Detection of Batrachochytrium dendrobatidis in Eleutherodactylus fitzingeri: effects of skin sample location and histologic stain.

Batrachochytrium dendrobatidis is a fungal pathogen that has been implicated in amphibian declines worldwide. Histopathologic techniques have been used to diagnose the disease, but their sensitivity has not been determined. It is also unclear whether the probability of detection varies between skin samples derived from different body parts. We examined 24 Fitzinger's rainfrogs (Eleutherodactylus fitzingeri) with chytridiomycosis. This is a common frog species with a broad range and high abundance throughout most of Costa Rica. We sampled 12 different body parts from each animal, and alternated the staining between a routinely used stain (hematoxylin and eosin [H&E]), and a more fungus-specific stain (periodic acid-Schiff [PAS]). The pelvic patch and the innermost finger of the hand were consistently the best places to detect the disease, although significant differences were found only with the gular area, the abdomen, and toes four and five. We found more positive samples using PAS than using H and E in all body parts, although significant differences were detected only in samples derived from the pelvic patch. Using the best combination of factors (stain and body part) and animals with the lightest infections (to test the sensitivity of the technique), we calculated that at least 17 sections are needed in order to reach 95% confidence that a frog is or is not infected. We conclude that the choice of stain and body part can significantly alter estimates of prevalence of B. dendrobatidis.