Abstract: hyaD is a gene involved in capsular polysaccharide synthesis of Pasteurella multocida type A. To investigate the effect of hyaD gene on P. multocida virulence and immune protection characteristics, an hyaD mutant (ΔhyaD) derived from bovine P. multocida type A strain CQ2 (PmCQ2) was constructed by homologous recombination. The results showed that, compared to the wild type strain, the capsule production and virulence of ΔhyaD were significantly reduced, and the bacterial loads in the mice organs infected with ΔhyaD were also decreased. The mutant strains were easier to adhere to mice peritoneal macrophage and were easily been swallowed, leading to the up-regulation of the expression of related inflammatory factors in macrophage. The hyaD gene deletion significantly down-regulated the expression of genes involved in capsule synthesis, LPS synthesis and transport, and iron transport. While significantly up-regulated the expression of protective antigen genes. The inactivated bacterin of wild type PmCQ2 and ΔhyaD strains were prepared by conventional methods, and the mice were immunized with both bacterin, respectively (one booster following the primary immunization). On the day 21 after first immunization, the immunized mice were challenged with homologous and heterologous serotypes P. multocida, the lungs of mice immunized with ΔhyaD had none or slight histopathological lesion at 24 h after challenged. Immunization of mice with ΔhyaD conferred significant protection against challenge from bovine P. multocida type A, type B and type F strains, as well as rabbit, porcine and avian P. multocida type A strains, with protection rate at 100%, 100%, 80%, 90%, 100%, and 100%, respectively; While immunization with similar doses of killed wild-type was failed to confer protections against heterologous and homologous serotypes excepting bovine P. multocida type A strains challenge, with protection rate above 80%. Taken together, the results show that the hyaD gene can affect the virulence of P. multocida by regulating capsule production and expression of virulence-related factors, and the hyaD gene deletion endows mutant cross-protection characteristics by up-regulating the expression of cross-protective antigens, and the mutant can be used as a candidate for the development of P. multocida vaccine. This study lays the foundation for the development of a universal vaccine against P. multocida infection.

Key words: Pasteurella multocida, hyaD, mutant, virulence, cross-protection