Experimental challenge of East Asian bullfrog Hoplobatrachus rugulosus tadpoles with tiger frog virus: dosage impact on disease severity

Ranaviruses are associated with epizootics in poikilothermic vertebrates worldwide. The ranavirus tiger frog virus (TFV) causes lethal, systemic disease in amphibians and reptiles. TFV was associated with mortality in East Asian bullfrogs Hoplobatrachus rugulosus (EAB) of all developmental stages in ranaculture facilities in Thailand between the late 1990s and late 2010s. However, experimental evidence of the pathogenic potential of TFV in EAB has not previously been reported. The objective of this study was to determine the susceptibility of EAB tadpoles to TFV under controlled laboratory conditions. We conducted bath challenges at 2 viral dosages, 5 × 104 and 5 × 106 TCID50 virus ml-1, and monitored cumulative mortality for 21 d. Low to moderate (33%) mortality was observed among tadpoles exposed to a low dosage inoculum, while high (71%) mortality was observed among tadpoles exposed to a high dosage. All tadpoles that died or were euthanized in the high dosage treatment during the 21 d study period tested positive by qPCR and virus isolation, with homogenates yielding high viral loads as measured by qPCR. Histopathology, aided by the development of a frog virus 3 (FV3)-specific in situ hybridization probe, revealed the expected microscopic lesions in EAB tadpoles exposed to TFV that died during the experiment, but they were absent in unexposed control tadpoles. The pathogenicity of TFV in this highly valued food species underscores the need for improved biosecurity measures and mitigation strategies to protect the Thai ranaculture industry from TFV.