Detection of Ranavirus by PCR and in situ hybridization in the American bullfrog (Rana catesbeiana) in Taiwan

Abstract

Between July 2007 and January 2012, researchers encountered higher than normal outbreaks of mortality (up to 10–70%) in tadpoles, metamorphs and adult American bullfrogs (Rana catesbeiana) in southern Taiwan. On gross examination, skin ulcerations were frequently found on the dorsal surface of the head of infected bullfrogs, although visceral lesions were absent. Based on ultrastructural examination, the cytoplasm of infected splenic phagocytes contained numerous Ranavirus particles about 120 nm in diameter. Using polymerase chain reaction (PCR) assessment for the detection of ranaviruses, an expected 1392-bp product specific for the major capsid protein (MCP) gene of Ranavirus was obtained. A comparison of amino acid sequences for the whole MCP gene of our isolates with ranaviruses from Asia, Australia, Europe, and America showed that the identities were between 96.1 and 99.8% similar. Our isolates appeared to be most closely related to Korea (99.8%) and Japan (99.6%) strains. A second PCR/restriction endonuclease analysis (REA) strategy based on two shorter fragments of the MCP gene was developed and correctly confirmed that these Taiwan, Japan and Korea isolates evolved from Frog virus 3 (FV3), and formed a new genotype group distributed in Asia. We deduced that Ranavirus is the main cause for the massive economic losses of in Taiwanese bullfrog aquaculture.