Ontogeny of the common carp (Cyprinus carpio L.) immune system

The production of farmed fish has increased considerably over the past years, and is now providing a significant contribution to the food market. Mortality however, can be high especially in young fish. The administration of immuno-stimulants via the food can be a sustainable approach especially in young animals because of its uncomplicated use, fast delivery, less stressful entry route and broad-spectrum effect. Extensive knowledge on the ontogeny of the fish immune system is required to facilitate the rational design and selection of immuno-stimulants.

In carp ontogeny, innate immune factors are the first components of the immune system to appear. Some mRNA's and proteins were transferred from mother to egg. Moreover, these mRNA's were also produced by the embryo long before hatching (which is at 2 days post fertilisation; dpf), indicating either involvement of these factors in development itself or can be seen more probably as a preparation of the immune system for the post-hatching period. In addition, complement factor 3 (C3) mRNA production was situated in the yolk syncytial layer in embryos, followed by the liver in larvae, providing a new series of C3 producing tissues in teleost development.

The second group of immune components appearing are the cells and myelopoietic tissues of the innate immune system. The development of myelopoietic tissues is described from the embryonic stage up to the adult situation. In addition, the first appearance of myeloid cells was associated with an immune response at the molecular level: 2 dpf embryos responded to lipopolysaccharide (LPS) injection with upregulation of a panel of immunological factors and down-regulation of others, implying a functional embryonic innate defence system.  

The generation of lymphoid cells during carp development was studied by analysing expression of the recombination activating ( rag ) genes. Rag was first expressed in the thymus at 4 dpf, while a cortex/medulla differentiation was observed from 7 dpf. Also from 1 week post fertilisation (wpf), rag was also expressed in kidney (together with immunoglobulin heavy chain expression), suggesting B cell recombination in kidney but not in spleen. Rag expression was detected in thymi of animals over 1 year old, but in kidney only at low levels, indicating life-long new formation of putative T cells but severely reduced formation of B cells in older fish.

The ontogeny of immune cells was also studied in mucosal organs (intestine, gills, and skin). In addition, rag expression was examined in the intestine to investigate extrathymic generation of lymphocytes. Intraepithelial lymphocytes (putative T cells) appeared in the intestine at 3 dpf, which is shortly after hatching but before feeding, implying an important function at early age. Together with the appearance of these cells before the thymus and rag expression in the intestine, this suggests that alike mammals at least part of these cells were generated in the intestine. Monocytes/macrophages appeared in the lamina propria of the intestine at 7 dpf, while B cells appeared in intestine and gills at 6-7 weeks, suggesting a functional mucosal immune system from this age onwards.

The effect of a two-week period of oral immuno-stimulation from the age of 2 or 6 weeks (before and after the first ability to produce antibodies) with LPS, yeast DNA or high-M alginate, was studied. Oral administration of immuno-stimulants from 6 wpf suggested suppressive effects consistent with oral or juvenile tolerance. After administration from 2 wpf, the effects were less pronounced. A challenge with Vibrio anguillarum (after oral immuno-stimulation) resulted in an initially higher cumulative mortality in the group fed with LPS, but lower mortality in the groups fed with yeast DNA or high-M alginate compared to the controls, providing a provisional warning for the use of pathogen-derived immuno-stimulants in larval and juvenile fish.

In conclusion, this thesis describes the appearance of the main components of the immune system in carp ontogeny, and adds to the knowledge of developmental immunology in teleost fish in general. Moreover, tools and data obtained in this thesis were applied for evaluation of oral immuno-stimulation, paving the way for more efficient production of this extensively cultured fish species.