Abstract

Batrachochytrium salamandrivorans (Bsal) is a chytrid fungus that infects amphibians, causing skin lesions and eventually death. To date, it has been thought that Bsal only leads to asymptomatic carrier states in frog species and does not cause disease as it does in susceptible salamander species. Therefore, we hypothesized that anurans would not develop disease when challenged with concentrations of Bsal that result in disease in salamanders. To test this hypothesis, we exposed invasive Cuban treefrogs (Osteopilus septentrionalis) to either a mock inoculation of autoclaved dechlorinated water or one of four concentrations of Bsal zoospores: 5x10³, 5x10⁴, 5x10⁵, or 5x10⁶ via water bath (n=4 per zoospore dose). These concentrations were chosen based on our experience in testing 23 species of salamanders with varying ranges of susceptibility and successfully eliciting disease. Frogs were swabbed every six days during water-change events to document the onset and progression of infection. Frogs exposed to 5x10⁶ zoospores tested positive for Bsal using qPCR starting four days post-exposure. Beginning two weeks post exposure, these frogs began showing signs consistent with Bsal chytridiomycosis, including darkened pigmentation on the dorsum, which progressed to red spots that appeared to be microhemorrhages (petechia). Infected animals also developed erythema and hemorrhage on the feet and ventral aspect of the abdomen, as well as excessive shedding on the feet. One frog in the 5x10⁶ group was euthanized and two died naturally. Histopathology was performed on all three cases; multiple erosive crater-like epidermal lesions were randomly distributed throughout the body and varied from superficial to full thickness epidermis. Lesions consisted of focal epidermal necrosis with numerous intralesional Bsal thalli that were evident when stained with H&E. This challenge experiment represents the first documentation of Bsal chytridiomycosis in an anuran species and emphasizes that the conservation threat of Bsal may extend beyond salamander species.

Acknowledgements

The authors thank Peter Iacono, Eric Suarez, and Brooke Talley of the Florida Fish and Wildlife Conservation Commission for collecting the Cuban treefrogs used in this study. The University of Tennessee (UT) and University of Florida histology laboratories assisted with slide preparations, and Melissa Brown helped with in situ hybridization. We thank Bobby Simpson and Alex Anderson of the UT Institute of Agriculture East Tennessee Research and Education Center for laboratory and logistic support. We also thank Bailee Augustino for assistance with animal care and data collection.