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.

Intra-muscular and oral vaccination using a Koi Herpesvirus ORF25 DNA vaccine does not confer protection in common carp (Cyprinus carpio L.).

Koi Herpes Virus (KHV or Cyprinid Herpesvirus 3, CyHV-3) is among the most threatening pathogens affecting common carp production as well as the highly valuable ornamental koi carp. To date, no effective commercial vaccine is available for worldwide use. A previous study reported that three intramuscular injections with an ORF25-based DNA vaccine, led to the generation of neutralizing antibodies and conferred significant protection against an intraperitoneal challenge with KHV. In the present study, we set out to optimize an ORF25-based DNA vaccination protocol that required fewer injections and would confer protection upon a challenge that better resembled the natural route of infection. To this end, ORF25 was cloned in pcDNA3 either as a soluble protein or as a full-length transmembrane GFP-fusion protein. We tested our ORF25-based DNA vaccines in multiple vaccination trials using different doses, vaccination routes (i.m. injection and oral gavage) and challenge methods (bath and cohabitation). Furthermore, we analysed local and systemic responses to the i.m. injected DNA vaccine through histological and RT-qPCR analysis. We observed a strong protection when fish received three injections of either of the two DNA vaccines. However, this protection was observed only after bath challenge and not after cohabitation challenge. Furthermore, protection was insufficient when fish received one injection only, or received the plasmid orally. The importance of choosing a challenge model that best reflects the natural route of infection and the possibility to include additional antigens in future DNA vaccination strategies against KHV will be discussed.

Massive mortality of Prussian carp Carassius gibelio in the upper Elbe basin associated with herpesviral hematopoietic necrosis (CyHV-2).

From 22 May to 10 June 2011 massive mortality of Prussian carp Carassius gibelio was observed in alluvial Lake Rehacˇka close to the Elbe River in the Czech Republic. More than 1400 kg of dead fish were collected and no other fish species were affected. Further molecular and cytogenetic investigation of fish (n = 232) revealed that the Rˇehacˇka population of Prussian carp consisted exclusively of gynogenetic triploid females. The causative agent was identified by means of molecular and electron microscopy as a herpesviral hematopoietic necrosis virus (Cyprinid herpesvirus 2, CyHV-2). This is the first report of CyHV-2 from the Czech Republic and the second finding worldwide of CyHV-2 causing mass mortality of C. gibelio. Some other localities in the upper Elbe River basin where C. gibelio was affected are also noted. We assume that the massive wave of deaths of all female gynogenetic Prussian carp can be attributed to limited genetic variation and the favourable conditions for development of viral disease.

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.

First detection of pike fry-like rhabdovirus in barbel and spring viraemia of carp virus in sturgeon and pike in aquaculture in the Czech Republic.

Rapid antigen detection enzyme-linked immunosorbent assay (ELISA) testing of cell cultures with organ homogenate from fish, collected from farms with a predominance of common carp or in natural aquaculture in the Czech Republic between 1995 and 2008, identified piscine vesiculovirus in 27 of 178 samples. Using reverse transcription semi-nested PCR, targeting a 550 nucleotide region of the glycoprotein (G) gene, piscine vesiculovirus was confirmed in 23 of the 27 organ samples diagnosed by ELISA as infected. PCR products were amplified and sequenced from 18 isolates from common carp Cyprinus carpio (family Cyprinidae), 2 isolates from northern pike Esox lucius (family Esocidae), and 1 isolate each from Siberian sturgeon Acipenser baerii (family Acipenseridae), common barbel Barbus barbus (family Cyprinidae), and koi carp Cyprinus carpio koi (family Cyprinidae). The sequences (based on 401 nucleotides) clustered into 2 genogroups. The majority of isolates (n = 22), including those from sturgeon and pike, grouped with the spring viraemia of carp virus (SVCV) Genogroup I and Subgroup Id. The 22 isolates could be further subdivided into 2 groups: Id1 (n = 20) and Id2 (n = 2). A marker (a non-conservative nucleotide substitution) for the Id1 SVCV group was identified. It was specifically found in all sequences of Id1 isolates when testing SVCV originating from different countries. The remaining isolate from barbel, was classified in the pike fry-like rhabdovirus Genogroup IV. This is the first confirmation of natural SVCV infection in sturgeon and pike, and pike fry-like rhabdovirus infection in barbel. In the case of the pike fry-like rhabdovirus, this is also its first identification in the Czech Republic. According to the presence/absence of evident clinical signs of rhabdoviral disease in the 3 infected hosts, only the sturgeon seemed to be susceptible to the monitored rhabdovirus.

Transcription of signal-3 cytokines, IL-12 and IFN alpha beta, coincides with the timing of CD8 alpha beta up-regulation during viral infection of common carp (Cyprinus carpio L).

Mammalian naïve CD8+ T cells are activated by antigen (signal 1) and CD28 costimulation (signal 2) to undergo several rounds of cell division, but programming for survival, effector function and memory requires a third signal that can be provided by IL-12 and/or type I interferons. Functional studies indicate that the route of antigen presentation and costimulation are conserved from fish to mammals. However, the potential of IL-12 and IFN alpha beta to act as signal-3 cytokines in infections inducing a CTL response has not been examined in fish. We report the cloning of CD8 alpha and CD8 beta homologues, each present in duplicate copies and of two TCR-C alpha isoforms in European common carp. The identification of (cytotoxic) T cell marker sequences and the availability of sequences coding for the signal-3 cytokines in the same fish species, allowed us to investigate by RT-qPCR their kinetics of gene expression during viral and parasitic infection. Our results show that transcription of signal-3 cytokines occurred concomitantly with CD8 alpha beta up-regulation exclusively at 4 days post-primary viral infection. No regulation of IL-12 and IFN alpha beta was observed after parasitic infection. Our data provide evidences for an evolutionary conservation of function for IL-12 and IFN alpha beta to act as third signal during CTL activation. In addition, we suggest that a CD8 alpha 2/beta1 and a p35p40b association could be the preferred combinations for the formation of a functional CD8 co-receptor and an IL-12p70 heterodimer during viral infection. The relevance of our findings to future vaccination strategies in fish is discussed.

Optimizing Genomic Prediction of Host Resistance to Koi Herpesvirus Disease in Carp.

Genomic selection (GS) is increasingly applied in breeding programs of major aquaculture species, enabling improved prediction accuracy and genetic gain compared to pedigree-based approaches. Koi Herpesvirus disease (KHVD) is notifiable by the World Organization for Animal Health and the European Union, causing major economic losses to carp production. GS has potential to breed carp with improved resistance to KHVD, thereby contributing to disease control. In the current study, Restriction-site Associated DNA sequencing (RAD-seq) was applied on a population of 1,425 common carp juveniles which had been challenged with Koi herpes virus, followed by sampling of survivors and mortalities. GS was tested on a wide range of scenarios by varying both SNP densities and the genetic relationships between training and validation sets. The accuracy of correctly identifying KHVD resistant animals using GS was between 8 and 18% higher than pedigree best linear unbiased predictor (pBLUP) depending on the tested scenario. Furthermore, minor decreases in prediction accuracy were observed with decreased SNP density. However, the genetic relationship between the training and validation sets was a key factor in the efficacy of genomic prediction of KHVD resistance in carp, with substantially lower prediction accuracy when the relationships between the training and validation sets did not contain close relatives.

Mapping and Sequencing of a Significant Quantitative Trait Locus Affecting Resistance to Koi Herpesvirus in Common Carp.

Cyprinids are the most highly produced group of fishes globally, with common carp being one of the most valuable species of the group. Koi herpesvirus (KHV) infections can result in high levels of mortality, causing major economic losses, and is listed as a notifiable disease by the World Organization for Animal Health. Selective breeding for host resistance has the potential to reduce morbidity and losses due to KHV. Therefore, improving knowledge about host resistance and methods of incorporating genomic data into breeding for resistance may contribute to a decrease in economic losses in carp farming. In the current study, a population of 1,425 carp juveniles, originating from a factorial cross between 40 sires and 20 dams was challenged with KHV. Mortalities and survivors were recorded and sampled for genotyping by sequencing using Restriction Site-Associated DNA sequencing (RADseq). Genome-wide association analyses were performed to investigate the genetic architecture of resistance to KHV. A genome-wide significant QTL affecting resistance to KHV was identified on linkage group 44, explaining approximately 7% of the additive genetic variance. Pooled whole genome resequencing of a subset of resistant (n = 60) and susceptible animals (n = 60) was performed to characterize QTL regions, including identification of putative candidate genes and functional annotation of associated polymorphisms. The TRIM25 gene was identified as a promising positional and functional candidate within the QTL region of LG 44, and a putative premature stop mutation in this gene was discovered.

Intramuscular DNA Vaccination of Juvenile Carp against Spring Viremia of Carp Virus Induces Full Protection and Establishes a Virus-Specific B and T Cell Response.

Although spring viremia of carp virus (SVCV) can cause high mortalities in common carp, a commercial vaccine is not available for worldwide use. Here, we report a DNA vaccine based on the expression of the SVCV glycoprotein (G) which, when injected in the muscle even at a single low dose of 0.1 µg DNA/g of fish, confers up to 100% protection against a subsequent bath challenge with SVCV. Importantly, to best validate vaccine efficacy, we also optimized a reliable bath challenge model closely mimicking a natural infection, based on a prolonged exposure of carp to SVCV at 15°C. Using this optimized bath challenge, we showed a strong age-dependent susceptibility of carp to SVCV, with high susceptibility at young age (3 months) and a full resistance at 9 months. We visualized local expression of the G protein and associated early inflammatory response by immunohistochemistry and described changes in the gene expression of pro-inflammatory cytokines, chemokines, and antiviral genes in the muscle of vaccinated fish. Adaptive immune responses were investigated by analyzing neutralizing titers against SVCV in the serum of vaccinated fish and the in vitro proliferation capacity of peripheral SVCV-specific T cells. We show significantly higher serum neutralizing titers and the presence of SVCV-specific T cells in the blood of vaccinated fish, which proliferated upon stimulation with SVCV. Altogether, this is the first study reporting on a protective DNA vaccine against SVCV in carp and the first to provide a detailed characterization of local innate as well as systemic adaptive immune responses elicited upon DNA vaccination that suggest a role not only of B cells but also of T cells in the protection conferred by the SVCV-G DNA vaccine.