ER. Experimental transmission and induction of ranaviral disease in Western Ornate box turtles (Terrapene ornata ornata) and red-eared sliders (Trachemys scripta elegans). Vet Pathol. 2007;44:285-297.

7. Johnson AJ, Pessier AP, Wellehan JF, Childress A, Norton TM, Stedman NL, et al. Ranavirus infection of free-ranging and captive box turtles and tortoises in the United States. J Wildl Dis. 2008;44:851-863.

8. MacLachlan NJ, Dubovi EJ, eds. Fenners Veterinary Virology. 4th ed. London, UK: Academic Press; 2011:172-175.

9. Whittington RJ, Becker JA, Dennis MM. Iridovirus infections in finfish - critical review with emphasis on ranaviruses. J Fish Dis. 2010;33:95-122.

Click the slide to view.

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1-1. Oral cavity

1-2. Tongue

1-3. Cross-section of skull

1-4. Oral cavity, hard palate

1-5. Lacrimal gland

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