Methodology   

Following harvest, left and right superficial cervical and subiliac LNs were removed from each carcass and pooled by type. LNs were placed in sterile bags and transported to 25 the Meat Science Laboratory at Texas A&M University in insulated containers with refrigerant materials. Upon arrival, all LNs were flame-sterilized and then trimmed of fat. LNs, once denuded, were again flame-sterilized. Each sterilized LN sample was weighed and placed in a sterile, labeled filter bag. A rubber mallet was used to pulverize the LNs samples. Pulverized LN samples were transported to the Texas A&M University Food Microbiology Laboratory for microbiological analyses. Pre-enrichment and isolation of Salmonella followed the USDA-FSIS Microbiology Laboratory Guidebook (MLG) 4.11. Samples were pre-screened using the Hygenia BAX Q7 with Real-Time Salmonella PCR Assay. A positive result was indicated by a plus-sign (+) on the results file. Negative results were indicated by a minus-sign (-) in the results file. All presumptive positives as indicated by the BAX were subjected to traditional cultural microbiological methods.    

From incubated plates exhibiting growth, at least three viable colonies were identified, and one was chosen and onto Lysine Iron Agar (LIA) and Triple Sugar Iron Agar slants. Salmonella-typical colonies were selected. Once an ideal colony was selected, half of the colony was removed via sterile disposable needle. Subsequently, the needle was stabbed into the butt of the TSI slant and upon removal, used to streak the angled agar surface. Using the same needle, this step was repeated for the paired LIA slant. The remaining half colony was picked using a disposable sterile loop (VWR) and was streaked on a Tryptic Soy Agar (TSA) plate. Slants and plates then were incubated at 35°C for 18 to 24 h and 35°C for 16 to 24 h, respectively. Following incubation, slants were pulled and screened for gas exchange representative of Salmonella spp. presence and presumptive-positive slants were identified. One representative presumptive-positive colony from the corresponding TSA plate then was selected and ½ of the colony was streaked onto a TSA nutrient slant. TSA slants were incubated at 35°C for 22 h before being prepped to ship. The other half of the colony was swabbed onto cryovials for storage and placed in the -80°C freezer located in the microbiology lab for reference. Incubated TSA slants were transported to USDA-APHIS National Veterinary Service Laboratory (NVSL) for confirmation via MALDI-TOF. Confirmed positive isolates were serotyped. Additionally, the minimum inhibitory concentration for 14 antibiotics was determined for each confirmed positive Salmonella isolate. NVSL utilized the Sensititre system with the National Antimicrobial Resistance Monitoring System (NARMS) Gram-negative CMV4AGNF plates.