Immuno-protective response of Asian seabass ( Lates calcarifer ) to inactivated vaccines against Streptococcus iniae and Vibrio harveyi

**Background:** Bacterial infections, particularly Streptococcosis (caused by Streptococcus iniae) and Vibriosis (caused by Vibrio harveyi), are major threats to Asian seabass aquaculture, leading to significant economic losses. Monovalent vaccines are becoming less cost-effective due to rising co-infections, prompting interest in polyvalent vaccines that reduce stress, cost, and provide broader protection. This study aimed to evaluate the immunogenicity and protective efficacy of monovalent and bivalent formalin-inactivated vaccines against S. iniae and V. harveyi in Asian seabass (Lates calcarifer), administered via injection or immersion.

**Methods:** A total of 1200 Asian seabass (mean weight 132.6 ± 25.4 g) were randomly divided into eight treatments in triplicate (50 fish per tank): S. iniae injection (SI), V. harveyi injection (VI), bivalent S. iniae and V. harveyi injection (SVI), S. iniae immersion (SIM), V. harveyi immersion (VIM), bivalent immersion (SVIM), phosphate-buffered saline injection (PBSI), and untreated control (CTRL). Vaccines were prepared by formalin inactivation of bacterial strains (S. iniae 9609NB and V. harveyi SB9612N4) at 10^10 CFU/mL. Injection groups received 0.1 mL intraperitoneally; immersion groups were exposed to 10^9 CFU/mL for 2 minutes. Blood and serum samples were collected on days 30 and 60 post-vaccination (9 fish per treatment). Hematological parameters (RBC, WBC, Hb, Hct, lymphocytes, neutrophils), serum biochemicals (total protein, albumin, globulin), non-specific immune parameters (respiratory burst via NBT, lysozyme activity, complement activity), serum bactericidal activity, and antibody titers against both pathogens were measured. On day 60, 30 fish per treatment were challenged intraperitoneally with LD50 doses of S. iniae (4.9 × 10^6 CFU/mL) or V. harveyi (6.8 × 10^7 CFU/mL), and mortality was recorded for 14 days to calculate relative percentage survival (RPS).

**Key Results:** Hematological parameters showed no significant differences among groups at either time point (P > 0.05). Serum total protein increased significantly on day 60 in VI and SVI groups compared to CTRL and PBSI (P < 0.05). Globulin increased significantly on day 30 in VI and SVI groups and on day 60 in the VI group (P < 0.05). Albumin and respiratory burst activity (NBT) showed no significant differences. Lysozyme activity increased significantly on day 30 in the SI group (P < 0.05) and on day 60 in the SVI group (P < 0.05). Complement activity increased significantly on day 60 in VI and SVI groups (P < 0.05). Serum bactericidal activity against V. harveyi was not significant. Antibody titers against S. iniae were significantly higher in SI, VI, SVI, and SVIM groups compared to controls (P < 0.05). Antibody titers against V. harveyi were significantly higher in SI, VI, SVI, VIM, and SVIM groups (P < 0.05). After challenge with S. iniae, RPS was significantly higher in SVI (86.6%), SI (83.3%), and VI (73.3%) groups compared to CTRL (43.3%) and PBSI (40%) (P < 0.05). After challenge with V. harveyi, RPS was significantly higher in VI (86.6%), SVI (83.3%), VIM (80%), and SVIM (76.6%) groups compared to CTRL (46.6%) and PBSI (50%) (P < 0.05).

**Clinical Implications:** The bivalent injection vaccine (SVI) demonstrated the highest immunogenicity and protection, with RPS of 86.6% against S. iniae and 83.3% against V. harveyi, significantly outperforming immersion methods and controls. The vaccine enhanced both humoral (antibody titers, complement) and non-specific (lysozyme) immune responses without adverse effects on hematological health. These findings support the use of bivalent formalin-inactivated vaccines administered by injection as a cost-effective and efficacious strategy to control co-infections in Asian seabass aquaculture, potentially reducing antibiotic use and economic losses.