BOVINE STAPHYLOCOCCUS AUREUS MASTITIS: FROM THE MAMMARY IMMUNE RESPONSE TO THE BACTERIA VIRULENCE GENES

Staphylococcus aureus (S. aureus) is one of the most important bacteria in veterinary medicine. In dairy herds, it is a contagious bacterium responsible mainly for subclinical mastitis in cattle, which frequently gives rise to persistent and chronic infection. Mastitis cause considerable economic losses due to i) decreased milk production, (ii) reduced milk quality, and (iii) treatment costs. Mastitis is also a public health problem. Indeed, the strains isolated from infected glands could produce enterotoxins. Three factors interact in mastitis: the host, the pathogen and the environment. This thesis focuses on two main aspects: the host immune response and the virulence factor of S. aureus. The first chapter of the thesis focused on the development of a new mammary gland model to study the innate immune response bacterial infection. The mammary gland is a complex organ, and the immune response is a consequence of the different cell population interactions. Continuous or primary epithelial cell lines have been extensively used to study the mammary gland immune response, but they are composed of a single cell population. Previous studies explored the tissues of lactating cows, unconsidering the possibility of an already triggered immune response. To investigate the innate immune response of the bovine mammary gland, we used an explant of healthy heifer gland. This model allowed us to: i) exclude previous exposure of the udder to microorganisms, which might have damaged the cells and/or triggered an immune response, and ii) consider the interaction of the challenging microorganism with the tissue cell populations. Our aim was to test whether this innovative model might be a valid model to investigate the innate immune response to infection. The study was carried out on 2 mm3-sections of heifer udders, in 2 consecutive trials, using LPS or LTA in the first trial and two different concentrations of S. aureus in the second. Treated and untreated sections were collected after 1h, 3h and 6h incubation; in the first trial, a final time-point at 18h was considered. The mRNA expression of TNFα, IL-1β, IL-6, IL-8 and LAP was analyzed by quantitative real-time PCR. Histological examination showed well-preserved morphology of the tissue, and apoptosis only showed a slight, not significant increase throughout the experiment. IL-1β and IL-6 were significantly up-regulated, in response to LPS or S. aureus, while TNF-α and IL-8 significantly increased only under LPS treatment. LAP expression showed a significant late increase when stimulated by LPS. The immunochemical staining of the sections demonstrated a higher number of T lymphocytes within the alveolar epithelium, in comparison with interstitial localization. Since the explants belonged to pubertal non-pregnant heifers, T cells may be regarded as resident cells, suggesting their participation in the regulation of mammary homeostasis. Therefore, applying our model would give new insights in the investigation of udder pathophysiology. The second chapter of the thesis focused on S. aureus in bovine intrammary infections. Previous literature on the S. aureus-intrammamary suggested that infection might be related to a combination of S. aureus virulence factors beyond host factors. The present study considered 169 isolates from different Italian dairy herds that were classified into four groups based on the prevalence of S. aureus infection at the first testing: low prevalence (< 5 %; LP), medium–low (5.1 - 24 %; MLP), medium–high (24.1 - 40 %; MHP) and high (> 40 %; HP). We aimed to correlate the presence of virulence genes with the herd prevalence of intramammary infections in order to develop new strategies for the control of S. aureus mastitis. Microarray data were statistically evaluated using binary logistic regression and correspondence analysis to screen the risk factors and the relationship between prevalence group and gene. The analysis showed: (1) 24 genes at significant risk of being detected in all the herds with infection prevalence >5%, including genes belonging to microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), immune evasion and serine proteases; and (2) a significant correlation coefficient between the genes interacting with the host immune response and HP isolates against LP ones. These results support the hypothesis that virulence factors, in addition to cow management, could be related to strain contagiousness, offering new insights into vaccine development.