Location: Food Safety and Enteric Pathogens Research
2025 Annual Report
Objectives
Objective 1: Identify genetic features of Salmonella outbreak isolates that influence fitness and impact prevalence in food animals.
Sub-objective 1a: Characterize unique genetic features of outbreak-associated Salmonella.
Sub-objective 1b: Evaluate colonization, dissemination and persistence of human outbreak-associated Salmonella in turkeys and/or swine.
Objective 2: Identify mechanisms of AMR gene transfer to food borne pathogens in poultry microbiota and test novel mitigation strategies to limit AMR gene mobility.
Sub-objective 2a: Identify commensal members of the microbiota harboring AMR and contributing to AMR transfer in young birds.
Sub-objective 2b: Test the efficacy of microbiota modulation at hatch to reduce AMR HGT.
Objective 3: Develop and evaluate non-antibiotic intervention strategies to limit Campylobacter and Salmonella colonization, persistence and/or shedding in food animals.
Sub-objective 3a: Test efficacy of dual-purpose recombinant attenuated Salmonella vaccine(s) (RASV) encoding Campylobacter antigens to reduce intestinal colonization of Campylobacter and Salmonella in turkeys.
Sub-objective 3b: Evaluate the efficacy of in-feed treatments to reduce intestinal colonization of Campylobacter and/or Salmonella in turkeys.
Approach
The goal of this project is to address research gaps in high priority, food safety research areas involving the most common causes of bacterial foodborne illness in the United States, Salmonella and Campylobacter. Limiting Salmonella and Campylobacter colonization as well as AMR transfer in food-producing animals can reduce foodborne pathogen carriage into the human food chain, decrease environmental contamination, diminish the cost of meat product recalls to producers, and limit AMR transmission. Experiments are planned to: 1) investigate genetic features and fitness factors that contribute to the emergence of Salmonella outbreak isolates associated with food animal products, 2) identify, characterize and displace commensal members of the poultry microbiome involved in transfer of antimicrobial resistance (AMR) to foodborne pathogens, and 3) develop and/or test non-antibiotic intervention strategies to reduce human foodborne pathogens at the first critical control point in the food animal production chain, namely on-farm colonization. Focusing on the ’who, when and how’ questions of these crucial food safety issues will support the advancement of applicable tools for targeted mitigations to control foodborne pathogens and AMR transmission, thereby providing the public with a safer food supply.
Progress Report
Salmonella causes an estimated 1.35 million cases of human illness annually, resulting in approximately 420 deaths and costing U.S. citizens an estimated $4.1 billion. Food-producing animals can harbor Salmonella in their intestinal tract, but Salmonella typically does not cause disease in these animals, resulting in unrecognized transmission to neighboring animals and contamination of the environment as well as the nation’s food supply during processing. To support the request from farmers for effective intervention strategies to control Salmonella in food animals, a vaccine created and patented by ARS researchers in Ames, Iowa, that was previously shown to reduce Salmonella in swine and poultry, was evaluated in cattle to address Objective 3, “Develop and evaluate non- antibiotic intervention strategies to limit Campylobacter and Salmonella colonization, persistence and/or shedding in food animals”. Vaccination prevented Salmonella colonization and fecal shedding in calves, whereas non- vaccinated calves were colonized by Salmonella in the intestinal tract and shed Salmonella in their feces. Current investigations will determine whether the immune response induced in the vaccinated calves can recognize other Salmonella strains, suggestive of cross-protection against additional Salmonella serotypes.
With greater than 2,600 serotypes of Salmonella, cross-protective vaccines that protect food animals against multiple Salmonella serotypes are needed by farmers. In support of Objective 3, “Develop and evaluate non- antibiotic intervention strategies to limit Campylobacter and Salmonella colonization, persistence and/or shedding in food animals”, a computer modelling approach called reverse vaccinology was used to develop a cross-protective vaccine against Salmonella. The study identified antigenic regions (sites where antibodies bind) from Salmonella proteins of clinically relevant, poultry-associated serotypes to design a multivalent vaccine, which protects against multiple variants of a pathogen. Cross-protection of the vaccine construct was predicted by comparing the sequence of the vaccine to >33,000 genome sequences from 136 Salmonella serotypes from human outbreaks. Future research will evaluate the vaccine in poultry.
Preventing Salmonella colonization on farms is critical for maintaining a safe food supply. While vaccination and probiotics (healthy bacteria) are currently used by the industry, they often offer limited protection across the many Salmonella serovars that contaminate poultry on farms. To develop a targeted yet broadly effective approach, ARS researchers in Ames, Iowa, created a defined mix of bacterial strains or microbial community (DC) designed to exclude Salmonella in poultry, supporting Sub-objective 2b, “Test the efficacy of microbiota modulation at hatch to reduce AMR HGT.” After administration to young chicks, the DC reduced Salmonella colonization and shedding by outcompeting the pathogen in the gut. DC treated birds also showed increased nutrient transporter gene expression and reduced inflammatory and immune gene expression, suggesting earlier microbiota maturation and lower host inflammation. Ongoing research is testing different DC combinations to optimize pathogen exclusion. This research supports the development of practical, microbiome-based strategies for poultry producers to reduce pathogen load at the source, ultimately benefiting food safety, and animal performance/welfare.
Efforts to further improve microbial consortia as an effective anti-pathogen intervention requires identifying beneficial bacteria that can outcompete pathogens or produce antimicrobial compounds that target pathogens. Selecting the right combinations from thousands of potential strains remains a major challenge. To address this, ARS researchers in Ames, Iowa, developed the Gene Ontology Overlap Profiler (GOOP), a predictive software tool that analyzes microbial genomes to identify strains with metabolic advantages against Salmonella and Campylobacter, in support of Sub-objective 2b, “Test the efficacy of microbiota modulation at hatch to reduce AMR HGT.” GOOP models microbial competition and
highlights the most promising candidates for further testing. Ongoing research focuses on validating its predictions to support development of targeted interventions. By potentially reducing the time for developing next-generation probiotics, this tool benefits scientists, probiotic developers, and poultry producers seeking effective, evidence- based methods to control foodborne pathogens at the farm level.
Campylobacter and Salmonella are leading causes of human bacterial foodborne illness in the U.S., yet can colonize the intestinal tract of poultry, including turkeys, without causing disease. Enhancing the immune system in the turkey to target Salmonella and Campylobacter requires understanding how different immune cells interact with these foodborne pathogens and how to change these interactions to generate a more effective immune response. However, foundational knowledge of gene expression patterns and cell surface markers that distinguish different immune cell types in the turkey is needed to be able to identify and improve their functions during the host response to pathogens. Greater knowledge can be gained at the gene expression level in turkeys, due to the limited immunological tools available for this species. Gene expression within individual immune cells from turkey was examined to contribute to this knowledge base and support efforts under Objective 3, “Develop and evaluate non-antibiotic intervention strategies to limit Campylobacter and Salmonella colonization, persistence and/or shedding in food animals”. Marker genes for specific cell types have been identified, as well as unique patterns of expression for genes important to implementation of the immune response. Immune cell isolation methods impact recovery and expression of certain cell types; each method had benefits depending on the cell types of interest. Future studies will use the identified marker genes to understand the role of specific turkey immune cells in the intestine and allow development of intervention strategies that can leverage target cell types to improve immune recognition and clearance of Salmonella and Campylobacter.
Accomplishments
1. Gene context matters for acquisition of antibiotic resistance genes by Salmonella in poultry. In the chicken gut, antibiotic resistance genes can transfer from non-pathogenic bacteria to pathogenic bacteria like Salmonella, potentially worsening the threat of drug-resistant foodborne illnesses. Identifying the source and frequency of transfer of antibiotic-resistant genes, in an agriculture setting, is critical to prevent pathogens from becoming more difficult to treat. To investigate this, ARS researchers in Ames, Iowa, raised chicks in controlled environments to test whether bacteria from eggshells or the barn environment contribute to antibiotic resistance spread. Although chicks were colonized by Salmonella, the bacteria did not acquire antibiotic resistance from test bacteria in either live chickens or under laboratory conditions. DNA analysis showed most antibiotic resistance genes were not easily transferable because of where they were located on the chromosome or key features for transfer to Salmonella was missing. These results suggest that antibiotic resistance genes may transfer to Salmonella from other bacteria less often than previously observed. This research benefits poultry producers, food safety regulators, and public health officials by helping target interventions where the risk of resistance transfer is greatest.
2. Vaccination reduces a multidrug-resistant Salmonella outbreak isolate in the turkey intestinal tract. A human foodborne outbreak from turkey products contaminated with multidrug-resistant (MDR) Salmonella enterica serotype Reading widely impacted the United States and Canada in 2017-2019, costing turkey producers millions of dollars to implement control strategies, investigate the outbreak, and recall contaminated products. To identify effective Salmonella control strategies for turkey producers, ARS researchers in Ames, Iowa, evaluated a commercially available vaccine and an internally developed vaccine to determine their effectiveness at protecting young turkeys against MDR Salmonella Reading. Both vaccines reduced Salmonella Reading in the intestinal tract and spleen. To address how vaccination may provide protection, the researchers showed that gut health-related gene expression in the cecal tonsil (part of the immune system located in the large intestine of poultry) was acutely decreased by Salmonella Reading. Immunization with either vaccine prevented these gene expression changes and supported normal intestinal barrier and transport functions in the turkeys. The results inform turkey producers on the efficacy of vaccination to reduce Salmonella Reading in turkeys and thereby improve food safety.
3. Salmonella vaccine improves pig health by decreasing the multidrug resistant foodborne pathogen. Salmonella is a human foodborne pathogen found in the intestinal tract of food-producing animals. In the United States, Salmonella serotype (I 4,[5],12:i:-) has emerged as the isolate most frequently associated with clinical illness in pigs. This strain is also one of the most common causes of human foodborne Salmonella outbreaks and is the most frequent multidrug resistant Salmonella isolate. Because potential contamination of pork products with Salmonella serotype I 4,[5],12:i:- is a food safety concern, ARS researchers in Ames, Iowa, investigated vaccination of pigs against this isolate and showed that a commercially available vaccine reduced clinical disease, intestinal colonization, and fecal shedding of Salmonella serotype I 4,[5],12:i:-, as well as improved average daily gain of vaccinated pigs compared to non-vaccinated pigs. Data on the vaccine informs pig producers of a non-antibiotic intervention strategy to control Salmonella on the farm to decrease contamination of the pig production environment and final pork products, thereby improving animal health and food safety.
Review Publications
Guernier-Cambert, V., Trachsel, J.M., Atkinson, B.M., Oladeinde, A.A., Anderson, C.L., Bearson, S.M., Monson, M.S., Looft, T.P. 2024. Tetracycline resistance gene transfer from Escherichia coli donors to Salmonella Heidelberg in chickens is impacted by the genetic context of donors. Veterinary Microbiology. 299. Article 110294. https://doi.org/10.1016/j.vetmic.2024.110294.
Monson, M.S., Gurung, M., Bearson, B.L., Whelan, S.J., Trachsel, J.M., Looft, T.P., Sylte, M.J., Bearson, S.M. 2024. Evaluating two live-attenuated vaccines against Salmonella enterica serovar Reading in turkeys: reduced tissue colonization and cecal tonsil transcriptome responses. Frontiers in Veterinary Science. 11. Article e2024. https://doi.org/10.3389/fvets.2024.1502303.
Rothrock Jr, M.J., Al Hakeem, W., Oladeinde, A.A., Looft, T.P., Li, X., Guard, J. 2024. Salmonella biomapping of a commercial broiler hatchery. Journal of Food Protection. https://doi.org/10.1016/j.jfp.2024.100347.
Oladeinde, A.A., Chung, T., Mou, C., Rothrock Jr, M.J., Li, G., Adeli, A., Looft, T.P., Reed, W., Abdo, Z., Plumblee Lawrence, J.R., Cudnik, D., Zock, G., Teran, J., Li, X. 2025. Broiler litter moisture and trace metals contribute to the persistence of Salmonella strains that harbor large plasmids carrying siderophores. Applied and Environmental Microbiology. 91(4). Article 0138824. https://doi.org/10.1128/aem.01388-24.
Leite, F.L., Arruda, P., Ford, B., Jordan, D., Gimenez-Lirola, L., Mora-Diaz, J., Bradshaw, D., Bearson, S.M. 2025. Oral live bivalent Salmonella vaccine reduces clinical disease, colonization and fecal shedding of multidrug resistant Salmonella enterica serovar I 4,[5],12:i:-. Vaccine. 62. Article 127540. https://doi.org/10.1016/j.vaccine.2025.127540.