Jan Zukal

Increasing Incidence of Geomyces destructans Fungus in Bats from the Czech Republic and Slovakia

Background White-nose syndrome is a disease of hibernating insectivorous bats associated with the fungus Geomyces destructans. It first appeared in North America in 2006, where over a million bats died since then. In Europe, G. destructans was first identified in France in 2009. Its distribution, infection dynamics, and effects on hibernating bats in Europe are largely unknown. Methodology/Principal Findings We screened hibernacula in the Czech Republic and Slovakia for the presence of the fungus during the winter seasons of 2008/2009 and 2009/2010. In winter 2009/2010, we found infected bats in 76 out of 98 surveyed sites, in which the majority had been previously negative. A photographic record of over 6000 hibernating bats, taken since 1994, revealed bats with fungal growths since 1995; however, the incidence of such bats increased in Myotis myotis from 2% in 2007 to 14% by 2010. Microscopic, cultivation and molecular genetic evaluations confirmed the identity of the recently sampled fungus as G. destructans, and demonstrated its continuous distribution in the studied area. At the end of the hibernation season we recorded pathologic changes in the skin of the affected bats, from which the fungus was isolated. We registered no mass mortality caused by the fungus, and the recorded population decline in the last two years of the most affected species, M. myotis, is within the population trend prediction interval. Conclusions/Significance G. destructans was found to be widespread in the Czech Republic and Slovakia, with an epizootic incidence in bats during the most recent years. Further development of the situation urgently requires a detailed pan-European monitoring scheme.

Histopathology confirms white-nose syndrome in bats in Europe.

White-nose syndrome, associated with the fungal skin infection geomycosis, caused regional population collapse in bats in North America. Our results, based on histopathology, show the presence of white-nose syndrome in Europe. Dermatohistopathology on two bats (Myotis myotis) found dead in March 2010 with geomycosis in the Czech Republic had characteristics resembling Geomyces destructans infection in bats confirmed with white-nose syndrome in US hibernacula. In addition, a live M. myotis, biopsied for histopathology during hibernation in April 2011, had typical fungal infection with cupping erosion and invasion of muzzle skin diagnostic for white-nose syndrome and conidiospores identical to G. destructans that were genetically confirmed as G. destructans.

NONLETHAL SCREENING OF BAT-WING SKIN WITH THE USE OF ULTRAVIOLET FLUORESCENCE TO DETECT LESIONS INDICATIVE OF WHITE-NOSE SYNDROME

Abstract Definitive diagnosis of the bat disease white-nose syndrome (WNS) requires histologic analysis to identify the cutaneous erosions caused by the fungal pathogen Pseudogymnoascus [formerly Geomyces] destructans (Pd). Gross visual inspection does not distinguish bats with or without WNS, and no nonlethal, on-site, preliminary screening methods are available for WNS in bats. We demonstrate that long-wave ultraviolet (UV) light (wavelength 366–385 nm) elicits a distinct orange–yellow fluorescence in bat-wing membranes (skin) that corresponds directly with the fungal cupping erosions in histologic sections of skin that are the current gold standard for diagnosis of WNS. Between March 2009 and April 2012, wing membranes from 168 North American bat carcasses submitted to the US Geological Survey National Wildlife Health Center were examined with the use of both UV light and histology. Comparison of these techniques showed that 98.8% of the bats with foci of orange–yellow wing fluorescence (n = 80) were WNS-positive based on histologic diagnosis; bat wings that did not fluoresce under UV light (n = 88) were all histologically negative for WNS lesions. Punch biopsy samples as small as 3 mm taken from areas of wing with UV fluorescence were effective for identifying lesions diagnostic for WNS by histopathology. In a nonlethal biopsy-based study of 62 bats sampled (4-mm diameter) in hibernacula of the Czech Republic during 2012, 95.5% of fluorescent (n = 22) and 100% of nonfluorescent (n = 40) wing samples were confirmed by histopathology to be WNS positive and negative, respectively. This evidence supports use of long-wave UV light as a nonlethal and field-applicable method to screen bats for lesions indicative of WNS. Further, UV fluorescence can be used to guide targeted, nonlethal biopsy sampling for follow-up molecular testing, fungal culture analysis, and histologic confirmation of WNS.

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.

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.

Heavy metals and metallothionein in vespertilionid bats foraging over aquatic habitats in the Czech Republic

There has been growing interest in the study and conservation of bats throughout the world. Declines in their absolute numbers in recent decades are due, in part, to the fact that insectivorous bats may bioaccumulate toxic pollutants. The purpose of the present study was to quantify heavy metal concentrations in kidney, liver, and pectoral muscle samples in relation to metallothionein (MT) levels. In total, 106 bats belonging to 11 European species (i.e., Myotis myotis, Myotis daubentonii, Myotis brandtii, Myotis nattereri, Myotis emarginatus, Myotis mystacinus, Pipistrellus pipistrellus, Pipistrellus nathusii, Pipistrellus pygmaeus, Nyctalus noctulla, Eptesicus serotinus) were used for the study. The highest MT levels were found in Pipistrellus pipistrellus. High MT levels were also found in juvenile bats and aquatic-insect-foraging species. Cadmium was found only in the liver and kidney of Myotis myotis, except for a solitary finding in Pipistrellus pipistrellus. Myotis myotis juveniles had significantly higher liver and kidney Zn concentrations than adults. Interestingly, the liver Pb concentration was approximately two times higher in females than in males of Myotis myotis. The same gender difference was found for kidney Zn concentration in Pipistrellus pipistrellus. The present study confirms exposure of vespertilionid bats to toxic heavy metals (Pb, Cd) in the Czech Republic and provides data on the essential element Zn and the metal-binding protein MT in bats foraging over aquatic, aquatic and terrestrial, and terrestrial habitats.

Emergence Behaviour of the Serotine Bat (Eptesicus Serotinus) Under Predation Risk

Emergence activity of a maternity colony of Eptesicus serotinus was monitored from May to August 1997 and 1998 at Střelice, Czech Republic. We focused mainly on the impact of predation risk on emergence parameters, but the effects of reproductive and climatic factors were assessed too. Observations were made with 10-days intervals during two consecutive nights from which the first was a control and the second a treatment night. On treatment nights a stuffed specimen of Tyto alba (in 1997) or Falco tinnunculus (in 1998) was placed close to the roost exits and recorded calls of the particular species were played back towards the roost. In 1997 the bats emerged earlier during lactation than during gravidity, while in 1998 the trend was opposite. This could be explained by a different course of food availability in each year. During poor weather the number of emerged bats decreased and bats probably used an alternative roost. The predation risk did not affect the values of any emergence parameter but induced changes in relationships among emergence parameters. When bats emerged earlier and thus were exposed to increased potential predation pressure, they increased their degree of clustering to decrease the probability of being attacked. The perception of predation risk was not affected by weather conditions or reproductive period.

Ectoparasites may serve as vectors for the white-nose syndrome fungus.

BackgroundVertebrate 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.MethodsWing mites collected from 33 bats at four hibernation sites in the Czech Republic were inspected for the presence and load of pathogens 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.ResultsAll 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 pathogens propagules between bats.ConclusionsMechanical 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.

Ecomorphometry of Myotis daubentonii and M. lucifugus (Chiroptera, Vespertilionidae) – a Palearctic-Nearctic comparison

The Palearctic Myotis daubentonii and the Nearctic M. lucifugus have traditionally been included into the subgenus Leuconoe due to their similar ecology and morphology. Recent molecular genetic analysis did not support this view since each of the two species was found to belong to another clade. The present paper is based on the analysis of eight external and 12 cranial characters in a sample of 158 M. daubentonii and 113 M. lucifugus, both species being represented by their nominate subspecies. Student t-test and principal component analysis (PCA) were used to test the variability and the impact of characters on interspecific differences. Significant sexual dimorphism was found mainly in M. daubentonii (females larger), hence in further analyses, males were separated from females. In both sexes, significant specific differences were found in most of the measurements, M. lucifugus having higher population averages than M. daubentonii. Only ca. 50% of variability was observed on the first two principal component axes in external characters but positive loadings suggested longer ears and tragi in M. lucifugus and negative loadings – longer feet and toe claws in M. daubentonii. PCA performed on cranial measurements provided ca. 70% of variability in each sexe. Insignificant specific differences on the first axis, influenced mainly by length variables of the skull including toothrows and mandible, suggest adaptations to analogous foraging strategies and diets. The projection of specimens onto the second and third principal components demonstrated significant differences between the species; these two axes were dominated by breadth and height of braincase, and interorbital constriction, respectively. The difference in the shape of neurocrania suggests different phylogenetic history in each of the species. The two bats are typical vicariants but their hunting habits do not seem to be identical, M. daubentonii appears to be more adapted to glean the water surface than M. lucifugus.

Establishment of Myotis myotis cell lines--model for investigation of host-pathogen interaction in a natural host for emerging viruses.

Bats are found to be the natural reservoirs for many emerging viruses. In most cases, severe clinical signs caused by such virus infections are normally not seen in bats. This indicates differences in the virus-host interactions and underlines the necessity to develop natural host related models to study these phenomena. Due to the strict protection of European bat species, immortalized cell lines are the only alternative to investigate the innate anti-virus immune mechanisms. Here, we report about the establishment and functional characterization of Myotis myotis derived cell lines from different tissues: brain (MmBr), tonsil (MmTo), peritoneal cavity (MmPca), nasal epithelium (MmNep) and nervus olfactorius (MmNol) after immortalization by SV 40 large T antigen. The usefulness of these cell lines to study antiviral responses has been confirmed by analysis of their susceptibility to lyssavirus infection and the mRNA patterns of immune-relevant genes after poly I:C stimulation. Performed experiments indicated varying susceptibility to lyssavirus infection with MmBr being considerably less susceptible than the other cell lines. Further investigation demonstrated a strong activation of interferon mediated antiviral response in MmBr contributing to its resistance. The pattern recognition receptors: RIG-I and MDA5 were highly up-regulated during rabies virus infection in MmBr, suggesting their involvement in promotion of antiviral responses. The presence of CD14 and CD68 in MmBr suggested MmBr cells are microglia-like cells which play a key role in host defense against infections in the central nervous system (CNS). Thus the expression pattern of MmBr combined with the observed limitation of lyssavirus replication underpin a protective mechanism of the CNS controlling the lyssavirus infection. Overall, the established cell lines are important tools to analyze antiviral innate immunity in M. myotis against neurotropic virus infections and present a valuable tool for a broad spectrum of future investigations in cellular biology of M. myotis.