LONG-TERM SURVIVAL OF PSEUDOGYMNOASCUS DESTRUCTANS AT ELEVATED TEMPERATURES

Novel diseases are emerging at a faster rate than at any point in history (Jones et al. 2008). Accordingly, emerging infectious diseases are increasingly being recognized as a threat to both human and animal health (Daszak et al. 2000; Pedersen et al. 2007). The emerging infectious diseases caused by fungal pathogens have been implicated in some of the most widespread and deadly disease outbreaks in wildlife (Fisher et al. 2012).

White-nose syndrome (WNS) is a cutaneous fungal disease that has caused catastrophic declines of several hibernating bat species throughout eastern North America (Blehert et al. 2009; Frick et al. 2010; Langwig et al. 2016). The disease is caused by the fungus Pseudogymnoascus destructans (PD; Lorch et al. 2011), first detected in New York State in 2006 (Blehert et al. 2009), and is believed to have been introduced into the US from Europe (Leopardi et al. 2015). During hibernation, PD proliferates on the skin of infected bats, resulting in damage to dermal tissues and the eponymous white growth around the animal's muzzle (Blehert et al. 2009). Individuals afflicted by WNS often succumb because of increased levels of arousal during hibernation and subsequent physiological disturbance (Reeder et al. 2012; Cryan et al. 2013; Verant et al. 2014).

Consistent with WNS only affecting bats during hibernation, PD is a psychrophilic fungus with a known optimal thermal range for growth between 12 and 15 C, with cessation of growth at 20 C (Verant et al. 2012). Fungal growth and spore production of PD positively correlate with relative humidity (RH) up to approximately 85%, above which no further increases in growth or sporulation occur (Marroquin et al. 2017). The fungus grows on a wide variety of substrates in a laboratory setting (Reynolds and Barton 2014a; Reynolds et al. 2015) and can persist in a viable state within the sediment of hibernacula in the absence of a bat host for at least 2 yr under cool and humid conditions (Lorch et al. 2013a, 2013b).

During hibernation, PD is likely transmitted through direct contact between bats, or from contaminated environments to bats (Lorch et al. 2011, 2013b; Hoyt et al. 2018); however, the mechanisms of spread between hibernacula are less well understood. The primary transmission mechanism is hypothesized to be bat mediated (Maher et al. 2012; Petit and Puechmaille 2015). Most hibernating bat species leave hibernacula in late spring to roost at maternity colonies. In the autumn, bats form mating swarms before returning to hibernacula (Langwig et al. 2015). The movement and mixing of bats across the landscape during this active period, and subsequent contact between infected bats from one hibernaculum and naïve bats from another, are believed to facilitate the spread of WNS (Langwig et al. 2015). However, the warmer temperatures outside of hibernacula and the higher body temperatures of active bats may limit dispersal of PD (Langwig et al. 2015). The prevalence of PD on bat skin during the summer months is relatively low (Dobony et al. 2011; Langwig et al. 2015). Furthermore, detection of PD on bats in summer is primarily restricted to PCR techniques, which could be detecting nonviable fungus. Dobony et al. (2011) were able to culture viable PD from the wings of bats sampled during the active period in May and August, but not from bats sampled in June and July. It is possible that these bats sampled early and later in the active period had only recently emerged from their hibernacula or may have begun to explore underground roosts after leaving the maternity colony.

Pseudogymnoascus destructans may persist for long periods within its thermal range (Lorch et al. 2013b; Hoyt et al. 2015), but the survival of PD conidia during prolonged exposure to temperatures above its upper thermal growth limit has not been studied. Knowledge of the influence of elevated temperatures on the survival of PD conidia will increase our understanding of PD dispersal, help to accurately predict how WNS may spread across North America (Reynolds and Barton 2014b), and aid in the development of management strategies to limit its range expansion. We investigated the ability of PD to survive at three elevated temperatures. We cultured PD at three temperatures on three artificial growth media containing varying levels of nutrients and on bat fur. We hypothesized that PD would survive longer when exposed to lower temperatures on media with higher levels of nutrients and on bat fur.

Experimental procedure: We tested the survival of PD at elevated temperatures using three different types of artificial growth media: Sabouraud dextrose agar (SD), brain–heart infusion agar (BHI), and BHI agar supplemented with 10% sheep (Ovis aries) blood (BHIB). We included SD agar because it is a commonly utilized medium for fungal cultivation, BHI because it is a complex medium that may more accurately represent the mammalian host environment, and BHIB because preliminary work demonstrated that PD has a unique morphology on this medium that is most consistent with what is observed during infection of bat skin. Conidia from PD (type isolate American Type Culture Collection #ATCC MYA-4855) were harvested following the protocol outlined by Lorch et al. (2011) from 60-d-old cultures grown at 7 C on SD. Inoculations occurred on multiple days and therefore involved multiple source cultures. The number of conidia per harvest was enumerated using a hemocytometer, and conidia were subsequently diluted in phosphate-buffered saline with 0.5% Tween 20 (Sigma-Aldrich, St. Louis, Missouri, USA) to final concentrations of 1, 10, and 100 conidia/µL. Preliminary data suggested that the number of conidia required to inoculate each plate and recover a distinguishable number of colony-forming units (CFUs) varied with incubation temperature and incubation time. Thus, plates were either inoculated with 100, 1,000 or 10,000 conidia; plates exposed to higher temperatures or longer incubation times were generally inoculated with a larger number of conidia (Supplementary Material Table S1). One hundred microliters of the appropriate conidial suspension were pipetted onto 25-mL petri dishes containing 20 mL of the respective medium. Conidial suspensions were spread over each plate using sterile glass spreading rods. Plates were lidded and completely sealed using Parafilm (Beemis Company Inc., Neenah, Wisconsin, USA) to ensure a RH of about 100%; all plates remained completely sealed throughout the duration of the experiment. Plates were incubated at 24, 30, or 37 C for 1, 5, 9, 15, 30, 60, 90, 120, or 150 d. These temperatures were selected as they are above the known thermal range of growth for PD but within the range of normal summertime temperatures for the continental US and representative of the skin or body temperatures of nonhibernating bats. At the end of the respective incubation period, plates were transferred to a 7 C incubator for 50 d before the number of visible CFUs was recorded. To account for varying levels of initial viability of PD conidia between the starting colonies used for each medium, we created a control group by inoculating plates of each medium as described above and immediately incubating at 7 C for 50 d before enumerating CFUs. For each combination of temperature+media+incubation duration, including controls, at least six replicate plates were inoculated, incubated, and enumerated (Table S1). The identity of PD colonies that were recorded at the longest incubation duration for a given medium or temperature were confirmed by sequencing of the internal transcribed spacer region of the ribosomal RNA gene (Lorch et al. 2013a) and compared with sequence data for the type isolate in GenBank. Counts of CFUs per plate were normalized for differences in the initial number of conidia plated through division by the appropriate correction factor (Table S1).

Statistical analysis: As the initial viability of inoculum (i.e., conidia) varied on the basis of medium and each plate represented a distinct unit of measurement (rather than repeated measures of the same individuals over time), traditional survival analyses were not appropriate (Moore 2016). Therefore, we investigated the significance of trends in our data using a generalized linear model implemented under a Bayesian framework. Our model used a Poisson error distribution: Y_i∼d_pois(λ_i), where Y_i was the observed CFUs of the ith plate and λ_i was the expected value of CFUs for the ith plate. We incorporated the effects of treatment (temperature and medium) and time using a log link and the process model: log(λ_i)=log(µ_i)+χ_iβ_i+t_iδ_j,t, where µ_i was the offset term of mean number of viable colonies per control group for the medium used on the ith plate (thus accounting for variation caused by the discrepancies in levels of initial conidia viability between the starting colonies used for each medium). The treatment of the ith plate, which in this case referred to temperature-by-medium interactions (a nine-level factor), was denoted by χ_i. The effect of the jth treatment was denoted by β_j, t_i indicating the days postinoculation (DPI) at which the ith plate was observed, and δ_j,twas the interaction between jth treatment on the tth day. We specified diffuse normal priors (µ=0,σ²=100) for each of the β_j effects. To account for potential temporal autocorrelation between observations at adjacent time points, we used a random walk prior for each δ_j vector. The prior for the precision term for the random walk prior was a gamma (1,1) prior. Additionally, we enforced a sum-to-zero constraint of each δ_j vector to ensure that all β_j effects remained identifiable. Our model was run using Markov chain Monte Carlo (MCMC) simulation incorporating three independent chains. Each MCMC chain was run for 100,000 iterations and we discarded the first 10,000 iterations as burn-in. We graphically inspected trace and density plots of posterior distributions for each chain for evidence of nonconvergence. We also calculated the Gelman–Rueben (GR) statistic (Gelman and Rubin 1992) of each chain. Trace and density plots and GR statistics (all GR values <1.1) of each chain indicated no evidence of nonconvergence of the MCMC chains. Differences in log-transformed CFU count between each medium over time at each temperature were calculated from the posterior distribution. Under a Bayesian framework, strongly supported differences between two measurements are defined as differences that are 95% certain to not be 0; therefore, significant differences in PD survival per treatment were inferred by differences in log CFUs per media per time point, with 95% credible intervals that did not incorporate 0 (i.e., no difference).

Although the primary site of PD infection and growth on hibernating bats is the skin, PD conidia are also known to adhere to bat hair (Wibbelt et al. 2010). Therefore, to examine PD survival on a more ecologically relevant substrate, we examined survival of PD at various temperatures and incubation durations on bat hair. Hair used in the experiment was removed from the dorsal surface of a little brown bat (Myotis lucifugus) that was found dead and submitted to the US Geological Survey, National Wildlife Health Center (Madison, Wisconsin, USA) for diagnostic testing. The bat originated from outside of the known range of WNS and was negative for WNS (on the basis of histopathologic examination; Meteyer et al. 2009) and PD (on the basis of real-time PCR; Muller et al. 2013). Small amounts of bat hair were placed in 1.5-mL microcentrifuge tubes and sterilized by autoclaving. Sterile bat hair was then manually placed, using sterile forceps, against a conidia-producing culture of PD (type isolate) that had been grown on SD for 60 d at 7 C. The conidia transferred to each piece of bat hair were not enumerated. However, inspection of hair samples under a light microscope confirmed that conidia were reliably transferred to the hair from the fungal culture.

Inoculated hair was placed into sterile 1.5-mL microcentrifuge tubes containing 20 µL of sterile water (to maintain about 100% RH within each tube) and sealed. Tubes were incubated in the dark at either 7, 24, 30, or 37 C for 5, 9, 15, 30, 60, 90, 120, 150, 180, 210, or 240 d. Each temperature and duration combination was performed in triplicate. At the end of the incubation period, the inoculated hair was removed from its tube using sterile forceps, placed onto the center of SD plates, and incubated at 7 C for 50 d. The internal transcribed spacer region of fungi growing from the hair was sequenced to confirm that it was PD.

Regardless of medium type, survival of PD was reduced at higher temperatures. Pseudogymnoascus destructans was not viable on any medium type after 60 d (DPI) at 30 C nor after 30 DPI at 37 C (Fig. 1). However, viable PD was detected up until 150 DPI at 24 C (Fig. 1). Increased survival of PD was highly specific to medium type, with BHIB performing best at all three incubation temperatures, followed by BHI, then SD. At all three temperatures (24, 30, and 37 C), no PD survived on SD past 30 DPI or on BHI past 60 DPI; however, viable PD was cultured on BHIB plates incubated at 24 C for 150 DPI, 30 C for 30 DPI, and 37 C for 15 DPI (Fig. 1). When the differences in posterior distributions of log CFU count generated by our Bayesian generalized model were compared between all three medium types, we found strong support for the increased survival of PD on BHIB versus the other artificial media.

When incubated at 24 C, BHIB demonstrated an increase in PD survival from day 15 onward as compared with BHI, and from day 5 onward compared with SD (Figs. 2, 3). Additionally, survival of PD was enhanced on BHI as compared with SD between 5 and 30 DPI (Figs. 2, 3).

Incubation at 30 C resulted in a less marked increase in survival on BHIB; however, increased survival was strongly supported at 9 and 30 DPI as compared with BHI and between 5 and 60 DPI when compared with SD (Figs. 2, 3). Survival of PD conidia at 30 C was better on BHI versus SD at 5 and 15 DPI (Figs. 2, 3).

When incubated at 37 C, the increase in PD survival on BHIB was still evident, with strongly supported increases detectable at 9 and 15 DPI compared with BHI and 1 to 15 DPI compared with SD (Figs. 2, 3). No difference in survival was evident between BHI and SD for any time point at 37 C (Figs. 2, 3).

Viable PD conidia persisted on bat fur incubated at 7 C for the entire duration of the experiment (240 d). However, PD also survived on bat fur at temperatures outside of its growth range. When incubated at 24 C, viable PD conidia survived until 180 DPI. When incubated at 30 C and 37 C, viable PD conidia survived for 60 DPI and 5 DPI, respectively (Fig. S1).

Our results provided systematically gathered evidence that PD is capable of survival for extended periods when exposed to temperatures outside of its thermal growth range. We tested multiple media because fungal spore viability is known to vary with medium composition (Darby and Mandels 1955; Wu et al. 2000). Although survival characteristics in our study were medium specific, we found evidence that PD conidia were capable of surviving for at least 15 d when incubated at 37 C, and for at least 30 d and 150 d when incubated at 30 and 24 C, respectively. Survival of PD conidia was highest on BHIB across all three incubation temperatures (24, 30, and 37 C). When grown on bat fur in vitro, PD conidia persisted in a viable state for 180 d at 24 C, 60 d at 30 C, and 5 d at 37 C. The parameters and mechanisms that influence the survival of fungal conidia are not well understood and the reasons for the observed media specificity in conidia survival remain unclear. However, varying nutrient content resulting in media-specific responses in osmoregulation (Basu et al. 2015) or gene expression (Shah et al. 2005) is a potential explanation. Additionally, it should be noted that we did not measure the RH of our different media plates. Differences in the RH of each medium could potentially explain substrate-specific survival, although we assume that the RH of all media was at or near 100% because of plates being sealed.

Although it is widely hypothesized that the primary facilitators of PD spread between hibernacula are the bats that it infects, current evidence suggests that the potential of this spread during the summer active period is limited (Langwig et al. 2015). This hypothesis, however, is based primarily on low detection rates of PD on skin swabs of bats sampled during the summer (Langwig et al. 2015) and the knowledge that PD does not proliferate at temperatures greater than 20 C (Verant et al. 2012). Our results provide evidence that the potential for infected bats to disperse PD across the landscape during the active season is greater than currently appreciated.

During the nonhibernal seasons, hibernating bat species disperse distances up to 500 km to reach roost sites (Fleming and Eby 2003). For most species, these seasonal roosts can consist of aboveground structures with temperatures that are not conducive to the proliferation of PD; however, some bats may also use underground sites that are more hospitable to PD (Altringham 2011). Since PD is known to persist in the environment year round in underground hibernacula (Lorch et al. 2013b), bats visiting such sites during the summer are likely carriers of PD conidia (Ballmann et al. 2017). Hibernating bat species generally show low fidelity to their summer roost sites (Lewis 1995; Fleming and Eby 2003) and move between roosts to reduce disturbance, parasitism, and time spent in unfavorable microclimates (Lewis 1995). Although primarily euthermic during nonhibernal seasons, North American bats are known to cease thermoregulation when not physically active and to allow their body temperatures to closely mirror those of the ambient environment (Henshaw and Folk 1966). Our results suggest that even if bats are regularly exposed to ambient temperatures that exceed the upper growth limit of PD, viable conidia can survive on bat fur for extended periods of time, potentially allowing spread of the fungus from one infested hibernaculum to multiple naïve hibernacula by a single animal. However, our study exposed a pure culture of PD to constant temperature and humidity conditions in complete darkness. These conditions are not likely to be representative of conditions in nature, where PD present on bats or in the environment is likely to be exposed to fluctuating temperature, humidity, and light levels throughout the active season. Additionally, the in vitro nature of our experiment did not consider host–pathogen interactions that may affect PD survival such as host immune response (Field et al. 2015) or antagonistic relationships with co-occurring microbial species of the bat microbiome (Lemieux-Labonté et al. 2017).

Ballmann et al. (2017) found that fresh fecal material was positive for the fungus 10 times more frequently than the skin. Indeed, several bats whose guano was PD positive did not harbor detectable cutaneous PD. White-nose syndrome-positive bats increase grooming frequency compared with uninfected bats (Brownlee-Bouboulis and Reeder 2013), and fungus is likely ingested by bats during grooming and subsequently excreted. The guano of infected bats is therefore a significant potential source of PD transmission. Ingested substances are known to pass through the digestive tract of insectivorous bats in less than 24 h (Staliński 1994; Roswag et al. 2012). Even disregarding that bats do not continually maintain a high body temperature (Henshaw and Folk 1966), our finding that PD conidia survived for 15 d at 37 C on BHIB agar suggested that PD conidia may be able to survive transit through the bat digestive system. Thus, bat guano may be a primary source for movement of viable PD. However, the ability of PD to survive transition through the digestive tract remains unevaluated, and conditions during gut transit, such as reduced pH and enzymatic activity, may affect fungal viability. Future studies should evaluate the impact of pH on PD survival or attempt to directly culture PD from fresh bat guano.

Human activity within bat hibernacula, whether for research or recreational purposes, has been implicated as a potential source of PD dispersal (Shelley et al. 2013; Ballmann et al. 2017). Equipment used both in and near bat hibernacula during nonhibernal seasons can become contaminated with PD (Ballmann et al. 2017). Our results suggested that survival of PD conidia on equipment may be greater than currently appreciated. Additionally, we found PD survival to be highly substrate specific, meaning that the true potential for human-mediated pathogen dispersal cannot be appreciated without a fuller exploration of how different materials may become fomites. Further research is needed to evaluate the survival of PD on a range of materials that comprise equipment and clothing used in caving pursuits, including plastics, metals, and soft materials. The potential of prolonged and substrate-specific survival of PD on equipment, even at elevated temperatures, highlights the need for adhering to decontamination and bio-safety practices aimed at preventing the movement of pathogens between contaminated and naïve locations.

In conclusion, our finding that PD spores can persist for extended periods of time in a viable state despite exposure to temperatures outside of the thermal growth range for the fungus suggested that the potential for bat- and fomite-mediated spread of PD during the warmer, active season is greater than is currently appreciated. Further work is needed to more fully assess the generality of these findings in natural systems (e.g., survival of conidia on various natural substrates, interactions between survival and temperature, substrate, and humidity). However, the implications for pathogen persistence and spread may be profound. Additionally, our work highlights the importance of continued research aimed at increasing our knowledge of the basic biology of emerging pathogens to construct more accurate epidemiological models and guide conservationists and resource managers in their decision making.

The authors thank Julia Lankton for providing histopathologic support, and Robin Russell and Kyle George for statistical consultation and analysis. This work was funded by the US Fish and Wildlife Service and the US Geological Survey. The use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the US Government. Analysis scripts used for this manuscript are available from the github repository http://www.github.com/zoolew/PdSurvival. Raw data associated with this manuscript is made freely available by the US Geological Survey at https://doi.org/10.5066/P9WCBGUQ.

Supplementary material for this article is online at http://dx.doi.org/10.7589/2019-04-106.