Intranasal delivery of a live attenuated vaccine confers efficient protection against Riemerella anatipestifer serotype 1 in ducklings.

Mucosal immunity serves as the first line of defense against pathogenic infections in animals. Respiratory mucosal vaccines elicit multi-layered immune responses that confer distinct advantages for disease prevention and control. Riemerella anatipestifer (RA) infection causes duck infectious serositis, leading to sustained economic losses in the duck industry. Commercially available inactivated RA vaccines are hampered by drawbacks such as stress induction and adjuvant residue. Targeting the RA-YM strain of serotype 1, the dominant epidemic RA serotype in China, this study employed markerless gene knockout technology to delete the virulence genes cas9 and sprA, thereby constructing the double-gene deletion mutant RA-YMΔcas9ΔsprA. The median lethal dose (LD₅₀) of this mutant was 2.9 × 10⁵-fold higher than that of the parental strain, indicating significant virulence attenuation. A single intranasal immunization of 7-day-old ducklings with the mutant induced high serum IgY level and mucosal secretory IgA (sIgA) antibodies, while also significantly upregulating the expression levels of key cytokines such as IFN-γ and IL-6. Thus, this immunization conferred effective protection against challenges with both the homologous parental strain and a heterologous virulent strain of the same serotype. Moreover, intranasal immunization proved to be superior to intramuscular injection. This safe and effective live- attenuated vaccine candidate provides a novel strategy for the control of RA infections.