MOLECULAR CHARACTERIZATION BY RT-REAL TIME PCR AND HIGH RESOLUTION MELTING ANALYSIS FOR FOOD SAFETY AND VETERINARY DIAGNOSTICS

This PhD thesis is the outcome of a range of activities and experimental results aimed to a better characterization of the risk that Escherichia coli and other microrganisms and parasites may pone to the health of animals and finally humans. One of the main activity of the present project was based on the test hypothesis that the virulence profile of E. coli strains toward bovine mammary gland can be modulated by the interaction with the host cells. These hypothesis were tested through a gene expression study of some virulence factors of six E. coli strains when co-cultured with a bovine mammary cell line, since the in vitro models represent both an essential tool to investigate the biological mechanistic of mastitis, and an efficient alternative to animal experiments. Preliminarly, a meta-analysis of existing literature studies on the available bovine mammary cell lines was performed, resulting in the selection of MAC-T as the most responsive cell line to bacteria causing mastitis. The E. coli strains used for the coculture experiments with MAC-T cells were isolated from different types of bovine mastitis (acute, chronic and undetermined) and from a VTEC food-borne strain associated to human clinical disease (O157). An upregulation of the virulence factor eae (intimin) in all but one the analyzed mastitis strains following co-culture with MAC-T cell line was detected through RT-reat time PCR, and also the adherence virulence factor ycd and the b12 gene were upregulated in some strains, overall suggesting the possibility that mastitic E. coli strains can acquire a more risky molecular profile when exposed to the bovine mammary cells. This finding may have clear implications on the risk assessment related to the E. coli strains in bovine mammary tissue and milk. In addition, with the aim to improve the current methodologies for foodborne risk analysis linked to E. coli, the project activity provided a preliminary research for the setup and validation of new protocols based on real time PCR-High Resolution Melting Analysis, a widely used technique to target sequence polymorphisms of the same gene in different species without the need to perform DNA sequencing or to use species-specific probes, to help the identification of putative verocytotoxic status in E. coli strains of O26 serogroup, and other serotypes, isolated from bovine milk. Since the applications of HRMA for the characterization of microorganisms can not be limited to food safety, but can be developed for a large number of issues linked to general veterinary diagnostics, among the objectives of this PhD project some new real-time PCR-HRMA coupled methods were also developed, providing a contribution to the advancement of the existing molecular tools for sensitive and effective species identification, or variant/mutation screening, applied to different foodborne and veterinary pathogens. Thus, new HRMA-based protocols were designed and tested for the identification of Pseudomonas spp responsible for chromatic alterations in mozzarella cheese, for the detection and differentiation of Dirofilaria repens and D. immitis in canine blood samples, for the detection of the mutation site associated to FQ resistance in Staphylococcus pseudintermedius isolated from canine diagnostic samples, and for discrimination of the two most common microsporidial parasites in honeybees, Nosema apis and N. ceranae. Overall, these new HRMA-based assays could represent additional tools for epidemiological studies, routine disease assessment and therapeutical decisions. The possibility to identify the presence of risk-predictive SNPs in E. coli isolates using these newly established HRMA-based protocols is a novel, and simpler, opportunity with respect to the current, and more complex, surveillance strategies that are based on the amplification of stx genes together with other virulence factors for the evaluation of VTEC status. In the future, a possible way forward of this research is represented, on one side, by the deeper assessment of the reciprocal modulation between E. coli mastitis-derived strains and immortalized MAC-T cells using high-throughput RNA sequencing, and on the other side by a large scale validation of the HRMA-based evaluation of risk-predictive SNPs in order to improve the current approaches. And overall, the established HRMA-based protocols when extensively validated would be highly suitable for routine veterinary diagnostics applied to field investigation, as quick and sensitive single step protocols allowing specific and sensitive detection of the targets with shorter analysis time and reduced cost, in parallel or in alternative to the classical approaches.