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Environmental stressors such as micro- and nanosized plastic particles (MNPs) or crude oil have a detrimental effect on aquatic animals; however, the impact upon the cardiovascular system of fish remains relatively under-researched. This study presents a novel approach for investigating the effect of crude oil and MNPs on the cardiac system of fish. We used salmonid larvae and cardiac cell cultures derived from hearts of salmonid fish and exposed them to environmental stressors. Following exposure to plastic particles or crude oil, the larvae exhibited some variation in contraction rate. In contrast, significant alterations in the contraction rate were observed in all cardiac cell cultures. The greatest differences between the control and treatment groups were observed in cardiac cell cultures derived from older brown trout. Following 7 days of exposure to MNPs or crude oil in Atlantic salmon larval hearts or cardiac cell cultures, there were only minor responses noted in mRNA expression of the selected marker genes. These findings show the use of a novel in vitro technique contributing to the existing body of knowledge on the impact of MNPs and crude oil on the cardiovascular system of salmonids and the associated risk., (© 2024 The Author(s). Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Replacing fishmeal, a finite resource with high market demand, in the diet of carnivorous rainbow trout with proteins from alternative sources may be a challenge for these fish. Therefore, this study investigated whether replacing fishmeal with protein derived from Hermetia illucens or Arthrospira platensis could promote disease susceptibility in local trout populations with different growth performance. This was assessed in vitro by measuring susceptibility to infection with the viral haemorrhagic septicaemia virus (VHSV) or the bacterium Yersinia ruckeri. Analysis of fin tissue explants and primary cell cultures from scales from the three trout populations infected in vitro with VHSV and gill explants infected with Y. ruckeri showed no significant differences in virus replication or bacterial counts. Evaluation of the virucidal or bactericidal effect of skin mucus showed a significant reduction in viral load and bacterial count for all samples with mucus addition, but no significant difference was observed between the experimental groups. This study documents no apparent impairment of innate immune mechanisms in the skin and gills of trout after feeding a diet replacing fishmeal with Arthrospira or Hermetia proteins. This underlines the potential of these alternative protein sources for the further development of sustainable trout aquaculture., (© 2023 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Several factors influence the susceptibility of cell lines to infection by different viruses. These can be related to tissue specificity of the viruses, physiological status of the cells, their differentiation level and their capacity to mount immune responses to combat viral infection. To study the influence of cell characteristics and immune responses on their susceptibility on virus infection, newly developed cell lines from common carp brain (CCAbre), fins (CCApin), gills (CCAgill), and heart (CCAcar) and the established common carp brain (CCB) cells were exposed to the carp infecting viruses cyprinid herpesvirus 3 (CyHV-3), carp oedema virus (CEV), and the yet not fully characterized common carp paramyxovirus (CCPV). The susceptibility of these cells to viral infection was measured by formation of a cytopathic effect (CPE), estimation of viral particles produced by the cells and presence of viral mRNA in the cells. Viral susceptibility of the cells was compared to cell characteristics, measured by mRNA expression of the epithelial cell markers cadherin 1, occludin, and cytokeratin 15 and the mesenchymal cell marker vimentin, as well as to the level of type I interferon (IFN) responses. All cell lines were susceptible to CyHV-3 and CCPV but not to CEV infection. The cell lines had different levels of type I IFN responses towards the viruses. Typically, CyHV-3 did not induce high type I IFN responses, while CCPV induced high responses in CCAbre, CCAcar, CCApin cells but no response in CCAgill cells. Consequently, the type I IFN response modulated cell susceptibility to CCPV but not to CyHV-3. Interestingly, when the three different passage levels of CCB cells were examined, the susceptibility of one passage was significantly lower for CyHV-3 and higher for CCPV infection. This coincided with a loss of epithelial markers and lower type I IFN responses. This study confirms an influence of cell characteristics and immune responses on the susceptibility of carp cell lines for virus infection. Depending on the vulnerability of the virus to type I IFN responses, cells with a lower IFN-response can be superior for replication of some viruses. Batches of CCB cells can differentiate and thus may have significantly different levels of susceptibility to certain viruses., (© 2022 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

The spread of viral diseases in eels is suggested to severely affect the European eel (Anguilla anguilla) panmictic population. The European Commission has initiated the Eel Recovery Plan (Council Regulation No. 1100/2007) to try to return the European eel stock to more sustainable levels within that measures eel restocking. However, scientific evidence evaluating the efficacy of stocking remains scarce. In addition, knowledge about the impact and contribution of eel stocking on the distribution of infectious diseases is insufficient. In this study, we aimed to investigate virus infections in batches of eels intended for restocking. We analysed samples of glass eels from certified fisheries and farmed European eels from different aquaculture farms. All analysed eels were purchased within a North Rhine Westphalian conservation program. Via a combination of cell culture and qPCR-based techniques, we detected infections of glass eels with the rhabdovirus Eel Virus European X and anguillid herpesvirus 1 infections in farmed eels (10-15 cm)., (© 2022 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Viral infections have been suggested to play a role in the decline of the panmictic population of the European eel (Anguilla anguilla). However, despite the importance of knowledge about pathogenic eel viruses, little is known about their spread in the wild European eel population and only a few eel pathogenic viruses have been described so far. In this study, we aimed to investigate the health status of the A. anguilla stock in North Rhine Westphalia (NRW) State of Germany. For this purpose, we examined tissue samples of 16 elvers, 100 yellow eels and 6 silver eels, sampled from the rivers Rhine, Lippe and Ems. Virus detection was performed via a combination of cell culture and PCR. Next to the detection of frequently encountered pathogenic eel viruses (anguillid herpesvirus 1 and eel virus European X (EVEX)), we isolated the eel picornavirus 1 (EPV-1) from tissue of yellow eels and elvers and demonstrate the distribution of EPV-1 in wild eel population in NRW., (© 2021 North Rhine Westphalian State Agency for Nature, Environment and Consumer Protection. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Tropical shrimp, like Litopenaeus vannamei, in land-based recirculating aquaculture systems (RAS) are often kept at low water salinities to reduce costs for artificial sea salt and the amount of salty wastewater. Although these shrimp are tolerant against low salinities, innate immunity suppression and changes in the microbial composition in the water can occur. As especially Vibrio spp. are relevant for shrimp health, alterations in the species composition of the Vibrio community were analysed in water from six RAS, run at 15‰ or 30‰. Additionally, pathogenicity factors including pirA/B, VPI, toxR, toxS, vhh, vfh, tdh, trh, flagellin genes and T6SS1/2 of V. parahaemolyticus were analysed. The Vibrio composition differed significantly depending on water salinity. In RAS at 15‰, higher numbers of the potentially pathogenic species V. parahaemolyticus, V. owensii and V. campbellii were detected, and especially in V. parahaemolyticus, various pathogenicity factors were present. A reduced salinity may therefore pose a higher risk of disease outbreaks in shrimp RAS. Because some of the detected pathogenicity factors are relevant for human health, this might also affect food safety. In order to produce healthy shrimp as a safe food for human consumption, maintaining high water salinities seems to be recommendable., (© 2020 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Common carp (Cyprinus carpio) is a very important fish species for warm-water aquaculture in Croatia. All Croatian carp farms are subjected to a surveillance programme for the presence of koi herpesvirus (KHV), causing a deadly disease called koi herpesvirus disease (KHVD). However, there is no surveillance for other viral pathogens of importance like carp edema virus (CEV), a causative agent of koi sleepy disease (KSD). During regular testing within the KHVD surveillance programme, we tested samples for CEV simultaneously. The screening indicated possible outbreaks of KHVD and KSD. During 2016, KHVD broke out in an isolated area and soon thereafter a KHV eradication programme was successfully performed. However, during 2018 and 2019, two additional mortality events occurred in lakes in the southern part of Croatia during the spring. Samples from both events tested positive for CEV. An epidemiological investigation confirmed the introduction of infected carps from an infected farm to one of the lakes. To prevent the spreading of CEV into open waters, it is of utmost importance to introduce CEV testing before fish movement or to perform regular testing of all carp farms in the country to determine CEV prevalence for the purpose of implementation of control measures., (© 2020 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Zebrafish (Danio rerio) is a laboratory model organism used in different areas of biological research including studies of immune response and host-pathogen interactions. Thanks to many biological tools available, zebrafish becomes also an important model in aquaculture research since several fish viral infection models have been developed for zebrafish. Here, we have evaluated the possible use of zebrafish to study infections with fish viruses that have not yet been tested on this model organism. In vitro studies demonstrated that chum salmon reovirus (CSV; aquareovirus A) and two alloherpesviruses cyprinid herpesvirus 1 (CyHV-1) and cyprinid herpesvirus 3 (CyHV-3) are able to replicate in zebrafish cell lines ZF4 and SJD.1. Moreover, CSV induced a clear cytopathic effect and up-regulated the expression of antiviral genes vig-1 and mxa in both cell lines. In vivo studies demonstrated that both CSV and CyHV-3 induce up-regulation of vig-1 and mxa expression in kidney and spleen of adult zebrafish after infection by i.p. injection but not in larvae after infection by immersion. CyHV-3 is eliminated quickly from fish; therefore, virus clearing process could be evaluated, and in CSV-infected fish, a prolonged confrontation of the host with the pathogen could be studied., (© 2019 John Wiley & Sons Ltd.)

Due to their pathogenic potential, identifying Vibrio species from recirculating aquaculture systems (RAS) for Pacific white shrimp (Litopenaeus vannamei) is of great importance to determine the risk for animal's as well as for the consumer's health. The present study compared identification results for a total of 93 Vibrio isolates, including type strains and isolates from shrimp aquaculture. Results from biochemical identifications, 16S rRNA sequencing, sequencing of the uridylate kinase encoding gene pyrH and analysis of the protein spectra assessed by MALDI-TOF MS were compared. The results achieved by these different methods were highly divergent for many of the analysed isolates and for several Vibrio spp difficulties in reliably identifying occurred. These difficulties mainly resulted from missing entries in digital databases, a low number of comparable isolates analysed so far, and high interspecific similarities of biochemical traits and nucleotide sequences between the closely related Vibrio species. Due to the presented data, it can be concluded that for identifying Vibrio spp. from samples in routine diagnostics, it is recommended to use MALDI-TOF MS analysis for a quick and reliable identification of pathogenic Vibrio sp. Nevertheless, editing the database, containing the main spectra of Vibrio is recommended to achieve reliable identification results., (© 2018 John Wiley & Sons Ltd.)

Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is an aetiological agent of a virulent and lethal disease in common and koi carp. In this study, we examined in vitro the anti-CyHV-3 activity of acyclovir (ACV), nucleoside analogue commonly used against human herpesviruses, as well as acyclovir monophospate (ACV-MP). The cytotoxicity of the ACV and the ACV-MP for two common carp cell lines, CCB (Common carp brain) and KF1 (Koi carp fin 1), was determined by means of MTT and crystal violet assays. In subsequent studies, the concentration of 66.67 μM was applied. The ACV and the ACV-MP (66.67 μM) inhibited a cytopathic effect (CPE) induced by the CyHV-3 virus in the CCB (ACV by 66%, ACV-MP by 58%) and the KF1 (ACV by 25%, ACV-MP by 37%). The viral load measured by the means of TaqMan qPCR was reduced in a range of 67%-93% depending on the analogue, the cell line and the time of incubation. The expression of viral genes (ORF149, ORF3, ORF134 and ORF78) in CCB cells infected with the CyHV-3 was strongly downregulated within the range of 78%-91%. In summary, both the ACV and the ACV-MP can inhibit CyHV-3 replication in vitro., (© 2018 John Wiley & Sons Ltd.)

Koi sleepy disease (KSD) is a disease with increasing importance in global common carp aquaculture. Despite the fact that carp edema virus (CEV) is most likely the causative agent of KSD, the disease often presents itself as multifactorial with several parasites and bacteria species present on gills, skin or in internal organs. Therefore, in this study, we analysed and presented initial results on an interaction of flavobacteria and CEV in the development of clinical KSD in carp suffering from proliferative gill disease. We examined selected field samples from Germany and Hungary and confirmed the presence of CEV and flavobacteria co-infections in subset of the samples. In several infection experiments, we studied the transfer and dynamics of both infections. Furthermore, we analysed which Flavobacterium species could be isolated from KSD-affected fish and concluded that Flavobacterium branchiophilum is a possible copathogen. Antibiotic treatment experiments showed that CEV seems to be the primary pathogen causing an insult to the gills of carp and by these enabling other pathogens, including F. branchiophilum, to establish co-infections. Despite the fact that F. branchiophilum co-infection is not required for the development of clinical KSD, it could contribute to the pathological changes recorded during the outbreaks., (© 2018 John Wiley & Sons Ltd.)

The effect of dietary β-glucan on the bacterial community in the gut of common carp (Cyprinus carpio) was examined after oral application of Aeromonas hydrophila. Carp received either feed supplemented with 1% MacroGard ® , a β-1,3/1,6-glucan, or a β-glucan-free diet. Fourteen days after feeding, half of the carp from each group were intubated with 10 9 colony-forming units (CFU) of a pathogenic strain of A. hydrophila. Gut samples were taken 12 hr to 7 days after application and analysed using microbiological and molecular biological techniques (NGS, RT-PCR-DGGE). The reaction of the mucosa and the microbiota to an A. hydrophila intubation differed in carp fed with β-glucan compared to carp from the control group. In β-glucan fed carp, the total bacterial amount was lower but the number of bacterial species was higher. Bacterial composition was different for carp from both treatment groups. The number of mucin filled goblet cells was reduced in carp fed the β-glucan diet. Mucus was obviously released from the goblet cells and was probably washed out of the gut together with high numbers of bacteria. This might be protective against pathogenic bacteria and, therefore, feeding with β-glucan may provide protection against infections of the gut in carp., (© 2018 John Wiley & Sons Ltd.)

Hungary is an important carp producer with intensive trading relationships with farms in other carp-producing areas in Europe. Carp in Europe were recently found infected with carp edema virus (CEV), a poxvirus which causes the koi sleepy disease (KSD) syndrome. Moribund carp were collected from 17 fish farms and angling ponds in different regions of Hungary. Histological analysis of gills from these carp revealed a proliferation of the interlamellar epithelium and an infiltration by eosinophilic cells. In 13 of 17 of these carp, CEV DNA was detected by qPCR and in seven fish more than 1 × 10 4 copies of virus-specific DNA sequences per 250 ng of DNA, which could be considered as clinically relevant and a cause of disease. A phylogenetic analysis of the sequences revealed that all three genogroups of CEV were present in Hungarian common carp with genogroup I being most abundant. These results support the hypothesis of a prolonged presence of CEV in European carp populations and suggest that previous outbreaks of KSD were not recorded or misdiagnosed. Hence, a testing of carp and koi for infection with CEV should be included into disease surveillance programmes to prevent further spreading of this disease., (© 2017 John Wiley & Sons Ltd.)

The Schlei fjord in northern Germany is the recipient water of a comprehensive eel, Anguilla anguilla (L.), stocking programme. Since 2015, stocked eels become alizarin red S marked, but to date no control mechanism is implemented in this stock enhancement measure to prevent anthropogenic spreading of diseases. Consequentially, it was possible that farmed stocking cohorts of 2015 and 2016 (in total ca. 1040 kg) were subsequently tested positive for anguillid herpesvirus 1 (AngHV 1). For this study, 100 eels [total length (TL) 24.3-72.9 cm, age ca. 1-6 years] were caught in 2016 and investigated with regard to AngHV 1 infection, parasite load (Anguillicoloides crassus) and body conditions. 68% of the eels were found to be virus positive while larger specimens were more often infected. In addition, a fitted generalized linear model (area under the curve = 0.741) demonstrated that an increase in individual TL is accompanied with an increased risk of clinically relevant virus loads. Anguillicoloides crassus turned out to be an important stressor for eels, because parasite and virus load revealed a significant positive correlation. The results of this study evidently show the urgent need of a disease containment strategy for eel stocking programmes., (© 2017 John Wiley & Sons Ltd.)