Research Project: Ecological Reservoirs and Intervention Strategies to Reduce Foodborne Pathogens in Cattle and Swine

Location: Food and Feed Safety Research

2021 Annual Report

Objectives
Objective 1: Identify the ecological niches or reservoirs for pathogenic and antimicrobial resistant foodborne bacteria and determine nutritional, immunological, biological and environmental factors impacting their ability to colonize, survive, and persist in the gut and environment of food producing animals using metagenomic and molecular characterization of competitiveness, resistance and virulence. 1.A: Determine the effect of dietary components, feedstuffs, phytochemical extracts, and organic acids on the intestinal microbiome and functional genomics of the gut, and the impact of these changes on enterohemorrhagic E. coli and Salmonella. 1.B: Characterize the effects of short chain nitrocompounds on hydrogen ecology, pathogen competitiveness and gene expression in E. coli, Salmonella, and Campylobacter. Objective 2: Characterize the biological factors affecting infection and maintenance of Salmonella in lymphatics of food producing animals and elucidate management practices to mitigate infection. 2.A: Determine the duration of Salmonella infection in the peripheral lymph nodes of cattle. 2.B: Determine the role of mucous membranes in uptake and distribution of Salmonella to the peripheral lymph nodes of cattle. 2.C: Determine the prevalence, antimicrobial susceptibilities, genetic relatedness, serotypes, and molecular characteristics of Salmonella isolated from head meat and trim intended for ground pork. Objective 3: Identify, develop, and test interventions, including exploring possible synergies of multiple interventions and alternatives to antibiotics that can kill pathogenic or antibiotic resistant foodborne pathogens or mitigate their virulence and resistance in the animal production environment. 3.A: Enhance the effectiveness of naturally occurring phytochemicals and organic acids in reducing E. coli and Salmonella in the animal gut. 3.B: Reduce-to-practice ß-D-thymol as a feed additive prebiotic pathogen control technology for swine. 3.C: Determine if feeding sodium chlorate will reduce populations of Salmonella within the peripheral lymph nodes. 3.D: Determine if application of a bacteriophage cocktail will reduce or eliminate Salmonella from the peripheral lymph nodes of experimentally-infected cattle. 3.E: Determine if killed, irradiated, or spent chemostatic effluent of a recombined porcine-derived competitive exclusion culture can stimulate in vitro and in vivo immune responses and characterize the production and efficacy of biofilms and bacteriocins associated with the culture. Objective 4: Investigate the ecology of antimicrobial and disinfectant resistance within the gut of food producing animals and their production environment and elucidate factors contributing to the acquisition, exchange, dissemination and maintenance of resistant elements in foodborne pathogens and commensal bacteria. 4.A: Determine association between multidrug resistance (MDR) and virulence traits in Escherichia coli and non-typhoidal Salmonella enterica serovars isolated from food producing animals that might provide a dissemination advantage.

Approach
Basic and applied research will be conducted to achieve project objectives. Studies employing metagenomic analysis will elucidate ecological niches or reservoirs where pathogens may exist, and when combined with traditional epidemiological and microbiological cultural methods, these studies will help reveal environmental, nutritional, and biological factors affecting fitness characteristics contributing to persistent colonization, survival, and growth of these pathogens in food animals and their production environment. Research involving both in vitro and in vivo methods will be used to assess and characterize adaptive responses microbes may exhibit to intrinsic and extrinsic stressors, such as those exerted by disinfectants and antimicrobials, as well as to learn how these stressors may influence pathogenicity, virulence, and resistance of the microbes. Animal studies conducted under clinical and field situations will be used to develop and evaluate interventions, thereby revealing specific metabolic endpoints, cellular mechanisms, and sites of action of cellular processes that may ultimately be exploited to decrease carriage and shedding of pathogens during production and at slaughter. When applicable, Cooperative Research and Development Agreements will be implemented with industry partners to aid in technology transfer.

Progress Report
This project expired on December 6, 2020, and was replaced by the bridging project 3091-32000-036-00D. Project work in Fiscal Year 2021 resulted in significant progress in identifying critical control points for the implementation of improved intervention strategies to minimize pathogen infection and dissemination in food-producing animals (Objective 3). Over the life of the project, major progress was made in elucidating mechanisms of antimicrobial resistance in microbial pathogens (Objective 4), in assessment of potentially practical alternatives to conventional antibiotics (Objective 1), and in development of management practices to reduce the carriage and environmental dissemination of pathogenic and antimicrobial-resistant microbes by food-producing animals (Objectives 2, 3). Work under Objective 1 defined critical inter-kingdom relationships that can be exploited to assure safe and effective carcass disposal so as to reduce risks of vector-borne and on-farm spread of pathogens such as E. coli, Salmonella, and Campylobacter. Project scientists identified potential mitigating interventions to prevent oral-fecal and intradermal infection/transmission of Salmonella in cattle and swine (Objective 2). Under Objective 3, project scientists and university collaborators discovered, patented, and licensed a naturally-selected bacterial probiotic for use in management of important livestock pathogens, and also for ruminal methane mitigation in dairy and beef cattle. Also under Objective 3, project scientists and collaborators developed a natural plant extract (from Nigella sativa) as an alternative to conventional antibiotics in swine; the product not only enhances colonization resistance against E. coli, but also improves growth efficiency in young pigs. Work under Objective 3 developed improved formulations of essential oils and nitro-based compounds for use as antibiotic alternatives and to enhance growth efficiency; industry partners are working to implement certain of these technologies into commercial food animal production protocols. Under Objective 4, project scientists developed best-use practices for disinfectants that will be of value to livestock producers and processors in their efforts to assure maintenance of effective disinfection practices in production and processing environments. Work by this project, overall, developed important new technologies and protocols that will help farmers and ranchers produce safer, more wholesome meat products for the American consumer.

Accomplishments
1. A carrier of antimicrobial resistance genes on a swine farm. Acquisition and transfer of antimicrobial resistance among livestock may contribute to broad dissemination of resistance genes within production environments, downstream watersheds, and by contamination of carcasses during processing. Microbial vectors contributing to dissemination of antimicrobial resistance genes are neither well known nor fully understood. During a study of swine farms experiencing an outbreak of neonatal diarrhea in swine, ARS researchers at College Station, Texas, isolated and identified an atypical bacterium, Aeromonas hydrophila strain CVM861, that has not previously been recognized as being common to swine production environments. This bacterium was found to exhibit multi-drug resistance; whole genome sequencing revealed the presence of a resistance plasmid and numerous resistance genes of relevance for food animal environments. This discovery provides important information on a previously unrecognized carrier of antimicrobial resistance genes in swine production facilities, and will guide development of strategies and protocols to reduce the spread of antibiotic resistance caused by Aeromonas and other bacteria residing within food animal environments. Minimizing antibiotic resistance in food animal production is critical to sustainable production of safe and wholesome meat and dairy products for the U.S. consumer.

Review Publications
Edrington, T.S., Arthur, T.M., Loneragan, G.L., Genovese, K.J., Hanson, D.L., Anderson, R.C., Nisbet, D.J. 2020. Evaluation of two commercially-available Salmonella vaccines on Salmonella in the peripheral lymph nodes of experimentally-infected cattle. Therapeutic Advances in Vaccines and Immunotherapy. 8:1-7. https://doi.org/10.1177/2515135520957760.
Beier, R.C., Andrews, K., Poole, T.L., Harvey, R.B., Crippen, T.L., Anderson, R.C., Nisbet, D.J. 2020. Interactions of organic acids with Staphylococcus aureus and MRSA strains from swine mandibular lymph node tissue, commercial pork sausage meat and feces. International Journal of Microbiology and Biotechnology. 5(4):165-183. https://doi.org/10.11648/j.ijmb.20200504.12.
Harvey, R.B., Norman, K.N., Anderson, R.C., Nisbet, D.J. 2020. A preliminary study on the presence of Salmonella in lymph nodes of sows at processing plants in the United States. Microorganisms. 8(10). Article 1602. https://doi.org/10.3390/microorganisms8101602.
Poole, T.L., Schlosser, W.D., Anderson, R.C., Norman, K.N., Beier, R.C., Nisbet, D.J. 2020. Whole genome sequence of Aeromonas hydrophila CVM861 isolated from diarrheic neonatal swine. Microorganisms. 8(11). Article 1648. https://doi.org/10.3390/microorganisms8111648.
Arzola-Alvarez, C., Hume, M.E., Anderson, R.C., Latham, E.A., Ruiz-Barrera, O., Castillo-Castillo, Y., Olivas-Palacios, A.L., Felix-Portillo, M., Armendariz-Rivas, R.L., Arzola-Rubio, A., Ontiveros-Magadan, M., Bautista-Martinez, Y., Salinas-Chavira, J. 2020. Influence of sodium chlorate, ferulic acid, and essential oils on Escherichia coli and porcine fecal microbiota. Journal of Animal Science. 98(3). Article skaa059. https://doi.org/10.1093/jas/skaa059.
Levent, G., Schlochtermeier, A., Ives, S.E., Norman, K.N., Lawhon, S.D., Loneragan, G.H., Anderson, R.C., Vinasco, J., Den Bakker, H., Scott, H.M. 2021. High-resolution genomic comparisons within Salmonella enterica serotypes derived from beef feedlot cattle: Parsing the roles of cattle source, pen, animal, sample type and production period. Applied and Environmental Microbiology. 18(12). Article e00485-21. https://doi.org/10.1128/AEM.00485-21.
Beier, R.C. 2021. Interactions and inhibition of pathogenic foodborne bacteria with individual dissociated organic acid species: A review. Journal of Food Chemistry & Nanotechnology. 7(1):4-17. https://doi.org/10.17756/jfcn.2021-0106.
Miranda, C.D., Crippen, T.L., Cammack, J.A., Tomberlin, J.K. 2021. Black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae), and house fly, Musca domestica L. (Diptera: Muscidae), larvae reduce livestock manure and possibly associated nutrients: An assessment at two scales. Environmental Pollution. 282. Article 116976. https://doi.org/10.1016/j.envpol.2021.116976.