Research Project: Prevention and Mitigation of Pathogen Transmission from Cattle and Swine to Food, Water, and Environment

Location: Meat Safety and Quality

2024 Annual Report

Objectives
Objective 1: Determine ecological and environmental factors associated with the levels and persistence of pathogens and antibiotic resistance in the host animal and transmission in the livestock production environment. Sub-objective 1.A: Determining sources and transmission of pathogens and antibiotic resistance in preharvest beef production environments. Sub-objective 1.B: Determining the development of AMR in commensal and pathogenic bacteria and their transmission in feedlot cattle and production systems. Sub-objective 1.C: Determining the impact of intestinal microbiome development on the longitudinal colonization and shedding of foodborne pathogens and antibiotic resistance in swine. Objective 2: Develop and evaluate intervention strategies that reduce or eliminate the occurrence, transmission, or persistence of foodborne pathogens in cattle, swine, their production systems, and the environment. Sub-objective 2.A: Evaluating the potential for dietary supplements to reduce pathogen and antibiotic resistance shedding in beef cattle feces and into the environment. Sub-objective 2.B: Identification of alternatives to antibiotics for use in nursery swine to reduce pathogens and AMR bacteria.

Approach
The overall goal of this project is to reduce the risk of foodborne illness, by providing information that can be used to reduce transmission of zoonotic pathogens and antibiotic resistance from cattle and swine production to food, water, and the environment. Cattle and swine remain important reservoirs for foodborne pathogen and antibiotic resistance, increasing the potential for transmission of foodborne pathogens to humans. Primary targets of the work include Escherichia coli O157:H7 and other Shiga-toxigenic E. coli, Salmonella, Campylobacter, and antibiotic resistant bacteria. Specific objectives are to (1) Determine ecological and environmental factors associated with the levels and persistence of pathogens and antibiotic resistance in the host animal and transmission in livestock production environments; and (2) Develop and evaluate intervention strategies that reduce or eliminate the occurrence, transmission, or persistence of foodborne pathogens in cattle, swine, their production systems, and the environment. Understanding the potential sources and transmission dynamics of pathogens in production environments is critical for identifying strategies to reduce their introduction and dissemination. Systems approaches examining multiple pathways and sample types will be used to identify the most important sources and transmission routes of pathogens and antibiotic resistance, using the U.S. Meat Animal Research Center (USMARC) preharvest beef and swine production environments. Moreover, research will determine how antibiotic use in cattle and swine affects pathogens and the development of antibiotic resistance in animals and their production environment. Research will also be conducted to identify alternatives to antibiotic use in cattle and swine. Expected outcomes are scientific information and management strategies that can be used to reduce 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. These outcomes will benefit U.S. agriculture and numerous stakeholders, including livestock producers, animal harvest and meat processing industries, regulatory agencies, and consumers.

Progress Report
Under Objectives 1 and 2, understanding the impact of antibiotic use in swine and cattle production is important to determining the potential for the development of antibiotic resistance in commensal and pathogenic bacteria. Under Sub-objective 1A, sampling and analysis in a long-term study was continued to identify factors that affect the occurrence and transmission dynamics of multiple pathogens and antibiotic resistance in cattle and waterways in pasture-based cattle production. Factors under study include wildlife, migratory waterfowl, and other environmental and seasonal effectors. Water, sediment, and feces were collected and analyzed, and weather and camera data were recorded. Preliminary analyses indicate that pathogen presence in the waterway might be impacted by season. Campylobacter were rarely detected in summer months and Salmonella were rarely detected in the winter months. Salmonella were regularly detected in feces from wildlife. Escherichia coli (E.coli) were always present but enumerations are highest in the summer. Although pastured cattle were in close proximity to the waterway throughout the year, E. coli O157:H7 were rarely detected. To better understand the spatial and temporal fluctuations of pathogens in the waterway samples, weekly sampling of the surface water was implemented. ARS scientists in Clay Center, Nebraska, were also participating in a multi-agency effort to determine bacterial antibiotic resistance and pathogen load in samples of surface water. Surface water was determined to be a suitable proxy for environmental loads of antibiotic resistance and pathogens. Different methods for detecting and isolating Salmonella and E. coli were evaluated. A modified filter-based method followed by selective enrichment was determined to provide the best recovery for Salmonella in 1-liter of surface water, and the method is currently being used to collect preliminary data for Salmonella distribution in the surface water of a mixed-use watershed. Additional studies are being completed to evaluate different methods for the enumeration and isolation of total E. coli and antibiotic resistant E. coli in surface waters. Results from these later studies will provide guidance for future research efforts studying E. coli in surface water. Feedlot cattle are treated with antibiotics at arrival to the feedlot to reduce the incidence of bovine respiratory illnesses, particularly with high-risk cattle that have been directly weaned and sold at sale barns. Under Sub-objective 1B, research with high-risk cattle was continued with collaborators at Texas Tech University (TTU), and University of Nebraska-Lincoln, to study how metaphylactic use of antibiotics might impact pathogens and antimicrobial-resistant (AMR) bacteria. A study with sale barn sourced cattle was completed at the TTU Burnett Center. The cattle were treated with saline, or one of three antibiotics commonly used for metaphylactic treatment in feedlot cattle, and fecal samples were collected over time and analyzed for pathogenic and AMR bacteria. Bacterial isolates were tested for antimicrobial susceptibilities according to National Antimicrobial Resistance Monitoring System (NARMS) protocols. The use of antibiotics on cattle at feedlot arrival had significant impact on the levels of some antimicrobial resistant bacteria in the feces early in the production phase but these levels transiently decreased after the first month. Many of the antimicrobial resistant bacteria increased after the second month in the feedlot and were at the highest levels at harvest. Salmonella in feces was also monitored over time and antibiotic treatment at arrival had a significant impact on levels and prevalence in the feces up to harvest. However, antibiotic use in feedlot cattle did not appear to increase multi-antibiotic resistant Salmonella. ARS scientists at location worked in collaboration with collaborators at West Texas A&M University, Texas Tech University (TTU), and USDA/ARS Lubbock, Texas, to characterize Salmonella isolates from the beef food chain continuum. Samples of feces were collected prior to harvest and samples of blood, liver, spleen, intestines, and peripheral lymph nodes were collected at harvest and tested for Salmonella. Salmonella was isolated, confirmed, and characterized for sensitivity to antimicrobials. Pure cultures of the Salmonella isolates were processed to purify DNA and whole genome sequenced. Preliminary results indicate that Salmonella isolates from internal tissues and fecal samples were genetically similar. For Sub-objective 2A, the environment was evaluated using samples of feed and of the feedlot surface material collected prior to and for 56 days after animals were placed into the pens. Antibiotic treatment appeared to impact the onset of bovine respiratory disease and the shedding of Salmonella and AMR Escherichia coli (E. coli) in the feces. Multidrug resistant Salmonella were routinely recovered from feedlot surface samples but were never recovered from bovine feces. Follow-up work will also determine if horizontal gene transfer occurs between multidrug resistant E. coli and Salmonella within the feedlot environment. An additional study related to this objective was completed in collaboration with scientists at Texas Tech University, South Dakota State University, and USDA-ARS Lubbock, Texas, to determine the impact of geographic location in the high plains on antimicrobial resistance and pathogen shedding in feces. Geographic location can impact the fecal shedding of antimicrobial resistant and pathogenic bacterial populations, and relocation of animals to regions of low incidence can significantly reduce fecal shedding of antimicrobial resistant and pathogenic bacteria. Reducing pathogen and AMR bacteria persistence and transmission from cattle and swine will require the development of intervention strategies that reduce their prevalence in the animal, the manure, and the production environment (Objective 2). Under Sub-objective 2A, one study was completed and a second study was continued with feedlot cattle to determine the impact of dietary antimicrobials on fecal shedding of AMR E. coli and Enterococcus spp. Fecal and environmental samples were collected prior to and after the supplementation of dietary antimicrobials. Under Sub-objective 2B, research with a potential fungal probiotic was completed at Clay Center, Nebraska, in collaboration with ARS scientists from Beltsville, Maryland. Piglets were orally treated with saline or the probiotic fungi on days 10 and 24 of age. Fecal samples were collected at weaning and at the end of the nursery phase to determine pathogen and AMR presence. Piglets were euthanized at weaning and end of the nursery phase to determine the distribution of pathogens and AMR bacteria throughout the gastrointestinal tract. Interestingly, AMR bacteria were observed at high levels in piglets regardless of age and the cecum appears to be a reservoir for AMR in the young piglet. Additional research was completed to determine transcriptomics of milk in nursing sows, and numerous genes associated with pathogen recognition and immune response were observed to be differentially expressed in sows of different age and milk type. Efforts are continuing to identify milk oligosaccharide compositions and determine relationships with pathogen colonization and shedding in piglets.

Accomplishments
1. Antimicrobial susceptibilities of antimicrobial resistant Escherichia (E. coli) from cattle. Antimicrobial use in feedlot cattle is a concern for development of antibiotic resistant bacteria. Antimicrobial resistant non-pathogenic Escherichia coli were isolated throughout the production cycle from feces of feedlot cattle treated with saline or one of three different antimicrobials at arrival. The antimicrobial resistant E. coli were tested by ARS researchers at Clay Center, Nebraska, for susceptibility to fourteen different antimicrobials. Across all treatments, E. coli exhibited increased multidrug resistance for up to two months after feedlot arrival but showed decreased multidrug resistance thereafter. Antimicrobial use at feedlots arrival can result in transient increases in antimicrobial resistance in E. coli but there was little impact on antimicrobial resistance in the fecal E. coli populations at harvest.

2. Regional effects on antimicrobial resistant and pathogenic bacteria from cattle. The United States Plains is the major cattle producing region in the U.S. and Salmonella are consistently observed more often in cattle from the southern plains when compared with the northern plains. ARS scientists in Clay Center, Nebraska, collaborated with ARS scientists in Lubbock, Texas, and colleagues at Texas Tech University, to determine if geographic location within the U.S. Plains impacted the fecal shedding of pathogenic or antimicrobial resistant bacterial populations. Cattle from the southern or northern plains were either kept at that location or shipped to the other location. Initially, cattle in the northern plains had lower fecal shedding of pathogenic and antimicrobial resistant bacteria, whereas cattle in the southern plains had higher shedding. Translocation of cattle to the opposite Plains locations resulted in shifts in the fecal shedding corresponding to that location. Finishing feedlot cattle in areas where pathogens are low, regardless of their original source location could result in lower fecal shedding of pathogenic and antimicrobial resistant bacteria at harvest.

3. Salmonella in surface water. Antibiotic-resistant Salmonella is a pathogen of critical concern in multiple food commodities. One route of contamination of antibiotic-resistant Salmonella onto fruits and vegetables is through agricultural or irrigation water. The National Antimicrobial Resistance Monitoring and Surveillance (NARMS), a collaboration between the Centers for Disease Control (CDC), Food and Drug Administration (FDA), and USDA-ARS, has developed consensus microbiological methods for the recovery of several antibiotic resistant pathogens from clinical, animal and food samples. Currently there is not a standardized method used to recover Salmonella from surface water or agricultural irrigation water. ARS scientists in California, Georgia, Maryland, and Nebraska, collaborated to modify a standard method that consistently recovered low levels of Salmonella from water samples at frequencies equivalent to a gold standard method. The modified standard method identified is now being used in Environmental Protection Agency National Rivers and Streams Assessment testing. This research benefits the NARMS program and its federal partners (FDA, CDC) as the monitoring of antibiotic resistant Salmonella is expanded in diverse waterways by providing a standard method capable of recovering the low levels of Salmonella normally present in surface and agricultural irrigation waters.

4. Quantifying antibiotic resistant E. coli in the environment. Some bacteria carry genes that can inactivate important medicines, resulting in untreatable infections from illness, injuries, or from procedures like heart surgeries or joint replacements. This means people are sick longer, or may even die, because the medicine that used to be effective against the infection has become drug resistant. These drug-resistance genes are easily shared among bacteria, and there is concern that they can be transferred to humans via water, soil, and air. Measuring these genes in the environment is difficult, time consuming, and expensive. ARS scientists in Clay Center, Nebraska, partnered with other federal agencies to develop and evaluate methods to detect and count Escherichia coli in water that carry genes for a particularly dangerous type of drug resistance, called extended spectrum-beta-lactamase resistance. The effort included a comparison with a global standard method, a modification of an Environmental Protection Agency standard method, and a modification of a widely used commercially available water-quality test. The results provided an effective method needed by the National Antibiotic Resistance Monitoring System (NARMS) for a new national survey of drug-resistant bacteria in surface waters.

Review Publications
Henniger, M.T., Rowan, T.N., Beever, J.E., Mulon, P., Smith, J.S., Voy, B.H., Wells, J.E., Kuehn, L.A., Myer, P.R. 2023. Validation of a minimally-invasive method for sampling epithelial-associated microorganisms on the rumen wall. Frontiers in Animal Science. 4. Article 1270550. https://doi.org/10.3389/fanim.2023.1270550.
Dornbach, C.W., Hales, K.E., Gubbels, E., Wells, J., Hoffman, A.A., Hanratty, A.N., Line, D.J., Smock, T.M., Manahan, J.L., McDaniel, Z.S., Kohl, K.B., Sanchez, N.C., Carroll, J.A., Warren, R., Smith, Z.K., Broadway, P.R. 2023. Longitudinal assessment of prevalence and incidence of Salmonella and Escherichia coli O157 resistance to antimicrobials in feedlot cattle sourced and finished in different regions of the U.S. Foodborne Pathogens and Disease. 20(8). https://doi.org/10.1089/fpd.2023.0009.
Harlow, K., Summers, K., Oliver, W.T., Wells, J., Ferguson, M., Crouse, M.S., Neville, B.W., Rempel, L.A., Rivera-Colon, I., Ramsay, T.G., Davies, C.L. 2024. Weaning transition, but not the administration of probiotic candidate Kazachstania slooffiae, shaped the gastrointestinal bacterial and fungal communities in nursery piglets. Frontiers in Veterinary Science. 10. Article e1303984. https://doi.org/10.3389/fvets.2023.1303984.
Lindholm-Perry, A.K., Keel, B.N., Hales, K.E., Wells, J.E., Kuehn, L.A., Keele, J.W., Crouse, M.S., Nonneman, D.J., Nagaraja, T.G., Lawrence, T.E., Amachawadi, R.G., Carroll, J.A., Burdick Sanchez, N.C., Broadway, P.R. 2024. Ileal epithelial tissue transcript profiles of steers with experimentally induced liver abscesses. Applied Animal Science. 40(3):414-420. https://doi.org/10.15232/aas.2023-02503.
Mcconn, B.R., Kraft, A.L., Durso, L.M., Ibekwe, A.M., Frye, J.G., Wells, J., Tobey, E.M., Ritchie, S.M., Williams, C.F., Cook, K.L., Sharma, M. 2024. An analysis of culture-based methods used for the detection and isolation of Salmonella spp., Escherichia coli, and Enterococcus spp. from surface water: a systematic review. Science of the Total Environment. 927. Article 172190. https://doi.org/10.1016/j.scitotenv.2024.172190.
Smock, T.M., Broadway, P.R., Sanchez, N.C., Carroll, J.A., Hoffman, A.A., Long, N.S., Manahan, J.L., McDaniel, Z.S., Theurer, M.E., Wells, J., Hales, K.E. 2023. Infrared thermography or rectal temperature as qualification for targeted metaphylaxis in high-risk, newly received beef steers and the effects on growth performance, complete blood count, and serum haptoglovin during a 42-day feedlot receiving period. Applied Animal Science. 39(4):213-226. https://doi.org/10.15232/aas.2022-02370.
Long, N.S., Hales, K.E., Berry, E.D., Legako, J.F., Woerner, D.R., Broadway, P.R., Carroll, J.A., Sanchez, N.C.B., Fernando, S.C., Wells, J.E. 2023. Antimicrobial susceptibility of trimethoprim-sulfamethoxazole and 3rd-generation cephalosporin-resistant Escherichia coli isolates enumerated longitudinally from feedlot arrival to harvest in high-risk beef cattle administered common metaphylactic antimicrobials. Foodborne Pathogens and Disease. 20(7):252-260. https://doi.org/10.1089/fpd.2023.0001.
Franklin, A.M., Weller, D.L., Durso, L.M., Bagley, M., Davis, B.C., Frye, J.G., Grim, C., Ibekwe, A.M., Jahne, M., Keely, S.P., Kraft, A.L., McConn, B.R., Mitchell, R., Ottesen, A., Sharma, M., Strain, E., Tadesse, D., Tate, H., Wells, J., Williams, C.F., Cook, K.L., Kabera, C., McDermott, P., Garland, J. 2024. A one health approach for monitoring antimicrobial resistance: Developing a national freshwater pilot effort. Frontiers in Water. 6. Article 1359109. https://doi.org/10.3389/frwa.2024.1359109.
Chatterjee, A., Anapalli, S.S. 2023. Comparison of cropping system models for simulation of soybean evapotranspiration with eddy covariance measurements in a humid subtropical environment. Water. 15(17). Article 3078. https://doi.org/10.3390/w15173078.