Outbreaks of koi sleepy disease (KSD) caused by carp edema virus (CEV) may seriously affect populations of farmed common carp, one of the most important fish species for global food production. The present study shows further evidence for the involvement of CEV in outbreaks of KSD among carp and koi populations: in a series of infection experiments, CEV from two different genogroups could be transmitted to several strains of naïve common carp via cohabitation with fish infected with CEV. In recipient fish, clinical signs of KSD were induced. The virus load and viral gene expression results confirm gills as the target organ for CEV replication. Gill explants also allowed for a limited virus replication in vitro. The in vivo infection experiments revealed differences in the virulence of the two CEV genogroups which were associated with infections in koi or in common carp, with higher virulence towards the same fish variety as the donor fish. When the susceptibility of different carp strains to a CEV infection and the development of KSD were experimentally investigated, Amur wild carp showed to be relatively more resistant to the infection and did not develop clinical signs for KSD. However, the resistance could not be related to a higher magnitude of type I IFN responses of affected tissues. Despite not having a mechanistic explanation for the resistance of Amur wild carp to KSD, we recommend using this carp strain in breeding programs to limit potential losses caused by CEV in aquaculture.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-017-0416-7) contains supplementary material, which is available to authorized users.

BackgroundInfections with carp edema virus, a pox virus, are known from Japanese koi populations since 1974. A characteristic clinical sign associated with this infection is lethargy and therefore the disease is called “koi sleepy disease”. Diseased koi also show swollen gills, enophthalmus, and skin lesions. Mortality rates up to 80 % are described. For a long period of time, disease outbreaks seemed to be restricted to Japan. However, during the last years clinical outbreaks of koi sleepy disease also occurred in the UK and in the Netherlands.Case presentationIn spring 2014 koi from different ponds showing lethargic behavior, skin ulcers, inflammation of the anus, enophthalmus, and gill necrosis were presented to the laboratory for diagnosis. In all cases, new koi had been purchased earlier that spring from the same retailer and introduced into existing populations. Eleven koi from six ponds were examined for ectoparasites and for bacterial and viral infections (cyprinid herpesviruses in general and especially koi herpesvirus (KHV) known formally as Cyprinid herpesvirus 3 (CyHV–3); and Carp Edema Virus). In most of the cases parasites were not detected from skin and gills. Only opportunistic freshwater bacteria were isolated from skin ulcers. In cell cultures no cytopathic effect was observed, and none of the samples gave positive results in PCR tests for cyprinid herpesviruses. By analyzing gill tissues for CEV in seven out of eleven samples by a nested PCR, PCR products of 547 bp and 180 bp (by using nested primers) could be amplified. An outbreak of Koi Sleepy Disease was confirmed by sequencing of the PCR products. These results confirm the presence of CEV in German koi populations.ConclusionA clinical outbreak of “koi sleepy disease” due to an infection with Carp Edema Virus was confirmed for the first time in Germany. To avoid transmission of CEV to common carp testing of CEV should become part of fish disease surveillance programs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0424-7) contains supplementary material, which is available to authorized users.

Gills of fish are involved in respiration, excretion and osmoregulation. Due to numerous interactions between these processes, branchial diseases have serious implications on fish health. Here, "koi sleepy disease" (KSD), caused by carp edema virus (CEV) infection was used to study physiological, immunological and metabolic consequences of a gill disease in fish. A metabolome analysis shows that the moderately hypoxic-tolerant carp can compensate the respiratory compromise related to this infection by various adaptations in their metabolism. Instead, the disease is accompanied by a massive disturbance of the osmotic balance with hyponatremia as low as 71.65 mmol L −1 , and an accumulation of ammonia in circulatory blood causing a hyperammonemia as high as 1123.24 µmol L −1 . At water conditions with increased ambient salt, the hydro-mineral balance and the ammonia excretion were restored. Importantly, both hyponatremia and hyperammonemia in KSD-affected carp can be linked to an immunosuppression leading to a four-fold drop in the number of white blood cells, and significant downregulation of cd4, tcr a2 and igm expression in gills, which can be evaded by increasing the ion concentration in water. This shows that the complex host-pathogen interactions within the gills can have immunosuppressive consequences, which have not previously been addressed in fish. Furthermore, it makes the CEV infection of carp a powerful model for studying interdependent pathological and immunological effects of a branchial disease in fish.

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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.

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.

The infection of common carp and its ornamental variety, koi, with the carp edema virus (CEV) is often associated with the occurrence of a clinical disease called 'koi sleepy disease'. The disease may lead to high mortality in both koi and common carp populations. To prevent further spread of the infection and the disease, a reliable detection method for this virus is required. However, the high genetic variability of the CEV p4a gene used for PCR-based diagnostics could be a serious obstacle for successful and reliable detection of virus infection in field samples. By analysing 39 field samples from different geographical origins obtained from koi and farmed carp and from all 3 genogroups of CEV, using several recently available PCR protocols, we investigated which of the protocols would allow the detection of CEV from all known genogroups present in samples from Central European carp or koi populations. The comparison of 5 different PCR protocols showed that the PCR assays (both end-point and quantitative) developed in the Centre for Environment, Fisheries and Aquaculture Science exhibited the highest analytical inclusivity and diagnostic sensitivity. Currently, this makes them the most suitable protocols for detecting viruses from all known CEV genogroups.

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