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ARS Home Midwest Area Ames, Iowa National Animal Disease Center Food Safety and Enteric Pathogens Research Research





- Kudva, Indira
- Loving, Crystal
- Byrne, Kristen
- Anderson, Christopher
- Maki, Joel
- 2023
- 2022
- 2021
- Publications
- Food Safety, (Animal and Plant Products) (108)

Research Project: Intestinal Microbial Ecology and Non-Antibiotic Strategies to Limit Shiga Toxin-Producing Escherichia coli (STEC) and Antimicrobial Resistance Transmission in Food Animals **Location:
                            Food Safety and Enteric Pathogens Research** 2023 Annual Report **Objectives**
                                

Objective 1:  Identify host and bacterial factors contributing to attachment and colonization of outbreak-associated Shiga toxin-producing Escherichia coli at gastrointestinal mucosa under various environmental conditions. 
Sub-objective 1.A: Characterize unique genetic features of outbreak-associated O157 promoting attachment and colonization in food animals.
Sub-objective 1.B1: Ascertain the impact of stress conditions on O157 to identify factors impacting virulence gene expression.
Sub-objective 1.B2. Ascertain impact of stress on host cells and local microbiota affecting O157 colonization at the rectoanal junction.
Objective 2:  Develop novel non-antibiotic intervention strategies to limit Shiga toxin-producing E. coli colonization, persistence and/or shedding from food animals.
Sub-objective 2.A: Identify novel E. coli with encoded bacteriocins, or competitive nutritional networks that interfere with O157 viability, growth, or attachment to food animal intestinal mucosa. 
Sub-objective 2.B: Evaluate efficacy of host and/or bacterial proteins to limit O157 mucosal attachment or colonization.  
Objective 3:  Identify intervention strategies to mitigate stress-induced dysbiosis, improve mucosal immunity, and minimize antimicrobial resistance gene transfer from food animal commensals to human pathogens.
Sub-objective 3.A: Define changes in host intestinal immune status, intestinal bacterial membership and function, and AMR mobile elements associated with weaning stress. 
Sub-objective 3.B: Identify non-antibiotic intervention strategies to minimize negative impact on gut dysbiosis and antimicrobial resistance gene transmission to human pathogens. **Approach**
                                

The goal of this project is to research practical solutions for food safety problems important to food production and public health sectors in the United States and globally. The research addresses food safety at the first link in the food production chain, namely the food animals on the farm. The research investigates the bacterial communities and the animal’s physiological response in the intestinal tract, as well as the interactions between microbiota and intestinal cells that lead to colonization with foodborne organisms and mobility of antimicrobial resistance genes into foodborne pathogens. Some microbiota members confer benefits to the host. Still others are benign to the animal but are harmful foodborne pathogens. The gut microbial consortium comprises a reservoir of antibiotic resistance genes of undefined composition and risk potential. Environmental factors, particularly stress, can modulate both host and bacteria, impacting the symbiotic relationship. Experiments are planned to:  1) examine the environmental, bacterial, and immunological factors affecting Shiga toxin-producing Escherichia coli (STEC) colonization of cattle; 2) identify unique genetic features of STEC impacting colonization and attachment; 3) define the impact of stress on bacterial and immunological events impacting bacterial colonization and antibiotic resistance gene transfer; and 4) examine novel, intervention strategies to reduce foodborne pathogen carriage and antibiotic resistance gene transfer in food animals. The combination of basic and applied research will supply knowledge and tools, as well as applicable strategies to control foodborne pathogens and antibiotic resistance gene mobility. **Progress Report**
                                

Shiga toxin-producing Escherichia coli (STEC) serotype O157:H7 (O157) contaminated meat and produce, are implicated in outbreaks of human infection. Identification of genetic patterns of O157 strains correlated with enhanced O157 survival in cattle or the environment may help predict outbreak potential of O157 isolates. In support of Objective 1, Sub-objective 1.A.: Characterize unique genetic features of outbreak-associated O157 promoting attachment and colonization in food animals, to better understand colonization and shedding dynamics in the asymptomatic bovine host, four O157 strains with differing bovine rectoanal junction (RAJ) squamous epithelial (RSE) cell-adherence, biofilm formation and toxin production phenotypes were experimentally assessed in cattle. There was no significant difference in the amount of O157 detected in recto-anal junction mucosa swabs or feces. The data was foundational in selection of additional isolates for in vitro testing to better understand the complex relationship between genotype and in vitro versus in vivo phenotype. Approximately 130 sequenced O157 isolates were received from Food Safety Inspection Service and the Center for Disease Control and Prevention, and in vitro assessment of phenotype commenced. 
Transportation stress is associated with increased O157 shedding in cattle, though the exact mechanism(s) associated with changes in O157 and the host animal are not clearly defined. Changes in the host mucosa and microbiota occurring in response to transportation stress likely influence O157 colonization and therefore are important to understand. Aligned with Objective 1, Sub-objective 1.B2.: Ascertain impact of stress on host cells and local microbiota affecting O157 colonization at the rectoanal junction, a group of cattle were subjected to transportation stress for approximately 2 hours; a non-transported cohort was included as a control. Blood, feces, rectoanal mucosal swabs (RAMS), and RAJ biopsies were collected at various timepoints before and after transport to assess the impact of transportation on microbiota, RAJ gene expression, and changes in cortisol levels and circulating immune cell populations. Transportation did not cause any major shifts in circulating immune cell populations, as the numbers of neutrophils, monocytes, and lymphocytes in the blood was the same in transported and non-transported animals even 30 minutes upon completion of transport. In addition, peripheral cortisol levels were not impacted by transportation. DNA was extracted from feces and RAMS and 16s rRNA V4 region sequenced to assess changes in the microbiota. Data analysis of microbial membership and transportation associated changes is ongoing. RNA was extracted was from RAJ biopsies, cDNA synthesis performed, and assessment of gene expression changes by real-time PCR is ongoing. 
Limiting O157 attachment to bovine intestinal epithelial cells could limit animal colonization and hence, could be explored as an intervention strategy. Thus, under Sub-objective 2.B.: Evaluate efficacy of host and/or bacterial proteins to limit O157 mucosal attachment or colonization, groups of cattle were parenterally vaccinated with one of two different inactivated mutant strains of O157 to assess the role of mucosal antibodies, specifically IgA, in limiting O157 adherence to bovine intestinal cells. Groups of cattle were vaccinated with adjuvanted, whole-cell O157 vaccine by the intramuscular route. Matched blood and fecal samples were collected at different time points following vaccination. While IgA was detected in the fecal extracts by ELISA, O157-specific IgA was not detected. Peripheral O157-specific IgG was detected, and specific to proteins associated with O157 cell wall. RNA was extracted from RAJ biopsies, cDNA synthesis performed, and assessment of changes in gene expression using real-time PCR is ongoing. 
Under Sub-objective 2.B.: Evaluate efficacy of host and/or bacterial proteins to limit O157 mucosal attachment or colonization, a total of 232 RAJ squamous epithelial (RSE) cell and follicle-associate epithelial (FAE) cell proteins, interacting with O157, were identified using proteomics. Likewise, a total of 173 O157 proteins that interact with RSE and FAE cells at the RAJ were identified. Identified O157 proteins are being analyzed in silico to shortlist proteins most relevant to O157 adherence to each RAJ cell type to design functional peptides from the proteins. Different analytical software is currently being evaluated for selecting optimal peptide sequences for synthesis. The proteins and/or peptides will be evaluated for their ability to block O157 adherence to RAJ cells in in vitro assays before testing in animals as potential therapeutics to prevent O157 colonization in cattle.
The short and long-term impact of swine management practices on the composition of the gastrointestinal microbiome, the acquisition of antimicrobial resistance genes (ARG) by foodborne pathogens, and changes in the immune status are not well understood. Weaning is associated with abrupt changes in the gastrointestinal microbial community and immune cell function. In support of Objective 3, Sub-objective 3.A.: Define changes in host intestinal immune status, intestinal bacterial membership, and function, and ARG mobile elements associated with weaning stress, we previously conducted a small pig animal trial to investigate the ARG transfer and immune responses following Salmonella challenge in weaning-aged piglets. Blood and tissues samples were collected from pigs at necropsy and analysis of gene expression in circulating blood cells indicated a rapid inflammatory response at day 2 post-inoculation that resolved by day 8. Peripheral blood mononuclear cells isolated from blood were submitted for single-cell RNA-sequencing (scRNA-seq) to better understand the acute immune response to Salmonella, which could be important for understanding how Salmonella eventually persists in lymphoid tissues of the pig.
Enrichment and isolation processes for Salmonella detection in routine animal samples are the cornerstones of surveillance programs, but recovery biases associated with culture media and sample types remain poorly understood. Culture media used to enrich Salmonella also increases the abundance of other Enterobacteriaceae known readily to ARG to Salmonella However, the transfer of ARG during enrichment has yet to be systematically investigated. In support of Objective 3, we observed the transfer of ampicillin resistance genes via plasmids to Salmonella during the pre-enrichment of swine cecal samples in buffered peptone water. Salmonella spiked into swine cecal samples from nursery-aged piglets resulted in the transfer of ampicillin resistance to 1-4% of recovered Salmonella. ARG transfer was not observed in selective enrichment media, such as Tetrathionate and Rappaport Vassiliadis Broth. Repeating this study with samples from 6–8-week-old pigs resulted in limited transfer of ampicillin resistance, suggesting findings are microbiota dependent because the ampicillin resistance gene abundance and genetic context is different across different groups of pigs. The transfer of ARG in pre-enrichment media is being further evaluated in finishing pigs. Overall, the findings could impact how we interpret genomic data and antimicrobial resistance profiles of recovered Salmonella isolates that have been enriched In certain media.
Previous research from our group demonstrated a highly fermentable resistant starch prebiotic can increase swine hindgut short-chain fatty acids (SCFA) production and reduce Salmonella shedding. However, the impact of the prebiotic starch on Salmonella shedding is variable and appears dependent on the microbial composition of the swine gastrointestinal tract. Combining probiotics with prebiotics represents a non-antibiotic intervention strategy that could minimize intestinal disturbances during the nursery period and promote colonization resistance to Salmonella. In support of Objective 3, Subobjective 3.B.: Identify non-antibiotic intervention strategies to minimize negative impact on gut dysbiosis and antimicrobial resistance gene transmission to human pathogens, 162 strains of Prevotella were isolated from nursey-aged pigs fed a prebiotic resistant starch. Prevotella are keystone species of the swine gastrointestinal microbiome and have been linked to decreased Salmonella shedding. Many of the isolates are the first cultured representative of a given Prevotella species and genomic analyses are revealing high intra-species diversity in carbohydrate substrate utilization. Further genomic analysis and in vitro assays will lead to candidate strains for mechanistic studies into how Prevotella enhances colonization resistance, and therefore, also decrease the dissemination of antimicrobial resistance into Salmonella.
Related to the production of SCFA, including butyrate, and its potentially beneficial effects to pig health and in support of Objective 3, Subobjective 3.A, a series of experiments evaluating the impact of butyrate on the response of monocytes to bacterial lipopolysaccharide (LPS) were performed. Monocytes migrate from the blood into the intestine and change their function in the context of local signals, which includes butyrate. LPS is often inflammatory, but data suggest butyrate reduces intestinal inflammation. Monocytes exposed to LPS produced proinflammatory cytokines TNF and IL-1beta; however, when butyrate was present production was limited. Instead, monocytes produced more IL-10 when exposed to LPS in the presence of butyrate. Salmonella can survive well in sites of inflammation and butyrate may limit the inflammatory response and thus, Salmonella colonization when butyrate levels are increased. **Accomplishments**
                                

1. Developed a laboratory-based model system for pre-screening STEC without having to use live animals. Shiga toxin-producing Escherichia coli (STEC) preferentially colonize the bovine rectoanal junction (RAJ) located at the terminal end of the animal’s intestinal tract. Studies evaluating STEC interactions with the RAJ are usually restricted to evaluation of cells from specific regions of the RAJ in the laboratory, or evaluation of the RAJ in live animals. Thus, information garnered from laboratory studies is limited and there is an increased reliance on live animal studies that can be expensive, particularly for studies needing to evaluate multiple different bacteria. Hence, ARS researchers in Ames, Iowa, developed a laboratory-based RAJ-in vitro organ culture (RAJ-IVOC) model system representing the entire RAJ site that would permit comprehensive bacterial adherence studies in the laboratory, without loss of tissue integrity under optimized conditions. The model system yields STEC adherence phenotype comparable to that observed in live animals. The RAJ-IVOC model developed here provides a convenient pre-screening system to evaluate interactions of multiple bacteria with the RAJ, prior to live animal experiments as needed, allowing for reduction of animal usage. STEC adherence factors that allow STEC persistence at the RAJ, identified using the RAJ-IVOC model, may be incorporated into anti-STEC therapies to reduce STEC carriage in cattle and thereby minimize STEC-contamination of cattle-derived food products. This in turn would directly impact the food industry and through them consumers. 2. Pig gut cells keep the balance. The gut is a unique part of the body as it balances letting in nutrients and keeping pathogens out. It is a complex tissue and a variety of cell types work together for optimal function and animal health. Food animal producers desire methods to enhance gut function, but development of strategies to enhance gut function are difficult to develop without a clear understanding of the function of all the individual cells in the gut. ARS researchers in Ames, Iowa, teamed up with Iowa State University collaborators and utilized cutting-edge technologies to deeply characterize more than 20,000 individual pig gut cells. A new immune cell type in pig gut important for creating a barrier to the outside environment was discovered, and a unique cell responding to stress signals was characterized. Overall, the data serve as a highly valuable resource, providing detailed descriptions of immune and epithelial cells in the pig gut and provide foundational baseline knowledge for feed producers to enhance gut health. Data is publicly available to the international research community as a resource for further exploration. 3. Oral Salmonella vaccine limits Salmonella in pigs with dietary additive. Application of commercially available vaccine for foodborne Salmonella can reduce the amount of Salmonella in pigs, an important control strategy to limit Salmonella in pork products. Dietary additives are often given to pigs for improving gut health, and are often given simultaneous to other intervention products. ARS researchers in Ames, Iowa, evaluated the impact of a common dietary additive used to modulate immune status of pigs (beta-glucan) on oral Salmonella vaccine immunogenicity and efficacy. Oral Salmonella vaccination significantly reduced the amount of Salmonella in the intestinal tract of pigs, and the dietary beta-glucan did not impact efficacy. In addition, Salmonella vaccination primed the immune system to respond rapidly with production of Salmonella-specific antibody and immune cells upon Salmonella exposure. Results are informative to producers using multiple strategies to improve gut health and limit carriage of foodborne Salmonella, and highlight the ability of commercial Salmonella vaccination to limit foodborne Salmonella in the pig. **Review Publications**
                            *Wiarda, J.E., Becker, S.R., Sivasankaran, S.K., Loving, C.L. 2022. Regional epithelial cell diversity in the small intestine of pigs. Journal of Animal Science. 101. Article eskac318. https://doi.org/10.1093/jas/skac318.*
                                *Wiarda, J.E., Watkins, H.R., Gabler, N.K., Anderson, C.L., Loving, C.L. 2023. Intestinal location- and age-specific variation of intraepithelial T lymphocytes and mucosal microbiota in pigs. Developmental and Comparative Immunology. 139. Article 104590. https://doi.org/10.1016/j.dci.2022.104590.*
                                *Loving, C.L., Bearson, S.M., Bearson, B.L., Kerr, B.J., Kiros, T.G., Shippy, D.C., Trachsel, J.M. 2022. Effect of dietary ß-glucan on intestinal microbiota diversity and Salmonella vaccine immunogenicity and efficacy in pigs. Veterinary Microbiology. 278. Article 109648. https://doi.org/10.1016/j.vetmic.2022.109648.*
                                *Kerr, B.J., Trachsel, J.M., Bearson, B.L., Loving, C.L., Bearson, S.M., Byrne, K.A., Pearce, S.C., Ramirez, S.M., Gabler, N.K., Schweer, W.P., Helm, E.T., De Mille, C.M. 2022. Evaluation of digestively resistant or soluble fibers, short- and medium-chain fatty acids, trace minerals, and antibiotics in nonchallenged nursery pigs on performance, digestibility, and intestinal integrity. Journal of Animal Science. 100(11). Article skac282. https://doi.org/10.1093/jas/skac282.*
                                *Kudva, I.T., Biernbaum, E.N., Cassmann, E.D., Palmer, M.V. 2023. Bovine rectoanal junction-in vitro organ culture model system to study Shiga toxin-producing Escherichia coli adherence. Microorganisms. 11(5). Article 1289. https://doi.org/10.3390/microorganisms11051289.*
                                *Wiarda, J.E., Trachsel, J.M., Sivasankaran, S.K., Tuggle, C.K., Loving, C.L. 2022. Intestinal single-cell atlas reveals novel lymphocytes in pigs with similarities to human cells. Life Science Alliance. 5(10). Article e202201442. https://doi.org/10.26508/lsa.202201442.*
                                *Corbett, R.J., Luttmann, A.M., Herrera-Uribe, J., Liu, H., Raney, N.E., Grabowski, J.M., Loving, C.L., Tuggle, C.K., Ernst, C.W. 2022. Assessment of DNA methylation in porcine immune cells reveals novel regulatory elements associated with cell-specific gene expression and immune capacity traits. Biomed Central (BMC) Genomics. 23(1). Article 575. https://doi.org/10.1186/s12864-022-08773-5.*
                                *Herrera Uribe, J., Lim, K., Byrne, K.A., Daharsh, L., Liu, H., Corbett, R.J., Marco, G., Schroyen, M., Koltes, J.E., Loving, C.L., Tuggle, C.K. 2023. Integrative profiling of gene expression and chromatin accessibility elucidates specific transcriptional networks in porcine neutrophils. Frontiers in Genetics. 14. Article 1107462. https://doi.org/10.3389/fgene.2023.1107462.*


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