Abstract

Inclusion body disease (IBD) is characterized by the formation of intracytoplasmic inclusions in neurons and in epithelial cells of various organs. It generally affects boid snakes. Clinically, it is commonly associated with central nervous system (CNS) disorders as well as regurgitation, stomatitis, and pneumonia. The disease is believed to be of viral etiology, and retroviruses have been implicated as a possible factor.^8,16 However, boa constrictors (Boa constrictor spp.) have also been shown to harbor endogenous retroviruses,¹³ making a definitive connection between retroviruses found in IBD-positive snakes, and disease difficult.

In the course of this study, tissues from 12 boa constrictors from Germany found to be IBD positive, were tested for cytopathogenic viruses (Table 1). These animals stemmed from three different captive populations and were all captive-bred in Germany. Two IBD-negative animals from populations with no history of IBD, were also included in the study (Table 1). Virus isolation was attempted on viper heart cells (VH2, ATCC CCL-140), terrapene heart cells (TH-1, ATCC CCL-50), and chick embryo fibroblasts (CEF)¹⁵ in animals from population A, and from primary cell cultures and on VH2, in animals from populations B and C and the two negative controls. A polymerase chain reaction (PCR) for retrovirus sequences was performed on tissues from the animals in the study as indicated in Table 1. The degenerated oligonucleotide primers 5'-GT(T/G) TTI (G/T)TI GA(T/C) ACI GGI (G/T)C-3' and 5'-ATI AGI A(G/T)(G/A) TC(A/G) TCI AC(A/G) T-3'^13,18 were used. These are specific for conserved motives in retroviral protease and reverse transcriptase genes. The same PCR was used to test the isolated cytopathogenic agents, as well as uninfected VH2.

Table 1. Animals tested

^aTissues collected for virus isolation: lu=lung, li=liver, int=intestine, br=brain, sp=spleen, pancr=pancreas, kidn=kidney, bm=bone marrow.
^bNot done.

Cytopathogenic viruses were isolated in VH2 from a total of five different animals from populations A, B, and C as shown in Table 2. Three of the isolates were identified as reoviruses on the basis of their cytopathic effect (CPE), chloroform stability, lack of inhibition by 5-iodo-2'-desoxyuridin (IUDR), which inhibits DNA polymerase, and morphology by electron microscopy. Two of the isolates were identified as adenoviruses by the same means. The growth of these isolates was inhibited by the addition of IUDR to the cell culture medium. The PCR for the detection of retroviral sequences was positive in several cases (Table 3). It was not positive on any of the cell cultures tested.

Table 2. Virus isolates

Table 3. Results of the PCR for retroviral sequences

Reoviruses^{1,2,11,12,14,19} and adenoviruses^{3,4-7,9,10,17} have frequently been reported in reptiles. Reoviruses have also been associated with CNS disorders in reptiles,^2,19 as have adenoviruses^3,7. The significance of the viruses isolated in this study for the IBD seen in the examined animals is unclear. One or both of the viruses found could play a role in the etiology of this disease. It is possible, however, that these viruses were opportunistic pathogens, and were only found because of an immune suppression caused by the IBD. Further studies are planned to clarify these questions.

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