Trond Ø. Jørgensen

Humoral immune parameters in Atlantic cod (Gadus morhua L.) I. The effects of environmental temperature

The effects of environmental temperature on certain humoral immune parameters in Atlantic cod (Gadus morhua L.) were studied. Serum samples were collected from captive cod, of wild origin, kept at different temperatures for 12 months. It was found that immunoglobulin and natural antibody levels increased with increasing temperature whereas the total serum protein concentration, anti-protease activity, iron concentration, unsaturated and total iron binding capacity decreased with increasing temperature. Haemolytic activity and percentage iron saturation also tended to decrease with increasing temperature although this was not statistically significant.

Immunoglobulin D (IgD) of Atlantic cod has a unique structure.

Abstract A new immunoglobulin heavy-chain gene with some homology to mammalian IgD was recently cloned from the channel catfish and Atlantic salmon, two species of teleost fish. We have cloned and sequenced a new H-chain gene from Atlantic cod (Gadus morhua L.) which has clear similarities to these genes, but which also differs in several ways. The similarities of catfish, salmon, and cod delta to the mammalian delta genes are sequence homology, location immediately downstream of IgM (mu), and expression by alternative splicing rather than class switching. A unique feature of catfish, salmon, and cod delta is the chimeric nature of the gene product, as the μ1 exon is spliced to the δ1 exon. Several unique features of cod IgD were found: (1) a deletion of the δ3, δ4, δ5, and δ6 domains described in catfish and salmon IgD, (2) a tandem duplication of a part of the delta locus including the δ1 and δ2 domains, (3) the presence of a truncated δ7 domain downstream of the δTM exons, and (4) the separation of the duplicated domains by a short exon (δy) which has homology to a conserved part of the transmembrane exon 1 (TM1) of some H-chain isotypes. This unique organization of the delta locus of cod probably developed after the evolutionary split from the catfish and salmon branches.

Ontogeny of lymphoid organs and immunoglobulin producing cells in atlantic cod (Gadus morhua L.)

The ontogeny of lymphoid organs and the development of cells expressing immunoglobulin heavy chain (IgH) mRNA as well as cells containing immunoglobulin (IgM) were studied in Atlantic cod (Gadus morhua L.), a marine teleost. Head kidney and spleen appeared as the first lymphoid organs, present at the time of hatching, whereas thymus was observed in 9 mm larvae. Fully developed lymphoid organs were not achieved until after metamorphosis. Cells expressing IgH mRNA were detected in paraffin sections of larvae and juveniles by in situ hybridization. Positive cells were not detected in fish smaller than 33 mm (58 days after hatching). IgH mRNA expression coincided with the first appearance of immunoglobulin-positive cells as revealed by immunohistochemistry in the same animals.

Humoral immune parameters in Atlantic cod (Gadus morhua L.) II. The effects of size and gender under different environmental conditions

The effects of size and gender on several humoral immune parameters in cod were examined under different environmental conditions. Serum samples were collected from wild cod of different sizes. Two samplings were undertaken: In the spring in relatively cold waters off the north west coast of Iceland and in the fall in relatively warm waters off the west coast of Iceland. Most of the parameters increased with increasing cod size, except the haemolytic activity which decreased. Higher serum protein levels were seen in cod sampled in the fall than in the spring. In cod sampled in the spring there was an apparent difference between specimens < 75 cm in length and the larger specimens with respect to haemolytic activity and iron concentration. None of the parameters were influenced by the gender of the cod.

Strongylocins, novel antimicrobial peptides from the green sea urchin, Strongylocentrotus droebachiensis

Sea urchins possess an innate immune system and are regarded as a potential source for the discovery of new antimicrobial peptides (AMPs). Here we report the purification and characterization of two novel antibacterial peptides (5.6 and 5.8kDa) from coelomocyte extracts of the green sea urchin, Strongylocentrotus droebachiensis. These are the first reported AMPs isolated from sea urchins. The cDNA encoding the peptides and genomic sequences was isolated and sequenced. The two peptides (named strongylocins 1 and 2) have putative isoforms (1b and 2b), similar to two putative proteins from the purple sea urchin S. purpuratus. The native strongylocins are cationic, defensin-like peptides (cysteine-rich), but show no similarity to other known AMPs concerning the cysteine distribution pattern. Strongylocin 1 consists of 83 amino acids that include a preprosequence of 35 amino acids, whereas strongylocins 2a and 2b are composed of 89 and 90 amino acids, respectively, where 38 amino acids represent a preprosequence. No introns were found in the cloned gene of strongylocin 1b, whereas three introns and four exons were found in strongylocins 1a and 2a/b. The latter gene organization was also found in genes coding for putative strongylocins in S. purpuratus. The molecular mass difference between the native peptide and the deduced strongylocin 2 suggests that the first amino acid is bromotryptophan. The native peptides display potent activities against Gram-negative and Gram-positive bacteria.

Molecular characterisation of a goose-type lysozyme gene in Atlantic cod (Gadus morhua L.).

Lysozymes are antibacterial enzymes important in the innate immune defense of several animal phyla. An Atlantic cod goose-type (g-type) lysozyme EST was identified in a suppression subtractive hybridisation (SSH) cDNA library and the full-length cDNA (codg1) was obtained by RACE-PCR. The lysozyme gene is organised in five exons and four introns similar to g-type lysozyme genes in other fish species. Two different cod lysozyme transcripts, named codg1 and codg2, seem to be produced by the use of alternative transcription start sites (TSS) in the lysozyme gene. The alternative TSS cause a different exon I usage where exon Ia transcripts possess a putative signal peptide (codg1) while exon Ib transcripts (codg2) lack this feature. Lysozyme without the signal peptide was produced recombinantly in Escherichia coli and displayed muramidase activity against Micrococcus luteus cells at an unusually low pH. Gene expression analysis of codg1 and codg2 showed that both were expressed in several tissues with highest expression in the head kidney, peritoneum and spleen. Codg1 and codg2 were differentially expressed in some tissues. In the non-immunised control group, codg2 was expressed significantly higher in the head kidney compared to codg1, while an opposite expression profile was observed in the gills. Compared to non-immunised fish, a significant up-regulation of codg2 transcripts was observed in the peritoneum and gills after injection of formalin inactivated Listonella anguillarum indicating a role for g-type lysozyme in the innate defense system of cod.

Identification, cloning and expression analysis of a hepcidin cDNA of the Atlantic cod (Gadus morhua L.)

Mammalian hepcidin is an antimicrobial peptide and a key regulator in the iron homeostasis. Here we report the identification and cloning of a hepcidin cDNA from Atlantic cod (Gadus morhua L.). The cod hepcidin cDNA was predicted to encode a prepropeptide of 98 amino acids (aa) with a signal peptide of 22 aa. A tentative RX(K/R)R motif for propeptide convertases was also identified suggesting a cleavage site located between Arg(72) and Gln(73). The deduced mature cod hepcidin sequence of 26 aa shows similarity to other reported hepcidins and the gene organization is also similar to corresponding genes in mammals and fish consisting of three exons and two introns. As reported for most other species, the expression level of cod hepcidin was highest in liver. However, high levels of hepcidin expression were also observed in the head kidney and peritoneum and an upregulation of hepcidin transcription was seen in all tissues examined 2 days after i.p. injection of formalin-inactivated Listonella (Vibrio) anguillarum. Poly I:C was also able to induce hepcidin transcription. In situ hybridization showed a leukocytic morphology and localization of hepcidin-positive cells in several tissues, and the expression data imply that cod hepcidin is an important component of the first-line defense against invading pathogens.

Two serotypes of Vibrio salmonicida isolated from diseased cod (Gadus morhua L.); virulence, immunological studies and vaccination experiments

Vibrio salmonicida is the causative agent of cold water vibriosis in Atlantic salmon ( Salmo salar ) in Norway and Scotland, and has more recently been isolated from Arcto-Norwegian cod. The present report deals with the serotyping of V. salmonicida isolated from cod, studies on its virulence, immunological properties, and the vaccination against cold water vibriosis in cod. Using a panel of monoclonal antibodies it was shown that two distinct sero-types of V. salmonicida exist, one of which is serologically identical to the serotype previously isolated from salmon. Vibrio salmonicida was shown by bacterial titration to be much more virulent in salmon as compared to cod, but immune responses determined by production of specific antibodies were negligible in cod compared to similar immunisation of salmon. Although the antibody production in cod was low, an immersion vaccine based on formalin inactivated V. salmonicida elicited 90–100% protection against cold water vibriosis in this species.

Expression of immunoglobulin heavy chain transcripts (VH-families, IgM, and IgD) in head kidney and spleen of the Atlantic cod (Gadus morhua L.)

Expression of the immunoglobulin (Ig) heavy chain transcripts in spleen and head kidney of the Atlantic cod (Gadus morhua L.) was investigated using in situ hybridization (ISH) and northern blotting. Specific detection of plasma cells was done with a probe for secretory IgM transcripts (mu 4). The plasma cells were often clustered close to blood vessels. Cells expressing surface IgM and IgD transcripts were detected using ISH with tyramide signal amplification (TSA). The positive cells were more abundant than plasma cells, had a lymphocyte-like morphology, and were evenly distributed throughout the tissues. This suggests that cod IgD mainly is expressed as a B-cell receptor akin to IgD in mammals. The VH-III family dominated the repertoire within the plasma cells, in agreement with data from cDNA cloning. Immunization with hapten-carrier antigen did not induce a systemic antibody response, and neither was any change in the clustering or distribution pattern of plasma cells within the tissues seen. A few clusters of plasma cells expressed only the rare VH-I and VH-II families, suggesting an ongoing clonal expansion and differentiation in these regions independently of immunization.

The specificity of atlantic salmon antibodies made against the fish pathogen Vibriosalmonicida, establishing the surface protein VS-P1 as the dominating antigen

The specificity of salmon (Salmo salar) antibodies made against the fish pathogen Vibrio salmonicida was studied. Salmon immunized with V. salmonicida emulisified in Freunds complete adjuvant produced antibodies which preferentially bound to a 40 KD outer surface molecule (VS-P1). Moreover, the bulk of the antibodies were specific for one particular epitope on VS-P1, defined by a mouse monoclonal antibody - as detected with a blocking ELISA. The data imply that salmon B cells mainly (90%) recognize one particular determinant on V. salmonicida and thus express a limited repertoire of antibody specificities against this pathogen.