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ARS Home » Plains Area » Clay Center, Nebraska » U.S. Meat Animal Research Center » Meat Safety and Quality » Research » Research Project #429923

Research Project: Assessment of Genotypic and Phenotypic Factors for Foodborne Pathogen Transmission and Development of Intervention Strategies

Location: Meat Safety and Quality

2020 Annual Report

Objective 1. Determine the genotypic and/or phenotypic factors associated with the levels and persistence of pathogens and antibiotic resistance in the host animal and the livestock production environment. Sub-objective 1.1 - Determine genotypic or phenotypic factors associated with persistence of E. coli O157:H7 in cattle production. Sub-objective 1.2 - Determine animal host genotypes that confer resistance/susceptibility to pathogen infection. Sub-objective 1.3 - Evaluate the influence of spatiotemporal, environmental, and wildlife factors on pathogen and antibiotic resistance occurrence and transmission dynamics in cattle and waterways in a pasture-based production system. Objective 2. Develop and evaluate intervention strategies that reduce or eliminate the occurrence, transmission or persistence of foodborne pathogens in host animals, including cattle and swine, and the environment. Sub-objective 2.1 - Develop a high-throughput procedure to identify and measure antibiotic resistance genes. Sub-objective 2.2 - Identify alternatives to antibiotics for use as growth promoters in production animals. Sub-objective 2.3 - Determine the effect of calcium hydroxide application to feedlot pens on E. coli O157:H7, total E. coli, and antibiotic resistance on feedlot pen surfaces and in cattle.

The overall goal of this project is to reduce the risk of human foodborne illness, by providing information that can be used to reduce the transmission of zoonotic pathogens and antibiotic resistance from cattle and swine production to food, water, and the environment. Primary targets of the work include Escherichia coli O157:H7 and other Shiga-toxigenic E. coli (STEC), Salmonella, Campylobacter, and antibiotic resistant bacteria in cattle and swine. Approaches for reducing these organisms include reduction of colonization and shedding by livestock, as well as reduction of pathogens and antibiotic resistant bacteria present in the manure and production environment. Persistent shedding, shedding in high numbers, and environmental persistence have been identified as important reasons for the prevalence and maintenance of zoonotic pathogens in livestock, and therefore are intervention targets to reduce these organisms. This project will focus on determining microbial, host, and environmental factors that contribute to these persistence mechanisms, and identify potential strategies for reduction of persistence. Furthermore, this project will develop molecular approaches to assess antibiotic resistance genes in cattle and swine production, and utilize these strategies to evaluate their distribution and abundance. In addition, understanding the potential sources and transmission dynamics of pathogens in livestock production environments is critical for identifying management strategies to reduce their introduction and dissemination. Expected outcomes are scientific information and procedures that will be used to reduce or eliminate foodborne pathogens and antibiotic resistance in livestock production, thus contributing to a safer food and water supply and a lower risk of human foodborne illness.

Progress Report
Reducing pathogens in cattle, swine, and their production environments will require the identification of host and environmental factors that are associated with their occurrence, levels, or persistence (Objective 1). In previous work, high-throughput sequencing of bovine colonic bacterial populations and bioinformatic analyses of the data identified bacteria that are associated with E. coli O157:H7 colonization and shedding by cattle. In ongoing work, host genomic associations with E. coli O157:H7 colonization and shedding are being determined. In addition, deeper sequencing into the fecal microbiome is ongoing to determine what associations can be made with colonic microbiota at lower thresholds. In similar studies with finishing pigs, sequencing of the DNA for determinations of swine colonic bacterial populations and antibiotic resistance is ongoing, and associations with swine colonic bacteria and pathogen colonization and shedding will be determined. In addition, sow immune function and transcriptomics assays to determine associations with piglet pathogen shedding have been completed. Pathogen characterization and confirmation have been completed for studies with nursery piglets to determine the potential for synergistic effects of a Lactobacillus acidophilus fermentation product and lysozyme to reduce the shedding of pathogens and antibiotic resistance. Also in Objective 1, studies were continued to identify factors that affect the occurrence and transmission dynamics of pathogens and antibiotic resistance in cattle and waterways in pasture-based cattle production. Potential factors include wildlife, migratory waterfowl, and environmental and seasonal effectors. Research to determine the immediate and long-term effects of flash grazing of cattle on the microbiological water quality of a riparian stream was completed. Reducing pathogen and antibiotic resistance persistence and transmission from cattle and swine will require the development of intervention strategies that reduce their prevalence and persistence in manure and the production environment (Objective 2). Likewise, novel approaches are needed to assay those resistance genes that are of concern to both animal and human health. In 2020, research was continued to develop a high-throughput sequence-based assay for detecting and quantifying the broad spectrum of bacterial antibiotic resistance genes. Also under Objective 2, research with scientists from the University of Nebraska-Lincoln was continued to determine the potential role for dietary and metaphylactic use of antibiotics to impact the shedding of antibiotic resistant bacteria and genes in feces and the persistence of these antibiotic resistance reservoirs in the environment, by using cattle fed conventionally and cattle never exposed to antibiotics. The background environmental populations were determined and monitored over the course of the two-year study. Results indicate that cattle never exposed to antibiotics shed antibiotic-resistant bacteria in the feces, and moreover, appear to shed a higher prevalence for some resistant bacteria than did cattle given antibiotics. Furthermore, these bacteria were also at a higher prevalence in the pen environment. The use of dietary antibiotics appeared to increase the shedding and environmental levels of antibiotic-resistant Enterococcus, whereas metaphylactic treatments using therapeutic levels of antibiotics appeared to increase antibiotic-resistant E. coli in feces and the environment. Samples are currently being analyzed for metagenomes, viromes, and plasmidomes, to determine how treatments altered these reservoirs for genes carrying antibiotic resistance. Fusobacterium is a bacterial pathogen that causes liver abscesses in cattle, and reduction of this microorganism is the main reason for feeding the macrolide antibiotic tylosin to beef cattle. Developing alternative antimicrobials targeted to control Fusobacterium in the rumen of cattle is needed to reduce dietary antibiotic use and the potential for antibiotic resistance development. Under Objective 2, studies with collaborators at the University of Nebraska were completed to determine if a direct fed microbial known to inhibit Fusobacterium will reduce the incidence of liver abscesses in cattle. In addition, collaborators in New Zealand have identified potential alternatives to antibiotics to reduce Fusobacterium in the rumen, and studies are being planned to further understand the mechanisms and develop animal studies to determine the efficacy of these compounds in the diet. Rumen microbiomes and rumen tissue transcriptomics from cattle with and without liver abscesses have been completed and associations with specific bacteria and gene transcription have been determined. Also under Objective 2, ARS researchers at Clay Center, Nebraska, completed collaborative studies with scientists at West Texas A&M University to determine the levels and prevalence of antibiotic resistant Escherichia coli and Salmonella in high-risk cattle. Cattle at high-risk for bovine respiratory disease are metaphylactically treated upon arrival to the feedlot to reduce the incidence of disease, but are also subject to subsequent therapeutic antibiotic treatments. In this work, dietary supplements were being evaluated to improve animal health, and a commercial product was identified that reduced Salmonella colonization of high-risk cattle following arrival at the feedlot. Studies will be initiated to determine if horizontal gene transfer occurs between multidrug resistant E. coli and Salmonella within these cattle. Follow-up research is being planned with collaborators at Texas Tech University and University of Nebraska-Lincoln to further study how metaphylatic use of antibiotics might impact pathogens and antibiotic-resistant bacteria.

1. Identification of E. coli O157:H7 colonization differences between low-shedding and super-shedding cattle. The bovine gastrointestinal tract is a source of the foodborne pathogen E. coli O157:H7, and cattle that shed this pathogen at high levels in their feces (super-shedders) make the greatest contribution to its dispersal in the production environment, increased transmission to other cattle, and subsequent contamination of beef. The factors that drive colonization and super-shedding are not well understood. ARS scientists at Clay Center, Nebraska, determined that low-shedders and super-shedders had similar levels of E. coli O157:H7 in samples collected from the mouth, but animals that were low-shedders had lower levels of E. coli O157:H7 in samples throughout the subsequent locations in the gastrointestinal tract, compared to animals that were super-shedders. These results suggested that low-shedding animals might exhibit a natural capacity to minimize E. coli O157:H7 occurrence, whereas super-shedders appear to allow greater colonization of this pathogen throughout the lower intestinal tract. This knowledge will facilitate identification of the specific factors that hinder E. coli O157:H7 colonization and should lead to strategies that producers could exploit to reduce this pathogen in cattle and beef, thereby reducing the risk of human foodborne illness.

Review Publications
Hansen, S., Messer, T., Mittelstet, A., Berry, E.D., Bartelt-Hunt, S., Abimbola, O. 2020. Escherichia coli concentrations in waters of a reservoir system impacted by cattle and migratory waterfowl. Science of the Total Environment. 705:135607.
Freetly, H.C., Dickey, A., Lindholm-Perry, A.K., Thallman, R.M., Keele, J.W., Foote, A.P., Wells, J.E. 2020. Digestive tract microbiota of beef cattle that differed in feed efficiency. Journal of Animal Science. 98(2):1-16.
Naderi Beni, N., Snow, D.D., Berry, E.D., Mittelstet, A.R., Messer, T.L., Bartelt-Hunt, S. 2020. Measuring the occurrence of antibiotics in surface water adjacent to cattle grazing areas using passive samplers. Science of the Total Environment. 726:138296.
Abimbola, O.P., Mittelstet, A.R., Messer, T.L., Berry, E.D., Bartelt-Hunt, S.L., Hansen, S.P. 2020. Predicting Escherichia coli loads in cascading dams with machine learning: An integration of hydrometeorology, animal density and grazing pattern. Science of the Total Environment. 722:137894.