<![CDATA[Graphical Abstract Highlight Research Adjuvanted polyvalent FKC elicited the strongest and most sustained multi-arm immune response in Osphronemus goramy compared with monovalent and non-adjuvanted vaccines. The lead formulation combined high agglutinating antibody titres with enhanced NBT respiratory burst, indicating synergistic humoral–innate activation against Aeromonas hydrophila. Polyvalent vaccines did not dilute immunogenicity; instead, strain combination plus adjuvant broadened and amplified immune responsiveness. Longitudinal profiling of il-1β and ifn-γ revealed a stable pro-inflammatory/Th1-like cytokine signature uniquely associated with the adjuvanted polyvalent FKC. The integrated immunological “fingerprint” supports the adjuvanted polyvalent FKC as a rational lead candidate for motile Aeromonas septicaemia control in warm-water gourami aquaculture. Abstract Bacterial septicaemia caused by Aeromonas hydrophila is an important constraint for giant gourami (Osphronemus goramy) culture in Southeast Asia. Inactivated whole-cell (bacterin) vaccines are widely used against bacterial diseases in aquaculture, but comparative data on monovalent versus polyvalent A. hydrophila vaccines, with and without oil-based adjuvant, remain scarce for this species. This study evaluated the safety, immunological responses, and protective efficacy of three formalin-killed cell (FKC) vaccines prepared from gourami-derived A. hydrophila isolates: a monovalent FKC (P2), a non-adjuvanted polyvalent FKC (P3), and an oil-adjuvanted polyvalent FKC (P4), using PBS (P1) as a control. Sub-adult giant gourami were vaccinated intraperitoneally and monitored for 42 days. Serum agglutinating antibody titres, nitroblue tetrazolium (NBT)-reducing activity, and splenic il-1β and ifn-γ mRNA expression were measured at multiple time points. At 21 days post-vaccination, a separate cohort was challenged intraperitoneally with virulent A. hydrophila Ah-S1, and survival was recorded for 14 days; relative percent survival (RPS) was calculated at day 14. All FKC formulations were clinically well tolerated, with only transient post-vaccination inappetence and no gross injection-site pathology. Vaccination induced clear, treatment- and time-dependent increases in agglutinating antibody titres, NBT-reducing activity, and splenic il-1β and ifn-γ transcription, with the strongest and most sustained responses in P4, intermediate responses in P3 and P2, and minimal changes in P1. Following homologous challenge, day-14 survival was 8.3% in P1, 61.7% in P2, 75.0% in P3, and 83.3% in P4, with corresponding RPS values of 58.2%, 72.7%, and 81.8%, respectively. Under these experimental conditions, the oil-adjuvanted polyvalent FKC (P4) produced the greatest enhancement of immune responses and protection against intraperitoneal A. hydrophila challenge in giant gourami. These findings support this formulation as a candidate for further vaccine development in O. goramy and highlight the need for dose optimisation, safety assessment, heterologous challenge, and field-validation studies before recommendations for large-scale use in aquaculture.]]>
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