Enhanced six-month protection and immune responses in four-finger threadfin following vaccination with formalin-killed Streptococcus iniae.

Streptococcus iniae is a major bacterial pathogen causing severe economic losses in marine and freshwater aquaculture worldwide. This study evaluated the long-term protective efficacy and immunostimulatory effects of a formalin-killed cell (FKC) vaccine, with and without Montanide ISA 763 adjuvant, against S. iniae infection in four-finger threadfin (Eleutheronema tetradactylum). Fish were assigned to three groups: control, FKC, and FKC combined with Montanide ISA 763 (FKC + ISA). Vaccinated fish received 1.5 × 109 CFU/fish and were challenged at 1, 3, and 6 months post-vaccination (mpv) using the virulent S. iniae GSI-111 strain. An additional heterologous challenge using the S. iniae T6 strain was performed at 6 mpv. Immune-related gene expression, including complement component 2 (C2), cluster of differentiation 8, tumor necrosis factor-α, interleukin-10, and interleukin-12 in spleen and kidney tissues, as well as serum lysozyme activity and S. iniae-specific immunoglobulin M (IgM) antibody responses, were evaluated throughout the experimental period. Both vaccinated groups exhibited significantly stronger immune responses than the control group, with the FKC + ISA group demonstrating the greatest and most sustained immunological activation. Elevated serum lysozyme activity and specific IgM antibody levels were maintained for up to 6 mpv. Following homologous challenge with the GSI-111 strain, the FKC + ISA group achieved 100% relative percent survival (RPS) under the present experimental conditions at all challenge time points, whereas the FKC group provided consistently high but slightly lower protection (92.9-97.2% RPS). During the heterologous challenge with the T6 strain at 6 mpv, the FKC + ISA group again achieved 100% RPS under the present experimental conditions, while the FKC group achieved 78.6% RPS. In contrast, the control group experienced high mortality, with survival rates ranging from 50 to 75%. Re-isolation analysis confirmed the absence or marked reduction of S. iniae in surviving vaccinated fish. These findings demonstrate that the Montanide ISA 763-adjuvanted FKC vaccine elicits robust and durable humoral and cellular immune responses, providing long-term homologous and heterologous protection against S. iniae infection in four-finger threadfin.