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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

2018 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 genotypic and/or phenotypic factors that are associated with their occurrence, levels, or persistence (Objective 1). Studies were continued to determine genotypic or phenotypic factors associated with persistence of the pathogen E. coli O157:H7 in cattle production. In addition, high-throughput sequencing of bovine colonic bacterial populations and summarizing the data for bioinformatics analyses were continued, with the goal to identify bacteria that are associated with E. coli O157:H7 colonization and shedding. In initial studies with finishing pigs, phenotypes for pathogen shedding were characterized, and DNA was isolated for determinations of swine colonic bacterial populations and antibiotic resistance genes. In addition, studies were initiated 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. Initial data indicate that pathogen shedding from piglets was variable across litters and potentially sow-dependent. Sow immune function will be assessed using transcriptomics, and associated with piglet pathogen shedding. In previous swine research, lysozyme was shown to reduce pathogen shedding and a Lactobacillus acidophilus fermentation product was shown to reduce fecal antibiotic resistance genes. Additional studies with nursery piglets have been initiated to determine the potential for synergistic effects of a Lactobacillus acidophilus fermentation product and lysozyme to reduce shedding of pathogens and antibiotic resistance. In 2018, 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 (e.g., raccoons, migratory waterfowl), insects, and seasonal and climatic effectors. Collaborations with scientists from the USDA APHIS National Wildlife Research Center, the University of Nebraska-Lincoln, and the USDA ARS Agroecosystem Management Unit have been established to expand this research. Collaborative work with engineers and hydrologists from the University of Nebraska includes the (1) hydrological modeling of processes of the USMARC semi-closed watershed-reservoir system, and (2) modeling the watershed-scale fate and transport of E. coli, which are anticipated to provide important tools that can be used to identify sources and manage risks associated with E. coli in agricultural watersheds. A related work was initiated to determine the presence, types, or concentrations of antibiotics in surface water within a cattle grazing area, and a separate study was continued to determine the immediate and long-term effects of flash grazing of cattle on the microbiological water quality of a riparian stream. 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 2018, research was continued to develop a high-throughput sequence-based assay for detecting and quantifying the broad spectrum of bacterial antibiotic resistance genes. A collaboration with scientists at 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 study was conducted in a recently renovated cattle-feeding facility that was cleaned and unused for a period of two years prior to the present study. The background environmental populations were determined and monitored over the course of the study. Initial results indicate that cattle never exposed to antibiotics shed antibiotic resistant bacteria in the feces, and moreover, 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 affected the shedding of resistant bacteria to some extent, but the metaphylactic treatments using therapeutic levels of antibiotics appeared to have a greater impact on fecal bacterial populations and antibiotic resistant bacteria. Samples are 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 2018, studies with collaborators at the University of Nebraska were initiated 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. In addition, studies were completed to determine the potential role for limonene in cattle diets to improve animal performance. Initial results suggest that limonene did not reduce liver abscesses and fecal shedding of Escherichia coli O157:H7. In 2018, collaborative studies were initiated with scientists at West Texas A&M University to determine how therapeutic use of antibiotics would influence fecal shedding of antibiotic resistant Escherichia coli and Salmonella from high-risk cattle. Cattle at high-risk of 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.

1. Bovine ruminal bacteria influence important cattle traits. The ruminant gastrointestinal tract is a complex system with bacterial ecosystems that can impact animal performance, health, and well-being. Despite advancements in sequencing technologies, scientists are just beginning to understand how to identify the relevant bacterial populations and how they interact to affect important cattle traits, particularly in the rumen which is the largest organ in the ruminant gastrointestinal tract. Scientists at Clay Center, Nebraska, in collaboration with scientists at University of Nebraska-Lincoln, used high-throughput DNA sequencing to study the bacterial ecology of the rumen of cattle fed different diets. They found that bacterial populations of the nearly 250 cattle analyzed are more complex and robust than previously reported in smaller studies. Moreover, diet was shown to have a significant impact on the structure and composition of the bacterial microbiota. In addition, previously unknown bacterial populations that directly impact animal performance were identified. This information is a solid foundation for future studies and will be useful to determine the influence of the ruminal microbiota on other traits, including the colonization and persistent shedding of foodborne pathogens such as Escherichia coli O157:H7. Greater understanding of these diverse and complex microbial populations in the bovine rumen is anticipated to lead to strategies to improve animal performance and well-being, and reduce pathogen carriage and shedding, which will benefit the animals, farmers, and consumers.

Review Publications
Wells, J.E., Berry, E.D., Kim, M., Shackelford, S.D., Hales, K.E. 2017. Evaluation of commercial ß-agonists, dietary protein, and shade on fecal shedding of Escherichia coli O157:H7 from feedlot cattle. Foodborne Pathogens and Disease. 14(11):649-655.
Kim, M., Kuehn, L.A., Bono, J.L., Berry, E.D., Kalchayanand, N., Freetly, H.C., Benson, A.K., Wells, J. 2017. The impact of the bovine faecal microbiome on Escherichia coli O157:H7 prevalence and enumeration in naturally infected cattle. Journal of Applied Microbiology. 123:1027-1042.
Spiehs, M.J., Berry, E.D., Wells, J., Parker, D.B., Brown-Brandl, T.M. 2017. Odorous volatile organic compounds, Escherichia coli, and nutrient concentrations when kiln-dried pine chips and corn stover bedding are used in beef bedded manure packs. Journal of Environmental Quality. 46(4):722-732.
Berry, E.D., Wells, J.E., Varel, V.H., Hales, K.E., Kalchayanand, N. 2017. Persistence of Escherichia coli O157:H7 and total Escherichia coli in feces and feedlot surface manure from cattle fed diets with or without corn or sorghum wet distillers grains with solubles. Journal of Food Protection. 80(8):1317-1327.
Paz, H.A., Hales Paxton, K.E., Wells, J., Kuehn, L.A., Freetly, H.C., Berry, E.D., Flythe, M.D., Spangler, M.L., Fernando, S. 2018. Rumen bacterial community structure impacts feed efficiency in beef cattle. Journal of Animal Science. 96(3):1045-1058.
Melchior, E.A., Hales, K.E., Lindholm-Perry, A.K., Freetly, H.C., Wells, J.E, Hemphill, C.A., Wickersham, T.A., Sawyer, J.E., Myer, P.R. 2018. The effects of feeding monensin on rumen microbial communities and methanogenesis in bred heifers fed in a drylot. Livestock Science. 212:131-136.
Hales, K.E., Wells, J., Berry, E.D., Kalchayanand, N., Bono, J.L., Kim, M.S. 2017. The effects of monensin in diets fed to finishing beef steers and heifers on growth performance and fecal shedding of Escherichia coli O157:H7. Journal of Animal Science. 95(8):3738-3744.