Prevalence and pathogen load estimates for the fungus Batrachochytrium dendrobatidis are impacted by ITS DNA copy number variation.

The ribosomal gene complex is a multi-copy region that is widely used for phylogenetic analyses of organisms from all 3 domains of life. In fungi, the copy number of the internal transcribed spacer (ITS) is used to detect abundance of pathogens causing diseases such as chytridiomycosis in amphibians and white nose syndrome in bats. Chytridiomycosis is caused by the fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), and is responsible for declines and extinctions of amphibians worldwide. Over a decade ago, a qPCR assay was developed to determine Bd prevalence and pathogen load. Here, we demonstrate the effect that ITS copy number variation in Bd strains can have on the estimation of prevalence and pathogen load. We used data sets from different amphibian species to simulate how ITS copy number affects prevalence and pathogen load. In addition, we tested 2 methods (gBlocks® synthetic standards and digital PCR) to determine ITS copy number in Bd strains. Our results show that assumptions about the ITS copy number can lead to under- or overestimation of Bd prevalence and pathogen load. The use of synthetic standards replicated previously published estimates of ITS copy number, whereas dPCR resulted in estimates that were consistently lower than previously published estimates. Standardizing methods will assist with comparison across studies and produce reliable estimates of prevalence and pathogen load in the wild, while using the same Bd strain for exposure experiments and zoospore standards in qPCR remains the best method for estimating parameters used in epidemiological studies.