Transcriptomic responses of bat cells to European bat lyssavirus 1 infection under conditions simulating euthermia and hibernation.

BACKGROUND: Coevolution between pathogens and their hosts decreases host morbidity and mortality. Bats host and can tolerate viruses which can be lethal to other vertebrate orders, including humans. Bat adaptations to infection include localized immune response, early pathogen sensing, high interferon expression without pathogen stimulation, and regulated inflammatory response. The immune reaction is costly, and bats suppress high-cost metabolism during torpor. In the temperate zone, bats hibernate in winter, utilizing a specific behavioural adaptation to survive detrimental environmental conditions and lack of energy resources. Hibernation torpor involves major physiological changes that pose an additional challenge to bat-pathogen coexistence. Here, we compared bat cellular reaction to viral challenge under conditions simulating hibernation, evaluating the changes between torpor and euthermia. RESULTS: We infected the olfactory nerve-derived cell culture of Myotis myotis with an endemic bat pathogen, European bat lyssavirus 1 (EBLV-1). After infection, the bat cells were cultivated at two different temperatures, 37 °C and 5 °C, to examine the cell response during conditions simulating euthermia and torpor, respectively. The mRNA isolated from the cells was sequenced and analysed for differential gene expression attributable to the temperature and/or infection treatment. In conditions simulating euthermia, infected bat cells produce an excess signalling by multitude of pathways involved in apoptosis and immune regulation influencing proliferation of regulatory cell types which can, in synergy with other produced cytokines, contribute to viral tolerance. We found no up- or down-regulated genes expressed in infected cells cultivated at conditions simulating torpor compared to non-infected cells cultivated under the same conditions. When studying the reaction of uninfected cells to the temperature treatment, bat cells show an increased production of heat shock proteins (HSPs) with chaperone activity, improving the bat's ability to repair molecular structures damaged due to the stress related to the temperature change. CONCLUSIONS: The lack of bat cell reaction to infection in conditions simulating hibernation may contribute to the virus tolerance or persistence in bats. Together with the cell damage repair mechanisms induced in response to hibernation, the immune regulation may promote bats' ability to act as reservoirs of zoonotic viruses such as lyssaviruses.

Species-Specific Molecular Barriers to SARS-CoV-2 Replication in Bat Cells.

Bats are natural reservoirs of numerous coronaviruses, including the potential ancestor of SARS-CoV-2. Knowledge concerning the interaction between coronaviruses and bat cells is sparse. We investigated the ability of primary cells from Rhinolophus and Myotis species, as well as of established and novel cell lines from Myotis myotis, Eptesicus serotinus, Tadarida brasiliensis, and Nyctalus noctula, to support SARS-CoV-2 replication. None of these cells were permissive to infection, not even the ones expressing detectable levels of angiotensin-converting enzyme 2 (ACE2), which serves as the viral receptor in many mammalian species. The resistance to infection was overcome by expression of human ACE2 (hACE2) in three cell lines, suggesting that the restriction to viral replication was due to a low expression of bat ACE2 (bACE2) or the absence of bACE2 binding in these cells. Infectious virions were produced but not released from hACE2-transduced M. myotis brain cells. E. serotinus brain cells and M. myotis nasal epithelial cells expressing hACE2 efficiently controlled viral replication, which correlated with a potent interferon response. Our data highlight the existence of species-specific and cell-specific molecular barriers to viral replication in bat cells. These novel chiropteran cellular models are valuable tools to investigate the evolutionary relationships between bats and coronaviruses. IMPORTANCE Bats are host ancestors of several viruses that cause serious disease in humans, as illustrated by the ongoing SARS-CoV-2 pandemic. Progress in investigating bat-virus interactions has been hampered by a limited number of available bat cellular models. We have generated primary cells and cell lines from several bat species that are relevant for coronavirus research. The various permissivities of the cells to SARS-CoV-2 infection offered the opportunity to uncover some species-specific molecular restrictions to viral replication. All bat cells exhibited a potent entry-dependent restriction. Once this block was overcome by overexpression of human ACE2, which serves at the viral receptor, two bat cell lines controlled well viral replication, which correlated with the inability of the virus to counteract antiviral responses. Other cells potently inhibited viral release. Our novel bat cellular models contribute to a better understanding of the molecular interplays between bat cells and viruses.

Higher white-nose syndrome fungal isolate yields from UV-guided wing biopsies compared with skin swabs and optimal culture media.

BACKGROUND: North American bat populations have suffered severe declines over the last decade due to the Pseudogymnoascus destructans fungus infection. The skin disease associated with this causative agent, known as white-nose syndrome (WNS), is specific to bats hibernating in temperate regions. As cultured fungal isolates are required for epidemiological and phylogeographical studies, the purpose of the present work was to compare the efficacy and reliability of different culture approaches based on either skin swabs or wing membrane tissue biopsies for obtaining viable fungal isolates of P. destructans. RESULTS: In total, we collected and analysed 69 fungal and 65 bacterial skin swabs and 51 wing membrane tissue biopsies from three bat species in the Czech Republic, Poland and the Republic of Armenia. From these, we obtained 12 viable P. destructans culture isolates. CONCLUSIONS: Our results indicated that the efficacy of cultures based on wing membrane biopsies were significantly higher. Cultivable samples tended to be based on collections from bats with lower body surface temperature and higher counts of UV-visualised lesions. While cultures based on both skin swabs and wing membrane tissue biopsies can be utilised for monitoring and surveillance of P. destructans in bat populations, wing membrane biopsies guided by UV light for skin lesions proved higher efficacy. Interactions between bacteria on the host's skin also appear to play an important role.

Bat-derived cells use glucose as a cryoprotectant.

Evolution of heterothermy in environments with variable temperatures has allowed bats to survive food scarcity during seasonal climatic extremes by using torpor as a hibernation strategy. The controlled reduction of body temperature and metabolism through complex behavioural and physiological adaptations at organismal, organ, cellular and molecular levels includes the ability of tissues and cells to adapt to temperature alterations. Based on the prediction that cells of different tissues cultured in vitro would differ in their ability to withstand freezing and thawing of the medium, we determined the survival rate of bat-derived cells following exposure to -20 °C for 24 h in media with no cryoprotective agents or medium supplemented by glucose in concentration range 0-3333 mM. Cell survival rates were determined in relation to availability of glucose in the medium, organ origin, cell concentration and bat species. In general, increased glucose helped cells survive at sub-zero temperatures, though concentrations up to 80-fold higher than those found in chiropterans were needed. However, cells in glucose-free phosphate buffered saline also survived, suggesting that other mechanisms may be contributing to cell survival at low temperatures. Highest in vitro viability was observed in nervus olfactorius-derived cell cultures, with high survival rates and rapid re-growth under optimal conditions after exposure to -20 °C. Kidney cells from different bat species showed comparable overall survival rate patterns, though smaller chiropteran species appeared to utilise lower glucose levels as a cryoprotectant than larger species. Our in vitro data provide evidence that cells of heterothermic bats can survive sub-zero temperatures and that higher glucose levels in important tissues significantly improve hibernation survival at extremely low temperatures.

Carp edema virus infection associated gill pathobiome: A case report.

Understanding disease aetiology and pathologic mechanisms is essential for fish health evaluation. Carp edema virus (CEV) is the causative agent of a disease (CEVD) responsible for high mortality rates in both wild and cultured common carp Cyprinus carpio. Inspection of two carp specimens from a pond with high fish mortality revealed CEV infection in both the host and its ectoparasite (Argulus foliaceus). In addition to flavobacteria, well known to be associated with gill lesions, we found that free-living eukaryotes (amoebae and ciliates) and a temporary parasite (Ichthyobodo spp.) colonizing the gills may also contribute to alterations in gill structure and/or function, either directly, through firm (Ichthyobodo) or weak (amoebae) attachment of trophozoites to the gill epithelium, or indirectly, through carriage of pathogenic bacteria. Bacterial assemblages rich in families and genera, with predominance of Cetobacterium spp. in low-intensity alteration of the gill tissue and of Flavobacterium spp. in gills with extensive necrotic lesions, were detected in gills and within the cytoplasm of associated amoebae using high-throughput sequencing. Quantitative PCR indicated F. swingsii as the prevailing flavobacterial species within amoebae from less affected gills and F. psychrophilum within amoebae from extensively affected gills. This case study suggests that eukaryotic organisms as part of the gill pathobiome may also contribute to irreversible gill lesions seen in CEVD. Emphasizing the complexity of mutual relationships between bacterial assemblages and eukaryotic co-pathogens, further studies regarding factors that trigger pathology and influence severity in the CEV-positive carp are needed.

Comparison of diagnostic methods for Tetracapsuloides bryosalmonae detection in salmonid fish.

Diagnostic accuracy of pathogen detection depends upon the selection of suitable tests. Problems can arise when the selected diagnostic test gives false-positive or false-negative results, which can affect control measures, with consequences for the population health. The aim of this study was to compare sensitivity of different diagnostic methods IHC, PCR and qPCR detecting Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonid fish and as a consequence differences in disease prevalence. We analysed tissue from 388 salmonid specimens sampled from a recirculating system and rivers in the Czech Republic. Overall prevalence of T. bryosalmonae was extremely high at 92.0%, based on positive results of at least one of the above-mentioned screening methods. IHC resulted in a much lower detection rate (30.2%) than both PCR methods (qPCR32: 65.4%, PCR: 81.9%). While qPCR32 produced a good match with IHC (60.8%), all other methods differed significantly (p < .001) in the proportion of samples determined positive. Both PCR methods showed similar sensitivity, though specificity (i.e., the proportion of non-diseased fish classified correctly) differed significantly (p < .05). Sample preservation method significantly (p < .05) influenced the results of PCR, with a much lower DNA yield extracted from paraffin-embedded samples. Use of different methods that differ in diagnostic sensitivity and specificity resulted in random and systematic diagnosis errors, illustrating the importance of interpreting the results of each method carefully.

Cold arousal - A mechanism used by hibernating bats to reduce the energetic costs of disturbance.

During the season of hibernation, temperate bats alternate between prolonged bouts of torpor with reduced body temperature and short arousals with a return to normothermy. Hibernating bats are sensitive to non-tactile stimuli and arouse following changes in microclimatic conditions or disturbance from other bats, potential predators, or humans. Here, we used temperature data loggers to register the skin temperature of 38 Myotis myotis bats over two winters (between January and March), during which regular visits were made to the hibernaculum. Two kinds of arousal were observed, normothermic (Tsk > 25 °C) and cold (Tsk < 15 °C). Although bats responded to the presence of a researcher by arousals of both kinds, cold arousals were more frequent (63.8%). We found that mass loss was not affected by the number of disturbances, however it was in positive relationship with the mass at the beginning of the observation and differed between sex and age categories. Furthermore normothermic bats crawling among cluster-mates initiated arousal cascades, which mainly consisted of cold arousals. We failed to detect any effect of age or sex on the number of arousals initiated by normothermic individuals. Warming by only a few degrees requires less energy than a normothermic arousal and we propose it is sufficient to activate the sensory system in order to assess the relevance of external stimuli. Our results indicate that cold arousals reflect a physiological and behavioural adaptation aimed at avoiding the energetic costs of disturbance that can lead to depletion of fat reserves.

Phagocyte activity reflects mammalian homeo- and hetero-thermic physiological states.

BACKGROUND: Emergence of both viral zoonoses from bats and diseases that threaten bat populations has highlighted the necessity for greater insights into the functioning of the bat immune system. Particularly when considering hibernating temperate bat species, it is important to understand the seasonal dynamics associated with immune response. Body temperature is one of the factors that modulates immune functions and defence mechanisms against pathogenic agents in vertebrates. To better understand innate immunity mediated by phagocytes in bats, we measured respiratory burst and haematology and blood chemistry parameters in heterothermic greater mouse-eared bats (Myotis myotis) and noctules (Nyctalus noctula) and homeothermic laboratory mice (Mus musculus). RESULTS: Bats displayed similar electrolyte levels and time-related parameters of phagocyte activity, but differed in blood profile parameters related to metabolism and red blood cell count. Greater mouse-eared bats differed from mice in all phagocyte activity parameters and had the lowest phagocytic activity overall, while noctules had the same quantitative phagocytic values as mice. Homeothermic mice were clustered separately in a high phagocyte activity group, while both heterothermic bat species were mixed in two lower phagocyte activity clusters. Stepwise regression identified glucose, white blood cell count, haemoglobin, total dissolved carbon dioxide and chloride variables as the best predictors of phagocyte activity. White blood cell counts, representing phagocyte numbers available for respiratory burst, were the best predictors of both time-related and quantitative parameters of phagocyte activity. Haemoglobin, as a proxy variable for oxygen available for uptake by phagocytes, was important for the onset of phagocytosis. CONCLUSIONS: Our comparative data indicate that phagocyte activity reflects the physiological state and blood metabolic and cellular characteristics of homeothermic and heterothermic mammals. However, further studies elucidating trade-offs between immune defence, seasonal lifestyle physiology, hibernation behaviour, roosting ecology and geographic patterns of immunity of heterothermic bat species will be necessary. An improved understanding of bat immune responses will have positive ramifications for wildlife and conservation medicine.

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.

Field study indicating susceptibility differences between salmonid species and their lineages to proliferative kidney disease.

Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea) is the causative agent of proliferative kidney disease (PKD), which affects both wild and farmed salmonid fish. The objective of this study was to outline differences in susceptibility to PKD in different salmonid species, hybrids and breeding lineages. Susceptibility to T. bryosalmonae infection was established based on cumulative mortality, pathological findings and detection of T. bryosalmonae in the kidney using immunohistochemistry and molecular methods. Determination of pure and hybrid individuals of different species in the genus Salvelinus, and dissimilarity of rainbow trout lineages, was performed using traditional polymerase chain reaction (PCR) and microsatellite analyses. Rainbow trout displayed higher disease severity compared with brook trout and Alsatian charr. Moreover, the results indicated differences in infection susceptibility, not only among different salmonid species but also among different lineages of charr and rainbow trout. Our study indicated that some salmonid species and even different lineages of the same species are more suitable for farming under PKD pressure.

Modelling invasive pathogen load from non-destructive sampling data.

Where microbes colonizing skin surface may help maintain organism homeostasis, those that invade living skin layers cause disease. In bats, white-nose syndrome is a fungal skin infection that affects animals during hibernation and may lead to mortality in severe cases. Here, we inferred the amount of fungus that had invaded skin tissue of diseased animals. We used simulations to estimate the unobserved disease severity in a non-lethal wing punch biopsy and to relate the simulated pathology to the measured fungal load in paired biopsies. We found that a single white-nose syndrome skin lesion packed with spores and hyphae of the causative agent, Pseudogymnoascus destructans, contains 48.93 pg of the pathogen DNA, which amounts to about 1560 P destructans genomes in one skin lesion. Relating the information to the known UV fluorescence in Nearctic and Palearctic bats shows that Nearctic bats carry about 1.7 µg of fungal DNA per cm2, whereas Palearctic bats have 0.04 µg cm-2 of P. destructans DNA. With the information on the fungal load that had invaded the host skin, the researchers can now calculate disease severity as a function of invasive fungal growth using non-destructive UV light transillumination of each bat's wing membranes. Our results will enable and promote thorough disease severity assessment in protected bat species without the need for extensive animal and laboratory labor sacrifices.

Reproductive toxicity of fluoroquinolones in birds.

BACKGROUND: While commercial poultry and captive birds are exposed to antimicrobials through direct medication, environmental pollution may result in contamination of wild birds. Fluoroquinolones are commonly used medications to treat severe avian bacterial infections; however, their adverse effects on birds remain understudied. Here, we examine toxicity of enrofloxacin and marbofloxacin during the egg incubation period using the chicken (Gallus Gallus domesticus) as a model avian species. Laboratory tests were based on eggs injected with 1, 10 and 100 µg of fluoroquinolones per 1 g of egg weight prior to the start of incubation and monitoring of chick blood biochemistry, reproductive parameters and heart rate during incubation. RESULTS: Eggs treated with fluoroquinolones displayed reduced hatchability due to embryonic mortality, particularly on day 13 of incubation. Total hatching success showed a similar pattern, with a significantly reduced hatchability in low and high exposure groups treated with both enrofloxacin and marbofloxacin. From 15 to 67% of chicks hatching in these groups exhibited joint deformities. Hatching one-day pre-term occurred with a prevalence of 31 to 70% in all groups treated with fluoroquinolones. Embryonic heart rate, measured on days 13 and 19 of incubation, increased in all enrofloxacin-treated groups and medium and high dose groups of marbofloxacin-treated eggs. Blood biochemistry of chicks sampled at hatch from medium dose groups showed hypoproteinaemia, decreased uric acid and increased triglycerides. Chicks from the enrofloxacin-treated group displayed mild hyperglycaemia and a two-fold rise in the blood urea nitrogen to uric acid ratio. Principal components analysis based on blood biochemistry clearly separated the control bird cluster from both enrofloxacin- and marbofloxacin-treated birds. CONCLUSIONS: Fluoroquinolones induce complex adverse effects on avian embryonic development, considerably reducing the performance of incubated eggs and hatching chicks. Cardiotoxicity, which quickens embryonic heart rate, meant that the total number of heart beats required for embryogenesis was achieved earlier than in the standard incubation period, resulting in pre-term hatching. Our data suggest that enrofloxacin has a higher potential for adverse effects than marbofloxacin. To conclude, care should be taken to prevent exposure of reproducing birds and their eggs to fluoroquinolones.

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.

White-nose syndrome detected in bats over an extensive area of Russia.

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.

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.

Ecotoxicity of selected antibiotics for organisms of aquatic and terrestrial ecosystems.

OBJECTIVES: The aim of this study was to assess the ecotoxicity of selected antibiotics (i.e. penicillin G, vancomycin and tetracycline) using ecotoxicological tests. Tests were conducted on organisms representing all trophic levels of the aquatic ecosystem, namely producers (green freshwater algae Pseudokirchneriella subcapitata), consumers (water fleas Daphnia magna) and decomposers (bacteria Vibrio fischeri). The effect of antibiotics on the representative of edaphon was measured by testing the inhibition of the reproduction of springtails Folsomia candida and earthworms Eisenia fetida. DESIGN: Methodologically, the procedure was carried out in accordance with the following standards: OECD 201 (Fresh water algal growth inhibition test), OECD 202 (Inhibition of the mobility of Daphnia magna), ISO 11348-2 (Inhibitory effect of antibiotics on the light emission of Vibrio fischeri), OECD 232 (Inhibition of reproduction of Collembola Folsomia candida) and OECD 222 (Inhibition of reproduction of Eisenia fetida). RESULTS: In aquatic organisms the highest level of toxicity was shown by tetracycline to algae (72hEC50 = 1.82 mg.l-1) and daphnia (48hEC50 = 8.16 mg.l-1). The least toxic for all test organisms was penicillin G. The results of the tests performed on the representative of edaphon, Folsomia candida, showed that its reproduction was most inhibited by penicillin G (28dEC50 = 328 mg.kg-1) and least by tetracycline (28dEC50 = 2560 mg.kg-1). Similar results were observed in Eisenia fetida (56dEC50 = 348 mg.kg-1 for penicillin G and 56dEC50 = 2735 mg.kg-1 for tetracycline. CONCLUSION: The ecotoxicity of antibiotics differed significantly depending on the test organism and testing conditions.

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.

Effect of Intramuscular Injection on Oxidative Homeostasis in Laboratory Guinea Pig Model.

In animal models, there was observed alteration of various physiological processes caused by microtraumas. Here reported experiment was aimed on the research of link between injection and development of an oxidative imbalance. Laboratory guinea pig was chosen as a suitable model for examining of the oxidative stress. Markers indicating oxidative homeostasis were assayed in the frontal, temporal and occipital brain lobe, cerebellum, liver, kidney, spleen and heart one hour after an intramuscular injection. Common biochemical parameters were measured in plasma samples as well. The most extensive effect was observed in the heart where the thiobarbituric acid reactive substances value was more than twice increased after the injection. The level of carbonylated proteins was significantly elevated in the kidney and ferric reducing antioxidant power value was increased in the brain compartments. The enzyme activities in the organs were not influenced except the activity of superoxide dismutase, which was moderately decreased in the brain. In the plasma samples, there was observed increase of the blood urea nitrogen. The results showed significant the influence of the intramuscular injection on a development of an oxidative insult. The injection can be considered as an adverse effect with quite extensive stress consequences.

Mixture toxicity of microcystin-LR, paraoxon and bromadiolone in Xenopus laevis embryos.

OBJECTIVES: Apart from infections and habitat loss, environmental pollution is another major factor of global decline of amphibians. Using the model of Xenopus laevis embryos, we test the hypothesis that combined exposure of amphibians to natural toxins and anthropogenic pollutants induces more pronounced adverse effects than single exposures. METHODS: Experimental procedures adhered to Frog Embryo Teratogenesis Assay - Xenopus standards (FETAX). Exposure groups included controls, solvent (dimethyl sulfoxide) controls, and embryos exposed for 96 h to single, double and triple action of paraoxon (P), bromadiolone (B), and microcystin-LR (M), added to the FETAX medium at a dose of 300, 350, and 500 µg.L(-1), respectively. Studied responses of X. laevis embryos included mortality and malformations, head-to-tail length, total antioxidant capacity, lipid peroxidation, and caspase-3 activity. RESULTS: The triple combination induced the highest mortality. Malformations in embryos significantly prevailed only in B-, and B+P-exposure groups. Apart from the single exposure to B, the tested substances and their combinations inhibited the embryonic growth. Triple exposure had the most pronounced effect both on the growth inhibition and total antioxidant capacity. Lipid peroxidation was increased after B+M exposure, while single and combined exposures to B and P had an opposite effect. CONCLUSIONS: This study helps to understand adverse effects of environmental pollution by natural toxins and agrochemicals in amphibians. The results allow for risk assessment of environmental pollution and findings of low concentrations of contaminants in aquatic environments. Further research to address issues such as mixture toxicity to metamorphosing and adult amphibians is necessary.

Impact of platinum group elements on the soil invertebrate Enchytraeus crypticus.

OBJECTIVES: The platinum group elements (PGE) platinum (Pt), palladium (Pd), and rhodium (Rh) are used in automobile catalytic converters, from which they have been emitted into the environment to an increasing degree over the last 20 years. Despite the bioavailability of these metals to plants and animals, studies determining the effects of PGE on organisms are extremely rare. Enchytraeids are ecologically relevant soil organisms, due to their activity in decomposition and bioturbation in many soil types worldwide. DESIGN: The experiments were carried out as described in the OECD Guideline 220 [CSN EN ISO 16387--Soil quality--Effects of pollutants on Enchytraeidae (Enchytraeus sp.)--Determination of effects on reproduction]. The reproductive effects of platinum (PtCl4), palladium (PdCl2) and rhodium (RhCl3) were examined. The concentrations of PGE tested were as follows: 5, 10, 25, 50 and 100 µmol x L(-1) PdCl2; 50, 100, 150, 200 and 250 µmol x L-1 PtCl4/RhCl3. The EC50 (medium effective concentration) was determined after 28 days of exposure. The inhibition of the reproduction of PGE-exposed enchytraeids was compared against controls. RESULTS: Values of 28dEC50 of PtCl4, PdCl2 and RhCl3 amounted to 161.9 µmol x L(-1), 70.0 µmol x L(-1) and 246.6 µmol x L(-1), respectively. We can confirm that the relative order of toxicities is Pd (II) >Pt(IV) >>Rh(III). CONCLUSION: To the best of our knowledge, this is the first study to use Enchytraeus crypticus as an indicator species to assess the risk of soil contamination by platinum, palladium and rhodium. Results of this study contribute important data on the ecotoxicity of a rarely studied elements.