Molecular Detection of Toxoplasma gondii, Neospora caninum and Encephalitozoon spp. in Vespertilionid Bats from Central Europe.

Bats may carry various viruses and bacteria which can be harmful to humans, but little is known about their role as a parasitic source with zoonotic potential. The aim of this study was to test wild bats for the presence of selected parasites: Toxoplasma gondii, Neospora caninum and microsporidia Encephalitozoon spp. In total, brain and small intestine tissues of 100 bats (52 Myotis myotis, 43 Nyctalus noctula and 5 Vespertilio murinus) were used for the DNA isolation and PCR detection of the abovementioned agents. Toxoplasma gondii DNA was detected by real-time PCR in 1% of bats (in one male of M. myotis), while all bats were negative for N. caninum DNA. Encephalitozoon spp. DNA was detected by nested PCR in 25% of bats, including three species (twenty-two M. myotis, two N. noctula and one V. murinus). Positive samples were sequenced and showed homology with the genotypes Encephalitozoon cuniculi II and Encephalitozoon hellem 2C. This is the first study on wild vespertilionid bats from Central Europe and worldwide, with a relatively high positivity of Encephalitozoon spp. detected in bats.

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.

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.

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.

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

BACKGROUND: Lead, 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. RESULTS: Soil 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 CaNa(2)EDTA 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 CaNa(2)EDTA 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. CONCLUSIONS: The reported blood parameters and reproduction impairment in captive birds may have implications for professionals dealing with lead exposure in wild birds.

Yew poisoning of olive baboons (Papio anubis) in captivity: laboratory diagnosis.

OBJECTIVES: Toxic effects of the yew have been known since ancient times. Yew toxicity is due to the content of cyanogenic glycosides and a mixture of alkaloids known as taxines. Taxine B is probably responsible for the most part of adverse effects in poisoned organisms. This particular taxoid is common in body fluids of the yew-poisoned. The present study is engaged with laboratory examination to confirm substances that lead to fatality of a pair of olive baboons (Papio anubis) following ingestion of yew seeds. When both cage mates (male and female) died suddenly, poisoning was suspected because many berries had fallen into the cage from a nearby fruiting yew tree (Taxus baccata) during the windy night before. METHODS: The analysis was performed using electrospray ionization quadrupole time-of-flight mass spectrometry. A flow injection analysis/mass spectrometry setting was prepared for this purpose. RESULTS: The above mentioned mass spectrometry analysis of taxoids confirmed poisoning by taxanes. The presence of taxin B/isotaxin B was confirmed in all investigated samples. Apparently in urine and bile there were concentrations ranging 150-220 ng.mL-1 and in blood serum concentrations 25-30 ng.mL-1. CONCLUSION: It follows from the results obtained that we confirmed that baboons were deadly intoxicated by yew fruits.

Clinical and Laboratory Parameters of Carp Edema Virus Disease: A Case Report.

In the present study, we describe a natural outbreak of carp edema virus disease (CEVD) in koi carp, concentrating on clinical manifestation, gross and microscopic pathology, immunological parameters, viral diagnostics, and phylogenetic analysis. Examination of white blood cell parameters showed increased monocyte and decreased lymphocyte counts in CEV-affected fish compared to healthy control fish. Regarding immune system functioning, the present work shows, for the first time, enhanced phagocytic activity in CEV-affected fish. Respiratory burst of phagocytes was strongly increased in diseased fish, the increase being attributed to an increased phagocyte count rather than enhancement of their metabolic activity. The present work also newly shows histopathological changes in the pancreatic tissue of diseased koi.

Case report: Filarial infection of a parti-coloured bat: Litomosa sp. adult worms in abdominal cavity and microfilariae in bat semen.

Background: Filarial infections have been understudied in bats. Likewise, little is known about pathogens associated with the reproductive system in chiropterans. While semen quality is critical for reproductive success, semen-borne pathogens may contribute to reproductive failure. Methods: For the first time we performed electroejaculation and used computer-assisted semen analysis to provide baseline data on semen quality in a parti-coloured bat (Vespertilio murinus). Results: The semen quality values measured in the V. murinus male appeared high (semen concentration = 305.4 × 106/mL; progressive and motile sperm = 46.58 and 60.27%, respectively). As an incidental finding, however, microfilariae were observed in the bat semen examined. At necropsy, eight adult filarial worms, later genetically identified as Litomosa sp., were found in the peritoneal cavity, close to the stomach, of the same particoloured bat male dying as a result of dysmicrobia and haemorrhagic gastroenteritis in a wildlife rescue centre. Histopathology revealed microfilariae in the testicular connective tissue and the epidydimal connective and fat tissues. A PCR assay targeting cytochrome c oxidase subunit 1 confirmed that adult worms from the peritoneal cavity and testicular microfilariae were of the same filarial species. Mildly engorged argasid mite larvae attached to the bat skin proved negative for filarial DNA and the adult filarial worms proved negative for endosymbiont Wolbachia. Conclusion: While the standard filarial life cycle pattern involves a vertebrate definitive host and an invertebrate vector, represented by a blood-sucking ectoparasite, our finding suggests that microfilariae of this nematode species may also be semen-borne, with transmission intensity promoted by the polygynous mating system of vespertilionid bats in which an infected male mates with many females during the autumn swarming. Presence of microfilariae may be expected to decrease semen quality and transmission via this route may challenge the success of reproductive events in females after mating. Further investigation will be necessary to better understand the bat-parasite interaction and the life cycle of this filarial worm.

Tacrine is implicated in oxidative stress in the laboratory guinea pig model.

OBJECTIVES: Tacrine was the first acetylcholinesterase inhibitor approved for the treatment of Alzheimer disease. The compound is not available for therapeutic purposes as it was withdrawn due to hepatotoxicity of its metabolites. The hepatotoxicity can be decreased by alternative ways of drug administration avoiding thus the first pass effect. The present study is aimed to investigate the influence of intramuscularly administrated tacrine on oxidative stress. METHODS: Laboratory guinea pigs were exposed to tacrine at doses of 0-800 µg/kg. The animals were euthanized 1 and 24 hours after the exposure. Parameters such as ferric reducing antioxidant power (FRAP), thiobarbituric acid reactive substances (TBARS), carbonylated proteins, caspase 3 activity, superoxide dismutase activity and glutathione reductase activity were assessed in the frontal, temporal and occipital lobe, cerebellum, liver, spleen, heart, and kidney. Moreover, levels of glucose, total and HDL cholesterol forms, triglycerides, blood urea nitrogen, creatinine, total bilirubin, total protein, albumin and activities of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and lactate dehydrogenase were assessed in plasma samples. RESULTS: Activities of the enzymatic markers, level of carbonylated proteins in organs and levels of biochemical markers in plasma were only slightly influenced by tacrine. Dose-dependent elevation of the FRAP value was recognized in the brain tissues and the liver. The TBARS value was increased in the kidney and heart 1 and 24 hours, respectively, after exposure. CONCLUSION: In the study, the effect of tacrine on markers of oxidative stress was proved. Possible positive effects of tacrine on the antioxidant defence in the brain tissue were discussed.

Melatonin influences antioxidant homeostasis and basal metabolism in the BALB/c mouse model.

OBJECTIVES: Melatonin is a hormone with strong antioxidant activity. It is biosynthesized in the pineal gland and serves in the biological signaling and control of the circadian rhythm. Though there is evidence of beneficial effects of melatonin, the substance was not investigated in greater details associated with specific regulation of oxidative stress in organs and tissues. DESIGN: The experiment is based on exposure of BALB/c mice to doses from 10 µg to 1mg of melatonin. Mice were euthanized after one and 24 hours, respectively. Biochemical markers in plasma, ferric reducing antioxidant power (FRAP), thiobarbituric acid reactive substances (TBARS) and activity of caspase-3 were examined in selected organs. RESULTS: We confirmed significant alteration in high-density lipoprotein and total cholesterols and glucose. After one day, levels of glucose and high-density lipoprotein cholesterol were decreased, while total cholesterol increased in a dose dependent manner. FRAP values increased in spleen, liver, kidney, heart, and brain indicating a growing potential of low molecular weight antioxidants 24 hours after exposure. However, TBARS values indicating oxidative stress were elevated in heart, kidney, and liver. CONCLUSIONS: Despite the antioxidant properties of melatonin, its effect on the organism is more complicated. It influences not only the oxidative homeostasis but also the basal metabolism, as represented by, e.g., cholesterol and glucose. This substance could thus be used for therapeutical purposes such as amelioration of pathologies associated with generation of reactive species or some metabolic dysfunctions.

Risk of single and combined exposure of birds to non-steroidal anti-inflammatory drugs and lead.

OBJECTIVES: Pharmaceuticals and heavy metals such as diclofenac and lead, respectively, have been identified as environmental contaminants toxic to birds and posing serious threats to declining populations of raptors worldwide. The aim of the present study was to test the hypothesis that a sublethal combination of non-steroidal anti-inflammatory drugs and lead induces more pronounced effects than single exposures in birds. METHODS: A total of 40 Japanese quails (Coturnix coturnix japonica) at the age of 2 months and average weight of 180g were on a random basis divided into four experimental groups of 10 specimens (i.e., control, diclofenac, lead, and lead+diclofenac exposures). Six lead shots in the total weight of 1.5 grams were inserted into the crop on day 0 of the experiment, while a total of 5 mg/kg of diclofenac administered intramuscularly were divided into treatments on days 0 and 5. Group responses were compared using haematology and biochemistry after 10 days. RESULTS: There was no mortality in control and both single and combined diclofenac and lead exposure groups, nor did the birds show any clinical signs of intoxication. Univariate analyses of blood parameters yielded a decrease in haematocrit in birds exposed to both substances when compared with the control, a lower haemoglobin level of the lead-exposed group, increased activity of aspartate aminotransferase in the NSAIDs-exposed group, increased activity of alkaline phosphatase in birds exposed to a combination of diclofenac and lead, and a higher phosphorus level in the lead-exposed group. The principal component analysis revealed no multivariate pattern of responses of blood parameters and did not allow separation of exposure groups from controls when the variables and samples were projected onto a two dimensional space. CONCLUSIONS: Results of the present study can enhance understanding of combination toxicity of veterinary drugs and heavy metals in birds, i.e. a scenario that has become environmentally relevant in recent decades. Fortunately, individual blood parameter effects prevailed and no joint mortal effects were recognised in Japanese quails exposed to a combination of sublethal doses of diclofenac and lead.

Risk of combined exposure of birds to cyanobacterial biomass containing microcystins, acetylcholinesterase inhibitor and anticoagulant.

OBJECTIVES: The objective of this study was to examine the hypothesis that a combination of cyanobacterial biomass containing microcystins, acetylcholinesterase inhibitor and anticoagulant can enhance avian toxic effects produced by single exposures only. METHODS: A total of 48 two-month-old Japanese quails (Coturnix coturnix japonica) with average body weight of 160 g were randomly divided into 8 experimental groups of six birds and sex ratio of 1:1. Experimental groups of control Japanese quails (C) and birds exposed to single and combined sub-lethal doses of paraoxon (P), bromadiolone (B), and microcystins in cyanobacterial biomass (M) included: C, P, P+B, B, B+M, P+M, M, and P+B+M. During the 10-day exposure birds in the respective groups received biomass containing 61.62 µg microcystins daily (i.e. 26.54 µg MC-RR, 7.62 µg MC-YR and 27.39 µg MC-LR), two 250 µg/kg doses of paraoxon, and two 500 mg/kg doses of bromadiolone. Group responses were compared using standard plasma biochemistry and antioxidant/oxidative stress parameters in tissues. RESULTS: While single and double combinations of toxicants induced responses in individual biochemical parameters measured and evaluated using univariate statistical analysis, those in the triple exposure were most extensive. The principal component analysis of antioxidant/oxidative stress parameters (glutathione reductase, lipid peroxidation, and ferric reducing antioxidant power) in tissues (liver, kidney, heart, brain, lungs, gonads, and pectoralis major muscle) clearly separated the triple group (P+B+M) from all single and double exposure groups and the control and indicated thus marked joint effects in the overall pattern of antioxidant/oxidative stress responses of this group. The separation was driven by the modification of the ferric reducing antioxidant power levels in heart and brain and the cardiac lipid peroxidation level, in particular. CONCLUSIONS: This experiment contributes to the understanding of the pathogenic mechanisms of combined sub-lethal exposure to natural toxins and agrochemicals and may be used for risk assessment of environmental pollution in birds.

Avian high-dose toxicity of cyanobacterial biomass.

OBJECTIVES: Previous studies using oral administration of environmentally relevant doses of cyanobacterial biomass containing microcystins (MCs) induced only sub-lethal effects in experimental birds. Therefore, the objective of this study was to obtain data on avian high-dose toxicity of MCs and compute LD50, if possible, following the natural oral route of administration. DESIGN: Responses of birds to single high-dose exposure to MCs were evaluated in fourteen-day old Japanese quail males (Coturnix coturnix japonica) with average body weight of 50 g which were randomly divided into five groups. Birds from four experimental groups were administered 7.5 ml of cyanobacterial biomass suspension containing increasing MCs quantities of 2500, 5000, 10000, and 20000 µg/kg using oral gavage. Controls received an equal dose of drinking water instead of the test substance. Birds were observed for clinical signs of acute toxicity. Survivors were killed on day 5 to obtain body and liver weights. A five-grade semi-quantitative system for histopathological liver damage scoring was used to compare cyanobacterial-biomass-exposed birds against controls. RESULTS: No mortality occurred during the period of five days post exposure in both control and MCs-exposed groups and this high-dose experiment failed to provide data to compute the LD50. Nevertheless, marked sub-lethal effects were recognised in the damage of liver that included dose-dependent changes in the body/liver ratios and morphological changes ranging from mild vacuolar dystrophy to focal liver necroses in the highest exposure group. Hepatic lesions were mainly observed in the pericentral area of the liver. CONCLUSIONS: Though maximum cyanobacterial biomass dose rates that could be administered to birds of the size were used in the present experiment and more pronounced hepatic lesions than after exposure to environmentally relevant doses were observed, birds would probably have survived unless killed for histopathology on day 5 of exposure. These results provide support to previously reported data on sub-lethal effects following exposure to cyanobacterial biomass containing MCs in birds and mortality occurring only in birds under combined action with other stressors.

Toxicological scoring of Alzheimer's disease drug huperzine in a guinea pig model.

Huperzine is a secondary metabolite in lycopods Huprzia and an inhibitor of acetylcholinesterase and antagonist of N-methyl-D-apartate receptor. Huperine is a suitable drug for the treatment of Alzheimer's disease as it is a part of traditional Chinese medicine. Currently, it undergoes clinical trials in the European Union and United States. The toxicological data about huperzine are missing and link between huperzine and oxidative stress has not been extensively investigated. For the above mentioned reasons, we organized experiment on a guinea pig model aimed at the investigation of adverse effects caused by huperzine. Guinea pigs were exposed to (-)-huperzine A in doses 5-625 µg/kg. Animals were sacrificed one day after exposure. Ferric reducing antioxidant power, thiobarbituric acid reactive substances, glutathione reductase, caspase 3 activity and selected biochemical markers (e.g. transaminases, blood urea nitrogen and glucose) were assayed. In frontal, parietal, temporal lobes and cerebellum, we found increase of antioxidants, glutathione reductase and oxidative stress markers in a dose dependent manner. Effects on liver, kidney and spleen were milder. We discuss ambivalent action of huperzine in the body and judge the huperzine action owing to recently reported experiments.