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White-nose syndrome (WNS) is a disease among hibernating North American bats caused by the psychrophilic fungus Pseudogymnoascus destructans. Since its discovery in New York state, US, in 2006, and as of 2020, WNS has rapidly spread to 34 American states and seven Canadian provinces, causing precipitous declines of native bat populations across North America. The rapid spread of this fungal pathogen has been facilitated by the social behavior of bats, as well as the ability of subterranean hibernacula to support a favorable environment for P. destructans, and is probably exacerbated by anthropogenic transmission events. Although many bat species roost in natural cave environments, bats also selectively use diverse structures for hibernacula. Certain areas of the US lack caves, forcing bats to select different winter roosting environments. Bats have been observed using roadway-associated structures, such as bridges and culverts, for roosting, especially in regions that lack natural cave environments. However, the potential for P. destructans transmission in such roadway-associated structures requires further investigation. Understanding potential pathogen transmission in these widely used anthropogenic structures is crucial to disease management and preventing further declines of imperiled bat populations. Our study investigated these structures as potential pathogen transmission corridors by surveying the use of these structures by Perimyotis subflavus and other susceptible bat populations and by measuring their temperature. The results suggest the environments of roadway-associated culverts are thermally conducive to the proliferation of P. destructans-even in regions with mild winters-and the development of WNS in susceptible bat populations. It is apparent these roadway-associated structures have the potential to spread P. destructans and exacerbate the effect of WNS on susceptible bat populations.

Pseudogymnoascus destructans is a fungal pathogen responsible for a deadly disease among North American bats known as white-nose syndrome (WNS). Since detection of WNS in the United States in 2006, its rapid spread and high mortality has challenged development of treatment and prevention methods, a significant objective for wildlife management agencies. In an effort to mitigate precipitous declines in bat populations due to WNS, we have developed and implemented a multi-year mitigation strategy at Black Diamond Tunnel (BDT), Georgia, singly known as one of the most substantial winter colony sites for tricolored bats (Perimyotis subflavus), with pre-WNS abundance exceeding 5000 individuals. Our mitigation approach involved in situ treatment of bats at the colony level through aerosol distribution of antifungal volatile organic compounds (VOCs) that demonstrated an in vitro ability to inhibit P. destructans conidia germination and mycelial growth through contact-independent exposure. The VOCs evaluated have been identified from microbes inhabiting naturally-occurring fungistatic soils and endophytic fungi. These VOCs are of low toxicity to mammals and have been observed to elicit antagonism of P. destructans at low gaseous concentrations. Cumulatively, our observations resolved no detrimental impact on bat behavior or health, yet indicated a potential for attenuation of WNS related declines at BDT and demonstrated the feasibility of this novel disease management approach.

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 precipitous declines of several species of cave-dwelling North American bats. While numerous advancements in the understanding of the disease processes underlying WNS have been made in recent years, there are still many aspects of WNS, particularly with respect to pathogen virulence, that remain unknown. In this preliminary investigation, we sought to further elucidate the disease cycle by concentrating on the pathogen, with specific focus on its ability to utilize lipids that compose bat wing sebum and are found in wing membranes, as a substrate for energy and growth. In vitro growth experiments were conducted with the three most common fatty acids that comprise bat sebum: oleic, palmitic, and stearic acids. None of the fatty acids were observed to contribute a significant difference in mean growth from controls grown on SDA, although morphological differences were observed in several instances. Additionally, as an accompaniment to the growth experiments, bat wing explants from Perimyotis subflavus and Eptesicus fuscus were fluorescently stained to visualize the difference in distribution of 16- and 18-carbon chain fatty acids in the wing membrane. Which substrates contribute to the growth of P. destructans is important to understanding the progressive impact P. destructans has on bat health through the course of the disease cycle.

Fungal pathogens are a growing worldwide concern. Declines in a number of economically and agriculturally important plant and animal species pose a significant threat to both biodiversity and food security. Although many effective antifungal agents have been identified, their toxicity often precludes their use with food products or sensitive animal species. This has prompted the exploration of natural products as effective treatment compounds. In the present study, several essential oils were tested for their capacity to limit the growth of the fungal pathogens Ascosphaera apis and Pseudogymnoascus destructans, the causative agents of chalkbrood disease among honey bee larvae and white-nose syndrome among bats, respectively. Essential oils of cinnamon bark, citronella, lemongrass, and orange were exposed to A. apis in contact-dependent oil-agar suspensions as well as in contact-independent shared airspaces. Essential oils of cinnamon bark, citronella, and lemongrass were exposed to P. destructans in contact-dependent oil-agar suspensions. All compounds were found to significantly inhibit mycelial growth at low concentrations, suggesting the potential for these natural products to be used for controlling these and other select fungal pathogens.

Iodinated X-ray contrast media (ICM) were investigated as a source of iodine in the formation of iodo-trihalomethane (iodo-THM) and iodo-acid disinfection byproducts (DBPs), both of which are highly genotoxic and/or cytotoxic in mammalian cells. ICM are widely used at medical centers to enable imaging of soft tissues (e.g., organs, veins, blood vessels) and are designed to be inert substances, with 95% eliminated in urine and feces unmetabolized within 24 h. ICM are not well removed in wastewater treatment plants, such that they have been found at elevated concentrations in rivers and streams (up to 100 μg/L). Naturally occurring iodide in source waters is believed to be a primary source of iodine in the formation of iodo-DBPs, but a previous 23-city iodo-DBP occurrence study also revealed appreciable levels of iodo-DBPs in some drinking waters that had very low or no detectable iodide in their source waters. When 10 of the original 23 cities' source waters were resampled, four ICM were found--iopamidol, iopromide, iohexol, and diatrizoate--with iopamidol most frequently detected, in 6 of the 10 plants sampled, with concentrations up to 2700 ng/L. Subsequent controlled laboratory reactions of iopamidol with aqueous chlorine and monochloramine in the absence of natural organic matter (NOM) produced only trace levels of iodo-DBPs; however, when reacted in real source waters (containing NOM), chlorine and monochloramine produced significant levels of iodo-THMs and iodo-acids, up to 212 nM for dichloroiodomethane and 3.0 nM for iodoacetic acid, respectively, for chlorination. The pH behavior was different for chlorine and monochloramine, such that iodo-DBP concentrations maximized at higher pH (8.5) for chlorine, but at lower pH (6.5) for monochloramine. Extracts from chloraminated source waters with and without iopamidol, as well as from chlorinated source waters with iopamidol, were the most cytotoxic samples in mammalian cells. Source waters with iopamidol but no disinfectant added were the least cytotoxic. While extracts from chlorinated and chloraminated source waters were genotoxic, the addition of iopamidol enhanced their genotoxicity. Therefore, while ICM are not toxic in themselves, their presence in source waters may be a source of concern because of the formation of highly toxic iodo-DBPs in chlorinated and chloraminated drinking water.

The recently identified causative agent of white-nose syndrome (WNS), Pseudogymnoascus destructans, has been implicated in the mortality of an estimated 5.5 million North American bats since its initial documentation in 2006 (Frick et al. in Science 329:679–682, 2010). In an effort to identify potential biological and chemical control options for WNS, 6 previously described bacterially produced volatile organic compounds (VOCs) were screened for anti-P. destructans activity. The compounds include decanal; 2-ethyl-1-hexanol; nonanal; benzothiazole; benzaldehyde; andN,N-dimethyloctylamine. P. destructans conidia and mycelial plugs were exposed to the VOCs in a closed air space at 15 and 4 °C and then evaluated for growth inhibition. All VOCs inhibited growth from conidia as well as inhibiting radial mycelial extension, with the greatest effect at 4 °C. Studies of the ecology of fungistatic soils and the natural abundance of the fungistatic VOCs present in these environments suggest a synergistic activity of select VOCs may occur. The evaluation of formulations of two or three VOCs at equivalent concentrations was supportive of synergistic activity in several cases. The identification of bacterially produced VOCs with anti-P. destructans activity indicates disease-suppressive and fungistatic soils as a potentially significant reservoir of biological and chemical control options for WNS and provides wildlife management personnel with tools to combat this devastating disease.

Background: The recently-identified causative agent of White-Nose Syndrome (WNS), Pseudogymnoascus destructans, has been responsible for the mortality of an estimated 5.5 million North American bats since its emergence in 2006. A primary focus of the National Response Plan, established by multiple state, federal and tribal agencies in 2011, was the identification of biological control options for WNS. In an effort to identify potential biological control options for WNS, multiply induced cells of Rhodococcus rhodochrous strain DAP96253 was screened for anti-P. destructans activity. Results: Conidia and mycelial plugs of P. destructans were exposed to induced R. rhodochrous in a closed air-space at 15°C, 7°C and 4°C and were evaluated for contact-independent inhibition of conidia germination and mycelial extension with positive results. Additionally, in situ application methods for induced R. rhodochrous, such as fixed-cell catalyst and fermentation cell-paste in non-growth conditions, were screened with positive results. R. rhodochrous was assayed for ex vivo activity via exposure to bat tissue explants inoculated with P. destructans conidia. Induced R. rhodochrous completely inhibited growth from conidia at 15°C and had a strong fungistatic effect at 4°C. Induced R. rhodochrous inhibited P. destructans growth from conidia when cultured in a shared air-space with bat tissue explants inoculated with P. destructans conidia. Conclusion: The identification of inducible biological agents with contact-independent anti- P. destructans activity is a major milestone in the development of viable biological control options for in situ application and provides the first example of contact-independent antagonism of this devastating wildlife pathogen.