Identification of immunoreactive proteins for Mycobacterium avium subsp. paratuberculosis detection

Background: Johne’s disease is a Mycobacterium avium subsp. paratuberculosis(MAP)-caused chronic enteritis of ruminants associated with enormous worldwide economic losses for the dairy cow- and goatrearing industries. Eradication programs and the management limitations for this disease have been hampered by the lack of simple and specific diagnostic tests for detecting the disease in subclinically infected (infected but symptom-free) animals. MAP is ubiquitous and it can be found in soil and water of infected farms, but actually there is no available method to find it. Aim of this project is to find, with proteomics, potential target proteins of MAP surface or secreted by MAP useful for developing a biosensor for its detection. Methods: Two different protein solubilization methods have been developed. The first one was developed using both freeze thaw cycles and bead beating and the second one using French press cycles and strong detergents for pellet solubilization. Results: According with obtained results it is possible to say that both are good methods for MAP protein extraction but bead beating is more convenient because of its higher protein recovery rate. This result is probably due to a better cell disruption obtained with the bead beating procedure. Moreover three immunogenic proteins were found with 2D immunoblotting, one of them is really interesting because of its similarity with a previously described immunogenic protein, the MAP2121c that is a 35 kDa membrane protein with a strong immunogenic activity and specificity. Conclusion: Obtained results demonstrate, according with previous findings, that, because of its abundance and MAP specificity, MAP2121c can be used as possible candidate for developing a biosensor.