Pasteurella haemolytica: Purification of saline-extractable proteins by isoelectrofocusing

Abstract

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 < 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.