Genomic analysis of Paenibacillus larvae isolates from the Czech Republic and the neighbouring regions of Slovakia.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybee larvae. In the Czech Republic, two large infested regions were recognised. This study aimed to analyse P. larvae strains occurring in the Czech Republic in the years 2016-2017 and to characterise the genetic structure of their population with the use of Enterobacterial Repetitive Intergenic Consensus genotyping (ERIC), multilocus sequence typing (MLST) and whole genome sequence (WGS) analysis. The results were complemented by the analysis of isolates collected in the year 2018 in areas of Slovakia located near the Czechia-Slovakia border. ERIC genotyping revealed that 78.9% of tested isolates belonged to the ERIC II genotype and 21.1% to ERIC I genotype. MLST showed six sequence types with ST10 and ST11 being the most frequent among isolates. Within six isolates we found discrepancies in correlations between MLST and ERIC genotypes. The use of MLST and WGS analysis of isolates revealed that each of the large infested geographic regions had its own dominating P. larvae strains. We assume that these strains represented primary sources of infection in the affected areas. In addition, the sporadic presence of strains identified by core genome analysis as genetically related was unveiled in geographically distant regions suggesting possible human-mediated transmission of AFB.

Sodium chloride treatment effects on rainbow trout suffering from proliferative kidney disease caused by Tetracapsuloides bryosalmonae.

The aim of the present study was to evaluate the effect of a long-term sodium chloride bath on rainbow trout Oncorhynchus mykiss naturally infected by Tetracapsuloides bryosalmonae. A total of 106 infected fish were divided into 2 groups. One group was left untreated and the other was treated with sodium chloride in increasing doses up a concentration of 0.8%. After 14 d, treatment was stopped and for a further 7 d the fish response to the sodium chloride bath was observed. Cumulative mortality was significantly lower in the treated group (19.2%) compared to the untreated group (31.5%) after 21 d. This corresponded to the lower but non-significant parasite intensity in kidney and spleen in the treated group after 14 d of treatment. However, lower prevalence of parasites in both tissues was recorded in the untreated group after 21 d of treatment, but a significant difference was observed only in spleen tissue. Furthermore, significant increases in leukocytes, hemoglobin, haematocrit, ferric reducing ability of plasma, and ceruloplasmin, and significant decreases in alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase activities were noticed in the treated group compared to the untreated group. In contrast, significant decreases in lysozyme concentration in the mucus and phagocyte oxidative burst in the blood were observed in the treated group. Histopathological examination revealed proliferative and reparative changes in parenchymatous tissues in the treated group. The 14- and 21-d salt bath used in rainbow trout with proliferative kidney disease was associated with a reduction in mortality and enhanced the reparative phase in the treated group.

Proliferative kidney disease in rainbow trout (Oncorhynchus mykiss) under intensive breeding conditions: Pathogenesis and haematological and immune parameters.

Proliferative kidney disease (PKD) is an endoparasitic disease of salmonid fish caused by Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea). This study presents a comprehensive view on PKD development in rainbow trout (Oncorhynchus mykiss) reared at an intensive fish breeding facility, with focus on mortality, pathology/histopathology, haematological findings and immune functions. Diseased and reference fish were sampled monthly and time course of natural infection was followed up from the onset of clinical signs (September 2014) to full recovery (January 2015). PKD- associated cumulative mortality was 30% with a peak value in October, while immunohistochemical testing indicated a continuous significant decrease in T. bryosalmonae numbers from September to December; with no parasites detected in January. During peak clinical infection, a significant decrease in red blood cell counts, haematocrit values, haemoglobin concentration, along with a reduction in lymphocytes and a significant phagocyte elevation corresponding with an increase in phagocyte oxidative burst were measured in comparison to control animals. Complement activity and total immunoglobulin plasma concentrations were also elevated, though only during the initial monitoring period (September). Individuals surviving PKD, recovered and were able to fully regenerate both renal structure and haematopoietic parameters to normal levels. Changes in the red blood cell parameters indicate anaemia and a decreased oxygen transportation capacity during the clinical disease phase. Together with an increased oxygen demand at higher temperatures and decreased oxygen solubility this could lead to decompensation and elevated mortality. The stimulation of immune parameters, and especially oxidative phagocytic burst, is likely to have had a strong effect on both, regeneration and elimination of the pathogenic agent.

Seasonal changes in immune parameters of rainbow trout (Oncorhynchus mykiss), brook trout (Salvelinus fontinalis) and brook trout × Arctic charr hybrids (Salvelinus fontinalis × Salvelinus alpinus alpinus).

Despite the high number of studies concerning seasonality of immune response in fish, information for some fish species is still scarce. Here, we assess seasonal changes in leukocyte counts and several immune parameters in three groups of farmed salmonids, i.e. brook trout (Salvelinus fontinalis), brook trout x Arctic charr hybrids (Salvelinus fontinalis x Salvelinus alpinus alpinus) and rainbow trout (Oncorhynchus mykiss) reared under the same conditions and fed with the same feed. Fish were sampled in five periods of the year (late April, early July, late August, early November and early February) and leukocyte counts, respiratory burst of blood phagocytes, lysozyme concentration in skin mucus and total complement activity were measured. Generalized linear models using fish body length as a continuous predictor and sampling period and fish species as categorical predictors, were significant for each of the parameters analysed. The highest seasonal variations in measured parameters were found in rainbow trout and lowest in hybrids. Our results confirm that measures of innate and adaptive immunity are strongly affected by season in all three groups of salmonids. The results will contribute to the improved assessment of immunocompetence in farmed fishes, essential for future sustainable development in aquaculture.

Degradation rate of praziquantel and fenbendazole in rainbow trout following oral administration.

OBJECTIVES: The aim of this study was to evaluate and compare the rate of degradation and elimination of praziquantel and fenbendazole antiparasitics following oral administration to salmonids. In addition, we determine whether the length of the legal withdrawal period is sufficient for complete elimination of antiparasitic residue from the body. The use of these drugs in fish is currently considered off-label and data on degradation are not available for rainbow trout. METHODS: The model species for this experiment was the rainbow trout (Oncorhynchus mykiss) and praziquantel and fenbendazole were chosen for experimental therapy. Both drugs were administered into the gastrointestinal tract using a stomach tube. Concentrations of fenbendazole and praziquantel were established through high performance liquid chromatography-tandem mass spectrometry. RESULTS: Our results show that concentrations of praziquantel and fenbendazole reach their maximum in the body within 24 hours of administration, with concentrations dropping sharply over the following 24 hours. With one exception, when trace amounts of both substances were found in blood plasma, the drugs were completely degraded and eliminated from the body by the end of the experiment (corresponding to 497.6 degree days). CONCLUSIONS: Praziquantel and fenbendazole both show a high rate of degradation and elimination from fish. As both substances were eliminated from the body within the required withdrawal period (i.e. within 500 degree days) they can be safely used based on current knowledge of their therapeutic effect for treating helminth infections.

Effect of arsenic and cyanobacterial co-exposure on pathological, haematological and immunological parameters of rainbow trout (Oncorhynchus mykiss).

OBJECTIVES: Under environmental conditions, fish are simultaneously exposed to multiple stressors. This study provides new knowledge on the effects of controlled exposure to multiple stressors, namely cyanobacterial biomass and food contaminated with arsenic. METHODS: Rainbow trout were divided into six groups of 25 fish and exposed to different contaminant combinations for 30 days: 1) control group, 2) cyanobacterial biomass, 3 & 4) two groups exposed to arsenic at concentrations of 5 mg.kg(-1) and 50 mg.kg(-1) fish feed, and 5 & 6) two groups exposed to cyanobacterial biomass and arsenic combined. We then evaluated pathological, haematological and immunological parameters at 10, 20 and 30 days after exposure. RESULTS: Marked gross pathological findings were present in groups exposed to arsenic and arsenic/cyanobacteria after 30 days. A strong decrease in haemoglobin concentration was observed in all experimental groups receiving arsenic after 10 days exposure. Total leukocyte count increased markedly in fish exposed to cyanobacterial biomass, and to higher arsenic concentrations by the end of the experiment. Neutrophils decreased significantly at the end of exposure. Similarly, exposure to cyanobacteria and/or arsenic led to suppression of opsonised zymosan particle-induced neutrophil respiratory bursts. CONCLUSIONS: Our results demonstrate that the effects of exposure to toxic cyanobacterial biomass and arsenic on fish are enhanced when the contaminants are combined. In particular, long-term exposure led to disturbances in the white blood-cell count. Modulation of phagocytosis, which is the first line of defence against invading pathogens, suggests that the combined action leads to a decreased ability to control infection.

Modulation of biochemical indices in common carp (Cyprinus carpio L.) under the influence of toxic cyanobacterial biomass in diet.

Cyanobacteria are producers of potent and environmentally abundant microcystins, representing an emerging global health issue. In the present study, we investigated the impact of cyanobacterial biomass on biochemical indices of common carp (Cyprinus carpio L., average weight of 246 ± 73 g) under laboratory conditions. The fish were fed a diet containing cyanobacterial biomass with microcystins in high concentration (0.4 mg/kg of fish weight and day) for 28 days. Statistical evaluation of the influence of the cyanobacterial biomass in food on the biochemical indices of the juvenile carp showed only minor differences. The activity of aspartate aminotransferase value and the urea concentration were significantly reduced compared to control group. The biochemical parameters of fish blood plasma significantly rose during the experiment in the control group as well as in the experimental group. This state was probably influenced by the environmental conditions and the fish diet. A significant rising value was established in calcium creatinine, total protein, phosphorus, lactate, urea and natrium. The present study demonstrates that the oral exposure of toxic cyanobacterial biomass has a minor influence on the biochemical indices of common carp and that the effect of other factors, e.g., nutrition is more visible.

Influence of arsenic and cyanobacteria co-exposure on plasmatic parameters of rainbow trout (Oncorhynchus mykiss W.).

OBJECTIVES: Fish can be exposed under environmental conditions to multiple stressors including natural toxins and environmental or feed contamination at the same time. This study brings new knowledge about the effects of controlled exposure to multiple stressors in fish. The aim of this study was to test the hypothesis that influence of cyanobacterial biomass and arsenic in feed can combine to enhance the effects on fish. METHODS: Rainbow trouts were sorted into six groups, each with 25 specimens: control group (fed with commercial feed), groups exposed to toxic cyanobacterial biomass (81 mg x kg(-1) MCs of feed), two groups exposed to arsenic (concentration of 5 mg x kg(-1), and 50 mg x kg(-1) of fish feed) and two groups exposed to combination of cyanobacterial biomass and arsenic in two concentrations mentioned above. The experiment lasted 30 days. During the experiment we evaluated the influence of co-exposure on plasmatic parameters mentioned above. Samples were collected on days 10, 20 and 30 of exposure. RESULTS: Biochemical analysis revealed a significant decrease in calcium (T20) and an increase in natrium (T10) and chlorides (T10) values in combined cyanobacterial and arsenic exposures. Our results showed a significant decrease in the values of magnesium after exposure to higher concentration of arsenic compared to control and feeding with addition of cyanobacterial biomass groups. The changes of other monitored plasmatic parameters were not significantly increased or decreased in comparison with controls. CONCLUSIONS: Our results confirmed the hypothesis that influence of toxic cyanobacterial biomass and a chemical agent represented by arsenic can combine to enhance the effects on fish. This work originally shows that while the single agents in sub-lethal doses do not cause changes in the plasmatic parameters, their co-exposure leads to the significantly decrease or increase of the electrolytes of rainbow trout.

Fish tapeworm Khawia sinensis: an indicator of environmental microcystins?

OBJECTIVES: Parasites have recently been recognized as accumulation indicators that take up and bio-concentrate substances from environmental pollution. Interestingly, helminths of fish are known to accumulate metals from the ambient environment and to contain several orders of magnitude higher concentrations than hosts. While the majority of reports mention inorganic toxin accumulation in parasites, studies concerning effects of organic pollution are infrequent and little is known about the potential of parasites to bio-accumulate microcystins. METHODS: The parasite-host system of tapeworm Khawia sinensis and common carp (Cyprinus carpio) was used to address this issue. Both the tapeworms and livers were dissected from experimental carps orally exposed to cyanobacterial biomass for 20 days. The total dose of microcystins amounted to 27 mg/kg of feed, i.e., 0.4 mg/kg of fish mass a day. Microcystin concentrations in tapeworms and carp liver tissues were measured using the LC-MS/MS method. RESULTS: Considering the three measured microcystin variants LR, YR and RR, only MC-RR was detected and its concentrations in tapeworms and carp liver tissue amounted to 5.78±3.78 ng/g and 2.11±0.74 ng/g fresh weight (p<0.05), respectively. CONCLUSIONS: Here we show accumulation of microcystin MC-RR in the tapeworm Khawia sinensis, a parasite of common carp (Cyprinus carpio). As this is the first report addressing this issue, further studies will be necessary to examine this specific parasite-host system.

In vivo effects of microcystins and complex cyanobacterial biomass on rats (Rattus norvegicus var. alba): changes in immunological and haematological parameters.

Toxic cyanobacteria represent a serious health and ecological problem in drinking and recreational waters worldwide. Some previous toxicological studies investigated effects of isolated microcystins on laboratory rodents including mice and rats. However, much less attention has been paid to more realistic exposure situations such as the effects of MCs accumulated in food. The objectives of the present study were to provide a simple model simulation of the food chain in order to evaluate impacts of microcystins (MCs) on rat immune and haematologicalparameters. Impacts of feeding experimental rats with a diet containing fish meat with and without microcystins and complex toxic biomass have been studied during a 28 day exposure. Red blood cell parameters (RBC counts, haematocrit values, MCH, MCV and MCHC) showed significant differences in experimental groups (p ≤ 0.05, p ≤ 0.01) in comparison with the control group. We also detected an immunomodulatory effect in the experimental groups. NK cells and γδ+ T lymphocytes were significantly increased in peripheral blood in the group exposed to isolated microcystin in the food. Significant change in the ratio of CD4+ and CD8+ cells (increase of CD4+ and a drop in CD8+) was found in the group with added cyanobacterial biomass with low concentration of MCs. The greatest changes in lymphoid organs were observed in the same groups. There was an increase of spleen subpopulations of γδ+ T lymphocytes as well as of IgM+ lymphocytes (B lymphocytes) and CD8+ T lymphocytes. Indeed, the modulation of CD4+ and CD8+ of peripheral lymphocytes was associated with similar changes in thymic lymphocytic subpopulations. In summary, food containing fish meat with considerable doses of microcystins (or toxic cyanobacterial biomass) induces significant changes in RBC parameters and influence preferably innate part of the immune system represented by NK cells and by gamma-delta T cells, which are known to play role as a bridge between adaptive and innate immune response.

Concentrations of microcystins in tissues of several fish species from freshwater reservoirs and ponds.

The aim of this study is to summarise the determination of concentrations of microcystins (MCs) in muscle and liver of freshwater fish species caught in stagnant waters of the Czech Republic. Within the years 2007-2009, 351 muscle samples and 291 liver samples of 16 freshwater fish species derived from four fishponds, and four water reservoirs were analysed. MCs were detected in 53 liver samples. The highest concentrations of microcystins were determined in liver samples of carnivorous fish species; 50.3 ng/g of fresh weight (FW) in perch (Perca fluviatilis) and 22.7 ng/g FW in pikeperch (Sander lucioperca). MCs in liver were detected in other five fish species; asp (Aspius aspius), pike (Esox lucius), common carp (Cyprinus carpio), grass carp (Ctenopharyngodon idella) and European eel (Anguilla anguilla). Concentrations of MCs in liver of nine fish species (European bream, whitefish, tench, silver carp, European catfish, roach, chub, crucian carp and rudd) were below the detection limit of 1.2-5.4 ng/g FW for different MC congeners. However, the concentrations of MCs in all muscle samples were below the detection limit. The assessment of MCs concentrations might be influenced by the detection method used. Due to the concentrations of MCs being below the detection limit in muscle samples of all fish species analysed, it seems that there might be a low potential threat for human health in case of fish muscle consumption.

Combined exposure of carps (Cyprinus carpio L.) to cyanobacterial biomass and white spot disease.

OBJECTIVES: Under environmental conditions, fish can be exposed to multiple stressors including natural toxins and infectious agents at the same time. This study brings new knowledge on the effects of controlled exposure to multiple stressors in fish. The aim of this study was to test the hypothesis that influence of cyanobacterial biomass and an infection agent represented by the white spot disease can combine to enhance the effects on fish. METHODS: Common carps were divided into four groups, each with 40 specimens for 20 days: control group, cyanobacterial biomass exposed group, Ichthyophthirius multifiliis-infected fish (Ich) and cyanobacterial biomass-exposed fish + Ichthyophthirius multifiliis-infected fish. During the experiment we evaluated the clinical signs, mortality, selected haematological parameters, immune parameters and toxin accumulation. RESULTS: There was no mortality in control fish and cyanobacterial biomass-exposed fish. One specimen died in Ichthyophthirius multifiliis-infected fish and the combined exposure resulted in the death of 13 specimens. The whole leukocyte counts (WBC) of the control group did not show any significant differences. Cyanobacteria alone caused a significant increase of the WBC on day 13 (p≤0.05) and on day 20 (p≤0.01). Also, I. multifiliis caused a significant elevation of WBC (p≤0.01) on day 20. Co-exposition resulted in WBC increased on day 13 and decrease on day 20, but the changes were not significant. It is evident from the differential leukocyte counts that while the increase of WBC in the group exposed to cyanobacteria was caused by elevation of lymphocytes, the increase in the group infected by I. multifiliis was due to the increase of myeloid cells. It well corresponds with the integral of chemiluminescence in the group infected by I. multifiliis, which is significantly elevated on day 20 in comparison with all other groups. CONCLUSIONS: We can confirm additive action of different agents on the immune system of fish. While single agents seemed to stimulate the immune response, the combination of both caused immunosuppression.

Influence of cyanobacteria on water activity and dry matter of muscles in the common carp (Cyprinus carpio L.) and rainbow trout (Oncorhynchus mykiss W.).

OBJECTIVES: Cyanobacteria are studied from the viewpoint of the issue of risks to water supply, agriculture and recreational activities for a long time. Cyanobacteria produce a wide range of substances which can be toxic and can influence the safety and quality of fish products. The aim of this study was to determine whether the diet with the content of cyanobacteria can affect the water activity and the dry matter of fish muscle and whether this diet can contribute significantly to the shelf life of fish muscles. METHODS: Common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) were used in this study. Both fish species were divided into two groups. The first group of fish was fed with feed with cyanobacteria (3% of dry matter), the second group with feed without cyanobacteria. The water activity and the dry matter were monitored immediately after sampling of the fish muscle on day 7, 14 and 21 (carp) or on day 10, 20 and 30 (rainbow trout) and seven days after every sampling and cold storage (6-8 °C). RESULTS: Feed with the content of cyanobacteria significantly decreased the water activity in muscles of both fish species on day 21 (in carp) and on day 30 (in rainbow trout). The dry matter of fish muscle significantly increased on day 7 and 21 (in carp) and on day 10 and 30, but decreased on day 20 (in rainbow trout). The cold storage significantly influenced the dry matter only. While the dry matter was increased in the common carp (7 days of cold storage after sampling on days 14 and 21), the dry matter decreased in the rainbow trout (7 days of cold storage after sampling on day 10). CONCLUSIONS: The decrease of water activity was found only after longer exposure in the both exposed fish species. The dry matter was influenced far greater and was mostly increased in the both exposed fish species.

Accumulation of microcystins in Nile tilapia, Oreochromis niloticus L., and effects of a complex cyanobacterial bloom on the dietetic quality of muscles.

The aim of this study was to investigate the kinetics of accumulation and elimination of microcystins in the tissues of Nile tilapia (Oreochromis niloticus) and to evaluate the effect of cyanobacterial exposure on fish muscle quality (levels of total fat and ash, protein, dry matter and the composition of saturated, monounsaturated and polyunsaturated fatty acids). Fish were exposed for 28 days to a natural cyanobacterial bloom with total microcystin concentration around 1,200 µg g⁻¹ biomass dry weight. The hepatopancreas accumulated microcystins up to 350 ng g⁻¹ fresh weight, but concentrations in muscle were generally below the detection limit (2 ng g⁻¹ fresh weight). Following the exposure, fish were moved to the clean water, but only minor microcystin removal from the hepatopancreas was observed during a 4 week depuration period. Exposures of tilapia to the complex cyanobacterial bloom had only minor and temporary impacts on the investigated parameters of dietetic quality.

Microcystin kinetics (bioaccumulation and elimination) and biochemical responses in common carp (Cyprinus carpio) and silver carp (Hypophthalmichthys molitrix) exposed to toxic cyanobacterial blooms.

Two species of common edible fish, common carp (Cyprinus carpio) and silver carp (Hypophthalmichthys molitrix), were exposed to a Microcystis spp.-dominated natural cyanobacterial water bloom for two months (concentrations of cyanobacterial toxin microcystin, 182-539 microg/g biomass dry wt). Toxins accumulated up to 1.4 to 29 ng/g fresh weight and 3.3 to 19 ng/g in the muscle of silver carp and common carp, respectively, as determined by enzyme-linked immunosorbent immunoassay. Concentrations an order of magnitude higher were detected in hepatopancreas (up to 226 ng/g in silver carp), with a peak after the initial four weeks. Calculated bioconcentration factors ranged from 0.6 to 1.7 for muscle and from 7.3 to 13.3 for hepatopancreas. Microcystins were completely eliminated within one to two weeks from both muscle and hepatopancreas after the transfer of fish with accumulated toxins to clean water. Mean estimated elimination half-lives ranged from 0.7 d in silver carp muscle to 8.4 d in common carp liver. The present study also showed significant modulations of several biochemical markers in hepatopancreas of fish exposed to cyanobacteria. Levels of glutathione and catalytic activities of glutathione S-transferase and glutathione reductase were induced in both species, indicating oxidative stress and enhanced detoxification processes. Calculation of hazard indexes using conservative U.S. Environmental Protection Agency methodology indicated rather low risks of microcystins accumulated in edible fish, but several uncertainties should be explored.

Effect of different cyanobacterial biomasses and their fractions with variable microcystin content on embryonal development of carp (Cyprinus carpio L.).

While numerous studies focused on the effects of microcystins, the role of other components of complex cyanobacterial water blooms in toxicity is poorly understood. In this study we have evaluated effects of various fractions of cyanobacterial biomass with different composition and microcystin content on embryolarval development of carp (Cyprinus carpio). The following samples (fractions) of four natural water blooms were prepared and tested: complex cyanobacterial biomass, crude aqueous extract of biomass, cellular pellet remaining from aqueous extract, permeate (i.e. microcystin-free fraction prepared during C-18 solid-phase extraction; SPE), and eluate (i.e. fraction prepared by SPE containing mostly microcystins). Complex biomass and the crude aqueous extract (regardless of microcystin content and/or microcystin variants present) in the sample were the most toxic. On the other hand, eluate fractions of all samples containing microcystins in concentrations 8-255 microgL(-1) induced no or only weak toxic effects. Exposures of fish to permeate fractions (with removed microcystins) of two samples dominated by Aphanizomenon sp. and Planktothrix sp. resulted in significant mortality, while other two samples dominated by Microcystis spp. induced minor effects. We have also observed significant inhibition of glutathione S-transferases (GST) at most fractions of the Aphanizomenon sp. and Planktothrix sp. dominated samples. Our data indicate that cyanobacterial water blooms as well complex biomass extracts induce significant embryolarval toxicity in common carp. However, these effects were independent of microcystin content, and the most pronounced effects were observed with the non-Microcystis dominated samples. Therefore, a critical examination of microcystin role in overall ecotoxicology of complex cyanobacterial blooms is needed.