IMMUNOBLOT AND ULTRASTRUCTURAL ANALYSIS OF ANTIGENS EXTRACTED FROM DICHELOBACTER-NODOSUS WITH POTASSIUM THIOCYANATE

The antigens extracted from Dichelobacter nodosus with potassium thioc yanate were analyzed by western blotting with sera from footrot-free s heep and from sheep infected with D. nodosus to identify components sp ecific for infection. Several components with molecular mass <33 kD we re associated with infection, particularly bands of 32.5 kD, 30.5 kD, and 28 kD. Components with molecular mass > 56 kD may be responsible f or false-positive reactions observed when sera of footrot-free sheep r eact with the potassium thiocyanate extract in solid phase enzyme-link ed immunosorbent assays (ELISAs). Bands of 62.5 kD, 56 kD, 40 kD, and 19 kD were recognized for most sheep regardless of their disease statu s or ELISA reactivity and therefore do not appear to be detected by th e ELISA. No components of the potassium thiocyanate extract were compl etely specific for infection. Antigens in the extract were identified primarily on the cell envelope by immunogold electron microscopy. Labe ling was concentrated in the periplasm, with minor labeling of the cel l surface. The extract consisted of tangled strands of particles with electron-dense cores, and few vesicular structures were observed.