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Diseases of Aquatic Organisms

    DAO prepress abstract   -  DOI: https://doi.org/10.3354/dao03621

    Extraction-free detection of amphibian pathogens from water baths

    Molly C. Bletz*, Brandon C. LaBumbard, Emily H. Le Sage, Douglas C. Woodhams

    *Corresponding author:

    ABSTRACT: Detecting and quantifying pathogens with quick, cost-efficient and sensitive methods is needed across disease systems for addressing pertinent epidemiological questions. Typical methods rely on extracting DNA from collected samples. Here we develop and test an extraction-free method from water bath samples that is both sensitive and efficient for two major amphibian pathogens – Batrachochytrium dendrobatidis and B. salamandrivorans. We tested both mock samples with known pathogen quantities as well as comparatively assessed detection from skin swabs and water baths from field sampled amphibians. Quantitative PCR directly on lyophilized water baths was able to reliably detect low loads of 10 and 1 zoospores, and detection rates were greater than that of swabs for field samples. Further concentration of samples did not improve detection and collection container type did not influence pathogen load estimates. This method of lyophilization (i.e. freeze-drying) followed by direct qPCR offers an effective and efficient tool for detecting amphibian pathogens which is crucial for surveillance efforts, and estimating shedding rates for robust epidemiological understanding of transmission dynamics. Furthermore, water bath samples have multiple functions and can be used to evaluate mucosal function against pathogens and characterize mucosal components. The multifunctionality of water bath samples and reduced monetary costs and time expenditures make this method an optimal tool for amphibian disease research and may also prove to be useful in other wildlife disease systems.