Develop subunit vaccines against Nocardia seriolae in hybrid snakehead (Channa maculata ♀ × Channa argus ♂) using two resuscitation-promoting factors.

Fish nocardiosis is a bacterial disease epidemic in aquaculture, and Nocardia seriolae is its main pathogen. The prevention and treatment of N. seriolae infection face multiple challenges, such as the unclear mechanism of chronic infection, resistance to phagocyte killing, and unsatisfied therapeutic efficacy of antibiotics. Resuscitation-promoting factors (Rpfs) are widely found in gram-positive bacteria, and have been reported to be vaccine candidates against tuberculosis and nocardiosis. In this study, RpfB and RpfD of N. seriolae ZJ0503 were obtained by prokaryotic expression and combined with Freund's Incomplete Adjuvant (FIA) as subunit vaccines, respectively. Finally, their efficacy were accessed in hybrid snakehead. The results showed that the relative survival rates (RPS) of subunit vaccines RpfB + FIA and RpfD + FIA were 77.78 % and 70.37 %, respectively. Histopathological examination revealed that hybrid snakehead did not show cytotoxic activity after intraperitoneal injection of RpfB + FIA and RpfD + FIA vaccines. In addition, both RpfB + FIA and RpfD + FIA vaccines increased the activity of multiple nonspecific immune parameters such as LYZ, CAT, ACP, AKP and SOD in the fish serum. The production of specific antibodies (IgM) and the expression of immune-related genes (tnfα, il-1β, cd4, cd8α, mhciα and mhciiα) were significantly increased compared with the control groups. In summary, RpfB and RpfD subunit vaccines were safe and can effectively protect hybrid snakehead from N. seriolae infection, and are ideal vaccine candidates for preventing fish nocardiosis.