Identification of immunogenic outer membrane proteins of Pasteurella multocida 3:A in rabbits

Abstract

Four groups of protective rabbit immune sera were used to identify Pasteurella multocida outer membrane immunogens by a radioimmunoprecipitation procedure and Western blot (immunoblot) analysis. These are rabbit hyperimmune sera against KSCN extract of P. multocida (group 1) and rabbit immune sera against the KSCN extract of P. multocida (group 2), the outer membrane of P. multocida (group 3), and live P. multocida cells (group 4). Rabbits mounted an antibody response to 18 proteins found in the outer membrane of P. multocida, and the major antibody activities were directed to the 27,000-molecular-weight outer membrane protein (27K protein), as well as the 37.5K, 49.5K, 58.7K, and 64.4K outer membrane proteins. These outer membrane immunogens appear to be exposed on the cell surface and accessible to antibodies, since adsorption of these immune sera with intact P. multocida cells resulted in a significant reduction of antibody activities directed against these proteins, especially the 37.5K protein. Antibodies eluted from immune serum-P. multocida cell complexes were reactive to the 37.5K immunogen, confirming that this protein is exposed on cell surface and accessible to antibodies. Western blot analyses with group 1, 3, and 4 immune sera confirmed that the 27K, 37.5K, 49.5K, 58.7K, and 64.4K proteins are the major outer membrane immunogens of P. multocida in rabbits. Lung lavages of immunized rabbits also contained similar antibody activities directed against several outer membrane proteins, with major activities against the 37.5K and 64.4K proteins.

Antibodies to outer membrane proteins but not to lipopolysaccharide inhibit pulmonary proliferation of Pasteurella multocida in mice

“…Preparation of P. multocida outer membranes. The outer membranes were prepared as described previously (11). The capsules of the organisms were removed enzymatically with hyaluronidase (11), since type A P. multocida is known to contain hyaluronic acid (3).…”

“…(iv) Purification of antibodies against P. multocida OMP. For determination of whether antibodies against OMP were the protective components of the immune sera, the effluent immune sera were adsorbed with intact P. multocida cells and the antibodies were eluted from the cell-antibody complex (11). In brief, freshly cultured homologous P. multocida cells (108 CFU in 0.5 ml of PBS) were mixed with an equal volume of serum and incubated for 1 h at 4°C with gentle agitation.…”

“…(i) Identification of antibodies against P. multocida OMP. The various fractions of rabbit immune sera were analyzed by Western blotting (11,13) with P. multocida outer membrane vesicles (14) as antigens.…”

Pasteurella multocida pathogenesis: 125 years after Pasteur

“…Early studies on the P. multocida outer membrane showed that a 37 kDa protein was among five identified as possible protective immunogens based firstly on radioimmunoprecipitation results using protective immune rabbit sera, and secondly on their location in the outer membrane (Lu et al, 1988). Monoclonal antibodies raised against the 37 kDa protein were able to passively protect rabbits and mice against infection with P. multocida with strong protection afforded against homologous strains, and some limited protection against heterologous strains (Lu et al, 1991).…”

Isolation of the Major Outer‐membrane Protein of Actinobacillus pleuropneumonias and Haemophilus parasuis

“…The immunoblot results and the N-terminal homologies suggest that p40 and p42 are at least related to porin proteins. Sarcosyl insoluble proteins are known to be OMPs, cell-surface exposed, and therefore accessible to antibodies (LUGTENBERG et al, 1986;LU et al, 1988). In addition to their capability of forming water-filled trans-outer-membrane channels (porins), which permit the passage of small hydrophilic molecules, OMPs, particularly MOMPs, are probably also involved in the infectious process.…”