the fungus trichophyton redellii sp. nov. causes skin ...the fungus trichophyton redellii sp. nov....

THE FUNGUS TRICHOPHYTON REDELLII SP. NOV. CAUSES SKIN

INFECTIONS THAT RESEMBLE WHITE-NOSE SYNDROME OF

HIBERNATING BATS

Jeffrey M. Lorch,1 Andrew M. Minnis,2 Carol U. Meteyer,3,5 Jennifer A. Redell,4 J. Paul White,4

Heather M. Kaarakka,4 Laura K. Muller,3 Daniel L. Lindner,2 Michelle L. Verant,1

Valerie Shearn-Bochsler,3 and David S. Blehert3,6

1 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 1656 LindenDrive, Madison, Wisconsin 53706, USA2 United States Forest Service, Northern Research Station, Center for Forest Mycology Research, One Gifford PinchotDrive, Madison, Wisconsin 53726, USA3 United States Geological Survey, National Wildlife Health Center, 6006 Schroeder Road, Madison, Wisconsin 53711, USA4 Bureau of Natural Heritage Conservation, Wisconsin Department of Natural Resources, 101 S Webster Street,Madison, Wisconsin 53707, USA5 Current address: United States Geological Survey National Center, Environmental Health, 12201 Sunrise Valley Drive,Reston, Virginia 20192, USA6 Corresponding author (email: [email protected])

ABSTRACT: Before the discovery of white-nose syndrome (WNS), a fungal disease caused byPseudogymnoascus destructans, there were no reports of fungal skin infections in bats duringhibernation. In 2011, bats with grossly visible fungal skin infections similar in appearance to WNSwere reported from multiple sites in Wisconsin, US, a state outside the known range of P.destructans and WNS at that time. Tape impressions or swab samples were collected from affectedareas of skin from bats with these fungal infections in 2012 and analyzed by microscopy, culture, ordirect DNA amplification and sequencing of the fungal internal transcribed spacer region (ITS). Apsychrophilic species of Trichophyton was isolated in culture, detected by direct DNAamplification and sequencing, and observed on tape impressions. Deoxyribonucleic acid indicativeof the same fungus was also detected on three of five bat carcasses collected in 2011 and 2012 fromWisconsin, Indiana, and Texas, US. Superficial fungal skin infections caused by Trichophyton sp.were observed in histopathology for all three bats. Sequencing of the ITS of Trichophyton sp.,along with its inability to grow at 25 C, indicated that it represented a previously unknown species,described herein as Trichophyton redellii sp. nov. Genetic diversity present within T. redelliisuggests it is native to North America but that it had been overlooked before enhanced efforts tostudy fungi associated with bats in response to the emergence of WNS.

Key words: Bat, dermatophyte, fungal infection, hibernation, Trichophyton, white-nosesyndrome.

INTRODUCTION

Dermatophytes (defined here on thebasis of taxonomy) are common on manygroups of free-ranging mammals (Manto-vani et al. 1982). Although dermatophyteshave been observed on hibernating bats(Courtin et al. 2010; Vanderwolf et al.2013b), they have not been linked todisease. Indeed, we are unaware of reportsof fungal skin infections in hibernatingbats in North America before the arrival ofwhite-nose syndrome (WNS), a deadlydisease caused by the fungus Pseudogym-noascus destructans (Blehert et al. 2009;Lorch et al. 2011). While it is possible thatbats are generally resistant to fungal

infections during hibernation (with theexception of P. destructans), it seems moreplausible that nonlethal fungal infectionshave gone undetected. Learning moreabout these fungi and how bats cope withthem could provide insight into why WNSis deadly to many North American bats.

Beginning in winter 2011, the Wisconsin(US) Department of Natural Resources(WDNR) reported hibernating bats withgrossly visible white fungus similar to someof the clinical signs that characterize WNS(Fig. 1A). However, Wisconsin was outsidethe known range of WNS at that time (USFish and Wildlife Service 2014). Potentiallyinfected animals were submitted to theUS Geological Survey–National Wildlife

DOI: 10.7589/2014-05-134 Journal of Wildlife Diseases, 51(1), 2015, pp. 36–47# Wildlife Disease Association 2015

36

Health Center (NWHC; Madison, Wiscon-sin, USA) for diagnostic testing and wereshown to be negative for WNS by histopa-thology and negative for P. destructans. Wedescribe the cause of these skin infectionsand provide information on how to distin-guish them from WNS.

MATERIALS AND METHODS

Sample collection

In winter 2011 and 2012, NWHC receivedbats that had visible fungal skin infections inthe field but subsequently were found nega-tive for WNS by histopathology and negativefor P. destructans by PCR (Muller et al. 2013)

FIGURE 1. Comparison between Trichophyton redellii infection (top panels) and Pseudogymnoascusdestructans infection (i.e., white-nose syndrome [WNS]; bottom panels) in bats. Bats infected with T. redelliihave visible white fungal growth on the ears, legs, wings, tail, or uropatagium (A); lesions may manifest as adistinct ring (arrow); the muzzle often lacks clinical signs of infection. Bats with WNS generally have visiblefungus on the muzzle in addition to other areas of unfurred skin (B). Fungal tape impressions collected frombats with clinical signs of T. redellii infection display radially symmetric obovate to pyriform microconidia (C),as opposed to the asymmetrical curved conidia typical of P. destructans (D). In histologic sections (preparedwith periodic acid–Schiff staining), the wing skin of bats with T. redellii infections (E) generally havesuperficial colonization and invasion of the keratin layers by the dermatophyte (arrow); aerial hyphae andfertile structures in the form of conidiophores and microconidia may also be present (arrowhead). In contrast,histologic cross sections of wing skin from bats with WNS (F) typically display cup-like aggregations of fungalhyphae (arrows), erosion of the epidermis, and occasional curved conidia (arrowheads). Scale bars520 mm.

LORCH ET AL.—DERMATOPHYTE INFECTION IN BATS 37

and culture analyses. These included a littlebrown bat (Myotis lucifugus) from Wisconsin;a little brown bat from Indiana, US; and threecave bats (Myotis velifer) from Texas, US(Table 1). The bats were euthanized bypersonnel of the WDNR, Indiana Departmentof Natural Resources, and Texas Parks andWildlife Department in accordance withapplicable state laws.

Nonlethal samples were collected from twosites in Wisconsin in March 2012 (WDNR’sAnimal Care and Use Committee Protocol 11-RedellD-01). PurFlockH sterile nylon flockedswabs (Puritan Medical Products CompanyLLC, Guilford, Maine, USA) moistened with15 mL sterile phosphate-buffered saline werewiped against the wing skin of hibernatingbats with white fungal growth and stored insterile 1.5-mL microcentrifuge tubes (n510).The forearms of animals without visible fungalgrowth were also swabbed to serve as controls(n52). Chilled swab samples were transport-ed to NWHC for processing within 24 h ofcollection. Fungal tape impressions (Fungi-TapeTM, Scientific Device Laboratory, DesPlaines, Illinois, USA) were also taken fromthree of the bats that were swabbed. The areaof the tape that contacted the skin was circledwith a marker, the tape was affixed to a

microscope slide, and the encircled areawas examined using a microscope (4003magnification).

Assessment of fungi on infected bat skin

Swab samples were subjected to cultureanalysis by streaking the swabs onto Sabour-aud dextrose agar plates with chloramphenicoland gentamicin (SDA; BD Diagnostic Sys-tems, Sparks, Maryland, USA) as described byLorch et al. (2010). The culture plates wereincubated at 7 C and checked once per weekfor 8 wk. Fungi isolated from initial plateswere thereafter maintained on SDA. Toidentify fungal isolates, we sequenced theinternal transcribed spacer region (ITS). Asterile toothpick was used to scrape myceliafrom pure cultures on fungal growth media.The scraped material was transferred to 0.2-mL strip tubes containing 20 mL microLYSISHsolution (Gel Company, San Francisco, Cali-fornia, USA), and the cells were lysedaccording to the manufacturer’s instructions.Polymerase chain reaction targeting ITS wasperformed using universal fungal primersITS1-F and ITS4 (Gardes and Bruns 1993;cycling conditions: 94 C for 3 min followed by40 cycles of 94 C for 1 min, 53 C for 1 min,and 72 C for 3 min, with a final extension for

TABLE 1. Bats sampled to characterize fungi that cause skin infections unrelated to white-nose syndrome.Identifiers represent US Geological Survey–National Wildlife Health Center case and accession numbers ofwhole bat carcasses, swabs, and fungal tape impressions submitted for testing. Clinical signs refer to thepresence (+) or absence (2) of visible fungus on the skin when the bats were observed in the field. Analysesperformed included culture (C) from swabs, direct amplification and sequencing of a portion of the fungalinternal transcribed spacer region (DAS) from swabs and carcass wing tissue, microscopic examination (M) offungal tape impressions collected directly from bat skin, and histopathologic examination (H) of wing tissue.

IdentifierMyotis

host species Collection locationaClinical

signs Sample typeAnalyses

performed

23489-01 lucifugus Greene Co., Indiana, USA + Carcass DAS, H23493-01 lucifugus Pierce Co., Wisconsin, USA + Carcass DAS, H23863-01 velifer Wheeler Co., Texas, USA + Carcass DAS, H23863-02 velifer Wheeler Co., Texas, USA + Carcass DAS, H23863-03 velifer Wheeler Co., Texas, USA + Carcass DAS, H44738-01 lucifugus Grant Co., Wisconsin, USA + Swab C, DAS44738-02 lucifugus Grant Co., Wisconsin, USA + Swab, tape impression C, DAS, M44738-03 lucifugus Grant Co., Wisconsin, USA + Swab, tape impression C, DAS, M44738-04 lucifugus Grant Co., Wisconsin, USA + Swab C, DAS44738-05 lucifugus Grant Co., Wisconsin, USA + Swab C, DAS44738-06 lucifugus Grant Co., Wisconsin, USA + Swab C, DAS44738-07 lucifugus Pierce Co., Wisconsin, USA + Swab C, DAS44738-08 lucifugus Pierce Co., Wisconsin, USA + Swab C, DAS44738-09 lucifugus Pierce Co., Wisconsin, USA + Swab, tape impression C, DAS, M44738-10 lucifugus Pierce Co., Wisconsin, USA + Swab C, DAS44738-11 lucifugus Pierce Co., Wisconsin, USA 2 Swab C, DAS44738-12 lucifugus Pierce Co., Wisconsin, USA 2 Swab C, DAS

a Co. 5 county.

38 JOURNAL OF WILDLIFE DISEASES, VOL. 51, NO. 1, JANUARY 2015

10 min at 72 C). All PCR reactions wereconducted using GoTaqH Flexi DNA poly-merase (Promega Corporation, Madison, Wis-consin, USA) according to the manufacturer’sinstructions, with 0.5-mL template per 25-mLreaction. Additional loci of two fungal isolatesof the bat-infecting fungus were sequenced:nuclear large subunit rRNA gene (LSU), DNAreplication licensing factor MCM7 (MCM7),RNA polymerase II second largest subunit(RPB2), and translation elongation factor EF-1a (TEF1) as described by Minnis andLindner (2013). The intergenic spacer region(IGS) was sequenced following the methods ofLorch et al. (2013), except that products weresequenced from both 59- (using primerCNL12 [Anderson and Stasovski 1992]; IGS-59) and 39- (using primer CNS1 [White et al.1990]; IGS-39) ends. All newly generatedsequences were deposited into GenBank.

After swabs were streaked on fungal growthmedium, each swab was returned to itsoriginal collection tube, and 50 mL of micro-LYSIS solution was added. The swab wasswirled in the solution for 30 s and allowed tosoak for an additional 5 min. Fungal cells inthe solution were lysed following the manu-facturer’s instructions. For whole carcassessubmitted for diagnostic testing, a small pieceof wing tissue was excised, and DNA wasextracted as described by Lorch et al. (2010).Polymerase chain reaction and double-strand-ed sequencing of PCR products were per-formed using primers ITS1-F and ITS4 asdescribed above. Histopathologic analyseswere performed on wing skin from the batcarcasses submitted for diagnostic evaluation(n55) following the methods of Meteyer et al.(2009).

Phylogenetic analysis

The ITS sequences of isolates most com-monly associated with the fungal infectionswere aligned with ITS sequences in GenBank(and newly generated sequences—see Results)of closely related fungi using the defaultsettings of the online version (http://mafft.cbrc.jp/alignment/server/) of MAFFT v.7 (Ka-toh and Standley 2013). Rambaut’s Se-ALv2.0a9 was used to adjust the resultingalignment, and areas with excessive gaps andambiguously aligned regions were deleted. Amaximum likelihood (ML) analysis was con-ducted with RAxML (Stamatakis 2006; Stama-takis et al. 2008) via RAxML-HPC2 v7.6.6using the CIPRES Science Gateway (Milleret al. 2010). All parameters were defaulted,but best scoring ML tree and bootstrap (BS)analyses were made in a single run, with 1,000

BS iterations and GTRGAMMA selected forthe bootstrapping phase and final tree. Parsi-mony-based BS analyses (Felsenstein 1985)with 1,000 BS replicates and maxtrees unlim-ited, 10 random taxon-addition sequences and100 trees held at each step, and all otherparameters at default were conducted withPAUP* (Swofford 2003) to address cladesupport using an additional method.

Characterization of fungal isolates

Representative isolates of taxa described asnew herein were incubated at 25 C and 10 Con SDA plates in the dark. Plates wereinoculated by transferring fungal material withan inoculating needle; all isolates were inocu-lated in triplicate. Measurements and obser-vations of cultures were made each week for4 wk. Terminology and sample referencecodes in parentheses for colony colorationare from Kornerup and Wanscher (1978).Microscopic observations were made of mate-rial from cultures mounted in 3% KOH orCotton Blue (Largent et al. 1997). At least 30conidia of each culture were measured.Length-to-width ratios are given as Q. Repre-sentative living cultures of taxa described asnew herein were deposited at the Center forForest Mycology Research (CFMR) in Madi-son, Wisconsin, and the Centraalbureau voorSchimmelcultures (CBS) in the Netherlands.

RESULTS

Assessment of fungi on infected bat skin

Swab samples from five of 10 infectedbats yielded pure, heavy growth of a singlefungus in culture after 10–30 d of incuba-tion; no microbial growth was observedfrom samples collected from the remainingfive animals with clinical signs or from thetwo bats without clinical signs (Table 2).Colony characteristics and microscopicobservations of all fungal isolates weresimilar and were morphologically consis-tent with Trichophyton. Attempts to re-culture skin samples from the five batcarcasses to specifically target Trichophy-ton were unsuccessful, possibly because ofthe fungus’ inability to survive freezing (batcarcasses were stored frozen).

Sequenced PCR products from directamplification of the fungal ITS of samplesfrom three of five bat carcasses mostclosely matched several undescribed


Trichophyton/Arthroderma spp. isolatedfrom cave soil (Lorch et al. 2013) andArthroderma quadrifidum (Brasch andGraser 2005) from the GenBank database.However, the ITS of A. quadrifidumdemonstrated only 98% identity with thatfrom the fungus detected on bats, indicat-ing that the bat-infecting fungus likelyrepresented a novel species. Three geneticvariants of the fungus were detected onthe three bats, differing from one anotherby one to four single nucleotide polymor-phisms (SNPs) across the ITS. Sequencingresults from direct PCR analysis from afourth bat most closely matched Debar-yomyces sp. (99% identity to Debaryo-myces marama, GenBank accessionJN942650), and that from the fifth animalwas not readable because of superimposedpeaks. The ITS of all fungal isolatesobtained from the swab samples wereidentical to two of the ITS sequencevariants of the putative Trichophyton

species obtained by direct PCR and DNAsequence analysis of skin samples from batcarcasses described earlier. Fungal ITS wasalso amplified and sequenced directly fromDNA extracted from swab samples, and all10 swabs yielded amplicons that wereidentical to the ITS sequences of the fungiisolated in culture. No amplicons wereproduced from direct PCR amplification ofthe samples collected from bats withoutclinical signs of fungal infection. Tapeimpressions taken from three bats con-tained dense fungal microconidia andhyphae morphologically consistent withthose observed in histopathology and inculture (Fig. 1C). The concordance ofmicroscopic, culture, and molecular evi-dence (Table 2) suggest that a putativeTrichophyton species was the cause of theskin infections observed in the hibernatingbats.

Sequences representing IGS-59, MCM7,RPB2, and TEF1 of the bat fungus were in

TABLE 2. Results of analyses conducted to investigate the cause of fungal skin infections in hibernating batsunrelated to white-nose syndrome. Culture results are based on DNA sequencing of the internal transcribedspacer region (ITS) of fungi cultured from a given sample. Molecular results are based on direct amplificationand DNA sequencing of the ITS from DNA extracted from a given tissue/swab sample. Microscopy results arebased on tape impressions of white fungal material observed on bats in the field; these tape impressions weresubsequently examined in the laboratory under a microscope, and fungi present were identified on the basis ofmorphology. All GenBank accession numbers represent sequences generated from fungal isolates except incases for which either culture analyses were not performed or there was no fungal growth on culture medium.

Identifier Culture resulta Molecular result Microscopy resultaITS GenBank

accessiona

23489-01 N/A Trichophyton redellii N/A KM09131223493-01 N/A Trichophyton redellii N/A KM09131323863-01 N/A Could not be interpretedb N/A N/A23863-02 N/A Debaryomyces sp. N/A KM09132023863-03 N/A Trichophyton redellii N/A KM09131444738-01 Trichophyton redellii Trichophyton redellii N/A KM09130844738-02 No fungal growth Trichophyton redellii Consistent with Trichophyton KM09131544738-03 Trichophyton redellii Trichophyton redellii Consistent with Trichophyton KM09130744738-04 No fungal growth Trichophyton redellii N/A KM09131644738-05 Trichophyton redellii Trichophyton redellii N/A KM09130944738-06 Trichophyton redellii Trichophyton redellii N/A KM09131044738-07 No fungal growth Trichophyton redellii N/A KM09131744738-08 Trichophyton redellii Trichophyton redellii N/A KM09131144738-09 No fungal growth Trichophyton redellii Consistent with Trichophyton KM09131844738-10 No fungal growth Trichophyton redellii N/A KM09131944738-11 No fungal growth No DNA amplification N/A N/A44738-12 No fungal growth No DNA amplification N/A N/A

a N/A 5 not applicable.b Superimposed peaks in the DNA sequence data prevented interpretation of results.


agreement with the ITS data, most closelymatching the few species of Trichophyton/Arthroderma (90–98% identity for IGS-59,82–85% identity for MCM7, 86–91%

identity for RPB2, and 90–92% identityfor TEF1) for which sequences of theseloci were available in GenBank. The IGS-39 sequence was more divergent, butportions of the sequence again mostclosely matched various species of Tricho-phyton/Arthroderma. Thus DNA se-quence data from multiple loci supportedthe placement of the bat-infecting funguswithin the dermatophyte group. The IGS-39 sequence of the bat-infecting fungusdemonstrated intraspecific variation at fiveSNPs over the 802-nucleotide regionsequenced, and MCM7 displayed similarlyhigh identity (i.e., two SNPs over 591nucleotides sequenced). However, IGS-59,LSU, RPB2, and TEF1 sequences wereidentical, indicating that the two variantslikely represent a single species.

Fungal elements were observed inhistopathology from all five bats examined.However, lesions diagnostic for WNS(Meteyer et al. 2009) and fertile structurescharacteristic of P. destructans (Fig. 1F;Gargas et al. 2009) were absent. Instead,fungi observed were associated with in-fections that were primarily superficial,with hyphae found on the surface of theskin, in the outer layers of keratin, andsometimes within intradermal pustules(Fig. 1E). Sessile, smooth-walled, obovateto pyriform microconidia borne laterallyand terminally on hyphae were present inassociation with hyphal colonization andinvasion of the keratin layer in three of thebats (cases 23489-01, 23863-01, 23863-03)and were suggestive of Trichophyton(Fig. 1E). Only vegetative hyphae lackingcharacteristics that allowed for morpho-logic identification were observed on theremaining two animals (cases 23493-01,23863-02).

Phylogenetic analysis

Internal transcribed spacer DNA se-quence data were used for the phylogenetic

analysis because the other loci sequenced(i.e., IGS, LSU, MCM7, RPB2, TEF1)were poorly represented for Trichophytonand related fungi in GenBank (completingmultigene phylogenetic analyses was notfeasible with existing data). Our samplingof dermatophyte ITS sequences for thisstudy followed that of Brasch and Graser(2005). New DNA sequences were alsogenerated from the original isolates usedto describe Trichophyton terrestre (Durieand Frey 1957) and its purported tele-omorph A. quadrifidum (Dawson andGentles 1961) using cultures UAMH 657and UAMH 2941 (from The University ofAlberta Microfungus Collection and Her-barium), respectively, because these datawere not previously available in GenBank(deposited for this study as GenBankaccessions KM091305 and KM091306,respectively). Deoxyribonucleic acid am-plification and sequencing for these iso-lates followed the methods describedearlier.

The best tree generated from the MLanalysis (Fig. 2) included a large, well-supported crown group consisting of theprimarily anthropophilic and zoophilicspecies of dermatophytes (groups 1 and2 as defined by Graser et al. [2000]), andthe remaining diversity included theprimarily geophilic species of dermato-phytes (group 3 species of Graser et al.[2000]) as basal to the crown group. Ingeneral, the topology of the best tree wasconsistent with earlier studies (Graser et al.2000; Brasch and Graser 2005), as was thegenerally poor resolution of relationshipsamong many taxa. However, sequencesthat represented isolates of the dermato-phyte recovered from bats in this studyresided within the geophilic group andformed a well-supported clade (87% MLBS, 88% parsimony-based BS) recognizedherein as Trichophyton redellii sp. nov.This clade is situated within another clade(86% ML BS, 96% parsimony-based BS)that includes A. quadrifidum, whichshares only 98% identity with T. redellii.Divergence in the sequences of IGS-59,


FIGURE 2. Best tree resulting from the maximum likelihood analysis. Bootstrap support values $70%

from 1,000 iterations from RAxML analysis are presented above branches, and bootstrap support values$70% from the 1,000 iterations from parsimony-based PAUP* analysis are bolded and presented belowbranches. Generic abbreviations are A. (Arthroderma), C. (Chrysosporium), M. (Microsporum), and T.(Trichophyton). GenBank accession numbers are followed by taxon name and, when appropriate, additionalidentifiers for each isolate. Scale bar50.05 nucleotide substitutions per site.


IGS-39, MCM7, RPB2, and TEF1 of A.quadrifidum obtained as described earlierfurther supported that T. redellii isdistinct from that species (IGS-59:87.1% identity among approximately 400nucleotides aligned; IGS-39: 85.8% iden-tity among 489 nucleotides; MCM7:93.7% identity among 552 nucleotides;RPB2: 97.2% identity among 861 nucle-otides; TEF1: 98.3% identity among 850nucleotides; A. quadrifidum [UAMH2941]. GenBank accessions KM091301,KM091304, KM091288, KM091292,KM091296, respectively).

The ITS-based phylogenetic analysis alsoindicated that T. terrestre is not conspecificwith its supposed teleomorph A. quadrifi-dum (Dawson and Gentles 1961; seeFig. 2). This was further supported byDNA sequence data from other locishowing significant divergence betweenthe two isolates (MCM7: 86.6% identityamong 552 nucleotides; RPB2: 92.0%

identity among 861 nucleotides; TEF1:95.1% identity among 850 nucleotides; T.terrestre [UAMH 657]. GenBank acces-sions KM091287, KM091291, KM091295,respectively).

Species Description

Trichophyton redellii: Minnis, J.M. Lorch,D.L. Lindner & Blehert, sp. nov. Fig. 3

MycoBank: MB809547

Diagnosis: This species is morphologicallysimilar to Trichophyton terrestre, butdiffers in its lack of growth at 25 C andoccurrence as an infectious agent onhibernating bats.

Holotype: US, Wisconsin, Grant Co., exwing of hibernating Myotis lucifugus, 23February 2012, collected by M. L. Verant,isolated by J. M. Lorch from NWHCNo. 44738-03, dried culture on SDA(holotype, CFMR 44738-03H); ex-type liv-ing culture CBS 134551 5 CFMR 44738-03; GenBank IGS (59 end: KM091299; 39

end: KM091302), ITS: KM091307, MCM7:KM091285, LSU: KM091297, RPB2:KM091289, TEF1: KM091293.

Etymology: The genitive species epithethonors the late David N. Redell for hisdiscovery of the infections caused by thisfungus and for his contributions to batconservation and research.

Description: Colonies exhibited no growthon SDA after 30 d at 25 C, ca. 3–5.5 mmin diameter on SDA after 14 d at 10 C,10.5–15.5 mm in diameter after 30 d at10 C; somewhat mounded at center;surface white to near yellowish white(4A2), velutinous; margin uncolored be-coming white, more or less even; reverselight yellow (3A5 or 4A5) to yellowishorange (4A7) near center and uncolored to

FIGURE 3. Trichophyton redellii (ex-type living culture): colony surface on Sabouraud dextrose agar(SDA) after 37 d incubated at 10 C in the dark (A); colony reverse on SDA after 37 d incubated at 10 C in thedark (B); conidia (C). Bar520 mm.


similarly colored as center to yellowishwhite (4A2) toward margin. Ascomata notobserved. Aerial hyphae abundant, 2–6 mmin diameter, septate, hyaline, walls thinand smooth. Thallic conidia borne singlyand laterally at right angles or terminallyon aerial hyphae, sessile or on short,simple pedicels, with macroconidia, mi-croconidia, and intermediate forms thatmake categorical distinction fairly inconse-quential. Conidia 6–65 mm 3 3–8 mm,length-to-width ratio (Q) 5 1.2–13.8(–21.7), ellipsoid, obpyriform, clavate,slightly apiosporous, cylindrical or irregu-lar, 0–5-septate and constricted or not atsepta, apices obtuse, bases slightly trun-cate with an annular frill, hyaline, wallsthin and smooth. Chlamydospores form-ing in vegetative hyphae or individualcells of conidia in older cultures, moreor less globose, hyaline, walls slightlythickened, up to approximately 22 mm indiameter.

Habitat and distribution: Found on hiber-nating Myotis lucifugus and Myotis veli-fer. Known from US: Indiana, Texas,Wisconsin.

Additional cultures examined: US, Wisconsin,Grant Co., ex wing of hibernating Myotislucifugus, 23 February 2012, collected byM. L. Verant, isolated by J. M. Lorch fromNWHC No. 44738-01, living culture CBS134550 5 CFMR 44738-01, GenBank ITS:KM091308; isolated by J. M. Lorch fromNWHC No. 44738-05, living culture CBS134552 5 CFMR 44738-05, GenBank ITS:KM091309; isolated by J. M. Lorch fromNWHC No. 44738-06, living culture CBS134553 5 CFMR 44738-06, GenBank ITS:KM091310; Pierce Co., ex wing of hiber-nating Myotis lucifugus, 19 March 2012,collected by M. L. Verant, isolated by J. M.Lorch from NWHC No. 44738-08; livingculture CBS 134554 5 CFMR 44738-08,GenBank IGS (59 end: KM091300; 39 end:KM091303), ITS: KM091311, MCM7:KM091286, LSU: KM091298, RPB2:KM091290, TEF1: KM091294.

DISCUSSION

The emergence of WNS highlighted ourlimited knowledge of fungi that colonizebat skin. Investigators have examinedfungal communities found on hibernatingbats (Johnson et al. 2013; Vanderwolf et al.2013b) and in sediment of bat hibernacula(Lorch et al. 2013). However, fungispecialized for growth on mammalian skinwere not prevalent in these studies. Themost common fungus detected on batswith clinical infections in our study wasthe previously undescribed dermatophyteTrichophyton redellii. Geophilic species ofTrichophyton/Arthroderma appear to beregular, although not necessarily abun-dant, inhabitants of caves and mines(Lorch et al. 2013; Vanderwolf et al.2013a) and have occasionally been report-ed as occurring on hibernating bats(Courtin et al. 2010; Vanderwolf et al.2013b). However, in previous studiesthere was no indication that these Tricho-phyton spp. were represented by anythingother than transitory conidia on bats. Theconcordance of microscopic, culture, mo-lecular, and histopathologic evidence inthis study suggests that T. redellii is indeedcapable of colonizing the skin of presum-ably healthy hibernating bats.

Although T. redellii has not beenpreviously described, it likely is native toNorth America. Introduced fungal patho-gens, like most exotic species, tend toexhibit very low genetic variation as aresult of founder effect (e.g., Engelbrechtet al. 2004; Dlugosch and Parker 2008).The genetic diversity in ITS, IGS, andMCM7 loci of T. redellii is inconsistentwith recent introduction. In contrast, therehas been no observed genetic variationamong North American isolates of the batpathogen P. destructans (Ren et al. 2012),a species strongly suspected to have beenintroduced to North America (Puech-maille et al. 2011). Infections causedby T. redellii were, instead, more likelyoverlooked before the discovery ofWNS. The threat of WNS prompted a


heightened awareness of fungal infections,and many state agencies (includingWDNR) more intensively monitored hi-bernating bat populations after the emer-gence of WNS. Thus, recent discovery ofT. redellii likely stemmed from increasedsurveillance.

Bats infected by T. redellii exhibitclinical signs that may be confused withWNS (Fig. 1A, B). Specifically, whitefungal growth is often evident on exposedskin. Bats with subtle signs generally havevisible infections limited to the elbows andwrists, whereas animals with more exten-sive colonization exhibit white on theforearms and, to a lesser extent, on thewing membranes, hind legs, uropatagium,tail, and ears. In contrast to WNS, clinicalmanifestation of T. redellii infection on themuzzle is apparently rare. With infectionsof T. redellii, the fungal colonizationpattern often has an active edge with acentral zone of clearing (Fig. 1A), similarto what is observed in classic humanringworm infection (which can be causedby other species of Trichophyton; Weitz-man and Summerbell 1995). While the‘‘ring’’ presentation is not typical of WNS,it is also not consistently observed with T.redellii; likewise, bats with WNS may lackvisible fungus on the muzzle. Thus, fieldsigns alone cannot reliably distinguish thetwo infections. However, the two condi-tions can be differentiated by performinga fungal tape impression of the whitematerial and examining fungal morpholo-gy under a microscope. While P. destruc-tans has distinctive asymmetrically curved,or crescent-shaped conidia borne at theends of verticillately branched conidio-phores (Gargas et al. 2009), T. redellii hasradially symmetric obovate to pyriformmicroconidia that attach laterally to thesides or ends of hyphae and are sessile oron very short pedicels (Fig. 1C, D).Trichophyton redellii does not form cup-like aggregates of fungal hyphae or causeskin erosion and ulceration characteristicof WNS (Meteyer et al. 2009); rather, T.redellii rarely penetrates beyond the

epidermis (Fig. 1E, F). Furthermore, batswith T. redellii infections have not beenobserved to exhibit any of the unusualbehaviors noted among bats with WNS,such as congregation near the entrances ofand premature egression from hibernacula(Turner and Reeder 2009). Infectionscaused by T. redellii were not observedto produce an orange fluorescence whenexposed to ultraviolet light (data notshown) as has been reported for P.destructans infections (Turner et al.2014). However, a larger sample size isrequired to determine whether this tech-nique is reliable for distinguishing be-tween the two infections.

Although P. destructans and T. redelliiare distantly related, the fungi shareseveral key characteristics. Both are slow-growing and psychrophilic, apparentlycapable of infecting bats only duringhibernation when the host’s body temper-ature is greatly reduced. Both may alsohave soil origins. The genus Pseudogym-noascus is well-represented in the sedi-ment of bat hibernacula, and P. destruc-tans likely evolved from a soil-dwellingancestor (Lorch et al. 2013). The closestrelatives to T. redellii have been found insoil, suggesting that the fungus may bederived from nonpathogenic soil-affiliatedspecies rather than existing pathogenicspecies that infect other mammalian hosts.Thus, T. redellii (along with P. destructans)may offer important insight into the factorsthat drive the evolution of fungal patho-gens. Additionally, T. redellii could prove tobe a valuable model for understanding theepidemiology of WNS because of itsbiologic and ecologic similarities to P.destructans. For example, both fungi mayshare common transmission routes, adap-tations for surviving when their hosts aremetabolically active (nonhibernal), andmechanisms that allow colonization of batskin.

In all sites where T. redellii has beenfound in Wisconsin, no mortality eventshave been observed and bat populationshave either increased or remained rela-


tively stable in the 3 yr that sites have beenmonitored by WDNR. Thus, it does notappear that T. redellii poses a significantthreat to populations of native bats.Although the abilities of both P. destruc-tans and T. redellii to act as infectiousagents could be related to a common traitof the host, such as hibernation, the twoinfections clearly have different outcomes.Future investigation of the relationshipbetween hibernating bats and T. redelliimay uncover mechanisms by which batscope with fungal infections during periodsof metabolic depression and further ex-plain why P. destructans is so deadly.

Infectious diseases in wildlife often shedlight on how little we know about hostspecies, pathogens, and the influence ofenvironmental factors on pathogenicity. Inthe case of WNS, deaths of millions of batsexposed our lack of understanding ofinteractions between hibernating batsand fungi that share a common environ-ment. We have demonstrated that fungalskin infections are not a novel occurrenceamong hibernating bats of North Americabut were likely overlooked before thearrival of P. destructans to the continent.Investigating other fungi such as T. redelliiand the infections they cause couldprovide an important means by which tostudy WNS pathogenesis, host and path-ogen ecology, and pathogen evolution, inaddition to better informing WNS surveil-lance and diagnostic efforts.

ACKNOWLEDGMENTS

This work was supported by the US Fishand Wildlife Service, the US GeologicalSurvey, the Wisconsin Department of NaturalResources, and the US Forest Service. Wethank Andrew Badje and Tyler Brandt fortheir help in collecting samples; D. Earl Greenfor contributing postmortem examinationand histopathologic findings; Brenda Ber-lowski-Zier, Douglas Berndt, Elizabeth Bo-huski, Kathryn Griffin, and Dottie Johnson forassistance with postmortem examinations orroutine diagnostic work; and Anne Ballmann,Jennifer Buckner, and C. LeAnn White forcoordinating sample submission. We thank AlHicks for providing a photograph of a bat with

white-nose syndrome (Fig. 1B). The use oftrade, product, or firm names is for descriptivepurposes only and does not imply endorse-ment by the US government or by the State ofWisconsin.

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Submitted for publication 20 May 2014.Accepted 23 July 2014.


the fungus trichophyton redellii sp. nov. causes skin ...the fungus trichophyton redellii sp. nov....

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