Bile aesculin agar

Enterococcus faecalis ATCC V583 strain was purchased from Cryosite (NSW, Australia) and maintained on Columbia blood agar (Oxoid, Melbourne, Australia) at 37 °C. Culture purity was periodically checked by culturing onto bile aesculin agar (Oxoid). About 1000 mL of sterile Todd Hewitt broth (THB) (Oxoid), was inoculated with 1 mL of an overnight broth and incubated at 37 °C for 3 days. Bacteria were harvested by centrifugation (6000 g), at 4 °C for 20 min. Cells were washed twice with saline (0.9% w/v) at 4 °C and cells were finally resuspended in 12 mL of ice cold saline. Cells were lysed by two passes (60 000 kPa) through a SLM Aminco French Press (Thermo Fisher, Waltham, MA, USA). Endogenous proteinase activity was controlled during lysis by the addition of 100 μL of bacterial protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Nucleic acids were then degraded by the addition of deoxyribonuclease I (2000 Units), ribonuclease A (1000 Units), and MgCl₂ (50 mm), and incubated on ice for 60 min. Intact cells were removed by centrifuging twice (8000 g at 4 °C for 5 min) and removing the supernatant.

All chickens that died during first week of life were collected at the farm and stored at − 20°C. A postmortem examination was performed on all chickens from the two flocks from day one to seven. Bacteriological sampling was done when macroscopic lesions indicated bacterial infection (increased vascularization, discoloration, exudations). The yolk sac or liver was sampled by a thin sterile cotton swab after sterilizing the surface with a hot iron. Samples were immediately plated on a BA and incubated aerobically overnight at 37°C. From plates showing dense growth of either presumptive E. coli (colony appearing as medium size, circular, convex, and greyish color) or E. faecalis (small, circular, convex and grey colonies) or both in mixture a single colony of presumptive E. coli or E. faecalis were subcultured on MacConkey agar (Oxoid, Basingstoke, UK) or bile aesculin agar (Oxoid, Basingstoke, UK), respectively. After overnight aerobic incubation at 37°C colonies on MacConkey agar with red/pink appearance were identified as E. coli and colonies on bile aesculin agar which colored the agar black were identified as Enterococcus spp..