Physicochemical characterization and immunostimulatory effects of mycogenic metal oxide nanoparticle-enriched feed-based bivalent vaccines in red hybrid tilapia (Oreochromis spp.).

Streptococcosis and motile Aeromonad septicemia (MAS) are major tilapia diseases causing significant economic losses. Feed-based vaccination offers a sustainable control strategy. However, the efficacy of oral vaccines in tilapia is largely constrained by inadequate adjuvant performance and associated side effects, while the safety, antigen interaction, and immunomodulatory potential of mycogenic metal oxide nanoparticles (MONPs) as a vaccine adjuvant remain insufficiently understood. This study evaluated the safety and immune responses of a novel feed-based bivalent vaccine (FBBV) containing formalin-killed Streptococcus agalactiae and Aeromonas hydrophila, enriched with mycogenic zinc oxide (ZnO-NPs) or aluminium oxide nanoparticles (Al2O3-NPs), in red hybrid tilapia. Four hundred and eighty red hybrid tilapia (10.0 ± 1.5 g) were randomly assigned to four groups (control, AquaBooster, FBBV/ZnO-NPs, and FBBV/Al2O3-NPs) and orally immunized at weeks 0, 2, and 6. Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM) analyses suggested interactions between mycogenic MONPs and formalin-killed whole cells (FKWC). Furthermore, spray-coating onto feed maintained the estimated DNA abundance of formalin-killed S. agalactiae while reducing that of A. hydrophila by approximately three-fold. Haematological parameters were largely stable, though white blood cell (WBC) and lymphocytes increased in all vaccinated groups. Histopathological examination revealed generally mild alterations in the control, AquaBooster, and FBBV/Al2O3-NPs groups, whereas the FBBV/ZnO-NP group exhibited marked renal mineralization. Meanwhile, spleen histology was comparable across groups. Moreover, early IgM induction occurred in FBBV/ZnO-NPs, while sustained IgM levels were seen in FBBV/Al2O3-NPs and AquaBooster groups. Gut-associated lymphoid tissue (GALT) area was significantly increased in all vaccinated groups compared with the control. Hindgut qPCR demonstrated comparable estimated DNA abundance of FKWC among the vaccinated groups, whereas hindgut RT-qPCR analysis revealed upregulation of innate (IL-1β, TNF-α) and adaptive (IgM, IgT) immune markers. Overall, both MONP formulations stimulated immune responses; however, their safety profiles differed. The Al2O3-NPs formulation exhibited minimal histopathological alterations, whereas the ZnO-NP formulation induced notable renal mineralization, indicating that this formulation warrants further dose optimization and safety evaluation.