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.

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.

Toxicity hazard of organophosphate insecticide malathion identified by in vitro methods.

OBJECTIVES: Malathion is generally not classified as toxic. However, the toxicity seems to be species-dependent. Local and systemic toxicity data for birds are rare, but a decrease of wild bird densities in areas where malathion was applied was reported. Aim of the study was to extend knowledge on malathion toxicity on cellular and organ level and to evaluate embryotoxicity and genotoxicity for birds using the chick embryo model HET-CAM. METHODS: Skin and eye irritation was determined using reconstructed skin and eye cornea tissues and the chorioallantoic membrane of chick embryo to simulate conjunctiva. Cytotoxicity in 3T3 Balb/c fibroblast culture was determined to estimate acute systemic toxicity. Chick embryo model was further employed to evaluate acute embryotoxicity for birds (mortality and genotoxicity). Data were analysed by means of general linear models. RESULTS: Malathion is not a skin and eye irritant. Cytotoxicity in vitro test provided LD50 value of 616 mg/kg suggesting higher toxic potential than is generally published based on in vivo tests on laboratory rodents. Embryotoxicity studies revealed dose and age dependent mortality of chick embryos. Genotoxicity was identified by means of micronucleus test in erythroid cells isolated from chorioallantois vascular system of chick embryos. CONCLUSIONS: Using in vitro alternative toxicological methods, a higher toxic potential of malathion was demonstrated than is generally declared. An increased health and environmental hazard may occur in areas with intensive agricultural production. The environmental consequences of delayed effects and embryotoxicity for bird populations in areas exposed to organophosphate insecticides, such as malathion, are obvious.

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.

Mycoplasma gallisepticum infection in the grey partridge Perdix perdix: outbreak description, histopathology, biochemistry and antioxidant parameters.

BACKGROUND: The grey partridge is an important game bird in Europe that has declined considerably over the last decades. The production and release of farm-bred birds can be threatened by infectious agents. The objective of this study was to describe the outbreak, pathology, and blood and tissue biochemical responses in a flock of grey partridges naturally infected with Mycoplasma gallisepticum. RESULTS: Morbidity and mortality rates were 100% and 60%, respectively. Necropsy revealed an accumulation of caseous exudate within the infraorbital sinuses, tracheitis, pneumonia and airsacculitis. There were significant increases in activities of lactate dehydrogenase, creatine kinase and amylase, and levels of total protein and glucose in Mycoplasma-infected birds when compared to control. Catalase showed significantly lower activity in the heart, lungs, liver and gonads of Mycoplasma-infected birds. Glutathione-S-transferase activity was elevated in the eye and the associated infraorbital sinus and kidneys, and decreased in the liver. Decreased levels of reduced glutathione were found in the heart, kidneys, liver and gonads. The activity of glutathione reductase was lower only in the lungs. Compared to healthy birds, mycoplasmosis in the grey partridge caused significant differences in the level of lipid peroxidation in lungs and plasma (p < 0.05), while the ferric reducing antioxidant power was lower in the heart and kidneys (p < 0.01). Significant correlations among responses of the antioxidant parameters were found namely in the heart, lungs, spleen, liver and plasma. There were also numerous significant inter-tissue correlations of all the studied antioxidant parameters. CONCLUSIONS: The present study demonstrates the high susceptibility of grey partridges to natural infection by M. gallisepticum, the severity of the disease based on histopathology, and the modulation of blood chemical profiles and oxidative stress-associated parameters in the avian hosts, thus enhancing the understanding of the pathogenesis of mycoplasmosis in birds. Moreover, the reported reference values can be useful for the evaluation of the state of health in grey partridges.

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.

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

BACKGROUND: Both 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. METHODS: Rodents 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. RESULTS: Bacterial 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. CONCLUSION: As 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.

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.

Tissue metallothionein response in the Japanese quail associated with exposure to cyanobacterial biomass, lead and the Newcastle disease virus.

OBJECTIVES: Here we tested the hypothesis that multiple toxic and infectious stressors combine in their adverse effects to produce higher tissue responses of metallothioneins (MTs) in birds. METHODS: We used Japanese quails as a model avian species. The study is based on data obtained from single and combined exposures of Japanese quails to cyanobacterial biomass containing microcystins, lead and a live Newcastle disease vaccination virus. Eight groups of 5 birds were exposed to single, double and triple combinations of these stressors and compared with controls. Birds were euthanized after the 30-day exposure to collect brain, liver, kidney, and pectoral muscle for MTs measurement. RESULTS: Baseline levels of MTs differed in avian tissues. The gradient of MTs in control quails was pectoral muscle < liver < brain < kidney. Double and triple exposures induced higher levels of MTs. While increases of MTs were driven mainly by dietary exposure to cyanobacterial biomass and/or lead, Newcastle disease vaccination induced the least response. Induction of brain MTs was dominated by exposure to lead. Patterns of MTs responses were similar in the liver and pectoral muscle as well as in the kidney and brain. CONCLUSIONS: Understanding better responses of birds to oxidative stress induced by toxic and infectious stressors may have implications for avian conservation issues and disease risk assessment.

Blood coagulation times in the European brown hare (Lepus europaeus).

BACKGROUND: Many causes of mortality in the European brown hare, such as bacterial and viral infections, anticoagulant poisoning, and trauma, may result in hemorrhage. There are, however, no reference values concerning blood clotting in this species. OBJECTIVES: The aim of this study was to determine reference values for blood coagulation times and related parameters in healthy European brown hares. METHODS: Blood samples from 30 clinically healthy adult hares (15 males and 15 females) were obtained. Hares were physically restrained for blood collection from the cephalic vein into tubes containing citrate and EDTA. RESULTS: Mean +/- SD were obtained for thrombin time (TT) (13.97 +/- 1.37 seconds), prothrombin time (PT) (13.32 +/- 2.15 seconds), activated partial thromboplastin time (APTT) (16.73 +/- 1.86 seconds), fibrinogen concentration (2.98 +/- 1.06 g/L), and platelet count (355.28 +/- 128.73 x 10(9)/L). CONCLUSIONS: Reference values for blood coagulation times and other parameters associated with blood clotting will be useful in the laboratory evaluation of hemorrhage in the European brown hare.

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.

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.