An equal amount of sterile sand was added to the contents in the mortar and ground. The products were then transferred to McCartney bottles and centrifuged at low speed of 3000 rpm for 10 minutes. Thereafter, the supernatant fluid was decanted off and 20 mls of sterile water was added to the sediment and mixed vigorously by vortexing to a uniform homogenate. The contents were again centrifuged at low speed of 3000 rpm for 20 minutes and the supernatant fluid was decanted. The sediments of these decontaminated homogenates were inoculated in duplicate Lowenstein-Jensen BIBF 1120 in vitro media slants supplemented with 0.4% sodium pyruvate to enhance the isolation of M. bovis and incubated aerobically at 37°C

for 8 weeks. The resulting cultures were tentatively identified as probable Mycobacterium tuberculosis-complex

by their slow growth and colony morphology. Purity and acid-fastness of the colonies were checked by Zhiel Neelsen staining. Preparation of lysates and molecular typing of isolates Cell lysates were prepared by suspending a loop full of bacterial colony in 250 μl of 1× TE buffer (10 mM Tris/HCl, pH8.0 and 1 mM EDTA in distilled water) in an Eppendorf tube. Bacterial cells were heat killed by incubation at 80°C for 1 hour in a temperature controlled water bath. After centrifuging the cells at 13000 rpm for 2 minutes, the supernatant was VX-680 datasheet discarded and the pellet resuspended in 500 μl of 150 mM sodium chloride. This step was repeated twice. Finally, the supernatant was discarded and the TGF-beta cancer pellet resuspended in 25 μl 1× TE buffer. These suspensions were used for spoligotyping as previously described [15]. Four microliters (4 μl) of the denatured bacterial suspension from each sample was used for amplification of the direct-repeat Aldehyde dehydrogenase (DR) region. The labelled amplicons were used as probes for hybridization with a set of 43 known oligonucleotide spacer sequences. The H37Rv M. tuberculosis, and M. bovis

BCG P3 strains, and purified water were included in each experiment as positive and negative controls, respectively. Bound PCR fragments were detected with a streptavidinhorseradish peroxidase-enhanced conjugate and an enhanced chemiluminescence (ECL) system, followed by exposure to ECL hyperfilms (Amersham Pharmacia-Biotech, Roosendael, The Netherlands). The expected patterns of the positive controls were observed and no reagent contamination was detected in all the negative controls. The spoligotypes were compared using the band-based Dice coefficient and clustering determined by the unweighted pair group algorithm with arithmetic averages (UPMGA) method, using the MIRU-VNTR plus software[36] Calculating the Discriminatory power Hunter-Gaston Discriminatory Index (HGDI) equation was used for the calculation of the discriminatory power for the set of strains that were used in this study [28, 29].