TaqMan real-time polymerase chain reaction for detection of Ophidiomyces ophiodiicola, the fungus associated with snake fungal disease

Abstract

Background: Fungal skin infections associated with Ophidiomyces ophiodiicola, a member of the Chrysosporium anamorph of Nannizziopsis vriesii (CANV) complex, have been linked to an increasing number of cases of snake fungal disease (SFD) in captive snakes around the world and in wild snake populations in eastern North America. The emergence of SFD in both captive and wild situations has led to an increased need for tools to better diagnose and study the disease.

Results: We developed two TaqMan real-time polymerase chain reaction (PCR) assays to rapidly detect O. ophiodiicola in clinical samples. One assay targets the internal transcribed spacer region (ITS) of the fungal genome while the other targets the more variable intergenic spacer region (IGS). The PCR assays were qualified using skin samples collected from 50 snakes for which O. ophiodiicola had been previously detected by culture, 20 snakes with gross skin lesions suggestive of SFD but which were culture-negative for O. ophiodiicola, and 16 snakes with no clinical signs of infection. Both assays performed equivalently and proved to be more sensitive than traditional culture methods, detecting O. ophiodiicola in 98% of the culture-positive samples and in 40% of the culture-negative snakes that had clinical signs of SFD. In addition, the assays did not cross-react with a panel of 28 fungal species that are closely related to O. ophiodiicola or that commonly occur on the skin of snakes. The assays did, however, indicate that some asymptomatic snakes (~6%) may harbor low levels of the fungus, and that PCR should be paired with histology when a definitive diagnosis is required.

Conclusions: These assays represent the first published methods to detect O. ophiodiicola by real-time PCR. The ITS assay has great utility for assisting with SFD diagnoses whereas the IGS assay offers a valuable tool for research-based applications.

Figure 1

Diagram of fungal ribosomal RNA regions and areas targeted for Ophidiomyces ophiodiicola-specific real-time PCR assays. One assay targets the 3′-end of the internal transcribed spacer region 2 (ITS-2) while the second assay targets the 3′-end of the intergenic spacer region (IGS). Expanded sequences (representing the type isolate of O. ophiodiicola) show the amplicons produced by each PCR and the primer and probe binding sites. Shaded boxes within the sequences represent nucleotide positions that vary between strains of O. ophiodiicola.

Figure 2

Standard curves for Ophidiomyces ophiodiicola real-time PCR assays. Genomic DNA (gDNA) isolated from a pure culture of the type isolate of O. ophiodiicola was quantified, serially diluted, and used as template. All samples were run in triplicate with all points depicting mean C_t values and error bars representing standard deviation. The assays targeting (A) the internal transcribed spacer region (ITS assay) and (B) intergenic spacer region (IGS assay) of O. ophiodiicola were both linear over seven logs, ranging from 5 fg to 5 ng template gDNA (ITS assay: R² = 0.998; IGS assay: R² = 0.997).