Rozi, Rozi and Tyasningsih, Wiwiek and Rahmahani, Jola and Aksono Herupradoto, Eduardus Bimo and Yunus, Muchammad and Anam Al Arif, Mohammad and Kuncorojakti, Suryo and Salleh, Annas and Suwarno, Suwarno (2025) Immunogenicity and protective efficacy of a native omp34 subunit vaccine against Aeromonas hydrophila in BALB/c mice: Identification of nitroblue tetrazolium as a correlate of protection within a One Health framework. Veterinary World, 18 (12). 4025 - 4045. ISSN 09728988

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Abstract

Background and Aim: Aeromonas hydrophila is a zoonotic, antimicrobial-resistant pathogen that causes significant losses in aquaculture and raises important One Health concerns. Outer membrane protein (OMP)-based subunit vaccines provide a targeted, antibiotic-sparing alternative to traditional bacterins, but validation across mammalian species remains limited. This study assessed the immunogenicity, safety, and protective effectiveness of a native ~34 kDa Omp34 (nOmp34) subunit vaccine in BALB/c mice, comparing it to a formalin-killed cell (FKC) vaccine, and examined immune factors that may predict survival. Materials and Methods: Female BALB/c mice (n = 13 per group) received subcutaneous injections of phosphate-buffered saline (PBS), FKC, FKC + incomplete Freundâs adjuvant (IFA), or native Omp34 + IFA on days 0, 14, and 28. Immune responses were assessed by measuring anti-Omp34 immunoglobulin (Ig)G2a levels via enzyme-linked immunosorbent assay, serum lysozyme activity, nitroblue tetrazolium (NBT) respiratory burst, and phagocytic activity at specified intervals up to day 42. On day 42, mice were challenged intraperitoneally with a lethal dose of A. hydrophila, causing 80 mortality, and observed for 14 days for survival, clinical scores, and body weight changes. Data analysis involved analysis of variance with Tukey post hoc tests, mixed-effects modeling, Spearman correlation, receiver operating characteristic curves, logistic regression, and Kaplan-Meier survival analysis. Results: By day 42, all immune biomarkers showed clear separation (nOmp34+IFA > FKC + IFA > PBS; p < 0.05). NBT demonstrated the strongest correlation with survival (Ï 0.90) and the highest predictive performance (Area under the curve AUC 0.80), exceeding IgG2a and phagocytosis (AUC 0.70). Post-challenge survival rates were 84.6% for nOmp34 + IFA, 61.5% for FKC + IFA, and 23.1% for PBS, corresponding to relative percent survival values of 80% and 50% compared to PBS. The direct comparison between nOmp34 and FKC revealed a favorable but not statistically significant survival benefit (p = 0.238). Vaccination was well-tolerated, with stable body weight, minimal reactogenicity, and no severe clinical events. Conclusion: The nOmp34 subunit vaccine elicited a strong, coordinated humoral and innate immune response, surpassing the matched bacterin in both efficacy and immune strength. NBT activity between days 35-42 proved to be a practical indicator of protection, aligning mechanistically with nicotinamide adenine dinucleotide phosphate -oxidase-mediated bacterial killing. These findings offer proof-of-concept for Omp34 as a scalable, antibiotic-sparing vaccine candidate and support its progression into aquaculture-relevant platforms within a One Health framework. © Rozi, et al. This article is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).

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