Merete Bjørgan Schrøder

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.

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.

Immunological differences in intestine and rectum of Atlantic cod (Gadus morhua L.).

The defence system of the distal gut (hindgut and rectum) of Atlantic cod, (Gadus morhua L.) was studied using (immuno)histochemical, electron microscopical and real-time quantitative PCR techniques. The uptake and transport of macromolecules in the intestinal epithelium was also investigated. In this study we observed that cod has many and large goblet cells in its intestinal epithelium and that IgM(+) cells are present in the lamina propria and their number is considerably higher in the rectum than in the intestine. Myeloperoxidase staining revealed low numbers of granulocytes in and under the epithelium of the distal intestine, whereas high numbers were found clustered in the submucosa of the rectum. Electron microscopy not only confirmed these observations, but also revealed the presence of lymphoid cells and macrophages within the intestinal epithelium. Acid phosphatase staining demonstrated more positive macrophage-like cells in the rectum than in the distal intestine. Antigen uptake studies showed a diffused absorption of horse radish peroxidase (HRP) and LTB-GFP, whereas ferritin uptake could not be detected. Basal gene expression of cytokines (IL-1beta, IL-8 and IL-10) and immune relevant molecules (hepcidin and BPI/LPB) were compared in both the intestine and rectum and revealed approximately 2-9 times higher expression in the rectum, of which IL-1beta expression showed the most prominent difference. The present results clearly indicate that intestinal immunity is very prominent in the rectum of cod.

Comparison of antibody responses in Atlantic cod (Gadus morhua L.) to Vibrio anguillarum, Aeromonas salmonicida and Francisella sp.

Bacterial diseases such as vibriosis, atypical furunculosis and francisellosis, are registered as an increasing problem in cod farming in Norway. In order to develop efficient vaccines against diseases it is of interest to investigate if the cod immune system differentiates between various serotypes of Vibrio anguillarum and variants of Aeromonas salmonicida associated with the diseases by raising specific antibody responses. Cod of the same origin were shown to raise significant responses to V. anguillarum, A. salmonicida and the intracellular bacteria Francisella sp. Individual responses to V. anguillarum or A. salmonicida varied from none to high responses, while all individuals immunised with Francisella revealed a significant response. The cod immune system appeared in some degree to distinguish between V. anguillarum serotypes and A. salmonicida variants. Although all bacteria had induced significant antibody responses detectable in whole cell ELISA, only some had induced antibodies with specificity to linear O-polysaccharide epitopes on blot.

Atypical furunculosis vaccines for Atlantic cod (Gadus morhua); vaccine efficacy and antibody responses

Atypical furunculosis caused by atypical Aeromonas salmonicida, is an emerging problem in farming of Atlantic cod (Gadus morhua) in Norway, and vaccines are needed. Atypical A. salmonicida comprises a heterogeneous group of bacteria differing in surface antigens such as the A-layer protein and lipopolysaccharides (LPS). Except for one of the experimental oil-adjuvanted whole cell vaccines based on various isolates they all resulted in moderate protection. No clear correlation between vaccine efficacies and the A-protein group or LPS type of the vaccine isolates was revealed, while a correlation between efficacy and the presence of cross-reacting LPS-specific antibodies is indicated.

Comparison of atypical furunculosis vaccines in spotted wolffish (Anarhicas minor O.) and Atlantic halibut (Hippoglossus hippoglossus L.)

Atypical furunculosis caused by atypical Aeromonas salmonicida is a problem in farming of Atlantic halibut (Hippoglossus hippoglossus) and spotted wolffish (Anarhicas minor) in Norway, and vaccines for marine fish species are not available. Susceptibility to atypical A. salmonicida infection and efficacy of furunculosis vaccines in various fish species indicate that the host responses differ. Here, spotted wolffish was shown to be highly susceptible to intraperitoneal challenge with atypical A. salmonicida compared to halibut, and the two species appeared to respond differently to identical vaccines. Grouping of the atypical A. salmonicida isolates used as vaccines was based on a variable region in the surface A-layer protein, but no clear correlation between vaccine efficacy and A-protein group was found.

Atypical furunculosis vaccines for Atlantic cod Gadhus morhua: impact of reattached Aeromonas salmonicida A-layer protein on vaccine efficacy.

Atypical furunculosis caused by atypical Aeromonas salmonicida bacteria is reported as an increasing problem in farmed Atlantic cod Gadus morhua in Norway. At present, furunculosis vaccines adapted for cod or other marine fish species are not available. To identify bacterial components important for inducing protection in cod, we compared oil-adjuvanted vaccines based on A. salmonicida isolates phenotypically differing in their major cell surface constituents, such as the A-layer protein and lipopolysaccharide O-chains. Also included was an A-layer-deficient isolate with physically reattached A-layer protein. Vaccines containing A. salmonicida A-layer-producing cells elicited significantly better protection than vaccines with A-layer-deficient cells or with a supernatant with secreted A-layer protein. The A. salmonicida cells with reattached A-layer-protein resulted in significant and equal protection to the A-layer-producing cells and protected significantly better than the A-layer-deficient isolate. These results indicate that the A-layer protein when attached to the cell surface plays a role in inducing protective immunity in cod.