Development of DNA-based assays for the detection of Batrachochytrium dendrobatidis in environmental samples and amphibians

Abstract

Chytridiomycosis is an emerging infectious disease affecting amphibians on a pandemic scale. The disease causing organism, Batrachochytrium dendrobatidis, has been recorded on a global scale to be the top causative driver behind the enigmatic declines in frog populations and species. Certain limitations do exist in the detection of chytridiomycosis, such as invasive sampling methods, low detection sensitivity and protocols being time intensive. The aim of this study was to develop a DNA-based assay, Loop-mediated Isothermal Amplification (LAMP) and conventional PCR, for the detection of B. dendrobatidis. In vitro tests involving live B. dendrobatidis cultures were undertaken to develop the LAMP assay. From given results the assay delivered promising data. Six LAMP primers were designed specific to B. dendrobatidis, labelled as Bd3F3, Bd3B3, Bd3FIP, Bd3BIP, Bd3LF and Bd3LB. A LAMP assay was developed which tested positive for both BdGPL and BdCAPE lineages from South Africa. The assay proved to be extremely sensitive for these isolates, yielding positive results up to a high DNA serial dilution of 5fg in 40 minutes and 0.1 zoospores in less than an hour which was only subjected to boiling. An additional PCR assay was also developed from the outer primers of the LAMP primer set. This additional PCR assay proved to be more sensitive than previously developed PCR assay for B. dendrobatidis, detecting positive DNA samples at 0.05pg and 0.1 zoospores per ml. In vitro tests were undertaken to test the viability and integrity of the developed LAMP assay. Mont Aux Sources was used as a field test since the population of Amietia hymenopus inhabiting its rivers are known to be infected with chytridiomycosis. LAMP successfully detected chytridiomycosis on both ventral skin swabs and toe clippings from several individuals across four rivers. Additional archived African samples of various frog species were tested for chytridiomycosis and revealed for the first time that Botswana frog populations are infected with chytridiomycosis in the Okavango Delta. The developed LAMP assay is a promising and powerful tool which can be implemented to overcome several short-comings that can be associated with histology, PCR and qPCR. Furthermore, LAMP has much higher reducibility with crude DNA samples.