Higher antibody titres against Pseudogymnoascus destructans are associated with less white-nose syndrome skin lesions in Palearctic bats.

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.

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.

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.

Hibernation temperature-dependent Pseudogymnoascus destructans infection intensity in Palearctic bats.

White-nose syndrome (WNS) is a fungal disease caused by Pseudogymnoascus destructans that is devastating to Nearctic bat populations but tolerated by Palearctic bats. Temperature is a factor known to be important for fungal growth and bat choice of hibernation. Here we investigated the effect of temperature on the pathogenic fungal growth in the wild across the Palearctic. We modelled body surface temperature of bats with respect to fungal infection intensity and disease severity and were able to relate this to the mean annual surface temperature at the site. Bats that hibernated at lower temperatures had less fungal growth and fewer skin lesions on their wings. Contrary to expectation derived from laboratory P. destructans culture experiments, natural infection intensity peaked between 5 and 6°C and decreased at warmer hibernating temperature. We made predictive maps based on bat species distributions, temperature and infection intensity and disease severity data to determine not only where P. destructans will be found but also where the infection will be invasive to bats across the Palearctic. Together these data highlight the mechanistic model of the interplay between environmental and biological factors, which determine progression in a wildlife disease.

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.

White-nose syndrome without borders: Pseudogymnoascus destructans infection tolerated in Europe and Palearctic Asia but not in North America.

A striking feature of white-nose syndrome, a fungal infection of hibernating bats, is the difference in infection outcome between North America and Europe. Here we show high WNS prevalence both in Europe and on the West Siberian Plain in Asia. Palearctic bat communities tolerate similar fungal loads of Pseudogymnoascus destructans infection as their Nearctic counterparts and histopathology indicates equal focal skin tissue invasiveness pathognomonic for WNS lesions. Fungal load positively correlates with disease intensity and it reaches highest values at intermediate latitudes. Prevalence and fungal load dynamics in Palearctic bats remained persistent and high between 2012 and 2014. Dominant haplotypes of five genes are widespread in North America, Europe and Asia, expanding the source region of white-nose syndrome to non-European hibernacula. Our data provides evidence for both endemicity and tolerance to this persistent virulent fungus in the Palearctic, suggesting that host-pathogen interaction equilibrium has been established.

Cytotoxicity of ketamine, xylazine and Hellabrunn mixture in liver-, heart- and kidney-derived cells from fallow deer.

OBJECTIVES: Chemical restraint of wild animals is practiced to accomplish intended procedures such as capture, clinical examination, collection of diagnostic samples, treatment and/or transport. Extra-label use of animal medicinal drugs is often necessary in wildlife because most approved therapeutics do not list wild species on the labelling. Here, we used cellular in vitro models, a cutting-edge tool of biomedical research, to examine cytotoxicity of anaesthetic agents in fallow deer and extrapolate these data for anaesthetic risks in wildlife. METHODS: We examined the cytotoxic effects of ketamine, xylazine, and ketamine-xylazine, i.e. the Hellabrunn mixture, on liver-, heart- and kidney-derived cell cultures prepared from a fallow deer (Dama dama) specimen. In line with preliminary studies we exposed cells to 10 µM, 50 µM, 100 µM, 1 mM, and 10 mM ketamine or xylazine. The combination of ketamine-xylazine was dosed at 0.025+0.02 mg/ml, 0.05+0.04 mg/ml, 0.75+0.06 mg/ml, 0.1+0.08 mg/ml, and 0.125+0.1 mg/ml per one well containing 10 000 cells. The quantification of cytotoxicity was based on lactate dehydrogenase activity released from damaged cells. RESULTS: Liver-derived cells show higher sensitivity to the cytotoxic effects of both ketamine and xylazine administered as single drugs when compared with cells cultured from the heart and kidney. The Hellabrunn mixture induced significantly higher cytotoxicity for kidney-derived cells ranging from 16.78% to 35.6%. Single and combined exposures to ketamine and xylazine resulted only in high-dose cytotoxicity in the heart-derived cells. CONCLUSIONS: Our results indicate that immobilization drugs significantly differ in their cytotoxic effects on cells derived from various organs of the fallow deer.

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.

Identification of monoclonal antibodies against 2,4-D herbicide by ELISA and DNA sequencing.

Seven hybridoma clones, E2/G2, E2/B5, E4/C2, G5/E10, F6/C10, B5/C3, and B7, produced within one fusion experiment in 1991 and the clone E4/C2 originated from 1995 were characterized by sequencing of genes coding for variable domains of the antibodies against 2,4-D herbicide. Amino acid sequences of selected antibodies, deduced from DNA analysis, were confirmed by mass spectrometry. Surprisingly, nucleotide sequence analysis of the clones E2/G2 and E2/B5, producing the most sensitive antibodies, proved to have sequence homology of their variable domains, although the IC(50) values determined for these antibodies 9 years prior to the DNA analysis were 2.0 and 8.2 ng/mL, respectively. The same findings arose from the comparison of the immunochemical to DNA data established for G5/E10, F6/C10, and B5/C3 clones producing antibodies with IC(50) values in the range of 26.3-43.1 ng /mL. The clone E4/C2, originating from the later fusion experiment, did not share nucleotide homology with any of the examined clones. Data obtained by ELISA, immunosensor, and DNA analysis within a 9 year period are discussed with respect to hybridoma stability, methodic artifacts, measurement reliability, and other possible factors influencing the result interpretation.