Rilevamento di Mycobacterium avium subsp. paratuberculosis (MAP) in campioni di latte ovino con un nuovo approccio biotecnologico

Paratuberculosis is an incurable gastroenteric condition among ruminants that is promoted by Mycobacterium avium subsp. paratuberculosis (MAP). In this thesis, we evaluated the functionality of several in-house and commercial diagnostic techniques to assess the presence of MAP and antibodies directed against it in unpasteurized milk samples. To estimate the presence of MAP shedder animals among the sheep herds, qPCR IS900 along with ELISA were carried out on two sources of milk samples, BTM and individual milk, and the specificity and sensitivity of both methods were estimated under different binary reference models (0,1). Accordingly, the incidence of MAP and antibodies directed against it were estimated higher at BTM level rather than individual. We innovatively used MAP-derived surface-exposed lipopeptids of L3P (specific in S-type) and L5P (specific in C-type) as capture antigens in an in-house milk ELISA test (H-MELISA). The result of this analysis depicted that H-MELISA L3P/L5P could enhance the discovery of MAP-positive milk samples among specimens that were potentially negative by commercial ELISA test. Interestingly, the overall positivity rates of H-MELISA L3P/L5P varied by the source of milk samples. In another step, the association between positivity in H-MELISA L3P/L5P and the type of MAP strains (S/C) was evaluated by strain typing using PCR IS1311-restriction enzyme analysis. The analysis depicted that all three MAP strains (S/C/Bison) existed among Sardinian sheep community. Further examination on the H-MELISA L3P/L5P positivity pattern of each C/S type MAP sample revealed that some samples had a reverse reactivity against both L3P and L5P. These might be the consequence of either cross-reactivity between L3P and L5P due to the similarity in the structures of the two epitopes or a within-herd mixed infection with both MAP strains of C and S types. In order to assess the viability of MAP in sheep and goat milk samples, a conventional and novel phage assay was optimized. Although, both methods comparably functioned on unpasteurized milk samples that contained no viable MAP, PBQ functioned more preferably, due to its lower LOD, rapidity, higher sensitivity, and lack of need for “intervention of other mycobacterial species such as M. smegmatis