Deliverables Identification of cellular and humoral defence mechanisms - Carp. Complement component C3, inducible nitrogen oxide synthetase (iNOS), interleukin-1 beta (IL-1beta), tumor necrosis alpha (TNF-alpha) were all present in the yolk sac embryo. This indicate that the carp embryo may have defence mechanisms that play important roles in eradication pathogens at a very early stage (2dpf). The expression of these genes could be modulated by microinjection of LPS in the eggs. - Sea bass. Two monoclonal antibodies against sea bass complement C3 have been obtained. These mAb immunostained in reducing and nonreducing western blot analysis C3 polypeptides in whole larval homogenates. No differences in C3 presence were observed between control and treated sea bass larvae. The sea bass C3 cDNA was cloned from aminoterminal analysis of purified C3 molecule. The results that will be obtained by studying its expression in sea bass larvae will be released after the end of the project. The use of molecular probes for complement factor C3 enables the R&D society to use complement expression system to monitor effects of e.g. vaccination and immunostimulation. - Halibut. Immune defence molecules such as complement component C3 and lysozyme was found in halibut eggs throughout the egg cycle. There may be a maternal transfer of these molecules and the antipathogenic significance of this presence is still not found. Cathepsin H and D was also found at low levels in halibut eggs. - Atlantic cod. Cod complement component C3 has been purified and characterised, anti cod C3 antiserum has been raised to be used in IH experiments. In addition, in-sity hybridisation probes, PCR primers have been made. With all these molecular probes it is possible for the R&D society to screen cod with respect to C3 contents, gene expression analysis that may be beneficial to study the cod immune system. - Spotted wolffish complement factor C3 was isolated and characterised. Using rabbit anti-spotted wolffish C3 antiserum there was a strong indication that C3 is maternally transferred to its offspring since C3 was found in unfertilised eggs. The wolffish embryo starts to produce its own C3 as the liver generations starts. The C3 contents in wolffish larvae could be increased by use of immunostimulants such as LPS and alginate. The exact significance of the presence of C3 in the eggs and embryos remains to be elucidated. The eggs also contained cathepsins that are important in metabolic/catabolic activities such as antigen degradation as well as peptide antigen expression on MHC II molecules. The activities of cathepsin increased during development. Identification of fish larvae cells active in endocytosis and phagocytosis Identification of fish larvae cells active in endocytosis and phagocytosis. - In the spotted wolffish, microinjection of tracer substances in the eggs with subsequent microscopic analysis of the embryos revealed that the embryo contained the tracer substances in the lumen of the intestine, intestinal wall, kidney and liver. - In the carp, microinjected tracer susbtances were not absorbed by embryonic cells. These results indicate that in wolffish there a phagocytic cells, but since the amount of tracer substances was low, it is speculative to conclude about the presence of phagocytic and endocytic cells in the fish embryo inside eggs. Modulation of the immune system of larval fish against infectious diseases by vaccination - Carp: No experiments done. - Atlantic cod: With few exceptions, cod does not appear to produce specific antibody response on challenge or vaccination/injection. - Spotted wolfish: A histopathological study has been performed on fish experimentally challenged with atypical A. salmonicida. In addition, mucosal uptake of antigens (vaccine) have been examined but no conclusive results have been reached. Immersion vaccination did not lead to increased survival of wolffish after experimental atypical A. salmonicida challenge. The reason may be due to vaccine potency. - Atlantic halibut and sea bass: Several attempts were made to vaccinate the sea bass and halibut larvae. No vaccination experiments were successful. However, several challenge protocols were developed using several fish bacterial species and strains. Modulation of fish larval defence mechanisms by immunostimulants - Carp. Adding immunostimulants to carp larval diets did not induce significant gene expressions (IL-1beta, TNF alpha and iNOS, C3, alpha-2-macroglobulin and SAA), whereas the contents of B cells, evaluated using flow cytometric analysis, in immunostimulated carp was lower compared to controls. Taken together, it appears that some immunostimulants affect leucocyte numbers and dynamics, whereas it is difficult to compared such effects with increase in gene expression of the selected genes. - Sea bass. The alginate (FMI) supplied by the project¿s co-ordinator, when administered by bathing or orally resulted in an apparent better growth of animals, although it not affected the cellular and molecular parameters studied. The A. salmonicida LPS had a detectable and reproducible physiological effect when administered orally to sea bass, and this effect did not affected larval viability and cellular and molecular parameters studied. In sea bass, LPS induced immunostimulant that appeared to be the most effective without any pathogen challenge. - Atlantic cod. So far, it seems that the immunostimulants in question did not induce disease resistance, elevated cellular defence mechanisms or humoral defence mechanisms. However, more work is required and is indeed in progress to evaluate the effects of immunostimulants. - Atlantic halibut. Difficult to interpret the results - Spotted wolffish. The addition of bioactive alginate in formulated feed induced higher specific growth rate compared to controls. No significant disease resistance was observed in immunostimulated fish. The use of immunostimulants as growth promoters may have high impact on the mass production of fish juveniles. Bioactive alginate is currently under investigation as a feed additive for use in several other fish species. Identification of cells participating in the defence against pathogens and cellular defence mechanisms during larval development - Carp immune cells The ontogenic appearance of carp leucocytes has been revealed. In brief, a very early presence (around hatching during the 2nd day p.f) of macrophages, granulocytes and at 3 dpf the putative "mucosal T cells" were found using antibody staining techniques. Leucocytes were cells found as early as 2 dpf in hematopoietic tissue in the tail region of carp. It appears that the haematopoietic tissue in the tail is far more extensive and contains considerable numbers of B cells and macrophages at an early age (2-4 days pf) compared to haematopoietic tissue in the (head) kidney. 'Mucosal T cells' were not detected in the hematopoietic tissue of the tail, but were found especially in the third segment from the gut, also from 3 dpf. Another monoclonal (WCI12) was used to study B cell ontogeny on a tissue level. Positive cells were found in (head) kidney and spleen from 2 weeks p.f., in gut from 5 weeks p.f., in gills from 6 weeks p.f., and in skin from 12 weeks p.f. To identify the location where and stage when granulocytes (both neutrophilic and basophilic) appear, electron microscopy was used. We found neutrophilic granulocytes between aorta and Vena cardinalis and the posterior blood island (around caudal part of Vena cardinalis) at 48 hours, but not at 24 hours. Basophilic granulocytes were found in tissue suspensions of 3-days-old animals, but in situ they were found somewhat later in 5-days-old animals. The results obtained shed light on the ontogenic appearance of lecocytes which, in turn, could contribute to the development of efficient vaccination and immunostimulation strategies for carp larvae in the future. - Sea bass immune cells The sea bass T cell were found to be present in the intestine and thymus 28 dph, head kidney 35 and spleen 45 dph. B cells were present in the head kidney and spleen 45 dph, and in the intestine and thymus 90 dph. This indicate that sea bass larvae can mount a specific immune response not before ca. 45 dph. This has significance on vaccination strategies for sea bass larvae. - Atlantic halibut immune cells Ontogenic development of halibut lecucoytes were not studied except the developmental cycle of halibut thymus. - Atlantic cod larval cells have been characterised by enzyme histochemistry and for their production of antimicrobial susbtances. Embryonic/larval cells were found to contain enzymes important for destruction of microbial components, and could be assayed for e.g. respiratory burst reaction that produces reactive oxygen radicals. - Spotted wolffish macrophages have been successfully isolated, cultivated and characterised. Simple methods for assaying microbial defence mechanisms have been done. Any presence of macrophages in wolffish embryo have not been found. For cod, halibut and spotted wolffish it is too early to give an indication of the timing of specific immune responses to occur. Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
