# Preliminary Screening for Ophidiomyces ophidiicola in Pet Snakes from Italy and Exploratory Evaluation of Droplet Digital PCR Assay

## TL;DR

This study tested pet snakes in Italy for a fungal disease and evaluated a new PCR method for detecting low levels of the pathogen.

## Contribution

The study introduces an exploratory evaluation of ddPCR for low-template diagnostics of Ophidiomyces ophidiicola in pet snakes.

## Key findings

- No Ophidiomyces ophidiicola was detected in 97 pet snakes from 31 species across Italy.
- Both PCR methods showed consistent amplification up to a 1:1000 dilution.
- ddPCR detected positive partitions at higher dilutions (up to 1:8000) compared to real-time PCR.

## Abstract

Ophidiomyces ophidiicola, the agent of ophidiomycosis, has recently been reported in wild snakes in Italy, but the status of captive populations remains unknown. We carried out an opportunistic survey of pet snakes from private collections and, in parallel, performed an exploratory evaluation of a droplet digital PCR (ddPCR) assay adapted from an established probe-based real-time PCR. Non-invasive skin swabs were collected by 32 private owners from 97 snakes, representing 31 species across ten Italian regions. All swabs tested negative for O. ophidiicola by both methods, including samples from four snakes that showed cutaneous lesions at the time of sampling. Both assays yielded consistent amplification up to the 1:1000 dilution (ddPCR 0.38 to 0.94 copies/µL for the culture-derived control and 0.24 to 0.33 copies/µL for the field-derived control at 1:1000), while ddPCR retained positive partitions in some replicates at higher dilutions (up to 1:8000). These results provide preliminary screening data for O. ophidiicola in an opportunistic sample of Italian pet snakes and suggest potential applicability of ddPCR as a complementary tool for low-template diagnostics, while highlighting the need for larger, standardised surveys and formal assay validation.