A polymerase chain reaction assay for improved determination of virulence of Dichelobacter nodosus, the specific causative pathogen for ovine footrot.

Three sets of oligonucleotide primers (Vf1 and Vr1, Vf2 and Vr2, Bf and Br) were derived from a D. nodosus virulent-specific clone pV470-13 (2146 bp) and a benign-specific clone pB645-335 (737 bp), respectively. Using the virulent-specific primers Vf1 and Vr1 in a polymerase chain reaction (PCR) enabled amplification of a DNA fragment of 460 bp in 25/27 virulent, 9/25 high intermediate, 9/24 low intermediate and 2/20 benign isolates of D. nodosus. On the other hand, using the second set of the virulent-specific primers Vf2 and Vr2 resulted in the production of either a fragment of 857 bp or 1300 bp in virulent, intermediate and 3/20 benign isolates. There appeared to be some correlation between the amplification of the smaller fragment (857 bp) and higher elastase activity and between the amplification of the larger fragment (1300 bp) and lower elastase activity of D. nodosus. The use of the benign-specific primers Bf and Br in PCR enabled amplification of a fragment of 609 bp in all 20 benign, 23/24 low intermediate, 20/25 high intermediate and 0/27 virulent isolates. The combination of the virulent-specific primers Vf2 and Vr2 and benign-specific primers Bf and Br in PCR would provide a rapid, specific and sensitive differentiation of strains of D. nodosus causing virulent, intermediate or benign footrot.