Isolation of a Bohle-like iridovirus from boreal toads housed within a cosmopolitan aquarium collection.

A captive 'survival assurance' population of 56 endangered boreal toads Anaxyrus boreas boreas, housed within a cosmopolitan collection of amphibians originating from Southeast Asia and other locations, experienced high mortality (91%) in April to July 2010. Histological examination demonstrated lesions consistent with ranaviral disease, including multicentric necrosis of skin, kidney, liver, spleen, and hematopoietic tissue, vasculitis, and myriad basophilic intracytoplasmic inclusion bodies. Initial confirmation of ranavirus infection was made by Taqman real-time PCR analysis of a portion of the major capsid protein (MCP) gene and detection of iridovirus-like particles by transmission electron microscopy. Preliminary DNA sequence analysis of the MCP, DNA polymerase, and neurofilament protein (NFP) genes demonstrated highest identity with Bohle iridovirus (BIV). A virus, tentatively designated zoo ranavirus (ZRV), was subsequently isolated, and viral protein profiles, restriction fragment length polymorphism analysis, and next generation DNA sequencing were performed. Comparison of a concatenated set of 4 ZRV genes, for which BIV sequence data are available, with sequence data from representative ranaviruses confirmed that ZRV was most similar to BIV. This is the first report of a BIV-like agent outside of Australia. However, it is not clear whether ZRV is a novel North American variant of BIV or whether it was acquired by exposure to amphibians co-inhabiting the same facility and originating from different geographic locations. Lastly, several surviving toads remained PCR-positive 10 wk after the conclusion of the outbreak. This finding has implications for the management of amphibians destined for use in reintroduction programs, as their release may inadvertently lead to viral dissemination.

Pathology, isolation and molecular characterisation of a ranavirus from the common midwife toad Alytes obstetricans on the Iberian Peninsula.

We describe the pathology, isolation and characterisation of a virus responsible for an outbreak of a systemic haemorrhagic disease causing high mortality in tadpoles of the common midwife toad Alytes obstetricans in the 'Picos de Europa' National Park in northern Spain. The virus, provisionally designated as the common midwife toad virus (CMTV), was isolated from homogenates of visceral tissue from diseased toad tadpoles following inoculation on epithelioma papulosum cyprini (EPC) cells. Molecular characterisation of the virus, including sequence analysis of the DNA polymerase and major capsid protein genes, showed that the isolated virus was a ranavirus with marked sequence identity to other members of the genus Ranavirus. A rabbit antiserum raised against purified virions was prepared and used to definitively demonstrate systemic distribution of the virus in diseased tadpoles, indicating that the isolated virus was the primary pathogen.

Experimental challenge and clinical cases of Bohle iridovirus (BIV) in native Australian anurans.

Ranaviruses have been observed with increasing frequency amongst poikilothermic vertebrate hosts. The impact of ranaviruses upon amphibian populations has remained largely unknown. A gene probe for Bohle iridovirus (BIV) based upon primers designed to detect epizootic haematopoietic necrosis virus (EHNV) was constructed. A PCR and dot-blot system was used successfully in screening for 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 and epizootics in captive frogs, juvenile Litoria caerulea, L. alboguttata, Cyclorana brevipes and Pseudophryne coriacea were acutely susceptible. High mortality (at or near 100%) resulted, usually occurring within 5 to 25 d depending on dose and method of exposure. Histopathological changes included mainly hepatic, renal and splenic necroses. Significant haemosiderosis was encountered in more chronically infected frogs. BIV could be reisolated from juvenile L. caerulea >40 d after inoculation, and >200 d after the first mortalities occurred in an epizootic in L. alboguttata. Adult L. rubella, L. inermis, L. caerulea, Cophixalus ornatus and Taudactylus acutirostris were less susceptible in trials ranging from 30 to > 100 d. There was some evidence of chronic infection, and BIV could be detected by PCR. Wild moribund adult L. caerulea from Townsville and captive juvenile Pseudophryne corieacea from Sydney undergoing mortality tested positive with 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. Ranaviruses could be having an impact on Australian herpetofauna.