Location: Food Safety and Enteric Pathogens Research
2024 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 human foodborne illness, but usually does not cause disease in food producing animals. With greater than 2,600 serotypes of Salmonella, vaccines that reduce colonization of various Salmonella serotypes in food animals are desirable. In support of Sub-objective 3, “Develop and evaluate non-antibiotic intervention strategies to limit Campylobacter and Salmonella colonization, persistence and/or shedding in food animals”, a vaccine study was conducted to investigate a live-attenuated vaccine created from a serogroup B Salmonella strain (Typhimurium) for cross-protection against a serogroup D Salmonella strain (Enteritidis) in broiler chickens. Preliminary data suggest the vaccine reduces intestinal colonization and dissemination to systemic organs by Salmonella serotype Enteritidis, one of the most significant serotypes transmitted from animals to humans globally.
Salmonella serotype Infantis has emerged as one of the most common serotypes in chickens and turkeys, and its emergence coincides with the increased presence of a megaplasmid called Emergent Salmonella Infantis (pESI) that contains numerous virulence and antimicrobial resistance genes. To determine if pESI enhances Salmonella serotype Infantis colonization in turkeys, turkeys were inoculated with Salmonella serotype Infantis strains with and without pESI, addressing Sub-objective 1B, “Evaluate colonization, dissemination and persistence of human outbreak-associated Salmonella in turkeys and/or swine”. Data are in the final stages of analysis, and the results indicate that the presence of pESI enhances tissue colonization and systemic dissemination in turkeys, which supports the concept that acquisition of the genes on pESI contributed to the emergence and persistence of the serotype in poultry production.
While pESI was originally noted in Salmonella serotype Infantis for both chickens and turkeys, recent indications show that pESI has been identified in other Salmonella serotypes isolated from turkeys but not chickens (serotypes Senftenberg, Agona, Schwarzengund, Muenchen and Alachua). To address Sub-objective 1A, “Characterize unique genetic features of outbreak-associated Salmonella”, publicly available whole genome sequence (WGS) data was used to determine pESI carriage, isolation source, and U.S. location of Salmonella serotype Senftenberg isolates. Current investigations are comparing the genetic content of Senftenberg pESI to Infanti pESI to evaluate genetic variation and content that may impact spread of pESI to other Salmonella serotypes.
Exposing chicks to cecal contents from mature birds can reduce Salmonella, potentially through a process called competitive exclusion. In support of “Objective 3B, Evaluate the efficacy of in-feed treatments to reduce intestinal colonization of Salmonella in poultry”, a study comparing the anti-Salmonella potential of mature cecal contents (CC) to a defined consortium (DC) of 15 commensal bacteria was conducted. Chicks were divided into three groups, and treated with DC, CC, or sterile PBS (control; CT), followed by Salmonella inoculation. The microbiota from CC showed differences in diversity compared to other groups for all timepoints. Salmonella counts from the CC group were below 10 CFU/g for all timepoints. A reduction in Salmonella was detected in DC compared to CT at 28 days post-inoculation. Both DC and CC microbial modulation led to differentially expressed chicken genes in the intestine, including nutrient transporters and immunological genes. Current analysis is ongoing to explore gene expression differences, but overall, microbiota modulation impacted Salmonella colonization and host gene expression in chickens.
Short chain fatty acids (SCFAs) are bacterial metabolites that promote gut health and reduce intestinal pH. These effects can lead to reduced colonization of poultry by Salmonella. Resistant starches can be fermented by the gut microbiota into SCFAs, and if given in feed as a prebiotic, may have potential to improve bird performance yet inhibit Salmonella. In support of “Objective 3B, Evaluate the efficacy of in-feed treatments to reduce intestinal colonization of Campylobacter and/or Salmonella in turkeys”, a study was performed to assess resistant potato starch as a dietary additive against Salmonella Infantis colonization in turkeys. Although initial colonization was similar, Salmonella Infantis load in cecal contents decreased sooner in poults fed a diet with resistant potato starch than poults on a non-amended diet. Ongoing research is examining how resistant potato starch impacts the turkey cecal microbiota and if this would suggest that the prebiotic could be strengthened by supplementing with specific SCFA-producing probiotics.
Understanding how the turkey immune system interacts with foodborne pathogens and how intervention strategies change these interactions requires the ability to distinguish different immune cell types. However, additional functional knowledge of specific immune cells in turkeys is still needed. As foundational support for “Objective 3, Develop and evaluate non-antibiotic intervention strategies to limit Campylobacter and Salmonella colonization, persistence and/or shedding in food animals”, genome-wide expression patterns were obtained for individual immune cells from turkey peripheral blood. Cell-type specific markers have been identified and will be utilized in the future to study immune cell responses to Salmonella or Campylobacter with the aim to better understand how these foodborne pathogens circumvent immune recognition and clearance in turkeys.
Accomplishments
1. Vaccination keeps multidrug-resistant Salmonella out of turkey bones. Multidrug-resistant (MDR) Salmonella enterica serotype Infantis has emerged as one of the most common Salmonella serotypes isolated from poultry in the U.S. ARS researchers in Ames, Iowa discovered that MDR Infantis can be found in the bone marrow of infected turkeys, which is not a common site to isolate Salmonella. A potential concern of MDR Infantis in turkey bone marrow is contamination of bone meal or mechanically-separated poultry meat during processing. Vaccination with a commercially available Salmonella vaccine protected against MDR S. Infantis in the bone marrow, in addition to reducing Salmonella in the intestinal tract, thereby informing producers of a method to reduce contamination of food products.
2. A highly pathogenic cattle-associated Salmonella outbreak isolate has enhanced attachment to host cells compared to low pathogenic isolates. Salmonella usually does not cause clinical disease in food animals. However, a 2015-2017 multistate outbreak of Salmonella Heidelberg associated with human disease was unique because the Salmonella isolate caused severe disease in dairy beef calves and frequently led to calf death. ARS researchers in Ames, Iowa investigated potential reasons for the increased disease severity of cattle-associated Salmonella Heidelberg and discovered that the highly pathogenic Salmonella Heidelberg isolate had increased expression of Salmonella genes involved in cell attachment, as well as enhanced Salmonella invasion of human and bovine epithelial cells relative to low pathogenic isolates. The data suggest that acquisition of genes involved in host cell attachment and invasion contributed to the increased pathogenicity and host range expansion of Salmonella Heidelberg in the bovine species, providing the cattle industry with a possible contributing factor to the Salmonella Heidelberg outbreak in dairy beef calves.
3. Toxin and antibiotic resistances in multidrug-resistant (MDR) Salmonella Reading outbreak isolates from turkeys and humans. Identifying how and why a Salmonella serotype emerges as a foodborne outbreak problem provides food animal producers and processors targets to improve control strategies. To investigate potential causes for a human foodborne outbreak of Salmonella enterica serotype Reading involving turkey products in the United States and Canada from 2017-2019, ARS scientists in Ames, Iowa compared the DNA make-up of MDR S. Reading isolates associated with the outbreak to Reading isolates collected before the outbreak period. Unique genetic traits were identified, including resistance to a toxin involved in the ability to compete with other bacteria in the intestine to survive (i.e., competitive exclusion). The results provide turkey stakeholders with information on genetic features within the emergent Reading outbreak isolates that could have promoted Salmonella dissemination within the turkey production environment and the subsequent human outbreak from contaminated products; the unique genetic features can be targeted for detection of the outbreak Salmonella Reading in turkey production and food products.
4. Fecal microbiota transplant limited Campylobacter levels in poultry. Chickens on farms that were consistently Campylobacter negative or -positive had microbiota differences that correlated with their Campylobacter status. To further determine if gut bacteria might influence Campylobacter status, ARS researchers in Ames, Iowa, in collaboration with Iowa State University, conducted two animal trials where newly hatched chickens were given intestinal contents from mature Campylobacter-free chickens (fecal microbiota transplantation (FMT)) to evaluate the effect on subsequent Campylobacter colonization. FMT significantly impacted the gut microbiota composition of birds and limited cecal Campylobacter colonization in birds that were inoculated with Campylobacter. These findings indicate that FMT can impact the development of the gut microbiota in young chickens and reduce Campylobacter colonization. The development of novel Campylobacter control strategies like FMT can be used by farmers to improve poultry food safety.
5. Campylobacter colonization status is associated with microbiota differences in commercial broilers. Controlling Campylobacter on farms is important because reducing Campylobacter in poultry reduces contamination risk of meat and the potential for infections in humans. To determine if microbiota composition influences Campylobacter colonization, ARS researchers in Ames, Iowa, in collaboration with Iowa State University, compared the intestinal microbiota of chickens from Campylobacter-positive and Campylobacter-negative farms. Key differences were detected in the microbiota of Campylobacter-positive flocks, including the increase of specific bacteria when Campylobacter was present and bacterial species that were present when Campylobacter was absent. The presence of specific microbes when Campylobacter is absent suggests they may compete with Campylobacter in the gut. Overall, these findings may be valuable for developing an evidence-based approach for researchers to design tailored gut microbial communities to reduce Campylobacter colonization in poultry and for producers to enhance food safety.
Review Publications
Burciaga, S., Trachsel, J.M., Sockett, D.C., Aulik, N.A., Monson, M.S., Anderson, C.L., Bearson, S.M. 2023. Genomic and phenotypic comparison of two variants of multidrug-resistant Salmonella enterica serovar Heidelberg isolated during the 2015-2017 multi-state outbreak in cattle. Frontiers in Microbiology. 14: Article 1282832. https://doi.org/10.3389/fmicb.2023.1282832.
Pang, J., Beyi, A., Looft, T.P., Zhang, Q., Sahin, O. 2023. Fecal microbiota transplantation reduces Campylobacter jejuni colonization in young broiler chickens challenged by oral gavage but not by seeder birds. Antibiotics. 12(10). Article 1503. https://doi.org/10.3390/antibiotics12101503.
Bearson, S.M., Monson, M.S., Bearson, B.L., Whelan, S.J., Byrd II, J.A., Burciaga, S. 2024. Commercial vaccine provides cross-protection by reducing colonization of salmonella enterica serovars infantis and hadar in turkeys. Vaccine. https://doi.org/10.1016/j.vaccine.2023.12.054.
Sivasankaran, S.K., Bearson, B.L., Trachsel, J.M., Nielsen, D.W., Looft, T.P., Bearson, S.M. 2024. Genomic and phenotypic characterization of multidrug-resistant salmonella enterica serovar reading isolates involved in a turkey-associated foodborne outbreak. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2023.1304029.
Jinji, P., Looft, T.P., Qijing, Z., Sahin, O. 2023. Deciphering the association between Campylobacter colonization and microbiota composition in the intestine of commercial broilers. Microorganisms. 11(7)1724. https://doi.org/10.3390/microorganisms11071724.