Description

Batrachochytrium dendrobatidis is a species of chytrid fungus responsible for the decline and extinction of amphibian populations worldwide (Longcore 1999 and others). Recently, B. dendrobatidis has been detected on western toads (Bufo boreas) in southwestern British Columbia (Deguise & Richardson 2009) and has therefore become a concern for conservationists across the province. Chytrid DNA can be extracted from tissue samples, skin swabs, and water bodies and subsequently detected using real time PCR techniques. It is currently unknown whether the chytrid fungus is affecting amphibian populations in the Kamloops area. This project aimed to first validate an effective real time PCR assay for detecting the presence of the amphibian pathogen B. dendrobatidis, and second to employ this real time PCR assay to detect B. dendrobatidis on experimental samples collected in the Kamloops region.
Specifically, spadefoots were captured during the summer of 2013 from the New Afton mining site located south of Kamloops, BC. A total of 78 spadefoots were captured, swabbed, and subsequently weighed, measured and released. Parameters for optimizing an effective real time PCR assay were tested to detect B. dendrobatidis in experimental samples; initially a Taqman assay was tried, but due to a lack of success, a SYBR Green assay was used. DNA was extracted from all samples and 30 of these samples were tested for B. dendrobatidis using the SYBR Green assay and gel electrophoresis.
B. dendrobatidis DNA was not detected in any of the 30 experimental samples. Melt curve analysis indicated the presence of multiple PCR products, products which were observed by gel electrophoresis at lengths less than 150 base pairs. It is possible that non-specific primer binding occurred, or that primer dimers formed, at low template DNA concentrations as these bands were not observed above 42 standard DNA copy numbers. Based on the results in hand, it is possible that (1) chytrid is not present in the ponds at New Afton mine, (2) chytrid is present but is not affecting the spadefoots, (3) the sensitivity of the SYBR Green assay was not sufficient to detect chytrid DNA, (4) PCR inhibitors interfered with the detection of chytrid DNA on samples, and (5) chytrid DNA did not adhere to the swabs that were used in this work. Future research should employ swabs with smaller tips and employ the more sensitive Taqman assay using a probe with a non-flourescent quencher (NFQ) as opposed to a black-hole quencher (BHQ-1) that was used here.