Development and application of a qPCR-based genotyping assay for Ophidiomyces ophidiicola to investigate the epidemiology of ophidiomycosis

By: , and 



Ophidiomycosis (snake fungal disease) is an infectious disease caused by the fungus Ophidiomyces ophidiicola to which all snake species appear to be susceptible. Significant variation has been observed in clinical presentation, progression of disease, and response to treatment, which may be due to genetic variation in the causative agent. Recent phylogenetic analysis based on whole-genome sequencing identified that Oophidiicola strains from the United States formed a clade distinct from European strains, and that multiple clonal lineages of the clade are present in the United States. The purpose of this study was to design a qPCR-based genotyping assay for Oophidiicola, then apply that assay to swab-extracted DNA samples to investigate whether the multiple Oophidiicola clades and clonal lineages in the United States have specific geographic, taxonomic, or temporal predilections. To this end, six full genome sequences of Oophidiicola representing different clades and clonal lineages were aligned to identify genomic areas shared between subsets of the isolates. Eleven hydrolysis-based Taqman primer-probe sets were designed to amplify selected gene segments and produce unique amplification patterns for each isolate, each with a limit of detection of 10 or fewer copies of the target sequence and an amplification efficiency of 90–110%. The qPCR-based approach was validated using samples from strains known to belong to specific clades and applied to swab-extracted Oophidiicola DNA samples from multiple snake species, states, and years. When compared to full-genome sequencing, the qPCR-based genotyping assay assigned 75% of samples to the same major clade (Cohen’s kappa = 0.360, 95% Confidence Interval = 0.154–0.567) with 67–77% sensitivity and 88–100% specificity, depending on clade/clonal lineage. Swab-extracted Oophidiicola DNA samples from across the United States were assigned to six different clonal lineages, including four of the six established lineages and two newly defined groups, which likely represent recombinant strains of Oophidiicola. Using multinomial logistic regression modeling to predict clade based on snake taxonomic group, state of origin, and year of collection, state was the most significant predictor of clonal lineage. Furthermore, clonal lineage was not associated with disease severity in the most intensely sampled species, the Lake Erie watersnake (Nerodia sipedon insularum). Overall, this assay represents a rapid, cost-effective genotyping method for Oophidiicola that can be used to better understand the epidemiology of ophidiomycosis.

Publication type Article
Publication Subtype Journal Article
Title Development and application of a qPCR-based genotyping assay for Ophidiomyces ophidiicola to investigate the epidemiology of ophidiomycosis
Series title PLoS ONE
DOI 10.1371/journal.pone.0289159
Volume 18
Issue 8
Year Published 2023
Language English
Publisher PLOS
Contributing office(s) National Wildlife Health Center
Description e0289159, 24 p.
Google Analytic Metrics Metrics page
Additional publication details