Phage Enumeration and Characterization from Fecal Samples

For enumeration of phages, C. jejuni strain NCTC 12662 (for phage NCTC 12673) and C. coli strain NCTC 12667 (for phage LmqsCPL1/1) were used. One gram of cecal content or feces was added to 9 mL of SM buffer (5.8 g NaCl, 2.0 g MgSO₄ × 7H₂O, 50 mL 1 M Tris, adjusted to pH 7.5, filled up with distilled water to 1000 mL) and shaken overnight at 4 °C. Subsequently, centrifugation (13,000×g for 10 min at 4 °C) and filtration with 0.2 µm filters (Sarstedt AG & Co. KG, Nümbrecht, Germany) were performed. Drinking water samples were filtered only. After preparing a tenfold dilution series in SM buffer, phage enumeration was conducted using the double agar overlay method described by Fischer et al.^{61 (link)}.
For calculating the number of phages applied per animal, the measured concentration in the drinking water and the volume of phage-dosed drinking water were used: $$\text{PFU/bird=}\frac{\text{phage concentration in drinking water}\left[\frac{\text{PFU}}{\text{mL}}\right]\text{*volume of phage-dosed drinking water [mL]}}{\text{number of birds per flock}}$$
Phages were investigated by restriction endonuclease analysis (REA) using HhaI and SwaI and pulsed-field gel electrophoresis (PFGE) as described elsewhere^{62 (link)}. However, for implementation of PFGE, GelRed (Biotium, Inc., Fremont, CA, USA) was used instead of ethidium bromide (EtBr).