Real-time PCR detected Pseudogymnoascus destructans associated with ectoparasites collected from three mist-netted free-flying bats (two gray bats, Myotis grisescens; one Indiana bat, Myotis sodalis) in late August to early September 2016 from Kentucky, US, a state impacted by white-nose syndrome. Presence of viable conidia could implicate ectoparasites as possible vectors of white-nose syndrome.

White-nose syndrome (WNS) continues to debilitate and kill North American bats due to infection with the psychrophilic fungus Pseudogymnoascus destructans. Either WNS lesions or P. destructans DNA has been detected on at least 12 eastern bat species (Flory et al. 2012; Bernard et al. 2015). Extreme mortality events have not yet been observed in some bats (e.g., gray bat, Myotis grisescens). Since the discovery of the causative agent, a number of diagnostic tests have been developed, including PCR (Lorch et al. 2010), real-time PCR (Muller et al. 2013), and quantitative PCR (Shuey et al. 2014), the latter of which can differentiate P. destructans from other closely related fungal species.

The availability of PCR has allowed detection of the fungus on biologic samples to the femtogram level with high sensitivity and specificity, essentially detecting the presence of a single conidium (Muller et al. 2013). Thus, Lučan et al. (2016) were able to determine that bat wing mites (Spinturnix myoti) collected from greater mouse-eared bats (Myotis myotis) captured in caves at the end of the hibernation season (March to April 2014) in the Czech Republic were capable of carrying the fungus. There have not been other studies similar in scope to the Lučan et al. (2016) analysis. More significantly, no freeflying bats have been trapped with subsequent removal and analysis of their ectoparasites in an attempt to detect the presence of P. destructans.

To determine if North American bat ectoparasites are potentially a contributing factor to the transmission of the causative agent of WNS from cave to cave, we used the Muller et al. (2013) PCR assay protocol targeting the intergenic spacer region of P. destructans to test for the presence of this fungus on arthropods recovered from freeflying bats and submitted to us. Field hygiene protocols followed US Fish and Wildlife Service WNS decontamination guidelines (US Fish and Wildlife Service 2016). Two groups of bats were assayed, one originating from known WNS sites in Kentucky prior to the start of winter hibernation (August to September 2016) and the second (control group) from a WNS-free region in California (July 2015). Of the 135 bats captured in Kentucky, ectoparasites were collected from 21 swarming gray bats and from one Indiana bat (Myotis sodalis). All ectoparasites from Kentucky bats were mites of the family Spinturnicidae, as determined by morphology using light microscopy (Krantz 1978). Ectoparasites from the same individual bat were pooled in sterile 1.5-mL microcentrifuge tubes in the field and contained up to 11 mites, although most had less than four. Eight of 60 bats captured in California had ectoparasites collected; four bats had only spinturnicid mites, and four bats had only hippoboscid flies (Diptera, Hippoboscoidea, Basilia sp.) verified by microscopic examination (Triplehorn and Johnson 2005).

We extracted DNA by using a commercial kit (DNeasy Blood & Tissue Kit, Qiagen Inc., Valencia, California, USA) following the manufacturer's protocol with unidirectional workflow to avoid cross contamination. Additionally, California samples were processed on a Monday, Kentucky samples the following Wednesday, and the fungal culture in a different laboratory under an exhaust hood the following week. Neither of the sample processing laboratories were previously exposed to P. destructans. Real-time PCR was performed on a Mastercycler ep realplex System (Eppendorf AG, Hamburg, Germany) with commercial master mix (QuantiFastProbe PCR+ROX Vial Kit, Qiagen) on 5-µL triplicates of DNA samples. California samples were loaded first, followed by Kentucky samples. Triplicates of genomic DNA isolated from a culture of P. destructans, as well as notemplate controls, were also included in the assay as positive and negative controls, respectively. Positive control wells were loaded last to minimize any risk of contamination. Forward and reverse primers were used at a final concentration of 0.4 µM and internal probe at 0.2 µM. Cycle threshold was automatically determined by the analysis software at 10× the SD above the noise of the baseline. Samples were considered positive if the reaction crossed the threshold baseline within 40 cycles for at least two of the three replicates.

No ectoparasites from the California bats tested positive for P. destructans, and none of the no-template controls did. Of the 22 samples from Kentucky bats, two originating from gray bats and one from an Indiana bat tested positive for the presence of P. destructans.

As with greater mouse-eared bats in Europe, both gray bats and Indiana bats form large colonies, with gray bats using caves both summer and winter (Hall and Wilson 1966), while Indiana bats primarily use caves for hibernation (Cope and Humphrey 1977). Proximity of the bats to one another and dense roosting clusters inside the cave would facilitate movement of ectoparasites between individual bats, as postulated by Lučan et al. (2016) for mouse-eared bats during hibernation. Detecting the presence of P. destructans on ectoparasites of gray and Indiana bats prior to hibernation has implications for the spread of the fungus, as mating occurs during swarming, with the potential to exchange ectoparasites. In addition, it is possible that bats of other species using the same caves, but with less cave site fidelity, are also exposed to contaminated ectoparasites and may move between multiple caves in a region inadvertently spreading the fungus.

The conidia of P. destructans remain viable for 8 d at 24 C but lose their ability to germinate after 8 mo at this temperature (Puechmaille et al. 2011). Bats visiting WNSpositive caves during the summer months have the potential to transport conidia on ectoparasites and transmit them to naïve bats within an 8-d time frame. Because ectoparasites testing positive for the fungus were present on gray and Indiana bats during summer months, fungal control measures should be considered that could happen outside caves, thereby lessening the impact on the ecology of caves and refining the methodologies for treatment. For example, the WNS-impacted little brown bat (Myotis lucifugus) could potentially benefit by treatment of the summer roosts and maternity colonies with insecticides or acaricides to control WNS vectors rather than attempting treatment for the fungus. It is also possible that passive insecticide applicators could be developed and affixed to cave gates or otherwise fastened to hibernation cave access points.

This project was funded by a grant from fightwns. We thank Janet Tyburec, Bat Survey Solutions, for providing the ectoparasites and Christopher Cornelison, Director, BioInnovation Laboratory, Kennesaw State University, for providing a culture of Pseudogymnoascus destructans.

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