ITS1 Copy Number Varies among Batrachochytrium dendrobatidis Strains: Implications for qPCR Estimates of Infection Intensity from Field-Collected Amphibian Skin Swabs

Ana V Longo,Patricia A Burrowes,Domingos Da Silva Leite,David Rodriguez,Kelly R Zamudio,Luís Felipe Toledo,Cinthya Mendoza Almeralla,Tom Coenye

doi:10.1371/journal.pone.0059499

Abstract

Genomic studies of the amphibian-killing fungus (Batrachochytrium dendrobatidis, [Bd]) identified three highly divergent genetic lineages, only one of which has a global distribution. Bd strains within these linages show variable genomic content due to differential loss of heterozygosity and recombination. The current quantitative polymerase chain reaction (qPCR) protocol to detect the fungus from amphibian skin swabs targets the intergenic transcribed spacer 1 (ITS1) region using a TaqMan fluorescent probe specific to Bd. We investigated the consequences of genomic differences in the quantification of ITS1 from eight distinct Bd strains, including representatives from North America, South America, the Caribbean, and Australia. To test for potential differences in amplification, we compared qPCR standards made from Bd zoospore counts for each strain, and showed that they differ significantly in amplification rates. To test potential mechanisms leading to strain differences in qPCR reaction parameters (slope and y-intercept), we: a) compared standard curves from the same strains made from extracted Bd genomic DNA in equimolar solutions, b) quantified the number of ITS1 copies per zoospore using a standard curve made from PCR-amplicons of the ITS1 region, and c) cloned and sequenced PCR-amplified ITS1 regions from these same strains to verify the presence of the probe site in all haplotypes. We found high strain variability in ITS1 copy number, ranging from 10 to 144 copies per single zoospore. Our results indicate that genome size might explain strain differences in ITS1 copy number, but not ITS1 sequence variation because the probe-binding site and primers were conserved across all haplotypes. For standards constructed from uncharacterized Bd strains, we recommend the use of single ITS1 PCR-amplicons as the absolute standard in conjunction with current quantitative assays to inform on copy number variation and provide universal estimates of pathogen zoospore loads from field-caught amphibians.

Highlights

Advances in quantitative polymerase chain reaction protocols and their application in detection and quantification of pathogens have contributed significantly to our understanding of disease dynamics in natural host populations [1,2]
We found a significantly negative relationship between the number of zoospores and cycle threshold number, Ct (B = 23.31, T = 244.93, P,0.000, Fig. 2)
Our results show that using zoospores counts (Standard Set A) to establish quantitative polymerase chain reaction (qPCR) standard curves can be problematic because Bd strains vary in their ITS1 copy number (Fig. 2)

Summary

Introduction

Advances in quantitative polymerase chain reaction (qPCR) protocols and their application in detection and quantification of pathogens have contributed significantly to our understanding of disease dynamics in natural host populations [1,2]. Recent genomic characterization of 20 global Bd strains indicates that Bd is composed of at least three divergent genetic lineages that differ in virulence [8] One of these lineages, the global panzootic lineage (GPL) is hypervirulent and has been implicated in the recent epizootics [8]. A novel Bd strain recently discovered in Brazil differs in DNA content compared to GPL strains from Panama and California [9] If these deeply-divergent strains carry polymorphisms at the primer or probe binding sites or if target ITS1 genes vary in copy number, qPCR efficiency and sensitivity among strains may vary [1,10], which will reduce the comparability of qPCR infection intensity estimates across sites

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Conclusion