Sodium thioglycolate

For the in vivo experiment, Salmonella enterica serovar Enteritidis (SE) was prepared and administered to all chicks on day of hatch as described by Shivaramaiah and co-authors (2011 (link)). Briefly, approximate concentration of SE was quantified spectrophotometrically (Spectronic 200E, Thermo Scientific, Madison, WI), followed by serial dilutions in sterile saline to reach an approximate concentration of 10⁴ CFU/chick. Exact concentration was retrospectively determined by serial dilution plating on TSA and determined to be 1.5 × 10⁴ CFU/chick. To prepare CP inoculum, a single frozen aliquot of wild-type CP TXAM0108 was inoculated into individual tubes with TSB containing 0.25% sodium thioglycolate (VWR, Radnor, PA), and incubated under anaerobic conditions at 37°C for up to 24 h. Post-incubation, cells were washed 3 times in sterile saline by centrifugation at 1,800 × g for 15 min. The approximate concentration of CP was quantified spectrophotometrically, followed by serial dilutions in sterile saline for the desired inoculum of 10⁷ CFU/chick. Exact concentration was determined retrospectively by serial dilution plating on TSA with sodium thioglycolate and determined to be 3.0 × 10⁷ CFU/chick.

A total of 74 hyper-immune sera were developed against 37 different peptides. Each serum was tested in replicates of 5, with 3 replicate experiments, and all data are presented as a composite of replications. In sterile borosilicate glass tubes (12 × 75 mm; Fisher Scientific, Hampton, NH), 10 µ L of 1 × 10⁶ CFU/mL of CP was added to 1 mL of 0.5X Luria-Bertani broth (Becton and Dickenson, Sparks, MD) containing 1.00% porcine stomach mucin (Sigma Aldrich, St. Louis, MO) and 20 µ L of chicken hyper-immune serum for a final concentration of 1 × 10⁴ CFU/mL of CP in each tube. All tubes were then incubated in anaerobic jars at 37°C. At the 4, 6, and 8 h marks of incubation, samples were collected for quantification of CFU by serial dilution plating on tryptic soy agar (TSA) with 0.25% sodium thioglycolate (VWR, Radnor, PA) and incubated in anaerobic jars at 37°C for 24 h. Growth (CFU/mL) of CP was calculated as a sample over negative ratio relative to the mean negative control and converted to a percentage.