Laboratory Maintenance and Culture of Pseudogymnoascus destructans, the Fungus That Causes Bat White‐Nose Syndrome

Abstract

Pseudogymnoascus destructans is a fungal pathogen that causes white-nose syndrome, an emerging and fatal disease of North American bats that has led to unprecedented population declines. As a psychrophile, P. destructans is adapted to infect bats during winter hibernation, when host metabolic activity and core body temperature are greatly reduced. The ability to maintain and cultivate isolates of P. destructans in the laboratory is necessary for conducting research with this fungus. This article describes protocols for culturing P. destructans from bat wing skin and soil, for cryopreserving the fungus, and for preparing liquid suspensions for laboratory experimentation.

Higher white-nose syndrome fungal isolate yields from UV-guided wing biopsies compared with skin swabs and optimal culture media

“…Understandably, the deeper the fungus invades the skin, the more difficult it becomes to detect, especially when non-invasive sampling techniques are used. In order to understand the pathology and progression of WNS in bats, samples are collected from bat cadavers or live hibernating bats using both non-invasive and invasive methods [42]. Use of non-lethal methods is especially important in the case of strictly protected European bat species and declining North American bat species, and is also advisable for bats being investigated for presence or absence of the WNS etiological agent in other temperate regions of the world [15,36,39].…”

“…A better alternative for the collection of samples for microbiological cultures may be skin biopsies, as the quantity of fungal elements within the tissue [45] allows the culture to continue growing from hyphae within the fungal biomass. While skin surface swabs probably contain higher amounts of conidia, it is still not known how the types of initial colony-forming units (i.e., conidia and/or hyphae) influence the likelihood of yielding a fungal isolate [42]. Other factors that may have impact on the culture yield include the intensity of infection, prevalence of WNS in the bat population under study, sample transport conditions, and growth characteristics, viability and persistence of the agent [11,36,42,44,[46][47][48][49].…”

“…A further problem is that overgrowth with competing microorganisms can frequently reduce the chances of producing a successful P. destructans culture. To address this, it is recommended to incubate the sample at temperatures between 7 to 10 °C in order to reduce the growth of other cultivable agents co-occurring in the sample [42]. Likewise, use of an antibiotic supplement (dosage and type may vary) in the culture media is highly recommended.…”

“…This observation may have been due to overall microbial overgrowth in both cases. In addition to physical competition for space, growth of microorganisms can also be limited by the temperature and length of incubation period, medium composition and aerobic conditions [42,[66][67][68].…”

White-Nose Syndrome Diagnostic Laboratory Network handbook

“…The WNS DxWG has taken several steps to address these challenges, starting with the establishment of a more formalized WNS Diagnostic Laboratory Network where standards for result interpretation are agreed upon by all members, and interlaboratory testing is done to maintain confidence in the assay's performance across a range of unique testing parameters (Blehert and Lorch, 2021). Second, the WNS case definition was updated to reflect the uncertainty inherent in testing samples at or ahead of the leading edge of the invasion front where pathogen loads are low and results may be more variable (Langwig and others, 2015b, c;Mosher and others, 2019;WNS DxWG, 2021).…”