1. Incubation and hatch occurred at Simmons Foods, Pineville, MO. On incubation d 19, eggs were vaccinated with Inovocox EM 1 vaccine (Zoetis Animal Health Global Health Poultry, Durham, NC) using the Embrex Inovoject System, and with Marek's disease virus vaccine (VAXXITEK HVT+IBD vaccine, Merial Animal Health Division, Gainesville, GA) before shipment to Colorado Quality Research. Spray vaccines for Newcastle disease and infectious bronchitis (Poulvac Aero vaccine, Pfizer Animal Health Global Poultry) were administered after arrival. All birds were cared for in a manner consistent with Guidelines for the Care and Use of Agricultural Animals in Research and Teaching, 3rd ed. (Federation of Animal Science Societies, Champaign, IL).
2. BMD Type A Medicated Article (bacitracin methylene disalicylate) came from Zoetis Animal Health Global Poultry, Durham, NC. STAFAC Antibiotic for poultry (virginiamycin) came from Phibro Animal Health Corporation, Teaneck, NJ.
3. Each composite litter sample was mixed and 100 ± 10 g of litter was weighed into 200 ± 20 mL of 0.1% peptone (dilution of 1:3) in a filter bag. The samples were thoroughly mixed and then 50 μL was spiral-plated onto phenylethanol agar containing 5% sheep blood, 100 μg/mL of sulfadiazine, and 10 IU/mL of polymyxin B for the initial screening of litter or Clostridium perfringens agar (Oxoid, Basingstoke, UK) for the remaining cycles. The culture plates were anaerobically incubated at 36 ± 2°C overnight. Colonies with typical morphology of C. perfringens were counted. The detection limit was 1.3 × 101cfu/g of litter (or slightly higher if additional diluent was required).
4. The doubling dilution tested had a concentration range of bacitracin and virginiamycin between 0.06 and 64 μg/mL for each compound. The bacitracin was obtained commercially (Sigma-Aldrich, St. Louis, MO). The virginiamycin was extracted from a confirmed potency premix. The media was supplemented with Brucella agar, as recommended by Clinical Laboratories Standards Institute document M11-A7, with an additional change of increasing the agar content to 1.7%. The agar plates were inoculated with a theoretical concentration of 105 cfu/spot and incubated anaerobically at 36 ± 2°C for 42 to 48 h. Growth-control plates (including an inoculated plate incubated aerobically to check for aerobic contamination) and a quality-control strain of Clostridium perfringens (American Type Culture Collection 13124, MRI code CL-3, Manassas, VA) were included. The quality control acceptable range of this organism was 0.5 to 2 μg/mL for bacitracin and 0.25 to 1 μg/mL for virginiamycin.
5. All statistical analysis was performed with SAS version 9.2 (SAS Institute, Cary, NC). Third-party names mentioned are or may be trademarks or registered trademarks of their respective owners.