Non-invasive sampling methods for the detection of Batrachochytrium dendrobatidis in archived amphibians

Abstract

Chytridiomycosis, an emerging infectious disease of amphibians caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), is associated with amphibian population declines worldwide. Investigation of the origin and spread of the pathogen requires examination of archived museum specimens of amphibians. Examination for Bd infection is usually done using histological techniques, which are often too destructive for valuable museum material. Three alternative methods for Bd detection (skin swabbing, brushing and scraping) were evaluated for ability to yield Bd DNA and destructiveness to specimens. Archived amphibians known to be Bd positive and which had been preserved in either formalin or ethanol for many years were used. Samples were analysed using a Bd-specific quantitative real-time Taqman PCR (qPCR) assay. There was no difference in the ability of each of the techniques to detect Bd infection, with the pathogen being detected in 75 to 81% of the 16 ethanol-fixed frogs examined. Visible evidence of sampling was left by scraping, but not by swabbing or brushing. The brush-qPCR technique detected higher counts of genomic equivalents than the other 2 sampling methods, although differences were not statistically significant. The qPCR assay did not detect Bd from any of the 6 formalin-fixed frogs examined, regardless of the sampling method. Nondestructive sampling techniques enable qPCR analysis of ethanol-preserved museum specimens for Bd. Recently, the incorporation of DNA cleanup steps allowed the detection of Bd in destructively sampled tissues from formalin preserved specimens. Further studies using nondestructive sampling incorporating DNA cleanup steps for the detection of Bd in formalin preserved specimens are warranted.