Pasteurellosis in laboratory rabbits: characterization of lipopolysaccharides of Pasteurella multocida by polyacrylamide gel electrophoresis, immunoblot techniques, and enzyme-linked immunosorbent assay

136

Infection with Campylobacter pyloridis has been strongly associated with gastritis in humans although its etiologic significance is currently undefined. We examined the structure and antigenicity of whole-cell, outer-membrane, acid-extractable surface protein, and proteinase K-treated whole cell lysate preparations from eight C. pyloridis strains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with homologous and heterologous immune rabbit serum. Whole-cell and outer-membrane profiles observed in all strains of C. pyloridis were nearly identical; none were similar to those of C. jejuni and C. fetus. Major whole-cell bands migrated at 26,000, 29,000, 56,000, and 62,000 molecular weights. The acid-extracted protein profiles of all C. pyloridis strains also were similar to one another and showed similarities with acid-extracted proteins from C. jejuni, with major bands migrating at 29,000, 48,000 to 53,000, and 62,000. All proteinase K-treated lysates showed different lipopolysaccharide (LPS) profiles, ranging from rough to smooth with multiple repeating side chains. Immunoblots of whole-cell and proteinase K-treated preparations of the C. pyloridis strains showed that there was antigenic cross-reactivity of proteins migrating at 62,000 and 56,000, but not in other regions, and cross-reactivity between LPS core regions but not side chains. These results suggest that C. pyloridis has both protein and core LPS group antigens and strain-specific protein and LPS side chain antigens.

Section: Discussionmentioning

confidence: 77%

Conservation and diversity of Campylobacter pyloridis major antigens

Pérez-Pérez¹,

Blaser²

1987

136

“…Purified P. multocida LPS were separated in a 15% separating gel containing 4 M urea (15), transferred to nitrocellulose paper, and reacted with various fractions of rabbit immune sera in a Western blot analysis.…”

Section: Methodsmentioning

confidence: 99%

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

Aguila

Lai

et al. 1991

The role of rabbit antibodies against Pasteurella multocida outer membrane proteins and lipopolysaccharides (LPS) in resistance remains unknown. Pooled immune sera against P. multocida outer membranes were prepared from specific-pathogen-free rabbits immunized with sucrose gradient-purified P. multocida outer membranes. Western immunoblotting showed that purified outer membrane protein antibodies reacted strongly against the outer membrane proteins but not the purified LPS. Affinity-purified LPS antibodies exhibited strong reactivity against purified LPS and very little reactivity against outer membrane vesicles. Mice were inoculated intranasally with immune serum or normal rabbit serum, challenged intranasally with 106 CFU of P. multocida, and euthanatized 48 h later to determine the number of P. multocida organisms in the lungs. Mice inoculated with pooled immune serum had a 3,300-fold reduction (P < 0.001) in the numbers of P. multocida in the lungs as compared with the controls. Similarly, mice inoculated with purified outer membrane protein antibodies had a 201-fold reduction (P < 0.001) in the numbers of P. multocida. Conversely, mice inoculated with affinity-purified LPS antibodies had a 1.1-fold reduction (P > 0.50) in the numbers of P. multocida. These results show that antibodies against the outer membrane proteins but not the LPS are the components of rabbit immune sera which inhibit P. multocida proliferation in mouse lungs. 1470 on July 10, 2020 by guest

“…Homologous P. multocida OM vesicles (13,19) or purified LPS were used as antigens (27). SDS-PAGE, electrophoretic transfer of P. multocida proteins and LPS to nitrocellulose membranes, and immunoblotting assays were performed as previously described (13,18,33). Nonspecific binding sites on nitrocellulose membranes were blocked in PBS solution containing 0.05% Tween 20 and 1% bovine serum albumin (pH 7.2).…”

mentioning

confidence: 99%

The outer membrane of Pasteurella multocida 3:A protects rabbits against homologous challenge

Lai

Pakes

et al. 1991

The protective efficacy of a vaccine purified from the Pasteurella multocida 3:A outer membrane (OM) was evaluated in rabbits by homologous challenge. Twenty-seven rabbits were divided into four groups: 1, vaccinated with OM and challenged; 2, nonvaccinated and challenged; 3, vaccinated with OM only; and 4, nonvaccinated and not challenged. Rabbits were immunized intranasally with 1 mg of OM protein on days 0, 7, 14, and 35, challenged intranasally on day 49, and killed on day 63. Mortality rates were 0, 67, 0, and 0% for groups 1 through 4, respectively. The prevalence of pneumonia was reduced from 73 (group 2) to 20% (group 1). The severity of pneumonia was reduced from 0.62 (group 2) to 0.07 (group 1), as measured by the group lesion index. The number of P. multocida in nasal cavities was reduced from 3.89 x 105 (group 2) to 6.19 x l92 (group 1). The geometric mean number of P. multocida in lungs was 8,360,000-fold less in group 1 than in group 2. Similarly, the prevalence of P. multocida colonization in nonrespiratory organs was reduced from 47 (group 2) to 4% (group 1). Furthermore, group 1 and 3 rabbits developed significantly elevated immunoglobulin A antibodies in nasal secretions and lung lavages and significantly elevated immunoglobulin G antibodies in lung lavages and sera. In addition, rabbit immune sera contained antibodies against P. multocida OM proteins and lipopolysaccharides and inhibited P. multocida proliferation in mouse lungs. These results indicate that a vaccine prepared from the OM of P. multocida provides a significant protection in rabbits against homologous challenge.