Quantification of S. Enteritidis and S. Typhimurium in broiler chickens inoculated with a cocktail of Salmonella serovars using novel duplex qPCR assays

Authors: Li, Xiang; Oladeinde, Adelumola; Rothrock Jr, Michael; Cho, Sohyun; AGGREY, SAMUEL - University Of Georgia; Plumblee Lawrence, Jodie

Submitted to: International Poultry Scientific Forum
Publication Type: Abstract Only
Publication Acceptance Date: 11/25/2024
Publication Date: 1/5/2025
Citation: Li, X., Oladeinde, A.A., Rothrock Jr, M.J., Cho, S., Aggrey, S., Plumblee Lawrence, J.R. 2025. Quantification of S. Enteritidis and S. Typhimurium in broiler chickens inoculated with a cocktail of Salmonella serovars using novel duplex qPCR assays. International Poultry Scientific Forum. p. T213.

Interpretive Summary: An advanced method for Salmonella detection has been developed that overcomes limitations of traditional testing. By using mathematical technique that includes an internal error-checking mechanism and complex statistical modeling, the authors created a highly sensitive test capable of accurately identifying two specific Salmonella types (Enteritidis and Typhimurium) in environmental environments like chicken farms. Their approach, which demonstrated good accuracy in detecting bacterial loads, offers a more reliable tool for monitoring potential health risks, potentially improving early detection of foodborne pathogens and supporting more effective public health surveillance strategies.

Technical Abstract: Precisely quantifying pathogen loads is essential for assessing environmental contamination levels and informing public health interventions. While singleplex quantitative polymerase chain reaction (qPCR) has been widely used due to its high sensitivity and specificity, it cannot identify PCR inhibitors in environmental samples, potentially leading to false-negative results or underestimation of pathogen concentrations. This study aimed to develop and validate a more robust method for quantifying Salmonella serovars of concern in environmental samples. We designed a duplex qPCR incorporating an internal amplification control (IAC) to monitor amplification interference, crucial for environmental sample testing. To account for experiment-to-experiment variation, replicate measurement variability, and uncertainties in related steps, we employed a Bayesian Markov chain Monte Carlo (MCMC) approach to generate master calibration curves for each duplex assay, using at least six independent instrumental runs. This method was applied to develop assays for S. Enteritidis and S. Typhimurium. Calibration curves for S. Enteritidis and S. Typhimurium assays exhibited R² values of 0.99, with amplification efficiencies of 1.04 and 0.93, respectively. Statistical analysis using Bayesian confidence intervals (95% BCI) revealed lower limits of quantification (LLOQs) of 36.85 and 36.02 Cq values for S. Enteritidis and S. Typhimurium, respectively. To validate the assays under real-world conditions, we tested cecal samples from experimental broiler chickens raised on fresh poultry litter for 7 days post-inoculation. Our qPCR found 6.83 – 7.55 and 6.90 – 7.68 Log10 gene copies/g of S. Enteritidis and S. Typhimurium in positive samples, respectively. In conclusion, our assays demonstrate decent LLOQs and amplification efficiencies, which are crucial parameters for accurate quantification methods. The successful quantification of S. Enteritidis and S. Typhimurium using Bayesian MCMC models supports the potential application of this approach in environmental settings. This method offers a more reliable and accurate tool for monitoring Salmonella contamination in complex environmental samples, potentially improving public health surveillance and risk assessment strategies.