Real-time pcr detection of Mycobacterium bovis in blood and lymph node aspirates of bovines positive in tuberculosis screening tests

Bovine tuberculosis is a chronic infectious disease affecting broad range of mammalian hosts. ESAT-6 is a low molecular weight immunodominant protein coded by esxA gene located on RD1 region of genome and is responsible for virulence of Mycobacterium bovis. Out of 200 animals screened for bovine Tuberculosis (bTB), 38 animals (19%) were found positive for Comparative intradermal tuberculin test (CITT) (32 cattle, 6 buffaloes) and 41 animals were tested positive by IFN-γ assay (29 cattle, 12 buffaloes). DNA extraction of blood (n=200) and lymph node aspirates (n=48) was done. Among 200 blood samples targeted for esxA (ESAT-6) gene, three samples (1.5%) whose C_T was between 23-34 were considered positive by real-time PCR. Out of 48 animals (lymph node aspirates) that were positive either by CITT or IFN-γ, one sample (2.08%) whose C_T was between 23-34 were considered positive by real-time PCR. Remaining samples whose C_T values were equal to or greater than 35 were considered negative. The sensitivity of esxA was 8 pg/μl by real time PCR.