Detection of virulence genes
PCR was performed to detect three virulence genes of M. bovis , including the variable surface lipoprotein gene (vsp ), cytadhesin (gap A) and the uvr C which encodes a protein involved in DNA excision and repair using the primers as described previously (Table 1). PCR reactions were performed in a 20 µl volume for each isolate as describe above. The PCR condition for detection of the vsp gene was initial denaturation at 940C for 5 min, followed by 35 cycles of denaturation at 940C for1 min, annealing at 550C for 1 min, and extension at 720C for1.5 min with a final extension at 720C for 10 min. The PCR condition for detection of the uvr C and gap A genes was initial denaturation at 940C for 2 min, followed by 35 cycles of denaturation at 940C for 30 sec, annealing at 560C for 30 sec for gap A and at 520C for 30 sec for uvr C, and extension at 720C for 2min with a final extension at 720C for 5 min.