ESTABLISH A FLOW CYTOMETRY ANALYSIS OF GROUPER LYMPHOCYTE DIFFERENTIATION UNDER IMMUNE STIMULATION

Grouper (Epinephelus sp.) are high economic value aquaculture species around subtropical regions especially in Southeast Asia. However, the disease control is still a serious problem in the recent years. To control the disease, using immunoprophylaxis method, such as vaccine is considered as an effective method to prevent disease outbreak and transmission. An ideal immune protection is contributed by humoral or cell-mediated immune responses mediated by helper T lymphocyte (T_H, CD4⁺/CD8^-) and cytotoxic T lymphocyte (T_C, CD4^-/CD8⁺), respectively. Humoral immunity predominately triggers neutralization of extracellular pathogens whereas cell-mediated immunity is mainly responsive to kill extracellular pathogens and virus. However, in teleost few tools were developed to evaluate the differentiation of these lymphocytes, so the effectiveness of vaccine is hard to detect. Flow cytometry analysis for immune cell differentiation is already used in mammals for years. It can detect multi-parametric cell characteristics simultaneously.

This project is aim to setup this technique for groupers, thus the antiserum for CD4 and CD8 were prepared and the specificity and sensitivity were also evaluated. Cytokines which induce T_H1 and T_H2 pathway, such as interferon-γ and interleukin-6 were used as standard to evaluate efficiency of established flow cytometry system in this project; finally, apply this system to the fish inoculated with different adjuvant formula and indicated that cytokine additives can control the direction of immune response and might increase the efficacy of fish vaccine.