Conference 15 - 2016 Case: 2 20170118

Signalment:

Four-year-old, male, red tail boa constrictor, (Boa constrictor constrictor).The patient initially presented on 10/29/201 4 for a 4-5 month history of anorexia. At that time, there was atrophy of the epaxial muscles and a firm, but compressible swelling expanding the cranial cervical region. Ultrasound of this area revealed a soft-tissue mass, for which the tissue of origin was unclear. The mass did not appear to be associated with the trachea or within the esophageal lumen. The patient represented in January 2015 for continued anorexia, progressive lethargy, and recent regurgitation after forced feeding. The patient had lost a significant amount of weight and on examination, there was a large accumulation of necrotic material in the mouth. During this exam, a second mass was appreciated just caudal to the previously described cervical mass. At the owner's request, the patient was euthanized.

Gross Description:

A 2 cm x 0.5 cm region of the hard palate is raised, irregularly surfaced, fleshy, and dark red. Extending from the esophageal wall and protruding into the lumen is a 2.5 cm x 1 cm x 1 cm, smooth, soft, pink and red mass, with a core of crumbly, brown-yellow material. Approximately 0.5 cm aboral to this mass is a 4.5 cm x 2.5 cm x 2.5 cm, smooth, firm, ovoid, pink and red mass that on section is composed of a central core of a crumbly, brown-yellow material (caseous necrosis) surrounded by a 0.7 cm wide rim of a fleshy, pink/red tissue. The corresponding eso-phageal serosa is firmly adhered to the local body wall. The esophageal mucosa, adjacent to the larger mass, has a 2 cm x 0.4 cm, poorly demarcated, mildly depressed, region composed of dozens of pinpoint red foci (erosion). Within the lumen of the aboral esophagus, approximately 3.5 cm from the larger mass, are three rough, ovoid aggregates of a mottled brown, yellow, and green, crumbly material. This material is not adhered to the mucosa.

Histopathologic Description:

ORAL CAVITY: Severely expanding the sub-mucosa, confluent with a large region of epithelial ulceration, and obliterating the adjacent lamellar bone is a densely packed population of foamy macrophages with fewer granulocytes, lymphocytes, and plasma cells. Unilaterally the alveolar bone is disrupted and replaced by a similar inflammatory cell population and the islands of retained bone has irregular, scalloped edges and are lined by numerous osteoclasts within distinct Howship's lacunae. The superficial osteoid matrix commonly contains a thin, irregular, basophilic line (reversal line). Similar inflammatory cells efface the dentin and invade the pulp of a tooth. The surface epithelium is entirely replaced by a band of necrotic cellular debris and fibrin. BRAIN, NOS: Occasional neurons contain discrete, intracytoplasmic, 1-10 um diameter, glassy, eosinophilic inclusions. NASAL CARTILAGE: Commonly, the respiratory epithelial cells and submucosal gland epithelial cells contain discrete, intracytoplasmic, 1 -1 0 um diameter, glassy, eosinophilic inclusions. Scattered throughout the submucosa are scant numbers of lymphocytes and plasma cells. ESOPHAGEAL MASSES (not submitted): Multifocally the esophageal wall is severely expanded by multiple unencapsulated, irregular masses composed of densely packed vacuolated macrophages mixed with fewer small lymphocytes, plasma cells, granulocytes, and multinucleated nucleated giant cells. Lymphocytes are occasionally present in small, poorly defined islands and cells contain discrete, intracytoplasmic, 1-4 um diameter, glassy, eosinophilic inclusions. The overlying epithelial cells contain discrete, 2-6 um, glassy, eosinophilic, intra-cytoplasmic inclusions. Additional findings include numerous discrete, 1-10um in diameter, glassy, eosinophilic, intracytoplasmic inclusions within multiple tissues including hepato-cytes, biliary epithelial cells, gastric mucosa, intestinal epithelium, tracheal epithelium, bronchiole epithelium, and the retinal ganglion cells. Concurrently within the liver, there were small numbers of randomly distributed macrophages, lymphocytes, and plasma cells.

Morphologic Diagnosis:

Oral cavity: Severe, diffuse, chronic, granulomatous and ulcerative stomatitis with numerous, eosinophilic, intracytoplasmic inclusion bodies
Esophageal mass (not submitted): Severe, multifocal, chronic, granulomatous and ulcerative esophagitis with numerous, eosinophilic, intracytoplasmic inclusion bodies
Stomach, intestine, trachea, lung, kidney, liver, nasal cavity, retina, and brain (not submitted): Severe, eosinophilic, intra-cytoplasmic inclusion bodies

Lab Results:

N/A

Condition:

Boid inclusion disease, stomatitis

Contributor Comment:

Inclusion body disease (IBD) is reported in multiple snake species, but most commonly within the family Boidae and Pythonidae. As the name implies, the characteristic finding in these cases are distinct, variably sized, eosinophilic, intracytoplasmic inclusions.² In one recent retrospective study, the prevalence of IBD in captive collections was approximately 19%. Although disease progression varies greatly between individuals and between species, the disease is classically associated with central nervous system signs, including head tremors, anisocoria, and opisthotonus.⁹ Commonly the animal succumbs to complications secondary to immunosuppression.¹⁵ In boas, the disease can have a more protracted progression of weeks to months⁹ and typically patients have a previous history of regurgitation.¹⁰ In addition, a proportion of boas can be subclinical carriers.^7-10 Meanwhile, pythons tend to display a more aggressive disease course of only a few weeks,⁹ with a more profound inflammatory reaction.¹⁰ Interestingly, regurgitation tends not to be a part of clinical disease in pythons.¹⁰ Gold standard testing remains the histologic demonstration of intracytoplasmic inclusions in multiple organs, most notably the liver, stomach, and esophageal tonsils, however, their absence does not rule out the disease.¹⁰ Although with further characterization of the underlying etiology, molecular testing and immunohisto-chemistry³ may be available in the future. Although long considered to have an underlying infectious etiology, the cause of IBD has been elusive. Originally the disease was thought to be associated with a retrovirus, however, recently divergent arenaviruses have been implicated as the cause of IBD.^2-4,8-10Of late, in vitro Koch's postulates have been met linking arenavirus to the development of IBD, however, in vivo studies have not be reported.⁹ Arenaviridae are enveloped, negative sense, single stranded, bipartite RNA viruses.^2,8 Arenaviruses have previously been thought to only affect rodents, with infrequent but possible transmission to other mammal species (e.g. humans and bats).² The viral genome is composed of a small (S) segment and a large (L) segment. The S segment encodes the viral nucleocapsid protein (NP) and the glycoproteins (GP1 and GP2) while the L segment encodes the viral RNA-dependent RNA polymerase and a small ring domain containing protein^2,9 The distinction intracytoplasmic inclusions consist of a unique 68KDa protein, that has been named "inclusion body disease protein" (IBDP).^3,9,10 This protein has been demonstrated to be the arenaviral NP protein.⁶ Boid-associated inclusion body arenaviruses tend to be highly divergent.^2,8,9
Proposed theories as to the mode of transmission include direct contact, possible arachnid vectors (i.e. the snake mite, Ophionyssus natricis), mammalian vectors (i.e. live prey), and vertical transmission.^8-10 As several other arenaviruses are able to cross the species barrier, it remains possible that the highly divergent boid inclusion body disease associated arenaviruses (BIBDAV) may as well. A recent report showed that BIBDAV was infective to tick cells lines (mite cell cultures were not available) and this data may support the role of Ophionyssus natricis infestation in disease transmission.⁸ Furthermore, maintenance of BIBDAV within mammalian (VERO E6) and boid cell lines appears to be temperature dependent, with strong growth at 30°C and inhibition of growth at 37°C. Thus, the authors purpose that transmission from a mammalian host is possibly hindered by the higher mammalian body temperature.⁸Boid snakes have large well-developed esophageal tonsils, which can be enlarged and abscessed. In this case, the large esophageal masses may represent severely inflamed esophageal tonsils.

JPC Diagnosis:

1.Oral cavity: Stomatitis, ulcerative and histiocytic, chronic, multifocal to coalescing, severe, with granulation tissue and bone resorption, red tail boa constrictor, Boa constrictor constrictor. Epithelial cells: epidermis, salivary gland, and nasal cavity: Intracytoplasmic protein inclusions of viral origin, numerous.

Conference Comment:

Boid inclusion body disease (BIBD) is considered by many to be the most important viral infection of captive boas and pythons, often causing progressive and rapidly fatal multisystemic disease.¹⁰ As mentioned by the contributor, the clinical presentation of BIBD is highly variable among individuals, especially in adult boas, where intracytoplasmic viral protein inclusions can be found in snakes without clinical signs.^2,6,10,13 However, the disease is still considered to be fatal due to severe impairment of immune function of leukocytes and myelopoietic cells, resulting in death due to opportunistic infections.^6,10 Interestingly, in this case, the conference moderator speculates that the extensive ulcerative stomatitis seen both grossly and histologically may be due to BIBD-induced starvation and regurgitation combined with the mechanical trauma from the reported forced feeding.
Typical gross findings associated with BIBD are usually limited to areas susceptible to secondary opportunistic infections, such as the oral cavity, gastrointestinal tract, lungs, liver, and kidney. Histologically, the hallmark of BIBD is the presence of numerous 1-4 um, pale, eosinophilic intra-cytoplasmic inclusion bodies in all major organs, especially the kidneys, liver, stomach, and brain. Inclusions are typically more prominent in the visceral organs of boas and central nervous system in pythons.¹⁰ In this case, inclusions are widely distributed throughout.
Conference participants discussed the composition of the highly distinctive inclusion bodies associated with this disease. The inclusions, which ultrastructurally are cytoplasmic aggregates of electron-dense material, composed of a n antigenically unique 68-kilodalton non-viral protein.^2,3,6,15 Recently, a novel group of arenaviruses were isolated from snakes with BIBD. In an in-vitro cell culture model, this arenavirus induces the pathognomonic inclusion bodies and was discovered to predominantly consist of arenaviral associated nuclear protein. This finding led to the suggestion of the formation of a novel genus called the Reptarenavirus and placing the remaining arenaviruses in the Mammarenavirus genus. There is still some controversy surrounding the formation of a new genus given the lack of in vivo confirmation.^3,9,11
Conference participants discussed the importance of arenaviruses as zoonotic pathogens associated with rodent host species. In humans, arenaviruses, such as Lassa and Machupo (Bolivian hemorrhagic fever), cause outbreaks of rapidly fatal viral hemorrhagic fevers, not unlike the Ebolavirus.¹ Additionally, hamsters are the primary source of lymphocytic chorio-meningitis (LCM) virus causing meningo-encephalitis in humans and callitrichid hepatitis in New World primates.¹³ Arenaviruses generally produce only mild or subclinical disease in their natural host species.

References:

1. Bell TM, Bunton TE, Shaia CI, Raymond JW, Honnold SP, Donnelly GC, Shamblin JD, Wilkinson ER, Cashman KA. Pathogenesis of Bolivian hemorrhagic fever in Guinea pigs. Vet Pathol. 2016; 53(1):190-199.
2. Bodewes, R, Kik, MJL, Raj S, et al. Detection of novel divergent arenaviruses in boid snakes with inclusion body disease in the Netherlands. J Gen Virol. 2013; 94:1206-1210.
3. Chang, L, Fu Am Wozniak, E, et al. lmmunohistochemical detection of a unique protein within cells of snakes having inclusion body disease, a world-wide disease seen in members of the families boidae and pythonidea. PLoS One. 2013. 8(12):1-16.
4. Charrel RN, de Lamballerie X. Zoonotic aspects of arenavirus infections. Vet Microbiol. 2010; 140:21320.
5. Drake, S, Marschang RE, Hetzel U, et al. Experimental infection of boa constrictor with an orthoreovirus isolated from a snake with inclusion body disease. J Zoo Wildl Med. 2014; 45(2):433-436.
6. Fowler ME, Miller RE. Fowler's Zoo and Wild Animal Medicine. Miller RE, Fowler ME eds. Vol 8. Philadelphia, PA; Saunders; 2014:70.
7. Hellebuyck, T, Pasmans F, Ducatelle, R, et al. Detection of arenavirus in a peripheral ondotogenic fibromyxoma in a red tail boa (Boa constrictor constrictor) with inclusion body disease. J Vet Diagn Invest. 2015; 27(2):245-248.
8. Hepojoki J, Kipar, A, Korzyukov, et al. Replication of boid inclusion body disease-associated arenaviruses is temperature sensitive in both boid and mammalian cells. J Virol. 2015; 89(2):1119-1128.
9. Hetzel U, Sironen T, Laurinmaki, P, et al. Isolation, identification, and characterization of novel arenaviruses the etiological agents of boid inclusion body disease. J Virol. 2013; 87(20):10918-10935.
10. Jacobson ER. Infectious Diseases and Pathology of Reptiles, Color Atlas and Text. Jacobson ER ed.1^st ed. CRC Press, Boca Raton, 2007:185, 410-412.
11. Keller S, Hetzel U, et al. Co-infecting reptarenaviruses can be vertically transmitted in boa constrictor. PLoS Pathog. 2017; 13(1):e1006197.
12. Pees M, Schmidt V, Marschang RE, et al. Prevalence of viral infections in captive collection of boid snakes in Germany. Vet Rec. 2010; 166:422-425.
13. Montali RJ, Connolly BM, Armstrong DL, Scanga CA, Holmes KV: Pathology and immunology of callitrichid hepatitis, an emerging disease of captive new world primates caused by lymphocytic choriomeningitis virus. Am J Path. 1995; 148(5):144-149.
14. Schillinger L, Selleri P, Frye FL. Lymphoblastic lymphoma and leukemia blood profile in a red-tail boa (Boa constrictor constrictor) with concurrent inclusion body disease. J Vet Diag Invest. 2011; 23:159-162.
15. Schmidt V, Marschang RE, Abbas MD, et al. Detection of pathogens in boidae and pythonidae with and without respiratory disease. Vet Rec. 2013; 172(9):236.