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.

Effects of sublethal exposure of European brown hares to paraoxon on the course of tularemia.

OBJECTIVES: The causative agent of tularemia Francisella tularensis is highly infectious and lagomorphs are important reservoirs and a source of human disease. The aim of the present study was to test the hypothesis that sublethal exposure to pesticides increases the susceptibility of hares to F. tularensis and modulates the course of the infection. METHODS: Experimental hares were allocated to a) control, b) paraoxon-treated, c) F. tularensis-treated, and d) paraoxon-and-F. tularensis-treated groups of five specimens on a random basis and subcutaneously inoculated with a wild F. tularensis subsp. holarctica strain (a single dose of 9 × 108 CFU pro toto) and/or injected a sublethal dose of paraoxon (100 µg/kg). Group differences were evaluated using survival curves, oxidative stress responses as well as caspase-3 and acetylcholinesterase activities in whole blood samples collected on day 2 post exposure. RESULTS: The paraoxon-and-F. tularensis-treated group showed a rapid onset of clinical signs and all deaths occurred on days 2 and 3 post exposure. F. tularensis-inoculated hares survived from 3 to 10 days, while only one hare died on day 12 in the paraoxon-treated group. Survival curves in the three exposed groups were significantly different from the control and median survival in F. tularensis-inoculated and paraoxon-and-F. tularensis-treated hares amounted to 7 and 2 days, respectively. Compared with controls, significant responses included an eight- and seven-fold activation of caspase-3 in F. tularensis-inoculated and paraoxon-and-F. tularensis-treated hares, respectively, and a 1.5-fold decrease of blood acetylcholinesterase activities in the paraoxon-treated and paraoxon-and-F. tularensis-treated groups. There was a 1.3- to 1.4-fold decrease of the ferric reducing antioxidant power in blood of F. tularensis-inoculated hares and the paraoxon-and-F. tularensis-treated group, respectively. The blood lipid peroxidation levels were of no differences among the four experimental groups. CONCLUSIONS: Results of this study can help understand the pathogenesis of tularemia and mortality of hares in agricultural habitats. Use of anticholinesterase agents in agriculture can pose a threat of infectious disease outbreaks and higher mortality in wildlife populations.

Biochemical responses and oxidative stress in Francisella tularensis infection: a European brown hare model.

BACKGROUND: The aim of the present study was to investigate biochemical and oxidative stress responses to experimental F. tularensis infection in European brown hares, an important source of human tularemia infections. METHODS: For these purposes we compared the development of an array of biochemical parameters measured in blood plasma using standard procedures of dry chemistry as well as electrochemical devices following a subcutaneous infection with a wild Francisella tularensis subsp. holarctica strain (a single dose of 2.6 × 109 CFU pro toto). RESULTS: Subcutaneous inoculation of a single dose with 2.6 × 109 colony forming units of a wild F. tularensis strain pro toto resulted in the death of two out of five hares. Plasma chemistry profiles were examined on days 2 to 35 post-infection. When compared to controls, the total protein, urea, lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase were increased, while albumin, glucose and amylase were decreased. Both uric and ascorbic acids and glutathione dropped on day 2 and then increased significantly on days 6 to 12 and 6 to 14 post-inoculation, respectively. There was a two-fold increase in lipid peroxidation on days 4 to 8 post-inoculation. CONCLUSIONS: Contrary to all expectations, the present study demonstrates that the European brown hare shows relatively low susceptibility to tularemia. Therefore, the circumstances of tularemia in hares under natural conditions should be further studied.

Modulation of ionising radiation generated oxidative stress by HI-6 (asoxime) in a laboratory rat model.

OBJECTIVES: HI-6 is an antidotum suitable for treatment of intoxication by nerve agents. The recent investigation appointed its modulation of inflammatory response as well as vegetative nervous system activity. However, the present experiments were carried out in order to assess the antioxidant effect of HI-6 in irradiated animals. METHODS: male Wistar rats were irradiated by ionizing radiation (7.5 Gy, LD50/30). Animals were divided into four groups: i.e. controls (A), irradiated (B), treated with HI-6 (C), and both irradiated and treated with HI-6 (D). Ferric reducing antioxidant power (FRAP), thiobarbituric acid reactive substances (TBARS) and glutathione reductase activity were assayed in liver, spleen, plasma, and whole blood. Clinical biochemistry markers were determined in plasma samples. RESULTS: We found significantly increased FRAP levels in liver, while its levels decreased in the spleen of B group animals. Ionising radiation (B group) also significantly elevated TBARS values in spleen. HI-6 reversed FRAP and TBARS values to control levels. Glutathione reductase activity was significantly elevated in spleen and liver of animals exposed to HI-6 (C and D groups). Clinical biochemistry markers were shifted only slightly. The in vitro test confirmed the inhibitory effect of HI-6 towards acetylcholinesterase. CONCLUSIONS: In conclusion, HI-6 is potent in suppressing oxidative stress and might be a promising drug in the field of radiation protection.

Effects of cyanobacterial biomass on avian reproduction: a Japanese quail model.

OBJECTIVES: The present study was aimed at evaluation of the response of Japanese quails to cyanobacterial biomass administered in feed using biochemical profiles and parameters of reproduction. DESIGN: Effects of cyanobacterial biomass were studied according to the OECD 206 Guideline on Avian Reproduction Toxicity. A total of 16 control and 16 experimental pairs (32 males and 32 females) were analyzed. The chronic exposure of parent birds lasted eight weeks with the daily sum of 61.62 microg MCs including 26.54 microg MC-RR, 7.62 microg MC-YR and 27.39 microg MC-LR. RESULTS: There was no mortality both in control and cyanobacterial-biomass-exposed adults during the present study. Nor did the birds show any clinical signs of intoxication. Lactate dehydrogenase activity was increased about three-fold in exposed birds. No other biochemical parameters were showing significant differences. A total of 824 and 821 eggs were laid by control and exposed birds, respectively, during the eight-week study period. Eggs laid by cyanobacterial-biomass-exposed hens had lower weight than in controls (11.99+/-1.13g and 12.40+/-1.27g, respectively; p<0.01). Egg viability, hatchability, and the effect of hatching in control and experimental birds were 79.6+/-9.3 and 86.8+/-8.2% (p<0.05), 83.2+/-12.6 and 90.1+/-9.3%, and 65.2+/-17.7 and 77.7+/-15.2% (p<0.05), respectively. There was also a statistically significant difference in the number of 14-day old survivors per hen per day in control and experimental birds (0.38+/-0.02 and 0.43+/-0.01 %, respectively). CONCLUSION: The lower weight of eggs produced by exposed parental hens was not reflected in their biological quality. On the contrary, reproductive parameters in cyanobacterial-biomass-exposed birds were better than in the control group. It might be hypothesized that compounds of hormonal activity could be present in the complex cyanobacterial biomass. However, further research into this issue is necessary.

Biochemical responses of juvenile and adult Japanese quails to cyanobacterial biomass.

OBJECTIVES: The aim of the present study was to evaluate differences between juvenile and adult Japanese quails in responses to the exposure to cyanobacterial biomass in the diet. DESIGN: The OECD 205 Guideline on Avian Dietary Toxicity Test (1984) was employed in the experiment. A total of 75 freshly hatched chicks and 30 adults were exposed to cyanobacterial biomass for 15 days and blood sampled daily and on days 5, 10 and 15, respectively. Japanese quail chicks and adults received the same daily dose of approximately 224.4 ng microcystins per gram of body weight. Biochemical responses were compared against controls. RESULTS: No Japanese quail chicks and adults died during the acute 15-day-cyanobacterial-biomass exposure. Biochemical responses to the biomass in diet were first observed from day 5 post exposure to cyanobacterial biomass both in chicks and adults and there were age-related differences in the parameters changed. The responses of adult birds included an increase in lactate dehydrogenase, a drop in glucose and the total antioxidant capacity as well as a 15 to 20 % inhibition of acetylcholinesterase activity. Japanese quail chicks exposed to cyanobacterial biomass for the first 15 days after hatching reacted by having hypoproteinaemia, increased concentrations of triglycerides, uric acid and the total antioxidant capacity and a drop in high-density lipoprotein cholesterol in the blood. CONCLUSIONS: Chicks were not found to be more susceptible to the effects of biomass exposure. It seems that, due to their physiological preparation for the oxidative stress associated with hatching, Japanese quail chicks were even better able to cope with the cyanobacterial-biomass-induced oxidative stress than adults.