Ontogeny of the immune system of fish using specific markers

A panel of monoclonal antibodies (mAbs) was used for the characterisation of leucocyte subpopulations during the ontogeny of common carp ( Cyprinus carpio , L.) and sea bass ( Dicentrarchus labrax , L.). In carp the leucocytes were monitored in different lymphoid organs by immunofluorescence and flow cytometry using specific mAbs for early T cells (WCL9), B cells (WCI12), monocytes/macrophages (WCL15) and thrombocytes (WCL6).Early T cells were very numerous (77 %) in thymus during the first weeks post fertilisation (p.f.), but also present in other organs, especially head kidney. Subsequently, these cells disappeared from all organs, except the thymus (40%). B cells appeared in the head kidney from the second week p.f., and later on in the spleen and blood, but their number remained low in the thymus and gut. Thrombocytes were detected in cell suspensions of spleen from the first week p.f. and their percentage increased until the 4 thweek (30%) and then decreased in spleen (10%), but increased in blood (30%). Monocyte/macrophage-like cells were present in all organs from the first week p.f. and their percentage gradually increased until the 8 thweek p.f..By using mAb WCL15 on fixed tissue the in situ distribution of monocytes/macrophages was studied in thymus, head kidney, spleen and gut from 2 days until 60 weeks p.f.. Macrophages were found from day 2 p.f. in the head kidney and in the dorsal part of the yolk sac epithelium. From 1 week onwards, macrophages were also found scattered in the thymus and gut and during the second week also in spleen. The number of macrophages increased in all lymphoid tissues until the 6 th-8 thweek p.f., then decreased except in the thymus, where they became localised mainly at the cortical-medullary boundary.By using mAb WCL38 increasing numbers of mucosal T cells were observed in cell suspensions of gills and intestine from the first week p.f. onwards. With immuno-histochemistry the early appearance of mucosal T cells could be confirmed in the gills and intestine but was also detected in skin. With mAb WCL9 the ontogeny of the carp thymus was studied and special attention was paid to the development of cortex and medulla. The differentiation between cortex and medulla started in the 4 thweek p.f.. Ultrastructural study of the developing thymus confirmed these data and permitted the analysis of the distribution and morphological differences of the epithelial cells. In addition, numerous apoptotic cells appeared in the cortex from the 4 thweek p.f. onwards, while lower numbers were observed in medulla.In sea bass, mAbs DLT15 and DLIg3 specific for T and B cells, respectively, were employed to describe the morphology and distribution of these cells. Enriched leucocyte fractions from different tissues (thymus, spleen, head kidney, intestine and blood) were used. High numbers of T cells were detected in thymus and intestine, whereas B cells were more numerous in blood, head kidney and spleen. Furthermore, an ontogenetic study with DLT15 revealed that the thymus was the first organ with T cells, followed by head kidney and spleen. Although some differences were observed between the two commercially important fish species studied, the data presented in this thesis are of phylogenetic interest and can be used for the development of vaccination strategies in young fish.