Determinants of Pseudogymnoascus destructans within bat hibernacula: implications for surveillance and management of white-nose syndrome.

1. Fungal diseases are an emerging global problem affecting human health, food security and biodiversity. Ability of many fungal pathogens to persist within environmental reservoirs can increase extinction risks for host species and presents challenges for disease control. Understanding factors that regulate pathogen spread and persistence in these reservoirs is critical for effective disease management. 2. White-nose syndrome (WNS) is a disease of hibernating bats caused by Pseudogymnoascus destructans (Pd), a fungus that establishes persistent environmental reservoirs within bat hibernacula, which contribute to seasonal disease transmission dynamics in bats. However, host and environmental factors influencing distribution of Pd within these reservoirs are unknown. 3. We used model selection on longitudinally collected field data to test multiple hypotheses describing presence-absence and abundance of Pd in environmental substrates and on bats within hibernacula at different stages of WNS. 4. First detection of Pd in the environment lagged up to one year after first detection on bats within that hibernaculum. Once detected, the probability of detecting Pd within environmental samples from a hibernaculum increased over time and was higher in sediment compared to wall surfaces. Temperature had marginal effects on the distribution of Pd. For bats, prevalence and abundance of Pd were highest on Myotis lucifugus and on bats with visible signs of WNS. 5. Synthesis and applications. Our results indicate that distribution of Pseudogymnoascus destructans (Pd) within a hibernaculum is driven primarily by bats with delayed establishment of environmental reservoirs. Thus, collection of samples from Myotis lucifugus, or from sediment if bats cannot be sampled, should be prioritized to improve detection probabilities for Pd surveillance. Long-term persistence of Pd in sediment suggests that disease management for white-nose syndrome should address risks of sustained transmission from environmental reservoirs.

DISPERSAL HAZARDS OF PSEUDOGYMNOASCUS DESTRUCTANS BY BATS AND HUMAN ACTIVITY AT HIBERNACULA IN SUMMER.

Bats occupying hibernacula during summer are exposed to Pseudogymnoascus destructans (Pd), the causative agent of white-nose syndrome (WNS), and may contribute to its dispersal. Furthermore, equipment and clothing exposed to cave environments are a potential source for human-assisted spread of Pd. To explore dispersal hazards for Pd during the nonhibernal season, we tested samples that were collected from bats, the environment, and equipment at hibernacula in the eastern US between 18 July-22 August 2012. Study sites included six hibernacula known to harbor bats with Pd with varying winter-count impacts from WNS and two hibernacula (control sites) without prior history of WNS. Nucleic acid from Pd was detected from wing-skin swabs or guano from 40 of 617 bats (7% prevalence), including males and females of five species at five sites where WNS had previously been confirmed as well as from one control site. Analysis of guano collected during summer demonstrated a higher apparent prevalence of Pd among bats (17%, 37/223) than did analysis of wing-skin swabs (1%, 4/617). Viable Pd cultured from wing skin (2%, 1/56) and low recapture rates at all sites suggested bats harboring Pd during summer could contribute to pathogen dispersal. Additionally, Pd DNA was detected on clothing and trapping equipment used inside and near hibernacula, and Pd was detected in sediment more readily than in swabs of hibernaculum walls. Statistically significant differences in environmental abundance of Pd were not detected among sites, but prevalence of Pd differed between sites and among bat species. Overall, bats using hibernacula in summer can harbor Pd on their skin and in their guano, and demonstration of Pd on clothing, traps, and other equipment used at hibernacula during summertime within the WNS-affected region indicates risk for pathogen dispersal during the nonhibernal season.

Optimized methods for total nucleic acid extraction and quantification of the bat white-nose syndrome fungus, Pseudogymnoascus destructans, from swab and environmental samples.

The continued spread of white-nose syndrome and its impacts on hibernating bat populations across North America has prompted nationwide surveillance efforts and the need for high-throughput, noninvasive diagnostic tools. Quantitative real-time polymerase chain reaction (qPCR) analysis has been increasingly used for detection of the causative fungus, Pseudogymnoascus destructans, in both bat- and environment-associated samples and provides a tool for quantification of fungal DNA useful for research and monitoring purposes. However, precise quantification of nucleic acid from P. destructans is dependent on effective and standardized methods for extracting nucleic acid from various relevant sample types. We describe optimized methodologies for extracting fungal nucleic acids from sediment, guano, and swab-based samples using commercial kits together with a combination of chemical, enzymatic, and mechanical modifications. Additionally, we define modifications to a previously published intergenic spacer-based qPCR test for P. destructans to refine quantification capabilities of this assay.