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

Abstract

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.

Comparative Pathology of Ranaviruses and Diagnostic Techniques

“…In amphibians, outbreaks of ranaviral disease are most often observed in larvae and recently metamorphosed animals (Green et al 2002 ;Docherty et al 2003 ;Balseiro et al 2009Balseiro et al , 2010; however, outbreaks that include adult animals are increasingly recognized (Cunningham et al 2007 ;Cheng et al 2014 ). Moreover, Earl and Gray ( 2014 ) demonstrated that ranavirus-associated mortality of larvae or metamorphs was suffi cient to cause population declines in highly susceptible species.…”

“…1a, b ). Hemorrhages are most often present on the ventral surfaces near the vent, rear legs, and gular regions, but can also be observed around the eyes, ear drum, tongue, tail, and feet (Balseiro et al 2009 ;Cheng et al 2014 ;Cunningham et al 2007 ;Docherty et al 2003 ;Geng et al 2010 ;Kik et al 2011 ;Meng et al 2014 ;Sutton et al 2014a ). Raised skin plaques or polyps have been described in tiger salamanders ( Ambystoma tigrinum ) and Chinese giant salamanders (Jancovich et al 1997 ;Bollinger et al 1999 ;Geng et al 2010 ).…”

Distribution and Host Range of Ranaviruses

“…Common midwife toad virus (CMTV) was fi rst isolated from common midwife toad tadpoles experiencing a mass mortality event in 2007 in northern Spain (Balseiro et al 2009 ). A second mass mortality event during 2008 in the same region of Spain involving CMTV affected common midwife toad tadpoles and juvenile alpine newts ( Mesotriton alpestris ; Balseiro et al 2010 ).…”

“…There are three recognized species of ranaviruses that are known to infect amphibians: FV3, ATV, and BIV. Also, there are several other ranaviruses that have been isolated from amphibians (e.g., common midwife toad virus, CMTV; Balseiro et al 2009 ), but are not currently recognized as ranavirus species. As discussed in Jancovich et al ( 2015 ), declaring a given isolate as a unique viral species is complex, and compounded by the fact that there is considerable sequence conservation, often greater than 95 % at the amino acid level among many ranavirus isolates.…”

Ecophysiology meets conservation: understanding the role of disease in amphibian population declines

“…Currently, three amphibian ranaviruses have been identified: frog virus 3 (FV3), Bohle iridovirus (BIV) and Ambystoma tigrinum virus (ATV) [57]. Mortality events and/or infections involving viruses have been detected in amphibian population across the globe [58][59][60][61][62][63][64][65][66][67][68]. While ranavirus infections have been detected in both adult and larval amphibians, the virus appears to be most lethal in larvae [69].…”