ORIGINAL PAPER

First isolation of Pseudogymnoascus destructansin bats from Portugal

Maria das Neves Paiva-Cardoso & Francisco Morinha & Paulo Barros &Hélia Vale-Gonçalves & Ana Cláudia Coelho & Lisete Fernandes & Paulo Travassos &Ana Sofia Faria & Estela Bastos & Mário Santos & João Alexandre Cabral

Received: 26 December 2013 /Revised: 20 May 2014 /Accepted: 3 June 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract The psychrophilic fungus Pseudogymnoascusdestructans (formerly known as Geomyces destructans) isconsidered the etiological agent of white-nose disease(WND), an emerging disease which affects bats during theirhibernation period. This disease is clinically characterized bythe growth of a white fungus on muzzle, ears, and wings’membranes of affected bats. This infection caused the death ofseveral million bats in North America. Conversely, Europeanbats show no evidence of significant mortality occurrencesassociated with P. destructans colonization. This fungus hasbeen isolated from bats in at least 15 European countries since2008, but was never before reported in the Iberian Peninsula.

This study describes the first case report of P. destructanscolonization in bats from Portugal. We isolated P. destructansfrom three hibernatingMyotis blythii (lesser mouse-eared bat)with visual signs of P. destructans colonization, during aroutine visit to a mine located in the Trás-os-Montes region,Northern Portugal.M. blythii is one of the rarest bat species inEurope, classified as critically endangered in Portugal.P. destructans was obtained from at least three different partsof the body of each specimen analyzed. The identification ofthe respective fungal isolates was based on the macroscopicand microscopic characterization of the cultures and con-firmed by PCR-based analysis. All nucleotide sequences ob-tained showed 100 % identity with previous data reported forP. destructans. This new finding improves the current knowl-edge about the European distribution of P. destructans, whichis of great interest for forthcoming studies on the fungusdispersion and impact among bat populations at regionaland/or global level.

Keywords Pseudogymnoascus destructans .White-nosedisease .WND . Bat .Myotis blythii . Portugal

Introduction

White-nose disease (WND), firstly known as white-nose syn-drome (WNS), is an emerging infectious disease that causedthe death of several million bats in eastern North America,with unprecedented devastating effects on the hibernatingpopulations (Cryan et al. 2013). The first evidence of WNDin North America was recorded in 2006 in east central NewYork caves, where some local hibernating bats exhibited anunusual white substance on their muzzles (Blehert et al. 2009).Due to its epizootic character, the disease spreads to otherareas, and WND has now been detected in 23 USA states and5 Canadian provinces (U.S. Fish and Wildlife Service 2014).

Communicated by C. Gortázar

Electronic supplementary material The online version of this article(doi:10.1007/s10344-014-0831-2) contains supplementary material,which is available to authorized users.

M. d. N. Paiva-Cardoso (*) :A. C. Coelho :A. S. FariaDepartment of Veterinary Sciences, School of Agrarian andVeterinary Sciences, Veterinary and Animal Science Centre(CECAV), University of Trás-os-Montes and Alto Douro (UTAD),Quinta de Prados, P.O. Box 1013, 5000-801 Vila Real, Portugale-mail: maria_neves_cardoso@msn.com

M. d. N. Paiva-Cardoso : F. Morinha :H. Vale-Gonçalves :M. Santos : J. A. CabralCentre for the Research and Technology of Agro-Environment andBiological Sciences (CITAB), UTAD, Vila Real, Portugal

F. Morinha : P. Barros :H. Vale-Gonçalves : P. Travassos :M. Santos : J. A. CabralLaboratory of Applied Ecology, UTAD, Vila Real, Portugal

F. Morinha : E. BastosInstitute for Biotechnology and Bioengineering, Centre of Genomicsand Biotechnology, UTAD (IBB/CGB-UTAD), Vila Real, Portugal

L. FernandesElectronic Microscopy Unit, UTAD, Vila Real, Portugal

Eur J Wildl ResDOI 10.1007/s10344-014-0831-2

The clinical presentation of WND is characterized by thepresence of conidia and hyphae of a white fungus on bats’muzzles, pinnae, and/or wings, which was isolated and sub-sequently classified as a novel species of the genusGeomyces,family Pseudeurotiaceae, designated Geomyces destructans(Gargas et al. 2009). More recently, a change of itstaxonomic classification to Pseudogymnoascus destructanshas been proposed by Minnis and Lindner (2013), based onthe phylogenetic analysis of eastern North AmericanGeomyces and allies isolates with evidences that the closestrelatives of this group are members of Pseudogymnoascusroseus species complex. P. destructans is a psychrophilicfungus, with an optimal in vitro growth temperature range of12.5–15.8 °C (upper limit of 19.0–19.8 °C), whose vitalproliferation is limited to cool environments such as thosetypically observed inside bat hibernacula caves (Verant et al.2012). Morphologically, P. destructans is characterized byasymmetrically curved conidea (Gargas et al. 2009). Fungalhyphae are branched and septated, with variable diameter andshape (parallel or irregular walls). Curved-shaped conidiameasuring 2.5 μm in width and 7.5 μm in length have uni-or bi-lateral blunted ends and a basophilic center (Meteyeret al. 2009).

Phylogenetic analysis of fungal isolates obtained from bathibernacula in eastern North America reinforces the hypothe-sis that this pathogen is non-native and invasive in easternNorth America since there are no closely related sister taxabetween P. destructans and other cohabitant fungi in thisgeographical area (Minnis and Lindner 2013).

In Europe, studies investigating potential samples ofP. destructans, collected between 2008 and 2010, confirmedthe occurrence of P. destructans in six countries (France,Germany, Switzerland, Czech Republic, Slovakia, and Hun-gary) (Martínková et al. 2010; Puechmaille et al. 2010;Wibbelt et al. 2010; Šimonovičová et al. 2011). Despite theadditional confirmation of P. destructans in association withbats of Austria, Belgium, Denmark, Estonia, The Netherlands,Poland, Romania, Turkey, and Ukraine, the full extent ofdistribution of P. destructans in Europe may not yet be known(Puechmaille et al. 2011).

Clinical signs of WND in bats include the characteristicpresence of white fungal proliferation on glabrous skin ofmuzzle, ears, and wings, anomalous behavior in winter (name-ly, frequent arousal from torpor), significant depletion of fatreserves, hypotonic dehydration, and concomitant electrolytedepletion (Foley et al. 2010; Reeder et al. 2012; Warneckeet al. 2012; Cryan et al. 2013). Histopathologically, the WNDlesions in North American bats reveal hyphae invasion ofepidermis, dermis, and underlying connective tissue, hairfollicles, and associated sebaceous glands, as well as thepresence of P. destructans characteristic conidiospores(Meteyer et al. 2009). Conversely, European bats presentfungal invasion of the skin restricted to the epidermis and

adnexa, and no cause–effect relationship between mortalityand P. destructans infection has been reported (Puechmailleet al. 2011; Wibbelt et al. 2013).

In the European context, only the species belonging to theMyotis genera have been recorded with P. destructans coloni-zation, despite the simultaneous presence of other bat speciesin the same caves (e.g.,Miniopterus schreibersii, Rhinolophushipposideros, and Rhinolophus ferrumequinum) (Puechmailleet al. 2011). Myotis is one of the most diverse genera, repre-sented by 103 species through the Holarctic region (Simmons2005; Stadelmann et al. 2007).

The present paper describes the first case report ofP. destructans colonization in bats from Portugal and theIberian Peninsula. P. destructans was isolated from lessermouse-eared bats (Myotis blythii, Tomes 1857) during thehibernation season. M. blythii is one of the rarest species inEurope, however, with a large Euro Asiatic distributionalrange (Dietz et al. 2009). In Portugal, the species is classifiedas critically endangered (CR), where the area of occupancy isvery restricted (less than 10 km2) and highly fragmented(Palmeirim et al. 1999; Rodrigues et al. 2003). The totalpopulation effective encompasses less than 2,500 mature in-dividuals (Cabral et al. 2006), mainly recorded in colonies(almost exclusively in caves) from the Southern (Algarve) andNorthern (Trás-os-Montes) regions of Portugal (Palmeirimet al. 1999).

Material and methods

Study area

During a routine census of hibernating bats, carried out inmines near Vila Real (41° 15.458′ N; 7° 53.405′ O), NorthernPortugal, three individuals of Myotis blythii were found, inMarch 2013, with visual signs of P. destructans colonization(Fig. 1a). Other six individuals of this species were alsoidentified in the same cave, an old mine called “Maria Isabel”,as well as three Plecotus auritus and five R. ferrumequinum,all without visual signs of fungal growth. The average airtemperature and humidity near the animals’ position were7.1 °C and 88 %, respectively.

Sample collection

Samples were collected from live bats using wet sterile cottonswabs (immersed in sodium chloride solution 0.9 %) bygently touching skin lesions compatible with P. destructanscolonization and promptly submerged in cap tubes with sterilesaline solution in order to avoid environmental contamination.They were collected from whitish lesions present on bats’muzzles, ears/pinnae, wings, and feet. These procedures fullyrespect the Guidelines of European Community Directive 92/

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43/EEC (http://ec.europa.eu/environment/nature/legislation/habitatsdirective/index_en.htm) and the Agreement on theConservation of Populations of European Bats (www.eurobats.org). No bat mortality was observed in the studycave.

A total of 15 samples were taken from the three specimensof M. blythii with obvious fungal growth on their bodies(Fig. 1a).

Culture, isolation, and microscopic identification

Each swab was then streaked into three Sabouraud dextrose(LiofilchemTM), Potato dextrose (DifcoTM), and Dermato-phyte Test Medium (MicrokitTM) agar plates each,subcultivated every 2 weeks and monitored regularly to phys-ically remove any fungal growth that was not morphologicallysimilar to P. destructans.

The plates were sealed with ParafilmTM and incubatedinverted in the dark at 12±2 °C, placed in plastic containerscontaining moist paper towels to maintain humidity.

The cultures were observed and photographed both mac-roscopically and microscopically. All isolates were observedin fresh preparation under darkfield microscopy and stainedwith lactophenol cotton blue for further observation by lightmicroscopy (Fig. 1b). Mix cultures were successivelysubcultivated until pure cultures were obtained. Samples iso-lated from pure cultures were submitted to a scanning electronmicroscope (SEM) analysis (FEI Quanta 400 ESEM). Thesamples were placed on aluminum stubs and fixed with

carbon glue. For visualization, the low vacuum mode wasused, with a partial pressure in the chamber of 7 mbar andan accelerating voltage of 20 kV. Pictures were taken duringthe experiment (Fig. 1c).

Molecular identification

The identification of isolated fungi was also accomplished byPCR analysis and subsequent sequencing of the obtained PCRproducts. The DNA extraction from culture isolates was per-formed using the ZR Fungal/Bacterial DNAMiniPrep (ZymoResearch) according to manufacturer’s instructions, withsome modifications: (a) cell disruption was carried out usinga standard vortex at maximum speed for 10 min and (b) DNAwas collected by two sequential elutions with 50 μl of DNAelution buffer and incubated for 5 min at room temperaturebefore each final centrifugation.

The genetic characterization of isolated fungi was per-formed by PCR-based analysis of the internal transcribedspacer (ITS) regions (ITS1, 5.8S, and ITS2) and the smallsubunit (SSU) of the rRNA gene. PCRs were performed in atotal volume of 10 μl containing 5 μl of 2× MyTaq HS Mix(Bioline), 2.5 pmol of each primer (Supplementary table), and0.5 μl DNA (20–50 ng). The amplification protocol for ITSregion was carried out with an initial denaturation at 95 °C for5 min; followed by 30 cycles of denaturation at 95 °C for 30 s,annealing at 57 °C for 45 s, and extension at 72 °C for 30 s;and a final extension at 60 °C for 10 min. These amplificationconditions were used for SSU region with a different

Fig. 1 Pseudogymnoascus destructans present in skin lesions of batsfrom Northern Portugal. a Fungal growth on muzzles, wings, and feet ofMyotis blythii (lesser mouse-eared bat); b Bright field microscope pho-tography of P. destructans isolate stained with lactophenol cotton blue—

conidiophores and curved conidia, magnification ×2500; c Scanningelectron micrograph (FEI Quanta 400 ESEM) of P. destructans iso-late—conidiophores and curved conidia, magnification ×5000

Table 1 Characterization ofPseudogymnoascus destructansisolates submitted to PCR-basedanalysis

Bat Isolate Body region GenBank Accessions

Number Species SSU ITS

1 Myotis blythii F1A Ear KF866376 KF866377

2 Myotis blythii F5A Nose KF866376 KF866378

3 Myotis blythii F8A Foot KF866376 KF866377

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annealing temperature (55 °C) and extension time (72 °C for45 s). The PCR products were purified with Illustra ExoStar 1-Step (GE Healthcare) and sequenced at Stab Vida (Lisbon,Portugal) (see sequencing primers in Supplementary Table).Sequence contigs were assembled and edited using theCodonCode Aligner version 4.2.3 (http://www.codoncode.com/aligner/) and submitted to GenBank database (Table 1).These sequences were compared with available genomic datausing BLASTN 2.2.28+ (Zhang et al. 2000).

Results and discussion

Fungal isolates morphologically similar toP. destructans (e.g.,asymmetrical curved conidia) were obtained from all 15swabs used to collect samples. Some of the initial culturesderived from samples were mixed cultures and other fungalspecies such as Verticillium leptobactrum, Tolypocladiumcylindrosporum, Histoplasma sp., Penicillium sp., andMucorsp. were identified (data not shown). The isolation rate ofP. destructans obtained by the cultural technique used was100 %, since all samples collected from suspect bats originat-ed cultures consistent with P. destructans microscopic mor-phology (Fig. 1b, c). The fungal colonies grew slowly andshowed phenotypic characteristics consistent with publisheddescriptions for P. destructans (Gargas et al. 2009). Moreover,we have obtained isolates compatible withP. destructans fromthe muzzle (bat no.1 and no.2), the ear (bat no.1), lesions onboth the dorsal (bat no.1, 2 and 3) and ventral (bat no.2 and 3)wing surfaces, and the foot (bat no. 3), which means that thefungus was simultaneously recovered from at least three dif-ferent parts of the body of each specimen ofM. blythii studied.

One characteristic isolate [e.g., conidea morphologicallyidentical to those of P. destructans as described by Gargaset al. (2009)] from each bat was chosen for subsequent PCRamplification and DNA sequence analyses. All three isolatesyielded identical SSU gene sequences that matched 100 % tothat of the type isolate of P. destructans (e.g., GenBankAccession GU350434). Two of the three isolates yieldedITS region sequences identical to that of the type isolate(e.g., GenBank Accession HM222616), while sequence fromthe third isolate (F5A) differed from that of the type by a singlenucleotide polymorphism. ITS region sequence from isolateF5A did, however, match that from other isolates ofP. destructans within the GenBank database (e.g., GenBankAccession HM584976).

To our best knowledge, this is the first time thatP. destructans is isolated from bats in Portugal. Microbiolog-ical and molecular analyses demonstrated the presence ofP. destructans in all three individuals (M. blythii) with externalsigns of P. destructans colonization. Although the presence ofother bat species was recorded in the same cave, such as

P. auritus and R. ferrumequinum, the clinical evidences ofP. destructans growth were only observed in specimens ofM. blythii. Since the first observation of the fungus in batsfrom the “Maria Isabel” mine (March 2013) no deaths wasobserved in this cave during the remaining hibernation period.This trend is corroborated by other studies conducted inEurope (Puechmaille et al. 2010; Wibbelt et al. 2010). In fact,mortality rates imputable to P. destructans colonization, withthe magnitude of those caused by WND in North America onhibernating bats, were never reported in Europe. In addition,all threeM. blythii specimens examined showed normal bodyweights for the species [bat no.1 (male), no.2 (female), andno.3 (female) with 25, 28, and 23 g, respectively], which is agood condition indicator for their survival success throughoutthe hibernation period (Dietz et al. 2009; Aşan and Albayrak2011; Barros 2012).

The identification of bats affected by P. destructans in over15 European countries, and now also in Northern Portugal,reinforces the hypothesis that P. destructans is widespreadacross Europe, including the Iberian Peninsula and, probably,other Mediterranean countries. Further studies are needed toclarify the extent of the distribution of P. destructans amongpopulations of bats in the Trás-os-Montes region and otherregions of Portugal, whose impact is far from being fullyknown and understood.

Acknowledgments This study was supported by funding from severalecological monitoring projects of the Laboratory of Applied Ecology(University of Trás-os-Montes and Alto Douro) and by Portuguese Foun-dation for Science and Technology (FCT) through the project PEst-OE/AGR/UI4033/2014 and the PhD Grant SFRH/BD/77872/2011.

Conflict of interest The authors declare that they have no conflict ofinterest.

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