Effect of two constant light regimens on antibody profiles and immune gene expression in Atlantic salmon following vaccination and experimental challenge with salmonid alphavirus.

Before seawater transfer, farmed Atlantic salmon are subjected to treatments that may affect the immune system and susceptibility to pathogens. E.g., exposure to constant light (CL) stimulates smoltification, which prepares salmon to life in sea water, but endocrine changes in this period are associated with suppression of immune genes. Salmon are vaccinated towards end of the freshwater period to safeguard that adequate vaccine efficacy is achieved by the time the fish is transferred to sea. In the present study, we investigated how the responses to vaccination and viral infection varied depending on the time of CL onset relative to vaccination. The salmon were either exposed to CL two weeks prior to vaccination (2-PRI) or exposed to CL at the time of vaccination (0-PRI). A cohabitant challenge with salmonid alphavirus, the causative agent of pancreatic disease, was performed 9 weeks post vaccination. The immunological effects of the different light manipulation were examined at 0- and 6-weeks post vaccination, and 6 weeks post challenge. Antibody levels in serum were measured using a serological bead-based multiplex panel as well as ELISA, and 92 immune genes in heart and spleen were measured using an integrated fluidic circuit-based qPCR array for multiple gene expression. The 2-PRI group showed a moderate transcript down-regulation of genes in the heart at the time of vaccination, which were restored 6 weeks after vaccination (WPV). Conversely, at 6WPV a down-regulation was seen for the 0-PRI fish. Moreover, the 2-PRI group had significantly higher levels of antibodies binding to three of the vaccine components at 6WPV, compared to 0-PRI. In response to SAV challenge, transcription of immune genes between 2-PRI and 0-PRI was markedly dissimilar in the heart and spleen of control fish, but no difference was found between vaccinated salmon from the two CL regimens. Thus, by using labor-saving high throughput detection methods, we demonstrated that light regimens affected antibody production and transcription of immune genes in non-vaccinated and virus challenged salmon, but the differences between the light treatment groups appeared eliminated by vaccination.

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.

Salmonid alphavirus-based replicon vaccine against infectious salmon anemia (ISA): impact of immunization route and interactions of the replicon vector.

A salmonid alphavirus (SAV)-based replicon encoding the infectious salmon anemia virus (ISAV) hemagglutinin-esterase (HE), pSAV/HE, is an efficacious vaccine against infectious salmon anemia (ISA). Delivered intramuscularly (i.m.), the replicon vaccine provides high protection against subsequent ISAV challenge in Atlantic salmon (Salmo salar), and induces a strong innate response locally at the injection site. This may be beneficial and could warrant reduced doses and improved efficacy compared to conventional DNA vaccines. In the present study, we found that intraperitoneal (i.p.) administration of the pSAV/HE replicon vaccine did not induce protection, neither alone or in combination with a sub-potent, inactivated low-dose ISAV vaccine given i.p. No significant differences between the two immunization routes regarding systemic immune responses could be observed. I.m. injection of the replicon vector encoding a non-viral gene or the protective glycoprotein (G protein) from the heterologous viral hemorrhagic septicemia virus (VHSV) induced no protection against ISA. Although the replicons without the ISAV HE did induce IFN-signaling pathways at the muscle injection site similar to the pSAV/HE replicon they did not improve the efficacy of a sub-potent inactivated low-dose ISAV vaccine delivered i.p. Moreover, there was a tendency for reduced efficacy of the pSAV/HE replicon vaccine injected i.m. when co-injected with the replicon encoding the VHSV G protein, which previously, after DNA vaccination, have been reported to induce cross-protection against heterologous virus challenge in fish.

Immune parameters correlating with reduced susceptibility to pancreas disease in experimentally challenged Atlantic salmon (Salmo salar).

Two strains of Atlantic salmon (Salmon salar) with different susceptibility to infectious salmon anaemia (ISA) were challenged with salmon pancreas disease virus (SPDV), the etiological agent of salmon pancreas disease (PD), by cohabitation. Serum and tissues were sampled at 0, 1, 3, 6 and 8 weeks post-challenge. Experimental challenge with SAV did not cause mortality, but virus loads and assessment of histopathology indicated that the fish more resistant to ISAV (ISAHi) also was more resistant to PD. Eight weeks post-challenge, the ISAHi strain had higher titres of SAV-neutralising antibodies than the less resistant strain (ISALo). Transcript levels of four adaptive and six innate immune parameters were analysed by real-time RT-PCR in heart, head kidney (HK) and gills of both strains. Secretory IgM (sIgM) and CD8 levels differed most between the two salmon strains. The ISAHi strain had significantly higher levels of sIgM in HK at all samplings, and significantly higher CD8 levels in gills at most samplings. In heart, both sIgM and CD8 levels increased significantly during the challenge, but the increase appeared earlier for the ISALo strain. By hierarchical clustering analysis of mRNA levels, a clear segregation was observed between the two strains prior to the virus challenge. As the viral infection developed, the clustering divide between fish strains disappeared, first for innate and later for adaptive parameters. At eight weeks post-challenge, the divide had however reformed for adaptive parameters. Possible pair-wise correlation between transcript levels of immune parameters was evaluated by a non-parametric statistical test. For innate parameters, the extent of correlation peaked at 3 wpc in all tissues; this came rapidly for ISALo and more gradual for ISAHi. The ISAHi strain tended to show higher correlation for innate parameters in heart and gill than ISALo at early sampling times. For adaptive immune parameters, little correlation was observed in general, except for ISAHi in heart at 6 wpc. Overall, the observed differences in immune parameters may provide important clues to the causes underlying the observed difference in susceptibility to PD.

Transcription of reference genes used for quantitative RT-PCR in Atlantic salmon is affected by viral infection.

Relative quantification using RT-qPCR is a widely used method for transcription profiling. Transcript levels of target genes in fish after experimental infection is often reported without documentation of stably transcribed reference genes. We present results demonstrating that transcription of typically used reference genes in Atlantic salmon is not stable during experimental infection with salmon pancreas disease virus (SPDV). Transcript levels 0 to 6 weeks after challenge revealed statistically significant changes between time-points that corresponded with a peak in viral load 3 weeks after challenge. The results emphasize the need for thorough method validation prior to transcriptional studies during viral infections.

Characterisation of two vitellogenins in the salmon louse Lepeophtheirus salmonis: molecular, functional and evolutional analysis.

The salmon louse Lepeophtheirus salmonis Krøyer affects a variety of wild salmonoid hosts, but is also an important pest in aquaculture, which is a globally important and rapidly growing industry. Salmon lice have large reproductive outputs, and knowledge of reproductive processes may be crucial for the control of this parasite. Here, we report on the characterisation of 2 vitellogenins (LsVit1 and LsVit2), which are the precursors of salmon-louse egg-yolk glycoprotein. The structure of LsVit1 and LsVit2 was examined and compared to that in other oviparous animals. Phylogenetic analysis of LsVit1 and LsVit2 confirmed the view that crustaceans are a polyphyletic group. Transcriptional and translational analysis demonstrated production of LsVit1 and LsVit2 in the subcuticular tissue of the adult female lice. LsVit1 and LsVit2 could also be found in maturing oocytes and developing embryos and early larval stages. LsVit2 was found to be processed into 2 smaller fragments, whereas LsVit1 was found to be full length when deposited into the oocytes. Degradation of LsVit1 and LsVit2 was characterised through embryogenesis and the early non-feeding larval stages. Finally, protein content and the level of free amino acids were analysed in embryos and larval stages and their role in nutrition and osmoregulation discussed. In conclusion, our results confirm the role of vitellogenins in reproduction as providers of embryonic and larval nutrition.

IgM+ and IgT+ B Cell Traffic to the Heart during SAV Infection in Atlantic Salmon.

B cells of teleost fish differentiate in the head kidney, and spleen, and either remain in the lymphatic organs or move to the blood and peripheral tissues. There is limited knowledge about piscine B cell traffic to sites of vaccination and infection and their functional roles at these sites. In this work, we examined the traffic of B cells in Atlantic salmon challenged with salmonid alphavirus (SAV). In situ hybridization (RNAScope) showed increased numbers of immunoglobin (Ig)M+ and IgT+ B cells in the heart in response to SAV challenge, with IgM+ B cells being most abundant. An increase in IgT+ B cells was also evident, indicating a role of IgT+ B cells in nonmucosal tissues and systemic viral infections. After infection, B cells were mainly found in the stratum spongiosum of the cardiac ventricle, colocalizing with virus-infected myocardial-like cells. From sequencing the variable region of IgM in the main target organ (heart) and comparing it with a major lymphatic organ (the spleen), co-occurrence in antibody repertoires indicated a transfer of B cells from the spleen to the heart, as well as earlier recruitment of B cells to the heart in vaccinated fish compared to those that were unvaccinated. Transcriptome analyses performed at 21 days post-challenge suggested higher expression of multiple mediators of inflammation and lymphocyte-specific genes in unvaccinated compared to vaccinated fish, in parallel with a massive suppression of genes involved in heart contraction, metabolism, and development of tissue. The adaptive responses to SAV in vaccinated salmon appeared to alleviate the disease. Altogether, these results suggest that migration of B cells from lymphatic organs to sites of infection is an important part of the adaptive immune response of Atlantic salmon to SAV.

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.

Salmon louse (Lepeophtheirus salmonis) transcriptomes during post molting maturation and egg production, revealed using EST-sequencing and microarray analysis.

BACKGROUND: Lepeophtheirus salmonis is an ectoparasitic copepod feeding on skin, mucus and blood from salmonid hosts. Initial analysis of EST sequences from pre adult and adult stages of L. salmonis revealed a large proportion of novel transcripts. In order to link unknown transcripts to biological functions we have combined EST sequencing and microarray analysis to characterize female salmon louse transcriptomes during post molting maturation and egg production. RESULTS: EST sequence analysis shows that 43% of the ESTs have no significant hits in GenBank. Sequenced ESTs assembled into 556 contigs and 1614 singletons and whenever homologous genes were identified no clear correlation with homologous genes from any specific animal group was evident. Sequence comparison of 27 L. salmonis proteins with homologous proteins in humans, zebrafish, insects and crustaceans revealed an almost identical sequence identity with all species. Microarray analysis of maturing female adult salmon lice revealed two major transcription patterns; up-regulation during the final molting followed by down regulation and female specific up regulation during post molting growth and egg production. For a third minor group of ESTs transcription decreased during molting from pre-adult II to immature adults. Genes regulated during molting typically gave hits with cuticula proteins whilst transcripts up regulated during post molting growth were female specific, including two vitellogenins. CONCLUSION: The copepod L.salmonis contains high a level of novel genes. Among analyzed L.salmonis proteins, sequence identities with homologous proteins in crustaceans are no higher than to homologous proteins in humans. Three distinct processes, molting, post molting growth and egg production correlate with transcriptional regulation of three groups of transcripts; two including genes related to growth, one including genes related to egg production. The function of the regulated transcripts is discussed in relation to post molting morphological changes in adult female salmon louse. There is clear evidence that transcription of the major yolk proteins is not induced before some of the post molting growth of abdomen and the genital segment has occurred. A hallmark for the observed growth is transcription of many putative cuticula proteins prior to the size increase.

DNA vaccine expressing the non-structural proteins of Piscine orthoreovirus delay the kinetics of PRV infection and induces moderate protection against heart -and skeletal muscle inflammation in Atlantic salmon (Salmo salar).

Piscine orthoreovirus (PRV) causes heart- and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). Erythrocytes are the main target cells for PRV. HSMI causes significant economic losses to the salmon aquaculture industry, and there is currently no vaccine available. PRV replicates and assembles within cytoplasmic structures called viral factories, mainly organized by the non-structural viral protein µNS. In two experimental vaccination trials in Atlantic salmon, using DNA vaccines expressing different combinations of PRV proteins, we found that expression of the non-structural proteins µNS combined with the cell attachment protein σ1 was associated with an increasing trend in lymphocyte marker gene expression in spleen, and induced moderate protective effect against HSMI.

Molecular characterization and classification of a clip domain containing peptidase from the ectoparasite Lepeophtheirus salmonis (Copepoda, Crustacea).

Clip domain containing serine peptidases (CSPs) include one or more N-terminal clip domain(s) and a C-terminal serine peptidase domain that shares traits with both chymotrypsin and trypsin. CSPs are found in arthropods and are involved in embryonic patterning, immune responses and blood clotting. Among crustaceans only one CSP, which activates prophenoloxidase in crayfish, have previously been reported. We here present LsCSP1, the first CSP found in copepods. LsCSP1 is expressed in the subcuticular tissue and the transcription appears to be upregulated during development. In conjunction with previous studies of CSPs, this study suggests that LsCSP1 may play a role in the immune responses of L. salmonis. Phylogenetic and structural analyses indicate that the CSPs and catalytically inactive CSP homologs (CSPHs) constitute a monophyletic lineage.

Relative transcript quantification by quantitative PCR: roughly right or precisely wrong?

BACKGROUND: When estimating relative transcript abundances by quantitative real-time PCR (Q-PCR) we found that the results can vary dramatically depending on the method chosen for data analysis. RESULTS: Analyses of Q-PCR results from a salmon louse starvation experiment show that, even with apparently good raw data, different analytical approaches 12 may lead to opposing biological conclusions. CONCLUSION: The results emphasise the importance of being cautious when analysing Q-PCR data and indicate that uncritical routine application of an analytical method will eventually result in incorrect conclusions. We do not know the extent of, or have a universal solution to this problem. However, we strongly recommend caution when analysing Q-PCR results e.g. by using two or more analytical approaches to validate conclusions. In our view a common effort should be made to standardise methods for analysis and validation of Q-PCR results.

Complete mutation screening and haplotype characterization of the BRCA1 gene in 61 familial breast cancer patients from Norway.

Mutations in the Breast-Cancer-1 (BRCA1) gene are the major cause of familial breast/ovarian cancer. Among familial breast cancer only, 15-20% have been suggested to have a deleterious mutation in BRCA1. A highly sensitive method (REF-SSCP) was applied to screen the open reading frame and the 5'UTRs of BRCA1 for mutations. The patient cohort comprised 61 unrelated moderate to high risk breast cancer patients from Western-Norway. Only one known deleterious BRCA1 mutation (c.816-817delGT) was found in two of the 61 patients (3.3%). Four haplotypes were established based on nine known single nucleotide polymorphisms. Two patients had a novel deletion (c.-33_-29delAAAAA) in the 5'UTR, and a novel amino acid substitution (L523W) was found in one patient. Size variations analysis in the 5'UTR was repeated in a cohort of 159 unrelated familial breast/ovarian cancer patients and 94 healthy blood donors. Two patients were identified with 5'UTR (c.-30 to -60) variations (CAAAA)5 and (CAAAA)7, instead of the (CAAAA)6-repeat. All of the identified 5'UTR size variations were localized between the start codon and the most stable secondary structures previously proposed for the exon 1b transcript. No such alterations were found among the healthy blood donors but association studies of the 5'UTR variations within the respective families were not conclusive.

Molecular characterisation of five trypsin-like peptidase transcripts from the salmon louse (Lepeophtheirus salmonis) intestine.

Four novel trypsin-like S1A peptidase transcripts (LsTryp2-5) from the marine parasitic copepod Lepeophtheirus salmonis were characterised based on analyses of 1918 expressed sequence tags from two adult female libraries. In addition, one previously described salmon louse trypsin, LsTryp1, has been further characterised. The five peptidases possessed all residues typically found in trypsins in correct sequence contexts. Interestingly, two cysteine residues, possibly involved in a disulphide bridge not previously reported in trypsins are conserved in all louse trypsin sequences. Phylogenetic analyses showed that the five louse peptidases form a monophyletic group with other crustacean trypsins (Brachyurin Ts). Quantitative PCR analyses demonstrated increased transcript levels from planktonic to early host-attached stages and from preadult to sexually mature adult stages. Furthermore, sex-specific differences in transcription regulation were found. In situ hybridisation demonstrated that all five trypsin-like peptidases are transcribed throughout the undifferentiated midgut, indicating a digestive function. The sequence characteristics, histological localisation and transcript regulation suggest that LsTryp1-4 encode typical digestive trypsins. LsTryp5, however, showed some sequence and regulatory peculiarities that rendered its function less clear. Our findings support earlier suggestions for the function of the midgut cells and suggest the existence of an additional undifferentiated cell-type.

Validation of reference genes for transcription profiling in the salmon louse, Lepeophtheirus salmonis, by quantitative real-time PCR.

The salmon louse Lepeophtheirus salmonis, a marine ectoparasitic copepod that feeds on salmonids, cause huge financial losses in the aquaculture industry through fish mortality, reduced growth and therapeutic costs. Detailed knowledge about the salmon louse life cycle, at the functional molecular level, is of fundamental importance to evaluate alternative therapeutic or prophylactic strategies. In quantitative real-time PCR, a powerful technique in biological studies of differentially expressed genes, the transcription level of a regulated gene can be measured relative to an unregulated reference "housekeeping" gene. In the present study we validate candidate reference genes for transcription profiling throughout the life cycle of the salmon louse. Our results show that the structural ribosomal protein S20 (RPS20) and the translation elongation factor 1alpha (eEF1alpha) are valid as reference genes showing less than two fold variation in transcript levels. The frequently used reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), however, is up to six fold regulated during the L. salmonis life cycle. Furthermore our results indicated that 18S RNA, although constitutively expressed, is not a convenient reference gene for relative quantification of most transcripts.

Salmonid alphavirus glycoprotein E2 requires low temperature and E1 for virion formation and induction of protective immunity.

Salmonid alphavirus (SAV; also known as Salmon pancreas disease virus; family Togaviridae) causes pancreas disease and sleeping disease in Atlantic salmon and rainbow trout, respectively, and poses a major burden to the aquaculture industry. SAV infection in vivo is temperature-restricted and progeny virus is only produced at low temperatures (10-15 °C). Using engineered SAV replicons we show that viral RNA replication is not temperature-restricted suggesting that the viral structural proteins determine low-temperature dependency. The processing/trafficking of SAV glycoproteins E1 and E2 as a function of temperature was investigated via baculovirus vectors in Sf9 insect cells and by transfection of CHSE-214 fish cells with DNA constructs expressing E1 and E2. We identified SAV E2 as the temperature determinant by demonstrating that membrane trafficking and surface expression of E2 occurs only at low temperature and only in the presence of E1. Finally, a vaccination-challenge model in Atlantic salmon demonstrates the biological significance of our findings and shows that SAV replicon DNA vaccines encoding E2 elicit protective immunity only when E1 is co-expressed. This is the first study that identifies E2 as the critical determinant of SAV low-temperature dependent virion formation and defines the prerequisites for induction of a potent immune response in Atlantic salmon by DNA vaccination.

Vaccine Adjuvants in Fish Vaccines Make a Difference: Comparing Three Adjuvants (Montanide ISA763A Oil, CpG/Poly I:C Combo and VHSV Glycoprotein) Alone or in Combination Formulated with an Inactivated Whole Salmonid Alphavirus Antigen.

Most commercial vaccines offered to the aquaculture industry include inactivated antigens (Ag) formulated in oil adjuvants. Safety concerns are related to the use of oil adjuvants in multivalent vaccines for fish, since adverse side effects (e.g., adhesions) can appear. Therefore, there is a request for vaccine formulations for which protection will be maintained or improved, while the risk of side effects is reduced. Here, by using an inactivated salmonid alphavirus (SAV) as the test Ag, the combined use of two Toll-like receptor (TLR) ligand adjuvants, CpG oligonucleotides (ODNs) and poly I:C, as well as a genetic adjuvant consisting of a DNA plasmid vector expressing the viral haemorrhagic septicaemia virus (VHSV) glycoprotein (G) was explored. VHSV-G DNA vaccine was intramuscularly injected in combination with intraperitoneal injection of either SAV Ag alone or combined with the oil adjuvant, Montanide ISA763, or the CpG/polyI:C combo. Adjuvant formulations were evaluated for their ability to boost immune responses and induce protection against SAV in Atlantic salmon, following cohabitation challenge. It was observed that CpG/polyI:C-based formulations generated the highest neutralizing antibody titres (nAbs) before challenge, which endured post challenge. nAb responses for VHSV G-DNA- and oil-adjuvanted formulations were marginal compared to the CpG/poly I:C treatment. Interestingly, heat-inactivated sera showed reduced nAb titres compared to their non-heated counterparts, which suggests a role of complement-mediated neutralization against SAV. Consistently elevated levels of innate antiviral immune genes in the CpG/polyI:C injected groups suggested a role of IFN-mediated responses. Co-delivery of the VHSV-G DNA construct with either CpG/polyI:C or oil-adjuvanted SAV vaccine generated higher CD4 responses in head kidney at 48 h compared to injection of this vector or SAV Ag alone. The results demonstrate that a combination of pattern recognizing receptor (PRR) ligands, such as CpG/polyI:C, increases both adaptive and innate responses and represents a promising adjuvant strategy for enhancing the protection of future viral vaccines.