Oral immunization of largemouth bass with recombinant Bacillus methylotrophicus expressing LMBV MCP induces...

Viral diseases have emerged as a critical bottleneck in the sustainable aquaculture of largemouth bass (Micropterus salmoides). Among these, largemouth bass virus (LMBV) has caused recurrent epizootics characterized by acute mortality and substantial economic losses, yet no licensed vaccine or effective antiviral treatment is currently available. To address this challenge, a probiotic Bacillus methylotrophicus strain with antagonistic activity against Aeromonas hydrophila was engineered as an oral vaccine carrier to express the major capsid protein (MCP) of LMBV. The recombinant B. methylotrophicus (BmP) exhibited stable fluorescence and efficient fusion protein expression verified by Western blotting. Following oral immunization, immunoglobulin M levels in serum increased by 1.8-2.4 fold compared with controls (P < 0.05). Activities of acid phosphatase and alkaline phosphatase were significantly elevated from 7 to 21 days post-immunization (dpi), lysozyme activity peaked at 14 dpi, and total superoxide dismutase activity reached its maximum (1.6-fold higher than control) at 16 dpi. Expression of il-1β and tnf-α in the head kidney and spleen increased by 3.5-5.2 fold, while antiviral and mucosal genes in the intestine were upregulated by 2.8-4.0 fold (P < 0.05). BmP remained detectable in the intestine for up to seven days after oral administration. Upon LMBV challenge, the high-dose group (HBmP) achieved a relative percent survival of 50%, while the low-dose group (LBmP) reached 30%. Collectively, these findings demonstrate that BmP expressing the heterologous MCP gene can activate systemic and intestinal immune responses, highlighting its potential as a promising, safe, and eco-friendly oral vaccine candidate for viral disease prevention in largemouth bass aquaculture.