Characterisation of salmon and trout CD8alpha and CD8beta.

The genes and corresponding cDNAs of both alpha and beta chains of the Atlantic salmon (Salmo salar) CD8 molecule have been sequenced and characterized. In addition, the cDNAs for alpha and beta chains of brown trout (Salmo trutta) and for the beta chain in rainbow trout (Oncorhynchus mykiss) have been sequenced. The cDNAs code for signal sequences which are preceded by short 5' UTRs. These are followed by typical immunoglobulin superfamily variable sequences all of which contain two conserved cysteines for the intra-chain disulphide bond. The hinge regions display conserved cysteines for dimerisation and several O-glycosylation motifs for each predicted protein. The domain sharing the highest sequence identity with mammals is the single pass transmembrane domain for all sequences. In salmon, each domain is predominantly coded for by a single exon except the cytoplasmic/3' UTR domains, which are coded for by 3 and 2 exons for the alpha and beta genes, respectively. In the alpha gene, the second cytoplasmic exon may be spliced out to form an alternative shorter transcript which if expressed would exhibit a truncated cytoplasmic tail. A splice variant found for the salmon beta gene introduces a stop codon after only 40 amino acids. Overall amino acid identities between salmonid sequences were higher than 90%, whereas they shared only 15-20% identity with species such as, chicken and human. Analysis of the expression patterns of the two salmon genes using quantitative RT-PCR shows a very high expression in the thymus. This is mirrored by the expression of the TCRalpha gene, which is known to be co-expressed with CD8 on mammalian T cells. This is the first report of a sequence for CD8beta in a teleost and together with the CD8alpha sequence, it encodes the ortholog of the CD8 co-receptor molecule on mammalian T cells.

Nonspecific immune response of rainbow trout (Oncorhynchus mykiss Walbaum) in relation to different status of vitamin E and highly unsaturated fatty acids.

This study was designed to examine the effects of dietary vitamin E (VE) on modulation of immune responses when supplied with two levels of n-3 highly unsaturated fatty acids (n-3 HUFA) in rainbow trout, Oncorhynchus mykiss. Six semipurified diets were prepared containing three levels of dietary VE (0, 100 or 1000 mg alpha-tocopheryl acetate kg(-1) diet) and n-3 HUFA either at 20 or 48% of dietary lipid provided from fish oil or docosahexaenoic acid (DHA) concentrated fish oil respectively. The diets were fed to rainbow trout (100 g initial mean weight) for 15 weeks. The VE, vitamin C (VC) content in plasma and tissues and the nonspecific immune responses, both humoral (alternative complement activity, total immunoglobulin) and cellular (phagocytosis, nonspecific cytotoxicity) were examined. VE contents in the kidney reflected the dietary input but were lower in fish fed 48% n-3 HUFA diets, and could have impaired some of immune responses compared to fish fed 20% n-3 HUFA. VC contents in kidney followed the same pattern as VE. Both humoral and cellular immune functions deteriorated in fish fed VE deficient diets whereas improvement in most of the parameters corresponded to its supplementation. However, the higher dose of dietary VE did not substantially enhance the responses assayed compared to the 100 mg dose. Besides clearly indicating the role of VE in maintaining the immune functions in fish in relation to dietary n-3 HUFA, this study has revealed that optimum health benefits could be achieved when VE is maintained slightly above the levels generally recommended for normal growth.

Cytotoxic T cell function in fish.

Fish possess immunoglobulins, major histocompatibility complex (MHC), T-cell receptors, and lymphocyte populations analogous to B and T cells and can evoke specific immune responses against a variety of antigens. However, T-cell subsets have yet to be demonstrated and the information on cell-mediated immunity is limited. Here we briefly review our recent studies on specific cell-mediated immunity, particularly on cytotoxic T-cell function employing isogeneic fish and cell lines. Analyses of the graft-versus host reaction (GVHR) and cell-mediated cytotoxicity (CMC) against allogeneic erythrocytes or cell lines show alloantigen-specific cytotoxicity in clonal ginbuna crucian carp. We also describe specific cytotoxicity against virus-infected cells using clonal ginbuna and a syngeneic cell line. Lastly, we report MHC-restriction in CMC against virus-infected cells using homozygous clonal rainbow trout and trout cell line which share the same MHC class I allele. These studies on CMC strongly suggest the presence of antigen specific cytotoxic T cells in teleosts and functional similarities between the immune systems of fish and higher vertebrates. Experimental model systems established in these studies can be applied to the investigation of protective antigens to induce cell-mediated immunity for the development of fish vaccines.

Specific cell-mediated cytotoxicity against a virus-infected syngeneic cell line in isogeneic ginbuna crucian carp.

In the present study, we attempted to determine whether virus-specific cell-mediated cytotoxicity occurs in fish. Experiments were conducted with clonal ginbuna crucian carp (Carassius auratus langsdorfii) (S3n strain), and the syngeneic cell line (CFS). Two infectious viruses were used: infectious pancreatic necrosis virus (IPNV) and eel virus from America (EVA). Peripheral blood leukocytes, which consisted almost entirely (95%) of lymphocytes and thrombocytes, from S3n ginbuna immunized with virus-infected CFS cells lysed the virus-infected CFS cells (immunogen) more completely than CFS cells infected with a different virus (non-immunogen). This specific cell-mediated cytotoxicity of ginbuna against virus-infected CFS cells was efficiently induced as a result of in vivo secondary immunization. A significant specific cytotoxic activity peaked during 7-10 days after the secondary immunization. In addition, effector cells immunized with IPNV-infected syngeneic cells did not lyse IPNV-infected xenogeneic cells. These results support the hypothesis that fish exhibit specific cytotoxicity against virus-infected cells, resembling the specific cytotoxicites of higher vertebrates.