Pasteurella haemolytica: purification of saline-extractable proteins by isoelectrofocusing

• Published in: Veterinary microbiology Vol. 10; no. 5; p. 465
• Main Authors: McKinney, K L, Confer, A W, Rummage, J A, Gentry, M J, Durham, J A
• Format: Journal Article
• Language: English
• Published: Netherlands 01-01-1985
• Subjects: Animals; Antibodies, Bacterial - analysis; Antibodies, Bacterial - biosynthesis; Antigens, Bacterial - immunology; Antigens, Bacterial - isolation & purification; Bacterial Proteins - immunology; Bacterial Proteins - isolation & purification; Cattle; Cattle Diseases - immunology; Enzyme-Linked Immunosorbent Assay; Female; Hydrogen-Ion Concentration; Immunization, Secondary; Isoelectric Focusing; Male; Mice; Molecular Weight; Pasteurella - immunology; Pasteurella Infections - immunology; Pasteurella Infections - microbiology; Pasteurella Infections - veterinary; Serotyping; Sodium Chloride; Vaccination
• ISSN: 0378-1135
• DOI: 10.1016/0378-1135(85)90028-8

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.