High immunogenicity of virus-like particles (VLPs) decorated with Aeromonas salmonicida VapA antigen in rainbow trout.

The Gram-negative bacterium A. salmonicida is the causal agent of furunculosis and used to be one of the most loss-causing bacterial infections in the salmonid aquaculture industry with a mortality rate of about 90% until the 1990s, when an inactivated vaccine with mineral oil as adjuvant was successfully implemented to control the disease. However, the use of this vaccine is associated with inflammatory side effects in the peritoneal cavity as well as autoimmune reactions in Atlantic salmon, and incomplete protection has been reported in rainbow trout. We here aimed at developing and testing a recombinant alternative vaccine based on virus-like particles (VLPs) decorated with VapA, the key structural surface protein in the outer A-layer of A. salmonicida. The VLP carrier was based on either the capsid protein of a fish nodavirus, namely red grouper nervous necrotic virus (RGNNV) or the capsid protein of Acinetobacter phage AP205. The VapA and capsid proteins were expressed individually in E. coli and VapA was fused to auto-assembled VLPs using the SpyTag/SpyCatcher technology. Rainbow trout were vaccinated/immunized with the VapA-VLP vaccines by intraperitoneal injection and were challenged with A. salmonicida 7 weeks later. The VLP vaccines provided protection comparable to that of a bacterin-based vaccine and antibody response analysis demonstrated that vaccinated fish mounted a strong VapA-specific antibody response. To our knowledge, this is the first demonstration of the potential use of antigen-decorated VLPs for vaccination against a bacterial disease in salmonids.

Epitope mapping of the monoclonal antibody IP5B11 used for detection of viral haemorrhagic septicaemia virus facilitated by genome sequencing of carpione novirhabdovirus.

The monoclonal antibody (mAb) IP5B11, which is used worldwide for the diagnosis of viral haemorrhagic septicaemia (VHS) in fish, reacts with all genotypes of VHS virus (VHSV). The mAb exceptionally also reacts with the carpione rhabdovirus (CarRV). Following next generation genome sequencing of CarRV and N protein sequence alignment including five kinds of fish novirhabdoviruses, the epitope recognized by mAb IP5B11 was identified. Dot blot analysis confirmed the epitope of mAb IP5B11 to be associated with the region N219 to N233 of the N protein of VHSV. Phylogenetic analysis identified CarRV as a new member of the fish novirhabdoviruses.

Efficacy of DNA Vaccines in Protecting Rainbow Trout against VHS and IHN under Intensive Farming Conditions.

Despite the negative impact of viral hemorrhagic septicemia (VHS) and infectious hematopoietic necrosis (IHN) on European rainbow trout farming, no vaccines are commercially available in Europe. DNA vaccines are protective under experimental conditions, but testing under intensive farming conditions remains uninvestigated. Two DNA vaccines encoding the glycoproteins (G) of recent Italian VHSV and IHNV isolates were developed and tested for potency and safety under experimental conditions. Subsequently, a field vaccination trial was initiated at a disease-free hatchery. The fish were injected intramuscularly with either the VHS DNA vaccine or with a mix of VHS and IHN DNA vaccines at a dose of 1 µg/vaccine/fish, or with PBS. At 60 days post-vaccination, fish were moved to a VHSV and IHNV infected facility. Mortality started 7 days later, initially due to VHS. After 3 months, IHN became the dominant cause of disease. Accordingly, both DNA vaccinated groups displayed lower losses compared to the PBS group during the first three months, while the VHS/IHN vaccinated group subsequently had the lowest mortality. A later outbreak of ERM caused equal disease in all groups. The trial confirmed the DNA vaccines to be safe and efficient in reducing the impact of VHS and IHN in farmed rainbow trout.

Antigenic variability of Vibrio anguillarum serotype O2a: A hurdle for vaccine efficacy against vibriosis in rainbow trout (Oncorhynchus mykiss).

Despite vaccination, outbreaks of vibriosis still occur in sea-reared rainbow trout in Denmark. Vibriosis outbreaks are caused mainly by V. anguillarum serotypes O1 and O2a, and bacterins of both serotypes are included in the commonly used vaccine against this disease in Danish aquaculture. However, while the strains belonging to serotype O1 are genetically similar, the strains belonging to serotype O2a are highly diverse. This work aimed first at examining how the antibody response and protection induced by bacterin-based vaccines were affected by the antigenic variability within V. anguillarum serotype O2a strains. Following vaccination of rainbow trout with either a commercial or an experimental vaccine, specific antibody reactivity in serum from vaccinated fish was examined by ELISA against 23 strains of V. anguillarum serotype O2a (VaO2a). The strains were divided into 4 distinct subgroups according to the observed detection pattern. Seven strains were strongly recognized only by sera from fish vaccinated with the experimental vaccine (EV-I antisera), while 13 other strains were primarily recognized by sera from fish vaccinated with the commercial vaccine (CV antisera). Two strains were recognized by both EV-I and CV antisera, but with intermediate reactivity, while one strain was not recognized at all. A partly similar recognition pattern was observed when purified lipopolysaccharide (LPS) was used as antigen in the examination of antibody reactivity in Western blotting. The level of protection was highly dependent on both the vaccine and the strain used for challenge and showed no consistent correlation with antibody reactivity. Secondly, we attempted to use a bacterin vaccine based on one of the V. anguillarum O2a strains intermediately recognized by both EV-I and CV antisera to investigate whether that could potentially provide protection across strain variability. The immunized fish did mount a cross-reactive antibody response, but protection still varied depending on the strain used for challenge. Interestingly, the grouping of strains according to antibody reactivity correlated not only with genotyping based on single nucleotides polymorphisms analysis (SNP) but also with variability in the accessory genome, indicating that presence or absence of protein antigens or proteins associated with the biosynthesis of antigenic epitopes may explain the observed distinct serological subgrouping within VaO2a strains by trout immune sera. In terms of vaccination against VaO2a, our results demonstrate that it is important to take (local) antigen variations into account when using bacterin-based vaccines but also that alternatives to traditional bacterin-based vaccines might be needed to induce protection against the highly virulent Vibrio anguillarum serotype O2a strains.

A density-based enrichment measure for assessing colocalization in single-molecule localization microscopy data.

Dual-color single-molecule localization microscopy (SMLM) provides unprecedented possibilities for detailed studies of colocalization of different molecular species in a cell. However, the informational richness of the data is not fully exploited by current analysis tools that often reduce colocalization to a single value. Here, we describe a tool specifically designed for determination of co-localization in both 2D and 3D from SMLM data. The approach uses a function that describes the relative enrichment of one molecular species on the density distribution of a reference species. The function reframes the question of colocalization by providing a density-context relevant to multiple biological questions. Moreover, the function visualize enrichment (i.e. colocalization) directly in the images for easy interpretation. We demonstrate the approach's functionality on both simulated data and cultured neurons, and compare it to current alternative measures. The method is available in a Python function for easy and parameter-free implementation.

Gill Transcriptomic Responses to Toxin-producing Alga Prymnesium parvum in Rainbow Trout.

The gill of teleost fish is a multifunctional organ involved in many physiological processes, including protection of the mucosal gill surface against pathogens and other environmental antigens by the gill-associated lymphoid tissue (GIALT). Climate change associated phenomena, such as increasing frequency and magnitude of harmful algal blooms (HABs) put extra strain on gill function, contributing to enhanced fish mortality and fish kills. However, the molecular basis of the HAB-induced gill injury remains largely unknown due to the lack of high-throughput transcriptomic studies performed on teleost fish in laboratory conditions. We used juvenile rainbow trout (Oncorhynchus mykiss) to investigate the transcriptomic responses of the gill tissue to two (high and low) sublethal densities of the toxin-producing alga Prymnesium parvum, in relation to non-exposed control fish. The exposure time to P. parvum (4-5 h) was sufficient to identify three different phenotypic responses among the exposed fish, enabling us to focus on the common gill transcriptomic responses to P. parvum that were independent of dose and phenotype. The inspection of common differentially expressed genes (DEGs), canonical pathways, upstream regulators and downstream effects pointed towards P. parvum-induced inflammatory response and gill inflammation driven by alterations of Acute Phase Response Signalling, IL-6 Signalling, IL-10 Signalling, Role of PKR in Interferon Induction and Antiviral Response, IL-8 Signalling and IL-17 Signalling pathways. While we could not determine if the inferred gill inflammation was progressing or resolving, our study clearly suggests that P. parvum blooms may contribute to the serious gill disorders in fish. By providing insights into the gill transcriptomic responses to toxin-producing P. parvum in teleost fish, our research opens new avenues for investigating how to monitor and mitigate toxicity of HABs before they become lethal.

Nervous Necrosis Virus-like Particle (VLP) Vaccine Stimulates European Sea Bass Innate and Adaptive Immune Responses and Induces Long-Term Protection against Disease.

The rapidly increasing Mediterranean aquaculture production of European sea bass is compromised by outbreaks of viral nervous necrosis, which can be recurrent and detrimental. In this study, we evaluated the duration of protection and immune response in sea bass given a single dose of a virus-like particle (VLP)-based vaccine. Examinations included experimental challenge with nervous necrosis virus (NNV), serological assays for NNV-specific antibody reactivity, and immune gene expression analysis. VLP-vaccinated fish showed high and superior survival in challenge both 3 and 7.5 months (1800 and 4500 dd) post-vaccination (RPS 87 and 88, OR (surviving) = 16.5 and 31.5, respectively, p < 0.01). Although not providing sterile immunity, VLP vaccination seemed to control the viral infection, as indicated by low prevalence of virus in the VLP-vaccinated survivors. High titers of neutralizing and specific antibodies were produced in VLP-vaccinated fish and persisted for at least ~9 months post-vaccination as well as after challenge. However, failure of immune sera to protect recipient fish in a passive immunization trial suggested that other immune mechanisms were important for protection. Accordingly, gene expression analysis revealed that VLP-vaccination induced a mechanistically broad immune response including upregulation of both innate and adaptive humoral and cellular components (mx, isg12, mhc I, mhc II, igm, and igt). No clinical side effects of the VLP vaccination at either tissue or performance levels were observed. The results altogether suggested the VLP-based vaccine to be suitable for clinical testing under farming conditions.

Long-Term Protection and Serologic Response of European Sea Bass Vaccinated with a Betanodavirus Virus-Like Particle Produced in Pichia pastoris.

Viral Nervous Necrosis (VNN) causes high mortality and reduced growth in farmed European sea bass (Dicentrarchus labrax) in the Mediterranean. In the current studies, we tested a novel Pichia-produced virus-like particle (VLP) vaccine against VNN in European sea bass, caused by the betanodavirus "Red-Spotted Grouper Nervous Necrosis Virus" (RGNNV). European sea bass were immunized with a VLP-based vaccine formulated with different concentrations of antigen and with or without adjuvant. Antibody response was evaluated by ELISA and serum neutralization. The efficacy of these VLP-vaccine formulations was evaluated by an intramuscular challenge with RGNNV at different time points (1, 2 and 10 months post-vaccination) and both dead and surviving fish were sampled to evaluate the level of viable virus in the brain. The VLP-based vaccines induced an effective protective immunity against experimental infection at 2 months post-vaccination, and even to some degree at 10 months post-vaccination. Furthermore, the vaccine formulations triggered a dose-dependent response in neutralizing antibodies. Serologic response and clinical efficacy, measured as relative percent survival (RPS), seem to be correlated with the administered dose, although for the individual fish, a high titer of neutralizing antibodies prior to challenge was not always enough to protect against disease. The efficacy of the VLP vaccine could not be improved by formulation with a water-in-oil (W/O) adjuvant. The developed RGNNV-VLPs show a promising effect as a vaccine candidate, even without adjuvant, to protect sea bass against disease caused by RGNNV. However, detection of virus in vaccinated survivors means that it cannot be ruled out that survivors can transmit the virus.

Zebrafish (Danio rerio) larvae as a model for real-time studies of propagating VHS virus infection, tissue tropism and neutrophil activity.

Viral haemorrhagic septicaemia virus (VHSV) is a negative-sense single-stranded RNA virus that infects more than 140 different fish species. In this study, zebrafish larvae were employed as in vivo model organisms to investigate progression of disease, the correlation between propagation of the infection and irreversibility of disease, cell tropism and in situ neutrophil activity towards the VHSV-infected cells. A recombinant VHSV strain, encoding "tomato" fluorescence (rVHSV-Tomato), was used in zebrafish to be able to follow the progress of the infection in the live host in real-time. Two-day-old zebrafish larvae were injected into the yolk sac with the recombinant virus. The virus titre peaked 96 hr post-infection in zebrafish larvae kept at 18°C, and correlated with 33% mortality and high morbidity among the larvae. By utilizing the transgenic zebrafish line Tg(fli1:GFP)y1 with fluorescently tagged endothelial cells, we were able to demonstrate that the virus initially infected endothelial cells lining the blood vessels. By observing the rVHSV-Tomato infection in the neutrophil reporter zebrafish line Tg(MPX:eGFP)i114 , we inferred that only a subpopulation of the neutrophils responded to the virus infection. We conclude that the zebrafish larvae are suitable for real-time studies of VHS virus infections, allowing in vivo dissection of host-virus interactions at the whole organism level.

A pentavalent vaccine for rainbow trout in Danish aquaculture.

Mariculture in Denmark is based on production of rainbow trout grown two years in fresh water followed by one growth season in sea cages. Although the majority of rainbow trout are vaccinated against the most serious bacterial pathogens - Aeromonas salmonicida subsp. salmonicida, Vibrio anguillarum and Yersinia ruckeri, by the use of commercially available vaccines, disease outbreaks requiring treatment with antibiotics still occur. The present study tested the potential of a new experimental multicomponent vaccine that is based on local bacterial strains, isolated from rainbow trout in Danish waters, and thus custom-designed for Danish rainbow trout mariculture. The vaccination with the multicomponent vaccine resulted in protection against three relevant bacterial diseases (yersiniosis, furunculosis, vibriosis) under experimental conditions. We showed that i.p. injection of the vaccine induced specific antibody responses in trout against the different bacterial antigens and regulated expression of genes encoding SAA, C3, IL-1ß, IL-6, IL-8, IgD and MHCII.

DNA vaccination for finfish aquaculture.

In fish, DNA vaccines have been shown to give very high protection in experimental facilities against a number of viral diseases, particularly diseases caused by rhabdoviruses. However, their efficacy in generating protection against other families of fish viral pathogens is less clear. One DNA vaccine is currently in use commercially in fish farms in Canada and the commercialisation of another was authorised in Europe in 2017. The mechanism of action of DNA vaccines, including the role of the innate immune responses induced shortly after DNA vaccination in the activation of the adaptive immunity providing longer term specific protection, is still not fully understood. In Europe the procedure for the commercialisation of a veterinary DNA vaccine requires the resolution of certain concerns particularly about safety for the host vaccinated fish, the consumer and the environment. Relating to consumer acceptance and particularly environmental safety, a key question is whether a DNA vaccinated fish is considered a Genetically Modified Organism (GMO). In the present opinion paper these key aspects relating to the mechanisms of action, and to the development and the use of DNA vaccines in farmed fish are reviewed and discussed.

Pichia pastoris yeast as a vehicle for oral vaccination of larval and adult teleosts.

Oral vaccination is of major interest because it can be used for mass vaccination of fish of various size and age. Given that their administration is relatively easy and stress-free, oral vaccines have both economic and animal welfare benefits. Yet, mostly due to their limited efficacy, only very few oral vaccines are available to aquaculture industry. Here we present a method for oral vaccine delivery based on the yeast Pichia pastoris. We could express a model antigen, green fluorescent protein (GFP), in this yeast and subsequently show delivery of the GFP protein to the intestine of juvenile flounder or adult carp and trout. We tested this approach in several commercially-relevant fish species, from juvenile to adult stage. To test the oral delivery of antigen to larval fish, the GFP-expressing Pichia pastoris was first fed to planktonic crustacean Daphnia or rotifers that served as 'bioencapsulation vehicles' and afterwards, fed to flounder larvae. Again, we could show delivery of intact GFP protein to the intestine. In rainbow trout, the orally-administered GFP-expressing yeast elicited a rapid local innate immune response in the intestine and a subsequent systemic response in the spleen. Our results show that Pichia pastoris is a good vehicle for oral antigen delivery and that it can be used in non-encapsulated form for older fish or in bioencapsulated form for larval fish. We discuss the immunomodulatory properties of the yeast itself, and its potential to enhance local immune responses and act as an adjuvant.

Time-course study of the protection induced by an interferon-inducible DNA vaccine against viral haemorrhagic septicaemia in rainbow trout.

The highly effective DNA vaccines against diseases caused by fish rhabdoviruses in farmed fish consist of a DNA plasmid vector encoding the viral glycoprotein under the control of a constitutive cytomegalovirus promoter (CMV). Among others, attempts to improve efficacy and safety of these DNA vaccines have focused on regulatory elements of plasmid vectors, which play a major role in controlling expression levels of vaccine antigens. Depending on the context, use of a fish-derived promoter with minimal activity in mammalian cells could be preferable. Another aspect related to the CMV promoter is that constitutive expression of the vaccine antigen may lead to rapid elimination of antigen expressing cells in the fish and thereby potentially reduce the long-term effects of the vaccine. In this study, we compared DNA vaccines with the interferon-inducible Mx promoter from rainbow trout and the CMV promoter, respectively. Plasmid constructs encoding the enhanced green fluorescent protein (EGFP) were used for the in vitro analysis, whereas DNA vaccines encoding the glycoprotein (G) of the viral haemorrhagic septicaemia virus (VHSV) were applied for the in vivo examination. The in vitro analysis showed that while the DNA vaccine with the CMV promoter constitutively drove the expression of EGFP in both fish and human cell lines, the DNA vaccine with the Mx promoter inducibly enhanced the expression of EGFP in the fish cell line. To address the impact on protection, a time-course model was followed as suggested by Kurath et al. (2006), where vaccinated fish were challenged with VHSV at 2, 8 and 78 weeks post-vaccination (wpv). The DNA vaccine with the CMV promoter protected at all times, while vaccination with the DNA vaccine containing the Mx promoter only protected the fish at 8 wpv. However, following induction with Poly (I:C) one week before the challenge, high protection was also evident at 2 wpv. In conclusion, the results revealed a more fish host dependent activity of the trout Mx promoter compared to the traditionally used cross species-active CMV promoter, but improvements will be needed for its application in DNA vaccines to ensure long term protection.

Virulence marker candidates in N-protein of viral haemorrhagic septicaemia virus (VHSV): virulence variability within VHSV Ib clones.

Four major genotypes of viral haemorrhagic septicaemia virus (VHSV), which have been isolated from many marine and freshwater fish species, are known to differ in virulence. While fast and low-cost genotyping systems based on monoclonal antibodies (MAbs) have been developed for typing of VHSV virulence, there is a need for supplementing the knowledge. In particular, 2 field isolates from viral haemorrhagic septicaemia (VHS) outbreaks in sea-reared rainbow trout Oncorhynchus mykiss in Sweden, SE-SVA-14 and SE-SVA-1033 (both genotype Ib), have yielded contradictory reactions. In the present study, upon cloning by limited dilution, both isolates appeared to be heterogeneous in terms of reactivity with nucleo (N)-protein-specific MAbs as well their gene sequences. Infection trials in rainbow trout further revealed differences in the virulence of these virus clones derived from the same primary isolate. Based on a comparative analysis of the entire genome of the clones tested, we suggest that the differences in virulence are tentatively linked to substitutions of amino acids (aa) in the N-protein region covered by aa 43-46 and aa position 168, or a combination of the two. The fact that such minor naturally occurring genetic differences affect the virulence implies that even low-virulent VHSV isolates in the marine environment should be considered as a potential threat for the trout farming industry. The described MAbs can represent useful tools for initial risk assessment of disease outbreaks in farmed trout by marine VHSV isolates.

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.

Influence of Temperature on the Efficacy of Homologous and Heterologous DNA Vaccines against Viral Hemorrhagic Septicemia in Pacific Herring.

Homologous and heterologous (genogroup Ia) DNA vaccines against viral hemorrhagic septicemia virus (genogroup IVa) conferred partial protection in Pacific Herring Clupea pallasii. Early protection at 2 weeks postvaccination (PV) was low and occurred only at an elevated temperature (12.6°C, 189 degree days), where the relative percent survival following viral exposure was similar for the two vaccines (IVa and Ia) and higher than that of negative controls at the same temperature. Late protection at 10 weeks PV was induced by both vaccines but was higher with the homologous vaccine at both 9.0°C and 12.6°C. Virus neutralization titers were detected among 55% of all vaccinated fish at 10 weeks PV. The results suggest that the immune response profile triggered by DNA vaccination of herring was similar to that reported for Rainbow Trout Oncorhynchus mykiss by Lorenzen and LaPatra in 2005, who found interferon responses in the early days PV and the transition to adaptive response later. However, the protective effect was far less prominent in herring, possibly reflecting different physiologies or adaptations of the two fish species. Received August 1, 2016; accepted March 10, 2017.

Can VHS Virus Bypass the Protective Immunity Induced by DNA Vaccination in Rainbow Trout?

DNA vaccines encoding viral glycoproteins have been very successful for induction of protective immunity against diseases caused by rhabdoviruses in cultured fish species. However, the vaccine concept is based on a single viral gene and since RNA viruses are known to possess high variability and adaptation capacity, this work aimed at evaluating whether viral haemorrhagic septicaemia virus (VHSV), an RNA virus and member of Rhabdoviridae family, was able to evade the protective immune response induced by the DNA vaccination of rainbow trout. The experiments comprised repeated passages of a highly pathogenic VHSV isolate in a fish cell line in the presence of neutralizing fish serum (in vitro approach), and in rainbow trout immunized with the VHS DNA vaccine (in vivo approach). For the in vitro approach, the virus collected from the last passage (passaged virus) was as sensitive as the parental virus to serum neutralization, suggesting that the passaging did not promote the selection of virus populations able to bypass the neutralization by serum antibodies. Also, in the in vivo approach, where virus was passaged several times in vaccinated fish, no increased virulence nor increased persistence in vaccinated fish was observed in comparison with the parental virus. However, some of the vaccinated fish did get infected and could transmit the infection to naïve cohabitant fish. The results demonstrated that the DNA vaccine induced a robust protection, but also that the immunity was non-sterile. It is consequently important not to consider vaccinated fish as virus free in veterinary terms.

Ultra-deep sequencing of VHSV isolates contributes to understanding the role of viral quasispecies.

The high mutation rate of RNA viruses enables the generation of a genetically diverse viral population, termed a quasispecies, within a single infected host. This high in-host genetic diversity enables an RNA virus to adapt to a diverse array of selective pressures such as host immune response and switching between host species. The negative-sense, single-stranded RNA virus, viral haemorrhagic septicaemia virus (VHSV), was originally considered an epidemic virus of cultured rainbow trout in Europe, but was later proved to be endemic among a range of marine fish species in the Northern hemisphere. To better understand the nature of a virus quasispecies related to the evolutionary potential of VHSV, a deep-sequencing protocol specific to VHSV was established and applied to 4 VHSV isolates, 2 originating from rainbow trout and 2 from Atlantic herring. Each isolate was subjected to Illumina paired end shotgun sequencing after PCR amplification and the 11.1 kb genome was successfully sequenced with an average coverage of 0.5-1.9 × 10(6) sequenced copies. Differences in single nucleotide polymorphism (SNP) frequency were detected both within and between isolates, possibly related to their stage of adaptation to host species and host immune reactions. The N, M, P and Nv genes appeared nearly fixed, while genetic variation in the G and L genes demonstrated presence of diverse genetic populations particularly in two isolates. The results demonstrate that deep sequencing and analysis methodologies can be useful for future in vivo host adaption studies of VHSV.

Sublethal concentrations of ichthyotoxic alga Prymnesium parvum affect rainbow trout susceptibility to viral haemorrhagic septicaemia virus.

Ichthyotoxic algal blooms are normally considered a threat to maricultured fish only when blooms reach lethal cell concentrations. The degree to which sublethal algal concentrations challenge the health of the fish during blooms is practically unknown. In this study, we analysed whether sublethal concentrations of the ichthyotoxic alga Prymnesium parvum affect the susceptibility of rainbow trout Oncorhynchus mykiss to viral haemorrhagic septicaemia virus (VHSV). During exposure to sublethal algal concentrations, the fish increased production of mucus on their gills. When fish were exposed to the algae for 12 h prior to the addition of virus, a marginal decrease in the susceptibility to VHSV was observed compared to fish exposed to VHSV without algae. If virus and algae were added simultaneously, inclusion of the algae increased mortality by 50% compared to fish exposed to virus only, depending on the experimental setup. We concluded that depending on the local exposure conditions, sublethal concentrations of P. parvum could affect susceptibility of fish to infectious agents such as VHSV.

Ichthyotoxicity of the microalga Pseudochattonella farcimen under laboratory and field conditions in Danish waters.

Blooms of the marine dictyochophyte Pseudochattonella farcimen have been associated with fish kills, but attempts to verify ichthyotoxicity of this microalga under experimental conditions have not been successful. In the early spring of 2009 and 2011, P. farcimen bloomed in the inner Danish waters. The blooms occurred at a seawater temperature of ~2°C and correlated with extensive kills of farmed salmonid fish (2009) and wild populations (2011). Several strains of P. farcimen were isolated from the 2009 bloom. However, exposure of rainbow trout Oncorhynchus mykiss to laboratory-grown P. farcimen cultures did not reveal any toxic effects. During the 2011 bloom, fish were exposed to bloom water under both laboratory and field conditions. While no clinical effect was observed on fish incubated in bloom water in the laboratory trial, a remarkable difference was seen in the field trial between rainbow trout kept in tanks supplied with a continuous flow of filtered versus non-filtered bloom water. Histological examination of the gill tissue revealed karyorrhexis and epithelial loosening in the affected fish. Microscopy analysis of algal cell morphology suggested that mucocysts detected on the cell surface only in freshly sampled bloom water might be associated with ichtyotoxicity.