Recombinant C5a Peptidase and Formalin‐Killed Cell: A Synergistic Vaccine Against <scp> <i>Streptococcus iniae</i> </scp> in Four‐Finger Threadfin Fish ( <scp> <i>Eleutheronema tetradactylum</i> </scp> )

Streptococcus iniae is a pathogenic bacterium that causes severe infections and high mortality rates in threadfin fish farms. Recurrent streptococcosis outbreaks underscore the urgent need for effective vaccination strategies. This study investigates an innovative recombinant protein vaccine incorporating a recombinant C5a peptidase encoding 1780 bp from S. iniae to enhance the immune response of the four-finger threadfin fish (Eleutheronema tetradactylum). Vaccine efficacy was assessed by evaluating specific antibody titres, lysozyme activity and the immune-related gene expression in a challenge test. The results demonstrated that fish immunised with C5a, FKC and C5a + FKC showed significantly higher specific antibody titres and lysozyme activity than the control group. Notably, rC5a + FKC displayed the highest IgM levels against the C5a antigen at 28 dpv expression. The rC5a-based vaccine significantly increased TNFα, IL-10 and IL-12 one day after immunisation, correlating with elevated serum antibody levels and the upregulated immune-related genes (C2, CD8). Fish immunised with the rC5a + FKC vaccine exhibited a stronger immune response and greater protective efficacy against the highly virulent S. iniae strain, with a relative survival rate of 97.5%. The combination rC5a + FKC RPS results are higher than those of the single antigens FKC (87.5%) and rC5a (30%), highlighting the potential of the rC5a and FKC vaccine combination as a promising method for preventing and treating S. iniae infections in threadfin fish, supporting healthier fish stocks and sustainable aquaculture practices.