Abstract
Live attenuated vaccines are known to induce robust immune responses and protective efficacy in fish without causing disease. Attenuation is typically achieved by subjecting pathogens to selective pressures that reduce their virulence while maintaining immunogenicity. In this study, Vibrio harveyi isolates were attenuated through serial sub-culturing on media containing rifampicin or novobiocin. The virulence of seven clonal isolates that had adapted to grow on antibiotic-supplemented media was evaluated via intraperitoneal injection of Asian seabass (Lates calcarifer) at a dose of 10⁷ CFU per fish. One isolate derived from a novobiocin medium, VH 2886, exhibited complete attenuation, with 0 % mortality during 21 days post-injection. The mucosal vaccine potential of VH 2886 was further evaluated through immersion immunization, with or without an oral booster. Asian seabass (∼4.5 g, n = 20) were immersed in VH 2886 (1.1 × 10⁸ CFU/mL) for 30 min. For the booster group, fish received feed containing VH 2886 (5 × 10⁸ CFU/g) for three consecutive days starting on day 20 post-immersion. Upon challenge with the virulent parental strain (VH-P), relative percent survival was 29.0 % for immersion-only and 72.0 % for the booster group. Serum from booster-vaccinated fish showed significantly higher V. harveyi-specific IgM levels and enhanced bactericidal activity. In vivo back-passage safety testing confirmed VH 2886 remained non-pathogenic after five serial passages. Sequencing of the gyrB gene identified two missense mutations in VH 2886 relative to the parental strain. This study highlights the VH 2886 mutant as a promising live attenuated vaccine candidate for mucosal vaccination to protect Asian seabass against V. harveyi infection.