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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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Low fertility is a major cause of lost productivity in the cattle industry. In addition, cattle provide a convenient model to study ovarian physiology in monovular species including humans. Our previous microarray studies in the bovine ovary showed the upregulation of two clusters, miR-212-132 and miR-183-96-182, in luteal relative to follicular tissues. The studies in this thesis were aimed at establishing the functional involvement of these miRNAs during the follicle-luteal transition using a bovine model as well as human tissues. The aim of the first study was to characterise the expression of miR-132 and miR-96 within luteal tissue using ISH and FACS. The expression of miR-132 was detected in most luteal compartments while miR-96 was not detectable using ISH. Further examination using FACS showed that miR-212-132 expression remained unchanged in sorted endothelia (CD144+) and steroidogenic (CD144-Nile Red (NR) +) cell fractions. In contrast, expression of miR-183 and miR-96 was significantly increased in CD144+ compared to CD144-NR+ fractions. To elucidate potential roles of these miRNAs in the CL, I used existing online databases to identify putative miRNA targets. I identified 3042 predicted bovine gene targets of these miRNAs as well as 174 miRNA targets that had been experimentally validated in human, mouse and/or rat. I also identified putatively targeted signalling pathways primarily involved in cell survival, proliferation and differentiation. For further investigation, I narrowed my list of targets to FOXO1 and ADCY6, the expression of which was naturally down- regulated during luteinisation. The second study used an in vitro model of bovine granulosa cell luteinisation. Levels of miR-183-96-182 and miR-212-132 increased significantly (P < 0.05) during the first 4 days of luteinisation in vitro. The function of miR-132 and miR-96 during luteinisation in vitro was studied. Transfection of bovine granulosa cells with specific miRNA inhibitors or mimics of miR-132 and miR-96 led, respectively, to abolished expression and a significant increase in the levels of these miRNAs (P < 0.01) within 4 days. These changes in miRNA levels did not have any effect on transcript levels of the predicted mRNA targets, FOXO1 and ADCY6, during luteinisation. However, progesterone production by luteinising granulosa cells decreased (P < 0.05) on day 2 after transfection with miR-132 inhibitor. The results demonstrated that putative miRNA target genes remained unchanged during in vitro luteinisation which was not consistent with in vivo results. The third study aimed to elucidate the effect of miRNA inhibition in bovine luteal cells in culture. The loss of miR-132 led to an increase (P < 0.05) in FOXO1 transcript but not protein levels. In contrast, inhibition of miR-96 increased protein but not transcript levels of FOXO1. Moreover, miR-96 inhibition induced an increase in the caspase 3/7 response of luteal cells to serum deprivation indicating an anti-apoptotic effect of this miRNA on these cells. In the fourth study, I investigated the role of miR-132 and miR-96 in human luteinised granulosa cells obtained from IVF patients. The levels of FOXO1 protein were significantly increased following depletion of miR-132 and miR-96, whereas caspase3/7 increased in response to miR-96 inhibition, regardless of whether cells had been serum deprived or not. Similarly, using Annexin V and Trypan blue staining an increase in numbers of apoptotic cells was observed in response to miR- 96 inhibition. In addition, reduction of FOXO1 with the siRNA inhibited the apoptotic effect of miR-96 inhibition. Interestingly, inhibition of pooled miR-132 and miR-96 reduced progesterone secretion. However, this effect was prevented by transfecting cells with FOXO1 siRNA. These results suggest that the effects of these miRNAs on cell survival and progesterone production are mediated through targeting FOXO1. In summary, my results identify miR-96 and miR-132 as potentially critical factors in ensuring luteal cell survival and steroidogenesis in both cattle and human.

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