Difco yeast extract

Samples (n = 2,126) were collected from 10 sheep farms situated within Nottingham, Derby and Northampton from animals with varying disease states. Sterile nylon flock swabs (E-swabs 480CE, Copan U.S.A.) were used for the collection of samples from the interdigital space of sheep and stored in liquid Amies solution at 5°C overnight. The swabs were inoculated onto Hoof Agar plates containing 4% w/v Bacto Eugon agar (BD, U.S.A.), 0.5% w/v Difco Yeast Extract (BD, U.S.A.), 1.5% w/v BBL Beef Extract (BD, U.S.A.), 1% Sodium Chloride and 6.6% w/v ovine hoof powder (Parker et al., 2005 (link)) and incubated anaerobically at 37°C. After 7 days plates were visually checked for putative D. nodosus and if present sub-cultured onto reduced agar (2%), hoof agar plates and incubated anaerobically at 37°C. Pure colonies were collected from plates in sterile PBS, washed by centrifugation and resuspended in molecular biology grade water (ThermoFisher, U.K.).

The bacterial isolation, DNA isolation, sequencing and analysis of sequence data in this study are as described previously in [1 ]. A total of 2126 Interdigital swabs (E-swabs 480 CE, Copan U.S.A.) were taken from ewes and lambs. The swabs were stored in liquid Amies solution at 5 °C overnight prior to being inoculation of hoof Agar plates containing 4% w/v Bacto Eugon agar (BD, U.S.A.), 0.5% w/v Difco Yeast Extract (BD, U.S.A.), 1.5% w/v BBL Beef Extract (BD, U.S.A.), 1% sodium chloride and 6.6% w/v ovine hoof powder [16 (link)]. and incubated under anaerobic conditions at 37 °C. Pure colonies were collected from plates in sterile PBS, washed by centrifugation and resuspended in molecular biology grade water (ThermoFisher, UK). Candidate colonies were identified by visual inspection (n = 83 isolates), bacteriodes spp and contaminated samples were identified by sequencing and eliminated from the analysis. Fifty six isolates remained and were available for further analysis.