Preparation of Cervid Feces Extracts

Cervid feces was prepared as described by Cheng et al. (9). Briefly, 1 g of previously collected fecal pellets was weighed and added into the feces extract buffer (20 mM sodium phosphate (pH 7.1), 130 mM NaCl, 0.05% Tween 20, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1X complete protease inhibitors (Roche) giving a final concentration of 10% (w/v). Then, the fecal pellets were homogenized in M tubes (GentleMACS M tubes) using a dissociator (GentleMACS, Miltenyi Biotec) for a minute with two to three repeats to ensure complete pellet disruption. The tubes were then placed onto an orbital shaker for 1 h at room temperature. After centrifugation at 18,000 × g for 5 min, supernatants were collected and stored at −80°C for further use.