Bovine mastitis is a significant disease affecting the dairy industry worldwide. The most frequently causative agent of contagious bovine mastitis is Staphylococcus aureus that produces numerous virulence factors contributing to its pathogenesis. However, it is not clearly defined which virulence factors play a critical role in bovine mastitis due to the heterogeneous virulence factor profiles in S. aureus isolated from different hosts and disease types. Among many virulence factors, it has long been postulated that staphylococcal cytotoxins and superantigens (SAgs) play an important role in the pathogenesis of S. aureus in bovine mastitis due to their potent toxicity toward host immune response. However, it has been a great challenge to determine the definite role of staphylococcal cytotoxins and SAgs in S. aureus pathogenesis due to the presence of multiple redundant cytotoxins and SAgs in a single S. aureus strain. Our longterm goal is to develop an effective vaccine to protect dairy cattle from S. aureus infection. The objective of this study is to; 1) determine the role of staphylococcal cytotoxins and superantigens in bovine mastitis; 2) develop a inducible and secretory expression vector and host system for a high production yield of recombinant protein; and 3) evaluate the protective effect of recombinant protein vaccine composed of staphylococcal cytotoxins and SAgs. The rationale of the proposed research is that development of an effective vaccine against S. aureus will prevent significant economic loss in the dairy industry and reduce the use of antibiotics in the dairy industry to prevent emergence of antibiotic resistance pathogens