Identification and characterization of outer membrane proteins of Pasteurella multocida involved in serotyping and pathogenesis

The studies described in this thesis are part of current research, globally aimed to identify and characterize outer membrane proteins (OMPs) of Pasteurella multocida involved in serotyping and pathogenesis.; In order to identify and characterize OMPs of P. multocida, a panel of monoclonal antibodies (MAbs) were produced by fusion of spleen cells of Balb/c mice immunized with OMPs, with SP2/0-AG 14 murine myeloma cells. Four MAbs designated MT1, MT2, MT3, and MT4.1 were produced and characterized. MAbs MT1 and MT4.1 identified respectively two major OMPs (MOMPs) of 32 (protein H; porin H; OmpH), and 28-37 kDa (heat-modifiable OMP; OmpA). MAbs MT2 and MT3 reacted with two distinct epitopes on a minor OMP (mOMP) of 32 kDa (protein W; OmpW) in competitive ELISA and immunoblot assay. The N-terminal amino acid sequence analysis of 32 and 28-37 kDa MOMPs demonstrated their marked similarity to families of porin and OmpA (heat-modifiable OMP) respectively of Gram-negative bacteria.; With a view to develop a classification method, based on OMPs by using MAbs, antigenicity as well as toxigenicity of P. multocida representing various serotypes, geographic origins and host species were studied by using SDS-PAGE, dot-ELISA, immunoblot, and polymerase chain reaction (PCR) assays. Based on the electrophoretic mobility of porin H, various OMP patterns were observed.; The "backpack" hybridoma tumor system was developed in Balb/c mice with a view to demonstrate the role and mechanism of protection of OmpH and OmpA. Mice bearing hybridoma tumors were continuously delivered IgG MAbs for the OmpH (MAb MT1) and OmpA (MAb MT4.1) in blood and peritoneal cavity, as detected by ELISA and immunoblot assay.; The results demonstrated that IgG MAbs against OmpH of P. multocida but not against OmpA, are involved in the protection of mice against lethal challenge infection by means of inhibition of proliferation and opsonophagocytosis of P. multocida as well as by rapid influx of neutrophils to the infection site.; In conclusion, this investigation focussed mainly on characterization of three OMPs of P. multocida. The results of antigenic characterization of P. multocida and other species showed that these proteins contain serotype, species or genus specific epitopes. It is further suggested that serotyping system based on proteins H and W by using MAbs, may be useful for identification of serotype D strains of P. multocida. A "backpack" hybridoma tumor system was developed in mice, with a view to demonstrate the role and mechanism of protection afforded by OmpH specific MAbs. It is suggested that the production of specific MAbs to proteins H and W of other serotypes of P. multocida may be useful for serotyping of P. multocida strains and that the strains with different OMP patterns should be incorporated in a multivalent vaccine for an effective protection against pasteurellosis. (Abstract shortened by UMI.)...