Comparative field assessment of nanobubble-assisted immersion versus injection vaccination against Streptococcus agalactiae serotypes Ia and III

Abstract Streptococcosis caused by Streptococcus agalactiae is a major source of economic loss in intensive tilapia aquaculture. Although vaccination is effective, its adoption is constrained by cost and logistical challenges. We previously developed a low-cost bivalent killed vaccine (combined serotypes Ia and III) that provides strong protection when administered by injection; however, injectable vaccination remains impractical for many farmers. Nanobubble-assisted immersion priming followed by oral boosting has shown promise as an alternative vaccination strategy, but its effectiveness for controlling streptococcosis in tilapia has not yet been optimized. This study evaluated two nanobubble-assisted immersion priming-oral boosting strategies for the bivalent S. agalactiae vaccine under laboratory conditions and subsequently compared its performance using a conventional prime–boost injection regimen protocol in field settings. In laboratory trials, nanobubble-assisted groups (O2NB-CS)-Vac and [O3NB-(O2NB-CS)-Vac] exhibited enhanced innate immune responses (lysozyme, IL1β, TNFα, IL6, HSP70) and higher protection, expressed as relative percent survival (RPS, 55.56–63.16%) than the non-nanobubble immersion group (RPS, 16.67–36.84%). Given their comparable efficacy, both nanobubble-assisted immersion groups were advanced to a farm trial. In the field trial, nanobubble-assisted immersion priming-oral boosting induced only negligible antibody IgM responses against both serotypes, consistent with laboratory results. In contrast, injection vaccination elicited strong IgM responses to serotype Ia, particularly after boosting. Immersion groups showed elevated lysozyme activity at days 30 and 60, and significant upregulation of gene IL1β and CD8 up to day 30, whereas no comparable immune responses were observed in the injection group relative to the non-vaccinated control. Laboratory challenge testing of field-vaccinated fish at day 60 post vaccination yielded lower protection in immersion groups (RPS, 10.39–42.86%) compared with injection (RPS, 85.71%). Harvest survival and production outcomes favored injection; however, interpretation of production-scale trial is limited by natural co-infections, coupled with potential environmental and husbandry variations. Overall, the nanobubble-assisted immersion strategy produced measurable innate immune activation and moderate protection under laboratory conditions, but it was less effective than injection in the current study. Further optimization of oral formulation and booster schedules are required to improve the immersion-prime oral boost strategies.