Ranaviruses In Amphibians
Brad R. Cullen and Lee Owens
Microbiology and Immunology, School of Biomedical and Molecular Biology, James Cook University, Townsville 4811, Australia
Email: bradford.cullen@jcu.edu.au
Ranaviruses of the family Iridoviridae have been observed with increasing frequency amongst poikilothermic vertebrate hosts. They have been known to cause systemic disease often resulting in high mortalities. The impact of ranaviruses upon amphibian populations has remained largely unknown.
Two gene probes for Bohle iridovirus (BIV) based upon primers designed to detect epizootic haematopoietic necrosis virus (EHNV) were constructed. One of 218bp, and a 595bp probe based on the sequence which codes for the viral capsid protein. The 218bp probe had 95.5% nucleotide identity with the corresponding EHNV sequence. For the capsid probe, sequence identity with other iridoviruses was as follows: FV3 93.3%, IV22 52.0%, WIV 51.8%, CzIV 51.2%, TIV 51.1%, IV6 46.9%, LCDV 45.1%. Digoxigenin labelled probes were used successfully for in situ hybridisation in BIV-infected cell cultures, whereas biotinylated probes encountered background staining problems. Digoxigenin labelled probes could not be used for in situ hybridisation in cane toads (Bufo marinus) due to the binding of anti-digoxigenin antibody to the steroidal endogenous digoxin-like factor in cane toad toxin. Biotinylated probes were effective and would be a useful tool in studying acute BIV disease. Other frog species would probably have to be individually tested for endogenous background staining. A PCR and dot blot system was used successfully for screening the presence of BIV nucleic acid in digested formalin-fixed, paraffin embedded amphibian tissues.
Juvenile frogs were more susceptible to BIV than adults. In experimental challenges, juvenile Litoria caerulea, Litoria alboguttata, Litoria latopalmata, Cyclorana brevipes, Limnodynastes terraereginae, Limnodynastes ornatus and Bufo marinus were acutely susceptible. Mortality was high, usually occurring within 5-25 days depending on dose and inoculation method. Histopathological changes included mainly hepatic, renal and splenic necroses. Adult Litoria caerulea, Litoria rubella, Litoria inermis and Bufo marinus were less susceptible in trials ranging from 30 to >100 days. There was some evidence of chronic infection, and BIV could be detected by PCR. Wild moribund adult Litoria caerulea from Townsville and captive juvenile Pseudophryne corieacea from Sydney undergoing mortality, tested positive for the BIV PCR. PCR and dot blot was more sensitive than viral isolation. PCR could detect BIV in amphibians long after BIV challenge, and in amphibians which appeared healthy.
Adult toads collected in Townsville in March 1997, which tested negative for BIV antibodies, were inoculated with 107TCID 50ml-1 BIV. The adult BIV-challenged toads became seropositive at 22-30 days p.i., but there were no mortalities. Of the unchallenged control toads, 13 out of the 29 toads (44.8%) were positive by the PCR and dot blot. Of the BIV inoculated toads, 19 of the 30 toads (63.3%) were positive by the PCR and dot blot. The prevalence of anti-Ranavirus antibodies in Bufo marinus serum collected at various sites around Queensland varied markedly between areas, and between collection times. No anti-Ranavirus antibodies were detected in any of 221 cane toads collected from Innisfail, Bundaberg, Brisbane or Beaconsfield (Mackay). Antibodies were detected in 6 out of 22 cane toads (27.3%) collected in Townsville in January 1996, but not in toads collected in Townsville at any other time. Prevalence rates at Gooseponds (Mackay) were 41.8% in January 1997, but dropped to 8.6% in April 1997. BIV could not be isolated in cell culture from any toads from Gooseponds. Anti-Ranavirus antibodies were consistently detected in toad collections from Hughenden from August 1995 to April 1997, with prevalence rates varying from 5.8% to 20.4% (overall prevalence rate 12.9%). BIV could not be isolated from 39 frogs collected from Mt Surprise in August 1995.
Ranaviruses have a broad host range, and are easily transmitted. Data on the geographical origin and time of emergence or introduction of ranaviruses is not known. Ranaviruses could be having an impact on herpetofauna globally, and may be contributing to the observed decline in amphibian numbers in some areas.