Evaluation of the efficacy of a bivalent inactivated whole-cell vaccine against Edwardsiella tarda and Vibrio anguillarum in Tilapia (Oreochromis niloticus)

Bacterial co-infections constrain tilapia aquaculture. We evaluated the efficacy of an adjuvant-free, formalin-inactivated bivalent whole-cell vaccine combining Edwardsiella tarda and Vibrio anguillarum in Nile tilapia. Fish received the bivalent formulation, matched monovalent vaccines, or phosphate-buffered saline. Systemic and skin-mucus immunoglobulin M levels were quantified by indirect enzyme-linked immunosorbent assay, and serum and mucus bactericidal activity were assessed at 1, 7, 14, and 21-days post-vaccination. At 21 days post-vaccination, fish were challenged intraperitoneally with E. tarda or V. anguillarum, or both. By 14–21 days post-vaccination, the bivalent vaccine induced strong systemic and mucosal immunoglobulin M responses and increased bactericidal activity; across readouts, responses were significantly greater than (p < 0.05) or equal to (p > 0.05) the corresponding monovalent vaccine. After challenge with E. tarda, 21-day post-challenge survival was higher in the bivalent (54.17%, Relative Percent Survival = 38.9%) than in the monovalent (29.17%) or control (25.00%) group (both p < 0.05). After V. anguillarum challenge, survival was similar in the bivalent (87.50%, Relative Percent Survival = 78.6%) and monovalent (75.00%) groups (p > 0.05), both of which showed greater survival than the control (41.67%) (p < 0.05). After mixed E. tarda + V. anguillarum challenge, survival was significantly higher (50.00%, Relative Percent Survival = 47.8%) in the bivalent group than in the control group (4.17%) (p < 0.05). No acute adverse reactions or behavioral abnormalities were observed. These findings indicate that an adjuvant-free bivalent vaccine elicits robust mid- to late-phase humoral responses and provides broad protection without evidence of antigenic interference, supporting a practical polyvalent strategy where E. tarda and V. anguillarum co-circulate.