White-nose syndrome (WNS) affects bats primarily in winter, with Pseudogymnoascus destructans, the fungus that causes WNS, growing on bats in colder climates as they are hibernating. As a result, nearly all disease investigations have been conducted on bats in the winter or as they are emerging in spring. Although P. destructans has been detected on bats during the summer season, the seasonal dynamics of infection during this period remain poorly understood. To test for the presence of P. destructans during the summer season, we sampled bats that were free flying from June 2017 to September 2017 and also sampled bats from a maternity roost in August and outside a known hibernaculum in September. We collected skin swabs from the muzzle and forearm of bats, and using real-time PCR methods, we detected P. destructans DNA on 16% (12/76) of bats sampled in Wisconsin, US, including juvenile little brown bats (Myotis lucifugus) from bat house maternity roosts, and free-flying adult bats of two species captured in June, the little brown bat and the migratory eastern red bat (Lasiurus borealis). These data illustrated the potential for P. destructans to be transferred and dispersed among bats during the summer and highlighted the complex seasonal dynamics associated with this pathogen.
White-nose syndrome (WNS), caused by the fungus Pseudogymnoascus destructans (Blehert et al. 2009), has produced unprecedented declines in hibernating bats in eastern North America (Frick et al. 2010). The disease affects bats primarily in winter, with P. destructans growing on bats in colder climates as they are hibernating, so nearly all disease research has been conducted on bats in winter or as they emerge in spring. Several studies have detected P. destructans during the summer (Dobony et al. 2011; Langwig et al. 2015; Carpenter et al. 2016; Ballmann et al. 2017). Reichard et al. (2014) stated that ecology and biology of WNS-affected bats during summer deserves continued attention; thus, we sought to contribute to one key aspect of the disease process, the persistence and transference of P. destructans on bats during summer. Here, we documented the summertime detection of P. destructans on bats in Wisconsin, US, including juvenile little brown bats (Myotis lucifugus) sampled from maternity roosts, and free-flying adult bats of two species captured in June: the little brown bat and the migratory eastern red bat (Lasiurus borealis).
We sampled free-flying bats during nine nights of mist netting (38-mm mesh, 2.6 m high, 6–12 m wide; Avinet, Dryden, New York, USA), from 13 June 2017 to 6 September 2017 in Grant County (Fig. 1). Nets were up before sunset and were attended continuously until closure (typically <3.5 h after sunset), with two nets used at most locations. We also sampled bats from a maternity roost including three bat houses on 15 August 2017 in Iowa County (Fig. 1), as well as outside a hibernaculum documented to have bats infected with P. destructans, in Dodge County on 30 August 2017 (Fig. 1). Bats were captured outside the bat houses with high mist-net systems (Bat Conservation and Management, Carlisle, Pennsylvania, USA) used in conjunction with tarps to direct the flight of bats. Bats were captured outside two entrances to the hibernaculum with harp traps (Bat Conservation and Management). Among data recorded for captured bats were species, age (adult or juvenile), and sex (Anthony 1988). We sampled bats for P. destructans by collecting skin swab samples from the forearm and muzzle following the procedures of Bernard et al. (2015). Specifically, we rolled a sterile, polyester-tipped swab that had been moistened in sterile, nuclease-free, deionized water five times across both the forearm and muzzle of each sampled bat. We stored swabs in sterile microtubes containing RNAlater tissue stabilization solution (Life Technologies, Grand Island, New York, USA) at ambient temperature while in the field and within 24 h, transferring them to 4 C storage until analysis. Decontamination protocols designed to reduce the unintentional spread of P. destructans were followed when handling bats and for cleaning equipment (US Fish and Wildlife Service 2016). Methods for capturing and handling bats followed the guidelines of the American Society for Mammalogists (Sikes et al. 2016) and were approved by the University of Wisconsin–Platteville Animal Care and Use Committee (protocol 0616-2017). Work was conducted under appropriate state and federal permits.
The extraction of DNA from skin swabs was performed at Northern Arizona University with Qiagen DNeasy 96 Blood & Tissue Kits (Qiagen Inc., Valencia, California, USA) using the manufacturer's supplementary protocol for yeast extractions. To test for the presence of P. destructans, we followed real-time PCR protocols established by Muller et al. (2013). Each extraction plate contained 16 negative control wells, consisting of extraction kit reagents but no swab. These extraction negative controls were distributed in two columns on opposite sides of the plate, as a means of identifying potential contamination of samples during processing. Each assay plate, run on an Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, California, USA), contained the following: swab extractions, 13 negative controls, and three serial dilutions of genomic DNA from P. destructans strain 20631-21 starting at 20 pg/µL. The 13 negative controls are a subset of the 16 extraction controls (the remaining three wells were used for the aforementioned positive controls) and contained both PCR MasterMix and extraction kit reagents from the respective well of the extraction negative controls. Due to the possibility of low fungal loads present in the samples collected and variable detectability of P. destructans by PCR when present in low amounts, PCR for each sample was performed in replicate on a separate assay plate immediately following the initial real-time PCR. The threshold baseline was set to 10% of the maximum fluorescence, as determined by the positive controls (Muller et al. 2013). We considered a sample positive for P. destructans DNA if the amplification crossed the threshold baseline within 40 cycles in at least one of the two replicates (Muller et al. 2013). We screened samples from 43 free-flying bats (24 females and 19 males), 23 bats (18 females and five males) from the maternity roost, and 10 bats (two females and eight males) from outside a hibernaculum (Table 1).
Of the 76 unique samples analyzed, samples from 11 little brown bats and one eastern red bat were positive for P. destructans (Table 2); P. destructans was not detected on any of 27 big brown bats (Eptesicus fuscus) or on the one northern long-eared myotis (Myotis septentrionalis) screened. Four of 43 free-flying bats screened for P. destructans were positive. All P. destructans–positive free-flying bats were adult males: three were little brown bats and one was an eastern red bat. Others have noted the preponderance of P. destructans–positive male bats in summer (Carpenter et al. 2016; Ballmann et al. 2017), which has been attributed to males using cooler summer roosting temperatures than reproductive females, possibly entering torpor more frequently, and sometimes selecting hibernacula where P. destructans is present for summer roost sites (Carpenter et al. 2016; Ballmann et al. 2017). Male bats may serve as both a summertime reservoir (Carpenter et al. 2016) and a dispersal agent of P. destructans (Ballmann et al. 2017), and our results were consistent with these views.
Bernard et al. (2015) documented P. destructans on migratory bats species, including the eastern red bat. Notably, their P. destructans–positive eastern red bats were bats captured leaving hibernacula from March and April (Bernard et al. 2015). We detected P. destructans on a free-flying, adult male eastern red bat captured on 14 June 2017 (Table 2), 4.8 km away from the closest known cave. This cave was determined to be negative for P. destructans when last surveyed (February 2015). The closest known P. destructans–positive site was a mine 10.8 km from our netting site. As Bernard et al. (2015) suggested, migratory bats, such as the eastern red bat, may be facilitating the spread of P. destructans, and again, our results supported that hypothesis.
Of the 23 little brown bats that we caught at the maternity colony on 15 August 2017, seven juveniles were P. destructans positive (five females and two males; Table 2). Unfortunately, we did not determine the specific house that bats exited from due to their close proximity; thus, we do not know whether P. destructans–positive bats were from one bat house or from multiple bat houses. Langwig et al. (2015) found that both prevalence and loads of P. destructans decreased during the summer and hypothesized that high body temperatures of bats occupying summer maternity colonies should prevent infection or limit growth of P. destructans. However, Dobony et al. (2011) detected P. destructans on three juvenile bats sampled on 19 August 2009 from a maternity colony and isolated conidia from live fungal growth on media plates from one of these samples. Whether P. destructans on these juveniles was transferred from females exposed to the fungus during hibernation, or through some other means (e.g., a visit to a P. destructans–positive hibernaculum), is unknown. Ballmann et al. (2017) also reported P. destructans–positive juvenile bats; however, these bats were caught outside known hibernacula, all of which were P. destructans positive at the time of the sampling or within the following season. Two sites known to have small populations of hibernating bats (<20 bats annually) exhibiting WNS were 0.8 and 1.2 km from the bat houses we sampled, leaving open the possibility that juveniles obtained P. destructans by visits to those sites or via adults that visited the sites and then subsequently transmitted P. destructans to the juveniles. The frequency of P. destructans–positive juveniles and the potential for P. destructans on these juveniles to lead to infection and impact their first-year survival deserves further research.
Only one of 10 bats (an adult, male little brown bat) trapped on 30 August 2017 outside a hibernaculum where WNS had been previously confirmed in the bat population was positive for P. destructans. Langwig et al. (2015 reported low P. destructans loads on bats at the end of summer and during autumn. Nevertheless, the fact that P. destructans levels were so low in our study as to be undetectable on most bats sampled outside this P. destructans–positive hibernaculum suggests that either P. destructans was entirely absent from some bats or the transfer of P. destructans from the environment of the hibernacula to the bats was insufficient for detection by PCR.
We documented P. destructans–positive bats in Wisconsin throughout the summer, including free-flying adult males, juveniles using bat houses, and a common migratory species. The occurrence of P. destructans on bats that have not yet hibernated, as in the case of juvenile little brown bats, or those species that do not use typical hibernation sites, as in the case of the eastern red bat (Dunbar and Tomasi 2006), suggested that P. destructans transmission can occur outside of caves and mines. The significance of these summer detections of P. destructans in the epizoology of WNS warrants further study: determining if P. destructans on bats in the summer is viable and establishing the biologic significance of P. destructans loads.
Funding for this research was provided by two grants from the University of Wisconsin–Platteville: a Pioneer Summer Research Grant to S. Hoerner from the Office of the Provost and a Scholarly Activity Improvement Fund grant to J. Huebschman from the Office of Research and Sponsored Programs. Thanks to David and Mary Hardyman, Phil Sealy and Karen Barton, Kathleen Gruentzel (park superintendent), and University of Milwaukee for access to their land or lands they manage. Field assistance was provided by Jennifer Redell (Wisconsin Department of Natural Resources) and employees and volunteers from US Geological Survey, National Wildlife Health Center. A special thanks to University of Wisconsin–Platteville biology students, Jacob Nottestad and Kiara Zurow, for their invaluable assistance in the field.