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Staphylococcus aureus is the major cause of bovine mastitis in European countries, a disease of major economic importance to the dairy industry. S. aureus isolates from intra-mammary infections were isolated from diverse clinical and geographical origins and were characterised by phenotypic and genotypic methods. S. aureus isolates were identified by their cultural and biochemical properties and were analysed by pulsed field gel electrophoresis (PFGE) after Smal digestion of DNA. A total of 382 S. aureus isolates from bovine intra-mammary infections, bovine skin lesions, milking personnel and non-farm-related human carriers were tested. Ten commercial dairy herds from NE Scotland were included in this study. S. aureus was not isolated from two of them. One herd showed the typical S. aureus infection (102 - 103 cfu ml-1) of low Total Bacterial Counts and low S. aureus numbers, while in the other seven herds S. aureus was isolated only in low numbers, indicating that elevated Bulk Tank Somatic Cell Counts were due to other organisms. S. aureus isolates were assigned by PFGE to 62 electrophoretic types (discriminatory index D = 0.91) and 17 antibiotypes (D = 0.68). The predominant electrophoretic type consisted of 81/382 isolates and its sub-type by 57/382 isolates. Clonal types of bovine intra-mammary infection S. aureus isolates were compared with isolates from other sites on the cows. In addition, epidemiologically unrelated S. aureus isolates from non-farm-related humans were also tested to compare the human and bovine reservoir of S. aureus. Certain clonal types prevailed in individual herds throughout the twelve-month sampling period, whereas the presence of other clonal types, were more sporadic. Certain clonal types were found in more than one herd suggesting that S. aureus isolates that belonged to these clonal types were particularly well-adapted to colonise and persist in the bovine mammary gland. Certain clonal types of S. aureus isolates recovered from cases of bovine mastitis had a broad geographical distribution, comprising isolates from N, NE, SW Scotland and Ireland. The findings of the present study confirm that certain clonal types have remained in circulation for the last 50 years. On the other hand, certain clonal types were found only in one particular geographical area. S. aureus was present in low numbers on bovine body sites from both beef and dairy cattle with the majority of isolates being recovered from the nasal cavity. S. aureus isolates recovered from cows' body sites belonged to different clonal types than those obtained from milk samples. S. aureus was not recovered from farm personnel. Furthermore, PFGE analysis of S. aureus bovine and non-farm-related human isolates suggested that there was no connection between these two sources. Sixty percent of S. aureus isolates tested in the present study were ?-lactamase positive. A high-level of tetracycline resistance was found among S. aureus isolates from a herd where the treatment used for eradication of the infection included tetracycline antibiotics. The induction of -S, aureus L-forms (cell wall-less forms) by antibiotics that inhibit cell wall synthesis was examined. Twelve isolates of S. aureus were treated with antibiotics. Ten were induced to L-forms, while two isolates produced no L-forms under the conditions used. Resistance to penicillin appeared to play no role in the induction of L-forms. S. aureus L-forms were not isolated from milk samples of dairy cattle infected with mastitis. The results of this study support the hypothesis that some S. aureus isolates are more persistent than others and widely distributed clonal types could be responsible for cases of bovine mastitis. Bovine skin lesions and the human reservoir probably are not able to serve as reservoirs of S. aureus isolates involved in bovine mastitis. Furthermore, the ability to induce L-forms in vitro suggests that these forms may have a role in the persistence of the disease.

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[No abstract submitted] / Land and Food Systems, Faculty of / Graduate

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