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First confirmation of Pseudogymnoascus destructans in British bats and hibernacula
  1. A. M. Barlow, BVSc, MSc, MRCVS1,
  2. L. Worledge, BSc, MSc4,
  3. H. Miller, BSc, MSc4,
  4. K. P. Drees, BSc, PhD5,6,
  5. P. Wright, BSc (Hons)7,
  6. J. T. Foster, BA, PhD5,6,
  7. C. Sobek, BSc5,
  8. A. M. Borman, BSc, PhD8 and
  9. M. Fraser, BSc, MSc8
  1. 1APHA-Starcross VI Centre, Staplake Mount, Starcross, Exeter, Devon EX6 8PE, UK
  2. 2Formerly AHVLA Langford, Langford House, Langford, Somerset BS40 5DX, UK
  3. 3Wildlife Network for Disease Surveillance, University of Bristol, School of Veterinary Science, Langford, Somerset BS40 5DU, UK
  4. 4Bat Conservation Trust, Quadrant House, 250 Kennington Lane, London SE11 5RD, UK
  5. 5Center for Microbial Genetics and Genomics, Northern Arizona University, 1298 S Knoles Dr, Flagstaff, Arizona 86011-4073, USA
  6. 6Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, New Hampshire 03824, USA
  7. 7APHA-Penrith VI Centre, Merrythought, Calthwaite, Penrith, Cumbria CA11 9RR, UK
  8. 8Mycology Reference Laboratory, Public Health England South-West Regional Laboratory, Myrtle Road, Kingsdown, Bristol BS2 8EL, UK
  1. E-mail for correspondence: alexmbarlow{at}btinternet.com

Abstract

White-nose syndrome (WNS) is a fatal fungal infection of bats in North America caused by Pseudogymnoascus destructans. P. destructans has been confirmed in Continental Europe but not associated with mass mortality. Its presence in Great Britain was unknown. Opportunistic sampling of bats in GB began during the winter of 2009. Any dead bats or samples from live bats with visible fungal growths were submitted to the Animal Health and Veterinary Laboratories Agency for culture. Active surveillance by targeted environmental sampling of hibernacula was carried out during the winter of 2012/2013. Six hibernacula were selected by their proximity to Continental Europe. Five samples, a combination of surface swabs or sediment samples, were collected. These were sent to the Center for Microbial Genetics and Genomics, Northern Arizona University, for P. destructans PCR. Forty-eight incidents were investigated between March 2009 and July 2013. They consisted of 46 bat carcases and 31 other samples. A suspected P. destructans isolate was cultured from a live Daubenton's bat (Myotis daubentonii) sampled in February 2013. This isolate was confirmed by the Mycology Reference Laboratory, Bristol (Public Health England), as P. destructans. A variety of fungi were isolated from the rest but all were considered to be saprophytic or incidental. P. destructans was also confirmed by the Center for Microbial Genetics and Genomics in five of the six sites surveyed.

  • Wildlife
  • Fungal diseases
  • Disease surveillance
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White-nose syndrome (WNS), a fatal fungal infection of hibernating insectivorous bats, has become one of the most catastrophic infectious wildlife diseases ever recorded in North America. The first case of WNS was in bats from Howes Cave near Albany, New York State, USA, in February 2006. The syndrome is caused by Pseudogymnoascus (previously Geomyces) destructans, a psychrophilic fungus. It is suggested that skin infection causes pathology leading to increased evaporative water loss and thus dehydration. This in turn causes energetically expensive arousal from hibernation, loss of body fat and starvation (Cryan and others 2010). WNS has since spread to a total of 25 states in the USA and five provinces in Canada. It has been confirmed in seven bat species, with P. destructans identified in an additional five species in North America. WNS has killed more than 5.7 million bats, and in some hibernacula 90–100 per cent mortality has been reported (http://whitenosesyndrome.org).

The presence of P. destructans was identified in Continental Europe in 2008 (Puechmaille and others 2010). It has now been confirmed in at least 14 European countries and is suspected in four more due to convincing photographic evidence (Frick and others, 2014) from 18 bat species (Martínková and others 2010, Wibbelt and others 2010, Puechmaille and others 2011, Zukal and others 2014). However, there has been no known incidence of mass mortality of bats due to P. destructans in Continental Europe (Puechmaille and others 2011). At present, the main European criteria for WNS are the presence of P. destructans, skin pathology as described by Meteyer and others (2009) and, crucially, mass mortality of bats. Paiva-Cardoso and others (2014) use the term white-nose disease, which would include the first two criteria above without evidence of mass mortality and thus would be applicable in both North America and Europe.

Transmission experiments carried out in North America comparing North American and European isolates in little brown bats (Myotis lucifugus) found that isolates from both regions caused similar dermal pathology and death (Warnecke and others 2012). The pathology included fungal hyphae causing cup-like epidermal erosions and ulceration of the skin without cellular inflammatory response, consistent with pathology diagnostic for WNS (Meteyer and others 2009).

A single clonal type of P. destructans has been recorded in North America (Rajkumar and others 2011). However, P. destructans is genetically diverse in Europe, and isolates with high genetic similarity (i.e. potential source populations) to those from North America have not yet been found (K. Drees and J. Foster, unpublished data). The wide distribution and genetic diversity of P. destructans in Continental Europe and the absence of mass mortalities in bats suggest that P. destructans has been present in Europe (and possibly Eurasia) for a prolonged period. European bats appear to have co-evolved with P. destructans and the introduction of P. destructans into North America may have been from Europe. As part of a project to predict the potential distribution of P. destructans throughout Europe, Puechmaille (unpublished data) indicated that environmental conditions should be suitable for the fungus in a large part of Great Britain.

No evidence of the fungus or mass mortalities had been previously reported in Great Britain. However, it was unknown if P. destructans was absent from Great Britain and whether British bat species would be susceptible to the fungus, or if they had innate resistance, as is the case with Continental bats.

Material and methods

Opportunistic sampling of bats

Opportunistic sampling began in 2009, carried out by licensed volunteers from the Bat Conservation Trust (BCT) during winter hibernacula visits for BCT's National Bat Monitoring Programme (www.bats.org.uk/nbmp). The number of sites surveyed each year ranged from 786 to 905. Any dead bats with visible fungal growth found during the surveys were submitted to Animal Health and Veterinary Laboratories Agency (AHVLA) Langford under the Diseases of Wildlife Scheme for initial European bat lyssavirus screening and fungal culture. Any live bats were sampled using a loop of sticky tape gently touched onto visible fungal growth (http://www.nwhc.usgs.gov/disease_information/white-nose_syndrome/WNS_sample_methods.pdf). These tape samples were stuck on to a piece of clear plastic and submitted to AHVLA Langford, where they were examined microscopically at 100 x magnification to screen for evidence of the characteristic P. destructans conidia. The tape samples were then detached from the plastic film for fungal culture. Swab and tape samples were plated on to Sabouraud Dextrose Agar plates (Oxoid, Ref PO0160A) and cultured at 5°C at AHVLA Penrith. The cultures were examined regularly for growth for up to 40 days. Genomic DNA was extracted from fungal isolates using pre-punched Flinders Technology Associates filters (Borman and others 2010) and subjected to PCR using primers ITS5 and ITS2 (White and others 1990) designed to amplify the internal transcribed spacer 1 (ITS1) region of the nuclear ribosomal RNA gene cassette. Sequences of the resulting PCR amplicons were used to search the GenEMBL database and a database of sequences generated by the Public Health England (PHE) Mycology Reference Laboratory (MRL) in Bristol.

It was reported from the field that there were considerable difficulties sampling fungal growth on live bats roosting in crevices in hibernacula using sticky tape. Therefore, during the summer of 2012, field workers were supplied with fine rayon-tipped wire shaft swabs (MWE Dryswab product MW142), which were in use during the winter of 2012/2013.

Active surveillance

By May 2012 no isolates of P. destructans had been recovered from the 31 submissions received. Therefore, targeted environmental sampling of hibernacula during the winter of 2012/2013 was initiated. Six hibernacula were selected for initial surveillance based on their proximity to Continental Europe (Kent—3; Sussex—3), whether any suspect cases had been found in the past (3) and the type of site to give a variety of hibernaculum substrates including chalk, sandstone and brick (Fig 1). The hibernacula were sampled in early 2013 and five environmental samples from each hibernaculum were collected. Sampling included any combination of surface substrate swabs and sediment samples. Each survey team was supplied with a kit including five sterile metal spatulas (SLS spatula 200 mm Micro Chattaway StSteel, Cat no. SPA1076), in individual sterile pouches, sterile plastic shaft rayon swabs (MWE Dryswab product code MW102), 10 ml plastic tubes containing sterile water and 2 ml sterile screw-cap tubes.

FIG 1:

South East England. Red star=positive environmental sample; blue star=positive environmental sample and positive swab from bat; black star=No positive results

Swabs were used on the wall surfaces of the site, close to where bats were or had previously been present. An area 10 cm×10 cm of the hibernaculum wall was sampled using sterile swabs dipped in sterile water. The swab tip was put into a sterile 2 ml screw-cap tube and the excess handle was broken off before capping. Sediment samples were taken from the floor of the hiberaculum away from the main entrances and those areas used by cavers or others entering the hibernaculum. Any surface debris was first cleared and approximately 2 g of superficial sediment with particles less than 2 mm in diameter were collected into a sterile 2 ml screw-cap tube using sterile metal spatulas. Each individual sample tube was labelled with the hibernaculum ID (name of site) and tube number to ensure easy identification. A sampling record sheet was kept to record the details of each site sampled with approximate location in the hibernaculum. The completed sample set (all five sample tubes) was put into a clean ziplock bag. The outside of this was wiped with fungicidal disinfectant (Byofresh Cage Clean Wipes) to prevent cross-contamination before being put into a further zip bag. The double-bagged samples from each hibernaculum were then put into an SLS 180 ml polypropylene pot (catalogue number CON8542) and transported to AHVLA Langford. Here, each sample tube was labelled with a unique laboratory reference number.

The samples were shipped to the Center for Microbial Genetics and Genomics at Northern Arizona University by FedEx for genetic analysis. Samples were put into a secondary ziplock bag, which was sprayed with 10 per cent bleach with a contact time of 10 minutes before drying. The complete batch of samples was put into a Pathopak 3 litre Biobottle (Intelsius product code PP013). This packaging is in compliance with the International Air Transport Association packing instructions 620 and 650 and UN 4GU/class 6.2/12/GB/5444. DNA was extracted directly from substrate swabs and sediment samples without culturing as previously described (Shuey and others 2014). A gene sequence specific for P. destructans was detected in the DNA extracts using the IGS-qPCR (Muller and others 2013). The TaqMan Exogenous Internal Positive Control (Life Technologies, Carlsbad, California, USA) was used to detect inhibitors in template DNA.

Results

Opportunistic sampling of bats

Fifty separate reports of bats with fungal growth were investigated between March 2009 and July 2013. 45 of these were of dead bats, mostly singles but up to four at any one site. 61 carcases with or without skin tape or swab samples, taken in situ in the hibernacula, were submitted from 44 of these. In one case (lesser horseshoe bat (Rhinolophus hipposideros)) only a tape sample was submitted. The bat species submitted, with number of carcases in brackets, included greater horseshoe bat (Rhinolophus ferrumequinum) (8), lesser horseshoe bat (18), brown long-eared bat (Plecotus auritus) (9), Natterer's bat (Myotis nattereri) (6), Daubenton's bat (Myotis daubentonii) (2), whiskered/Alcathoe/Brandt's bat (1), unidentified Myotis species (2), Pipistrelle species (12) and unidentified bat (3). The other five were of single reports in live bats with skin fungal growths including two Daubenton's bats and single cases of whiskered/Alcathoe/Brandt's bat, Noctule and a Pipistrelle species. The majority of these reports were from the south and west of England with only two from Wales and one from Scotland.

A suspected P. destructans isolate was cultured from a white focus (approximately 2.5 mm diameter) on the ear of a live Daubenton's bat that had been sampled with a fine rayon-tipped wire shaft swab in a Kent hibernaculum in February 2013. This site was also part of the active surveillance study, with all five samples taken from the hibernacula walls. A variety of other fungi were isolated from the carcases, and tape/swab samples submitted from the other species report case submissions, including Rhizopus species, Penicillium species, Scopulariopsis species, Mucor species, Gliocladium species, Paeciliomyces species and Myceliophthora species. All these were considered to be saprophytic or incidental findings.

The suspected P. destructans isolate was submitted to the MRL, Bristol (PHE), for further identification. The organism was incapable of growth at temperatures above 20°C, but grew readily at 4°C. Colonies on Sabouraud's dextrose agar (10 mm in 14 days at 4°C) were restricted with white aerial mycelium and a dark, drab brown reverse colony colour. Upon extended incubation at 4°C, the colony centre developed a drab grey colouration with extensive sporulation. Microscopic examination of cultures revealed asymmetrically curved, reniform conidia born singly and occasionally in short chains on penicilliately branched conidiophores. Conidia were initially thin walled, smooth, hyaline and measured 5–10 cm x 2–3 cm (see Fig 2); with age, conidia became conspicuously thick walled.

FIG 2:

Light microscopy of the isolate of P. destructans (National Collection of Pathogenic Fungi 7867) (lacto-fuchsin) (Bar=10 μm)

Sequencing of the isolate cultured from the Daubenton's bat in the Kent hibernaculum using the extraction and PCR methods described in Borman and others (2006, 2010) demonstrated that the current isolate shared 100 per cent nucleotide homology with P. destructans isolates when compared with sequences from GenBank (Accession numbers KF866378; HM584970; HM584976). The morphology of the fungal isolate (Fig 2) was also consistent with P. destructans. The sequence from the P. destructans isolate described has been deposited at GenBank under accession number HG798544 and the viable isolate has been stored in the National Collection of Pathogenic Fungi (NCPF) housed at the PHE MRL under the unique identifier NCPF 7867. Although no histopathology was performed to confirm active infection of bats, this is the first confirmed isolate of P. destructans in Great Britain.

Active surveillance

P. destructans was detected in substrate swabs and sediment via IGS-qPCR in five of the six sites surveyed (Table 1). Site B, from which the P. destructans NCPF 7867 isolate was obtained during opportunistic sampling of bats, tested positive for P. destructans in one of the five surface substrate swab samples collected.

TABLE 1:

Results of environmental sampling

Discussion

Seven of the 17 species of resident and breeding British bats are UK Biodiversity Action Plan priority species. If WNS was to occur in Great Britain it could have serious consequences for some of these bat species, many of which use underground structures for hibernation. This surveillance was important to determine if P. destructans was present and if so, whether it was associated with bat mortality due to WNS.

The opportunistic sampling and targeted active surveillance work have confirmed the presence of P. destructans in Great Britain for the first time. The environmental sampling approach provides an effective means of determining the presence of the fungus without unnecessary disturbance to hibernating bats.

The results of this opportunistic and active surveillance work indicate that, similar to other reports from Europe, P. destructans is present in Great Britain, but is not causing mortality. There is room for cautious optimism as a consequence of these latest findings. However, further active surveillance work, in addition to ongoing opportunist sampling, is required to understand the distribution of P. destructans across Great Britain and a fuller understanding of disease and/or conservation threats to British bats.

Acknowledgments

This work was funded by a grant from Bat Conservation International and by the Bat Conservation Trust and AHVLA Diseases of Wildlife Scheme (AHVLADoWS). The authors are grateful to the volunteers who took part in the active surveillance project and to those bat workers who have submitted swabs and dead bats for testing as part of the opportunistic sampling. Also, we would like to thank Tina Kelly for laboratory support at AHVLA Langford.

References

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Footnotes

  • Provenance: not commissioned; externally peer reviewed

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