Dynamic distribution of formalin-inactivated Edwardsiella tarda in olive flounder (Paralichthys olivaceus) post intramuscular injection.

Intramuscular (i.m.) injection is one of the common delivery methods of vaccination in aquaculture, which could induce an ideal immune protection to fish. In the present study, the olive flounders (Paralichthys olivaceus) were injected intramuscularly with 200 μl of three concentrations of formalin-inactivated Edwardsiella tarda bacterin (107, 108, 109 CFU ml-1) to investigate the transportation and dynamic distribution of antigen uptake in tissues by absolute real-time quantitative PCR (qPCR). The amount of uptaken antigen increased firstly, and then decreased. The peak occurred first in the blood at 6-9 h after i.m. injection, and in the spleen and head kidney at 9-15 h, then in the liver, gill and muscle at 15-24 h, finally in the skin and intestine at 36 h. The amount of uptaken antigen was highest in the head kidney, followed by in the spleen, blood, gill, and liver, and lowest in the muscle, skin and intestine. Among the three dose groups, the amount of uptaken antigen in all tested tissues became higher with the increasing dose of injected bacterin. Moreover, the tissue distribution of antigen uptake was investigated by indirect immunofluorescence assay (IIFA) at 15 h after i.m. injection with 200 μl of 108 CFU ml-1E. tarda bacterin. The distribution of antigen was mainly observed in the head kidney, then in the spleen, blood, liver, gill and muscle, and least in the skin and intestine, which correlated with the results of absolute qPCR detection. Furthermore, the expression levels of MHC Iα, MHC IIα, CD4-1 and CD8α were detected by RT-qPCR. The expression of these four genes peaked highest in the head kidney, followed by in the spleen, liver, blood and gill, and lowest in the muscle, skin and intestine, and the levels increased in parallel with the increasing dose of injected vaccine. All these results provided an important insight into the dynamic transportation of antigen uptake, and also deepened the understanding of immune response to the i.m. injection.