Year: 2026
Type: article
Abstract: The amphibian disease chytridiomycosis, caused by the waterborne chytrid fungus, Batrachochytrium dendrobatidis (Bd), has been linked to severe declines in anuran species and populations globally. Tadpoles can carry low-intensity, nonlethal Bd infections and thus could play an important role in maintaining this pathogen within ecosystems by serving as disease reservoirs. However, Bd can be challenging to detect at low levels; therefore, comprehensive, standardized methods are needed to accurately detect and diagnose infections at the tadpole life stage. Although traditional visual techniques are still employed, quantitative polymerase chain reaction (qPCR) assays have become the most common method used to identify and quantify Bd loads by amplifying DNA, and samples are typically analyzed in triplicate. Yet, studies conducting qPCR assays often utilize different sampling techniques, replicate numbers, or diagnostic criteria. This study investigates the importance of conducting triplicate qPCR assays in the detection and diagnosis of low-intensity Bd infections in tadpoles. We evaluated the relationship between Bd loads and the number of wells that test positive and assessed redetection rates by analyzing samples in two separate runs, in which each sample was analyzed in triplicate. We found that most tadpoles harbored low pathogen loads, which were primarily detected in just one out of three replicates and were rarely detected in both runs. Conversely, tadpoles with high pathogen loads yielded three positive wells and were consistently redetected. Next, we compared prevalence outcomes based on whether at least one or at least two positive wells were required for a positive diagnosis and calculated the proportion of samples misclassified as negative when single positive wells were excluded. Our results suggest that all samples yielding one or more positive wells should be considered Bd-positive and that including at least three replicates is necessary to capture low-intensity infections. Using this approach, we identified three potential reservoir species out of the seven species investigated. Bd loads differed significantly among species as well as between early and late development stage tadpoles. We recommend that future field and experimental studies assessing Bd infection in tadpoles apply these criteria to ensure that individuals, species, and life stages with low pathogen burdens are included in disease prevalence assessments.
Source: Herpetologica
Institutions: McGill University, Smithsonian Tropical Research Institute
Language: English
Domain: Life Sciences
Open Access status: hybrid
Funders: Smithsonian Institution, McGill University, Secretaría Nacional de Ciencia, Tecnología e Innovación, Smithsonian Conservation Biology Institute, Smithsonian Tropical Research Institute, Natural Sciences and Engineering Research Council of Canada, Fonds de recherche du Québec – Nature et technologies
OpenAlex ID: W7204106968