Pasteurella haemolytica: purification of saline-extractable proteins by isoelectrofocusing.
A procedure was developed for separating antigens associated with a saline extract of Pasteurella haemolytica serotype 1. Seven antigens were identified by immunoelectrophoresis to be associated with the extract. The extract was subjected to preparative isoelectrofocusing in a pH range of 3-10. The majority of extracted proteins were found to have pI's of 4-6, whereas the carbohydrate antigen(s) were distributed over a pI range of 3.0-8.0. The fractions that were of interest were pooled and refocused in a narrower pH range to improve resolution of the protein antigens. Specific antigens from defined pH ranges were pooled to form 6 antigen groups. These antigen groups were examined further by immunoelectrophoresis, analytical isoelectrofocusing, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The molecular weights of the proteins found in the capsular extracts ranged from 33 k to greater than 80 k. Injection of mice with capsular extract or antigen Groups 1-6 in Freund's incomplete adjuvant resulted in a serum antibody response to the various antigens as detected by an enzyme-linked immunosorbent assay. Significant protection (P less than 0.05) against challenge with virulent P. haemolytica was seen in mice injected with antigen Groups 2 and 4. Six calves were immunized with saline extract. These calves had greater resistance to experimental pneumonic pasteurellosis than did 6 non-vaccinated calves. A serum antibody response to the crude extract and to each antigen group was detected in vaccinated calves by an enzyme-linked immunosorbant assay.
D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis / Q000096:biosynthesis; D000942:Antigens, Bacterial / Q000276:immunology / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000276:immunology / Q000302:isolation & purification*; D002417:Cattle; D002418:Cattle Diseases / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006863:Hydrogen-Ion Concentration; D007117:Immunization, Secondary; D007525:Isoelectric Focusing; D008297:Male; D051379:Mice; D008970:Molecular Weight; D010325:Pasteurella / Q000276:immunology*; D010326:Pasteurella Infections / Q000276:immunology / Q000382:microbiology / Q000662:veterinary; D012703:Serotyping; D012965:Sodium Chloride; D014611:Vaccination
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D012965:Sodium Chloride