Comparison of surface protein extraction methods and immunogenicity.

is the major contagious bovine mastitis pathogen and has no effective vaccine. Strain variation and limited knowledge of common immunogenic antigen/s are among major constraints for developing effective vaccines. cell surface proteins that are exposed to the host immune system constitute good vaccine candidates. The objective of this study was to compare two novel surface protein extraction methods with biotinylation method and evaluate immune-reactivity of extracted proteins. Surface proteins were extracted from nine genetically distinct strains from cases of bovine mastitis. After extraction, bacterial cell integrity was examined by Gram staining and electron microscopy to determine if extraction methods caused damage to cells that may release non-surface proteins. The extracted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and evaluated for immune-reactivity using western blot. Results showed that all three extraction methods provided multiple protein bands on SDS-PAGE. Western blot result showed several immunoreactive surface proteins, in which some proteins strongly (well-resolved, thick, dark, and intense band) reacted across the nine strains tested. The three methods are valid for the extraction of surface proteins and hexadecane, and cholic acid methods are more feasible than biotinylation since both are easier, cheaper, and have minor effects on the bacterial cell. Strongly immune-reactive surface proteins may serve as potential candidates for a vaccine to control mastitis in dairy cows.