Defensive response of European sea bass (Dicentrarchus labrax) against Listonella anguillarum or Photobacterium damselae subsp. piscicida experimental infection.

Sea bass were experimentally infected with Listonella anguillarum or Photobacterium damselae subsp. piscicida (Phdp). At 24 and 72h post-infection, the expression analysis of immune-relevant genes (IL-1ß, IL-6, IL-8, Hepcidin), the transcriptional level and detection of HSP70, and the quantification of serum iron were investigated in association with the histological analysis and the bacterial recognition in tissues by immunohistochemistry. At 15 days post-infection, the specific antibody response was detected in surviving fish, as well as the transcriptional levels of TcR and BcR sequences. Both experimental infections were characterized by a similar acute response, whereas different histological and immunohistochemistry evidences were observed. In particular, the early reaction appeared suitable for the clearance of L. anguillarum, thus limiting the histological lesions, the bacterial dissemination and the further development of acquired immunity in surviving fish. On the contrary, the innate response appeared not enough to resolve the Phdp infection, which was characterized by tissue damage, bacterial widespread and substantial detection of specific humoral immunity in surviving fish, also associated to lymphocytes clonal expansion. Besides the opportunistic conditions involved in fish vibriosis and pasteurellosis, the comparison between these experimental infection models seems to suggest that the rate of development of the acquired immunity is strictly linked to the activation of the host innate response combined to the degree of bacterial virulence.

Apoptosis in thymus of teleost fish.

The presence and distribution of apoptotic cells during thymus development and in adult were studied by in situ end-labelling of fragmented DNA in three temperate species carp (Cyprinus carpio), sea bass (Dicentrarchus labrax) and dusky grouper (Epinephelus marginatus) and in the adult thymus of three Antarctic species belonging to the genus Trematomus spp. During thymus development some few isolated apoptotic cell (AC) firstly appeared in the central-external part of the organ (carp: 5 days ph; sea bass: 35 days ph grouper: 43 days ph). Initially the cells were isolated and then increased in number and aggregated in small groups in the outer-cortical region of the thymus larvae. The high density of apoptotic cells was observed in the junction between cortex and medulla from its appearance (border between cortex and medulla, BCM). ACs decreased in number in juveniles and adult as well as the ACs average diameter. In late juveniles and in adulthood, the apoptosis were restricted to the cortex. In Antarctic species the thymus is highly adapted to low temperature (high vascularisation to effort the circulation of glycoproteins enriched plasma and strongly compact parenchyma). The apoptosis process was more extended (4-7 fold) as compare with the thymus of temperate species, even if the distribution of ACs was similar in all examined species. Data suggested a common process of T lymphocyte negative-selection in BCM of thymus during the ontogeny. The selection process seems to be still active in adult polar fish, but restricted mainly in the cortex zone.

Antigen-dependent T lymphocytes (TcRß+) are primarily differentiated in the thymus rather than in other lymphoid tissues in sea bass (Dicentrarchus labrax, L.).

All jawed vertebrates share lymphocyte receptors that allow the recognition of pathogens and the discrimination between self and non-self antigens. The T cell transmembrane receptor (TcR) has a central role in the maturation and function of T lymphocytes in vertebrates via an important role in positive selection of the variable region of TcR αß/γδ chains. In this study, the TcRß transcript expression and TcRß(+) cell distribution during the ontogeny of the immune system of sea bass (Dicentrarchus labrax, L.) were analysed. RT-PCR analysis of larvae during early development demonstrated that the ß chain transcript is expressed by 19 days post-fertilisation (p.f.). RNA probes specific for the ß chain were synthesised and used for in situ hybridisation experiments on 30 day p.f. to 180 day old juvenile larvae. A parallel immunohistochemical study was performed using the anti-T cell monoclonal antibody DLT15 developed in our laboratory [Scapigliati et al., Fish Shellfish Immunol 1996; 6:383-401]. The first thymus anlage was detectable at 32-33 days p.f. (Corresponding to about 27 days post-hatch). DLT15(+) cells were detected at day 35 p.f. in the thymus whereas TcRß(+) cells were recognisable at day 38 p.f. in the thymus and at day 41 p.f. in the gut. TcRß(+) cells were observed in capillaries from 41 to 80 days p.f. At day 46 p.f., TcRß(+) cells were identified in the head kidney and were detected in the spleen 4 days later. The present results demonstrate that TcRß(+) cells can be differentiated first in the thymus and then in other organs/tissues, suggesting potential TcRß(+) cell colonisation from the thymus to the middle gut. Once the epithelial architecture of the thymus is completed with the formation of the cortical-medullary border (around 70-75 days p.f.), DLT15(+) cells or TcRß(+) cells are confined mainly to the cortex and cortical-medullary border. In particular, a large influx of TcRß(+) cells was observed at the cortical-medullary border from 72 to 90 days p.f., suggesting a role in positive selection for this thymic region during the ontogeny of the fish immune system. This study provides novel information about the primary differentiation and distribution of TcRß(+) cells in sea bass larvae and juveniles.

Majority of TcRbeta+ T-lymphocytes located in thymus and midgut of the bony fish, Dicentrarchus labrax (L.).

Real-time polymerase chain reaction (PCR) and in situ hybridization analyses were performed to investigate the occurrence and distribution of T-lymphocytes expressing TcRbeta in intestine and lymphoid tissues of the bony fish, Dicentrarchus labrax (sea bass). Immunohistochemistry with the monoclonal antibody DLT15 (pan-T-cell marker) was carried out to compare the cytology, distribution and number of T-cells and TcRbeta+ cells in the various sampled lymphoid organs. The highest TcRbeta expression was revealed by real-time PCR in the thymus, with high levels also being found in the gut. In the thymus, DLT15+ and TcRbeta+ cell populations were concentrated in the cortex and TcRbeta+ cells were notably reactive at the cortical-medullary border, suggesting a specialized role of this region in thymocyte selection. The density of DLT15+ T-cells increased from the anterior to posterior intestine, whereas TcRbeta+ lymphocytes were more numerous in the middle intestine compared with other segments. The existence, in fish thymus, of a medulla and a cortex comparable with those of mammals is revealed by this study. The concentration of TcRbeta+ cells in the sea bass midgut also strongly suggests a special role of this intestinal segment in antigen-specific cellular immunity. The large population of TcRbeta(-)/DLT15+ T-cells in the posterior gut can probably be ascribed to the TcRgammadelta phenotype fraction.