Location: Food and Feed Safety Research
Project Number: 3091-32420-001-018-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jun 1, 2026
End Date: May 31, 2028
Objective:
The objective of this collaborative research project is to determine how prior exposure to moderately acidic conditions influences the ability of Salmonella to invade, survive, and replicate within bovine macrophages, and to define how these changes affect host cellular and transcriptional responses. The project will compare acid-adapted and non-adapted Salmonella across multiple serotypes relevant to cattle and food safety to identify serotype-specific differences in association, invasion, and intracellular persistence. A second objective is to characterize bacterial and host transcriptional responses using dual RNA-seq, focusing on stress-response pathways, virulence-associated genes, and inflammatory responses. This analysis will determine whether acid adaptation alters the regulatory programs of Salmonella or modifies macrophage immune signaling. A third objective is to integrate phenotypic outcomes with transcriptomic data to identify mechanisms through which acid adaptation influences intracellular survival and host-pathogen interactions. The results will support improved understanding of serotype-specific persistence in bovine systems and inform future strategies aimed at reducing Salmonella risk in livestock populations.
Approach:
This project will use a bovine macrophage model to evaluate how different bacterial preparations influence host-pathogen interactions. Macrophage cultures will be maintained under conditions that support stable growth and functionality, and passages will be limited to ensure consistent cellular responses. Initial optimization will establish appropriate macrophage density, viability, and infection conditions prior to experimental runs. Control strains representing typical, invasion-defective, and replication-defective phenotypes will be included to validate the infection model and benchmark assay performance. Multiple multiplicities of infection (MOIs) will be tested to identify conditions that provide measurable but controlled levels of association, invasion, and intracellular replication. Antimicrobial susceptibility assays will be used to determine effective antibiotic concentrations for eliminating extracellular bacteria while maintaining intracellular integrity. Bacterial preparations will be standardized using widely accepted methods for assessing culture density, including comparison to turbidity standards and plate-based quantification. Bacteria exposed to mildly acidic conditions will be compared to those grown under standard conditions to assess whether prior environmental stress affects host-cell interactions. Representative serotypes relevant to animal health and food safety will be included, and purity will be confirmed prior to infection. Macrophages will be infected and sampled over multiple timepoints to measure invasion, replication, and persistence. Standardized lysis and plating procedures will be used to quantify intracellular bacteria. Macrophage counts and viability assessments will support normalization of bacterial recovery and allow evaluation of potential cytotoxic effects. Sampling across early, intermediate, and late timepoints will help capture dynamic shifts in both bacterial behavior and host response. For transcriptomic analysis, RNA will be collected from infected cells using established extraction and purification workflows. RNA quality will be evaluated using spectrophotometric and fluorometric methods. Ribosomal RNA depletion and strand-specific library preparation will be used to generate samples suitable for combined host-pathogen transcriptomic analysis. Sequencing will be performed on an appropriate platform, followed by quality control and trimming of raw reads. Bioinformatic workflows will include alignment of reads to host and bacterial reference sequences, enabling separate characterization of each transcriptome. Phenotypic results (e.g., CFU, invasion, replication) will be integrated with differential gene expression analyses to examine how prior environmental exposure influences bacterial regulatory pathways and host immune responses. Particular attention will be given to genes involved in stress adaptation, virulence, and inflammation. Overall, this integrated phenotypic and transcriptomic approach will determine whether prior exposure to environmental stressors influences bacterial survival, host responses, and serotype-specific infection outcomes.