Elastase test.

The virulence of a D. nodosus isolate was assessed using the elastase test, as described previously (38). Virulent D. nodosus type strain A1001 (elastase positive at 4 to 8 days postinoculation) was used as a virulent control. Plates were incubated in anaerobic jars containing an anaerobic gas pack (Gas Pak; BD, Cockeysville, MD, USA) and an anaerobic indicator (Oxoid, Hampshire, United Kingdom) at 37°C. Plates were examined after 4, 8, 12, 16, 20, 24, and 28 days of incubation and reincubated anaerobically after each examination. An isolate was classified as elastase positive (virulent) if growth was observed and a zone of clearing was observed within 12 days. If a zone of clearing was observed later or not at all, the isolate was classified as elastase negative (benign), in accordance with the dichotomous classification system used by Australian animal health agencies (19).