Sigrun Espelid

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.

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.

Immunoglobulin producing cells in the spotted wolffish (Anarhichas minor Olafsen): localization in adults and during juvenile development

The presence of immunocompetent cells was studied in the larval and adult stages of the spotted wolffish, Anarhichas minor. In situ hybridization with a probe complementary to the secretory Igmu-chain was used to localize immunoglobulin producing cells or plasma cells in organs from adult fish and the appearance of these cells in lymphoid tissues during juvenile development. Plasma cells were located in pronephros, spleen, gut, gills and skin of adult wolffish. In juveniles, the first plasma cells were detected in the kidney 1 week post-hatching and the appearance in other lymphoid organs was in the order spleen, gut and thymus. No plasma cells were detected in skin and gills during the sampling period of juveniles (<10 cm). Our study confirmed that plasma cells are present in both the systemic and mucosal compartments of adult fish but during ontogeny there is an earlier appearance of plasma cells in the gut compared to gill and skin compartments.

Oral administration of lipopolysaccharide to Atlantic salmon (Salmo salar L.) fry. Uptake, distribution, influence on growth and immune stimulation

Abstract Atlantic salmon fry were fed Aeromonas salmonicida lipopolysaccharide (LPS)-coated feed for 62 days and then challenged with virulent A. salmonicida bacteria. The fry were fed LPS-coated feed also after challenge. Fry that were fed LPS feed (0.1% LPS) showed a higher mortality throughout the challenge period (accumulated mortality of 57% after 42 days) compared with fry fed control feed (accumulated mortality of 36.5% after 42 days). Fry receiving LPS-coated feed showed an increase in mean weight of 10.7% at day 62 compared with fry receiving control feed. The increase was, however, not statistically significant. Sixty days of feeding with LPS-coated feed did not result in measurable amounts of specific antibodies against A. salmonicida LPS in the homogenised fry. The total amount of immunoglobulins (Igs) was, however, slightly increased. Studies of immunohistochemical localisation and radioactive LPS distribution revealed high levels of LPS in the intestinal epithelial cells. Head kidney, liver and heart showed low levels of radioactivity and no immunohistochemical staining. In another set of experiments, Atlantic salmon fry were fed LPS-coated feed (0.03% and 0.01%, respectively) for 64 days and then challenged with A. salmonicida and Vibrio anguillarum . The accumulated mortality in fry challenged with A. salmonicida was 40.9%, 34.3% and 30.8% after feeding 0.03% and 0.01% LPS and control feed, respectively. The accumulated mortality in fry challenged with V. anguillarum was 47.0%, 55.0% and 55.1% after feeding 0.03% and 0.01% LPS and control feed, respectively. Fry receiving 0.03% and 0.01% LPS-coated feed showed a statistically significant increase in mean weight at day 64 compared with fry receiving control feed.

The humoral immune response in Atlantic salmon (Salmo salar L.) against the hapten carrier antigen NIP-LPH; the effect of determinant (NIP) density and the isotype profile of anti-NIP antibodies

The humoral immune response in Atlantic salmon ( Salmo salar L) against the semi-synthetic antigen NIP-LPH (4-hydroxy-3-iodo-5-nitrophenyl-acetic acid conjugated to Limulus polyphemus haemocyanin) was studied. The effect of the conjugation ratio on the antibody activity, affinity, specificity and shift of isotypic determinants of anti-NIP antibodies was tested. No affinity maturation of the antibody response similar to mammalian responses could be seen, except for the antigen having the lowest conjugation ratio (NIP 6 LPH) which demonstrated a moderate (two-fold) increase, of the average intrinsic association constant ( K 0 ) value from day 59–134 after immunisation. The NIP 6 LPH antigen also elicited anti-NIP antibodies having a 15-fold higher K 0 than the more substituted NIP 19 LPH or NIP 37 LPH. By using a panel of monoclonal antibodies binding determinants on salmon Igs, a different spectrum of such determinants could be recorded on anti-NIP molecules in normal and immune salmon sera. Although the specificity of these monoclonal is unproven (i.e. react with the C or V regions on salmon Ig) these data suggest that Atlantic salmon have more than one antibody isotype and possess the ability to alter the composition of antibody classes during an ongoing immune response, akin to higher vertebrates.

The spotted wolffish (Anarhichas minor Olafsen) complement component C3 : isolation, characterisation and tissue distribution

The complement component C3 was isolated from spotted wolffish (Anarhichas minor Olafsen) serum by polyethylene glycol precipitation, anion exchange chromatography and gel filtration. Silver staining in SDS-PAGE and rabbit anti-wolffish C3 antiserum used in Western blotting revealed that spotted wolffish C3 contains two polypeptide chains, M(r)65 and 115kDa, respectively. The high molecular weight alpha-chain of the C3 incorporated 14C-methylamine suggesting that it contained a reactive thioester group. The deduced amino acid sequence, after screening a liver cDNA expression library, showed that the wolffish C3 contained key amino acids for binding C3 convertase, factor H, I and properdin. Also, high degree of homology to other vertebrate C3 was found in the beta-alpha junction site. Phylogenetic tree analysis indicated that the Japanese flounder and spotted wolffish that belong to order pleuronectiformes and perciformes, respectively, are phylogenetically close species. Immunohistochemical experiments showed that liver hepatocytes and blood contained C3, and in situ hybridisation experiments revealed that liver hepatocytes expressed C3.

Immunoglobulin VHfamilies and light chain isotypes in the spotted wolffish (Anarhichas minor Olafsen)

The spotted wolffish (Anarhichas minor Olafsen) is a species of the Perciformes, the most diverse and numerous order of all fish. A cDNA library from head kidney tissue was screened for immunoglobulin (Ig) heavy and light chain transcripts, and showed highest identity to Ig sequences from other perciform species. So far only one Ig class is described in spotted wolffish, but three V(H)families were identified among the heavy chain transcripts. Highest diversity was located at the CDR3 region and demonstrates the importance of this gene element to the antibody repertoire. Two V(L)families were identified among the light chain clones and three distinct isotypes were present. Use of polymerase chain reaction and in situ hybridisation techniques revealed individual variations in the relative expression of the three isotypes of light chains.

Vaccination and immune responses against atypical Aeromonas salmonicida in spotted wolffish (Anarhichas minor Olafsen) juveniles.

To study the effect of early vaccination, wolffish juveniles of size 50 and 90 mm, respectively, were vaccinated with an oil-adjuvanted atypical A. salmonicida bacterin. Vaccination resulted in significant protection after challenge with the homologous bacterial strain and specific antibody responses were demonstrated against whole bacteria as well as purified A-layer protein and LPS by ELISA and Western blotting but individual variation in immune responses was apparent. The A-protein was the most immunogenic bacterial component. In addition, higher numbers of immunoglobulin producing cells were detected by in situ hybridisation in kidney and spleen of vaccinated fish compared to non-vaccinated fish. Plasma cells were also present in gut and gills in equal numbers irrespective of treatment. No plasma cells were found in the skin. Finally, the frequencies of expressed V(H)families and C(L)isotypes of wolffish immunoglobulins were shown by PCR. The relative expression of the three variable regions of the Ig heavy chain and the three isotypes of the Ig light chain in the spotted wolffish spleen seemed to be unaffected by immunisation with a complex antigen like the A. salmonicida bacterin.

Antigen processing of Vibrio salmonicida by fish (Salmo salar L.) macrophages in vitro

Macrophages from Atlantic salmon (Salmo salar L.) were cultured in vitro and their ability to phagocytose the fish pathogen Vibrio salmonicida was studied. The intracellular degradation of the pathogen was detected by use of monoclonal antibodies against two different surface components of the bacterium, LPS and a 24 kDa molecule. The bacterium itself and the 24 kDa protein antigen were shown to be highly susceptible to phagocytic digestion compared to the more resistant LPS components of the bacterial membrane, which persisted internally for several weeks. The phagocytic and degradative processes were also shown to be inhibited by the ionophore monensin whereas NH4Cl blocked only the intracellular processing of V. salmonicida.

Identification and cloning of immunogenic Aliivibrio salmonicida Pal-like protein present in profiled outer membrane and secreted subproteome.

Aliivibrio salmonicida is the aetiological agent of cold water vibriosis affecting farmed fish species, a disease that today is fully controlled by vaccination. However, the molecular mechanisms behind the successful vaccine are largely unknown. In order to gain insight into the possible mechanisms of A. salmonicida vaccines, we report here the profiles of both the outer membrane and secreted subproteomes of A. salmonicida LFI315. The 2 subproteomes were resolved by 2-dimensional electrophoresis that identified a total of 82 protein entries. Monoclonal antibodies specific to an unidentified protein antigen were utilized in the immunoproteomic analysis of both outer membrane proteins and extracellular proteins. The immunogenic protein was located in both subproteomes and identified as a 20 kDa peptidoglycan-associated lipoprotein (Pal). The identity of the antigen was verified by heterologous expression of the cloned A. salmonicida pal gene (VSAL_I1899). It is likely that the immunogenic Pal-like protein is among the constituents that act as a protective antigen in the successful vaccine used today. In view of this, it may be considered a potentially useful component in future vaccine development and pathogenicity studies.