Can the examination of different types of hive samples be a non-invasive method for detection and quantification of viruses in honey bee (Apis mellifera L.) colonies?

Introduction: Honey bee viruses have been shown to negatively affect the vigour and longevity of European honey bees (Apis mellifera L). In the present work, beehive materials were tested for their potential to serve as non-invasive samples for honey bee virus detection. Material and Methods: Honey, pollen, hive debris, hive grid smears and forager honey bees were collected from 24 hives at four locations in the Czech Republic. Deformed wing virus (DWV), acute bee paralysis virus (ABPV), sacbrood virus (SBV) and black queen cell virus (BQCV) were detected using a reverse transcription PCR (RT-PCR) and real-time quantitative RT-PCR and the results for bees and alternative materials compared. Results: All forager bee samples contained DWV, BQCV and SBV and 54.2% had ABPV. When comparing beehive materials to bees, the most promising results were obtained from honey and pollen samples, with BQCV and SBV detected in all honey samples and ABPV in 12.5%. Detection of SBV was achieved in 91.6% of pollen samples, detection of BQCV in 87.5% and detection of DWW in 75%. The results for debris and smears were less consistent with the viral profile of the forager samples. Conclusion: The best candidate materials for honey bee virus detection in a non-invasive technique are honey and pollen.

Nephrocalcinosis in farmed salmonids: diagnostic challenges associated with low performance and sporadic mortality.

Disease conditions that involve multiple predisposing or contributing factors, or manifest as low performance and/or low-level mortality, can pose a diagnostic challenge that requires an interdisciplinary approach. Reaching a diagnosis may also be limited by a lack of available clinical profile parameter reference ranges to discriminate healthy fish from those affected by specific disease conditions. Here, we describe our experience investigating poorly performing rainbow trout (Oncorhynchus mykiss) in an intensive recirculation aquaculture, where reaching a final diagnosis of nephrocalcinosis was not as straightforward as one would wish. To list the issues making the diagnosis difficult, it was necessary to consider the creeping onset of the problem. Further diagnostic steps needed to ensure success included obtaining comparative data for fish blood profiles and water quality from both test and control aquacultural systems, excluding infections with salmonid pathogenic agents and evaluating necropsy findings. Major events in the pathophysiology of nephrocalcinosis could be reconstructed as follows: aquatic environment hyperoxia and hypercapnia → blood hypercapnia → blood acid-base perturbation (respiratory acidosis) → metabolic compensation (blood bicarbonate elevation and kidney phosphate excretion) → a rise in blood pH → calcium phosphate precipitation and deposition in tissues. This case highlights the need to consider the interplay between water quality and fish health when diagnosing fish diseases and reaching causal diagnoses.

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

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

Carp edema virus infection associated gill pathobiome: A case report.

Understanding disease aetiology and pathologic mechanisms is essential for fish health evaluation. Carp edema virus (CEV) is the causative agent of a disease (CEVD) responsible for high mortality rates in both wild and cultured common carp Cyprinus carpio. Inspection of two carp specimens from a pond with high fish mortality revealed CEV infection in both the host and its ectoparasite (Argulus foliaceus). In addition to flavobacteria, well known to be associated with gill lesions, we found that free-living eukaryotes (amoebae and ciliates) and a temporary parasite (Ichthyobodo spp.) colonizing the gills may also contribute to alterations in gill structure and/or function, either directly, through firm (Ichthyobodo) or weak (amoebae) attachment of trophozoites to the gill epithelium, or indirectly, through carriage of pathogenic bacteria. Bacterial assemblages rich in families and genera, with predominance of Cetobacterium spp. in low-intensity alteration of the gill tissue and of Flavobacterium spp. in gills with extensive necrotic lesions, were detected in gills and within the cytoplasm of associated amoebae using high-throughput sequencing. Quantitative PCR indicated F. swingsii as the prevailing flavobacterial species within amoebae from less affected gills and F. psychrophilum within amoebae from extensively affected gills. This case study suggests that eukaryotic organisms as part of the gill pathobiome may also contribute to irreversible gill lesions seen in CEVD. Emphasizing the complexity of mutual relationships between bacterial assemblages and eukaryotic co-pathogens, further studies regarding factors that trigger pathology and influence severity in the CEV-positive carp are needed.

Low-level pathogen transmission from wild to farmed salmonids in a flow-through fish farm.

While the potential effects of pathogens spread from farmed fish to wild populations have frequently been studied, evidence for the transmission of parasites from wild to farmed fish is scarce. In the present study, we evaluated natural bacterial and parasitic infections in brown trout (Salmo trutta m. fario) collected from the Cerná Opava river (Czech Republic) as a potential source of infections for rainbow trout (Oncorhynchus mykiss) reared in a flow-through farm system fed by the same river. The prevalence of bacterial and protozoan infections in farmed fish was comparable, or higher, than for riverine fish. Despite this, none of the infected farmed fish showed any signs of severe diseases. Substantial differences in metazoan parasite infections were observed between wild and farmed fish regarding monogeneans, adult trematodes, nematodes, the myxozoan Tetracapsuloides bryosalmonae found in riverine fish only, and larval eye-fluke trematodes sporadically found in farmed fish. The different distribution of metazoan parasites between brown and rainbow trout most probably reflects the availability of infected intermediate hosts in the two habitats. Despite the river being the main water source for the farm, there was no significant threat of parasite infection to the farmed fish from naturally infected riverine fish.

Cyanobacteria Microcystis aeruginosa Contributes to the Severity of Fish Diseases: A Study on Spring Viraemia of Carp.

Fish are exposed to numerous stressors in the environment including pollution, bacterial and viral agents, and toxic substances. Our study with common carps leveraged an integrated approach (i.e., histology, biochemical and hematological measurements, and analytical chemistry) to understand how cyanobacteria interfere with the impact of a model viral agent, Carp sprivivirus (SVCV), on fish. In addition to the specific effects of a single stressor (SVCV or cyanobacteria), the combination of both stressors worsens markers related to the immune system and liver health. Solely combined exposure resulted in the rise in the production of immunoglobulins, changes in glucose and cholesterol levels, and an elevated marker of impaired liver, alanine aminotransferase (ALT). Analytical determination of the cyanobacterial toxin microcystin-LR (MC-LR) and its structurally similar congener MC-RR and their conjugates showed that SVCV affects neither the levels of MC in the liver nor the detoxification capacity of the liver. MC-LR and MC-RR were depurated from liver mostly in the form of cysteine conjugates (MC-LR-Cys, MC-RR-Cys) in comparison to glutathione conjugates (LR-GSH, RR-GSH). Our study brought new evidence that cyanobacteria worsen the effect of viral agents. Such inclusion of multiple stressor concept helps us to understand how and to what extent the relevant environmental stressors co-influence the health of the fish population.

Carp Edema Virus Infection Is Associated With Severe Metabolic Disturbance in Fish.

Significant mortalities associated with emerging viral diseases are challenging the economy of common carp aquaculture. As such, there is an increased need to disentangle how infected fish cope with progressive disease pathology and lose the ability for homeostatic maintenance of key physiological parameters. A natural carp edema virus (CEV) infection outbreak at a carp fish farm provided an opportunity to examine diseased and healthy carp in the same storage pond, thereby contributing to our better understanding of CEV disease pathophysiology. The disease status of fish was determined using PCR-based virus identification combined with analysis of gill pathology. Compared with healthy control carp, the blood chemistry profile of CEV-infected fish revealed major disruptions in electrolyte and acid-base balance (i.e., hyponatraemia, hypochloraemia, hyperphosphatemia, elevated pH, base excess, and anion gap and decreased partial dissolved carbon dioxide). In addition, we recorded hyperproteinaemia, hyperalbuminaemia, hypotonic dehydration, endogenous hyperammonaemia, and decreased lactate along with increased creatinine, alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase. Red blood cell associated hematology variables were also elevated. The multivariate pattern of responses for blood chemistry variables (driven by sodium, pH, partial dissolved carbon dioxide, ammonia, and albumin in the principal component analysis) clearly discriminated between CEV-infected and control carp. To conclude, we show that CEV infection in carp exerts complex adverse effects and results in severe metabolic disturbance due to the impaired gill respiratory and excretory functioning.

Comparison of diagnostic methods for Tetracapsuloides bryosalmonae detection in salmonid fish.

Diagnostic accuracy of pathogen detection depends upon the selection of suitable tests. Problems can arise when the selected diagnostic test gives false-positive or false-negative results, which can affect control measures, with consequences for the population health. The aim of this study was to compare sensitivity of different diagnostic methods IHC, PCR and qPCR detecting Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonid fish and as a consequence differences in disease prevalence. We analysed tissue from 388 salmonid specimens sampled from a recirculating system and rivers in the Czech Republic. Overall prevalence of T. bryosalmonae was extremely high at 92.0%, based on positive results of at least one of the above-mentioned screening methods. IHC resulted in a much lower detection rate (30.2%) than both PCR methods (qPCR32: 65.4%, PCR: 81.9%). While qPCR32 produced a good match with IHC (60.8%), all other methods differed significantly (p < .001) in the proportion of samples determined positive. Both PCR methods showed similar sensitivity, though specificity (i.e., the proportion of non-diseased fish classified correctly) differed significantly (p < .05). Sample preservation method significantly (p < .05) influenced the results of PCR, with a much lower DNA yield extracted from paraffin-embedded samples. Use of different methods that differ in diagnostic sensitivity and specificity resulted in random and systematic diagnosis errors, illustrating the importance of interpreting the results of each method carefully.

Field study indicating susceptibility differences between salmonid species and their lineages to proliferative kidney disease.

Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea) is the causative agent of proliferative kidney disease (PKD), which affects both wild and farmed salmonid fish. The objective of this study was to outline differences in susceptibility to PKD in different salmonid species, hybrids and breeding lineages. Susceptibility to T. bryosalmonae infection was established based on cumulative mortality, pathological findings and detection of T. bryosalmonae in the kidney using immunohistochemistry and molecular methods. Determination of pure and hybrid individuals of different species in the genus Salvelinus, and dissimilarity of rainbow trout lineages, was performed using traditional polymerase chain reaction (PCR) and microsatellite analyses. Rainbow trout displayed higher disease severity compared with brook trout and Alsatian charr. Moreover, the results indicated differences in infection susceptibility, not only among different salmonid species but also among different lineages of charr and rainbow trout. Our study indicated that some salmonid species and even different lineages of the same species are more suitable for farming under PKD pressure.

Carp oedema virus disease outbreaks in Czech and Slovak aquaculture.

This work describes the first confirmed cases of carp oedema virus disease (CEVD) in Slovakia and the Czech Republic and the phylogenetic analysis of Czech and Slovak carp oedema virus (CEV) isolates. Four cases of disease outbreak in the Czech Republic are described, the oldest dating from mid-May 2013 and one case from Slovakia dating from May 2019. In all cases, virus presence was confirmed using nested PCR. PCR products were sequenced and compared with 357-bp nucleotide sequences encoding the CEV P4a protein in GenBank. In four cases of disease outbreak (three common carp breeding facilities and one koi garden pond), CEV detected belonged to genogroup I. In one case (koi garden pond), fish were confirmed as infected with CEV from genogroup II. This work complements data on CEV occurrence in European countries and contributes to a better understanding of the pathways leading to transmission of the virus throughout Europe.

Health Surveillance of Wild Brown Trout (Salmo trutta fario) in the Czech Republic Revealed a Coexistence of Proliferative Kidney Disease and Piscine Orthoreovirus-3 Infection.

The population of brown trout (Salmo trutta fario) in continental Europe is on the decline, with infectious diseases confirmed as one of the causative factors. However, no data on the epizootiological situation of wild fish in the Czech Republic are currently available. In this study, brown trout (n = 260) from eight rivers were examined for the presence of viral and parasitical pathogens. Salmonid alphavirus-2, infectious pancreatic necrosis virus, piscine novirhabdovirus (VHSV) and salmonid novirhabdovirus (IHNV) were not detected using PCR. Cell culturing showed no viruses as well, and serological analysis of 110 sera did not detect any specific antibodies against VHSV or IHNV. Fish from two rivers were positive for the presence of piscine orthoreovirus-3 (PRV-3), subtype PRV-3b. However, none of the PRV-3-positive fish showed gross pathologies typically associated with PRV infections. By far the most widespread pathogen was Tetracapsuloides bryosalmonae which was confirmed in each of the examined locations, with a prevalence of up to 65% and 100%, as established by immunohistochemistry and PCR, respectively. Furthermore, up to 43.8% of fish showed signs of proliferative kidney disease caused by T. bryosalmonae, suggesting that this parasite is a main health challenge for brown trout in the Czech Republic.

Effects of trichothecene mycotoxin T-2 toxin on haematological and immunological parameters of rainbow trout (Oncorhynchus mykiss).

The aim of this study was to assess the effects of T-2 toxin-contaminated feed (at concentrations of 1.0 and 1.8 mg/kg) on the rainbow trout immune system by studying non-specific cellular and humoral immune responses and its effect on red and white blood cells. Consumption of T-2 toxin at both concentrations resulted in significantly increased erythrocyte counts and a decrease in mean corpuscular volume. While a significant decrease in mean corpuscular haemoglobin was observed at both experimental concentrations, the decrease in plasma haemoglobin was only significant at the higher T-2 toxin concentration. Higher T-2 toxin concentrations resulted in a significant increase in leukocyte and lymphocyte count, while absolute phagocyte count and counts of less mature neutrophil granulocyte forms remained unchanged at both concentrations. Non-specific humoral immunity (bactericidal activity measured as complement activation) decreased significantly in both experimental groups when compared with the control. The results of this study show that T-2 toxin in feed at a concentration range of 1.0-1.8 mg/kg influences the immunological defence mechanisms of rainbow trout.Trial registration number, MSMT-3876/2014-14; date of registration, 31/1/2014.

Effect of Feeding Honey Bee (Apis mellifera Hymenoptera: Apidae) Colonies With Honey, Sugar Solution, Inverted Sugar, and Wheat Starch Syrup on Nosematosis Prevalence and Intensity.

Here, we present the results of a 2-year field trial aimed at testing the effect of overwintering on different feeds on the course of Nosema ceranae infection. In August 2015, four experimental bee colony groups were established. After the last honey harvest, each colony was provided with 20 kg of feed, either honey, sugar (3:2 solution in tap water), inverted syrup made of sucrose, or wheat starch syrup. Samples of live bees were collected from each beehive in August (before feeding), November, and May. The following year, feeding and sampling were performed in the same way. Bees were examined microscopically to estimate the percentage of Nosema-infected individuals in the sample and the spore number per bee. Fitness parameters were also measured in all colonies. In all hives, presence of N. ceranae was confirmed through polymerase chain reaction. Nosema apis was not detected in the apiary. Significant differences in nosematosis prevalence and/or intensity were observed between the experimental groups. For most parameters, best results were recorded in the group fed with honey. Worst fitness and highest nosematosis prevalence and intensity were found in colonies fed with wheat starch syrup.

Stable-isotope dilution LC-MS/MS method for quantitative determination of microcystin conjugates with cysteine and glutathione in biotic matrices.

Microcystins are cyclic peptide toxins with hepatotoxic and tumor-promoting properties, which are produced in significant quantities (up to tens of µg/L) in freshwater cyanobacterial water blooms. Several studies reported microcystin accumulation in fish with possible food transfer to humans. These compounds are further metabolized to cysteine and glutathione conjugates which can be present in tissues in significant concentrations. In this study, we focused on the development and evaluation of robust and highly sensitive SPE-LC-MS/MS method for the analysis of microcystin conjugates in fish tissue samples. For the first time, we demonstrate the use of isotopically labeled internal standards which are essential for accurate and precise determination of analytes in complex biotic matrices. LLOQs of respective microcystin conjugates (signal-to-noise ratio; S/N > 10, peak-to-peak method) ranged from 3.3 to 5.0 ng/g of tissue fresh weight (FW). The calibration was linear within a range of concentrations from 1 to 70 ng/mL for all analyzed conjugates. The precision and repeatability of the method were very good with recoveries in the range of 88.5-107.6% and relative standard deviations between 8.8 and 13.2% for all analytes. In the follow-up study, fully validated method was used for the determination of microcystin conjugate levels in common carp exposed to microcystin-containing cyanobacterial biomass under controlled conditions. Significant amounts of microcystin conjugates (up to 55 ng/g) were found in the tissues of fish after 7 weeks of exposure. Our method was shown to be robust, sensitive, selective, and suitable for the determination of trace levels of microcystin conjugates in fish tissues.

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.

Effect of T-2 toxin-contaminated diet on common carp (Cyprinus carpio L.).

The T-2 toxin, a fungal metabolite produced by Fusarium molds, occurs in a range of agriculture products. Reduced availability of fish meal has led to increasing use of cereals as a source of protein in commercial aquaculture feeds, which has increased the potential for mycotoxin contamination. The purpose of this study was to investigate toxicity of T-2 toxin intake in common carp (Cyprinus carpio L.) using haematological, biochemical and immunological parameters and oxidative stress indices. In a four-week feeding trial, fish were fed a commercial diet with 5.3 mg/kg T-2 toxin added. Ingestion of contaminated diet did not lead to mortality of fish, probably due to lower feed intake. On the other hand, it significantly affected haematological variables such as haematocrit, haemoglobin, red blood cell counts leading to anemia and white blood cell counts leading to leukopenia due to lymphopenia. Plasma glucose concentration and alanine amino transferase activity showed a significant increase while triglycerides concentration decreased. Activity of ceruloplasmin was significantly decreased in plasma. Further, liver glutathione S-transferase activity was significantly increased and catalase activity decreased, in parallel with a significant increase in caudal kidney catalase activity and a decrease in glutathione peroxidase activity. Finally, lipid peroxidation (detected as malondialdehyde) was significantly increased in the liver and caudal kidney. Changes in non-specific immune response and cytokine levels in head kidney indicated immune system sensitivity to T-2 toxin. Overall, the results demonstrate that this feed-borne mycotoxin is able to induce anaemia and oxidative stress and cause changes in the immune response of common carp.

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.