Comparative Evaluation of Oral Biofilm and Killed Cell Vaccines Against <scp> <i>Streptococcus iniae</i> </scp> in Four‐Finger Threadfin Fish ( <scp> <i>Eleutheronema tetradactylum</i> </scp> ): Immune Response and Protection Efficacy

Mariculture, a significant component of the maritime industry that focuses on marine food production, faces challenges in maintaining productivity during bacterial disease outbreaks, particularly in high-value aquaculture such as the four-finger threadfin fish in Taiwan. Streptococcosis, caused by Streptococcus iniae, is a major contributor to the mortality of the four-finger threadfin (Eleutheronema tetradactylum). Recurrent streptococcosis outbreaks have highlighted the pressing need for highly effective vaccination strategies. Given its safety, environmental friendliness, and protective effects, vaccination is widely acknowledged as an effective means of preventing aquatic diseases. An innovative approach involves using biofilm-forming S. iniae as vaccine candidates for aquaculture. This study presents an effective approach for developing a biofilm-based vaccine by cultivating S. iniae on chitosan particles, facilitating robust biofilm formation and enhancing immune responses in four-finger threadfin fish. For comparison, a formalin-killed cell (FKC) vaccine, prepared from whole-cell S. iniae, was evaluated. Immune responses were examined in the blood, mucus, and gut lavage from both the vaccinated and control groups. These responses include immune-related gene expression, antibody titers, and lysozyme activity. At 30 days post-vaccination, the biofilm vaccine group exhibited elevated antibody titers, with values of 0.23 ± 0.02 in serum, 0.09 ± 0.01 in mucus, and 0.16 ± 0.01 in gut lavage. Following vaccination, both the FKC and biofilm vaccines significantly upregulated the expression of key proinflammatory cytokines (tumour necrosis factor-α, interleukin [IL]-10, IL-12) in the spleen and kidney, indicating robust activation of the innate immune response. However, the biofilm vaccine induced markedly higher expression of these cytokines, highlighting its stronger stimulation of innate immune responses. These results suggest that the biofilm-based formulation stimulates early immune signalling pathways that are critical for protection against S. iniae infection. In the challenge experiments, the relative percent survival was of 22.85% for the biofilm and 42.8% for the FKC vaccine groups. This study demonstrates that while both FKC and biofilm vaccines activated innate and adaptive immunity, the FKC vaccine provided higher protection (RPS 42.8% vs. 22.85%), indicating that strong immunogenicity does not always translate into effective protection and that oral vaccine strategies require further refinement. Further optimisation of oral vaccine formulations is required to improve the protective efficacy of biofilm-based vaccines in aquaculture.