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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

2021 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
This is the final report for Project 3040-32000-032-00D, which expired in 2021 and was replaced with new Project 3040-32000-035-00D. Additional information can be found in the Annual Report for Project 3040-32000-035-00D. Substantial progress was made over the life of this project to identify factors that affect pathogen and antibiotic resistance occurrence and survival in both the host animal and in cattle and swine production environments (Objective 1). This and other information was used in the development of strategies to reduce the occurrence and transmission of foodborne pathogens and antibiotic resistance in these livestock and their production environments (Objective 2). In previous work, high-throughput sequencing of bovine colonic bacterial populations identified bacteria that are associated with Escherichia coli O157:H7 (E. coli O157:H7) in cattle. In work supporting Sub-objective 1.1, host genomic associations with E. coli O157:H7 colonization and shedding were determined and currently are being validated. Also in Sub-objective 1.1, shedding of E. coli O157:H7 in the gastrointestinal tracts of 40 cattle was characterized in detail. 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. In particular, the rumen appears to be a barrier to E. coli O157:H7 passage. In collaboration with scientists at University of Nebraska-Lincoln, high-throughput DNA sequencing was used to study the bacterial ecology of the bovine rumen. Diet was shown to have a significant impact on the structure and composition of the ruminal bacterial microbiota. This information will be useful to determine the influence of the ruminal microbiota on other traits, including E. coli O157:H7 shedding. Fusobacterium necrophorum is a primary cause of liver abscesses in beef cattle. In support of Sub-objective 1.2, collaborative research with scientists at the University of Nebraska-Lincoln identified differentially expressed genes in cattle with and without liver abscesses, that are potential candidates for genetic selection. In addition, differences in bacterial populations in rumen epithelial-attached bacteria were identified, which are potential candidates for the development of probiotics to reduce liver abscesses. In swine, research efforts have targeted preweaned and nursery piglets and finishing pigs. Fecal shedding of pathogens and antibiotic resistance has been characterized, and associations with the fecal microbiota and host genotypes are being evaluated. In preweaned piglets, milk transcriptomics of the sow have been determined. Reduction in pathogen colonization after parturition would have long-term benefits in the swine production environment. Under Sub-objective 1.3, a long-term study was conducted to identify factors that affect the occurrence and transmission dynamics of pathogens and antibiotic resistance in cattle and waterways in pasture-based cattle production. This study will be continued under the new Project 3040-32000-035-00D. Factors under study include wildlife, migratory waterfowl, and other environmental and seasonal effectors. A number of closely related experiments were completed that support this long-term research, including a study that determined the effects of management-intensive grazing of cattle on the water quality of a riparian stream. Additional related experiments included collaborative research with engineers from the University of Nebraska-Lincoln: (1) the development of predictive hydrological models for the concentrations of E. coli in water in a semi-closed agricultural watershed-reservoir system; (2) the determination of trends in E. coli concentrations in water associated with cattle grazing rotations and the presence of migratory waterfowl; (3) the development of a simulation in the watershed-scale Soil and Water Assessment Tool (SWAT) to assess the effectiveness of proposed best management practices to reduce E. coli, nitrogen, phosphorus, and atrazine at the watershed outlet; and (4) a study examining the presence and concentrations of antibiotics in a stream that flows through a cattle grazing area. These studies provide important tools that can be used to identify sources and manage risks associated with antibiotic resistance and E. coli in agricultural watersheds. Also under Sub-objective 1.3, we 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 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. In support of Sub-objective 2.1 to develop a high-throughput sequence-based assay for detecting and quantifying antibiotic resistance genes, databases of antibiotic resistance genes were compiled and binned based upon sequence relatedness. Groups of related genes were aligned based on sequence, and redundant primers were designed to specifically target each gene group. Gene assays are being validated for specificity. Under Sub-objective 2.2, studies were completed comparing conventionally raised cattle to cattle that have never been treated with antibiotics, to discern the contribution of conventional feedlot practices to the development, transmission, and persistence of antibiotic resistance. A collaboration with scientists at the University of Nebraska-Lincoln determined the potential role for 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. Results indicate that cattle never exposed to antibiotics shed antibiotic resistant bacteria in the feces and shed a higher prevalence for some resistant bacteria than did cattle given antibiotics. 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 had a greater impact on fecal bacterial populations and antibiotic resistant bacteria. Also under Sub-objective 2.2, collaborative studies with scientists at West Texas A&M University were completed 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 were metaphylactically treated upon arrival to the feedlot to reduce the incidence of disease but were also subjected to subsequent therapeutic antibiotic treatments. As part of this work, a commercial Bacillus probiotic was identified that reduced Salmonella colonization of cattle following arrival at the feedlot. A separate project examined the use of limonene as a potential alternative to tylosin, by testing its ability to reduce liver abscesses and E. coli O157:H7. Contrary to previous reports, limonene feeding did not reduce either liver abscesses or the shedding of E. coli O157:H7. Additional studies with collaborators at the University of Nebraska were completed to determine if direct fed microbials known to inhibit Fusobacterium will reduce the incidence of liver abscesses in cattle. We did not see any significant impact of feeding these direct fed microbials on the incidence of liver abscesses in cattle, however, subsequent work demonstrated that the direct fed microbials were antagonistic to each other, so future work will examine the direct fed microbials separately and in the absence of dietary monensin. Understanding how dietary supplements impact the shedding of E. coli O157:H7 by cattle is important. Dietary monensin did not appear to impact fecal presence or absence for E. coli O157:H7 in cattle. Dietary protein does not appear to be a factor in fecal shedding of E. coli O157:H7, but supplementation with soybean meal may be beneficial to reducing fecal shedding of this pathogen by cattle. Beta-agonists are fed to cattle and previous research had proposed that these compounds reduced the fecal shedding of E. coli O157:H7. No significant effect of feeding either of the beta-agonists zilpaterol or ractopamine on E. coli O157:H7 fecal shedding was observed. In swine, a Lactobacillus fermentation extract was evaluated as an alternative to antibiotic growth promotants. In preliminary studies, feeding the fermentation product had little impact on pathogen shedding, but was shown to reduce the prevalence of fecal antibiotic resistance genes. Additional studies with nursery piglets determined the potential for synergistic effects of the Lactobacillus fermentation product and lysozyme. Pathogen prevalence was lower for both the Lactobacillus product and lysozyme when supplemented individually. The combination of the two treatments also reduced pathogen shedding, but animal growth was significantly lower. In support of Sub-objective 2.3, we conducted studies to determine if the use of hydrated calcium hydroxide (lime) can be used to reduce pathogens and antibiotic resistant bacteria on feedlot pen surfaces. Calcium hydroxide did not effectively reduce E. coli O157:H7, total E. coli, and antibiotic resistance on feedlot pen surfaces.

1. Probiotic feeding reduces Salmonella in beef cattle. Feedlot cattle that are purchased from auction barns are often at high risk for disease and pathogens due to stresses from weaning, transportation, and handling, all of which can affect the immune function of the calf. As a preventative measure, antimicrobials have been used as mass treatments to reduce disease, especially bovine respiratory disease, when these high-risk cattle arrive at the feedlot. Scientists at Clay Center, Nebraska, in collaboration with scientists at West Texas A&M University, examined the use of commercial alternatives to antibiotics for mass treatment of high-risk animals upon arrival at the feedlot and determined their effects on various health parameters and Salmonella shedding. Chromium propionate treatments improved some white blood cell and immune function parameters but had no effect on Salmonella levels in cattle feces. Cattle treated with a Bacillus probiotic did not exhibit improvements in white blood cell parameters, however these animals shed reduced levels of Salmonella following treatment. This is the first report of an effective treatment to reduce Salmonella shedding in feedlot cattle and identifies a potential dietary approach for reducing the risk of human foodborne illness associated with beef consumption.

2. Reducing liver abscesses in feedlot cattle. Liver abscesses in cattle are due to bacterial infection and result in liver condemnation at an estimated cost of $64 million annually to the beef industry. The bovine rumen is believed to be the primary source of Fusobacterium necrophorum that causes these infections, and antibiotics are commonly fed to feedlot cattle to control this bacterium. Scientists at Clay Center, Nebraska, in collaboration with scientists at the University of Nebraska-Lincoln, identified genes in the rumen and specific ruminal bacteria that were associated with the presence or absence of severe liver abscesses in feedlot cattle. Over 200 genes were differentially expressed between animals with and without liver abscesses, and these genes could be useful to develop markers to identify animals that are more susceptible to liver abscesses. The community structure of the overall rumen microbiota was not associated with liver abscesses, but differences were identified amongst the bacterial communities attached to the rumen. This study identified new mechanisms in the rumen that may be contributing to liver abscesses, which may lead to alternative treatments and reduction of the use of antibiotics to control liver abscesses in feedlot cattle.

Review Publications
Wells, J., Berry, E.D., Kim, M., Bono, J.L., Oliver, W.T., Kalchayanand, N., Wang, R., Freetly, H.C., Means, W.J. 2020. Determination of gastrointestinal tract colonization sites from feedlot cattle transiently shedding or super-shedding Escherichia coli O157:H7 at harvest. Journal of Applied Microbiology. 129:1419-1426.
Abimbola, O., Mittelstet, A., Messer, T., Berry, E., van Griensvan, A. 2020. Modeling and prioritizing interventions using pollution hotspots for reducing nutrients, atrazine and E. coli concentrations in a watershed. Sustainability. 13:103.
Smock, T.M., Samuelson, K.L., Wells, J.E., Hales, K.E., Hergenreder, J.E., Rounds, P.W., Richeson, J.T. 2020. Effects of Bacillus subtilis PB6 and/or chromium propionate supplementation on serum chemistry, complete blood count, and fecal Salmonella spp. count in high-risk cattle during the feedlot receiving and finishing periods. Translational Animal Science. 4(3):1-11.
Abbas, W., Keel, B.N., Kachman, S.D., Fernando, S.C., Wells, J.E., Hales, K.E., Lindholm-Perry, A.K. 2020. Rumen epithelial transcriptome and microbiome profiles of rumen epithelium and contents of beef cattle with and without liver abscesses. Journal of Animal Science. 98(12):1-13.
Abbas, W., Howard, J.T., Paz, H.A., Hales, K.E., Wells, J., Kuehn, L.A., Erickson, G.E., Spangler, M.L., Fernando, S.C. 2020. Influence of host genetics in shaping the rumen bacterial community in beef cattle. Scientific Reports. 10. Article 15101.