INTRODUCTION: Serological tests can be used to test whether an animal has been exposed to an infectious agent, and whether its immune system has recognized and produced antibodies against it. Paired samples taken several weeks apart then document an ongoing infection and/or seroconversion. METHODS: In the absence of a commercial kit, we developed an indirect enzyme-linked immunosorbent assay (ELISA) to detect the fungus-specific antibodies for Pseudogymnoascus destructans, the agent of white-nose syndrome in bats. RESULTS AND DISCUSSION: Samples collected from European Myotis myotis (n=35) and Asian Myotis dasycneme (n=11) in their hibernacula at the end of the hibernation period displayed 100% seroprevalence of antibodies against P. destructans, demonstrating a high rate of exposure. Our results showed that the higher the titre of antibodies against P. destructans, the lower the infection intensity, suggesting that a degree of protection is provided by this arm of adaptive immunity in Palearctic bats. Moreover, P. destructans infection appears to be a seasonally self-limiting disease of Palearctic bats showing seroconversion as the WNS skin lesions heal in the early post-hibernation period.

• Keywords
• Myotis bat species, adaptive antifungal immunity, antibody prevalence, disease severity, emerging wildlife infection, indirect ELISA,
• MeSH
• Ascomycota MeSH
• Chiroptera * MeSH
• Skin Diseases * MeSH
• Mycoses * epidemiology   veterinary   MeSH
• Seroepidemiologic Studies MeSH
• Syndrome MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Palearctic bats host a diversity of lyssaviruses, though not the classical rabies virus (RABV). As surveillance for bat rabies over the Palearctic area covering Central and Eastern Europe and Siberian regions of Russia has been irregular, we lack data on geographic and seasonal patterns of the infection. RESULTS: To address this, we undertook serological testing, using non-lethally sampled blood, on 1027 bats of 25 species in Bulgaria, the Czech Republic, Poland, Russia and Slovenia between 2014 and 2018. The indirect enzyme-linked immunosorbent assay (ELISA) detected rabies virus anti-glycoprotein antibodies in 33 bats, giving an overall seroprevalence of 3.2%. Bat species exceeding the seroconversion threshold included Myotis blythii, Myotis gracilis, Myotis petax, Myotis myotis, Murina hilgendorfi, Rhinolophus ferrumequinum and Vespertilio murinus. While Myotis species (84.8%) and adult females (48.5%) dominated in seropositive bats, juveniles of both sexes showed no difference in seroprevalence. Higher numbers tested positive when sampled during the active season (10.5%), as compared with the hibernation period (0.9%). Bat rabies seroprevalence was significantly higher in natural habitats (4.0%) compared with synanthropic roosts (1.2%). Importantly, in 2018, we recorded 73.1% seroprevalence in a cave containing a M. blythii maternity colony in the Altai Krai of Russia. CONCLUSIONS: Identification of such "hotspots" of non-RABV lyssavirus circulation not only provides important information for public health protection, it can also guide research activities aimed at more in-depth bat rabies studies.

• Keywords
• Chiroptera, Europe, Siberia, blood samples, rabies, seroprevalence,
• MeSH
• Chiroptera virology   MeSH
• Ecosystem MeSH
• Rhabdoviridae Infections epidemiology   MeSH
• Caves MeSH
• Lyssavirus isolation & purification   MeSH
• Antibodies, Viral blood   MeSH
• Seasons MeSH
• Seroepidemiologic Studies MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Europe epidemiology   MeSH
• Russia epidemiology   MeSH
• Names of Substances
• Antibodies, Viral MeSH

Understanding how context (e.g., host species, environmental conditions) drives disease susceptibility is an essential goal of disease ecology. We hypothesized that in bat white-nose syndrome (WNS), species-specific host-pathogen interactions may partly explain varying disease outcomes among host species. We characterized bat and pathogen transcriptomes in paired samples of lesion-positive and lesion-negative wing tissue from bats infected with Pseudogymnoascus destructans in three parallel experiments. The first two experiments analyzed samples collected from the susceptible Nearctic Myotis lucifugus and the less-susceptible Nearctic Eptesicus fuscus, following experimental infection and hibernation in captivity under controlled conditions. The third experiment applied the same analyses to paired samples from infected, free-ranging Myotis myotis, a less susceptible, Palearctic species, following natural infection and hibernation (n = 8 sample pairs/species). Gene expression by P. destructans was similar among the three host species despite varying environmental conditions among the three experiments and was similar within each host species between saprophytic contexts (superficial growth on wings) and pathogenic contexts (growth in lesions on the same wings). In contrast, we observed qualitative variation in host response: M. lucifugus and M. myotis exhibited systemic responses to infection, while E. fuscus up-regulated a remarkably localized response. Our results suggest potential phylogenetic determinants of response to WNS and can inform further studies of context-dependent host-pathogen interactions.

• Keywords
• Eptesicus fuscus, Myotis lucifugus, Myotis myotis, Pseudogymnoascus destructans, Disease ecology, emerging infectious diseases, host–pathogen interactions, susceptibility, virulence,
• MeSH
• Ascomycota genetics   pathogenicity   MeSH
• Chiroptera classification   microbiology   MeSH
• Dermatomycoses microbiology   veterinary   MeSH
• Species Specificity MeSH
• Phylogeny MeSH
• Host-Pathogen Interactions genetics   MeSH
• Wings, Animal microbiology   pathology   MeSH
• Nose microbiology   pathology   MeSH
• Gene Expression Profiling * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The greater mouse-eared bat (Myotis myotis) is a flagship species for the protection of hibernation and summer maternity roosts in the Western Palearctic region. A range of pathogenic agents is known to put pressure on populations, including the white-nose syndrome fungus, for which the species shows the highest prevalence and infection intensity of all European bat species. Here, we perform analysis of blood parameters characteristic for the species during its natural annual life cycle in order to establish reference values. Despite sexual dimorphism and some univariate differences, the overall multivariate pattern suggests low seasonal variation with homeostatic mechanisms effectively regulating haematology and blood biochemistry ranges. Overall, the species displayed a high haematocrit and haemoglobin content and high concentration of urea, while blood glucose levels in swarming and hibernating bats ranged from hypo- to normoglycaemic. Unlike blood pH, concentrations of electrolytes were wide ranging. To conclude, baseline data for blood physiology are a useful tool for providing suitable medical care in rescue centres, for studying population health in bats adapting to environmental change, and for understanding bat responses to stressors of conservation and/or zoonotic importance.

• MeSH
• Chiroptera blood   physiology   MeSH
• Hematocrit standards   MeSH
• Hematologic Tests standards   MeSH
• Hibernation MeSH
• Climate MeSH
• Reference Values MeSH
• Seasons MeSH
• Sentinel Species physiology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Arctic Regions epidemiology   MeSH

Resistance and tolerance allow organisms to cope with potentially life-threatening pathogens. Recently introduced pathogens initially induce resistance responses, but natural selection favors the development of tolerance, allowing for a commensal relationship to evolve. Mycosis by Pseudogymnoascus destructans, causing white-nose syndrome (WNS) in Nearctic hibernating bats, has resulted in population declines since 2006. The pathogen, which spread from Europe, has infected species of Palearctic Myotis for a longer period. We compared ecologically relevant responses to the fungal infection in the susceptible Nearctic M. lucifugus and less susceptible Palearctic M. myotis, to uncover factors contributing to survival differences in the two species. Samples were collected from euthermic bats during arousal from hibernation, a naturally occurring phenomenon, during which transcriptional responses are activated. We compared the whole-transcriptome responses in wild bats infected with P. destructans hibernating in their natural habitat. Our results show dramatically different local transcriptional responses to the pathogen between uninfected and infected samples from the two species. Whereas we found 1526 significantly upregulated or downregulated transcripts in infected M. lucifugus, only one transcript was downregulated in M. myotis. The upregulated response pathways in M. lucifugus include immune cell activation and migration, and inflammatory pathways, indicative of an unsuccessful attempt to resist the infection. In contrast, M. myotis appears to tolerate P. destructans infection by not activating a transcriptional response. These host-microbe interactions determine pathology, contributing to WNS susceptibility, or commensalism, promoting tolerance to fungal colonization during hibernation that favors survival.

• Keywords
• Host–pathogen interaction, Infection, Opportunistic pathogen, Resistance, Tolerance,
• MeSH
• Chiroptera * MeSH
• Hibernation * MeSH
• Mycoses * MeSH
• RNA MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Europe MeSH
• Names of Substances
• RNA MeSH

BACKGROUND: Spatiotemporal distribution patterns are important infectious disease epidemiological characteristics that improve our understanding of wild animal population health. The skin infection caused by the fungus Pseudogymnoascus destructans emerged as a panzootic disease in bats of the northern hemisphere. However, the infection status of bats over an extensive geographic area of the Russian Federation has remained understudied. RESULTS: We examined bats at the geographic limits of bat hibernation in the Palearctic temperate zone and found bats with white-nose syndrome (WNS) on the European slopes of the Ural Mountains through the Western Siberian Plain, Central Siberia and on to the Far East. We identified the diagnostic symptoms of WNS based on histopathology in the Northern Ural region at 11° (about 1200 km) higher latitude than the current northern limit in the Nearctic. While body surface temperature differed between regions, bats at all study sites hibernated in very cold conditions averaging 3.6 °C. Each region also differed in P. destructans fungal load and the number of UV fluorescent skin lesions indicating skin damage intensity. Myotis bombinus, M. gracilis and Murina hilgendorfi were newly confirmed with histopathological symptoms of WNS. Prevalence of UV-documented WNS ranged between 16 and 76% in species of relevant sample size. CONCLUSIONS: To conclude, the bat pathogen P. destructans is widely present in Russian hibernacula but infection remains at low intensity, despite the high exposure rate.

• Keywords
• Chiroptera, Distribution, Hibernation, Prevalence, Pseudogymnoascus destructans,
• MeSH
• Ascomycota * MeSH
• Chiroptera * classification   genetics   MeSH
• Dermatomycoses epidemiology   veterinary   MeSH
• Animals, Wild MeSH
• Species Specificity MeSH
• Hibernation MeSH
• Molecular Typing MeSH
• Nose * MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Russia epidemiology   MeSH

In underground hibernacula temperate northern hemisphere bats are exposed to Pseudogymnoascus destructans, the fungal agent of white-nose syndrome. While pathological and epidemiological data suggest that Palearctic bats tolerate this infection, we lack knowledge about bat health under pathogen pressure. Here we report blood profiles, along with body mass index (BMI), infection intensity and hibernation temperature, in greater mouse-eared bats (Myotis myotis). We sampled three European hibernacula that differ in geomorphology and microclimatic conditions. Skin lesion counts differed between contralateral wings of a bat, suggesting variable exposure to the fungus. Analysis of blood parameters suggests a threshold of ca. 300 skin lesions on both wings, combined with poor hibernation conditions, may distinguish healthy bats from those with homeostatic disruption. Physiological effects manifested as mild metabolic acidosis, decreased glucose and peripheral blood eosinophilia which were strongly locality-dependent. Hibernating bats displaying blood homeostasis disruption had 2 °C lower body surface temperatures. A shallow BMI loss slope with increasing pathogen load suggested a high degree of infection tolerance. European greater mouse-eared bats generally survive P. destructans invasion, despite some health deterioration at higher infection intensities (dependant on hibernation conditions). Conservation measures should minimise additional stressors to conserve constrained body reserves of bats during hibernation.

• MeSH
• Ascomycota physiology   MeSH
• Chiroptera blood   microbiology   physiology   MeSH
• Hibernation * MeSH
• Body Mass Index MeSH
• Host-Pathogen Interactions * MeSH
• Skin Diseases blood   microbiology   pathology   veterinary   MeSH
• Mycoses blood   microbiology   pathology   veterinary   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

While white-nose syndrome (WNS) has decimated hibernating bat populations in the Nearctic, species from the Palearctic appear to cope better with the fungal skin infection causing WNS. This has encouraged multiple hypotheses on the mechanisms leading to differential survival of species exposed to the same pathogen. To facilitate intercontinental comparisons, we proposed a novel pathogenesis-based grading scheme consistent with WNS diagnosis histopathology criteria. UV light-guided collection was used to obtain single biopsies from Nearctic and Palearctic bat wing membranes non-lethally. The proposed scheme scores eleven grades associated with WNS on histopathology. Given weights reflective of grade severity, the sum of findings from an individual results in weighted cumulative WNS pathology score. The probability of finding fungal skin colonisation and single, multiple or confluent cupping erosions increased with increase in Pseudogymnoascus destructans load. Increasing fungal load mimicked progression of skin infection from epidermal surface colonisation to deep dermal invasion. Similarly, the number of UV-fluorescent lesions increased with increasing weighted cumulative WNS pathology score, demonstrating congruence between WNS-associated tissue damage and extent of UV fluorescence. In a case report, we demonstrated that UV-fluorescence disappears within two weeks of euthermy. Change in fluorescence was coupled with a reduction in weighted cumulative WNS pathology score, whereby both methods lost diagnostic utility. While weighted cumulative WNS pathology scores were greater in the Nearctic than Palearctic, values for Nearctic bats were within the range of those for Palearctic species. Accumulation of wing damage probably influences mortality in affected bats, as demonstrated by a fatal case of Myotis daubentonii with natural WNS infection and healing in Myotis myotis. The proposed semi-quantitative pathology score provided good agreement between experienced raters, showing it to be a powerful and widely applicable tool for defining WNS severity.

• MeSH
• Ascomycota genetics   physiology   MeSH
• Chiroptera metabolism   microbiology   MeSH
• DNA, Fungal genetics   MeSH
• Phylogeny MeSH
• Skin Diseases microbiology   pathology   MeSH
• Wings, Animal microbiology   pathology   radiation effects   MeSH
• Linear Models MeSH
• Optical Imaging MeSH
• Severity of Illness Index MeSH
• Ultraviolet Rays MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Fungal MeSH

Pathogenic and non-pathogenic related microorganisms differ in secondary metabolite production. Here we show that riboflavin overproduction by a fungal pathogen and its hyperaccumulation in affected host tissue exacerbates a skin infection to necrosis. In white-nose syndrome (WNS) skin lesions caused by Pseudogymnoascus destructans, maximum riboflavin concentrations reached up to 815 μg ml(-1), indicating bioaccumulation and lack of excretion. We found that high riboflavin concentrations are cytotoxic under conditions specific for hibernation, affect bats' primary fibroblasts and induce cell detachment, loss of mitochondrial membrane potential, polymerization of cortical actin, and cell necrosis. Our results explain molecular pathology of WNS, where a skin infection becomes fatal. Hyperaccumulation of vitamin B2 coupled with reduced metabolism and low tissue oxygen saturation during hibernation prevents removal of excess riboflavin in infected bats. Upon reperfusion, oxygen reacts with riboflavin resulting in dramatic pathology after arousal. While multiple molecules enable invasive infection, riboflavin-associated extensive necrosis likely contributes to pathophysiology and altered arousal pattern in infected bats. Bioaccumulation of a vitamin under natural infection represents a novel condition in a complex host-pathogen interplay.

• MeSH
• Ascomycota classification   genetics   pathogenicity   MeSH
• Cell Adhesion MeSH
• Chiroptera microbiology   MeSH
• Dermatomycoses microbiology   MeSH
• Microscopy, Electron MeSH
• Virulence Factors metabolism   MeSH
• Fibroblasts cytology   metabolism   microbiology   MeSH
• Phylogeny MeSH
• Host-Pathogen Interactions MeSH
• Wings, Animal cytology   microbiology   ultrastructure   MeSH
• Cells, Cultured MeSH
• Membrane Potential, Mitochondrial MeSH
• Riboflavin metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Virulence Factors MeSH
• Riboflavin MeSH

BACKGROUND: Vertebrate ectoparasites frequently play a role in transmission of infectious agents. Pseudogymnoascus destructans is a psychrophilic fungus known to cause white-nose syndrome (WNS), an emerging infectious disease of bats. It is transmitted with direct contact between bats or with contaminated environment. The aim of this study was to examine wing mites from the family Spinturnicidae parasitizing hibernating bats for the presence of P. destructans propagules as another possible transmission route. METHODS: Wing mites collected from 33 bats at four hibernation sites in the Czech Republic were inspected for the presence and load of pathogen's DNA using quantitative PCR. Simultaneously, wing damage of inspected bats caused by WNS was quantified using ultraviolet light (UV) transillumination and the relationship between fungal load on wing mites and intensity of infection was subjected to correlation analysis. RESULTS: All samples of wing mites were positive for the presence of DNA of P. destructans, indicating a high probability of their role in the transmission of the pathogen's propagules between bats. CONCLUSIONS: Mechanical transport of adhesive P. destructans spores and mycelium fragments on the body of spinturnicid mites is highly feasible. The specialised lifestyle of mites, i.e., living on bat wing membranes, the sites most typically affected by fungal growth, enables pathogen transport. Moreover, P. destructans metabolic traits suggest an ability to grow and sporulate on a range of organic substrates, including insects, which supports the possibility of growth on bat ectoparasites, at least in periods when bats roost in cold environments and enter torpor. In addition to transport of fungal propagules, mites may facilitate entry of fungal hyphae into the epidermis through injuries caused by biting.

• MeSH
• Ascomycota genetics   physiology   MeSH
• Chiroptera microbiology   MeSH
• Hibernation MeSH
• Wings, Animal parasitology   MeSH
• Skin parasitology   MeSH
• Mycelium MeSH
• Nose parasitology   MeSH
• Mites microbiology   MeSH
• Spores, Fungal MeSH
• Ultraviolet Rays MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic MeSH