The United States Department of Agriculture Food Safety and Inspection Service (USDA FSIS) does not maintain a zero-tolerance policy for Salmonella in poultry and poultry products, despite being a known food safety hazard throughout the poultry industry. In 2016, USDA FSIS established performance standards for a 52-week moving window with the maximum acceptable percent positive for comminuted turkey (325 g sample) at 13.5% (7 of 52 samples). Based upon FSIS verification sampling results from one 52-week moving window, the Salmonella prevalence for each poultry establishment in category 1 (below limit), 2 (meeting limit), or 3 (exceeding limit) are published for public viewing. Moreover, many poultry producers continue to have post-intervention samples test positive. Therefore, the use of quantification would be more valuable to determine the efficacy of process control interventions, corrective actions, and final product Log CFU/g of Salmonella to make rapid, within shift, food safety decisions. Therefore, the objectives of these studies are to develop, verify, and validate a rapid and reliable quantification method utilizing RT-PCR to enumerate Salmonella in the poultry industry from flock to final product and to utilize the method in an application study. BAX^® System SalQuant^® is an application of the BAX^® System Real-Time PCR Assay for Salmonella to enumerate low levels of Salmonella with shortened enrichment times. Curve development encompassed inoculating poultry matrix samples at four levels with an ATCC strain of Salmonella, with three biological replicates per inoculation level, and five technical replicates being run on the BAX^® System for various timepoints, gathering the data, and creating a linear-fit equation. A linear-fit equation was provided for each timepoint. The ideal timepoint, based on the statistical parameters surrounding the equation (R2 > 0.80, Log RMSE < 0.60, and enumerable range 0.00 to 4.00 Log CFU/mL (g)) that most accurately estimate Salmonella compared to most probable number (MPN), was chosen to be utilized for further studies.

The goal of this study was to develop a rapid RT-PCR enumeration method for Salmonella in pork and beef lymph nodes (LNs) utilizing BAX^®-System-SalQuant^® as well as to assess the performance of the methodology in comparison with existing ones. For study one: PCR curve development, pork, and beef LNs (n = 64) were trimmed, sterilized, pulverized, spiked with 0.00 to 5.00 Log CFU/LN using Salmonella Typhimurium, and then homogenized with BAX-MP media. Samples were incubated at 42 °C and tested at several time points using the BAX^®-System-RT-PCR Assay for Salmonella. Cycle-Threshold values from the BAX^®-System, for each Salmonella concentration were recorded and utilized for statistical analysis. For study two: Method comparison; additional pork and beef LNs (n = 52) were spiked and enumerated by (1) 3M™EB-Petrifilm™ + XLD-replica plate, (2) BAX^®-System-SalQuant^®, and (3) MPN. Linear-fit equations for LNs were estimated with recovery times of 6 h and a limit of quantification (LOQ) of 10 CFU/LN. Slopes and intercepts for LNs using BAX^®-System-SalQuant^® when compared with MPN were not significantly different (p < 0.05), while the same parameters for 3M™EB-Petrifilm™ + XLD-replica plate were significantly different (p > 0.05). The results support the capability of BAX^®-System-SalQuant^® to enumerate Salmonella in pork and beef LNs. This development adds support to the use of PCR-based quantification methodologies for pathogen loads in meat products.

Poultry is the primary reservoir of Campylobacter, a leading cause of gastroenteritis in the United States. Currently, the selective plating methodology using selective agars, Campy Cefex and Modified Charcoal Cefoperazone Deoxycholate agar, is preferentially used for the quantification of Campylobacter spp. among poultry products. Due to the specific nature of Campylobacter, this methodology is not sensitive, which can lead to skewed detection and quantification results. Therefore, Campylobacter detection and quantification methods are urgently needed. The objective was to develop a shortened enrichment-based quantification method for Campylobacter (CampyQuant™) in post-chill poultry rinsates using the BAX^® System Real-Time PCR assay for Campylobacter. The specificity and sensitivity for the detection of C. jejuni, C. coli, and C. lari in pure culture were determined. The BAX^® System Real-Time PCR assay consistently detected and identified each species 100% of the time with an enumeration range of 4.00 to 9.00 Log₁₀ CFU/mL. Enrichment time parameters for low-level concentrations (0.00, 1.00, and 2.00 Log₁₀ CFU/mL) of Campylobacter using the BAX^® System Real-Time PCR assay were elucidated. It was determined that an enrichment time of 20 h was needed to detect at least 1.00 Log₁₀ CFU/mL of Campylobacter spp. Using the BAX^® System Real-Time PCR assay for Campylobacter. As a result, time of detection, detection limits, and enrichment parameters were used to develop the CampyQuant™ linear standard curve using the detected samples from the BAX^® System Real-Time PCR assay to quantify the levels in post-chill poultry rinsates. A linear fit equation was generated for each Campylobacter species using the cycle threshold from the BAX^® System Real-Time PCR assay to estimate a pre-enrichment of 1.00 to 4.00 Log₁₀ CFU/mL of rinsates detected. The statistical analyses of each equation yielded an R² of 0.93, 0.76, and 0.94 with a Log₁₀ RMSE of 0.64, 1.09, and 0.81 from C. jejuni, C. coli, and C. lari, respectively. The study suggests that the BAX^® System Real-Time PCR assay for Campylobacter is a more rapid, accurate, and efficient alternative method for Campylobacter enumeration.

Shiga toxin–producing Escherichia coli (STEC) and Salmonella are foodborne pathogens commonly harbored in the gastrointestinal tract of sheep. These pathogens can be on the hide of sheep and transferred to the carcass, causing a foodborne hazard. Salmonella can also be found in the lymph nodes of sheep, creating a biological hazard during harvest and processing. Developing countries lack baseline data on the presence of these foodborne pathogens on meat products, specifically sheep meat. Therefore, the objective of this study was to evaluate the presence of STEC and Salmonella on sheep hides, preevisceration carcasses, and final carcasses and Salmonella in subiliac lymph nodes from two small Honduran harvest facilities, plants A and B. Sponge swabs from the foreshank region of hides and carcasses and subiliac lymph node samples were collected from 96 sheep (86 at plant A; 10 at plant B). Microbial detection of STEC and Salmonella was performed by using the BAX System to screen for prevalence, and suspect samples were subjected to conventional culture isolation for confirmation. Overall baseline contamination on hides were 34.4 and 10.4% present for STEC and Salmonella; however, through implementation of sanitary procedures, preevisceration and final carcass samples were significantly decreased from the hide for both pathogens (P = 0.05). Moreover, overall plant A had significantly higher rates (P = 0.05) of STEC and Salmonella at each carcass sampling site compared with plant B. After each sampling was performed, recommendations were provided to each facility on the basis of pathogen presence, performance techniques, and contamination risks. Through recommendations and implementation of hazard analysis and critical control points, good manufacturing practices, and sanitation standard operating procedures in each facility, the meat supply in Honduras will become safer and more wholesome. Therefore, the development of a baseline with continued sampling is crucial to understand the risk of foodborne pathogens to consumers in the Honduran sheep meat supply. Salmonella and STEC were reduced from sheep hides and carcasses with applied interventions. Implemented recommendations reduced Salmonella and STEC presence over time. Proper SSOPs and GMPs remain critical for reducing foodborne pathogen presence. Salmonella was recovered only in 1.0% of Honduran sheep lymph nodes.

The accurate quantification of Campylobacter jejuni in poultry samples is critical for ensuring food safety and compliance with regulatory standards. This study evaluated the performance of three enrichment media—Mueller–Hinton Broth (MHB), Bolton’s Blood-Free Broth 2x (BFBB2x), and Buffered Peptone Water (BPW)—in supporting C. jejuni detection and quantification using the BAX^® Q7-RT PCR system and traditional plate count methods. Results demonstrated high reliability across all media types, with BFBB2x and MHB showing the strongest correlations (R² = 0.99) for the BAX^® system. BFBB2x exhibited the lowest RMSE (0.13), while MHB balanced precision (RMSE = 0.4) with sensitivity. For plate counts, MHB and BPW achieved the highest correlations (R² = 0.99) and precision (RMSE = 0.26), with MHB demonstrating the lowest detection limit (2.56 log₁₀ CFU/mL) compared to BFBB2x (2.93 log₁₀ CFU/mL) and BPW (3.31 log₁₀ CFU/mL). The findings underscore MHB’s robustness as an enrichment medium, offering consistent performance across both molecular and culture-based methods. The current study supports MHB as the more effective medium for the reliable and precise quantification of C. jejuni in poultry-associated matrices, highlighting its utility in minimizing contamination risks and enhancing food safety. Future research should explore its applicability in diverse poultry products and production environments.