Hana Bandouchova

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.

Detoxification and oxidative stress responses along with microcystins accumulation in Japanese quail exposed to cyanobacterial biomass

The cyanobacterial exposure has been implicated in mass mortalities of wild birds, but information on the actual effects of cyanobacteria on birds in controlled studies is missing. Effects on detoxification and antioxidant parameters as well as bioaccumulation of microcystins (MCs) were studied in birds after sub-lethal exposure to natural cyanobacterial biomass. Four treatment groups of model species Japanese quail (Coturnix coturnix japonica) were exposed to controlled doses of cyanobacterial bloom during acute (10 days) and sub-chronic (30 days) experiment. The daily doses of cyanobacterial biomass corresponded to 0.2-224.6 ng MCs/g body weight. Significant accumulation of MCs was observed in the liver for both test durations and slight accumulation also in the muscles of the highest treatment group from acute test. The greatest accumulation was observed in the liver of the highest treatment group in the acute test reaching average concentration of 43.7 ng MCs/g fresh weight. The parameters of detoxification metabolism and oxidative stress were studied in the liver, heart and brain. The cyanobacterial exposure caused an increase of activity of cytochrome P-450-dependent 7-ethoxyresorufin O-deethylase representing the activation phase of detoxification metabolism. Also the conjugation phase of detoxification, namely the activity of glutathione-S-transferase, was altered. Cyanobacterial exposure also modulated oxidative stress responses including the level of glutathione and activities of glutathione-related enzymes and caused increase in lipid peroxidation. The overall pattern of detoxification parameters and oxidative stress responses clearly separated the control and the lowest exposure group from all the higher exposed groups. This is the first controlled study documenting the induction of oxidative stress along with MCs accumulation in birds exposed to natural cyanobacterial biomass. The data also suggest that increased activities of detoxification enzymes could lead to greater biotransformation and elimination of the MCs at the longer exposure time.

Ferric reducing antioxidant power and square wave voltammetry for assay of low molecular weight antioxidants in blood plasma: performance and comparison of methods.

The purpose of the present study was to employ two methods—square wave voltammetry (SWV) performed on screen printed sensors and ferric reducing antioxidant power (FRAP)—as suitable tools for the assay of low-molecular-weight antioxidants (LMWAs). LMWAs were assayed by both methods and the resulting data were statistically compared. Plasma samples from five Cinereous vultures accidentally intoxicated with lead were used to represent real biological matrices with different levels of LMWAs. Blood was collected from the birds prior to and one month after treatment with Ca-EDTA. SWV resulted in two peaks. The first peak, with the potential value of 466 ± 15 mV, was recognized as ascorbic and uric acids, while the second one (743 ± 30 mV) represented glutathione, tocopherol, ascorbic acid and in a minor effect by uric acid, too. Contribution of individual antioxidants was recognized by separate assays of LMWA standards. Correlation between peaks 1 and 2 as well as the sum of the two peaks and FRAP was analysed. While peak 1 and the sum of peaks were in close correlation to FRAP results (correlation coefficient of 0.97), the relation between peak 2 and FRAP may be expressed using a correlation coefficient of 0.64. The determination of thiols by the Ellman assay confirmed the accuracy of SWV. Levels of glutathione and other similar structures were stable in the chosen model and it may be concluded that SWV is appropriate for assay of LMWAs in plasma samples. The methods employed in the study were advantageous in minimal sample volume consumption and fast acquisition of results.

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.

Tularemia induces different biochemical responses in BALB/c mice and common voles

BackgroundBoth BALB/c mice and common voles (Microtus arvalis) are considered highly susceptible to tularemia. However, the common vole is reported to harbour Francisella tularensis in European habitats as well as to survive longer with chronic shedding of the bacterium. The purpose of the present study was to compare the response of these two rodents to a wild Francisella tularensis subsp. holarctica strain infection.MethodsRodents were evaluated for differences in the total antioxidant capacity derived from low-molecular-weight antioxidants, biochemistry including lipid metabolism, tissue bacterial burdens and histopathology following experimental intraperitoneal infection with 160 colony forming units (CFU) pro toto.ResultsBacterial burdens in common voles started to develop later post-exposure and amounted to lower levels than in BALB/c mice. Elevation of liver function enzymes was more pronounced in mice than common voles and there were marked differences in lipid metabolism in the course of tularemia in these two species. Hypertriglyceridemia and hypercholesterolemia developed in mice, while physiologically higher levels of triglycerides and cholesterol showed a decreasing tendency in common voles.On the other hand, the total plasma antioxidant capacity gradually dropped to 81.5% in mice on day 5 post-infection, while it increased to 130% on day 6 post-infection in common voles. Significant correlations between tissue bacterial burdens and several biochemical parameters were found.ConclusionAs differences in lipid metabolism and the total antioxidant capacity of highly susceptible rodent species were demonstrated, the role of triglycerides, cholesterol and antioxidants in tularemic sepsis should be further investigated.

Combined exposure to cyanobacterial biomass, lead and the Newcastle virus enhances avian toxicity.

Under environmental conditions, wild birds can be exposed to multiple stressors including natural toxins, anthropogenic pollutants and infectious agents at the same time. This experimental study was successful in testing the hypothesis that adverse effects of cyanotoxins, heavy metals and a non-pathogenic immunological challenge combine to enhance avian toxicity. Mortality occurred in combined exposures to naturally occurring cyanobacterial biomass and lead shots, lead shots and Newcastle vaccination as well as in single lead shot exposure. Mostly acute effects around day 10 were observed. On day 30 of exposure, there were no differences in the liver accumulation of lead in single and combined exposure groups. Interestingly, liver microcystin levels were elevated in birds co-exposed to cyanobacterial biomass together with lead or lead and the Newcastle virus. Significant differences in body weights between all Pb-exposed and Pb-non-exposed birds were found on days 10 and 20. Single exposure to cyanobacterial biomass resulted in hepatic vacuolar dystrophy, whereas co-exposure with lead led to more severe granular dystrophy. Haematological changes were associated with lead exposure, in particular. Biochemical analysis revealed a decrease in glucose and an increase in lactate dehydrogenase in single and combined cyanobacterial and lead exposures, which also showed a decreased antibody response to vaccination. The combined exposure of experimental birds to sub-lethal doses of individual stressors is ecologically realistic. It brings together new pieces of knowledge on avian health. In light of this study, investigators of wild bird die-offs should be circumspect when evaluating findings of low concentrations of contaminants that would not result in mortality on a separate basis. As such it has implications for wildlife biologists, veterinarians and conservationists of avian biodiversity.

Lead toxicosis of captive vultures: case description and responses to chelation therapy

BackgroundLead, a serious threat for raptors, can hamper the success of their conservation. This study reports on experience with accidental lead intoxication and responses to chelation therapy in captive Cinereous (Aegypius monachus) and Egyptian (Neophron percnopterus) Vultures.ResultsSoil contamination by lead-based paint sanded off the steel aviary resulted in poisoning of eight Cinereous and two Egyptian Vultures. A male Egyptian Vulture developed signs of apathy, polydipsia, polyuria, regurgitation, and stupor, and died on the next day. Liver, kidney and blood lead concentrations were 12.2, 8.16 and 2.66 μg/g, respectively. Laboratory analyses confirmed severe liver and kidney damage and anaemia. Blood Pb levels of Pb-exposed Cinereous Vultures were 1.571 ± 0.510 μg/g shortly after intoxication, decreased to 0.530 ± 0.165 μg/g without any therapy in a month and to 0.254 ± 0.097 μg/g one month after CaNa2EDTA administration. Eight months later, blood lead levels decreased to close to the background of the control group. Blood parameters of healthy Pb-non-exposed Cinereous Vultures were compared with those of the exposed group prior to and after chelation therapy. Iron levels in the lead-exposed pre-treatment birds significantly decreased after chelation. Haematocrit levels in Pb-exposed birds were significantly lower than those of the controls and improved one month after chelation. Creatine kinase was higher in pre-treatment birds than in the controls but normalised after therapy. Alkaline phosphatase increased after chelation. A marked increase in the level of lipid peroxidation measured as thiobarbituric acid reactive species was demonstrated in birds both prior to and after chelation. The ferric reducing antioxidant power was significantly lower in pre-treatment vultures and returned to normal following chelation therapy. Blood metallothionein levels in lead-exposed birds were higher than in controls. Reduced glutathione dropped after CaNa2EDTA therapy, while oxidised glutathione was significantly lower in both pre- and post-treatment birds. A chick in an egg produced by a Cinereous Vulture female two months after lead toxicosis died on day 40 of artificial incubation. Lead concentrations in foetal tissues were consistent with levels causing avian mortality.ConclusionsThe reported blood parameters and reproduction impairment in captive birds may have implications for professionals dealing with lead exposure in wild birds.