Conference 08 - 2011 Case: 02 20111102

Signalment:

An approximately 1-year-old male Boa constrictor.Three boid snakes were purchased from a breeder and incorporated into a private reptile collection. Although apparently normal at the time of purchase, within a few days this snake developed sluggish behavior with twisting, ataxia, and opisthotonus. Clinical signs persisted for a week prior to death.

Gross Description:

The body of an 823 g, 129 cm male boa was in thin body condition with decreased fat stores and mildly atrophied skeletal muscle. A moderate pericardial effusion consisting of 2-3 ml of serous to faintly serosanguinous fluid was present as well as serous atrophy of epicardial fat. Excessive clear mucus was present in the mouth, and the stomach was empty. The kidneys appeared somewhat pale and ureters were prominent and filled with uric acid.

Histopathologic Description:

Liver: There is widespread hepatocellular necrosis associated with large, amorphic, amphophilic to basophilic intranuclear inclusion bodies resulting in karyomegaly and margination of nuclear chromatin. Occasional hepatocytes and numerous biliary epithelial cells contain one or more rounded, brightly eosinophilic intracytoplasmic inclusion bodies. Loose fibrin thrombi, high numbers of heterophils and scattered lymphocytes are present within sinusoids throughout the liver.

Stomach: Numerous mucosal epithelial cells contain rounded, brightly eosinophilic intracytoplasmic inclusion bodies. Occasional epithelial cells contain amphophilic intranuclear inclusion bodies. Additional lesions not included on the slide include brightly eosinophilic intracytoplasmic inclusion bodies within neurons and glial cells in the brain (occasionally associated with gliosis); within epithelial cells of the adrenal gland, thyroid follicles, bronchi, renal tubules, acinar pancreatic cells, and intestinal enterocytes; within lymphocytes in the spleen; and occasionally within cardiomyocytes. Low numbers of intestinal enterocytes also contain amphophilic intranuclear inclusion bodies.

Morphologic Diagnosis:

Liver: Severe, diffuse acute necrotizing hepatitis with intralesional inclusion bodies.
Stomach: Intracytoplasmic and intranuclear inclusion bodies.

Lab Results:

Routine fecal flotation revealed no parasite eggs or oocysts.

Condition:

Boid inclusion body disease

Contributor Comment:

Death in this case was determined to be due to dual viral infection with histologic lesions consistent with concurrent inclusion body disease and adenovirus infection, although confirmation of the latter infection by virus isolation was not attempted. In the absence of ancillary testing, herpesviral infection could not be ruled out as a cause of hepatic necrosis.

Inclusion body disease is an important infection in boid snakes, having emerged during the last 30 years on several continents.(13) Although snakes may remain chronic carriers of the disease, once clinical signs develop, the disease is fatal within weeks or months.(2,13) Clinical signs are variable and can include neurologic, digestive or respiratory systems.(6,12,13) Antemortem diagnosis can be achieved by biopsy of the liver, esophageal tonsil, gastric mucosa or skin; or by detection of typical cytoplasmic inclusion bodies within peripheral blood leukocytes, although this method yields higher false positives and false negatives.(2,3) Histologically, there are large, brightly eosinophilic intracytoplasmic inclusion bodies within a wide range of tissues (particularly in the liver and pancreas), often in the absence of a tissue or cellular response.(1,2,6,14)

Retroviruses have been isolated from boids with inclusion body disease; however, Kochs postulates have yet to be completely fulfilled.(5,13,16) The bloodsucking snake mite, Ophionyssus matrices, may act as a vector.(13) Concurrent infectious disease is common, possibly due to lymphoid depletion and resultant immunosuppression.(6) Pneumonia, ulcerative stomatitis(6), entamoebic colitis(10), and neoplastic diseases such as lymphoma(11) have been reported in conjunction with inclusion body disease.

Adenoviral hepatitis has previously been described in boid snakes, and is characterized by severe necrotizing hepatitis with basophilic to eosinophilic intranuclear inclusions within hepatocytes, accompanied by heterophils and small mononuclear cells.(4,8,13) Other lesions previously attributed to adenovirus or adenoviral-like infection in snakes include enteritis, splenitis, nephritis, pneumonia and encephalopathy.(7), The ability for latent adenoviral infections to induce clinical disease following immunosuppression has been demonstrated in species other than snakes(15), and in the case presented here it is tempting to speculate that inclusion body disease was involved in that respect.

Major viral diseases in boid snakes
Agent	Typical lesions	Inclusions
Adenovirus	Necrotizing hepatitis	Amphophilic (occasionally eosinophilic), intranuclear
Herpesvirus	Necrotizing hepatitis, glomerulonephritis	Eosinophilic to amphophilic intranuclear inclusions
Inclusion body disease virus (suspected retrovirus)	Inclusions in the absence of a tissue or cellular response	Eosinophilic, intracytoplasmic
Paramyxovirus	Hemorrhagic to necrotizing pneumonia, pancreatitis, occasionally CNS disease	Occasional, eosinophilic, intranuclear or cytoplasmic
Adenovirus, parvovirus, picornavirus, herpesvirus	Viral-associated gastrointestinal disease - causal relationship not established

JPC Diagnosis:

1. Liver: Hepatic atrophy, diffuse, severe.
2. Liver: Necrosis, hepatocellular, multifocal, with rare intranuclear viral inclusions.
3. All cell types: Intracytoplasmic inclusions.
4. Stomach: Diffuse loss of parietal cell granules.

Conference Comment:

The moderator attributed the hepatic atrophy and gastric parietal cell granular degeneration to the poor nutritional status of the animal, which is a common finding in inclusion body disease. The rare intranuclear inclusions in hepatocytes are likely due to the concurrent adenoviral infection. The conference moderator also noted the presence of melanin pigment within the liver, which is normal in reptiles.

The moderator discussed the composition of the inclusions seen in inclusion body disease, which remains controversial. The inclusion material was initially thought to result from the associated retrovirus, but recent research suggests that this condition may be a storage disease, similar to the transmissible spongiform encephalopathies. The intracytoplasmic inclusions, which ultrastructurally are either non-membrane limited aggregates of granular electron-dense material, or membrane-bound aggregates of electron-dense material with membrane-like fragments, are composed of a distinct 68-kd protein, and typically cause no inflammatory response, as opposed to necrosis and inflammation associated with viral infections. Nonviral inclusions have been demonstrated in cells infected with other retroviruses, such as avian leukosis virus, visna virus, and simian immunodeficiency virus.

Boid inclusions stain with PTAH and toluidine blue, and are eosinophilic with H&E, suggesting protein composition; they do not stain with Congo red or thioflaven-T, suggesting that the material in not amyloid. Boid inclusions have a similar staining pattern to Mallory bodies, non-viral inclusions composed of cytokeratin filaments. The inclusions may also be overproduced or poorly degraded components of the virus that accumulates in the host cell cytoplasm.(16)

As this is one of the most important viral diseases of boas and pythons with no known treatment, diagnosis of IBD is imperative. Antemortem diagnosis is often easily obtained through biopsy, since the inclusions are present in all tissues. Preferring the liver, the moderator discussed various high-yield biopsy locations. The skin is a poorer option because inclusions are more widely distributed, necessitating multiple biopsies to decrease false positive results. Cytology is another option, but the slides must be stained with H&E in order to differentiate the inclusions from other structures, such as lipid or intracellular parasites. Currently, immunohistochemical staining is available for frozen tissue, which may lead to a serologic test in the future.

References:

1. Carlisle-Nowak MS, Sullivan N, Carrigan M, et al. Inclusion body disease in two captive Australian pythons (Morelia spilota variegata and Morelia spilota spilota). Aust Vet J. 1998;76:98-100.
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11. Schilliger L, Selleri P, Frye FL. Lymphoblastic lymphoma and leukemic blood profile in a red-tail boa (Boa constrictor) with concurrent inclusion body disease. J Vet Diagn Invest. 2011;23:159-162.
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13. Schumacher J, Jacobson ER, Homer BL, et al. Inclusion body disease in boid snakes. J Zoo Wild Med. 1994;25:511-524.
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15. Ward JM, Young DM. Latent adenoviral infection of rats: intranuclear inclusions induced by treatment with a cancer chemotherapeutic agent. JAVMA. 1976;169:952-953.
16. Wozniak E, McBride J, DeNardo D, et al. Isolation and characterization of antigenically distinct 68-kd protein from nonviral intracytoplasmic inclusions in boa constrictors chronically infected with the inclusion body disease virus (IBDV: Retroviridae). Vet Pathol. 2000;37:449-459.