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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

2019 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 livestock and their production environment will require the identification of host and environmental factors that are associated with their occurrence, levels, or persistence (Objective 1). Studies were continued to determine genotypic or phenotypic factors that are associated with persistence of the pathogen E. coli O157:H7 in cattle production. In addition, high-throughput sequencing of bovine colonic bacterial populations and bioinformatics analyses of the data were completed, and bacteria that associated with E. coli O157:H7 colonization and shedding have been identified and reported. Host genomic associations with E. coli O157:H7 colonization and shedding are being determined. In studies with finishing pigs, phenotypes for pathogen shedding were characterized, and sequencing of the DNA for determinations of swine colonic bacterial populations and antibiotic resistance genes has been initiated. In addition, studies were completed to determine the potential role for oligosaccharides in swine milk to reduce pathogen colonization and shedding in feces of piglets after farrowing and prior to the nursery. Oligosaccharides in milk have been shown to result in the selection of beneficial microorganisms in the gastrointestinal tract, and reduction in pathogen colonization after parturition would have long-term benefits in the swine production environment. Sow immune function and transcriptomics assays are in progress to determine associations with piglet pathogen shedding. Additional studies with nursery piglets have been completed to determine the potential for synergistic effects of a Lactobacillus acidophilus fermentation product and lysozyme to reduce shedding of pathogens and antibiotic resistance. Pathogen characterization and confirmation have been initiated for samples from these studies. In 2019, 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. In addition, research to determine the immediate and long-term effects of flash grazing of cattle on the microbiological water quality of a riparian stream was continued. Several collaborative studies with engineers and hydrologists from the University of Nebraska were completed, including: (1) a study examining the presence, types, and concentrations of antibiotics in surface water within a cattle grazing area; (2) the hydrological modeling of the U.S. Meat Animal Reserach Center (USMARC) semi-closed watershed-reservoir system; and (3) modeling the watershed-scale fate and transport of E. coli in the USMARC semi-closed watershed-reservoir system. These works are anticipated to provide important tools that can be used to identify sources and manage risks associated with agricultural antibiotics and E. coli in agricultural watersheds. 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 2019, research was continued to develop a high-throughput sequence-based assay for detecting and quantifying the broad spectrum of bacterial antibiotic resistance genes. 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 viromes and plasmids, to determine how treatments altered these reservoirs for genes carrying antibiotic resistance. Fusobacterium is a bacterial pathogen that causes liver abscesses, 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. In 2019, studies with collaborators at the University of Nebraska were continued 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 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 analyses to determine relationships to liver abscess development are currently underway. In 2019, we continued 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. Little is known about the potential risk of using metaphylactic and therapeutic antibiotics on the development of antibiotic resistant Salmonella in cattle. Studies are being planned to determine if horizontal gene transfer occurs between multidrug resistant E. coli and Salmonella among these cattle.

1. E. coli O157:H7 transmission by cattle pest flies found in leafy greens. Leafy greens are a leading source of Escherichia coli O157:H7 causing human foodborne illness. Pest flies can carry this pathogen, and may transmit it to leafy greens and other fresh produce. ARS scientists at Clay Center, Nebraska, determined the occurrence of E. coli O157:H7-positive flies in leafy greens planted up to 600 feet from a cattle feedlot, and assessed their potential risk to transmit this pathogen to leafy greens. E. coli O157:H7 carriage rates of house, face, flesh, and blow flies were similar to each other and were greater than the carriage rate of stable flies. E. coli O157:H7 carriage rates were not different in flies found at different distances from the feedlot, ranging from 0 to 600 feet. Genetic subtyping showed that the majority of the E. coli O157:H7 found in the flies were of the same predominant subtypes found in the feedlot pen surface manure and the leafy greens, indicating the potential role for flies to transmit E. coli O157:H7 to the leafy greens. Due in part to this work and previous research conducted by ARS scientists at Clay Center, Nebraska, the produce industry has revised their guidelines for growers to increase the set-back distance between leafy greens fields and concentrated animal feeding operations. This information is critical for understanding the food safety risks associated with growing leafy greens in close proximity to cattle production, and for determining safe distances between cattle feedlots and fresh produce that will reduce preharvest contamination and protect public health.

Review Publications
Berry, E.D., Wells, J.E., Durso, L.M., Friesen, K.M., Bono, J.L., Suslow, T.V. 2019. Occurrence of Escherichia coli O157:H7 in pest flies captured in leafy greens plots grown near a beef cattle feedlot. Journal of Food Protection. 82(8):1300-1307.