Sequential PLGA-Encapsulated Selenium Microsphere Pretreatment Enhances Protective Efficacy of PLGA-Encapsulated Oral Subunit Vaccine Against Largemouth Bass Ranavirus in Micropterus salmoides

Background: Largemouth bass ranavirus (LMBV) triggers heavy mortality in farmed Largemouth bass (Micropterus salmoides). Oral subunit vaccines are suitable for mass fish immunization yet often yield unsatisfactory immunity. Intestinal microbiota strongly modulates mucosal immune competence. This study assessed whether PLGA/selenium nanoparticle (P/Se) pretreatment enhances protection afforded by a PLGA-based oral subunit vaccine (P/V) against LMBV. Methods: A 2 × 2 factorial design was applied. Fish received 28-day oral P/Se pretreatment followed by three oral doses of P/V. Material characterization, 16S rRNA high-throughput sequencing combined with microbial assembly analysis (diversity, NST, Sloan neutral model), qRT-PCR for immune related genes, serum specific antibody ELISA, LMBV immersion challenge and histopathological examination were conducted. Results: P/Se and P/V microspheres were successfully fabricated with acceptable particle size, and loading efficiency. P/Se pretreatment significantly elevated gut microbial α-diversity and enriched Actinobacteriota, in addition to increasing the relative contribution of stochastic processes to gut community assembly. The combined pretreatment–vaccine group markedly up-regulated intestinal mucosal immune genes (IL-1β, IL-8, IFN-γ, TNF-α, IgT) and systemic IgM. Consistently, higher serum-specific IgM antibody levels, improved survival rate, and alleviated virus-induced pathological injuries in trunk kidney and liver were observed in this combined group. Vaccination alone only conferred limited protection, and pretreatment alone produced no obvious antiviral protection. Conclusions: P/Se sequential intervention remodels intestinal microbiota and enhances the protective potency of oral subunit vaccine. This sequential feeding strategy offers a feasible way to optimize oral-vaccine performance for cultured largemouth bass.