IDENTIFICATION OF BIOMARKERS FOR THE ASSESSMENT OF FARM RUMINANT HEALTH STATUS

The aim of this PhD thesis was to identify biomarkers for evaluating the health of farm ruminants and possible the quality of products. A rapid identification and quantification of acute phase proteins (APPs) can provide useful information on the health status of animal. From a clinical perspective, the availability of a quick, and reliable, diagnostic marker specific for inflammatory diseases is of paramount importance. APPs can be employed in the assessment of animal health and welfare both ante- and post-mortem as an aid to meat inspection. This work can be divided into two principal studies: the first one focused on the localization of APPs in extra hepatic tissues; in particular we have analyzed the distribution of acute phase proteins in the bovine forestomachs and abomasums and then the expression of SAA and Hp RNA in bovine tissues (and evaluation of suitable reference genes). The second part was more focused on the search for specific inflammatory biomarkers; in turn, this part of the thesis can be sub-divided into 2 studies: Escherichia coli lipopolysaccharides and Staphylococcus aureus enterotoxin B relationship with inflammatory microRNAs in bovine monocytes and the development of a highly sensitive sandwich ELISA to quantify the goat and bovine AGP and SAA in serum. Regarding the first part of our investigations we demonstrated the expression of the four APPs’ mRNA. These results were confirmed by Western blot analysis followed by localization through immunohistochemistry. We have also identified SAA and Hp’s mRNA in all analyzed tissues in non pathological conditions. In the second part of the thesis, we investigated the effect of Escherichia coli lipopolysaccharide (LPS) and Staphylococcus aureus enterotoxin B (SEB) on the expression of five miRNAs involved in the inflammatory response, including miR-9, miR-125b, miR-155, miR-146a and miR-223, in bovine CD14+ cells (monocytes). We obtained an up-regulation both miR-155 and miR-146a when cells were stimulated with LPS and a down-regulation of miR-125b, miR-155 and miR-223 after SEB stimulation, suggesting miRNAs as potential inflammatory biomarkers. The last part of the study was devoted to ELISA detection of APP. A precise and sensitive test to determine AGP content was developed as sandwich ELISA in serum of bovine and goats. A good analytical sensitivity was demonstrated. The test can detect a wide range of AGP concentrations. Yet, neither in goats nor in bovine species, the test managed to identify the animals with disease. Regarding SAA, a novel immunoassay to specifically detect SAA in ruminants' serum was developed, but the present results must still be regarded as preliminary.