Study Of Protectivity Of Capsular Proteins, Out Membrane Proteins And Recombinant Cpm39 Protein Of Avain Pasteurella Multocida Cultured In Vivo And In Vitro

Fowl cholera is a highly contagious disease of domestic poultry and wild birds caused by capsular serogroup A and somatic serotypes 1, 3 and 4 of Pasteurella multocida (P. multocida) strains. The capsular 39 kDa protein (Cp39) of avian P. mutocida serotype A strain had been previously characterized as an adhesive protein. However, some investigations indicated that the cultural condition determined cross-protective activity of the Cp39 protain. The objective of this study was to determine cultural conditions that may affect expression of the 39 kDa adhesive protein or influence its cross-protective activity.Avian P. multocida strains C48-3, C51-3, X-73 and P-1059 propagated in chick embryo allantoic cavity and cultured DSA plates, respectively. After collected the bacterial cells by centrifugation used for prepare of capsular crude extracts (CCEs) and outer membrane proteins (OMPs). SDS-PAGE of CCEs and OMPs from both in vivo-grown and in vitro-grown P. mutocida strains demonstrated that six protein bands of 28, 39, 43, 56, 66 and 97 kDa, whereas the 102 kDa protein expressed only for in vivo-propagated P. multocida strains. Western blot analysis indicated that the mice antiserum against the purified 39 kDa adhesive protein (Cp39) of strain C48-3 reacted to 39 kDa protein of CCEs and OMPs from both in vivo-grown and in vitro-grown P. multocida strains. On the other hand, antiserum against the purified Cp39 was recognized three protein bands of 27, 56 and 58 kDa of CCEs and OMPs from both in vivo-grown and in vitro-grown P. multocida strains. This result indicated that the 39 protein is a cross-reacting antigen of P. multocida capsular serogroup A strains.The mature adhesive protein (Cpm39) of avian P. multocida strain C48-3 expressed in E. coli and its immunogenicity was detected with SDS-PAGE and Western blot. A fragment of cpm39 gene of C48-3 strain was cloned into prokaryotic expression vector pMAL-p2X and expressed in E. coli BL21(DE3)by IPTG induction. The SDS-PAGE analysis revealed a single protein band with a molecular weight of 78 kDa was expressed in E. coli by IPTG inducing, and Western blot results showed that the fusion protein of MBP-Cpm39 was recognized specifically by an antiserum against the Cp39 protein of C48-3 strain, suggesting that the fusion protein of MBP-Cpm39 possessed high immunogenicity.Sera from vaccinated mice were collected before challenge, and the antibody titers of these sera against recombinant protein MBP-Cpm39 was measured by ELISA. The MBP-Cpm39 and CCEs of both in vivo-grown and in vitro-grown C48-3 strain induced very high titers of antibodies against fusion protein of MBP-Cpm39, but low titiers of antibodies against fusion protein of MBP-Cpm39 in the OMPs of both in vivo-grown and in vitro-grown C48-3 strain. The MBP-Cpm39 in E. coli and CCEs of in vivo-grown C48-3 strain provided 100% protection against homologous serotype C48-3 strain challenge, and these proteins provided 60-80% protection against heterologous serotype C51-3 strain. Protection rate of the OMPs from in vivo-grown C48-3 strain to homologous serotype C48-3 strain and to heterologous serotype C51-3 strain were 60-80% and 40-60%, reaspectively. Protection rate of the CCEs and OMPs of in vivo-grown C48-3 strain to homologous serptype C48-3 strain were 80-100% and 60-80%, respectively, whereas 40-60% protection against heterologous serotype C51-3 strain. These results demonstrated that the Cpm39 protein is a cross-protective antigen of P. multocida serogtype A strains, and the Cpm39 protein could be useful to provide cross-protective immunity for controlling the prevalence of fowl cholera.