Nutritional Capability of and Substrate Suitability for Pseudogymnoascus destructans,the Causal Agent of Bat White-Nose Syndrome

Pseudogymnoascus destructans, the causal agent of bat white-nose syndrome, has caused nearly six million deaths in North American bats since its introduction into the United States in 2006. Current research has shown that caves can harbor P. destructans even after the infected bats are removed and bats no longer visit or inhabit previously infected caves. Our research focuses on elucidating reservoir requirements by investigating the nutritional capabilities of and substrate suitability requirements for six different P. destructans isolates from various localities including Illinois, Indiana, New York (Type specimen), and Pennsylvania. Enzyme assays implicate that both urease and b-glucosidase appear to be constitutive, lipase and esterase activity were more rapid than proteinase activity on 6% gelatin, gelatin degradation was accompanied by medium alkalinization, the reduction of thiosulfate generated hydrogen sulfide gas, chitinase and manganese dependent peroxidase activity were not visually demonstrated within eight weeks, and keratinase activity was not evident at pH 8 within eight weeks. We demonstrate that all P. destructans isolates are capable of growth and sporulation on dead fish, insect, and mushroom tissues. Sole nitrogen source assays demonstrated that all P. destructans isolates exhibit Class 2 nitrogen utilization and that growth-dependent interactions occur among different pH and nitrogen sources. Substrate suitability assays demonstrated that all isolates could grow and sporulate on media ranging from pH 5–11 and tolerated media supplemented with 2000 mg/L of calcium and 700 mg/L of three separated sulfur compounds: thiosulfate L-cysteine, and sulfite. All isolates were intolerant to PEG-induced matric potential with delayed germination and growth at −2.5 MPa with no visible germination at −5 MPa. Interestingly, decreasing the surface tension with Tween 80 permitted germination and growth of P. destructans in −5 MPa PEG medium within 14 days suggesting a link between substrate suitability and aqueous surface tension altering substances.     

Bacteria Isolated from Bats Inhibit the Growth of Pseudogymnoascus destructans,the Causative Agent of White-Nose Syndrome

Emerging infectious diseases are a key threat to wildlife. Several fungal skin pathogens have recently emerged and caused widespread mortality in several vertebrate groups, including amphibians, bats, rattlesnakes and humans. White-nose syndrome, caused by the fungal skin pathogen Pseudogymnoascus destructans, threatens several hibernating bat species with extinction and there are few effective treatment strategies. The skin microbiome is increasingly understood to play a large role in determining disease outcome. We isolated bacteria from the skin of four bat species, and co-cultured these isolates with P. destructans to identify bacteria that might inhibit or kill P. destructans. We then conducted two reciprocal challenge experiments in vitro with six bacterial isolates (all in the genus Pseudomonas) to quantify the effect of these bacteria on the growth of P. destructans. All six Pseudomonas isolates significantly inhibited growth of P. destructans compared to non-inhibitory control bacteria, and two isolates performed significantly better than others in suppressing P. destructans growth for at least 35 days. In both challenge experiments, the extent of suppression of P. destructans growth was dependent on the initial concentration of P. destructans and the initial concentration of the bacterial isolate. These results show that bacteria found naturally occurring on bats can inhibit the growth of P. destructans in vitro and should be studied further as a possible probiotic to protect bats from white-nose syndrome. In addition, the presence of these bacteria may influence disease outcomes among individuals, populations, and species.     

The Effects of Cutaneous Fatty Acids on the Growth of Pseudogymnoascus destructans,the Etiological Agent of White-Nose Syndrome (WNS)

White Nose Syndrome (WNS) greatly increases the over-winter mortality of little brown (Myotis lucifugus), Indiana (Myotis sodalis), northern (Myotis septentrionalis), and tricolored (Perimyotis subflavus) bats. It is caused by a cutaneous infection with the fungus Pseudogymnoascus destructans (Pd). Big brown bats (Eptesicus fuscus) are much more resistant to cutaneous infection with Pd, however. We thus conducted analyses of wing epidermis from hibernating E. fuscus and M. lucifugus to determine their fatty acid compositions, and laboratory Pd culture experiments at 4.0–13.4°C to determine the effects of these fatty acids on Pd growth. Our analyses revealed that the epidermis of both bat species contain the same 7 fatty acid types (14:0, 15:0, 16:0. 16:1, 18:0, 18:1, & 18:2), but the epidermis of M. lucifugus contains: a) more stearic (18:0) acid, b) less palmitoleic (16:1) acid, c) less myristic (14:0) acid, and, d) less oleic (18:1) acid than that of E. fuscus. The growth of Pd was inhibited by: a) myristic and stearic acids at 10.5–13.4°C, but not at 4.0–5.0°C, b) oleic acid at 5.0–10.6°C, c) palmitoleic acid, and, d) linoleic (18:2) acid at 5.0–10.6°C. One set of factors that enables E. fuscus to better resist cutaneous P. destructans infections (and thus WNS) therefore appears to be the relatively higher myristic, palmitoleic, and oleic acid contents of the epidermis.     

Mycobiome of the Bat White Nose Syndrome Affected Caves and Mines Reveals Diversity of Fungi and Local Adaptation by the Fungal Pathogen Pseudogymnoascus (Geomyces) destructans

Current investigations of bat White Nose Syndrome (WNS) and the causative fungus Pseudogymnoascus (Geomyces) destructans (Pd) are intensely focused on the reasons for the appearance of the disease in the Northeast and its rapid spread in the US and Canada. Urgent steps are still needed for the mitigation or control of Pd to save bats. We hypothesized that a focus on fungal community would advance the understanding of ecology and ecosystem processes that are crucial in the disease transmission cycle. This study was conducted in 2010–2011 in New York and Vermont using 90 samples from four mines and two caves situated within the epicenter of WNS. We used culture-dependent (CD) and culture-independent (CI) methods to catalogue all fungi (‘mycobiome’). CD methods included fungal isolations followed by phenotypic and molecular identifications. CI methods included amplification of DNA extracted from environmental samples with universal fungal primers followed by cloning and sequencing. CD methods yielded 675 fungal isolates and CI method yielded 594 fungal environmental nucleic acid sequences (FENAS). The core mycobiome of WNS comprised of 136 operational taxonomic units (OTUs) recovered in culture and 248 OTUs recovered in clone libraries. The fungal community was diverse across the sites, although a subgroup of dominant cosmopolitan fungi was present. The frequent recovery of Pd (18% of samples positive by culture) even in the presence of dominant, cosmopolitan fungal genera suggests some level of local adaptation in WNS-afflicted habitats, while the extensive distribution of Pd (48% of samples positive by real-time PCR) suggests an active reservoir of the pathogen at these sites. These findings underscore the need for integrated disease control measures that target both bats and Pd in the hibernacula for the control of WNS.     

Potent Inhibition of Pseudogymnoascus destructans,the Causative Agent of White-Nose Syndrome in Bats,by Cold-Pressed,Terpeneless, Valencia Orange Oil

The causative agent of White-nose Syndrome (WNS), Pseudogymnoascus destructans, has been shown to be fatal to several species of bats in North America. To date, no compounds or chemical control measures have been developed which eliminates the growth of the fungus in the environment or in affected animals. In the current study, we evaluated the activity of cold-pressed, terpeneless orange oil (CPT) against multiple isolates of P. destructans in vitro. For all assays, a modified Kirby-Bauer disk diffusion assay was used. Standardized spore suspensions were prepared, adjusted to a specific optical density, and used to plate fungal lawns. Plates were incubated at either 15°C or 4°C for up to 6 months and checked at regular intervals for growth. Once controls had grown, zones of inhibition were measured (mm) on test plates and compared to those obtained using current antifungal drugs. All P. destructans isolates were completely inhibited by 100% CPT (10 μL) at 1 month of incubation regardless of temperature (4°C and 15°C). Complete inhibition persisted up to 6 months following a single exposure at this concentration. Of the standard antifungals, only amphotericin B demonstrated any activity, resulting in zone diameters ranging from 58 mm to 74 mm. CPT, at the highest concentration tested (100%), had no significant effect against a variety of other environmental organisms including various filamentous fungi, bacteria and aerobic actinomycetes. Given that CPT is relatively non-toxic, the possibility exists that the all-natural, mixture could be used as an environmental pre-treatment to eradicate P. destructans from bat habitats. Additional studies are needed to assess any undesirable effects of CPT on bat behavior and health and overall impacts on other members of the interconnected ecosystem(s).     

Novel Trichoderma polysporum Strain for the Biocontrol of Pseudogymnoascus destructans,the Fungal Etiologic Agent of Bat White Nose Syndrome

White-nose syndrome (WNS), an emerging disease of hibernating bats, has rapidly spread across eastern North America killing millions of bats. Pseudogymnoascus destructans (Pd), the sole etiologic agent of WNS, is widespread and persistent in bat hibernacula. Control of Pd in the affected sites is urgently needed to break the transmission cycle while minimizing any adverse impact on the native organisms. We isolated a novel strain of Trichoderma polysporum (Tp) from one of the caves at the epicenter of WNS zoonotic. Detailed experimental studies revealed: (1) Tp WPM 39143 was highly adapted to grow at temperatures simulating the cave environment (6°C-15°C), (2) Tp WPM 39143 restricted Pd colony growth in dual culture challenges, (3) Tp WPM 39143 caused four logs reduction of Pd colony forming units and genome copies in autoclaved soil samples from one of the WNS affected caves, (4) Tp WPM 39143 extract showed specific fungicidal activity against Pd in disk diffusion assay, but not against closely related fungus P. pannorum (Pp), (5) Tp WPM 39143 extract retained inhibitory activity after exposure to high temperatures, light and proteinase K, and (6) Inhibitory metabolites in Tp WPM 39143 extract comprised of water-soluble, high polarity compounds. These results suggest that Tp WPM 39143 is a promising candidate for further evaluation as a biocontrol agent of Pd in WNS affected sites.     

DNA-based detection of the fungal pathogen Geomyces destructans in soils from bat hibernacula

White-nose syndrome (WNS) is an emerging disease causing unprecedented morbidity and mortality among bats in eastern North America. The disease is characterized by cutaneous infection of hibernating bats by the psychrophilic fungus Geomyces destructans. Detection of G. destructans in environments occupied by bats will be critical for WNS surveillance, management and characterization of the fungal lifecycle. We initiated an rRNA gene region-based molecular survey to characterize the distribution of G. destructans in soil samples collected from bat hibernacula in the eastern United States with an existing PCR test. Although this test did not specifically detect G. destructans in soil samples based on a presence/absence metric, it did favor amplification of DNA from putative Geomyces species. Cloning and sequencing of PCR products amplified from 24 soil samples revealed 74 unique sequence variants representing 12 clades. Clones with exact sequence matches to G. destructans were identified in three of 19 soil samples from hibernacula in states where WNS is known to occur. Geomyces destructans was not identified in an additional five samples collected outside the region where WNS has been documented. This study highlights the diversity of putative Geomyces spp. in soil from bat hibernacula and indicates that further research is needed to better define the taxonomy of this genus and to develop enhanced diagnostic tests for rapid and specific detection of G. destructans in environmental samples.     

First Isolation of Pseudogymnoascus destructans,the Fungal Causative Agent of White-Nose Disease,in Bats from Italy

Mycopathologia - White-nose disease, caused by the dermatophyte Pseudogymnoascus destructans, is a devastating pathology that has caused a massive decline in the US bat populations. In Europe, this...     

Increasing incidence of Geomyces destructans fungus in bats from the Czech Republic and Slovakia

Background

White-nose syndrome is a disease of hibernating insectivorous bats associated with the fungus Geomyces destructans. It first appeared in North America in 2006, where over a million bats died since then. In Europe, G. destructans was first identified in France in 2009. Its distribution, infection dynamics, and effects on hibernating bats in Europe are largely unknown.

Methodology/Principal Findings

We screened hibernacula in the Czech Republic and Slovakia for the presence of the fungus during the winter seasons of 2008/2009 and 2009/2010. In winter 2009/2010, we found infected bats in 76 out of 98 surveyed sites, in which the majority had been previously negative. A photographic record of over 6000 hibernating bats, taken since 1994, revealed bats with fungal growths since 1995; however, the incidence of such bats increased in Myotis myotis from 2% in 2007 to 14% by 2010. Microscopic, cultivation and molecular genetic evaluations confirmed the identity of the recently sampled fungus as G. destructans, and demonstrated its continuous distribution in the studied area. At the end of the hibernation season we recorded pathologic changes in the skin of the affected bats, from which the fungus was isolated. We registered no mass mortality caused by the fungus, and the recorded population decline in the last two years of the most affected species, M. myotis, is within the population trend prediction interval.

Conclusions/Significance

G. destructans was found to be widespread in the Czech Republic and Slovakia, with an epizootic incidence in bats during the most recent years. Further development of the situation urgently requires a detailed pan-European monitoring scheme.     

The Resistance of a North American Bat Species (Eptesicus fuscus) to White-Nose Syndrome (WNS)

White-nose Syndrome (WNS) is the primary cause of over-winter mortality for little brown (Myotis lucifugus), northern (Myotis septentrionalis), and tricolored (Perimyotis subflavus) bats, and is due to cutaneous infection with the fungus Pseudogymnoascus (Geomyces) destructans (Pd). Cutaneous infection with P. destructans disrupts torpor patterns, which is thought to lead to a premature depletion of body fat reserve. Field studies were conducted at 3 WNS-affected hibernation sites to determine if big brown bats (Eptesicus fuscus) are resistant to Pd. Radio telemetry studies were conducted during 2 winters to determine the torpor patterns of 23 free-ranging E. fuscus hibernating at a site where Pd occurs. The body fat contents of free-ranging E. fuscus and M. lucifugus during hibernation at 2 different WNS-affected sites were also determined. The numbers of bats hibernating at the same site was determined during both: a) 4–7 years prior to the arrival of Pd, and, b) 2–3 years after it first appeared at this site. The torpor bouts of big brown bats hibernating at a WNS-affected site were not significantly different in length from those previously reported for this species. The mean body fat content of E. fuscus in February was nearly twice that of M. lucifugus hibernating at the same WNS-affected sites during this month. The number of M. lucifugus hibernating at one site decreased by 99.6% after P. destructans first appeared, whereas the number of E. fuscus hibernating there actually increased by 43% during the same period. None of the E. fuscus collected during this study had any visible fungal growth or lesions on their skin, whereas virtually all the M. lucifugus collected had visible fungal growth on their wings, muzzle, and ears. These findings indicate that big brown bats are resistant to WNS.     

Pan-European distribution of white-nose syndrome fungus (Geomyces destructans) not associated with mass mortality

Background

The dramatic mass mortalities amongst hibernating bats in Northeastern America caused by “white nose-syndrome” (WNS) continue to threaten populations of different bat species. The cold-loving fungus, Geomyces destructans, is the most likely causative agent leading to extensive destruction of the skin, particularly the wing membranes. Recent investigations in Europe confirmed the presence of the fungus G. destructans without associated mass mortality in hibernating bats in six countries but its distribution remains poorly known.

Methodology/Principal Findings

We collected data on the presence of bats with white fungal growth in 12 countries in Europe between 2003 and 2010 and conducted morphological and genetic analysis to confirm the identity of the fungus as Geomyces destructans. Our results demonstrate the presence of the fungus in eight countries spanning over 2000 km from West to East and provide compelling photographic evidence for its presence in another four countries including Romania, and Turkey. Furthermore, matching prevalence data of a hibernaculum monitored over two consecutive years with data from across Europe show that the temporal occurrence of the fungus, which first becomes visible around February, peaks in March but can still be seen in some torpid bats in May or June, is strikingly similar throughout Europe. Finally, we isolated and cultured G. destructans from a cave wall adjacent to a bat with fungal growth.

Conclusions/Significance

G. destructans is widely found over large areas of the European continent without associated mass mortalities in bats, suggesting that the fungus is native to Europe. The characterisation of the temporal variation in G. destructans growth on bats provides reference data for studying the spatio-temporal dynamic of the fungus. Finally, the presence of G. destructans spores on cave walls suggests that hibernacula could act as passive vectors and/or reservoirs for G. destructans and therefore, might play an important role in the transmission process.     

Recovery of little brown bats (Myotis lucifugus) from natural infection with Geomyces destructans, white-nose syndrome

Geomyces destructans produces the white fungal growth on the muzzle and the tacky white discoloration on wings and ears that characterize white-nose syndrome (WNS) in cave-hibernating bats. To test the hypothesis that postemergent WNS-infected bats recover from infection with G. destructans, 30 little brown bats (Myotis lucifugus) were collected in May 2009 from a WNS-affected hibernation site in New Jersey. All bats were confirmed to be infected with G. destructans using a noninvasive fungal tape method to identify the conidia of G. destructans and polymerase chain reaction (PCR). The bats were then held in captivity and given supportive care for 70 days. Of the 26 bats that survived and were humanely killed after 70 days, 25 showed significant improvement in the external appearance of wing membranes, had no microscopic evidence of infection by G. destructans, and had wing tissue samples that were negative for G. destructans by PCR. A subset of the bats was treated topically at the beginning of the rehabilitation study with a dilute vinegar solution, but treatment with vinegar provided no added advantage to recovery. Provision of supportive care to homeothermic bats was sufficient for full recovery from WNS. One bat at day 70 still had both gross pathology and microscopic evidence of WNS in wing membranes and was PCR-positive for G. destructans. Dense aggregates of neutrophils surrounded the hyphae that remained in the wing membrane of this bat.     

蝙蝠传染病白鼻综合症研究进展

于2006年爆发的蝙蝠传染病白鼻综合症(WNS)使大量的蝙蝠物种面临死亡威胁。研究发现,蝙蝠白鼻综合症是由好寒性真菌锈腐假裸囊子菌(Pseudogymnoascus destructans)引起,通过侵染皮肤表面而使冬眠期蝙蝠表现出一系列异常行为,导致其储存的能量和脂肪提早耗尽,最终死亡。本文对白鼻综合症发病症状、传播规律、致病机理和免疫遗传反应等研究进行了论述,并进一步提出了研究展望。     

Psychrophilic and Psychrotolerant Fungi on Bats and the Presence of Geomyces spp. on Bat Wings Prior to the Arrival of White Nose Syndrome

Since 2006, Geomyces destructans, the causative agent of white nose syndrome (WNS), has killed over 5.7 million bats in North America. The current hypothesis suggests that this novel fungus is an invasive species from Europe, but little is known about the diversity within the genus Geomyces and its distribution on bats in the United States. We documented the psychrophilic and psychrotolerant fungal flora of hibernating bats prior to the arrival of WNS using culture-based techniques. A total of 149 cultures, which were obtained from 30 bats in five bat hibernacula located in four caves and one mine, were sequenced for the entire internal transcribed spacer (ITS) nuclear ribosomal DNA (nrDNA) region. Approximately 53 operational taxonomic units (OTUs) at 97% similarity were recovered from bat wings, with the community dominated by fungi within the genera Cladosporium, Fusarium, Geomyces, Mortierella, Penicillium, and Trichosporon. Eleven Geomyces isolates were obtained and placed in at least seven distinct Geomyces clades based on maximum-likelihood phylogenetic analyses. Temperature experiments revealed that all Geomyces strains isolated are psychrotolerant, unlike G. destructans, which is a true psychrophile. Our results confirm that a large diversity of fungi, including several Geomyces isolates, occurs on bats prior to the arrival of WNS. Most of these isolates were obtained from damaged wings. Additional studies need to be conducted to determine potential ecological roles of these abundant Geomyces strains isolated from bats.     

Antifungal testing and high-throughput screening of compound library against Geomyces destructans, the etiologic agent of geomycosis (WNS) in bats

Bats in the northeastern U.S. are affected by geomycosis caused by the fungus Geomyces destructans (Gd). This infection is commonly referred to as White Nose Syndrome (WNS). Over a million hibernating bats have died since the fungus was first discovered in 2006 in a cave near Albany, New York. A population viability analysis conducted on little brown bats (Myotis lucifugus), one of six bat species infected with Gd, suggests regional extinction of this species within 20 years. The fungus Gd is a psychrophile ("cold loving"), but nothing is known about how it thrives at low temperatures and what pathogenic attributes allow it to infect bats. This study aimed to determine if currently available antifungal drugs and biocides are effective against Gd. We tested five Gd strains for their susceptibility to antifungal drugs and high-throughput screened (HTS) one representative strain with SpectrumPlus compound library containing 1,920 compounds. The results indicated that Gd is susceptible to a number of antifungal drugs at concentrations similar to the susceptibility range of human pathogenic fungi. Strains of Gd were susceptible to amphotericin B, fluconazole, itraconazole, ketoconazole and voriconazole. In contrast, very high MICs (minimum inhibitory concentrations) of flucytosine and echinocandins were needed for growth inhibition, which were suggestive of fungal resistance to these drugs. Of the 1,920 compounds in the library, a few caused 50%--to greater than 90% inhibition of Gd growth. A number of azole antifungals, a fungicide, and some biocides caused prominent growth inhibition. Our results could provide a theoretical basis for future strategies aimed at the rehabilitation of most affected bat species and for decontamination of Gd in the cave environment.     

Lipolytic Activity and the Utilization of Fatty Acids Associated with Bat Sebum by Pseudogymnoascus destructans

Mycopathologia - Pseudogymnoascus destructans is the causative agent of a fungal infection of bats known as white-nose syndrome (WNS). Since its discovery in 2006, it has been responsible for...     

Aerial Transport of Keratinaceous Substrate and Distribution of the Fungus Geomyces pannorum in Antarctic Soils

Abstract Aerobiological sampling was conducted year-round at three sites on Signy Island, maritime Antarctic, between December 1992 and January 1994, and during the summer at a fourth coastal site. The concentrations of keratinaceous particles were monitored. Feather fragments were the most abundant of all particle types trapped. Seal hairs were also common, particularly on the coast. Numbers of both particle types were most common in the air in summer. The Antarctic soil fungus Geomyces pannorum, which is able to utilize keratin-based substrates, was also present in aerobiological samples on Signy Island. G. pannorum was only found during early winter on the year-round sampling sites. It was more abundant during summer in the air at the coastal site, which was more heavily influenced by seals, birds and humans. Skuas were swabbed when they returned to the island early in summer. G. pannorum was recovered in culture, suggesting that these birds may act as vectors for the transport of microorganisms between Antarctica and more northern landmasses. Organic material deposited on snow was concentrated on the soil at the edge of the ice cap by melt wash, making additional keratin and other organic substrates available to soil microorganisms. Received: 4 January 1998; Accepted: 25 February 1998