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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Biochemical and histopathological responses of Wistar rats to oral intake of microcystins and cyanobacterial biomass.

OBJECTIVES: Cyanobacteria are producers of potent and environmentally abundant microcystins, representing an emerging global health issue. In the present study, we investigated the impact of pure microcystins and cyanobacterial biomass on laboratory rats (Wistar albino rats, males, 30 days old) under different exposure scenarios. METHODS: The rats were fed diets containing fish meat with microcystins in various concentrations and forms (cyanobacterial biomass and isolated microcystins) for 28 days. RESULTS: Although considerable amounts of microcystins (MCs) were administered to the rats, all levels of MCs in the liver were close to the detection limit (3-5 ng/g fresh weight) using liquid chromatography - tandem mass spectrometry. Only rats exposed to cyanobacterial biomass had clearly higher hepatic and splenic somatic indexes while markers of oxidative stress (glutathione-S-transferase, glutathione reductase, lipid peroxidatio) were significantly increased in the group exposed to the high dose of MCs. Most of the analysed biochemical parameters did not show clear differences among groups. Levels of bilirubin and lipases were significantly increased only after exposure to cyanobacterial biomass and MCs, respectively. Considering microscopic findings in the liver, kidney, thymus, spleen and brain, histopathology was dominated by alterations in the hepatic parenchyma and renal cortical tubular system. CONCLUSIONS: The present study demonstrates that oral exposure to MCs and cyanobacterial biomass may induce biochemical and detoxification responses associated with damage to liver and kidneys and in the laboratory rat.

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.

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.

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.