Road-killed common toads (Bufo bufo) in Flanders (Belgium) reveal low prevalence of ranaviruses and Batrachochytrium dendrobatidis.

Road-killed common toads (Bufo bufo; n51,740) from Flanders, Belgium, were tested for ranavirus and Batrachochytrium dendrobatidis (Bd) using polymerase chain reaction. Both infections were present at a very low prevalence (,0.2% with a confidence interval of 95% for ranavirus and 0.63% for Bd). Healthy amphibians can be asymptomatic carriers of diseases responsible for devastating amphibian population declines. Most importantly, ranavirus and Batrachochytrium dendrobatidis (Bd) are primary pathogens of amphibians, and disease outbreaks are believed to be driven by environmental factors (Walker et al., 2010). A thorough risk analysis to predict future scenarios in indigenous amphibian species should include the identification of principle pathogen reservoirs. The difficulty in identifying amphibian, clinically healthy reservoirs for both agents is that asymptomatically infected animals often have low-grade infections. Although noninvasive sampling (e.g., skin swabs) enables the detection of both pathogens, tissue samples provide a higher detection probability. However, conservation concerns hamper tissue sampling of wild amphibians. Traffic kills many amphibians on roads yearly, especially during spring migration (Elzanowski et al., 2009). These road victims could provide a good opportunity for monitoring ranaviruses and Bd in native amphibians. In Flanders (Belgium), the common toad (Bufo bufo) provides the most obvious choice to monitor both infections for four reasons: Common toads are commonly killed by traffic during spring migration; migrating toads are counted on numerous sites in Flanders, enabling an estimate of population trends; the common toad is a suitable host for both ranaviruses and Bd (Bosch and Marinez-Solano, 2006; Cunningham et al., 2007); and toad populations have been found to be declining in several regions (Loras et al., 2011). Our goal was to assess ranavirus and Bd infections in common toads using tissues of road-killed specimens during the migration season. Between 27 February 2011 and 7 April 2011, 1,740 road-killed common toads were collected from 104 migration sites in all five Flemish provinces (Fig. 1). Each toad was placed in a plastic bag that was sealed and labeled. For detection of Bd DNA, a quantitative polymerase chain reaction (qPCR; Boyle et al., 2004) was performed on a skin sample of each toad. For detection of ranavirus infections, a liver sample was examined by PCR using the primers MCP 4 and MCP 5 (Mao et al., 1997). To assess whether the presence of one or both pathogens in a toad population coincides with obvious population declines, if available, numbers of migratory toads of 2011 and previous years were extracted using the database obtained from the working group Natuurpunt Hyla (Hyla Werkgroep, 2012). None of the samples were PCR-positive for ranaviruses. At nine sites (three in the province of East Flanders, three in Flemish Brabant, and three in Limburg), 11 Bd-positive toads were found (0.63%