A Dna-Based Assay Identifies Batrachochytrium Dendrobatidis in Amphibians

Abstract

Chytridiomycosis caused by Batrachochytrium dendrobatidis (Chytridiomycota) has been implicated in declines of amphibian populations on four continents. We have developed a sensitive and specific polymerase chain reaction-based assay to detect this pathogen. We isolated B. dendrobatidis from captive and wild amphibians collected across North America and sequenced the internal transcribed spacer regions of the rDNA cassette of multiple isolates. We identified two primers (Bd1a and Bd2a) that are specific to B. dendrobatidis under amplification conditions described in this study. DNA amplification with Bd1a/Bd2a primers produced a fragment of approximately 300 bp from B. dendrobatidis DNA but not from DNA of other species of chytrids or common soil fungi. The assay detected 10 zoospores or 10 pg of DNA from B. dendrobatidis and detected infections in skin samples from a tiger salamander (Ambystoma tigrinum), boreal toads (Bufo boreas), Wyoming toads (Bufo baxteri), and smooth-sided toads (Bufo guttatus). This assay required only small samples of skin and can be used to process a large number of samples.

Health and Habitat Quality Assessment for the Eastern Hellbender (Cryptobranchus Alleganiensis Alleganiensis) in Indiana, Usa

“…Cotton swabs were used to sample the ventrum, feet, and mouth for amphibian chytrid zoospores following the protocol by Briggs and Vredenburg (2007). Swab samples were stored in 95% ETOH until submission to Pisces Molecular LLC (Boulder, Colorado, USA) for quantitative polymerase chain reaction (qPCR) analysis (Annis et al, 2004).…”

Experimental Infection of Native North Carolina Salamanders With Batrachochytrium Dendrobatidis

“…Inoculum preparation and high-dose infection in P. glutinosus and D. orestes A B. dendrobatidis inoculum was made by growing strain BD 197 (Annis et al, 2004) on tryptone agar. When zoospores were abundant, plates were flooded with a small amount of sterile water, and the zoospores (and a few contaminating thalli) were transferred to a 15-ml, conical centrifuge tube.…”

Pre-emptive National Monitoring Plan for Detecting the Amphibian Chytrid Fungus in Madagascar

“…An agarose-based PCR assay will be used to analyze the swab samples for the presence of Bd-DNA (Annis et al 2004), and Proteinase K digestion (10 mg/ml concentration) followed by a standard salt extraction protocol (Bruford et al 1992) will be used to extract total genomic DNA. Presence of the pathogen will be detected using Bdspecific primers: Bd1a (5 0 -CAGTGTGCCATATGTCACG-3 0 ) and Bd2a (5 0 -CATGGTTCATATCTGTCCAG-3 0 ) (Annis , designed from the ITS1 and ITS2 regions, respectively.…”