Isolation of two novel Fusobacterium necrophorum variants from sheep in Australia

Abstract

The detection of three novel Fusobacterium necrophorum leukotoxin (lktA) sequences (B, C and D) in sheep, goats, cattle (Zhou et al., 2009) and pigs (Zhou et al., 2010), suggests the existence of un-identified strains or subspecies of F. necrophorum. However, these strains or subspecies have not been isolated, and hence their identities await confirmation. We have recently isolated five strains of F. necrophorum from sheep hoof scrapings collected into Stuarts transport agar (0.6% agar) and cultured onto Wilkins-Chalgren agar (Oxoid, Australia) containing 5% horse blood. These were incubated over five days at 37 ⁰C in an anaerobic atmosphere (BD GaspakTM EZ anaerobe container system, Becton, Dickinson & Co. USA). Biochemical identification using the Vitek 2 Anaerobe and Corynebacterium (ANC) identification card on the Vitek 2 Compact (Biomerieux) gave a confidence interval (CI) for F. necrophorum ranging from 93% to 99%. Bacterial DNA was isolated using the QIAamp DNA mini kit (Qiagen, Australia) with the bacterial pellet protocol and using the Qiacube DNA extraction robot.