Construction of Heme‐Binding Protein Deleted Strain and Using It as an Attenuated Vaccine Against <i>Nocardia seriolae</i> in Hybrid Snakehead (<i>Channa maculata</i> ♀ × <i>Channa argus</i> ♂)

Nocardia seriolae is the main pathogen causing fish nocardiosis, which can infect various aquatic freshwater and marine fish species. We have previously confirmed that the heme-binding protein of N. seriolae (NsHBP) is a secreted protein relating to bacterial virulence. In the present study, a ΔNsHBP mutant of N. seriolae was successfully generated by deleting NsHBP gene, and it was further used for attenuated vaccine development and evaluation in hybrid snakehead (Channa maculata ♀ × Channa argus ♂). The LD₅₀ of strain ΔNsHBP by intraperitoneal injection in hybrid snakehead was presented at 5.47 × 10⁷ CFU/mL, significantly (p < 0.01) higher than that of the wild-type strain at 5.28 × 10⁵ CFU/mL, indicating the virulent reduction of strain ΔNsHBP in comparison to wild-type strain. The live attenuated vaccine used strain ΔNsHBP at sub-clinical dosages (LD₁₅, 1.26 × 10⁷ CFU/mL) to immunise the fish by intraperitoneal injection for 56 days could provide a relative percentage survival (RPS) at 82.40% against artificial challenge with wild-type strain ZJ0503. Additionally, serum enzymes activities of lysozyme (LZM), peroxidase (POD), acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD) as well as specific antibody (IgM) titers were induced in fish following ΔNsHBP vaccination. Furthermore, the expression of immune-related genes (TNFα, IL-1β, MHCIα, MHCIIα, CD4 and CD8α) was significantly increased in comparison to the control group during immunisation period with ΔNsHBP in fish. Taken together, it is revealed that the deleted strain ΔNsHBP could be a potential candidate for live attenuated vaccine development to control fish nocardiosis in aquaculture.