Isolation and characterization of flagellar filament from zoospores of Dermatophilus congolensis

- Culture conditions for zoospores from Dermatophilus congolensis were determined.
- A method for isolating flagellar filament from zoospores was established.
- Strain variations were detected in flagellin size and antigenicity.

Highly motile zoospores from Dermatophilus congolensis bovine isolates from clinical dermatophilosis in Japan were obtained by culturing at 27 °C in an ambient atmosphere on heart infusion agar supplemented with 5% defibrinated sheep blood for 72 h or in heart infusion broth for 48 h with gentle shaking. After vigorous mechanical agitation of the zoospore suspension, the flagellar filaments detached from motile zoospores and were isolated in the clear gelatinous part of the final pellet by differential centrifugation. Typical morphology of a flagellar filament, with a width of approximately 15 nm, was observed in the isolated flagellar filament by electron microscopy. A single major protein (flagellin) band with an apparent molecular mass of 35 kDa was detected in the flagellar filament of D. congolensis strain AM-1 and that of 33 kDa was detected in strain IT-2 by SDS-PAGE. In immunoblot analysis of whole-cell proteins from seven isolates of D. congolensis, antiserum to strain AM-1 zoospores reacted with the 35-kDa antigen band of strain AM-1, but not with any antigen band of other strains in a similar molecular mass range. In contrast, antiserum to strain IT-2 zoospores reacted with antigen bands at 33 kDa from six strains, except strain AM-1. Similar strain-specific reactions of these anti-zoospore sera with isolated flagellar filaments from strains AM-1 and IT-2 were confirmed by immunoblot, indicating the presence of antigenic variations of flagellins of D. congolensis zoospores.