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Out of 262 swab samples from feet of clinically footrot affected sheep from Jammu and Kashmir, 135 (52.0%) detected positive for Dichelobacter nodosus by 16S rRNA gene specific PCR. Out of these135 positive samples, 82 (61.74%) tested positive for intA gene carried by virulent strains. A mulplex PCR for simultaneous detection and virulence characterization of D. nodosus in clinical samples was therefore devised. The test detected 77 (57.03%) samples positive for virulent D. nodosus. The results were comparable and the success of the multiplex PCR was established. Out of 30 randomly selected isolates subjected to gelatin gel test, 24 isolates with intA gene produced thermostable protease while six isolates without intA gene revealed the production of thermolabile protease. This indicated a good co-relation between presence of intA gene and gelatine gel test in determination of the D. nodosus virulence.