Relationship Between The Capsule And OmpH Of Avian Pasteurella Multocida C48-3and Their Pathogenicity

To investigate the relationship between capsule and outer membrane protein H(OmpH) in the pathogenicity of Pasteurella multocida type A strains, the hexABC geneknock-out mutant (C48-3hexABC) and the ompH gene knock-out mutant(C48-3ompH) of avian P. multocida C48-3were constructed by homologousrecombination, and the DNA replacement was confirmed by PCR, RT-PCR and DNAsequencing. The growth ability of C48-3ΔhexABC and C48-3ompH were relativelyincreased compared to the parent strain C48-3. Electron microscopy imaging ofC48-3ΔhexABC revealed the absence of capsular material compared to the parent strainC48-3, and a little capsular material in the cell surface of C48-3OmpH were observed.SDS-PAGE and Western blot demonstrated that the capsule mutation did not affect theexpression of OmpH. RT-PCR confirmed that the ompH mutation affects thetranslocation of capsule polysaccharide to bacterial cell surface.To further investigate the role of capsule and OmpH protein involved in thepathogenicity of P. multocida type A strains, we were designed experiments to comparethe biological properties between C48-3, C48-3ΔhexABC, C48-3ΔompH and itscomplemented strain C48-3C, including the investigation of virulence, adhesion ability,serum resistance and antiphagicytic activity. Virulence assay showed that theC48-3ΔhexABC was significantly attenuated in mice, and the C48-3ΔompH wasrelatively attenuated in mice by intraperitoneal injection. The adhesion assay revealedthat the number of C48-3ΔompH that adhered to CEF cells was significantly lower thanthat of C48-3, C48-3ΔhexABC and C48-3C (P<0.01), respectively. Pretreatment of thebacterial cells with the anti-rOmpH rabbit serum significantly inhibited the adherence ofstrains C48-3ΔhexABC, C48-3and C48-3C as compared to treatment with normalrabbit serum, but mutant strain C48-3ΔompH was not affected. Phagocytosis assayrevealed that the two mutant strains of C48-3ΔhexABC and C48-3ΔompH were readilytaken up by mouse peritoneal macrophages, but parent strain C48-3and complementedstrain C48-3C were significantly resistant to phagocytosis, while the pretreatment ofC48-3ΔhexABC and P-1059with anti-rOmpH rabbit serum were significantly increasedthe phagocytosis by mouse peritoneal macrophages. These results demonstrated thatcapsule is a important virulent factor in avian P. multocida type A strains, andconfirmed that the OmpH is a major adherence factor of the organisms.The results of this study for further investigate it that pathogenic mechanism ofcapsular and OmpH in avian P. multocida type A strains. These were contribution forthe prevention and treatment of fowl cholera, as well as for further research efficient,safe, attenuated vaccine candidate strains.