Polystyrene microparticles can affect the health status of freshwater fish - Threat of oral microplastics intake.

Plastic waste pollution is considered one of the biggest problems facing our planet. The production and use of these materials has led to huge amounts of plastic waste entering the aquatic environment and affecting aquatic life. In our experiment, the effect of polystyrene microparticles (PS-MPs; 52.5 ± 11.5 µm) on individual juvenile rainbow trout (Oncorhynchus mykiss) was tested at three different dietary concentrations of 0.5, 2 and 5 % for six weeks. At the end of the experiment, various health parameters of exposed organisms were compared with the control group. The haematological profile revealed an immune response by a decrease in lymphocyte count with a concurrent increase in the number of neutrophil segments at the highest concentration of PS-MPs (5 %). Biochemical analysis showed significant reductions in plasma ammonia in all tested groups, which may be related to liver and gill damage, as determined by histopathological examination and analysis of inflammatory cytokines expression. In addition, liver damage can also cause a significant decrease in the plasma protein ceruloplasmin, which is synthesized in the liver. PS-MPs disrupted the antioxidant balance in the caudal kidney, gill and liver, with significant changes observed only at the highest concentration. In summary, PS-MPs negatively affect the health status of freshwater fish and represent a huge burden on aquatic ecosystems.

Non-steroidal anti-inflammatory drugs caused an outbreak of inflammation and oxidative stress with changes in the gut microbiota in rainbow trout (Oncorhynchus mykiss).

One of the main contributors to pharmaceutical pollution of surface waters are non-steroidal anti-inflammatory drugs (NSAIDs) that contaminate the food chain and affect non-target water species. As there are not many studies focusing on toxic effects of NSAIDs on freshwater fish species and specially effects after dietary exposure, we selected rainbow trout (Oncorhynchus mykiss) as the ideal model to examine the impact of two NSAIDs - diclofenac (DCF) and ibuprofen (IBP). The aim of our study was to test toxicity of environmentally relevant concentrations of these drugs together with exposure doses of 100× higher, including their mixture; and to deepen knowledge about the mechanism of toxicity of these drugs. This study revealed kidneys as the most affected organ with hyalinosis, an increase in oxidative stress markers, and changes in gene expression of heat shock protein 70 to be signs of renal toxicity. Furthermore, hepatotoxicity was confirmed by histopathological analysis (i.e. dystrophy, congestion, and inflammatory cell increase), change in biochemical markers, increase in heat shock protein 70 mRNA, and by oxidative stress analysis. The gills were locally deformed and showed signs of inflammatory processes and necrotic areas. Given the increase in oxidative stress markers and heat shock protein 70 mRNA, severe impairment of oxygen transport may be one of the toxic pathways of NSAIDs. Regarding the microbiota, an overgrowth of Gram-positive species was detected; in particular, significant dysbiosis in the Fusobacteria/Firmicutes ratio was observed. In conclusion, the changes observed after dietary exposure to NSAIDs can influence the organism homeostasis, induce ROS production, potentiate inflammations, and cause gut dysbiosis. Even the environmentally relevant concentration of NSAIDs pose a risk to the aquatic ecosystem as it changed O. mykiss health parameters and we assume that the toxicity of NSAIDs manifests itself at the level of mitochondria and proteins.

Do foodborne polyethylene microparticles affect the health of rainbow trout (Oncorhynchus mykiss)?

Due to the fact that plastic pollution is a global environmental problem of modern age, studies on the impact of these synthetic materials on aquatic, and especially fish organisms, are an important part of the ecosystem and human nutrition. In our study, the toxicity of pristine polyethylene (PE) microparticles (approx. 50 µm) on rainbow trout (Oncorhynchus mykiss) was tested in three different dietary concentrations - 0.5%, 2% and 5%. After six weeks of exposure, various health indices were evaluated. Electron microscopy of the intestine revealed the disintegration of PE particles to <5 µm in size, and thus we concluded that microplastics are able to reach tissues. The haematological profile revealed changes in total red blood cells count and haematocrit (5% PE) which could be associated with spleen congestion observed histologically. The marker of lipid peroxidation was increased in gills suggesting the disruption of balance in antioxidant enzymes capacity and histopathological imaging revealed inflammation in higher PE concentrations. In addition, ammonia was decreased and calcium elevated in biochemical profile, confirming the gill damage. Electron microscopy of the gills showed lesions of lamellae and visible rings around the mucinous cell opening indicating their higher activity. Another injured was the liver tissue, as confirmed by hepatodystrophies and increased expression of pro-inflammatory genes in 2% PE. Impaired innate immunity was confirmed by an increased presence of mucinous cells and a decrease in leukocytes. Kidney damage manifested itself by higher expression of pro-inflammatory cytokines and histopathology. The damage in gills, liver and kidney together correlated with the increased antioxidant capacity of plasma. In conclusion, PE microparticles are able to affect health indices of O. mykiss. The potential problem for aquatic ecosystems and even human consumption should be considered.

Could the musk compound tonalide affect physiological functions and act as an endocrine disruptor in rainbow trout?

In the present study, the effect of polycyclic musk compound tonalide (AHTN) in two concentrations was studied in male rainbow trout (Oncorhynchus mykiss, Walbaum 1792). A feeding trial was conducted with AHTN incorporated into feed granules. One concentration was environmentally relevant (854 µg/kg); the second one was 10× higher (8699 µg/kg). The fish were fed twice a day with the amount of feed at 1 % of their body weight. After an acclimatization period, the experimental phase in duration of six weeks followed. At the end of the experiment, fish were sampled and the biometrical data were recorded. Subsequently, hematological and biochemical tests, histopathological examination, analysis of oxidative stress markers and evaluation of endocrine disruption using plasma vitellogenin were performed. In conclusion, an increase of hematocrit for both AHTN concentrations was found, but no significant changes were observed in biochemical profile. Moreover, AHTN caused lipid peroxidation in caudal kidney tissue, which was confirmed by histopathological images. The long-lasting AHTN exposure could thus be harmful for maintaining homeostasis in the rainbow trout organism. However, the vitellogenin concentration seemed not to be affected by AHTN.

Factors Affecting Antioxidant Response in Fish from a Long-term Mercury-Contaminated Reservoir.

The objective of this work was to evaluate antioxidant defence and oxidative damage in organs (liver, gills, kidney, and brain) of five fish species (Aspius aspius, Esox lucius, Sander lucioperca, Abramis brama, Rutilus rutilus) from the long-term mercury-contaminated Skalka Reservoir in the Czech Republic. Special emphasis was placed on a comprehensive assessment of the factors that may affect the antioxidant response to mercury in fish. Antioxidant enzymes (glutathione reductase, glutathione peroxidase, and glutathione-S-transferase) did not significantly respond to mercury contamination. Levels of the analysed enzymes and oxidative damage to lipids were predominantly determined by a separate organ factor or species factor, or by the combination of both (p < 0.001). Levels of total glutathione and the reduced/oxidized glutathione ratio were influenced by mercury contamination in combination with their specific organ distribution (p < 0.001). Our results suggest that species and type of organ alone or in combination are more important factors than chronic exposure to mercury contamination with respect to effects on antioxidant defence in fish under field conditions. Our findings suggest that the main antioxidant defensive mechanism in fish from the studied long-term mercury contaminated site was the inter-tissue distribution of glutathione.

Neopterin and biopterin as biomarkers of immune system activity associated with crating in broiler chickens.

Neopterin and biopterin belong to a group of unconjugated pterin derivates. These biomolecules are present in many animal species and perform several functions. Pterin concentrations may provide additional information on the effect of stress on immune system activity. This study focused on an investigation of the effect of crating on plasma concentrations of neopterin and biopterin in broilers. The effects of 2 crating periods (2 and 4 h) were monitored in Hubbard broilers (n = 90) aged 42 d. After a given crating period, randomly selected chickens from each group were sampled immediately and the remaining chickens were sampled after 24 h. Plasma corticosterone increased (P < 0.001) immediately after 2 and 4 h crating, but no difference between the crated and the control noncrated broilers was found 24 h later. Immediately after crating, neopterin in 2- and 4-h broilers did not differ from the control, but 24 h later a decrease (P = 0.011) in plasma neopterin was found in 4-h broilers compared with the control. Simultaneously, 24 h after crating, neopterin levels in 2- and 4-h broilers decreased (P < 0.001) in comparison with the levels immediately after crating. Plasma biopterin was higher (P < 0.001) in 4-h broilers than in the control immediately after the crating. A time of sampling effect (P = 0.016) was found for the heterophil-to-lymphocyte ratio, with heterophil-to-lymphocyte ratio higher 24 h after crating in comparison with its level immediately after the crating. This study shows that crating may significantly affect the immune system of broiler chickens. This is corroborated by the increase in plasma biopterin concentrations in broilers immediately after crating and the decrease in plasma neopterin concentrations in broilers 24 h after crating. The correlations were found for widely used indicators of acute and chronic stress in birds [i.e., plasma corticosterone concentrations (biopterin) and the heterophil-to-lymphocyte ratio (neopterin), respectively].

Recovery ability of common carp (Cyprinus carpio) after a short-term exposure to terbuthylazine.

Effects of a high terbuthylazine concentration (3.3 mg/1) on Cyprinus carpio were studied using a commercial herbicide formulation Click 500 SC (terbuthylazine 500 g/l). The fish were exposed to the pesticide for 24 h and allowed to recover for 6 days. Biometric parameters, plasma biochemical parameters and biomarkers of oxidative stress as well as histopathological changes in selected tissues were assessed on day 1 and 7. After a 24-h exposure, there were significant alterations found in the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as well as in the plasma concentrations of glucose, natrium, chlorides, calcium and phosphorus. Hepatosomatic index, plasma albumin and lactate reflected the treatment with a delay. Ion levels and ALT were found to be restored after a 6-day recovery period, which was too short for AST activity and glucose to diminish to the control levels. The histopathological examination revealed disorders in the gills of the exposed fish, however, the changes were not detected after a 6-day recovery period. The study shows high regeneration potential of the fish.

Alkylphenol ethoxylates and alkylphenols--update information on occurrence, fate and toxicity in aquatic environment.

Alkylphenols and their precursors, alkylphenol etoxylates, are a group of manmade chemicals used mainly as surfactants in domestic and industrial applications worldwide. It has been well established that they have endocrine disruption activity, hepatotoxic, genotoxic and other negative effects on animal and human health. In spite of the effort to reduce their use, they persist in the environment not only in industrial but also in remote regions, and were detected in the variety of natural matrices including air, water, soil as well as food products, and human blood and urine worldwide. This article summarizes their occurrence, fate in natural conditions, and toxicity including mode of action. A subject of our concern was the aquatic environment as the most important reservoir and target of their deleterious impact.

Effects of subchronic exposure to atrazine on zebrafish (Danio rerio).

The aim of this study was to investigate the effects of subchronic exposure to atrazine on fish growth and the development of histopathological changes in selected organs (gill, kidney, liver) in Danio rerio. Juvenile growth tests were performed on D. rerio according to OECD method No. 215. For 28 days, fish at an initial age of 30 days were exposed to the environmental atrazine concentration commonly detected in Czech rivers (0.3 microg/L) and a range of sublethal concentrations of atrazine (3.0, 30.0 and 90.0 microg/L). The results showed decreasing growth rates and morphological changes in the liver (dystrophic lesions of hepatocytes) at 90.0 microg/L of atrazine. The environmental concentration of atrazine in Czech rivers did not have any effect on fish growth and development of histopathological changes in D. rerio. The value of NOEC was 30.0 microg/L and the value of LOEC was 90.0 microg/L.

Neopterin and biopterin as biomarkers of immune system activation associated with castration in piglets.

Recent reports have shown that stressful situations may affect the production of unconjugated pterins (neopterin and biopterin). The aim of the study was to investigate the effect of castration on neopterin and biopterin plasma concentrations in piglets, using 2 groups of 12 piglets allocated to castrated and uncastrated (control) groups. Pterin concentrations were determined by HPLC with fluorescence detection. Blood samples were also analzyed for leukocyte profiles and plasma cortisol concentrations. A time × treatment interaction (P < 0.05) was detected for neopterin concentrations, such that neopterin was greater (P < 0.01) at 1 h after surgery in castrated piglets compared with precastration concentrations, and neopterin was greater (P = 0.05) in castrated than in control piglets at 1 h. Castration had no effect on biopterin concentration (P > 0.1). Time effects (P < 0.05) for neutrophil and lymphocyte concentrations and neutrophil-to-lymphocyte ratios were found. A time × treatment interaction (P < 0.01) was detected for plasma cortisol concentrations, such that cortisol was greater (P < 0.01) at 1 and 24 h after surgery in castrated piglets compared with precastration concentrations and was greater (P < 0.01) in castrated than in control piglets at 1 and 24 h. This study showed that castration activated the immune system of piglets as demonstrated by an increase in plasma neopterin concentrations.

Changes in selected biochemical indices related to transport of broilers to slaughterhouse under different ambient temperatures.

The effect of transport distance on selected biochemical parameters (corticosterone, uric acid, triglycerides, total protein, glucose, and lactate) under various ambient temperatures was monitored in a group of unsexed Ross 308 broilers aged 42 d. Broilers were transported to the slaughterhouse over 3 different travel distances (10, 70, and 130 km). They were sampled before and after each transportation in 3 various periods with different ambient temperatures (-5 to +5°C, 10 to 20°C, and 25 to 35°C), which approximately correspond to temperature conditions during transport in individual seasons of the year (winter, fall, summer). The changes in biochemical parameters were specific in their dependence on the travel distance and the ambient temperature under which the broilers were transported. The highest corticosterone concentration was found in broilers before transport (i.e., immediately after catching, crating, and loading) at all ambient temperatures. The concentration of corticosterone was higher at winter temperatures than at summer and fall temperatures. Triglycerides decreased with travel distance, although this effect was detected under summer temperatures only. The concentration of total protein was higher only after 10 km of transport and then it decreased with travel distance at all monitored ambient temperatures. A highly significant decrease (P < 0.01) in the glucose level of broilers was observed after 130 km of transport when compared with broilers before transport at fall and winter temperatures. The effect of travel distance on lactate concentrations was the same at all monitored ambient temperatures, with the lactate level decreasing with travel distance. The results obtained indicate that pretransport handling procedures (catching, crating, and loading) may be more stressful for broilers than the transport itself. To improve broiler meat quality, it is necessary to meet the need for broilers to recover before being slaughtered. With regard to different seasons of the year, we can assume that transport under conditions of low ambient temperatures in winter represents a more stressful event than transport during fall and summer.

Effects of subchronic nitrite exposure on rainbow trout (Oncorhynchus mykiss).

Subchronic toxicity of nitrite in rainbow trout (Oncorhynchus mykiss; mean mass ± S.D., 18.9 ± 1.3g) was assessed in a 28-day trial. The influence of nitrite on fish mortality, growth rate, haematology, blood biochemistry, and gill histology was observed. Survival was not affected by exposures up to 1 mg l(-1) NO(2)(-) (at 10 mg l(-1) Cl(-)). On the basis of growth rate inhibition data, the values of NOEC (28 d LC(0)) and LOEC (28 d LC(10)) were estimated at 0.01 and 0.2 mg l(-1) NO(2)(-), respectively. At 0.01 mg l(-1) NO(2)(-) (the lowest concentration tested), there was segmental hyperplasia of the respiratory epithelium of secondary lamellae and elevated glucose and decreased potassium. Elevated nitrite concentrations were found in blood plasma of fish exposed to concentrations of 1.0 mg l(-1) NO(2)(-) and higher, and in muscle tissue at the highest concentration 3.0 mg l(-1) NO(2)(-). Plasma and muscle nitrite levels were lower than those in the ambient water in all experimental groups.

Stress in broilers resulting from shackling.

The aim of this study was to assess stress response of broilers to different periods of shackling. Stress effects of shackling were monitored in a group of male Ross 308 broilers (total number: 400) aged 42 d. Three shackling treatments were used in our experiment: shackling of broilers for 30 s (group T30), 60 s (group T60), and 120 s (group T120). Corticosterone plasma concentration was elevated in T60 broilers (P<0.05) and in T120 birds (P<0.01); glucose plasma concentration was increased (P<0.05) in both T60 and T120 broilers when compared with nonshackled control. Lactate concentrations increased in T30 birds (P<0.05) and in both T60 and T120 birds (P<0.01). Furthermore, T120 broilers exhibited an increase (P<0.01) in heterophil counts and heterophil:lymphocyte ratio. Duration of tonic immobility was increased (P<0.05) in T60 and T120 broilers. Number of attempts to induce tonic immobility decreased (P<0.01) in all test groups (T30, T60, T120). Duration of shackling period was positively correlated (P<0.001) with corticosterone, glucose and lactate level, tonic immobility duration, and heterophil:lymphocyte ratio. The number of inductions was negatively correlated (P<0.001) with duration of the shackling period. According to the results of our study, the act of shackling is a considerable traumatic procedure for broilers, and its stress effect is markedly dependent on duration of shackling period that the broiler chickens experience. It follows from our study that the optimal shackling period should be less than 60 s.

Four years of monitoring antibiotic resistance in microorganisms from bacteremic patients.

From the second semester of 2002 to the end of the first semester of 2005, a total of 2544 bacterial strains were isolated from the blood stream of patients with clinical sepsis and bacteremia hospitalized in six University Hospitals in the Slovak Republic. Almost 30% of strains were coagulase-negative staphylococci (CONS), about 14% were Staphylococcus aureus and, of the Gram-negative bacteria, up to 9% were Klebsiella pneumoniae. All CONS, S. aureus and Enterococcus spp. strains were found to be still susceptible to vancomycin, but the resistance of CONS and/or S. aureus to macrolides and fluoroquinolones dramatically increased during the period of this study. Among Gram-negative bacteria, increasing levels of resistance to higher generation cephalosporins, to fluoroquinolones resistance in Pseudomonas aeruginosa and Acinetobacter spp. to meropenem was recorded, which is alarming. The results were periodically submitted to cooperating hospitals with proposals for rationalizing the prophylactic and general use of indicated antibiotics as well as for improving hospital hygiene measures and anti-epidemic practices.

Monitoring of antibiotic resistance in bacterial isolates from bacteremic patients.

The aim of the study was to monitor the prevalence of pathogens and development of resistance in bacteria isolated from bacteremic patients. Five University Clinics and/or Regional Hospitals in the Slovak Republic participated in the study and a total of 421 isolates were collected in the second half of the year 2002. The most prevalent organisms were coagulase-negative staphylococci (CONS) (19%), Staphylococcus aureus (18.3%), among Gram-negative bacteria Escherichia coli (13.3%), Klebsiella pneumoniae (11.4%) and Pseudomonas aeruginosa (7.8%) followed by enterococci, Acinetobacter baumannii and Enterobacter sp. All CONS and S. aureus were susceptible to vancomycin; resistance to oxacillin was observed for 55% of the CONS and only for 4% of S. aureus isolates. A higher prevalence of resistance to erythromycin, clindamycin, gentamicin and ofloxacin was found in CONS in comparison to S. aureus. Enterococcus sp. isolates were fully susceptible to vancomycin and teicoplanin. Gentamicin, amoxicillin/clavulanate, third generation cephalosporins and ciprofloxacin showed good activity against E. coli. Although 17% of K. pneumoniae isolates were resistant to ciprofloxacin, it was the most effective drug against K. pneumoniae; the prevalence of resistance to other antibiotics was rather higher. Gentamicin and ciprofloxacin were the most active against Enterobacter sp. isolates and ceftazidime and meropenem against P. aeruginosa.

First occurrence of transferable extended-spectrum beta-lactamase hydrolyzing cefoperazone in multiresistant nosocomial strains of Klebsiella pneumoniae from two hospitals in Czech and Slovak Republics.

The occurrence of positive synergy between antibiotic discs of amoxicillin/clavulanate and cefoperazone was registered in two Klebsiella pneumoniae strains, isolated from hospitals in Czech and Slovak Republic, indicating the presence of genes coding for an extended-spectrum beta-lactamase active also against cefoperazone, a broad-spectrum cephalosporin. Sulbactam inhibited the hydrolysis of cefoperazone by cell-free lysates of these strains which substantiates its use in combination with cefoperazone. Resistance to cephalothin, cefotaxime, ceftazidime, cefoperazone, cefepime and aztreonam was transferred from K. pneumoniae isolates to Escherichia coli K-12 3110 and to Proteus mirabilis P-38 recipient strains.

[New aspects of antibiotic resistance and possibilities of its prevention]. / Novsie aspekty rezistencie na antibiotiká a niektoré moznosti jej limitácie.

New phenomena of the antibiotic resistance in bacteria have recently appeared. The may hold present explosive development of resistance and prevent its transferability from multiple drug resistant bacteria to still sensitive ones. They may prevent the production of so-called extended-spectrum beta-lactamases (ESBLs) among Enterobacteriaceae producing resistance virtually to all penicillins and cephalosporins with exception of those antibiotics potentiated by clavulanic acid or sulbactam, the resistance to vancomycin in enterococci and staphylococce, and the resistance of Stenotrophomonas maltophilia. Factors participating on the development of resistance include: a) transferability of resistance genes among bacteria which explosively change susceptible strains to resistant ones, b) dosage and types of antibiotics which cause the selection pressure to certain species of bacteria, c) level of organization and strict adherence to hygienic and anti-epidemic regimen starting with the entry of patients into the hospital. Analyses are necessary to check whether the patient brings resistant bacteria with a transferable resistance (with ESBLs) into the hospital. Preventive measures would be strictly applied to stop the clonal spread of resistant strains among the patients and/or hospital environment, which occurs if these strains have such opportunity. Last, but not least to be considered is the dosage, composition and rationality of administration of antibacterials, mainly in post-operative prophylaxis in intensive care units, in so-called empirical usage, etc. At the same time, it would be highly unethical to hesitate with application of antibacterials to patients when it is justified, necessary and rational. Hospital antibiotics policy should rationally decide between these alternatives in each application of antibiotics or their combinations.