Investigation of the mycobiome and emerging fungal pathogens in Northern Arizona
Black-footed ferrets, Mustela nigripes, are highly endangered predators in the Western United States. Black-footed ferrets were believed to be extinct until a colony was found in 1980. This colony was used as the founder population for a reintroduction program. One specific reintroduction location was Aubrey Valley in Northern Arizona, and this colony appeared to be successful until the ferret population crashed in 2015, and the cause was proposed to be a pathogen. Prairie dogs are the main prey for ferrets and also provide the burrows that ferrets occupy. Prairie dog fleas were tested for plague and were found to be negative. Because no ferret carcasses could be found, Arizona Game and Fish harvested twenty prairie dogs and examined their lungs. Histopathology of the prairie dog lungs revealed fungal structures that were consistent with coccidioidomycosis or adiaspiromycosis. To identify the fungi present, DNA was extracted from four quadrants of each lung. The extracted DNA was subjected to CocciDx (measures Cocci DNA) and Fungiquant (measured fungal DNA) qPCR, followed by ITS2 amplicon sequencing to define the fungal communities present in the lungs. Other remaining sections of the lungs were subsequently cultured for 10 days to attempt to grow and isolate pure fungal colonies, and DNA was then extracted from the purified colonies. Fungal species were identified using Sanger DNA sequencing, and the sequences were queried on the NCBI database. CocciDx revealed no Coccidioides DNA; however, the result of the ITS2 sequencing suggested that Coccidioides DNA was present. From Sanger sequencing, many different species of Onygenales fungi were identified as well as uncultured fungi, but no Coccidioides species. This research is important because fungal pathogens may be the underlying cause of the population decrease of endangered black-footed ferrets. We hypothesize that uncharacterized fungal pathogens are found in the ferret-prairie dog ecosystem, and may reflect selection for a mycobiome that supports the evolution of novel emerging fungal pathogens that could impact human health in the future.