Pseudogymnoascus destructans, the causal agent of white-nose syndrome (WNS), is responsible for widespread mortality of hibernating bats across eastern North America. To document P. destructans exposure and infections on bats active during winter in the southeastern US, we collected epidermal swabs from bats captured during winters 2012–13 and 2013–14 in mist nets set outside of hibernacula in Tennessee. Epidermal swab samples were collected from eight Rafinesque's big-eared bats (Corynorhinus rafinesquii), six eastern red bats (Lasiurus borealis), and three silver-hair bats (Lasionycteris noctivagans). Using real-time PCR methods, we identified DNA sequences of P. destructans from skin swabs of two Rafinesque's big-eared bats, two eastern red bats, and one silver-haired bat. This is the first detection of the WNS fungus on Rafinesque's big-eared bats and eastern red bats and the second record of the presence of the fungus on silver-haired bats.

The white-nose syndrome (WNS) epizootic, caused by the psychrophilic fungus Pseudogymnoascus destructans (Lorch et al. 2011; Minnis and Lindner 2013), has caused severe mortality of hibernating bats throughout eastern North America since its discovery in New York in winter 2006–07 (Blehert et al. 2009; Turner et al. 2011). The fungus has been reported from 28 states and five Canadian provinces on 10 species of North American bats, with seven species experiencing population losses due to WNS (Turner et al. 2011; USFWS 2014). Histologic evidence of WNS involving fungal invasion of tissue (Meteyer et al. 2009) has been identified on seven North American bat species, including two endangered species (gray bats [Myotis grisescens] and Indiana bats [M. sodalis]), with others being considered for federal listing as a result of WNS. Over the past seven winters, WNS has killed an estimated 5.7 million bats and now threatens once abundant species with regional extinction (Reeder and Turner 2008; Frick et al. 2010; Langwig et al. 2012; USFWS 2014).

White-nose syndrome is a disease of hibernating bats, as infection and mortality occur when bats are torpid during hibernation (Lorch et al. 2011; Warnecke et al. 2012). In healthy individuals, torpor bouts typically last 12–15 d (Reeder et al. 2012; Brownlee-Bouboulis and Reeder 2013), and bouts are separated by brief arousal periods that serve various physiologic purposes (Geiser 2004; Moore et al. 2011; Jonasson and Willis 2012). Extensive torpor bouts may facilitate the growth of P. destructans on bats, ultimately causing lesions that disrupt torpor resulting in the premature depletion of energy reserves (Warnecke et al. 2012). Critical physiologic processes such as gas exchange and water balance are also affected (Cryan et al. 2010; Warnecke et al. 2013). As of January 2014, three additional species, Virginia big-eared bat (Corynorhinus towsendii virginianus), Southeastern bat (M. austroriparius), and a silver-haired bat (Lasionycteris noctivagans), have been found positive for P. destructans, despite showing no histologic evidence or clinical signs of WNS (USFWS 2014). Here we document the first cases of the WNS fungus on the Rafinesque's big-eared bat (Corynorhinus rafinesquii) and eastern red bat (Lasiurus borealis) and the second case of P. destructans on a silver-haired bat.

We conducted this study to assess the relationship between winter activity and variation in load and prevalence of P. destructans on bats in southern latitudes of the US. During winters 2012–13 and 2013–14 (n = 70 nights, October–April each year), we used mist nets (75/2, 2.6 m high, 4 shelves, black polyester for bats, 6–12 m wide; Avinet, Dryden, New York, USA) to capture bats leaving five hibernacula in eastern Tennessee. Although most bats captured were Myotis species and obligate hibernators (n = 712/947, results to be published elsewhere), we also captured eight Rafinesque's big-eared bats (five males, three females), six eastern red bats (three males, three females), and three silver-haired bats (two males, one female). All capture and handling techniques were approved by the University of Tennessee Institutional Animal Care and Use Committee (IACUC 2026-0514) and were consistent with the guidelines issued by the American Society of Mammalogists (Sikes et al. 2011). We obtained both federal (USFWS TE 71613A) and state (Tennessee Wildlife Resource Agency [TWRA 3716] and Tennessee Department of Environment and Conservation [TDEC 2011-031]) permits to capture and handle bats at winter hibernacula.

Biometric information and epidermal swab samples were collected from each individual. Sterile cotton-tipped swabs were dipped in sterile deionized water and rubbed across the forearm of the folded right wing and muzzle five times. We extracted DNA from all swab samples using Qiagen DNeasy 96 Blood & Tissue kits (Qiagen, Valencia, California, USA). The samples were tested for P. destructans using a real-time PCR assay targeting the IGS region, with a sample considered positive if the fluorescence crossed the threshold in fewer than 40 cycles for either replicate following the methods of Muller et al. (2013). Detections of P. destructans in only one replicate are common when quantities are near the detection limit of the assay. Plates were run in duplicate with 16 negative controls distributed throughout each plate. We then used a standard curve generated according to Muller et al. (2013) to calculate the fungal DNA concentration (ng).

Of 17 swab samples collected, two Rafinesque's big-eared bats, two eastern red bats, and one silver-haired bat were positive for P. destructans DNA (Table 1). All P. destructans positive individuals were captured during the mid- to late hibernation period (January–April, Table 1). Sites where positive individuals were captured were also inhabited by WNS-susceptible species such as northern long-eared bats (Myotis septentrionalis), little brown bats (Myotis lucifugus), and Indiana bats. Due to the small sample size, we were unable to determine prevalence or demographic and seasonal trends related to the presence of P. destructans on Rafinesque's big-eared, eastern red, and silver-haired bats. However, all three species remain active during winter in the southern portions of their range and have been found foraging during warm winter nights (Boyles et al. 2006; Dunbar et al. 2007; Falxa 2007; Perry et al. 2010; Johnson et al. 2012), which could help maintain the fat stores necessary for winter survival.

Table 1.

PCR results of Pseudogymnoascus destructans (Pd) swab samples from bats captured in mist-nets in Tennessee during winter (October–April) 2012–13 and 2013–14. Pd+ indicates the sample contained P. destructans DNA. * Indicates samples where Pd DNA was only detected in one PCR replicate.

PCR results of Pseudogymnoascus destructans (Pd) swab samples from bats captured in mist-nets in Tennessee during winter (October–April) 2012–13 and 2013–14. Pd+ indicates the sample contained P. destructans DNA. * Indicates samples where Pd DNA was only detected in one PCR replicate.
PCR results of Pseudogymnoascus destructans (Pd) swab samples from bats captured in mist-nets in Tennessee during winter (October–April) 2012–13 and 2013–14. Pd+ indicates the sample contained P. destructans DNA. * Indicates samples where Pd DNA was only detected in one PCR replicate.

Our results show that Rafinesque's big-eared bats and eastern red bats are exposed to the fungal pathogen P. destructans. However, we note that PCR detection of the pathogen from epidermal swabs does not demonstrate whether these species have developed characteristic skin lesions that define the disease. Our results are important for assessing overall risk of exposure to the pathogen, but there are currently no indications of population declines or that these bats are susceptible to WNS. Winter activity of Rafinesque's big-eared bats, eastern red bats, and silver-haired bats in and near hibernacula may facilitate infection with P. destructans on these species. However, survival strategies of these species that tend to be more active during winter may reduce disease progression. While continued activity throughout the winter may prevent WNS in these species, these bats may be facilitating the spread of the fungus to roost sites and colonies not yet colonized by P. destructans.

Our project was supported by the University of Tennessee Institute of Agriculture Center for Wildlife Health and the Department of Ecology and Evolutionary Biology. Fungal swab collection and real-time PCR methods used for this project were developed under a National Science Foundation Grant (DEB-1115895). We thank our interns, Anna Chow, Reilly Jackson, Devin Jones, Mariah Patton, and Ana Reboredo-Segovia, for assisting with fieldwork; Colin Sobek for assisting with lab work; and the Tennessee Wildlife Resources Agency (Jim Evans, Chris Ogle, Chris Simpson, Dustin Thames), Great Smoky Mountains National Park (James Carr, Bill Stiver, Rick Varner, Ryan Williamson), Oak Ridge National Laboratory (Neil Giffen, Angelina Hayes, Kitty McCracken, Kelly Roy), and the Tennessee Chapter of The Nature Conservancy (Cory Holliday) for access to sites and assistance in the field. We acknowledge the WNS community for comments and information regarding observations of silver-hair bats and eastern red bats at hibernacula throughout winter.

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