The aim of this project was to study the dynamics of intramammary infection and resulting inflammation in Staphylococcus aureus mastitis in cows. Four cows with naturally occurring subclinical mastitis in one or more quarters, caused by S. aureus, were used in this study. The strains of S. aureus causing infection in the cows were determined by restriction enzyme fragmentation pattern (REFP) analysis. Two cows were infected with one strain of S. aureus, designated strain A, and two cows were infected with a different strain of S. aureus, designated strain B. The cows were subsequently challenged by the intramammary route in a novel cross-over design with large numbers (108-109) of either the indigenous strain or the non-indigenous strain of S. aureus. Quarters which were naturally infected with S. aureus and then challenged by the intramammary route with S. aureus remained persistently infected three weeks following challenge in three of the four cows, irrespective of the challenge strain. The fourth cow required treatment with antibiotics due to an increase in the severity of mastitis. The results of this study showed that in one cow, intramammary challenge of a subclinically infected quarter with the indigenous strain of S. aureus failed to induce an anamnestic immune response which might have instigated clearance of the original infection from the quarter; while in the other cow, the indigenous strain was replaced with three distinct isolates. It also showed that intramammary challenge of a subclinically infected quarter with the non-indigenous strain of S. aureus, in two cows, did not result in permanent replacement of the indigenous strain with the recently infused non-indigenous strain. In addition to the original strains A and B, eight distinct strains isolated from the four cows, were recognised by REFP analysis, indicating that a cow, or even a quarter, may be infected simultaneously by more than one strain of S. aureus. This study showed that the examination of multiple colonies per milk sample was required in order to identify the diversity of S. aureus strains present and to maximise the benefit of bacterial strain identification as an epidemiological tool in mastitis investigations. As REFP analysis is a time consuming method, a repetitive extragenic palindromic polymerase chain reaction (REP-PCR) was developed during this project to facilitate more rapid identification of the ten S. aureus strains originally identified by REFP. To investigate the acute phase response as an indicator of inflammation in mastitis, mammary secretions from the four cows with naturally occurring subclinical mastitis caused by S. aureus were collected prior to, and following, intramammary challenge with S. aureus. Serum amyloid A (SAA) was isolated and purified from the serum of bovine clinical cases by ultracentrifugation, gel electrophoresis and electro-elution. Serum amyloid A was identified by enzyme linked immunosorbent assay and immunoblotting using a polyclonal rabbit anti-human SAA antibody. The purified SAA was used to immunise mice in an attempt to produce a monoclonal antibody specifically against bovine SAA but this proved to be unsuccessful even when the SAA was bound to a carrier protein. Increased levels of haptoglobin were identified in mammary secretions from a single quarter of one cow. This quarter had been originally uninfected and then challenged by the intramammary route with strain B. In the absence of a polyclonal or monoclonal antibody to bovine SAA, sodium dodecyl sulphate polyacrylamide gel electrophoresis was used to identify proteins in mammary secretions with a similar molecular weight to SAA. A protein of similar molecular weight to SAA was identified in the same quarter in which the increased haptoglobin levels were measured
