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Appendix A Contributed Manuscripts A1 THE EMERGENCE OF CRYPTOCOCCUS GATTII IN BRITISH COLUMBIA AND THE PACIFIC NORTHWEST1 Karen H. Bartlett, Sarah E. Kidd, and James W. Kronstad2 An unprecedented emergence of cryptococcal infections in animals and oth- erwise healthy humans was recognized in 1999 on the east coast of Vancouver Island, British Columbia. Unexpectedly, these infections were caused by Cryp- tococcus gattii, a species closely related to the AIDS-associated fungal pathogen Cryptococcus neoformans. Human cases have continued over the past 8 years and now total approximately 170 with eight deaths. Extensive environmental 1 Reprinted with kind permission from Springer Science+Business Media: Current Infectious Dis- eases Reports, The emergence of Cryptococcus gattii in British Columbia and the Pacific Northwest, 10, 2008, p. 108–115, Karen H. Bartlett, Sarah E. Kidd, and James W. Kronstad. Current Infectious Disease Reports 2008, 10:58–65 Current Medicine Group LLC ISSN 1523-3847 Copyright © 2008 by Current Medicine Group LLC Papers of particular interest, published recently, have been highlighted as: + Of importance ++ Of major importance 2 Karen H. Bartlett, PhD, Sarah E. Kidd, PhD, and James W. Kronstad, PhD. Corresponding author: James W. Kronstad, PhD, The Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, V6T 1Z4, Canada. Email: kronstad@interchange.ubc.ca. 101

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102 FUNGAL DISEASES sampling, coupled with detailed molecular typing of isolates, revealed areas of permanent and transient colonization with primarily three genotypes of the fungus. C. gattii was found in air, soil, water, and in association with numerous tree species. Importantly, there is solid evidence for human-mediated dispersal of the pathogen, and C. gattii has now been detected in the environment on the mainland of British Columbia and in the Pacific Northwest. Associated animal and human cases are now being reported and further spread of the pathogen may be inevitable. Introduction The basidiomycetous yeast Cryptococcus neoformans has a global distribu- tion and has achieved prominence in recent decades because of its propensity to infect immunocompromised people (Casadevall and Perfect, 1998). In fact, cryptococcosis is recognized as an AIDS-defining illness, and in the absence of highly active antiretroviral therapy, the disease is a significant cause of death in individuals with HIV infection (Bicanic and Harrison, 2005; Bicanic et al., 2005). People and animals acquire the fungus via the inhalation of desiccated yeast cells or basidiospores from environmental sources such as avian guano, soil, and trees. Pulmonary infection often results in dissemination to the central nervous system and C. neoformans is the leading cause of fungal meningitis (Casadevall and Perfect, 1998). Isolates of C. neoformans have previously been divided into three varieties known as grubii, neoformans, and gattii and into serotypes (A–D and hybrids such as AD) defined by antigenic differences in the capsular polysaccharide that is the major virulence factor (Casadevall and Perfect, 1998). The gattii variety is now recognized as a separate species based on phenotypic and molecular traits, and mating (Kwon-Chung et al., 2002). Thus the current view is that the species C. neoformans (var grubii and neoformans) contains strains of serotypes A, D, and AD, and the distinct species C. gattii contains isolates of the B and C sero- types (Kwon-Chung and Varma, 2006). An excellent review of the differences be- tween C. gattii and C. neoformans has been published by Sorrell (Sorrell, 2001). Extensive surveys have been performed over the past 10 years to characterize the genotypes and distribution of C. neoformans and C. gattii isolates (Barreto de Oliveira et al., 2004; Boekhout et al., 2001; Boukhout et al., 1997; Fraser et al., 2005+; Kidd, 2003; Kidd et al., 2004 ++; Kidd et al., 2005+; Meyer et al., 1999; Meyer et al., 2003). These surveys used a variety of DNA-based typing methods to provide detailed classifications of isolates into molecular types. Thus, isolates of C. neoformans var grubii (serotype A) are represented by the VNI, VNII, and VNB (Litvintseva et al., 2006) molecular types, var neoformans (serotype D) is represented by the VNIV type, and isolates of the AD hybrid serotype are the VNIII type. Four molecular types are recognized for C. gattii isolates (designated VGI–VGIV) and further divisions within the molecular types have been identified

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103 APPENDIX A FIGURE A1-1 Map of the forecasted ecologic niche and region of emergence of C. gattii Figure A1-1.eps in British Columbia (BC). The optimal, potential, and unsuitable ecologic niches of C. bitmap gattii in BC are indicated based on biogeoclimatic data for the region (Mak, 2007). Note that the distribution of human and animal cases and the locations of positive environmental samples coincide primarily with the optimal ecologic niche. The information on human and animal cases, and environmental sampling, from Washington (WA) is not included. (Fraser et al., 2005+; Kidd et al., 2005+; Kidd et al., 2007++). For example, VGII strains can be further classified into VGIIa and VGIIb subtypes, as well as other less-well characterized subtypes (Kidd et al., 2004; MacDougall et al., 2007++). There is currently an intense focus on C. gattii due to the unprecedented emergence of the VGI, VGIIa, and VGIIb molecular types as primary pathogens of humans and animals on Vancouver Island in British Columbia (BC) (Kidd et al., 2004; MacDougall et al., 2007++) (Fig. A1-1). Remarkably, the major- ity of human cases have occurred in people without recognized immunologic defects, thus highlighting the unusual pathogenicity of C. gattii relative to C. neoformans. The purpose of this review is to summarize recent progress in the investigation of this fascinating emergence with regard to human and animal exposure, environmental colonization, isolate characterization, and the potential for further dispersal.

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104 FUNGAL DISEASES Overview of Veterinary and Clinical Aspects of the Emergence of C. gattii in BC Animal sentinels played a key role in the study of the emergence of C. gattii in BC and in particular contributed to our understanding of the range of environ- mental niches for the pathogen. A single veterinary pathology laboratory handled clinical specimens from the majority of southern BC veterinary practices, and this allowed early detection and monitoring of C. gattii in the animal population. In addition, the BC Provincial Animal Health Branch Laboratory was able to per- form necropsies on porpoises that were found stranded and dead on Vancouver Island and nearby islands, and these became index cases (Stephen et al., 2002). Beginning prior to the first documented human case in 1999 and continuing to the present, veterinary cases have been diagnosed two to three times more frequently than human cases (Lester et al., 2004); this disparity is likely an underestimate given that only those animals seen by a veterinarian are diagnosed and that infec - tions in wildlife are not considered. The diagnosed cases have primarily been in companion animals (dogs, cats, and ferrets) but also include other domesticated species such as llamas, horses, mink, and psittacine birds (Duncan et al., 2006b; Lester et al., 2004; Stephen et al., 2002). Sampling in the environs of these animal cases has been particularly productive for identifying sources of C. gattii (Kidd et al., 2007a++; MacDougal et al., 2007++). Unlike the colonized koalas of Australia (Krockenberger et al., 2002), no sig- nificant wild animal host or reservoir has been identified in BC. Limited surveys of wild animals were performed between 2003 and 2007 with the examination of necropsy samples of nares, lung, anus or cloacae, and brain for C. gattii. In two surveys, all fatally injured animals turned into rescue facilities were studied. In the first study, 91 animals (14 species) were examined, and only two eastern gray squirrels were positive (Duncan et al., 2006a). In the second study, only one great blue heron was found to have a pulmonary C. gattii infection of 226 animals necropsied (Bartlett, unpublished data). Additionally, 18 river otters were trapped in early spring 2007, but none showed signs of disease or colonization with C. gattii (Bartlett and Balke, unpublished data). Duncan et al. (2005b) established sentinel veterinary practices in areas known to have exposure to airborne C. gattii and found positive C. gattii cultures from nasal swabs of asymptomatic animals in 4.3% of 94 cats, 1.1% of 280 dogs, and 1.5% of 351 horses. Additionally, six cats and two dogs were found to have cryptococcal antigen titers of greater than 1:2. Of seven cats and five dogs that were selected from the asymptomatic but culture- or antigen-positive cohorts and followed over 27 months, only two cats progressed to clinical disease, suggesting that the majority of animals exposed to C. gattii may naturally clear the organism (Duncan et al., 2005a). In the first years of recognition of both the emergence of C. gattii disease and the stability of the pathogen’s environmental niche, it appeared that all hu - man and animal cases had some contact with Vancouver Island. MacDougall and Fyfe (MacDougall and Fyfe, 2006) were able to identify human cases of disease

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105 APPENDIX A with historic travel to Vancouver Island and to determine a likely incubation period (median 6–7 months) based on isolated exposure. In addition, Hoang et al. (Hoang et al., 2004) performed a retrospective chart review examining all cases of cryptococcosis identified between 1997 and 2002 at the largest teach - ing hospital located on the BC mainland. They discovered that there had been a sudden increase in cryptococcal cases of all origins (C. neoformans var grubii, C. n. var neoformans, C. gattii, and C. laurentii), but all C. gattii cases (3/26 charts) reported travel history to Vancouver Island (Hoang et al., 2004). The first cases of mainland-acquired C. gattii infection were identified in animals (ferret, llama, and cats) in 2003, and three cases in cats in Washington were reported in 2005. Eight off-island human cases with no travel history to an endemic area were documented (five in BC and two in Oregon) in 2004 to 2005 (MacDougall et al., 2007++). Upton et al. (Upton et al., 2007+) recently reported the first con - firmed human case in Washington presenting in 2006, and the Whatcom County Public Health Department has now identified four additional cases diagnosed in 2007 (Stern, personal communication). Unlike in BC, cryptococcosis is not yet a reportable disease in Washington, although public health officials are actively soliciting case studies. The VGIIa genotype accounted for 78% of the examined veterinary cases and 87% of the human cases; all off-island veterinary cases to date had the VGIIa genotype (Bartlett, unpublished data) (MacDougall et al., 2007++). Environmental and Dispersal Studies on Vancouver Island Competing theories have been proposed regarding the origin of C. gattii on Vancouver Island (eg, recent introduction, long-term colonization, specific imported vectors). Suffice it to say, the colonization pattern and dispersal of the organism argues against a one-time introduction to Vancouver Island, particularly if the timeline extends only to the first animal and human cases (1998–1999). The first systematic sampling performed on Vancouver Island in 2002 mapped the colonization of C. gattii along a 200 km north-south and a 40 km east-west corridor. This study revealed that C. gattii is not homogeneously spread in the environment, with central Vancouver Island having a higher percentage of colo - nized trees and higher concentration of the organism in soil. The heterogeneous pockets of colonization could explain why limited-sampling strategies may miss the organism. Additionally, even though C. gattii has been found to be perma- nently colonized in some areas, it appears to be transiently colonized in others. The permanently colonized sites have yielded C. gattii repeatedly over the last 5 years, although transiently positive results may be due to limits of detection or failure of the organism to establish true colonization (Kidd et al., 2007a++). As well, sites that initially appeared to be negative for C. gattii have more recently yielded positive environmental samples (Bartlett, unpublished data). It has been shown that in addition to the airborne spread of propagules, wood products,

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106 FUNGAL DISEASES soil, water, vehicles, and shoes can act as dispersal mechanisms for the organ - ism (Kidd et al., 2007a++). These mechanisms are consistent with the findings of a veterinary case-control study, where statistically significant risk factors for disease in cats and dogs related to soil disturbance within 10 km of cases, log - ging within 10 km, travel to Vancouver Island, or owner hiking within 6 months of diagnosis (Duncan et al., 2006c). Although limited environmental sampling in the San Juan Islands, Olympic Peninsula, and Oregon has not yielded C. gat- tii (Fraser et al., 2006; Kidd et al., 2007b++; Upton et al., 2007+). Kidd et al. (2007a++,2007b++) reported finding positive environmental samples from is - lands in the Georgia Strait and in northern Washington. A rather surprising finding was that co-isolated C. gattii strains are heteroge- neous. The first isolates distributed to the research community were mostly from one sampling site (central Vancouver Island) and may have unduly influenced our thinking about the composition of the BC outbreak strains (Kidd et al., 2004++; Fraser et al., 2005+; Fraser et al., 2003). In the initial analysis of the C. gattii isolates from this site, Kidd et al. (Kidd et al., 2004++) used polymerase chain reaction (PCR)-fingerprinting to demonstrate that 5% represented the VGI molecular type and 95% belonged to VGII (90% of these were VGIIa and 10% were VGIIb based upon a one polymorphic band in the PCR-fingerprint profiles). Subsequent work revealed that the composition of the C. gattii population var- ies in different regions where detailed molecular subtyping of isolates has been undertaken. In the southern extreme of Vancouver Island, VGIIa accounts for 91% of the isolates and the remainder are VGIIb, whereas at another site VGIIa accounts for only 66% of the isolates, with VGIIb and VGI at 19% and 15%, respectively (Bartlett and Kidd, unpublished data). Of course, the genotype frequencies are likely to be dynamic, and repeated sampling is important. Also, additional diagnostic tools sensitive enough to detect and differentiate isolates directly in environmental samples (eg, PCR on soil samples) would facilitate a better understanding of the population structure and mechanisms of spread of the organism. Already heightened awareness of changing ecologic niches has resulted in an expansion of knowledge of the environmental origins of other cryptococcal species (Filion et al., 2006). Molecular Characterization of Isolates from BC and the Pacific Northwest Following the initial analyses of genotype frequency described above, Kidd et al. (2005+) used multilocus sequence typing (MSLT) and gene genealogy analyses with four genes to examine patterns of molecular variation as well as population structure of the isolates from Vancouver Island compared with a worldwide sample of C. gattii strains. This work demonstrated that the VGIIa and VGIIb genotypes originally established by PCR-fingerprinting (Kidd et al., 2004++) corresponded to specific MLST profiles. Similar MLST results with additional genes were obtained by Fraser et al. (Fraser et al., 2005+). Of specific

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107 APPENDIX A interest from these studies was the identification of isolates from other areas of the world with identical or similar genotypes to the VGIIa (as represented by isolate A1MR265) and VGIIb (represented by isolate A1MR272) strains from Vancouver Island. For example, the VGIIa genotype was also shared by the NIH444 strain (from a patient in Seattle, ca 1971), CBS7750 (from a Eucalyp - tus tree in San Francisco, ca 1990) and with isolates from other parts of North America (KB10455 and KB9944) (Fraser et al., 2005+; Kidd et al., 2005+). A Brazilian clinical isolate, ICB107, differed from the VGIIa genotype at only one of 22 loci (Fraser et al., 2005+). The VGIIb genotype was also observed among environmental isolates from Australia (eg, Ram002, Ram005, WM1008), clini - cal isolates from Australia (eg, NT-6, NT-13), as well as a clinical isolate from Thailand (MC-S-115) (Fraser et al., 2005+; Kidd et al., 2005+). A Caribbean strain 99/473 of the VGIIb type was also found to differ at only one of 22 loci (Fraser et al., 2005+). Intriguingly, two isolates from human cases in Oregon (2004) were recently found to represent subtypes within the VGII genotype that have not identified among any other strains to date (MacDougall et al., 2007++). The VGIIa and VGIIb isolates from Vancouver Island have been obtained from both clinical and environmental sources. However, the situation is more complex for strains of the VGI genotype from clinical and environmental sources. Specifically, Kidd et al. (2005+) characterized six VGI isolates from Vancouver Island and identified four different genotypes by MLST analysis. Two of these were environmental isolates with a different genotype from the clinical isolates. Thus, in contrast to the VGII types, it was not possible to establish an epidemio - logic link between environmental and clinical isolates of the VGI type. However, recent analysis of further environmental VGI isolates from Vancouver Island indicated that they were highly similar to a porpoise isolate (A1MF2863), being identical at four MLST loci (Kidd and Bartlett, unpublished data). It is possible that the clinical isolates of the VGI type represent strains acquired during travel outside of Vancouver Island. Overall, Kidd et al. (2005+) found that the Vancouver Island isolates were part of a predominately clonal population with little evidence of sexual recom - bination occurring between them. Fraser et al. (2005+) also presented evidence that the VGIIa and VGIIb strains from Vancouver Island were related in that they shared 14 identical loci out of the 30 examined and proposed that the genotypes represent either siblings arising from a past mating event, or that one may be the parent of the other, perhaps as the result of same-sex mating between MATα par- ents. Selected isolates from Vancouver Island and other parts of the world have been tested for mating competence. These studies revealed that the VGII isolates are generally fertile whereas VGI strains are not (Campbell et al., 2005; Fraser et al., 2003; Kidd et al., 2004++). In general, the ability of C. gattii isolates to mate has implications for recombination events that might generate strains with different virulence properties and environmental adaptability.

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108 FUNGAL DISEASES The Global Distribution of C. gattii Prior to the emergence of C. gattii on Vancouver Island, it was commonly accepted that this species was restricted to tropical and subtropical regions of the world, and that infection was associated with exposure to Eucalyptus trees (Ellis and Pfeiffer, 1990; Kwon-Chung and Bennett, 1984; Sorrell et al., 1996). The idea of a limited geographic distribution came from a study that surveyed a worldwide collection of clinical isolates (Kwon-Chung and Bennett, 1984). This survey revealed that C. gattii was prevalent only in regions with tropical and subtropical climates (22%–50% of isolates) relative to C. neoformans (50%–71% of isolates). However, this study also reported that 13% of the strains from North America, and 3.3% of the strains from Europe were C. gattii (without reference to travel histories). More recent surveys have focused on identifying the molecular types of C. gattii found in collections from various regions. In this regard, VGI appears to be the most widely distributed type worldwide (Kidd, 2003; Meyer et al., 2003), and this type is also found most frequently among clinical and en - vironmental isolates in Australia (Campbell et al., 2005). Strains of the VGII type are also found in parts of Australia as well as in North and South America (Fraser et al., 2005+; Kidd, 2003; Kidd et al., 2004++; Kidd et al., 2005+; Meyer et al., 2003). In a recent, large-scale study of IberoAmerican isolates, VGIII predomi - nated, and this type has also been found in India and the United States (Kidd, 2003; Meyer et al., 2003). The VGIV type has been found in Central America and South Africa (Kidd, 2003; Meyer et al., 2003). Notably, the VGIII and VGIV types were not found in the collections from Vancouver Island suggesting that these genotypes may have a more limited distribution. More recently, Meyer et al. (2007) have surveyed 160 VGII strains recov- ered globally since 1986 using PCR-fingerprinting, amplified fragment length polymorphism analysis and MLST with eight loci. This work revealed that the VGIIa genotype from Vancouver Island is also found among Brazilian isolates and that Colombian isolates are closely related. Interestingly, the majority of the latter isolates are mating type a in contrast to mating type α for the Vancouver Island strains (Escandon et al., 2006), and mating was demonstrated between the Colombian MATa strains and VGIIa MATα strains from Brazil and Vancouver Island. This work suggests that the VGIIa genotype was present in South America as early as 1986 and it sheds additional light on the potential mating interactions for VGII types of C. gattii that may be relevant for the situation on Vancouver Island. Overall, these surveys provide an interesting view that the genotypes of C. gattii (at least for VGI and VGII) are likely to have a worldwide distribution and the concomitant potential for permanent colonization of suitable environments. This view highlights the need for more extensive environmental sampling glob- ally to generate a detailed picture of genotype frequency over time and location. The most extensive view is now available from the work on Vancouver Island and the lessons learned from this work can be applied in other locations (Kidd

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109 APPENDIX A et al., 2007a++), especially with regard to the need for extensive multisource sampling over many years. The wide distribution of C. gattii genotypes should also be considered in light of recent reports that infections with this species are occurring in patients with AIDS (South Africa [Morgan et al., 2006], Southern California [Chaturvedi et al., 2005a]). Therefore, it will be important to identify the endemic areas for specific C. gattii genotypes in order to monitor human and animal disease. Origin of the C. gattii in BC and the Pacific Northwest: Aboriginal Species or Landed Immigrant? It is fun to speculate about the origin of the genotypes on Vancouver Island, and this activity has consumed much energy in the research community. How - ever, the extent of global strain dispersal has been demonstrated to be significant (Kidd et al., 2005+, Xu et al., 2000), making it difficult to accurately determine a specific origin of any given genotype. It is possible that the species has been a long-term resident of BC and that changing conditions (eg, climate or land use) or improved surveillance are responsible for the current level of awareness. Alterna- tively, it has been suggested that the emergence is due to the recent introduction of a particularly virulent genotype that may be well adapted to the local condi - tions such that large numbers of infectious cells are propagated (Fraser et al., 2005+). Although it may be difficult to garner strong evidence for a given theory, it is clear that much more information is needed about the C. gattii genotypes on Vancouver Island and worldwide and about the disease caused by C. gattii in immunocompetent hosts. Below, we discuss some of the studies that are needed to generate a more detailed view of C. gattii that may help in infection control. Ecologic adaptability, colonization, and dispersal The environmental sampling revealed a high level of soil colonization on Vancouver Island, and it would be interesting to examine soil persistence and competition in laboratory and field settings. These types of experiments may be relevant to addressing how the fungus becomes aerosolized and the nature of the infectious particle. An investigation of conditions required for the propagation of the infectious particles in soil/trees would also be highly relevant to understand - ing the factors that influence exposure of humans/animals. It is likely that no one factor can explain the dramatic emergence of C. gattii on Vancouver Island, and there may be interplay between soil conditions, temperature, and moisture. Current weather station data are insufficient to ad - equately describe the microclimates in areas colonized by the pathogen. Climate oscillations driven by alternating El Niño and La Niña currents have produced both drier and wetter than normal summer conditions in BC over the last few decades. Outbreaks of another fungal disease, coccidioidomycosis, have been

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110 FUNGAL DISEASES shown to follow soil disruption in California (Zender and Talamantes, 2006). Data gathered from the BC environment conclusively show that C. gattii is well adapted to survive in dry, low nutrient soil and is more likely to be airborne dur- ing dry summer weather (Kidd et al., 2007a++). The stability of the colonization of soil and trees at permanently colonized sites suggests that the pathogen can effectively compete with resident soil microflora. Longer cycles of meteorology patterns and finer tools of climate measurement will be needed to understand the complex relationship of microbe, climate, and ecologic niche. Additional sampling around the world is needed to investigate predicted favorable climate/soil/water conditions that might allow colonization by C. gat- tii. Mak (2007) has recently developed ecologic niche models that predict the probable extent of environmental colonization of C. gattii based on human, ani- mal, and environmental data and climate projections for the Pacific Northwest (Fig. A1-1). Areas that may eventually be impacted include the Lower Mainland of BC with a population base of approximately 2 million people. These projec - tions could be used by public health officials on both sides of the US-Canada border to plan strategies for risk communication and anticipated morbidity and mortality (Mak, 2007). Clinical considerations Perhaps the most relevant topics regarding the emergence of C. gattii have to do with identifying risk factors for people, designing ways to limit exposure, and developing effective methods to treat the infections that do occur. It is common to see statements in the literature that C. gattii is a primary pathogen that infects immunocompetent people, and that C. neoformans is an opportunistic pathogen that infects immunocompromised people. The distinction may be less clear given that C. gattii is now being found in AIDS patients and C. neoformans can infect seemingly immunocompetent people (Chaturvedi et al., 2005a; Morgan et al., 2006; Speed and Dunt, 1995). There is clearly a need for retrospective studies of patients to determine host risk factors as well as prospective case studies to determine efficacy of treatments. The number of cases continuing to occur on Vancouver Island (and among tourists [Lindberg et al., 2007]) would allow this type of investigation. An interesting consideration in terms of exploring possible virulence differ- ences for C. gattii versus C. neoformans is whether mouse virulence studies have relevance for human disease. For example, the strains with the VGIIa and VGIIb genotypes from Vancouver Island both cause disease in humans, but laboratory studies revealed virulence differences between the two strains tested (Fraser et al., 2005+). The more virulent strain, A1MR265, of the VGIIa genotype showed equal virulence in the mouse model to strain H99 that is representative of the most common VNI type of C. neoformans (var grubii). It is possible that these re- sults reflect the fact that only one isolate of each genotype from Vancouver Island

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111 APPENDIX A was tested and the isolates selected may not be representative. It is clear, however, that strains of C. gattii show virulence differences (Kronstad, unpublished data) (Chaturvedi et al., 2005b; Fraser et al., 2005+) and that multiple isolates from Vancouver Island and worldwide collections need to be tested. The same is true for C. neoformans as demonstrated by the range of virulence detected by Clancy et al. (2006). Thus, we need to develop better models to assess differences in virulence and to explore possible differences that may be relevant to infection of immunocompetent versus immunocompromised hosts. Applications of genomic approaches to develop a detailed understanding of C. gattii The emergence of C. gattii provided the impetus to sequence the genomes of isolates representing the VGI (WM276) and VGIIa (A1MR265) genotypes (Michael Smith Genome Sciences Center, 2007++; The Broad Institute, 2007++). These are important resources for the next steps in characterizing the virulence of C. gattii, the genetic diversity of the species and the interactions of the fungus with the environment. One can imagine, for example, using the genomes for transcriptome and proteome studies to identify differences in expression for C. gattii relative to C. neoformans. Some of these differences may reveal factors that contribute to the primary pathogenesis of C. gattii relative to C. neoformans. The two C. gattii genomes also provide a platform for more detailed analyses of genotypes and comparative studies of genome variability. In the latter case, comparative hybridization or genome resequencing approaches can be used to study the microevolution of genomes in strains in the environment and clinical strains during passage through human and animal hosts (eg, during relapse or drug therapy). Comparative genome hybridization experiments with the VGI and VGIIa genomes have been initiated to identify genomic changes in mutants that have lost virulence and to examine genome variation in strains representing the VGI, VGIIa, and VGIIb genotypes (Kronstad, unpublished data). The declining cost of sequencing will also allow further genome-sequencing projects to provide a deeper view of genome content and variability. The more detailed information may eventually lead to the separation of the molecular types of C. gattii into distinct varieties or species. Media Coverage of the Emergence of C. gattii Any emerging infectious disease represents a challenge to the public health system. The system must respond to educate caregivers about appropriate in - terventions while balancing the message to allow the public to make informed choices. For example, the lay press recently reported concern by members of the public in Alabama where experimental plots of genetically engineered Eucalyptus trees will be grown; the fear being that C. gattii will be imported into the environ-

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392 FUNGAL DISEASES Kubátová & Dvořák (2005) investigated fungi associated with insects hibernating in underground sites but did not find Geomyces species. To our knowledge, only one study in Europe has investigated fungi present in bats’ skin and hair samples where, based on our current knowledge, G. destructans is most likely to be found. During the winter 1999/2001, Larcher et al. (2003) collected 25 samples of hair and skin swabs from six species, including three Myotis Myotis, but did not find any Geomyces species. It is important to note that most fungal cultures have been carried out at temperatures above 24–25°C, temperatures at which G. destructans does not grow (Gargas et al., 2009; Chaturvedi et al., 2010), which could explain why although present, this fungal species had never been reported in Europe before the study of Puechmaille et al. (2010). Combining previously published data from France, Germany, Switzerland, Hungary, The Czech Republic and Slovakia (Puechmaille et al., 2010; Martínková et al., 2010; Wibbelt et al., 2010), additional data collected from France, Germany and Hungary (this study), and new data from Belgium, The Nether-lands, Poland, Estonia and Ukraine (this study), we demonstrate here that G. destructans is widespread in Europe. We consider the photographic evidence of bats with white fungus matching the characteristic growth pattern (e.g., Figure A21-2; pictures from Romania and Turkey) to most likely represent G. destructans, because so far all tested live European bats with such white fungal growth on their nose, simi - lar to Figure A21-2, have been confirmed to carry that species of fungus. These findings further support the fact that G. destructans is widespread across Europe. However, to confirm the presence of G. destructans in Europe prior to 2008, his- torical collections of bat specimens (or cave soil samples), especially specimens collected during the hibernation period, should be screened for the fungus. As depicted in Figure A21-1, most cases of bats with G. destructans (con- firmed and suspected) have been found from North-eastern France through Bel - gium, The Netherlands, Germany and the Czech Republic. However, it is not clear whether this pattern reflects an actual higher occurrence and/or prevalence of the fungus in these regions or if it is at least partly due to sampling bias, whereby the fungus is more likely to be detected in regions with a higher number of underground sites visited every winter or in regions were the fungus is specifi - cally sought. In our opinion, it is most likely that this large-scale pattern is due to a sampling bias. For example, the largest number of sites with G. destructans in any European country was reported from the Czech Republic (76 localities with suspected or confirmed G. destructans) were most sites have been searched for signs of the fungus (>800 hibernacula) (Martínková et al., 2010). G. destructans growth on bats The clear seasonal peak in the number of observations of bats with white fungal growth indicates an increasing prevalence or detectability of G. destruc- tans as winter passes. This suggests that bats might acquire G. destructans late

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393 APPENDIX A during the hibernation period or that the fungus is carried by bats at the onset of hibernation but needs time to develop the visible white fungal growth due to the phenology of the fungus. Therefore, the absence of visible white fungal growth on bats when observed with the naked eye may not directly reflect the absence of G. destructans, but rather just the absence of visible fungal colonies. Further complicating matters, our ability to detect G. destructans growth on bats can substantially differ with proximity to the bats (i.e., low ceiling versus high ceil - ing) or the location of the bat (ceiling versus crevices). Our results confirm the suggestion of Martínková et al. (2010) by showing that during the hibernation period, bats can remove the fungus from their snout, ears and wings to a point where the fungus is no longer visible to the naked eye, although some spores might still be present on their skin. During hibernation, bats arouse every two weeks on average (Brack and Twente, 1985; Twente et al., 1985) and if bats consistently groom off the fungus on these occasions, our ability to visually detect the fungus, if present, will be considerably reduced. We also showed that towards the end of the hibernation period, bats were emerging from the hibernaculum without visible signs of the fungus despite showing visible white fungal growth from two weeks to five days before leaving the hibernacu- lum. It would be important to investigate whether bats carry spores out of hiber- nacula and as a result could possibly contaminate maternity roosts and maternity mates as suggested by Hallam and McCracken (2011). Factors affecting G. destructans prevalence Although it is not possible to clearly identify the mechanism responsible for the sudden increase in the prevalence of G. destructans in late February and March, these data suggest that shorter winter periods should be associated with lower prevalence. This prediction seems to hold as in the Mediterranean region, where hibernation periods are shorter (Rodrigues, 2003), no bats with visually conspicuous fungal growth have yet been reported during winter cave surveys. The case reported from Southern France (June 25th 2010, Figure A21-2) was found in the Pyrenees mountains at ca. 1700 m a.s.l. and hence, is not considered typical of the Mediterranean climate. It is nevertheless too early to conclude on this association between G. destructans prevalence and the hibernation duration, as other factors would need to be considered such as for example, the higher temperature observed in hibernacula in the Mediterranean region compared to other regions in Europe (Rodrigues, 2003). Higher temperatures in hibernacula have been associated with more frequent arousals in Rhinolophus ferrumequinum (Ransome, 1971; Arlettaz et al., 2000; Park et al., 2000). Considering that this association holds for other species, as a consequence of more frequent arousals, bats are expected to groom more often and therefore, reduce the probability of a visible fungal growth to develop. More surveys and strategic sampling efforts are needed to uncover whether the length of the hibernation period and/or climatic

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394 FUNGAL DISEASES conditions have a direct or indirect effect on the growth rates, prevalence, and detectability of G. destructans on bats. It is crucial that the change in prevalence or detectability over the hiberna - tion period is considered when comparing prevalence across sites and/or years. Our results from monitoring one site throughout the hibernation period over two consecutive years as well as reported cases from multiple sites in multiple years show that bats with fungal growth are first seen in January, the number of cases slowly increases into February and peaks in March, then in April when bats emerge from hibernation it drops again. Our results are in agreement with recent results from the Czech Republic where in the winter 2009/2010, the num - ber of sites with bats with white fungal growth increased from 4.1% in January/ February (33/800 sites; regular bat monitoring) to 77.5% in late February/March (76/98 sites; additional inspections) (Martínková et al., 2010) The Czech study reported that this increase in G. destructans prevalence was ‘‘suggestive of an epi- zootic spread of the fungus’’ (Martínková et al., 2010); we propose an alternative explanation whereby the increase in prevalence of G. destructans in late winter (March) might regularly (yearly) occur in Europe but has gone unnoticed. Nearly all hibernation counts in previous years were carried out between December and mid-February when prevalence/detectability of G. destructans is low, but not in March (Battersby, 2010) when the prevalence/detectability of G. destructans is at its highest (Figure A21-3). Although the total numbers of bats in the hibernacula decreased through April as bats left for the maternity colonies, our results show that there is a high probability of fungal growth developing on the remaining individuals. This further supports our hypothesis proposed above and links the duration of the hibernation period with the prevalence of G. destructans. By increasing the sample size, some cases might be reported earlier in the hiberna - tion season or later through the summer, but we expect that the general pattern observed will not change. Despite these difficulties in assessing the occurrence of the fungus on bats, our data are consistent with other studies (Puechmaille et al., 2010; Martínková et al., 2010; Wibbelt et al., 2010), and also demonstrate that the most commonly encountered bat species with G. destructans in Europe is the largest species of Myotis on the continent, Myotis myotis. In countries/regions (i.e., the Netherlands, Northwest Germany) where M. dasycneme is more com- monly encountered in hibernacula, G. destructans prevalence can reach high lev- els in that species. It is interesting to note that neither Pipistrellus pipistrellus nor Miniopterus schreibersii have been observed with G. destructans (Puechmaille et al., 2010; Martínková et al., 2010; Wibbelt et al., 2010), although these two species are known to hibernate in aggregations of tens of thousands of individu- als, especially the latter (Furman and Özgül, 2004; Nagy and Postwana, 2011; Benda et al., 2003; Serra-Cobo et al., 1998). Although rare, hibernacula of a few thousands and up to about 34,000 individuals are also known for species of Myo- tis in Europe (Furman and Özgül, 2004; Nagy and Postwana, 2011; Kokurewicz, 2009; Arthur and Lemaire, 2009; Sachanowicz et al., 2006; Dietz et al., 2009).

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395 APPENDIX A G. destructans outside of the hibernation period We observed three individual bats with white fungal growth around their nose (one confirmed as G. destructans) from May and June, when they were still torpid in cold underground sites. This represents the first mention of individu - als with G. destructans colonisation outside of the hibernation period and raises questions about the role of these individuals in the persistence of the fungus in bat populations. Pipistrellus pipistrellus During the summer period, while females ag- gregate in colonies to raise their young, it remains largely unknown where males are roosting (e.g., Senior et al., 2005). Furthermore, during the swarming season in late summer/autumn, large numbers of individuals aggregate in caves, mines or tunnels and come in close contact with each other (chasing, mating) (Senior et al., 2005; Parsons and Jones, 2003; Parsons et al., 2003a; Parsons et al., 2003b; Rivers et al., 2006; Rivers et al., 2005), which could represent an opportunity for G. destructans to be transmitted between individuals. We isolated G. destructans from the environment surrounding hibernating bats. The presence of viable spores of G. destructans on the surfaces of hiberna- tion sites has huge implications for the understanding of disease transmission mechanisms and disease modelling (Hallam and McCracken, 2011) It seems likely that cave walls could serve as a passive vector and/or reservoir for G. de- structans spores. It is not yet known how long these spores can remain viable but fungal spores generally remain viable for extended periods. Bats entering these sites in autumn (for swarming and/or hibernation) could become contaminated with spores of G. destructans left from bats infected during the previous winter. In North-America, Lindner et al. (2010) successfully amplified ITS sequences identical to G. destructans DNA from soil samples collected during the winter 2008–2009 at three bat hibernacula and stressed the importance of considering the environment as a reservoir for G. destructans and in the dynamics of WNS transmission. Our results confirm this and further suggest that more work is needed to understand the persistence of G. destructans on hibernacula walls (reservoir or passive vector) where they are in physical contact with bats. Insights into the origin of G. destructans and WNS The wide distribution of G. destructans in Europe and the absence of associ- ated mortality supports the hypothesis that G. destructans has co-evolved with European bats and only recently arrived in North America where it is causing unprecedented mass mortalities (Puechmaille et al., 2010; Blehert et al., 2009; Martínková et al., 2010; Wibbelt et al., 2010). Alternatively, G. destructans could have been present on both continents and a virulent strain could have evolved in North-America. Until the relationships between G. destructans populations across continents are clarified, precautions should be taken to minimise the chances of transcontinental movement of viable G. destructans (Puechmaille et al., 2011).

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396 FUNGAL DISEASES During the two years monitoring at one site in Germany where G. destructans prevalence reached high levels in March-April, not a single dead bat was found. This is in agreement with previous studies (Puechmaille et al., 2010; Martínková et al., 2010; Wibbelt et al., 2010) reporting that the presence of G. destructans in bats from Europe is not associated with mass mortality. This sharply contrasts with mass mortalities reported in North America where hundreds or thousands of dead bats are found in hibernacula towards the end of the hibernation period. Recent pathological investigations of bats dying from WNS in North America led Cryan et al. (2010) to propose that mortality was caused by important disruptions of wing-dependant physiological functions due to infection by G. destructans. In North America, the fungus deeply invades wings tissues (Meteyer et al., 2009) and causes damages that are thought to alter homeostasis and water balance, resulting in more frequent arousals than bats can afford with their fat reserves, leading to death by starvation (Cryan et al., 2010). The pathology associated with G. destructans colonisation in Europe is not yet known. We believe that the first step in understanding mortality differences between bats from Europe and North America rely on understanding pathological differences incurred by the fungus on the bats’ wings. As a result, we urge the necessity to carry out pathological investigation of live bats from Europe colonised by G. destructans. Despite the absence of mortality associated with the presence of G. destructans in Europe, it would be necessary to investigate whether chronic infections with the fungus are compromising the health of individuals, especially in M. Myotis and M. dasycneme, which show high prevalence of the fungus towards the end of the hibernation period. Phylogeographic studies of European bat species have shown that in the last 100,000 years, some species colonised Europe from Western Asia (Flanders et al., 2009), including Myotis blythii (Berthier et al., 2006; Currat et al., 2008) which has been found with G. destructans (Wibbelt et al., 2010). Assuming that G. destructans can be transported over long distances by bats, we speculate that the distribution of G. destructans is probably not limited to Europe and possibly extends eastwards into Russia, Western and Central Asia. Further surveys are necessary to clarify the global distribution of G. destructans. Conclusions We have shown here that G. destructans, the most likely causative agent of WNS in North America, is widespread in Europe, but is not associated with mass mortality. The prevalence of visible fungal growth on bats increases in February/ March before sharply decreasing when bats emerge from hibernation. We also isolated viable G. destructans from the walls of an underground site suggesting that the hibernacula could act as passive vectors and/ or reservoirs for G. de- structans and therefore, might play an important role in the transmission process. Further research is needed to clarify the global prevalence of G. destructans and

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397 APPENDIX A identify variables (e.g., temperature, humidity and hibernation length) explaining regional differences. Finally, further research is needed in different parts of the globe, especially temperate region of the Northern and Southern hemispheres, to precisely determine the global distribution of G. destructans. Materials and Methods Sample collection During ongoing population censuses carried out at hibernacula in different countries across Europe and during additional hibernacula surveys carried out for the purpose of this study, information on bats with visible white fungal growth on snouts and/or ears was recorded. Whenever possible, sterile dry cotton swabs (Puechmaille et al., 2010) or adhesive tape touch imprints (Wibbelt et al., 2010) were used to collect fungal material from the bats. In Estonia, samples were collected from the wall of the tunnel where a bat with characteristic white fun - gus was observed nine days prior to the sampling. Where no sample collection was possible, a photograph was taken of the bat (photographic record). In cases where neither sample collection nor photographic evidence was obtained, the record was classified as visual observation. Live hibernating bats with powdery, white fungal growth on their noses were considered suspects of infection by G. destructans (Gd-suspects) but not suspected of having WNS. There is presently no data supporting the occurrence of WNS in Europe and the co-occurrence of the fungus with lesions characteristic of WNS (Meteyer et al., 2009) has not (yet) been reported in Europe (Wibbelt et al., 2010; Barlow et al., 2005). Although, prevalence of G. destructans can reach high levels in some European species (i.e., Myotis myotis, M. dasycneme) in late winter (especially in March), it can be expected that by chance alone some bats dying from causes unrelated to the presence of G. destructans will also be carrying the fungus. Unless the criteria for the diagnosis of WNS are met (confirmation by histo-pathology and PCR) (Meteyer et al., 2009) WNS should not be assumed as a cause of mortality in dead bats found in hibernacula of Europe. Various species of fungi have been identified on dead bats (Wibbelt et al., 2010; Voyron et al., 2011), most of them likely being saprophytes that colonise bat carcasses post-mortem. Fungal cultures In the laboratory, samples were treated as in Puechemaille et al. (2010) for swabs and following Wibbelt et al. (2010) for touch imprints. Briefly, swabs were streak-plated onto plates of Sabouraud’s agar, supplemented with 0.1% mycological peptone. For touch imprints, small areas with fungal conidia char- acteristic of G. destructans were identified by light microscopy and the tape was disinfected and excised before being transferred for culture to Sabouraud’s agar.

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398 FUNGAL DISEASES The plates were sealed with parafilm and incubated inverted in the dark at 10°C. A fungal growth developed within 14 days, from which single spore cultures were established. Molecular identification Each culture was sequenced for one molecular marker, the rRNA gene internal transcribed spacer (ITS, ca. 930 bp.) region (ITS1, 5.8S, and ITS2) to further confirm species identity. The DNA extraction, PCR amplification and DNA sequencing followed protocols described in Puechmaille et al. (2010). Briefly, DNA was extracted using the Qiagen Blood and Tissue kit following the manufacturer’s instructions with slight modifications (after step 3, we added an incubation time of 10 minutes at 70°C). PCR reactions were carried out in 25 mL containing 1 mL of DNA extract (at 10–75 ng/mL), 1.5 mmol/L MgCl2, 0.4 mmol/L each primer (Forward: ITS4, 5′-TCCTCCGCTTATTGATATGC – 3′; Reverse: ITS5, 5′-GGAAGTAAAAGTCGTAACAAGG – 3′; (White et al., 1990), 0.2 mmol/L dNTP, 1x PCR buffer and 1 U Platinum Taq DNA Polymerase High Fidelity (Invitrogen). PCR cycling conditions were: initial step 15′ at 95°C, then 10 cycles of 30″ at 95°C, 1′45″ at 60°C (reduce of 2°C every 2 cycles), 1′ at 72°C, following by 30 cycles of 30″ at 95°C, 1′45″ at 50°C and 1′ at 72°C. PCR products were purified and sequenced by Macrogen Inc. (Seoul, Korea) in both directions using the PCR primers. Complementary sequences were assembled and edited for accuracy using CodonCode Aligner 3.0.3 (www.codoncode.com/ aligner/download.htm). Monitoring of visible fungal growth on bats One site situated in Northwest Germany (Latitude: 52.1; Longitude: 8.2) near the city of Osnabrück was monitored over two consecutive winters, 2006/2007 (5th September until 19th May) and 2007/2008 (28th August until 23rd April). The monitoring consisted of counting the total number of bats at the site as well as the number of bats with visible white fungal growth similar to the pictures presented in Figures A21-2 and A21-4. The counts were done by the same person (V. Korn) every 4 days on average during the first year and every 2.5 days on average during the second year. The procedures complied with guidelines of the American Society of Mammalogists and were carried out under permit number FBD7.2 60 from the Administration of the County of Osnabrück, Department of Environment.

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399 APPENDIX A Supporting Information FIGURE A21-S1 Monitoring of bats at an hibernaculum in Germany during (A) the win- ter 2006/2007 (September 5th Figure A21-S1.eps and (B), the winter 2007/2008 2006 until April 19th 2007) bitmap (August 28th 2007 until April 23rd 2008). The blue line represents the total number of bats counted whereas the green line represents the number of bats with visible white fungal growth (Gd-suspects). Dotted vertical lines separate counts from each month. Note that the number of counts per month was not equal between months. In (B), the black line rep - resents the total number of bats counted whereas the blue line represents the total number of bats bar one portion of the hibernaculum where bats grouped densely (ca. 20 individu - als) and did not allow a reliable identification of the number of bats with white fungal growth. The green line represents the number of bats with visible white fungal growth (Gd-suspects) counted at the hibernaculum without considering individuals densely group - ing at one place in the hibernaculum. The group of about 20 individuals formed while the hibernaculum was partially flooded, likely as a result of bats changing position to avoid drowning. Note that the right Y-axis scale is different between (A) and (B).

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400 FUNGAL DISEASES Acknowledgments We would like to thank Dóczy Annamária, Andriy-Taras Bashta, Frédéric Blanc, Sándor Boldogh, Gaby Bollen, Thomas Chatton, Emrah Coraman, Jére Csaba, Simon Dutilleul, Mehmet Doker, Oliver Gebhardt, Lena Godlevska, René Janssen, Daniel Lefèvre, Barti Levente, Vadim Martyniuk, Gerhard Mascher, Mykola Matveev, Bernd Ohlendorf, Rian Pulles, Tony Rock, Wolfgang Rackow, Sébastien Roué, Bücs Szilárd, Abigel Szodoray-Parádi, Farkas Szodoray-Parádi and Julien Vittier for providing us with their field observations. The comments of Paul Cryan, Paul Racey, Natalia Martínková and an anonymous reviewer helped to improve a previous version of the manuscript. Author Contributions Conceived and designed the experiments: SJP GW VK ECT. Performed the experiments: SJP GW HF VK KM AK. Analyzed the data: SJP GW. Contributed reagents/materials/analysis tools: SJP GW VK HF KM AK FF WB CB TB TC MD TG AJH FH GH MH CJ YLB LL MM BM KP MS AW UZ ECT. Wrote the paper: SJP GW. References A national plan for assisting states, federal agencies, and tribes in managing white-nose syndrome in bats. 2010 Draft v. 10.21.2010.16 p. Arlettaz R, Ruchet C, Aeschimann J, Brun E, Genoud M, et al. (2000) Physiological traits affecting the distribution and wintering strategy of the bat Tadarida teniotis. Ecology 81:1004–1014. Arthur L, Lemaire M (2009) Les Chauves-souris de France, Belgique, Luxembourg et Suisse: Mèze: Biotope, Paris: Muséum national d’Histoire naturelle. 576 p. Barlow A, Ford S, Green R, Morris C, Reaney S (2009) Investigation into suspected white-nose syndrome in two bat species in Somerset. Vet Rec 165:481–482. Bastian F, Alabouette C, Saiz-Jimenez C (2009) The impact of arthropods on fungal community structure in Lascaux Cave. J Appl Microbiol 106:1456–1462. Battersby J (2010) Guidelines for surveillance and monitoring of European bats. Bonn, Germany. 95 p. Benda P, Ivanova T, Horáček I, Hanák V, Cerveny J, et al. (2003) Bats (Mammalia: Chiroptera) of the Eastern Mediterranean. Part 3. Review of bat distribution in Bulgaria. Acta Soc Zool Bohem 67:245–357. Berthier P, Excoffier L, Ruedi M (2006) Recurrent replacement of mtDNA and cryptic hybridization between two sibling bat species Myotis Myotis and Myotis blythii. Proc R Soc B 273:3101–3109. Blehert DS, Hicks AC, Behr M, Meteyer CU, Berlowski-Zier BM, et al. (2009) Bat white-nose syn - drome: an emerging fungal pathogen? Science 323: 227. Brack V, Twente JW (1985) The duration of the period of hibernation of three species of vespertilionid bats. I. Field studies. Can J Zoolog 63:2952–2954. Cabrera A (1904) Ensayo monogra´fico sobre los quirópteros de España. Mem Soc Españ Hist Nat 2:249–292. Chaturvedi V, Springer DJ, Behr MJ, Ramani R, Li X, et al. (2010) Morphological and molecular characterizations of psychrophilic fungus Geomyces destructans from New York bats with White Nose Syndrome (WNS). PLoS ONE 5: e10783.

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