Characterization of, and immune responses of mice to, the purified OmpA-equivalent outer membrane protein of Pasteurella multocida serotype A:3 (Omp28).
Pasteurella multocida A:3 is a major cause of bovine pneumonia. A major antigenic heat-modifiable 28kDa outer membrane protein (Omp28) was previously identified. The purpose of this study was to purify and characterize Omp28 immunologically and structurally. Omp28 was extracted from N-lauroylsarcosine-insoluble protein preparations by a combination of detergent fractionation with Zwittergent 3-14 and chromatography. Partial N-terminal amino acid sequence confirmed Omp28 as a member of the OmpA-porin family. However, porin activity could not be demonstrated in a lipid-bilayer assay. Heat modifiability of purified Omp28 was demonstrated, and Omp28 was found in outer membrane fraction of P. multocida. Surface exposure of Omp28 was demonstrated by partial protease digestion of intact bacteria, by binding of anti-Omp28 polyclonal ascites fluid to the bacterial surface, and by partial inhibition of anti-outer membrane antiserum binding by previous incubation of the bacteria with anti-Omp28 serum. CD-1 mice vaccinated with purified Omp28 developed a significant antibody titer (P<0.05) compared to the control treatment group but were not protected from a homologous intraperitoneal bacterial challenge. By contrast, treatment groups vaccinated with P. multocida outer membrane, formalin-killed P. multocida or a commercial vaccine were significantly protected from challenge. In vitro complement-mediated killing of P. multocida was observed in post-vaccination sera of outer membrane, formalin-killed P. multocida, and commercial vaccine-treatment groups, but not with sera from the Omp28-treatment group. In conclusion, although Omp28 is surface exposed and antigenic, it may not be a desirable immunogen for stimulating immunity to P. multocida.
Gatto
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NT
Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, 250 McElroy Hall, Stillwater 74078, USA.
Dabo
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Hancock
R E
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Confer
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AW
D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology* / Q000302:isolation & purification; D002417:Cattle; D002418:Cattle Diseases / Q000276:immunology*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015151:Immunoblotting; D008051:Lipid Bilayers / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D010326:Pasteurella Infections / Q000276:immunology*; D016979:Pasteurella multocida / Q000276:immunology*; D011897:Random Allocation; D020539:Sequence Analysis, Protein; D014611:Vaccination / Q000592:standards / Q000662:veterinary
D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D008051:Lipid Bilayers; C078167:OMPA outer membrane proteins