Comparison of Staphylococcus aureus surface protein extraction methods and immunogenicity

Abstract

Staphylococcus aureus 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. S. aureus 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 S. aureus surface protein extraction methods with biotinylation method and evaluate immune-reactivity of extracted proteins. Surface proteins were extracted from nine genetically distinct S. aureus 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 S. aureus mastitis in dairy cows.

Development of a coagglutination kit as a rapid test for diagnosing Newcastle disease in poultry

“…The resulting pellet was heated using a water bath at 80°C for 15 min and then cooled rapidly. The pellet was mixed with PBS and made into a suspension at 10% concentration (w/v) [ 8 ]. Next, the purified rabbit immunoglobulin was added to the pellet and the mixture was incubated overnight at room temperature.…”

Evaluation of Streptococcus uberis Surface Proteins as Vaccine Antigens to Control S. uberis Mastitis in Dairy Cows

“…SUSP was extracted using a method previously developed by our group [ 33 ] with slight modifications. Briefly, nine S. uberis strains isolated from cases of mastitis in the United States and other countries were used.…”

“…One pure colony was inoculated into 500 mL Todd Hewitt broth (THB) (BD and Company, Sparks, MD, USA) and incubated at 37 °C, and grown to mid-log phase. All subsequent procedures were the same as that of Abdi et al [ 33 ].…”

“…The SDS-PAGE protocol described by Abdi et al [ 33 ] was used. Briefly, 20 µg of SUSP in 10 µL of phosphate buffered saline (PBS 7.4) was mixed with 10 µL of 2× Laemmli buffer with 5% beta-mercaptoethanol (Bio-Rad Lab., Hercules, CA, USA).…”

“…Western blot was conducted as described by Abdi et al [ 33 ]. Briefly, the SUSP were separated by electrophoresis and transferred to nitrocellulose membrane (Bio-Rad) at a constant 100 V with 400 mA for 60 min by a wet method (Bio-Rad).…”