Storage of samples at high temperatures reduces the amount of amphibian chytrid fungus Batrachochytrium dendrobatidis DNA detectable by PCR assay

Chytridiomycosis, caused by the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), is an emerging infectious disease responsible for amphibian declines on several continents. In laboratory conditions, optimal temperatures for Bd growth and survivorship are between 17 and 25 degrees C. We investigated the effect of different storage temperatures, both in field and laboratory conditions, on detection of Bd from swabs stored for 7 d. We sampled 52 wild Litoria wilcoxii males for Bd by simultaneously running 2 cotton swabs along the skin of the frog. One group of swabs was stored in a freezer within 2 h of sampling and the other was kept in a car in an exposed environment for 7 d before being stored in the freezer. In the laboratory experiment, swabs were inoculated with zoospores of Bd and underwent one of 4 treatments: immediate DNA extraction, or storage at 27, 38 or 45 degrees C for 7 d prior to DNA extraction. Swabs from all treatments were analyzed by quantitative (real-time) PCR test. Though prevalence of Bd did not differ significantly between swabs that were frozen and those that remained in a car for 7 d (19.2 vs. 17.3%, respectively), the number of Bd zoospores detected on car swabs taken from infected frogs was, on average, 67% less than that detected on the corresponding frozen swab. In the laboratory experiment, the number of zoospore equivalents varied significantly with treatment (F(3,35) = 4.769, p = 0.007), indicating that there was reduced recovery of Bd DNA from swabs stored at higher temperatures compared with those stored at lower temperatures or processed immediately. We conclude that failure to store swabs in cool conditions can result in a significant reduction in the amount of Bd DNA detected using the PCR assay. Our results have important implications for researchers conducting field sampling of amphibians for Bd.