| Turbot (Scophthalmus maximus) is an economically important fish and recently culturing has become popular in China. However, with the development of intensive culture, infectious diseases have become a major obstacle to the cultivation of it and caused a big loss. The swollen abdomen is the most important disease of turbot, and its agent is the Edwardsiella tarda. Problems, such as drug resistance, food and environmental security, have emerged due to over using of aquacultural drug to prevent and cure infectious diseases. Therefore, immunization has become the most important method in preventing and curing fish diseases. In this study, with methods of micro observation, immunohistochemistry, indirect fluorescence analyze technology, flow cytometry and agglutination tires analyze, the lymphoid tissues and cells were observed and compared in mucosal and systemic lymphoid organ in turbot, Ig+ cells and plasma antibody level were measured and compared in unvaccinated turbots and vaccinated ones by inactivated E. tarda and OPM via direct immersion (d.i.), intraperitoneal injection (i.p.) and anal incubation (a.i.). The immune responses were evaluated in mucosal and systemic lymphoid tissues in turbot vaccinated by different antigen via different route. The independent immunological role of mucosal tissues was discussed. The following are the details.(1) The tissue observation of mucosal and systemic immune organs of turbot. Lymphocytes presented in skin, gill, hindgut, head kidney, spleen and peripheral blood (PBL) after micro observation to tissue sections. The immunoglobulin-positive cell (Ig+ cell) could be locally observed in these tissues above except in the skin by immunohistochemistry with a monoclonal antibody (mouse-anti-turbot IgM). Ig+ cells of gill were located in filament sinusoid between the epithelial cells and the support cells. Ig+ cells of hindgut presented within the lamina propria or the lower layers of lamina epithelialis. Ig+ cells in PBL often appeared among lymphocytes. Ig+ cells were often distributed throughout the parenchyma in spleen, but tended to concentrate around melano-macrophage centres (MMCs). Ig+ cells scattered in the interstitial tissue in head kidney. The Ig+ cells isolated from skin, gills, hindgut kidney and PBL were measured in healthy turbots (aged 2.5 and 10 months post-fertilization, mpf) by flow cytometry. Results were that the percentage of Ig+ cells respectively was 0±0.00% of skin, 1.58±0.40% of gills, 2.53±0.70% of hindgut, 17.05±0.39% of PBL, 21.06±1.79% of kidney in turbot aged 2.5 mpf and was 0.94±0.74% of skin, 1.60±0.71% of gills, 3.91±1.06% of hindgut, 28.95±0.39% of PBL, 21.54±0.90% of kidney in turbot aged 10 mpf.(2) Isolation and identification of E. tarda and preparation of antigens. The main pathogenic bacteria was isolated from turbot suffered swollen abdomen. Biochemical identification and 16S rRNA gene sequence analysis were used to identify the main pathogenic bacteria. The results showed that it was identified as E.tarda. Then the killed cell antigen was prepared with method of 0.5% formalin-killing. The major OPM antigen was also prepared with the extraction method of Sarkosyl. (3) The immune response of mucosal and systemic lymphoid tissues in turbot after immunization with inactivated E. tarda. After d.i., a 7-day advanced phase in peaking time of number of Ig+ cells was observed in gill compared with in PBL or kidney from immunized turbot. A high elevation of number of Ig+ cells was observed in the gill, and this was significantly different from the control group's. However, a slight elevation in the number of Ig+ cells was observed in the kidney and PBL in vaccinated turbots via bath, and this was insignificantly different from their respective control group's. After i.p., the peaks of number of Ig+ cells in gills, PBL and kidney reached on the same time point from immunized turbot. A slight elevation of Ig+ cells was observed in the gills from turbot vaccinated by i.p., and the increase was not significant compared with its control's and was significantly lower than vaccinated turbot via bath. But, a clear increase of the number of Ig+ cells was observed in kidney and PBL in turbot vaccinated by i.p., and a significant difference was observed in the kidney and PBL compared with their respective controls. In addition, the peak of agglutination tire in plasma was on the same time point as the peak of the number of Ig+ cells in gill in turbot vaccinated by d.i, and it was higher significantly than its control's. This showed that immune response to E .tarda was significant and mainly from gill in turbot vaccinated by d.i..(4) The immune response to different antigen in turbot. After immunization with inactivated E. tarda and major OPM via d.i., i.p. and a.i., the followings were obtained: The number of Ig+ cells in PBL and the plasma agglutination tire was respectively insignificantly lower in turbot vaccinated by OPM than in one vaccinated by inactivated E. tarda via i.p.. The number of Ig+ cell in PBL and the plasma agglutination tire was respectively insignificantly higher in turbot vaccinated by OPM than in one vaccinated by inactivated E. tarda via d.i.. The number of Ig+ cell in hindgut and PBL as well as the plasma agglutination tire was respectively insignificantly lower in turbot vaccinated by inactivated E. tarda than in one vaccinated by major OPM via a.i..Therefore, our results clearly supported the existence of mucosal and systemic compartments in immune system, and the mucosal compartment had tissue and cell conditions to conduct a independent special immune response to an antigen in turbot. The administration route could play an important role in inducing immune response of the two immune compartments, the d.i mainly elicited mucosal immune, the i.p. could induce a more intensive systemic immune, and the a.i. almost had a same role to induce responses in gut mucosal and systemic tissue. Additionally, the different antigen had different immunological role upon mucosal and systemic compartment in fish. These are essential for designing vaccination strategies in fish. |
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