Inclusion Body Disease of Boid Snakes
Inclusion body disease of boid snakes has been recognized since the mid 1970′s. It is named for the characteristic intracytoplasmic inclusions that are seen in epidermal cells, oral mucosal epithelial cells, visceral epithelial cells, and neurons. In the 1970′s, through the late 1980′s, this disease was most commonly seen in Burmese pythons, Python molusus bivittatus. Starting in the late 1980′s until present, it has been seen most commonly in boa constrictors, Boa constrictor.
Inclusion Body Disease (IBD) has been identified in boid snakes including multiple subspecies of the boa constrictor (Boa constrictor), green anaconda (Eunectes murinus), Haitian boa (Epicrates striatus), Burmese python (Python molurus bivittatus), Indian python (P. m. molurus), reticulated python (P. reticulatus), ball python (P. regius), carpet python (Morelia spilota variegata) and diamond pythons (M. s. spilota). In addition, a disease resembling IBD was diagnosed in an eastern king snake (Lampropeltis getulus) that was housed with boa constrictors and in palm vipers (Bothriechis marchi). Tissues to be used in the proposed study are already in the PI’s tissue repository.
Worldwide in captive boid snakes. Its occurrence in the wild is unknown. Several cases have recently been seen in captive pythons in Australia, Canary Islands, and Italy. The transport of captive snakes in the pet trade and transport between different zoologic institutions probably account for the spread around the world.
IBD has been identified primarily in adult snakes. However, all age groups should be considered susceptible. There are anecdotal reports of infection in neonates.
A retrovirus has been isolated from boa constrictors with IBD and may be the causative agent (Figure 1). However, we currently do not know if the isolated virus is the cause of IBD. Research is ongoing to determine the relationship, if any, between this virus and the formation of inclusions in cytoplasm of cells in infected snakes.
Clinical signs are quite variable. Regurgitation and signs of central nervous system disease (Figure 2; Figure 3; Figures 4a and 4b) are commonly seen in boa constrictors. Stomatitis, pneumonia, undifferentiated cutaneous sarcomas (Figure 5), lymphoproliferative disorders, and leukemia has all been seen. Burmese pythons generally show signs of central nervous system disease without manifesting any other clinical signs; regurgitation is not seen in Burmese pythons.
IBD is typically diagnosed in post-mortem and biopsy specimens, using light microscopy. In hematoxylin and eosin (H&E) stained tissue sections of a wide variety of epithelial and neuronal cells, characteristic intracytoplasmic inclusions are seen. Several snakes have been seen with proliferative pneumonia (Figure 6). While inclusions are commonly seen in the liver, kidney, and pancreas (Figure 7; Figure 8; Figure 9), we have seen cases where there are very few inclusions. Thus, inclusions may be missed by pathologists in cases having few inclusions in the brain or other tissues. While the presence of characteristic inclusions is diagnostic for the disease, the absence of inclusions does not necessarily mean the snake is disease or IBD free. In some cases many tissue sections have to be examined to find inclusions. While cells having inclusions may show mild degenerative changes, inflammation is rarely seen in visceral tissues. In the brain, mild to severe encephalitis, with lymphocytic perivascular cuffing may be seen. Several snakes with lymphoproliferative disorders have been identified with lymphoid infiltrates in multiple organs.
Inclusions can also be seen in lymphocytes and other circulating blood cells in peripheral blood films stained with Wright-Giemsa or Hematoxylin and Eosin stained slides.
We now isolate and H&E stain white blood cells from 1mL of whole blood using a cytopsin protocol. Staining of these cells with H&E is far superior than using Wright-Giemsa staining. With Wright-Giemsa staining the inclusions stain basophilic and may not be readily recognizable. With H&E staining, inclusions stain exactly the same as in tissues embedded in paraffin, sectioned and stained with H&E. Blood films should first be examined before tissue biopsies are obtained and submitted for histopathology. However, we do not know how often inclusions are seen in a peripheral blood film of a snake with IBD. It must be remembered that absence of inclusions in a blood film does not necessarily mean the snake is free of IBD.
Immunohistochemical (IHC) Staining
A study by Wozniak and coworkers demonstrated that inclusion bodies in IBD are composed of an antigenically unique 68-kd protein. It is unknown whether all IBD cases in different snake species consist of exactly the same protein or if variability occurs that is species dependent. Recently, we have isolated inclusions (Figure 10) from a boa constrictor with IBD and, using hybridoma technology, have produced a new anti-IBDP MAB that stains inclusions in paraffin-embedded tissues. This MAB is currently going through rigid testing and validation. We are also validating a blood based test using IHC staining. Peripheral white blood cells (PWBC) can be isolated from 1ml of whole blood, and using a cytospin protocol, WBCs are spun onto a microscopic slide, where IHC staining is performed.
Interpretation of Findings
Inclusions in some boas may only be found in the brain or limited to a few organs. For the IHC blood test, we do not know how many snakes that are infected will have inclusions in peripheral blood. Based on a small sample size, all snakes we received having inclusions internally also had inclusions in peripheral blood cells. We feel very comfortable about having few if any false-positives. However, there may be false-negatives. We will not know the sensitivity and specificity of this test until validation studies are finalized and when we have a greater number of cases with blood films matched with tissues.
The big question is: What to do with positive snakes?
This will depend on whether they are ill or not. Those that are ill should be euthanized. Those that are healthy should be separated from ill snakes and followed over time. It may take more than one blood test to confirm if a snake is positive or negative. At this point we recommend 2 blood samples 90 days apart. We have selected 90 days since this is the length of quarantine we recommend for new animals coming into an established collection. Mites have been associated with outbreaks so be aware of this.
Exact route of transmission has not been identified. Possibly by: 1) direct contact; 2) intrauterine transmission to developing embryos in viviparous species and eggs in oviparous species; 3) venereal transmission. The snake mite, Ophionyssus natricis has been implicated as a vector for the virus since mite infestations are commonly seen in epizootics of IBD.
Currently, a presumptive diagnosis is based on the light microscopic identification of intracytoplasmic inclusions in tissue sections. Some snakes have numerous inclusions in tissues and others have few. A more sensitive and specific molecular diagnostic test is needed such as IHC staining, western blot or ELISA to screen individuals and colonies of snakes at risk. We are working toward an immune-based blood screening test to allow animals at risk to do a rapid screening for IBD. We believe that sequencing the IBD unique 68 kda protein is the key to understand the pathogenesis of IBD, which will help us determine a better strategy to prevent IBD. We are also working on obtaining the sequence of retroviruses isolated from infected snakes that will help us understand the relation between the virus and IBD.
Past research support has come from several primate donors, the National Reptile Breeders’ and Mid-Atlantic Reptile Expos. Currently we have support from Morris Animal Foundation to develop better diagnostic tests. Dr. Rita Chang is a doctoral graduate student who is now working on this project.
References including link to Inclusion body disease of boid snakes review
- Axthelm MK. 1985. Viral encephalitis of boid snakes. Int Colloq Pathol Reptiles Amphib 3:25. (Abstract)
- Carlisle-Nowak MS, Sullivan N, Carrigan M, Knight C, Ryan C, and Jacobson ER. 1998. Inclusion body disease in two captive Australian pythons (Morelia spilota variegata and Morelia spilota spilota). Aust Vet J 76:98-100.
- Chang L, Jacobson ER. 2010. Inclusion body disease, a worldwide infectious disease of boid snakes: a review. J Exo Pet Med 19(3): 216–225.
- Jacobson ER, Klingenberg RJ, Homer BL, Mader DR. 1999. Inclusion body disease. Bull of the Assoc Reptil Amphib Vet. 9:18-25.
- Jacobson ER, Oros J, Tucker S, Pollock D, Vaughn K, Munn RJ, Lock B, Mergia A, Yamamoto JK. 2001. Isolation and characterization of retroviruses from boid snakes with inclusion body disease. Am J Vet Res 62:217-224.
- Oros J, Tucker S, Jacobson ER. 1998. Inclusion body disease in two captive boas in the Canary islands. Vet Rec 143:283-285.
- Raymond JT, Garner MM, Nordhausen RW, Jacobson ER. 2001. A disease resembling includion body disease of boid snakes in captive palm vipers (Bothriechis marchi). J Vet Diagn Investig 13:82-86.
- Schumacher J, Jacobson ER, Homer BL, Gaskin JM. 1994. Inclusion body disease in boid snakes. J Zoo and Wildlife Med 25(4):511-524.
- Wozniak E, McBride J, DeNardo D, et al. Isolation and characterization of an antigenically distinct 68-kd protein from nonviral intracytoplasmic inclusions in boa constrictors chronically infected with the inclusion body disease virus (IBD: retroviridae). Vet Pathol 37:449-459.
For More Information, contact
Dr. Elliott Jacobson
Department of Small Animal Clinical Sciences
College of Veterinary Medicine
University of Florida
PO Box 100126
Gainesville, FL 32610-0126