A Universal and Efficient Detection of Chytridiomycosis Infections in Amphibians Using Novel Quantitative PCR Markers

Abstract

Chytridiomycosis is an infectious disease in amphibians caused by two chytrid fungi, Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal), and is the worst infectious disease known in wildlife so far. Worldwide spread of the disease has caused unprecedented loss of global amphibian diversity. Although some lineages of Bd are enzootic and are not as deadly as the pandemic lineage, nearly 40% of amphibian species are still declining globally due to chytridiomycosis. Efcient surveillance and monitoring of chytridiomycosis are the immediate safeguard against rapid declines or extinctions of amphibian populations. Previous studies showed that existing diagnostic assays were not sensitive to certain Bd haplotypes like those from Korea, China, India, Japan, and Brazil and thereby, there is a need for a universal, sensitive, specifc, reproducible, and afordable diagnostic assay. We designed a one-step SYBR green-based quantitative polymerase chain reaction (nSYBR qPCR) for robust detection of Bd. It amplifes an 82 base-pair segment between the 5.8S rRNA and ITS2 of the Bd genome. Te primer pair was tested in-silico on 40 isolates from four known Bd lineages. Using skin swab samples of wild amphibians and cultured zoospores from Australia and Panama, we compared the clinical specifcity and sensitivity of the newly described primers to the existing TaqMan-based qPCR assay. From India, we used samples which had been previously tested with Nested PCR to validate the new primer pairs. Te newly described primer pair was then tested on swab samples from Anura, Caudata and Gymnophiona from India. We report widespread chytridiomycosis with varying infection loads on them. Te new assay showed comparable efciency to the TaqMan-based qPCR assay. Tis diagnostic assay can facilitate widespread surveillance of chytridiomycosis where it has been previously absent, which may reveal several reservoirs of the pathogen and can improve our understanding of this important wildlife disease.