Active surveillance for antibodies confirms circulation of lyssaviruses in Palearctic bats.

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.

Low seasonal variation in greater mouse-eared bat (Myotis myotis) blood parameters.

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.

Numerous cold arousals and rare arousal cascades as a hibernation strategy in European Myotis bats.

Hibernating bats optimise the duration of torpor bouts and arousals in relation to hibernaculum microclimatic conditions and fat reserves. Clustering has significant physiological and ecological benefits, promoting successful hibernation of individuals. Such aggregations may help maintain optimal temperatures, allowing better energy utilisation than in solitarily bats. However, aroused bats in a cluster could conceivably disturb those still hibernating, starting an energy-demanding arousal process. Our study was conducted over two winters in two different hibernacula (cave and mine) in the Czech Republic, where Greater mouse-eared bats (Myotis myotis) have previously been diagnosed with white-nose syndrome. In 118 arousal episodes we recorded 193 individual arousals in which a warming phase was observed, 135 (69.9%) being cold arousals, where bats ceased increasing their body temperatures at ≤ 10 °C. The remaining arousals were standard normothermic arousals, where body (fur) surface temperatures reached > 20 °C. Cold arousals occurred during the mid- and late hibernation periods, suggesting they were a response to disturbance by a neighbour in the same cluster. Arousal cascades, where bats aroused in series, were rare (12.7%) and reached a maximum in mid-January. Our data suggest that Myotis bats prolong their torpor bouts using numerous cold arousals but few arousal cascades. Upon arrival of a bat, the clustered bats show tolerance to disturbing by conspecifics.

Alterations in the health of hibernating bats under pathogen pressure.

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.

White-nose syndrome pathology grading in Nearctic and Palearctic bats.

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.

Deeply torpid bats can change position without elevation of body temperature.

Because body temperature is tightly coupled to physiological function, hibernating animals entering deep torpor are typically immobile. We analysed thermal behaviour and locomotory activity of hibernating greater mouse-eared bats Myotis myotis and found two types of movement behaviour related to body temperature, i.e. movement at high fur temperature and at low fur temperatures (Tflow; <5°C). First Tflow movements appeared at the beginning of March and often occurred during long torpor bouts. In most cases, Tflow events represented slow displacements between clusters of bats. In several cases, however, departure or arrivals from and into clusters was also recorded without any elevation in body temperature. Distance travelled, flight duration and speed of locomotion during Tflow events was lower than in high fur temperature events. Such behaviour could allow bats to save energy long-term and prolong torpor bouts. Tflow movement in torpid bats significantly changes our understanding of basic hibernation principles and we strongly recommend further studies on the subject.

White-nose syndrome fungus: a generalist pathogen of hibernating bats.

Host traits and phylogeny can determine infection risk by driving pathogen transmission and its ability to infect new hosts. Predicting such risks is critical when designing disease mitigation strategies, and especially as regards wildlife, where intensive management is often advocated or prevented by economic and/or practical reasons. We investigated Pseudogymnoascus [Geomyces] destructans infection, the cause of white-nose syndrome (WNS), in relation to chiropteran ecology, behaviour and phylogenetics. While this fungus has caused devastating declines in North American bat populations, there have been no apparent population changes attributable to the disease in Europe. We screened 276 bats of 15 species from hibernacula in the Czech Republic over 2012 and 2013, and provided histopathological evidence for 11 European species positive for WNS. With the exception of Myotis myotis, the other ten species are all new reports for WNS in Europe. Of these, M. emarginatus, Eptesicus nilssonii, Rhinolophus hipposideros, Barbastella barbastellus and Plecotus auritus are new to the list of P. destructans-infected bat species. While the infected species are all statistically phylogenetically related, WNS affects bats from two suborders. These are ecologically diverse and adopt a wide range of hibernating strategies. Occurrence of WNS in distantly related bat species with diverse ecology suggests that the pathogen may be a generalist and that all bats hibernating within the distribution range of P. destructans may be at risk of infection.