The influence of concentration of inactivated Edwardsiella tarda bacterin and immersion time on antigen uptake and expression of immune-related genes in Japanese flounder ( Paralichthys olivaceus )

Our previous work has demonstrated that the immune response of Japanese flounder was associated with the concentration of formalin-inactivated Edwardsiella tarda and immersion time. In order to further investigate the influence of immersion vaccine dose and bath time on the antigen uptake, formalin-killed Edwardsiella tarda bacterin was prepared and adjusted to four concentrations (10 9, 10 8, 10 7, 10 6  cfu ml −1 ) for 30, 60 and 90 min immersion in Japanese flounder model, respectively. Absolute quantitative real-time PCR was employed to examine the bacterin uptake in gill, skin, spleen and kidney at 3 and 6 h post vaccination. The results showed that the antigen uptaken in gills and skin were significant higher than spleen and kidney, and the antigen amounts in gill and skin both declined from 3 to 6 h, whereas the antigen amounts in spleen and kidney gradually increased. Significant higher antigen amounts were detected in 10 9 -30, 10 9 -60, 10 8 -60, 10 8 -90 and 10 8 -90 groups than other groups (P < 0.05), especially the 10 8 -60min group displayed the highest antigen uptaken. Meanwhile, the expression profiles of antigen recognization and presentation genes (MHCⅡα, TcRα, CD4-1), immunoglobulins (IgM, IgT), inflammatory cytokines (IL-1β, IL-6), heat shock protein 70 (HSP70) and c-type lysozyme were analyzed using real-time PCR. On the whole, the transcription levels of the eight genes exhibited to be higher in 10 7 -90, 10 8 and 10 9  cfu ml −1 groups than other groups (P < 0.05), especially the 10 8 -60 group displayed the highest up-regulation. These results demonstrated that immersion with formalin-inactivated E. tarda, especially under 10 8 -60 min condition could efficiently enhance the antigen uptake and the expression of immune-related genes, which provided evidences for an enhanced vaccination effects under an optimized combination of vaccine dose and immersion time.