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1 October 2016 Molecular Detection of Pseudogymnoascus destructans (Ascomycota: Pseudeurotiaceae) and Unidentified Fungal Dermatitides on Big Brown Bats ( Eptesicus fuscus) Overwintering inside Buildings in Canada
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Abstract

Big brown bats (Eptesicus fuscus) overwintering outside the underground environment are not believed to play a role in the epidemiology of the disease white-nose syndrome (WNS), caused by the fungus Pseudogymnoascus destructans (Pd). Using quantitative real-time PCR (qPCR), we provide molecular evidence for Pd on four big brown bats overwintering in heated buildings in New Brunswick, Canada. Two of the affected individuals also had very mild, focal, pustular, fungal dermatitis identified microscopically. A third bat, which was qPCR Pd-negative, had similar fungal lesions. Despite determining that these fungal lesions were caused by a suspected ascomycete, the intralesional fungi were not confirmed to be Pd. These findings demonstrate that bats overwintering in heated buildings and other above-ground sites may have subclinical or preclinical WNS, or be contaminated with Pd, and could play a role in local dispersal of Pd. Our inability to determine if the ascomycetes causing pustular lesions were Pd highlights the need for ancillary diagnostic tests, such as in situ hybridization or immunohistochemistry, so that Pd can be detected directly within a lesion. As the host–pathogen relationship for Pd evolves, and where bat species are exposed to the fungus under varying temperature regimes, lesions may become less stereotypic and such tests could help define these changes.

White-nose syndrome (WNS), caused by the fungus Pseudogymnoascus destructans (Pd), is decimating populations of eastern North American bats. First detected in New York in 2006–07, it is believed Pd was introduced from Europe (Leopardi et al. 2015). Estimates suggest >6.7 million bats have died from WNS (US Fish and Wildlife Service 2012), and several species may be at risk of regional extinction (Frick et al. 2010). White-nose syndrome is associated with bats in underground hibernacula during periods of extended torpor (hibernation), when they fail to show a cellular immune response to Pd invasion (Meteyer et al. 2011, 2012) and where environmental conditions provide high humidity and the range of temperatures at which Pd grows (Verant et al. 2012).

More than 50% of the 45 North American bat species hibernate underground (Foley et al. 2011), most above 40°N. Other bats may enter torpor for short or extended periods while roosting in buildings, hollow trees, and under bark. As winter approaches some migratory bats leave the northern portion of their range, possibly avoiding the need for torpor. Although WNS is reported in cave-roosting big brown bats (Eptesicus fuscus) and Pd prevalence on this species can be high (Langwig et al. 2014), we are not aware that Pd or WNS has been documented in hibernating bats overwintering outside underground hibernacula. We present incidents where E. fuscus overwintering in buildings had Suspect (b) category diagnoses of WNS (Canadian Wildlife Health Cooperative [CWHC] 2014), based on premature egression from hibernacula (a clinical or field sign associated with WNS) and cryptic Pd infections as determined by quantitative real-time PCR (qPCR) for Pd, but with no external signs of the fungus (see Janicki et al. 2015). The affected individuals also lacked grossly visible or microscopic lesions consistent with WNS (Meteyer et al. 2009), providing further evidence of the cryptic nature of these preclinical or subclinical infections. Although Pd on the surface of bat skin or fur could yield positive qPCR without infection, molecular detection of Pd on multiple bats during hibernation <195 km from Pd-positive underground hibernacula strongly suggests infection. In addition, some affected bats had a fungal dermatitis caused by unidentified ascomycetes. We discuss epidemiologic implications and diagnostic significance of these results.

Surveillance for WNS and Pd in bats in Atlantic Canada follows a standardized necropsy protocol (CWHC 2014) and has been underway since winter 2008–09. The disease was first recorded in New Brunswick in 2011. Most postmortem submissions of bats include little brown bats (Myotis lucifugus) and northern long-eared bats (Myotis septentrionalis) which overwinter in underground hibernacula in the region. However, 10 big brown bats were submitted from New Brunswick during mid-November to late May 2013–14 (Table 1). Six of these bats were discovered active and alive inside the living spaces of human-occupied, heated buildings in Fredericton (45°57′N–66°38′W). The remaining four were found dead or alive outside, near buildings, where they may have been overwintering, in Fredericton (n=3) and near Royalton (46°28′N–67°45′W), 85–190 km north of the closest known underground hibernaculum. Prior to necropsy, swabs for Pd were taken with one sterile, polyester swab from the dorsal and ventral wing membranes, ears, and muzzle of each bat (CWHC 2014). Swabs were frozen at −80 C and sent to the Animal Health Laboratory, University of Guelph (Ontario) to test for Pd using qPCR for WNS surveillance (Muller et al. 2013). Skin from all wing membranes, ears, and two cross-sections of the muzzle were fixed in 10% neutral-buffered formalin, dehydrated in alcohol and xylene, and embedded in paraffin; 5-μm sections were stained with H&E and periodic acid-Schiff to detect fungi.

Table 1.

Findings in big brown bats (Eptesicus fuscus) collected in or adjacent to buildings, during winter in New Brunswick, Canada, and submitted to Canadian Wildlife Health Cooperative (CWHC) November 2013–May 2014. Included are quantitative real-time PCR (qPCR) results for Pseudogymnoascus destructans (Pd), white-nose syndrome status based on histologic evaluation, whether fungal elements were seen histologically, and minimum outdoor temperature on day of collection.

i0090-3558-52-4-902-t01.tif

a 

O = outside; B = inside building.

b 

M = male; F = female.

c 

Imm = immature.

d 

N = negative; P = positive; Cq = quantification cycle.

e 

Environment Canada.

f 

Data not available for 15 April; 16 April data presented.

Cause of death for the 10 bats included rabies (n=1), possible hypothermia (n=1), and euthanasia of emaciated (n=7) and apparently healthy individuals (n=1). Four of the 10 bats had positive qPCR results for Pd (Table 1) and two of these also had a pustular, focal dermatitis with intralesional, unidentified ascomycetes. Fungi were identified as ascomycetes based on the presence of septa without clamp connections, the typical morphologic feature distinguishing the group (Alexopoulos et al. 1996). Although the monokaryon hyphae of basidiomycetes share this feature, most filamentous fungal infections are caused by ascomycetes and most pathogenic infections associated with basidiomycetes present solely in the single-celled yeast state (de Hoog et al. 2000). A third bat with similar, multifocal lesions was qPCR Pd-negative based on a quantification cycle >40, the cut-off for WNS surveillance protocol (Muller et al. 2013). Although Pd qPCR was positive in four of 10 E. fuscus, lesions consistent with WNS (Meteyer et al. 2009) were not present. Additionally, fungal hyphae or conidia morphologically consistent with the typical growth described for intralesional Pd in bats with WNS (Meteyer et al. 2009) were not identified microscopically. Skin lesions caused by the unidentified ascomycetes were only found on the muzzle and were minor and not associated with significant disease.

In the first case (CWHC 107769; Table 1), the fungus was on the epidermal surface in a solitary, intraepidermal, microscopic pustule on the muzzle of an adult female in good body condition that had been found alive inside a building. The fungal hyphae within the pustule were narrow (2.5-μm diameter) and septate with random branching. In contrast, the hyphae within the epidermis were often bulbous and almost torulose, with diameters <5.0 μm (Fig. 1A). In the second case (CWHC 107774), the fungus was in a solitary, microscopic, intraepidermal pustule in the muzzle skin of an immature female in poor body condition, also found alive inside a building. The fungal hyphae were septate with prominent constrictions at their frequent septa and with random branching. The hyphal walls bulged slightly and varied from 2.5–5.0 μm in diameter (Fig. 1B). In the last case (CWHC 110014), there were morphologically different fungi in two separate microscopic, intraepidermal pustules in the muzzle skin of an immature female that was emaciated and found alive outside, adjacent to a building. The hyphae of one fungus were branched with infrequent septation and were variable in diameter (2.5–7.5 μm; Fig. 1C); these hyphae were not confined to the intraepidermal pustule but extended into the surrounding dermis. The morphology of the second fungus varied depending on its location in the lesion. On the epidermal surface, and within the intraepidermal pustule, there were slender (1.8–2.5 μm diameter), unbranched, septate hyphae. However, where they extended into the surrounding dermis, the diameters of the hyphae were 2.5–5.0 μm (Fig. 1D). Conidiogenesis was not observed from any of the fungi.

Figure 1.

Suppurative dermatitis associated with unidentified fungal hyphae (suspect ascomycete) in histologic sections of Canadian big brown bat (Eptesicus fuscus) muzzles stained with periodic acid-Schiff. Canadian Wildlife Health Cooperative (CWHC) numbers 107769 (A) and 107774 (B) were both found alive inside buildings, and were quantitative real-time PCR (qPCR)-positive for Pseudogymnoascus destructans (Pd), the fungal agent of white-nose syndrome. Fungal hyphae associated with inflammation were restricted to the epidermis of the muzzle. (C, D) Images from the muzzle of CWHC 110014, found alive outside and qPCR Pd-negative. The hyphae penetrate the dermis.

i0090-3558-52-4-902-f01.tif

Outside New Brunswick, big brown bats share caves with Myotis spp. and tricolored bats (Perimyotis subflavus). However, big brown bats are known to hibernate only in heated buildings in New Brunswick (McAlpine et al. 2002). Such behavior is common in the northern US (Whitaker and Gummer 2000). It is unknown how the Pd-positive big brown bats reported here might have acquired Pd. Although big brown bats may participate in swarming behavior at New Brunswick hibernacula, there is no evidence to support this.

Verant et al. (2012) found the optimal Pd growth temperature to be 12.5–15.8 C, with atypical fungal morphologies observed above 12 C; fungal morphologies typical for Pd were displayed between 0–7 C. Vanderwolf et al. (2012) report average winter dark zone temperatures of 5.1±1.1 C for New Brunswick underground hibernacula. While the descriptions of Verant et al. (2012) for atypical Pd morphologies share some similarities with the unidentified fungi we describe, and big brown bats were hibernating in buildings that likely provided the higher temperatures associated with atypical growth of Pd, there is no in situ or immunohistochemical diagnostic test available to confirm that these unidentified fungi are Pd. Nonetheless, our data demonstrate that Pd may occur on E. fuscus overwintering in buildings, although risk of the subsequent development of WNS is unclear. In spite of temperature conditions in buildings that are more conducive for Pd growth than are underground hibernacula, we could not confirm WNS in any of the Pd-infected big brown bats, consistent with Frank et al. (2014) findings that big brown bats have some resistance to WNS. Langwig et al. (2014) suggested that even occasional movements among underground hibernacula during winter could be important in Pd spread. Although big brown bats do not appear to use underground hibernacula in New Brunswick, these bats can regularly move in and out of winter roosts in buildings and caves (Beer 1955; Whittaker and Gummer 2000).

We can neither conclude nor discount that the fungi in the big brown bat lesions were Pd. Ancillary diagnostic tests, such as in situ hybridization or immunohistochemistry, are needed so that Pd can be detected directly within a microscopic lesion, particularly in the absence of identifying features such as conidia. As the host–pathogen relationship for Pd evolves, and where bats are exposed to the fungus under varying temperature regimes, lesions may become less stereotypic, and such tests could help define these changes.

Acknowledgements

We are grateful to the following members of the public for submitting bat specimens for testing: John Boudreau, Cara Hazelton, Wendell Price (Canadian Border Services Agency), Joe Robichaud, and Larry Tannahill. We are especially grateful to Melissa Behr, University of Wisconsin, School of Veterinary Medicine; Carol Meteyer, US Geological Survey, National Wildlife Health Center; and David Overy, Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, who graciously provided constructive comments on the pathology as it related to the fungal infections reported here, as well as David Groman with Aquatic Diagnostic Services, Atlantic Veterinary College, for preparation of photomicrographs and Hamid Haghighi and Patricia Bell-Rogers with Animal Health Laboratory, University of Guelph, for qPCR testing of our samples.

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© Wildlife Disease Association 2016
Donald F. McAlpine, Scott McBurney, Mary Sabine, Karen J. Vanderwolf, Allysia Park, and Hugh Y. Cai "Molecular Detection of Pseudogymnoascus destructans (Ascomycota: Pseudeurotiaceae) and Unidentified Fungal Dermatitides on Big Brown Bats ( Eptesicus fuscus) Overwintering inside Buildings in Canada," Journal of Wildlife Diseases 52(4), 902-906, (1 October 2016). https://doi.org/10.7589/2015-03-076
Received: 26 March 2015; Accepted: 1 January 2016; Published: 1 October 2016
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