Refining ranavirus surveillance: evaluating non-lethal sampling of larval amphibians

Abstract Emerging infectious diseases threaten wildlife globally, yet standard surveillance often relies on lethal sampling, an approach that can conflict with conservation priorities. The recognised threat of Ranavirus ( Rv ) to amphibian populations exemplifies this challenge, highlighting the urgent need for refined, non-lethal surveillance methods. Using a stepwise approach, we conducted four sequential experiments spanning laboratory infections and wild populations to evaluate buccal swabbing as a non-lethal alternative for Rv detection in larval amphibians. We compared detection rates and viral loads among buccal swabs, oral, liver and tail tissues in larvae from five species across two orders (Anura and Caudata). Our work demonstrates that, although no method shows perfect detectability, buccal swabbing matched lethal liver samples for Rv detection and quantification. Swabs consistently outperformed tail tissues across all experimental contexts, which fell short, yielding false negatives in up to 50% of infected individuals. Lower total DNA from swabs did not reduce sensitivity for viral detection or quantification, with DNA quality deemed adequate for downstream applications. Validating buccal swabbing as a refined, non-lethal tool that aligns with the 3Rs principles reduces welfare impacts and offers broad transferability for pathogen surveillance in diverse wildlife and captive settings.