Bat white-nose disease fungus diversity in time and space.

White-nose disease (WND), caused by the psychrophilic fungus Pseudogymnoascusdestructans, represents one of the greatest threats for North American hibernating bats. Research on molecular data has significantly advanced our knowledge of various aspects of the disease, yet more studies are needed regarding patterns of P.destructans genetic diversity distribution. In the present study, we investigate three sites within the native range of the fungus in detail: two natural hibernacula (karst caves) in Bulgaria, south-eastern Europe and one artificial hibernaculum (disused cellar) in Germany, northern Europe, where we conducted intensive surveys between 2014 and 2019. Using 18 microsatellite and two mating type markers, we describe how P.destructans genetic diversity is distributed between and within sites, the latter including differentiation across years and seasons of sampling; across sampling locations within the site; and between bats and hibernaculum walls. We found significant genetic differentiation between hibernacula, but we could not detect any significant differentiation within hibernacula, based on the variables examined. This indicates that most of the pathogen's movement occurs within sites. Genotypic richness of P.destructans varied between sites within the same order of magnitude, being approximately two times higher in the natural caves (Bulgaria) compared to the disused cellar (Germany). Within all sites, the pathogen's genotypic richness was higher in samples collected from hibernaculum walls than in samples collected from bats, which corresponds with the hypothesis that hibernacula walls represent the environmental reservoir of the fungus. Multiple pathogen genotypes were commonly isolated from a single bat (i.e. from the same swab sample) in all study sites, which might be important to consider when studying disease progression.

Population genetics as a tool to elucidate pathogen reservoirs: Lessons from Pseudogymnoascus destructans, the causative agent of White-Nose disease in bats.

Emerging infectious diseases pose a major threat to human, animal, and plant health. The risk of species-extinctions increases when pathogens can survive in the absence of the host. Environmental reservoirs can facilitate this. However, identifying such reservoirs and modes of infection is often highly challenging. In this study, we investigated the presence and nature of an environmental reservoir for the ascomycete fungus Pseudogymnoascus destructans, the causative agent of White-Nose disease. Using 18 microsatellite markers, we determined the genotypic differentiation between 1497 P. destructans isolates collected from nine closely situated underground sites where bats hibernate (i.e., hibernacula) in Northeastern Germany. This approach was unique in that it ensured that every isolate and resulting multilocus genotype was not only present, but also viable and therefore theoretically capable of infecting a bat. The distinct distribution of multilocus genotypes across hibernacula demonstrates that each hibernaculum has an essentially unique fungal population. This would be expected if bats become infected in their hibernaculum (i.e., the site they spend winter in to hibernate) rather than in other sites visited before they start hibernating. In one hibernaculum, both the walls and the hibernating bats were sampled at regular intervals over five consecutive winter seasons (1062 isolates), revealing higher genotypic richness on walls compared to bats and a stable frequency of multilocus genotypes over multiple winters. This clearly implicates hibernacula walls as the main environmental reservoir of the pathogen, from which bats become reinfected annually during the autumn.

Seasonal patterns of Pseudogymnoascus destructans germination indicate host-pathogen coevolution.

Emerging infectious diseases rank among the most important threats to human and wildlife health. A comprehensive understanding of the mode of infection and presence of potential reservoirs is critical for the development of effective counter strategies. Fungal pathogens can remain viable in environmental reservoirs for extended periods of time before infecting susceptible individuals. This may be the case for Pseudogymnoascus destructans (Pd), the causative agent of bat white-nose disease. Owing to its cold-loving nature, this fungal pathogen only grows on bats during hibernation, when their body temperature is reduced. Bats only spend part of their life cycle in hibernation and do not typically show signs of infection in summer, raising the question of whether Pd remains viable in hibernacula during this period (roughly six months). If so, this could facilitate the re-infection of bats when they return to the sites the following winter. In a laboratory experiment, we determined the germination rate of Pd spores kept under constant conditions on a wall-like substrate, over the course of two years. Results showed that the seasonal pattern in Pd germination mirrored the life cycle of the bats, with an increased germination rate at times when hibernating bats would naturally be present and lower germination rates during their absence. We suggest that Pd is dependent on the presence of hibernating bats and has therefore coupled its germination rate to host availability. Furthermore, we demonstrate that Pd spores survive extended periods of host absence and can remain viable for at least two years. There is, however, a strong decrease in spore viability between the first and second years (98%). Pd viability for at least two years on a solid mineral-based substrate establishes the potential for environmental reservoirs in hibernacula walls and has strong implications for the efficacy of certain management strategies (e.g. bat culling).