Saline-extracted antigens of Pasteurella haemolytica: separation by chromatofocusing, preliminary characterization, and evaluation of immunogenicity

Saline extracts of logarithmic-phase Pasteurella haemolytica, serotype 1, were separated by chromatofocusing. The resulting fractions were analyzed by immunodiffusion and an enzyme-linked immunosorbant assay, and six antigen groups (AG's) were identified. AG 1 did not bind to the column, AG's 2, 3 and 4 were eluted with a decreasing pH gradient, and AG's 5 and 6 were eluted with an increasing NaC1 gradient. Fractions containing each AG were pooled and further purified by gel filtration. The AG's were subsequently characterized as to protein, carbohydrate and 2-keto-3-deoxyoctanate (KDO) content. AG's 1, 5, and 6 had higher carbohydrate contents than AG's 2, 3 and 4. Only AG 5 contained detectable levels of KDO. The AG's were also analyzed by SDS-PAGE and immunoblotting. Each AG produced a characteristic pattern of proteins and antigens, although two antigenic proteins were common to all AG's. AG 1 contained the greatest number of antigenic proteins. Immunization of mice with each AG in Freund's incomplete adjuvant resulted in a strong antibody response to the homologous AG for four of the six AG's. Limited protection against a P. haemolytica challenge was observed in mice that were immunized with AG 2 or 4.