Development of gene probes of Dichelobacter nodosus for differentiating strains causing virulent, intermediate or...

Seven Dichelobacter nodosus genomic DNA clones including six specific for virulent and one for benign strains were identified. A collection of 96 footrot isolates, which in turn comprised 27 virulent isolates showing elastase activity at 7 days, 25 high intermediate isolates with elastase activity at 14 days, 24 low intermediate isolates with elastase activity at 21-28 days and 20 benign isolates with no elastase activity at up to 28 days, were used to assess these clones. Of the six virulent specific clones, five (pV238-83, pV405-239, pV470-65, pV470-145 and pV470-178) reacted with 27 virulent isolates, and 12 of 25 high intermediate isolates, but none of 24 low intermediate isolates and 20 benign isolates in dot blot hybridization. The other virulent-specific clone (pV470-13) recognized all 27 virulent and 25 intermediate isolates, and 22 of 24 low intermediate isolates and three of 20 benign isolates in dot blot hybridization. By contrast, the benign-specific clone (pB645-335) detected all 20 benign isolates and 24 low intermediate isolates, and also 13 of 25 high intermediate isolates, but none of 27 virulent isolates in dot blot hybridization. Southern hybridization analysis indicated that whereas clones pV238-83, pV405-239 and pV470-178 bound a Sau3A band of 0.5 kb, clones pV470-65 and pV470-145 recognized two Sau3A bands of 0.7 and 0.5 kb in virulent strains of serogroups A to I. However, clone pV470-13 detected a Sau3A band pattern in virulent strains different from those recognized by the other five virulent specific-clones. Besides showing a distinct Sau3A band pattern in intermediate strains, pV470-13 also reacted with three benign strains that showed binding with it in dot blot hybridization. The benign-specific clone pV645-335 detected a Sau3A band of 0.5 kb in both intermediate and benign strains of serogroups A to I. Thus the combination of pV470-13 and pB645-335, or any other virulent-specific clone, would clearly differentiate among D. nodosus strains causing virulent, intermediate or benign footrot.