Immune response of flounder (Paralichthys olivaceus) was associated with the concentration of inactivated Edwardsiella tarda and immersion time.

In the optimization of immersion strategy, vaccine concentration and immersion time are two key factors needed to be considered, which largely determined the immune efficacy. In this work, the healthy flounder were vaccinated in formalin-killed Edwardsiella tarda at four concentrations (10(6), 10(7), 10(8), 10(9) CFU ml(-1)) for three immersion times (30, 60, 90 min), respectively. At the 6th week post vaccination, the flounders were challenged with live E. tarda, and the relative percent survival (RPS) of flounder in 10(9)-30, 10(8)-60, 10(8)-90 and 10(7)-90 min groups were 70%, 78%, 74% and 65%, respectively, which were much higher than the other vaccination groups. Meanwhile, the sIg(+) cells in the leucocytes of peripheral blood (PBL), spleen (SL), head kidney (HKL) were monitored by flow cytometry, and the specific sera and mucosal antibodies were measured by indirect ELISA for 6 weeks. The results showed that the proportions of sIg(+) cells in PBL, SL and HKL of vaccinated fish were significantly higher than the untreated fish since the 2nd week (P<0.05), and the fish in 10(9)-30, 10(8)-60, 10(8)-90 and 10(7)-90 min groups exhibited stronger responses than other groups, especially the 10(8)-60 min group displayed the strongest response, which reached the peaks (54.63% in PBL, 37.21% in SL, 36.51% in HKL) at the 5th week. ELISA assay showed that the kinetics of specific antibodies in sera were similar to the variation of sIg(+) cells, and the higher antibody levels were also detected in the four groups with higher RPS and stronger sIg+ response, whereas the mucosal antibody showed a faster response, which were significantly higher than the control since the 1st week (P>0.05). These results demonstrated that the higher RPS was closely associated with stronger immune response, and immersion with formalin-inactivated E. tarda under 10(8) CFU ml(-1) for 60 min induced the highest immune response of flounder against E. tarda bacterin, which might be applied for the control of edwardsiellosis in flounder.