Consequences of Haemorrhagic Smolt Syndrome (HSS) for the Immune Status of Atlantic salmon (Salmo salar L.) (Case Study).

Haemorrhagic smolt syndrome (HSS) is a disorder of unknown aetiology causing losses in the fresh water phase of Atlantic salmon farming. Normally, the mortality is limited and symptoms disappear upon seawater exposure. In this case study, classical HSS pathology with internal organ haemorrhages and nephrocalcinosis was diagnosed, and the losses were substantial. Microarray analyses of head kidney revealed association between HSS and enhanced expression of stress genes and proteins reducing bioavailability of iron, heme, and retinol. In parallel, suppression of multiple metabolic pathways was observed. Up-regulation of genes encoding acute phase proteins, complement, and lectins indicated mild inflammation but without characteristic features of viral or bacterial infections. Microarray analyses highlighted several members of tumor necrosis factor receptor superfamily that may control development of B-cell immunity. Examination of IgM at the mRNA and protein levels showed the impact of HSS on vaccine responses. In fish without HSS symptoms (non-HSS), titres of vaccine specific antibodies to A-layer of Aeromonas salmonicida subsp. salmonicida and Moritella viscosa and antibodies binding to DNP-keyhole limpet hemocyanin (DNP-KLH), which are presumably polyreactive, were respectively four- and 14-fold higher than in HSS-diseased fish. Parallel sequencing of variable regions of immunoglobulin Mrevealed a larger size of most abundant clonotypes shared by multiple individuals in the non-HSS group. The results of the current case study indicated that, in addition to direct damage, HSS suppresses humoral immune responses including the production of specific and polyreactive antibodies.

A time-course study of gene expression and antibody repertoire at early time post vaccination of Atlantic salmon.

The majority of studies of vaccine responses in Atlantic salmon have focused on several weeks after vaccination, and employed a limited number of marker genes. In this study, novel techniques were used to examine a broad panel of expressed genes and antibody repertoire of Atlantic salmon following vaccination. Salmon parr were vaccinated with a multivalent oil-based vaccine, and blood plasma and head kidney were sampled at several time-points between 0-35 days post vaccination. Saline-injected fish were used as control at all time-points. Microarray analyses showed increased expression of immune genes from the first day to the end of study in the head kidney of vaccinated fish. Genes up-regulated in the late phase included several leukocyte markers and components of the oxidative burst complex. A suite of genes that can take part in B cells differentiation were up-regulated from day 14, at which time secretory IgM transcripts also peaked. This coincided with marked increased plasma titres of non-vaccine specific antibodies binding to a hapten-carrier antigen DNP-KLH, while antibodies to bacterial components of the vaccine, Moritella viscosa and Aeromonas salmonicida, first showed significantly elevated antibody levels at day 21, and at a markedly lower magnitude than the non-vaccine specific titres. Sequencing of the variable region of IgM heavy chain (CDR3) revealed higher cumulative frequencies of unique clonotypes in vaccinated salmon starting from day 14 when specific antibodies were first detected. Reduced sequence variance of CDR3 suggested expansion of recently emerged clonotypes. Overall, the results presented here follow a broad panel of gene expression, immunoglobulin sequencing and plasma antibody titres in the first few weeks after vaccination of Atlantic salmon, pointing to a potentially important contribution of non-vaccine specific antibody responses early in the vaccine response.

Vaccination against pancreas disease in Atlantic salmon, Salmo salar L., reduces shedding of salmonid alphavirus.

Salmon pancreas disease virus, often referred to as salmonid alphavirus (SAV), causes pancreas disease (PD) in European salmonids. SAV transmits horizontally from fish shedding virus into the water and ocean currents are believed to be a main contributor of viral spread between marine farms. Vaccination against PD is previously shown to reduce mortality and severity of clinical PD. In this study, we demonstrate that vaccination against PD significantly reduces viral shedding from infected individuals. The results suggest that PD vaccination can be an important tool to reduce the infection pressure, a known key risk for PD outbreaks at neighbouring farms.

Phenotypic and genetic characterization of Piscirickettsia salmonis from Chilean and Canadian salmonids.

BACKGROUND: The study presents the phenotypic and genetic characterization of selected P. salmonis isolates from Atlantic salmon and rainbow trout suffering from SRS (salmonid rickettsial septicemia) in Chile and in Canada. The phenotypic characterization of the P. salmonis isolates were based on growth on different agar media (including a newly developed medium), different growth temperatures, antibiotics susceptibility and biochemical tests. RESULTS: This is the first study differentiating Chilean P. salmonis isolates into two separate genetic groups. Genotyping, based on 16S rRNA-ITS and concatenated housekeeping genes grouped the selected isolates into two clades, constituted by the Chilean strains, while the Canadian isolates form a branch in the phylogenetic tree. The latter consisted of two isolates that were different in both genetic and phenotypic characteristics. The phylogenies and the MLST do not reflect the origin of the isolates with respect to host species. The isolates included were heterogeneous in phenotypic tests. CONCLUSIONS: The genotyping methods developed in this study provided a tool for separation of P. salmonis isolates into distinct clades. The SRS outbreaks in Chile are caused by minimum two different genetic groups of P. salmonis. This heterogeneity should be considered in future development of vaccines against this bacterium in Chile. Two different strains of P. salmonis, in regards to genetic and phenotypic characteristics, can occur in the same contemporary outbreak of SRS.

A trypsin-like protease with apparent dual function in early Lepeophtheirus salmonis (Krøyer) development.

BACKGROUND: Trypsin-like serine proteases are involved in a large number of processes including digestive degradation, regulation of developmental processes, yolk degradation and yolk degradome activation. Trypsin like peptidases considered to be involved in digestion have been characterized in Lepeophtheirus salmonis. During these studies a trypsin-like peptidase which differed in a number of traits were identified. RESULTS: An intronless trypsin-like serine peptidase (LsTryp10) from L., salmonis was identified and characterized. LsTryp10 mRNA is evenly distributed in the ovaries and oocytes, but is located along the ova periphery. LsTryp10 protein is deposited in the oocytes and all embryonic cells. LsTryp10 mRNA translation and concurrent degradation after fertilization was found in the embryos demonstrating that LsTryp10 protein is produced both by the embryo and maternally. The results furthermore indicate that LsTryp10 protein of maternal origin has a distribution pattern different to that of embryonic origin. CONCLUSION: Based on present data and previous studies of peptidases in oocytes and embryos, we hypothesize that maternally deposited LsTryp10 protein is involved in regulation of the yolk degradome. The function of LsTryp10 produced by the embryonic cells remains unknown. To our knowledge a similar expression pattern has not previously been reported for any protease.

Vaccines for fish in aquaculture.

Vaccination plays an important role in large-scale commercial fish farming and has been a key reason for the success of salmon cultivation. In addition to salmon and trout, commercial vaccines are available for channel catfish, European seabass and seabream, Japanese amberjack and yellowtail, tilapia and Atlantic cod. In general, empirically developed vaccines based on inactivated bacterial pathogens have proven to be very efficacious in fish. Fewer commercially available viral vaccines and no parasite vaccines exist. Substantial efficacy data are available for new fish vaccines and advanced technology has been implemented. However, before such vaccines can be successfully commercialized, several hurdles have to be overcome regarding the production of cheap but effective antigens and adjuvants, while bearing in mind environmental and associated regulatory concerns (e.g., those that limit the use of live vaccines). Pharmaceutical companies have performed a considerable amount of research on fish vaccines, however, limited information is available in scientific publications. In addition, salmonids dominate both the literature and commercial focus, despite their relatively small contribution to the total volume of farmed fish in the world. This review provides an overview of the fish vaccines that are currently commercially available and some viewpoints on how the field is likely to evolve in the near future.

Protection against Atlantic halibut nodavirus in turbot is induced by recombinant capsid protein vaccination but not following DNA vaccination.

Fish nodaviruses (betanodaviruses) are small, non-enveloped icosahedral single-stranded positive-sense RNA viruses that can cause viral encephalopathy and retinopathy (VER) in a number of cultured marine teleost species, including Atlantic halibut (Hippoglossus hippoglossus). A recombinant protein vaccine and a DNA vaccine were produced, based on the same capsid-encoding region of the Atlantic halibut nodavirus (AHNV) genome, and tested for protection in juvenile turbot (Scophthalmus maximus). Vaccine efficacy was demonstrated in the fish vaccinated with recombinant capsid protein but not in the DNA-vaccinated fish, despite the fact that in vivo expression of the DNA vaccine-encoded antigen was confirmed by RNA in situ hybridisation and immunohistochemistry. Combined DNA and recombinant vaccine administration did not improve the effect of the latter. Surprisingly, fish vaccinated with 50 microg recombinant protein demonstrated a threefold lower survival rate than the two groups that received 10 microg recombinant protein. Neither the recombinant protein vaccine nor the DNA vaccine induced anti-viral antibodies 9 weeks after immunisation, while antibodies reactive with the recombinant protein were detectable mainly in fish vaccinated with 50 microg recombinant protein. The study also demonstrates evidence of viral replication inside the myocytes of intramuscularly challenged fish.

Complete sequence of RNA1 and subgenomic RNA3 of Atlantic halibut nodavirus (AHNV).

The Nodaviridae are divided into the alphanodavirus genus, which infects insects, and the betanodavirus genus, which infects fishes. Betanodaviruses are the causative agent of viral encephalopathy and retinopathy (VER) in a number of cultivated marine fish species. The Nodaviridae are small non-enveloped RNA viruses that contain a genome consisting of 2 single-stranded positivesense RNA segments: RNA1 (3.1 kb), which encodes the viral part of the RNA-dependent RNA polymerase (RdRp); and RNA2 (1.4 kb), which encodes the capsid protein. In addition to RNA1 and RNA2, a subgenomic transcript of RNA1, RNA3, is present in infected cells. We have cloned and sequenced RNA1 from the Atlantic halibut Hippoglossus hippoglossus nodavirus (AHNV), and for the first time, the sequence of a betanodaviral subgenomic RNA3 has been determined. AHNV RNA1 was 3100 nucleotides in length and contained a main open reading frame encoding a polypeptide of 981 amino acids. Conservative motifs for RdRp were found in the deduced amino acid sequence. RNA3 was 371 nucleotides in length, and contained an open reading frame encoding a peptide of 75 amino acids corresponding to a hypothetical B2 protein, although sequence alignments with the alphanodavirus B2 proteins showed only marginal similarities. AHNV RNA replication in the fish cell-line SSN-1 (derived from striped snakehead) was analysed by Northern blot analysis, which indicated that RNA3 was synthesised in large amounts (compared to RNA1) at an early point in time post-infection.

A DNA vaccine directed against a rainbow trout rhabdovirus induces early protection against a nodavirus challenge in turbot.

A DNA vaccine encoding the envelope glycoprotein from a fish rhabdovirus, viral hemorrhagic septicemia virus (VHSV), has previously been shown to induce both early and long time protection against the virus in rainbow trout. Challenge experiments have revealed that the immunity established shortly after vaccination is cross-protective against heterologous fish rhabdoviruses. In this study, we show that the DNA vaccine encoding the VHSV glycoprotein also induces early protection against a non-enveloped, positive-sense RNA virus belonging to the Nodavirus family, the Atlantic halibut nodavirus (AHNV). In a vaccine efficacy test using juvenile turbot as model fish, the fish injected with the VHSV vaccine were completely protected against a nodavirus challenge performed 8 days post vaccination, while the cumulative mortality in the control group reached 54%. A DNA vaccine carrying the gene encoding the capsid protein of AHNV revealed no protective properties against the nodavirus challenge. Histological examination of muscle tissue sections from the vaccine injection site showed that the DNA vaccine against VHSV triggered a pronounced inflammatory response in turbot similar to what has earlier been observed in rainbow trout.