Location: Livestock Issues Research
Project Number: 3096-32000-009-028-T
Project Type: Trust Fund Cooperative Agreement
Start Date: Feb 1, 2021
End Date: Jan 31, 2026
Objective:
The objective of this proposal is to determine how Salmonella populations are influenced in cattle supplemented with a Bacillus subtillis probiotic feed additive.
Approach:
Fecal samples will be collected by Kemin and/or Cactus personnel at 4 time points between trial initiation and harvest. Samples will be obtained from all pens from each of the 2 treatments. Convenience grab samples (n = 2; 50g minimum/sample) will be aseptically collected from fresh manure (fresh manure will be a subjective decision based on the individual collecting samples) on the floor of each pen and placed into a single sealed and labeled bag. Bags will be labeled to identify pen number and date. Individuals collecting samples should change gloves and disinfect their hands between pens following aseptic collection procedures to the extent possible. Bagged samples will be placed in coolers containing enough ice packs sufficient to keep the manure samples cool. Samples will be shipped or transported to USDA-ARS in Lubbock within 24 h of collection to ensure processing within 48 h of sample collection.
Upon arrival at the USDA-ARS, 2 aliquots from each pen sample (25 g/sample) will be weighed into strainer bags and homogenized with 225 mL phosphate buffered saline (PBS) in a stomacher (230 rpm for 2 min.). From the strainer bag, 1 mL of the homogenate will be removed and placed into a 1.5 mL microcentrifuge tube and subjected to serial dilution by transferring 100 uL of homogenate to 900 uL of PBS. Then, 100 uL of selected dilutions will be plated via stick-spreading on Brilliant Green and/or XLT4 agar with or without novobiocin and incubated overnight at 37°C. After incubation, phenotypical colonies will be counted to determine Salmonella concentrations defined as CFU/g based on the dilution factor. Duplicate counts will be averaged to determine the final Salmonella concentration for each pen. Phenotypic colonies from each plate will be pulled and subjected to a Salmonella latex agglutination test or other biochemical test for positive confirmation. In conjunction to quantification, 1 mL of homogenate will be placed in a 1:10 dilution of Tetrathionate Broth with iodine, and 1 mL will also be placed in Rappaport Vassiladus enrichment broth and placed in 37°C and 42°C incubators, respectively overnight. From the enriched cultures, a 1 uL loop will be utilized to streak the enriched media onto brilliant green and XLT4 agars. Similarly, latex agglutination tests will be performed on phenotypic colonies.
Upon harvest of the subject cattle, trained personnel from West Texas A&M University will collect subiliac lymph nodes (n=~300) from the carcass after hide removal and evisceration. Samples will be collected in labeled, sealable bags and packaged with ice packs and packing material. Following shipment and receipt of the lymph node samples by USDA-ARS, the lymph nodes will be denuted, the surface sanitized in boiling water, macerated, and homogenized in strainer bags with a 1:10 w/v of PBS. Isolation and quantification of Salmonella will occur as described above for the fecal samples. Following data collection, all samples and isolates will be destroyed via chemical and/or steam sterilization, and no isolates will be serotyped throughout the study.
