DESIGN AND OPTIMIZATION OF A POLYPROTEIN FOR DIAGNOSIS OF BOVINE PARATUBERCULOSIS

ROMANO MARIA ISABEL; ALONSO MARIA NATALIA; ROBERTO DAMIÁN MOYANO; MON MARIA LAURA; GRIFFA NATANAEL; ROMERO MAGALI; ALVARADO PINEDO, MARÍA FIORELLA; MARIA DE LA PAZ SANTANGELO; TRAVERIA, GABRIEL EDUARDO

Cancun

Congreso; 14th International Colloquium on Paratuberculosis; 2018

International Association for Paratuberculosis

Paratuberculosis control requires identification and removal of infected animals from the herd. To emphasize the PTB controls on health systems it is necessary to develop new solutions to diagnosis. Currently, the gold standard technique is bacterial culture; however, it is a complex strategy given the slow growth of these mycobacteriaIn this context, the aim of the present work is to develop a diagnostic tool based on an ELISA and a lateral flow immunochromatography (LFIC) that allows the specific diagnosis of paratuberculosis.For that purpose, antigens from Mycobacterium avium subspecie paratuberculosis previously identified and characterized by our group were designed and synthesized as polyprotein tagged with His. The expression of the construction was performed in E coli BL21 and subsequently purified using a nickel column. For the development of the ELISA, 100ul of the polyprotein (0,25mg/ml) was used and 98 sera were evaluated corresponding to healthy animals, paratuberculosis-infected cattle and tuberculosis -infected cattle. In parallel, the same sera were analyzed with another ELISA frequently used in the laboratory for the diagnosis of paratuberculosis using PPA-3 as antigen.Our findings showed that while ELISA-PPA-3 reacts with sera from animals infected with M. bovis, while the polyprotein was found to be sensitive and specific for diagnosis of bovine paratuberculosis. For the development of the LFIC, G protein was conjugated to colloidal gold, purified bovine immunoglobulins (0, 05μg/μl) was used as control line and the polyprotein (0, 5 μg/μl) was used as test line. The same sera were evaluated and a correlation was achieved with the results of the ELISA using the polyproteinFinally, the results obtained so far show that the constructed polyprotein can be used for the specific diagnosis of paratuberculosis since it did not induce reactions with sera from tuberculosis-infected cattle or from healthy cattle, one of the main problems of the methods used nowadays.