Virus-specific antibody secreting cells reside in the peritoneal cavity and systemic immune sites of Atlantic salmon (Salmo salar) challenged intraperitoneally with salmonid alphavirus.

The development and persistence of antibody secreting cells (ASC) after antigenic challenge remain inadequately understood in teleosts. In this study, intraperitoneal (ip) injection of Atlantic salmon (Salmo salar) with salmonid alphavirus (WtSAV3) increased the total ASC response, peaking 3-6 weeks post injection (wpi) locally in the peritoneal cavity (PerC) and in systemic lymphoid tissues, while at 13 wpi the response was only elevated in PerC. At the same time point a specific ASC response was induced by WtSAV3 in PerC and systemic tissues, with the highest frequency in PerC, suggesting a local role. Inactivated SAV (InSAV1) induced comparatively lower ASC responses in all sites, and specific serum antibodies were only induced by WtSAV3 and not by InSAV1. An InSAV1 boost did not increase these responses. Expression of immune marker genes implies a role for PerC adipose tissue in the PerC immune response. Overall, the study suggests the Atlantic salmon PerC as a secondary immune site and an ASC survival niche.

There and back again? A B cell's tale on responses and spatial distribution in teleosts.

Teleost B cells are of special interest due to their evolutionary position and involvement in vaccine-induced adaptive immune responses. While recent progress has revealed uneven distribution of B cell subsets across the various immune sites and that B cells are one of the early responders to infection, substantial knowledge gaps persist regarding their immunophenotypic profile, functional mechanisms, and what factors lead them to occupy different immune niches. This review aims to assess the current understanding of B cell diversity, their spatial distribution in various systemic and peripheral immune sites, how B cell responses initiate, the sites where these responses develop, their trafficking, and the locations where long-term B cell responses take place.

B-1 plasma cells require non-cognate CD4 T cell help to generate a unique repertoire of natural IgM.

Evolutionarily conserved, "natural" (n)IgM is broadly reactive to both self and foreign antigens. Its selective deficiency leads to increases in autoimmune diseases and infections. In mice, nIgM is secreted independent of microbial exposure to bone marrow (BM) and spleen B-1 cell-derived plasma cells (B-1PC), generating the majority of nIgM, or by B-1 cells that remain non-terminally differentiated (B-1sec). Thus, it has been assumed that the nIgM repertoire is broadly reflective of the repertoire of body cavity B-1 cells. Studies here reveal, however, that B-1PC generate a distinct, oligoclonal nIgM repertoire, characterized by short CDR3 variable immunoglobulin heavy chain regions, 7-8 amino acids in length, some public, many arising from convergent rearrangements, while specificities previously associated with nIgM were generated by a population of IgM-secreting B-1 (B-1sec). BM, but not spleen B-1PC, or B-1sec also required the presence of TCRαß CD4 T cells for their development from fetal precursors. Together, the studies identify important previously unknown characteristics of the nIgM pool.

The importance of the Atlantic salmon peritoneal cavity B cell response: Local IgM secreting cells are predominant upon Piscirickettsia salmonis infection.

The intraperitoneal route is favored for administration of inactivated and attenuated vaccines in Atlantic salmon. Nevertheless, the immune responses in the teleost peritoneal cavity (PerC) are still incompletely defined. In this study, we investigated the B cell responses after intraperitoneal Piscirickettsia salmonis (P. salmonis) challenge of Atlantic salmon, focusing on the local PerC response versus responses in the lymphatic organs: spleen and head kidney. We observed a major increase of leukocytes, total IgM antibody secreting cells (ASC), and P. salmonis-specific ASC in the PerC at 3- and 6-weeks post infection (wpi). The increase in ASC frequency was more prominent in the spleen and PerC compared to the head kidney during the observed 6 wpi. The serum antibody response included P. salmonis-specific antibodies and non-specific antibodies recognizing the non-related bacterial pathogen Yersinia ruckeri and the model antigen TNP-KLH. Finally, we present evidence that supports a putative role for the adipose tissue in the PerC immune response.

Salmonid Alphavirus Subtype 3 Induces Prolonged Local B Cell Responses in Atlantic Salmon (Salmo salar) After Intraperitoneal Infection.

B cell responses are a crucial part of the adaptive immune response to viral infection. Infection by salmonid alphavirus subtype 3 (SAV3) causes pancreas disease (PD) in Atlantic salmon (Salmo salar) and is a serious concern to the aquaculture industry. In this study, we have used intraperitoneal (IP) infection with SAV3 as a model to characterize local B cell responses in the peritoneal cavity (PerC) and systemic immune tissues (head kidney/spleen). Intraperitoneal administration of vaccines is common in Atlantic salmon and understanding more about the local PerC B cell response is fundamental. Intraperitoneal SAV3 infection clearly induced PerC B cell responses as assessed by increased frequency of IgM+ B cells and total IgM secreting cells (ASC). These PerC responses were prolonged up to nine weeks post-infection and positively correlated to the anti-SAV3 E2 and to neutralizing antibody responses in serum. For the systemic immune sites, virus-induced changes in B cell responses were more modest or decreased compared to controls in the same period. Collectively, data reported herein indicated that PerC could serve as a peripheral immunological site by providing a niche for prolonged maintenance of the ASC response in Atlantic salmon.

Detection of specific Atlantic salmon antibodies against salmonid alphavirus using a bead-based immunoassay.

Salmonid alphavirus (SAV) is the etiological cause of pancreas disease (PD) in Atlantic salmon (Salmo salar). Several vaccines against SAV are in use, but PD still cause significant mortality and concern in European aquaculture, raising the need for optimal tools to monitor SAV immunity. To monitor and control the distribution of PD in Norway, all salmonid farms are regularly screened for SAV by RT-qPCR. While the direct detection of SAV is helpful in the early stages of infection, serological methods could bring additional information on acquired SAV immunity in the later stages. Traditionally, SAV antibodies are monitored in neutralization assays, but they are time-consuming and cumbersome, thus alternative assays are warranted. Enzyme-linked immunosorbent assays (ELISAs) have not yet been successfully used for anti-SAV antibody detection in aquaculture. We aimed to develop a bead-based immunoassay for SAV-specific antibodies. By using detergent-treated SAV particles as antigens, we detected SAV-specific antibodies in plasma collected from both a SAV challenge trial and a field outbreak of PD. Increased levels of SAV-specific antibodies were seen after most fish had become negative for viral RNA. The bead-based assay is time saving compared to virus neutralization assays, and suitable for non-lethal testing due to low sample size requirements. We conclude that the bead-based immunoassay for SAV antibody detection is a promising diagnostic tool to complement SAV screening in aquaculture.

Infection and microbial molecular motifs modulate transcription of the interferon-inducible gene ifit5 in a teleost fish.

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are involved in antiviral defense. Members of this protein family contain distinctive multiple structural motifs comprising tetratricopeptides that are tandemly arrayed or dispersed along the polypeptide. IFIT-encoding genes are upregulated by type I interferons (IFNs) and other stimuli. IFIT proteins inhibit virus replication by binding to and regulating the functions of cellular and viral RNA and proteins. In teleost fish, knowledge about genes and functions of IFITs is currently limited. In the present work, we describe an IFIT5 orthologue in Atlantic salmon (SsaIFIT5) with characteristic tetratricopeptide repeat motifs. We show here that the gene encoding SsaIFIT5 (SsaIfit5) was ubiquitously expressed in various salmon tissues, while bacterial and viral challenge of live fish and in vitro stimulation of cells with recombinant IFNs and pathogen mimics triggered its transcription. The profound expression in response to various immune stimulation could be ascribed to the identified IFN response elements and binding sites for various immune-relevant transcription factors in the putative promoter of the SsaIfit5 gene. Our results establish SsaIfit5 as an IFN-stimulated gene in A. salmon and strongly suggest a phylogenetically conserved role of the IFIT5 protein in antimicrobial responses in vertebrates.

Intracellular distribution and transcriptional regulation of Atlantic salmon (Salmo salar) Rab5c, 7a and 27a homologs by immune stimuli.

Rab GTPases control trafficking of intracellular vesicles and are key regulators of endocytic and secretory pathways. Due to their specific distribution, they may serve as markers for different endolysosomal compartments. Since Rab GTPases are involved in uptake and trafficking of endocytosed ligands and cell receptors, as well as secretion of immune mediators, they have been implicated in diverse immunological processes and their functions are often exploited by intracellular pathogens such as viruses. While Rab proteins have been studied extensively in mammals, their functions in vesicle trafficking in teleosts are not well known. In the present work, Atlantic salmon Rab5c, Rab7a and Rab27a homologs were studied in terms of intracellular distribution and gene expression. Structured illumination microscopy demonstrated that transgenic, GFP-tagged salmon Rab5c and Rab7a are, predominantly, located within early endosomes and late endosomes/lysosomes, respectively. In contrast, Rab27a showed a broader distribution, which indicates that it associates with diverse intracellular vesicles and organelles. Infection of salmon with Salmonid alphavirus subtype 3 (SAV3) enhanced the mRNA levels of all of the studied Rab isoforms in heart and head kidney and most of them were upregulated in spleen. This may reflect the capacity of the virus to exploit the functions of these rab proteins. It is also possible that the transcriptional regulation of Rab proteins in SAV3-infected organs may play a role in the antiviral immune response. The latter was further supported by in vitro experiments with adherent head kidney leukocytes. The expression of Rab5c and Rab27a was upregulated in these cells following stimulation with TLR ligands including CpG oligonucleotides and polyI:C. The expression of most of the analyzed Rab isoforms in the primary leukocytes was also enhanced by stimulation with type I IFN. Interestingly, IFN-gamma had a negative effect on Rab7a expression which may be linked to the priming activity of this cytokine on monocytes and macrophages. Overall, these data demonstrate that the intracellular distribution of Rab5c, Rab7a and Rab27a is phylogenetically conserved within vertebrates and that these molecules might be implicated in viral infections and the regulation of the antiviral immune response in Atlantic salmon.

Microbial Danger Signals Control Transcriptional Induction of Distinct MHC Class I L Lineage Genes in Atlantic Salmon.

Antigen processing and presentation by major histocompatibility complex (MHC) molecules is a cornerstone in vertebrate immunity. Like mammals, teleosts possess both classical MHC class I and multiple families of divergent MHC class I genes. However, while certain mammalian MHC class I-like molecules have proven to be integral in immune regulation against a broad array of pathogens, the biological relevance of the different MHC class I lineages in fish remains elusive. This work focuses on MHC class I L lineage genes and reveals unique regulatory patterns of six genes (Sasa-lia, Sasa-lda, Sasa-lca, Sasa-lga, Sasa-lha, and Sasa-lfa) in antimicrobial immunity of Atlantic salmon (Salmo salar L.). Using two separate in vivo challenge models with different kinetics and immune pathologies combined with in vitro stimulation using viral and bacterial TLR ligands, we show that de novo synthesis of different L lineage genes is distinctly regulated in response to various microbial stimuli. Prior to the onset of classical MHC class I gene expression, lia was rapidly and systemically induced in vivo by the single-stranded (ss) RNA virus salmonid alpha virus 3 (SAV3) but not in response to the intracellular bacterium Piscirickettsia salmonis. In contrast, lga expression was upregulated in response to both viral and bacterial stimuli. A role for distinct MHC class I L-lineage genes in anti-microbial immunity in salmon was further substantiated by a marked upregulation of lia and lga gene expression in response to type I IFNa stimulation in vitro. Comparably, lha showed no transcriptional induction in response to IFNa stimulation but was strongly induced in response to a variety of viral and bacterial TLR ligands. In sharp contrast, lda showed no response to viral or bacterial challenge. Similarly, induction of lca, which is predominantly expressed in primary and secondary lymphoid tissues, was marginal with the exception of a strong and transient upregulation in pancreas following SAV3 challenge Together, these findings suggest that certain Atlantic salmon MHC class I L lineage genes play important and divergent roles in early anti-microbial response and that their regulation, in response to different activation signals, represents a system for selectively promoting the expression of distinct non-classical MHC class I genes in response to different types of immune challenges.

Differences in smolt status affect the resistance of Atlantic salmon (Salmo salar L.) against infectious pancreatic necrosis, while vaccine-mediated protection is unaffected.

In today's aquaculture of Atlantic salmon (Salmo salar L.), a majority of viral disease outbreaks occur after seawater transfer. A relevant question is how the parr-smolt transformation influences the efficacy of viral vaccines and the innate resistance against viral diseases. In this study, vaccinated and unvaccinated A. salmon parr were exposed to different photoperiodic regimens (1-, 3- or 6-week continuous light-WCL). Fish groups at different stages in the smoltification process were induced, as demonstrated by differences in morphological and physiological smolt parameters. At the time of seawater transfer, the 6-WCL group had reached a more pronounced stage in the smoltification process than the 1-WCL group. In unvaccinated fish, the subsequent cohabitation challenge with infectious pancreatic necrosis virus (IPNV) gave a significantly higher accumulated mortality in the 6-WCL group (87%) compared to the 1-WCL group (39%). In the vaccinated groups, this effect was not apparent and there were no differences in accumulated mortality between the 1 WCL, 3 WCL and 6-WCL groups. These data suggest that the resistance to IPN in A. salmon was negatively influenced by smoltification, while vaccine-mediated protection to IPN was maintained equally well irrespective of smolt status.

Profiling the Atlantic Salmon IgM+ B Cell Surface Proteome: Novel Information on Teleost Fish B Cell Protein Repertoire and Identification of Potential B Cell Markers.

Fish immunology research is at a pivotal point with the increasing availability of functional immunoassays and major advances in omics approaches. However, studies on fish B cells and their distinct subsets remain a challenge due to the limited availability of differentially expressed surface markers. To address this constraint, cell surface proteome of Atlantic salmon IgM+ B cells were analyzed by mass spectrometry and compared to surface proteins detected from two adherent salmon head kidney cell lines, ASK and SSP-9. Out of 21 cluster of differentiation (CD) molecules identified on salmon IgM+ B cells, CD22 and CD79A were shortlisted as potential markers based on the reported B cell-specific surface expression of their mammalian homologs. Subsequent RT-qPCR analyses of flow cytometry-sorted subpopulations from head kidney leukocytes confirmed that both cd22 and cd79a genes were highly expressed in IgM+ lymphoid cells but were observed in barely detectable levels in IgM- non-lymphoid suspension and adherent cells. Similarly, significantly high cd22 and cd79a mRNA levels were observed in IgM+ or IgT+ lymphoid cells from the spleen and peritoneal cavity, but not in their corresponding IgM- IgT- non-lymphoid fractions. This suggests that the B cell restrictive expression of CD22 and CD79A extend down to the transcription level, which was consistent across different lymphoid compartments and immunoglobulin isotypes, thus strongly supporting the potential of CD22 and CD79A as pan-B cell markers for salmon. In addition, this study provides novel information on the salmon B cell surface protein repertoire, as well as insights on B cell evolution. Further investigation of the identified salmon CD molecules, including development of immunological tools for detection, will help advance our understanding of the dynamics of salmon B cell responses such as during infection, vaccination, or immunostimulation.

Author Correction: Profiling Atlantic salmon B cell populations: CpG-mediated TLR-ligation enhances IgM secretion and modulates immune gene expression.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Profiling Atlantic salmon B cell populations: CpG-mediated TLR-ligation enhances IgM secretion and modulates immune gene expression.

While TLR-activated pathways are key regulators of B cell responses in mammals, their impact on teleost B cells are scarcely addressed. Here, the potential of Atlantic salmon B cells to respond to TLR ligands was shown by demonstrating a constitutive expression of nucleic-acid sensing TLRs in magnetic sorted IgM+ cells. Of the two receptors recognizing CpG in teleosts, tlr9 was the dominating receptor with over ten-fold higher expression than tlr21. Upon CpG-stimulation, IgM secretion increased for head kidney (HK) and splenic IgM+ cells, while blood B cells were marginally affected. The results suggest that CpG directly affects salmon B cells to differentiate into antibody secreting cells (ASCs). IgM secretion was also detected in the non-treated controls, again with the highest levels in the HK derived population, signifying that persisting ASCs are present in this tissue. In all tissues, the IgM+ cells expressed high MHCII levels, suggesting antigen-presenting functions. Upon CpG-treatment the co-stimulatory molecules cd83 and cd40 were upregulated, while cd86 was down-regulated under the same conditions. Finally, ifna1 was upregulated upon CpG-stimulation in all tissues, while a restricted upregulation was evident for ifnb, proposing that salmon IgM+ B cells exhibit a type I IFN-response.

Isolation and characterisation of two cDNAs encoding transglutaminase from Atlantic cod (Gadus morhua).

Two cDNAs encoding transglutaminase (TG) were identified in a subtractive cDNA library prepared from the head kidney of poly I:C stimulated Atlantic cod (Gadus morhua). Full-length TG-1 and TG-2 cDNA were cloned from the head kidney by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The deduced amino acid (aa) sequence for TG-1 was 695 aa with an estimated molecular mass of 78.3 kDa, while TG-2 was a 698 aa protein with an estimated molecular mass of 78.8 kDa. The two proteins were named TG-1 and TG-2 and both possess transglutaminase/protease-like homologous domains (TGc) and full conservation of amino acids cysteine, histidine, and aspartate residues that form the catalytic triad. Sequence analysis showed high similarity (93.1%) with Alaska pollock TG, and the TGs were grouped together with TGs from chum salmon, Japanese flounder, Nile tilapia, and red sea bream in addition to Alaska pollock in phylogenetic analysis. Interestingly, they showed different tissue distribution with highest constitutive expression in reproductive and immunological organs, indicating important roles in these organs. Furthermore, the up-regulation of TG-1 and TG-2 in head kidney after stimulating Atlantic cod with poly I:C suggested a role of TGs in immune response in Atlantic cod.

Genomic analysis of the host response to nervous necrosis virus in Atlantic cod (Gadus morhua) brain.

Genome sequencing combined with transcriptome profiling promotes exploration of defence against pathogens and discovery of immune genes. Based on sequences from the recently released genome of Atlantic cod, a genome-wide oligonucleotide microarray (ACIQ-1) was designed and used for analyses of gene expression in the brain during infection with nervous necrosis virus (NNV). A challenge experiment with NNV was performed with Atlantic cod juveniles and brain samples from virus infected and uninfected fish were used for microarray analysis. Expression of virus induced genes increased at 5 days post challenge and persisted at stable level to the last sampling at 25 days post challenge. A large fraction of the up-regulated genes (546 features) were known or expected to have immune functions and most of these have not previously been characterized in Atlantic cod. Transcriptomic changes induced by the virus involved strong activation of genes associated with interferon and tumour necrosis factor related responses and acute inflammation. Up-regulation of genes involved in adaptive immunity suggested a rapid recruitment of B and T lymphocytes to the NNV infected brain. QPCR analyses of 15 candidate genes of innate immunity showed rapid induction by poly(I:C) in Atlantic cod larvae cells suggesting an antiviral role. Earliest and greatest expression changes after poly I:C stimulation was observed for interferon regulatory factors IRF4 and IRF7. Comparative studies between teleost species provided new knowledge about the evolution of innate antiviral immunity in fish. A number of genes is present or responds to viruses only in fish. Innate immunity of Atlantic cod is characterized by selective expansion of several medium-sized multigene families with ribose binding domains. An interesting finding was the high representation of three large gene families among the early antiviral genes, including tripartite motif proteins (TRIM) and proteins with PRY-SPRY and NACHT domains. The latter two with respectively 52 and 114 members in Atlantic cod have gone through expansions in different groups of fish. These proteins most likely have ligand binding properties and their propagation could be linked to the loss of MHC class II in the Atlantic cod genome.

Characterization of arrangement and expression of the beta-2 microglobulin locus in the sandbar and nurse shark.

Beta 2 microglobulin (beta2m) is an essential subunit of major histocompatibility complex (MHC) type I molecules. In this report, beta2m cDNAs were identified and sequenced from sandbar shark spleen cDNA library. Sandbar shark beta2m gene encodes one amino acid less than most teleost beta2m genes, and 3 amino acids less than mammal beta2m genes. Although sandbar shark beta2m protein contains one beta sheet less than that of human in the predicted protein structure, the overall structure of beta2m proteins is conserved during evolution. Germline gene for the beta2m in sandbar and nurse shark is present as a single locus. It contains three exons and two introns. CpG sites are evenly distributed in the shark beta2m loci. Several DNA repeat elements were also identified in the shark beta2m loci. Sequence analysis suggests that the beta2m locus is not linked to the MHC I loci in the shark genome.

Susceptibility of Atlantic cod Gadus morhua juveniles to different routes of experimental challenge with infectious pancreatic necrosis virus (IPNV).

Atlantic cod Gadus morhua L. juveniles weighing 40 g were challenged with infectious pancreatic necrosis virus (IPNV) by intraperitoneal (i.p.) or intramuscular (i.m.) injection or by bath. The amount of infectious virus was determined over 6 wk in head kidney, heart and pylorus tissues. No mortality or clinical signs were observed in either of the challenged groups. However, 6 wk after challenge virus was still present in the fish, which shows that IPNV can persist asymptomatically in cod. I.p. and i.m. injections were the most efficient routes of challenge giving the highest virus recovery. The prevalence of individuals with a viral titre > or = 500 infectious units g(-1) tissue was lower in the group of fish challenged by bath; thus bath was a less efficient route of challenge than injection. Our data also show that pylorus and head kidney are target organs for IPNV in cod, and levels of virus recovery were not considerably different between these 2 organs. Challenged by injection, the cod heart is also a target organ for IPNV. Compared to head kidney and pylorus, the heart seems to have a minor role in virus multiplication. Virus was also recovered from cohabiting fish, demonstrating that covertly infected cod may represent a reservoir of infectious IPNV for surrounding fish populations. Expression analysis of selected cod immune genes showed that i.p. injection of IPNV induced gene expression of ISG15 and LGP2, markers for the innate antiviral defence, while expression of markers for the inflammatory response (interleukins IL-1 beta, IL-8, IL-10) was not significantly increased.

Atlantic cod (Gadus morhua L.) possesses three homologues of ISG15 with different expression kinetics and conjugation properties.

Two new interferon stimulated gene 15 (ISG15) family members were identified in a subtractive cDNA library constructed from a mixture of head kidney and spleen of Atlantic cod (Gadus morhua) stimulated with polyinosinic:polycytidylic acid (poly I:C). Two full-length Atlantic cod (Ac) ISG15-2 and AcISG15-3 cDNAs were cloned with rapid amplification of cDNA ends (RACE). The cDNA sequence of AcISG15-2 encodes a 16.9kDa protein and AcISG15-3 encodes a 18.4kDa protein, both of which possess the characteristic structural features of two tandem ubiquitin-like domains and the LRGG motif necessary for conjugation. Furthermore, the AcISG15-3 protein is expressed with a C-terminal extension in common with the human ISG15 protein. Gene expression analysis using quantitative reverse transcriptase PCR (RT-qPCR) showed that AcISG15-1, AcISG15-2, and AcISG15-3 transcripts were up-regulated in head kidney after poly I:C stimulation, suggesting that these proteins may be involved in the cod immune response. However, transient expression of myc-tagged AcISG15 proteins revealed differences in their abilities to form conjugates in vitro. We show that AcISG15-2 forms covalent conjugates to a range of cellular protein as a response to poly I:C, recombinant Atlantic salmon IFNa1 (rSasaIFNa1) and infectious pancreatic necrosis virus (IPNV), whereas conjugation was absent for AcISG15-1 and AcISG15-3. Thus, these results suggest there are three ISG15 homologues in Atlantic cod and that the three proteins may play different roles in innate immunity.

Characterization of arrangement and expression of the T cell receptor gamma locus in the sandbar shark.

Ig and T cell receptor (TCR) genes consist of separate genomic elements, which must undergo rearrangement and joining before a functional protein can be expressed. Considerable plasticity in the genomic arrangement of these elements has occurred during the evolution of the immune system. In tetrapods, all Ig and TCR chain elements are arranged as translocons. In teleosts, the Ig heavy and TCR chains are translocons, but light chain genes may occur as clusters. However, in chondrichthyes, all of the Ig light and heavy chain genes are arranged as clusters. These clusters vary in number from <10 to several hundred, depending on isotype and species. Here, we report that the germ-line gene for the TCR gamma chain in a chondrichthyan, the sandbar shark (Carcharhinus plumbeus), is present as a single locus arranged in a classic translocon pattern. Thus, the shark utilizes 2 types of genomic arrangements, the unique cluster organization for Ig genes and the "conventional" translocon organization for TCR genes. The TCR gamma translocon contains at least 5 V region genes, 3 J segment genes, and 1 C segment. As expected, the third hypervariable segment (CDR3), formed by the rearrangement of the Vgamma and Jgamma segments, contributed the major variability in the intact V region structure. Our data also suggest that diversity may be generated by mutation in the V regions.

Characterisation and expression analysis of the interleukin genes, IL-1beta, IL-8 and IL-10, in Atlantic cod (Gadus morhua L.).

The mammalian interleukins IL-1beta and IL-8 are important pro-inflammatory cytokines often used as markers of an activated inflammatory response, while IL-10 is regarded as an anti-inflammatory cytokine and plays a crucial role in the regulation of inflammation. Few cytokines from gadoid fish have been described, and herein the sequence and expression of these interleukin genes are studied in Atlantic cod (Gadus morhua L.). IL-1beta, IL-8 and IL-10 from cod show similarities in gene organisation and predicted protein sequences, compared to counterpart genes in other teleosts. Gene expression was studied using quantitative real time PCR in response to several treatments both in vitro and in vivo. In adherent head kidney cells, infectious pancreatic necrosis virus (IPNV) and lipopolysaccharide (LPS) significantly stimulated gene expression of IL-1beta. The expression of IL-1beta was not increased after treatment with a viral imitator (poly I:C), and neither IL-8 nor IL-10 responded to any of these agents in vitro. However, in vivo administrated poly I:C and formalin-killed Vibrio anguillarum (In-V.ang) induced interleukin expression, varying in intensity between different organs. IL-1beta and IL-10 gene expression profiles showed an opposite induction pattern in the in vivo experiments. Injection of In-V.ang highly induced IL-1beta expression, while a low induction was evident for IL-10, whereas the opposite was observed after injection of poly I:C. This pattern was particularly marked in spleen, where also IL-8 followed the expression pattern of IL-1beta. The opposite expression profiles indicate a suppressive role for IL-10 on the transcription of IL-1beta, and to a lesser extent on IL-8 transcription. Along with the identification of important promoter regulatory motives, these results provide tools for studying inflammatory responses in cod.