Laboratory Maintenance and Culture of Pseudogymnoascus destructans , the Fungus That Causes Bat White‐Nose Syndrome

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
David S. Blehert ◽  
Jeffrey M. Lorch
Keyword(s):  
Pseudogymnoascus Destructans ◽  
White Nose Syndrome ◽  
Laboratory Maintenance

scholarly journals Pseudogymnoascus destructans growth in wood, soil and guano substrates

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jenny Urbina ◽  
Tara Chestnut ◽  
Jennifer M. Allen ◽  
Taal Levi
Keyword(s):  
Dna Extraction ◽  
Emerging Infectious Diseases ◽  
Transmission Risk ◽  
Pseudogymnoascus Destructans ◽  
White Nose Syndrome ◽  
Management Interventions ◽  
Pathogen Persistence ◽  
Tenfold Increase ◽  
And Control

AbstractUnderstanding how a pathogen can grow on different substrates and how this growth impacts its dispersal are critical to understanding the risks and control of emerging infectious diseases. Pseudogymnoascus destructans (Pd) causes white-nose syndrome (WNS) in many bat species and can persist in, and transmit from, the environment. We experimentally evaluated Pd growth on common substrates to better understand mechanisms of pathogen persistence, transmission and viability. We inoculated autoclaved guano, fresh guano, soil, and wood with live Pd fungus and evaluated (1) whether Pd grows or persists on each (2) if spores of the fungus remain viable 4 months after inoculation on each substrate, and (3) whether detection and quantitation of Pd on swabs is sensitive to the choice to two commonly used DNA extraction kits. After inoculating each substrate with 460,000 Pd spores, we collected ~ 0.20 g of guano and soil, and swabs from wood every 16 days for 64 days to quantify pathogen load through time using real-time qPCR. We detected Pd on all substrates over the course of the experiment. We observed a tenfold increase in pathogen loads on autoclaved guano and persistence but not growth in fresh guano. Pathogen loads increased marginally on wood but declined ~ 60-fold in soil. After four months, apparently viable spores were harvested from all substrates but germination did not occur from fresh guano. We additionally found that detection and quantitation of Pd from swabs of wood surfaces is sensitive to the DNA extraction method. The commonly used PrepMan Ultra Reagent protocol yielded substantially less DNA than did the QIAGEN DNeasy Blood and Tissue Kit. Notably the PrepMan Ultra Reagent failed to detect Pd in many wood swabs that were detected by QIAGEN and were subsequently found to contain substantial live conidia. Our results indicate that Pd can persist or even grow on common environmental substrates with results dependent on whether microbial competitors have been eliminated. Although we observed clear rapid declines in Pd on soil, viable spores were harvested four months after inoculation. These results suggest that environmental substrates and guano can in general serve as infectious environmental reservoirs due to long-term persistence, and even growth, of live Pd. This should inform management interventions to sanitize or modify structures to reduce transmission risk as well early detection rapid response (EDRR) planning.

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

PLoS ONE ◽  
2015 ◽  
Vol 10 (4) ◽  
pp. e0121329 ◽  
Author(s):  
Joseph R. Hoyt ◽  
Tina L. Cheng ◽  
Kate E. Langwig ◽  
Mallory M. Hee ◽  
Winifred F. Frick ◽  
...  
Keyword(s):  
Causative Agent ◽  
Pseudogymnoascus Destructans ◽  
White Nose Syndrome

scholarly journals Hepatic lipid signatures of little brown bats (Myotis lucifugus) and big brown bats (Eptesicus fuscus) at early stages of white-nose syndrome

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Evan L. Pannkuk ◽  
Nicole A. S.-Y. Dorville ◽  
Yvonne A. Dzal ◽  
Quinn E. Fletcher ◽  
Kaleigh J. O. Norquay ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Fatty Acid Distribution ◽  
Eptesicus Fuscus ◽  
Myotis Lucifugus ◽  
Pseudogymnoascus Destructans ◽  
Hepatic Lipid ◽  
Early Stages ◽  
White Nose Syndrome ◽  
Big Brown Bats ◽  
Little Brown Bats

AbstractWhite-nose syndrome (WNS) is an emergent wildlife fungal disease of cave-dwelling, hibernating bats that has led to unprecedented mortalities throughout North America. A primary factor in WNS-associated bat mortality includes increased arousals from torpor and premature fat depletion during winter months. Details of species and sex-specific changes in lipid metabolism during WNS are poorly understood and may play an important role in the pathophysiology of the disease. Given the likely role of fat metabolism in WNS and the fact that the liver plays a crucial role in fatty acid distribution and lipid storage, we assessed hepatic lipid signatures of little brown bats (Myotis lucifugus) and big brown bats (Eptesicus fuscus) at an early stage of infection with the etiological agent, Pseudogymnoascus destructans (Pd). Differences in lipid profiles were detected at the species and sex level in the sham-inoculated treatment, most strikingly in higher hepatic triacylglyceride (TG) levels in E. fuscus females compared to males. Interestingly, several dominant TGs (storage lipids) decreased dramatically after Pd infection in both female M. lucifugus and E. fuscus. Increases in hepatic glycerophospholipid (structural lipid) levels were only observed in M. lucifugus, including two phosphatidylcholines (PC [32:1], PC [42:6]) and one phosphatidylglycerol (PG [34:1]). These results suggest that even at early stages of WNS, changes in hepatic lipid mobilization may occur and be species and sex specific. As pre-hibernation lipid reserves may aid in bat persistence and survival during WNS, these early perturbations to lipid metabolism could have important implications for management responses that aid in pre-hibernation fat storage.

Characterization of PdCP1, a serine carboxypeptidase from Pseudogymnoascus destructans, the causal agent of White-nose Syndrome

2018 ◽  
Vol 399 (12) ◽  
pp. 1375-1388 ◽  
Author(s):  
Chapman Beekman ◽  
Zhenze Jiang ◽  
Brian M. Suzuki ◽  
Jonathan M. Palmer ◽  
Daniel L. Lindner ◽  
...  
Keyword(s):  
Pathogenic Fungus ◽  
Hydrolytic Enzymes ◽  
Carboxyl Terminus ◽  
Serine Carboxypeptidase ◽  
Pseudogymnoascus Destructans ◽  
Fluorescent Substrate ◽  
White Nose Syndrome ◽  
Species Comparisons ◽  
Bona Fide ◽  
Multiple Species

Abstract Pseudogymnoascus destructans is a pathogenic fungus responsible for White-nose Syndrome (WNS), a disease afflicting multiple species of North American bats. Pseudogymnoascus destructans infects susceptible bats during hibernation, invading dermal tissue and causing extensive tissue damage. In contrast, other Pseudogymnoascus species are non-pathogenic and cross-species comparisons may therefore reveal factors that contribute to virulence. In this study, we compared the secretome of P. destructans with that from several closely related Pseudogymnoascus species. A diverse set of hydrolytic enzymes were identified, including a putative serine peptidase, PdCP1, that was unique to the P. destructans secretome. A recombinant form of PdCP1 was purified and substrate preference determined using a multiplexed-substrate profiling method based on enzymatic degradation of a synthetic peptide library and analysis by mass spectrometry. Most peptide substrates were sequentially truncated from the carboxyl-terminus revealing that this enzyme is a bona fide carboxypeptidase. Peptides with arginine located close to the carboxyl-terminus were rapidly cleaved, and a fluorescent substrate containing arginine was therefore used to characterize PdCP1 activity and to screen a selection of peptidase inhibitors. Antipain and leupeptin were found to be the most potent inhibitors of PdCP1 activity.

scholarly journals Determinants of Pseudogymnoascus destructans within bat hibernacula: Implications for surveillance and management of white-nose syndrome

2018 ◽  
Vol 55 (2) ◽  
pp. 820-829 ◽  
Author(s):  
Michelle L. Verant ◽  
Elizabeth A. Bohuski ◽  
Katherine L. D. Richgels ◽  
Kevin J. Olival ◽  
Jonathan H. Epstein ◽  
...  
Keyword(s):  
Pseudogymnoascus Destructans ◽  
White Nose Syndrome

scholarly journals Experimental Infection ofTadarida brasiliensiswithPseudogymnoascus destructans, the Fungus That Causes White-Nose Syndrome

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
Author(s):  
Michelle L. Verant ◽  
Carol U. Meteyer ◽  
Benjamin Stading ◽  
David S. Blehert
Keyword(s):  
Animal Model ◽  
Experimental Animal ◽  
Experimental Infection ◽  
Abundant Species ◽  
Experimental Animal Model ◽  
Pseudogymnoascus Destructans ◽  
Tadarida Brasiliensis ◽  
Content Type ◽  
White Nose Syndrome ◽  
Management Actions

ABSTRACTWhite-nose syndrome (WNS) is causing significant declines in populations of North American hibernating bats, and recent western and southern expansions of the disease have placed additional species at risk. Understanding differences in species susceptibility and identifying management actions to reduce mortality of bats from WNS are top research priorities. However, the use of wild-caught susceptible bats, such asMyotis lucifugus, as model species for WNS research is problematic and places additional pressure on remnant populations. We investigated the feasibility of usingTadarida brasiliensis, a highly abundant species of bat that tolerates captivity, as the basis for an experimental animal model for WNS. Using methods previously established to confirm the etiology of WNS inM. lucifugus, we experimentally infected 11T. brasiliensisbats withPseudogymnoascus destructansin the laboratory under conditions that induced hibernation. We detectedP. destructanson all 11 experimentally infected bats, 7 of which exhibited localized proliferation of hyphae within the epidermis, dermis, and subcutaneous tissue, similar to invasive cutaneous ascomycosis observed inM. lucifugusbats with WNS. However, the distribution of lesions across wing membranes ofT. brasiliensisbats was limited, and only one discrete “cupping erosion,” diagnostic for WNS, was identified. Thus, the rarity of lesions definitive for WNS suggests thatT. brasiliensisdoes not likely represent an appropriate model for studying the pathophysiology of this disease. Nonetheless, the results of this study prompt questions concerning the potential for free-ranging, migratoryT. brasiliensisbats to become infected withP. destructansand move the fungal pathogen between roost sites used by species susceptible to WNS.IMPORTANCEWhite-nose syndrome (WNS) is a fungal disease that is causing severe declines of bat populations in North America. Identifying ways to reduce the impacts of this disease is a priority but is inhibited by the lack of an experimental animal model that does not require the use of wild-caught bat species already impacted by WNS. We tested whetherTadarida brasiliensis, one of the most abundant species of bats in the Americas, could serve as a suitable animal model for WNS research. WhileT. brasiliensisbats were susceptible to experimental infection with the fungus under conditions that induced hibernation, the species exhibited limited pathology diagnostic for WNS. These results indicate thatT. brasiliensisis not likely a suitable experimental model for WNS research. However, the recovery of viable WNS-causing fungus from experimentally infected bats indicates a potential for this species to contribute to the spread of the pathogen where it coexists with other species of bats affected by WNS.

scholarly journals Destructin-1 is a collagen-degrading endopeptidase secreted by Pseudogymnoascus destructans, the causative agent of white-nose syndrome

2015 ◽  
Vol 112 (24) ◽  
pp. 7478-7483 ◽  
Author(s):  
Anthony J. O’Donoghue ◽  
Giselle M. Knudsen ◽  
Chapman Beekman ◽  
Jenna A. Perry ◽  
Alexander D. Johnson ◽  
...  
Keyword(s):  
Causative Agent ◽  
Genetic Manipulation ◽  
Hydrolytic Enzymes ◽  
Collagen Degradation ◽  
Mammalian Host ◽  
Peptidase Activity ◽  
Pseudogymnoascus Destructans ◽  
White Nose Syndrome ◽  
Proteolytic Activities ◽  
The Impact

Pseudogymnoascus destructans is the causative agent of white-nose syndrome, a disease that has caused the deaths of millions of bats in North America. This psychrophilic fungus proliferates at low temperatures and targets hibernating bats, resulting in their premature arousal from stupor with catastrophic consequences. Despite the impact of white-nose syndrome, little is known about the fungus itself or how it infects its mammalian host. P. destructans is not amenable to genetic manipulation, and therefore understanding the proteins involved in infection requires alternative approaches. Here, we identify hydrolytic enzymes secreted by P. destructans, and use a novel and unbiased substrate profiling technique to define active peptidases. These experiments revealed that endopeptidases are the major proteolytic activities secreted by P. destructans, and that collagen, the major structural protein in mammals, is actively degraded by the secretome. A serine endopeptidase, hereby-named Destructin-1, was subsequently identified, and a recombinant form overexpressed and purified. Biochemical analysis of Destructin-1 showed that it mediated collagen degradation, and a potent inhibitor of peptidase activity was identified. Treatment of P. destructans-conditioned media with this antagonist blocked collagen degradation and facilitated the detection of additional secreted proteolytic activities, including aminopeptidases and carboxypeptidases. These results provide molecular insights into the secretome of P. destructans, and identify serine endopeptidases that have the clear potential to facilitate tissue invasion and pathogenesis in the mammalian host.

Prevalence and phenology of white-nose syndrome fungus Pseudogymnoascus destructans in bats from Poland

2014 ◽  
Vol 9 (4) ◽  
pp. 437-443 ◽  
Author(s):  
Konrad Sachanowicz ◽  
Arkadiusz Stępień ◽  
Mateusz Ciechanowski
Keyword(s):  
North America ◽  
Mass Mortality ◽  
Parasitic Fungus ◽  
Pseudogymnoascus Destructans ◽  
Southern Poland ◽  
White Nose Syndrome ◽  
Two Samples ◽  
Myotis Myotis ◽  
Myotis Daubentonii ◽  
Fungal Dna

AbstractPseudogymnoascus destructans (Pd), a parasitic fungus (being responsible for a disease known as white-nose syndrome, WNS) that caused mass mortality of cave-dwelling, hibernating bats in North America, appears to be native of Europe, where it also occurs on wintering bats, but no similar outbreaks of WNS have been recorded. Herein, we provide the first account on prevalence and phenology of P. destructans in Poland. Bats were counted once per month, from October or January to May (2010-2013), in an abandoned ore mine in southern Poland. Presence of P. destructans in two samples was confirmed by sequencing of isolated fungal DNA. Observations of phenotypically identical mycosis on bats hibernating at this site in March 2006 are likely to be the first known records of P. destructans from Poland. All Pd-suspected individuals were Myotis myotis with an exception of one Myotis daubentonii. The first Pd-suspected bats were noted in mid-February, but their number was the highest in March, what overlapped with maximum numbers of hibernating M. myotis. The prevalence in March was 7%–27% of M. myotis individuals. No mass mortality of bats was observed in the mine, with only three dead individuals found in the hibernaculum which hosted up to 130 bats, representing 6–7 species.

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