Pseudogymnoascus destructans, the causal agent of white-nose syndrome (WNS), is commonly found on bats captured both inside and outside caves during hibernation, a time when bats are most vulnerable to infection. It has not been documented in the southeast US on bats captured outside caves or on the landscape in summer. We collected 136 skin swabs from 10 species of bats captured at 20 sites on the Tennessee side of Great Smoky Mountains National Park, 12 May–16 August 2015. Three swabs were found positive for P. destructans, one from a male tricolored bat (Perimyotis subflavus) and two from male big brown bats (Eptesicus fuscus). This detection of P. destructans on free-flying male bats in the southeast US during summer has potential repercussions for the spread of the fungus to novel bat species and environments. Our finding emphasizes the need to maintain rigorous year-round decontamination of field clothing and equipment until more is understood about the viability of P. destructans found on bats captured outside hibernacula during summer, about the potential for males to act as reservoirs of the fungus, and the risk of fungal transmission and spread.

Since its discovery in New York in the winter of 2006–07, white-nose syndrome (WNS) has caused devastating declines in cave-hibernating bat populations across eastern North America (Frick et al. 2010; Langwig et al. 2012). The disease is known to affect seven bat species of which the tricolored bat (Perimyotis subflavus), northern long-eared myotis (Myotis septentrionalis), little brown myotis (Myotis lucifugus), and Indiana myotis (Myotis sodalis) have experienced the greatest rates of mortality, with the latter three being at risk of regional extinction (Frick et al. 2010; Langwig et al. 2012). An additional five species carry Pseudogymnoascus destructans, the fungal pathogen that causes WNS (Minnis and Lindner 2013), but have yet to develop the disease (Bernard et al. 2015).

White-nose syndrome affects torpid bats during hibernation. Pseudogymnoascus destructans is psychrophilic, growing at temperatures of approximately 0–20 C, with optimal growth at 12.5–15.8 C, within the temperature range of many winter cave hibernacula and of bats during winter torpor (Langwig et al. 2012; Verant et al. 2012). The pathogen initially colonizes and invades bats' epidermal tissues and, later, deeper dermal tissues, resulting in erosion and ulceration. The tissue damage, in combination with associated disruption of homeostatic processes, alteration of normal hibernation behavior, and reduction in energy reserves is frequently fatal (Verant et al. 2014). However, if individuals survive until spring emergence, they can clear the infection when they become metabolically active and their body temperature, and the ambient temperature, exceeds 19.8 C, the upper limit for fungal growth (Fuller et al. 2011; Verant et. al 2014; Langwig et al. 2015).

The presence and growth of P. destructans on substrates and in sediments of infected hibernacula have been confirmed, even in the absence of hibernating bats (Raudabaugh and Miller 2013; Reynolds et al. 2015). Most bat species abandon caves during summer (Harvey et al. 2011) but individuals frequently re-enter caves for short periods (Hall and Brenner 1968; Briggler and Prather 2003), providing the opportunity for contact with P. destructans within the cave. While the manifestation of WNS may be impeded by increased temperatures and metabolic activity during summer (Verant et al. 2014; Langwig et al. 2015), the fungus could still be transmitted to and between bats. In the northeast US, P. destructans has been documented during summer on female bats occupying maternity caves (Langwig et al. 2015) and emerging from a maternity colony in a bat house (Dobony et al. 2011). There have been no documented occurrences of free-flying bats positive for P. destructans captured on the landscape during the summer (May–August) in the southeast US. The southeast US is far in distance and climate from where WNS effects have been more extensively studied and where a greater understanding of the ability of the fungus to infect bats could help us understand the effects of geographic variation on the disease. We report the detection of P. destructans from individuals of two of 10 bat species captured on the landscape during the summer in the southeast US.

Over 29 nights in May–August 2015, we used 1–8 mist nets (Avinet, Dryden, New York, USA; 38 mm mesh size, 2.6 m high, 4–12 m wide), in various single-, double-, and triple-high arrangements, to capture bats at 20 stream and flight corridor locations on the Tennessee side of Great Smoky Mountains National Park (GRSM; 35°41′0′′N, 83°32′0′′W). Contamination of GRSM caves by P. destructans was confirmed in 2010; in 2011 bats in GRSM were histologically determined to have WNS (Carr et al. 2014). We deployed mist nets 30 min before civil sunset and kept them open for 5 h. For each bat captured, we determined sex, examined wings for WNS-related damage (Reichard and Kunz 2009), and collected skin swab samples to determine presence and load of P. destructans by firmly rubbing a single, sterile, polyester-tipped swab, dipped in sterile deionized water, five times across both the muzzle and the forearm of the bat's folded right wing. We stored swabs in microtubes containing RNAlater® tissue stabilization solution (Life Technologies, Grand Island, New York, USA) at −4 C. We decontaminated all equipment and changed nitrile gloves between processing each bat. All capture and handling protocols were approved by the University of Tennessee Institutional Animal Care and Use Committee (IACUC 2026-0515) and followed the guidelines issued by the American Society of Mammalogists (Sikes and Gannon 2011). Scientific collection permits were obtained from the US Fish and Wildlife Service (TE35313B-2) and National Park Service (GRSM-2015-SCI-1228).

We sent fungal swab samples to the University of New Hampshire for analysis. They performed DNA extractions on all swabs using Qiagen DNeasy 96 Blood and Tissue kits (Qiagen Inc, Valencia, California, USA). They tested extractions, plus 16 negative control wells, for P. destructans on plates using a real-time PCR assay targeting the IGS region of the rRNA gene complex. They ran all plates in duplicate, using aliquots from the same extractions, and considered a sample positive for P. destructans DNA if it crossed the positive P. destructans threshold baseline in fewer than 40 cycles for either replicate. Detection of P. destructans in only one replicate occurs when quantities are near the detection limit of the assay. For positive samples, we used a standard curve and the cycle threshold value to calculate the fungal DNA load (Muller et al. 2013).

We captured 146 bats of 10 species and collected skin swabs from 136 individuals (Table 1). Three of the swabs were positive for P. destructans DNA: two from male big brown bats (Eptesicus fuscus) and one from a male tricolored bat (Table 1). All three positive individuals were captured during early to midsummer (May–June) and showed no signs of WNS-induced wing damage. The positive tricolored bat was captured on 14 May 2015 at a trailhead 2.03 km from the nearest known cave. Both positive big brown bats were captured within 100 m of the entrance of a cave on 6 June. The fungal loads for these male bats were similar to those reported on female big brown bats swabbed at caves in summer in the northeast US (Langwig et al. 2015; Table 1).

Table 1.

The PCR results of 136 Pseudogymnoascus destructans swab samples from bats captured in mist nets at 20 locations on the Tennessee side of Great Smoky Mountains National Park, USA during Summer 2015 (May–August). Pd+ indicates samples contained P. destructans DNA; Pd− indicates samples were negative for P. destructans DNA.

The PCR results of 136 Pseudogymnoascus destructans swab samples from bats captured in mist nets at 20 locations on the Tennessee side of Great Smoky Mountains National Park, USA during Summer 2015 (May–August). Pd+ indicates samples contained P. destructans DNA; Pd− indicates samples were negative for P. destructans DNA.
The PCR results of 136 Pseudogymnoascus destructans swab samples from bats captured in mist nets at 20 locations on the Tennessee side of Great Smoky Mountains National Park, USA during Summer 2015 (May–August). Pd+ indicates samples contained P. destructans DNA; Pd− indicates samples were negative for P. destructans DNA.
a

M = male; F = female.

b

Species with ≤5 P. destructans swabs collected not listed: evening bat (Nycticeius humeralis, n=4); hoary bat (Lasiurus cinereus, n=1); eastern small footed bat (Myotis leibii, n=2); little brown bat (Myotis lucifugus, n=1). All swab samples were negative for P. destructans DNA.

Our results indicate that bats in the southeast US can be positive for P. destructans during the summer. Similar to observations of disease transmission in other wildlife (Alitzer et al. 2006), the presence of P. destructans on bats during their most active period could result in transmission of the fungus to novel individuals and caves, potentially increasing the spatial spread of WNS. While the possibility exists for capturing individuals carrying the fungus when mist netting near contaminated caves where bats may have recently had contact with contaminated substrate or sediments, we also captured a bat positive for P. destructans a considerable distance from any known caves.

The three bats found positive for P. destructans were males. Many bat species show dramatic sex differences in thermoregulatory behavior during summer, with males typically using torpor more frequently and maintaining a lower minimum body temperature during torpor than do females. In addition, male bats use cooler solitary roosts, where their body temperatures remain lower than females clustering in warmer maternity colonies (Dietz and Kalko 2006; Johnson and Lacki 2013). Therefore, body temperatures maintained by males during summer are more likely to fall within the range needed for growth of P. destructans (Verant et al. 2012), potentially allowing greater persistence of the fungus on males than on females and allowing males to serve as a reservoir for fungal transmission.

Winter is considered the most crucial time for researchers to follow strict decontamination measures to avoid spread of P. destructans, due to increased fungal loads and heightened propensity for WNS infection (Langwig et al. 2015). Our findings indicate that stringent decontamination protocols (US Fish and Wildlife Service 2012) should be observed year-round, whether netting at cave sites or on the landscape. To fully understand the risk of spread of P. destructans, we need to determine the viability of the fungus when found on bats during the summer and the role of males as reservoirs of the fungus.

Our research was supported by the National Park Service and University of Tennessee Institute of Agriculture Center for Wildlife Health and the Department of Forestry, Wildlife and Fisheries. We thank Michael Barnes and Shelby Cotham, field technicians with the University of Tennessee; Ryan Williamson, Wildlife Technician with Great Smoky Mountains National Park; Chris Ogle, Wildlife Surveys Manager with Tennessee Wildlife Resources Agency; and numerous volunteers for providing support in the field. Finally, we thank Jeff Foster and Katy Parise for analyzing our fungal swab samples at the University of New Hampshire.

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