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United States Department of Agriculture

Agricultural Research Service


Location: Food and Feed Safety Research

2013 Annual Report

1a. Objectives (from AD-416):
1. Determine particular ecological niches/reservoirs for pathogenic/antimicrobial-resistant bacteria, identifying nutritional/biological/environmental factors affecting ability to colonize/survive/persist within gut of food-producing animals & their production environment. a) Determine effect of feeding distillers grains & other diets & organic acids on gut microbial ecology; b) Determine effects of feeding diets high in rumen undegradable intake protein & of feeding monensin on colonization/carriage/shedding of Campylobacter in cattle; c) Evaluate mechanistic effects of short chain nitrocompounds & free fatty acids on hydrogen balance/fitness of foodborne pathogens; d) Determine if specific or shared reservoirs exist for Clostridium difficile in populations of swine & humans in integrated agri-business operations; phenotypically/genotypically characterize Cl. difficile isolates; e) Determine effects of lactoferrin & whey-protein concentrate on fecal shedding & gut populations of Salmonella Typhimurium in weaned piglets; f) Determine influence of vitamin D supplement on fecal prevalence/concentrations of E. coli O157:H7 in cattle. 2. Evaluate ways to better characterize/control Salmonella, in particular multi-drug-resistant Salmonella, for use on commercial dairy farms to enhance food safety. a) Develop Salmonella serotype-specific real-time PCR method; b) Determine if Salmonella serotype changes due to changing environment or acquisition of antimicrobial resistance; c) Determine if feeding sodium chlorate, with/without nitroethane, is effective in reducing populations of Salmonella as well as E. coli O157:H7 & generic E. coli in milk-replacer fed calves & cull dairy cattle. 3. Develop interventions that prevent/mitigate colonization of gut of food-producing animals (particularly lower GI tract before slaughter) or that reduce pathogenic/antimicrobial-resistant bacteria in production environment. a) Determine if porcine-derived competitive exclusion culture can stimulate innate immune responses in neonatal gnotobiotic piglets; b) Develop/characterize bactericidal effectiveness of thymol & diphenyliodonium chloride products bound to clay-based adsorbents; c) Establish feasibility of fresh/dried citrus peel & purified essential oils as feed additives to reduce pathogen populations in ruminants; d) Develop targeted management interventions to facilitate exclusion of antimicrobial-resistant bacteria from gut of treated animals. 4. Develop understanding of microbial adaption to intrinsic/extrinsic stressors on acquisition/exchange/expression of incompatibility plasmids & antimicrobial resistance elements in foodborne pathogens in production/processing environments. a) Determine if number/size of plasmids possessed by different wildtype E. coli affects growth rate in nonselective & mixed culture environments, and if these plasmids are lost during long-term maintenance without selection pressure; b) Determine if IncN/Incl1 can mobilize IncA/C in tri-parental conjugations; c) Determine if occurrence of bacterial resistance to disinfectants is important in disinfectant control or impacts antibiotic resistance in microorganisms of animal origin.

1b. Approach (from AD-416):
Basic and applied research will be conducted to achieve project objectives. Ecological studies utilizing metagenomic analysis will elucidate 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 related to the persistent colonization, survival, and growth of these pathogens in food animals and their production environment. Research involving both in vitro and in vivo experimentation will be used to measure and characterize adaptive responses microbes may exhibit to intrinsic and extrinsic stressors, such as those exerted by disinfectants and antimicrobials, as well as to determine the role these stressors may play in pathogenicity, virulence, and resistance. 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. Opportunities for mitigation will be validated in the field. In some cases, Cooperative Research and Development Agreements will be implemented with industry partners to aid in technology transfer.

3. Progress Report:
Work under this project in FY 2013 resulted in significant progress in evaluating and developing new strategies and best-use applications of existing strategies for the control of foodborne pathogens in cattle and swine. Project work has provided new knowledge on routes of Salmonella infection and the application of an array of novel feed additives to reduce pathogen colonization in swine and cattle. When formulated appropriately, the feeding of naturally occurring plant compounds such as citrus acids, tannins, or essential oil compounds like thymol can help reduce the carriage and shedding of foodborne and animal health pathogens. Project work also helped elucidate new, important information on management and environmental factors influencing the carriage and potential dissemination of foodborne pathogens as well as disinfectant and antimicrobial-resistant bacterial populations. The work established that environmentally ubiquitous organisms, such as Pseudomonas aeruginosa, may be an important reservoir of disinfectant and antimicrobial resistance in agricultural environments. Work on the development of practical, cost-effective interventions or management practices to reduce the carriage and environmental dissemination of pathogenic and antimicrobial-resistant microbes by food-producing animals is ongoing. New technology and protocols developed from this work will help U.S. farmers and ranchers produce safer and more wholesome meat products at lower costs for the consumer.

4. Accomplishments
1. Development of a more relevant method to study Salmonella infection in cattle. Salmonella is a foodborne pathogen that can invade the peripheral lymph nodes of cattle and can evade post-harvest interventions aimed at preventing contamination of ground meat. It had long been thought that Salmonella translocates to the lymph from the gut, but experimental models could not consistently replicate this mechanism of infection. ARS scientists at College Station, Texas, developed a new and more reliable model of Salmonella translocation using a skin allergy instrument to inoculate calves via intra- and/or transdermal injection at various abdominal and pelvic regions of the skin. This new model makes it possible to reproducibly and predictably determine the route and duration of lymph node infection and to evaluate candidate interventions that can eliminate the infection in food-producing animals, thus enhancing microbial food safety of beef products reaching the consumer.

2. Clostridium difficile is not readily transmitted from pigs to their human caretakers. Clostridium difficile is a bacterium that causes disease and death in humans. Historically, the infection was acquired during hospital stays, but more virulent strains that are transmissible outside of hospital environments have recently emerged. Although the origin of the new strains is unknown, some speculate they could have come from animals or meat. ARS scientists at College Station, Texas, working with Texas A&M University researchers, established the distribution and antimicrobial resistance characteristics of C. difficile in human wastewater and swine feces from populations of humans and swine having close geographical and occupational contact with each other. The work established that C. difficile isolated from swine sources had less antimicrobial resistance than those isolated from human sources and that resistance in C. difficile isolated from sources of human swine workers was no different than from non-swine workers. This is important because it provides strong evidence that C. difficile is not transferred from swine to humans, and that swine are not the source of C. difficile antibiotic resistance in humans.

Review Publications
Tedeschi, L.O., Callaway, T.R., Muir, J.P., Anderson, R.C. 2011. Potential environmental benefits of feed additives and other strategies for ruminant production. Revista Brasileira de Zootecnia. 40:291-309.

Edrington, T.S., Farrow, R.L., Hume, M.E., Anderson, P.N., Hagevoort, G.R., Caldwell, D.J., Callaway, T.R., Anderson, R.C., Nisbet, D.J. 2012. Evaluation of the potential antimicrobial resistance transfer from a multi-drug resistant Escherichia coli to Salmonella in dairy calves. Current Microbiology. 66:132-137.

Callaway, T.R., Block, S., Genovese, K.J., Anderson, R.C., Harvey, R.B., Nisbet, D.J. 2012. Impact of by-product feedstuffs in Escherichia coli O157:H7 and Salmonella Typhimurium in pure and mixed ruminal and fecal culture in vitro. Agriculture, Food and Analytical Bacteriology. 2:139-148.

Broadway, P.R., Callaway, T.R., Carroll, J.A., Donaldson, J.R., Rathmann, R.J., Johnson, B.J., Cribbs, J.T., Durso, L.M., Nisbet, D.J., Schmidt, T.B. 2012. Evaluation of the ruminal bacterial diversity of cattle fed diets containing citrus pulp pellets. Agriculture, Food and Analytical Bacteriology. 2:297-308.

Edrington, T.S., Farrow, R.L., Carter, B.H., Islas, A., Hagevoort, G.R., Callaway, T.R., Anderson, R.C., Nisbet, D.J. 2012. Age and diet effects on fecal populations and antibiotic resistance of a multi-drug resistant Escherichia coli in dairy calves. Agriculture, Food and Analytical Bacteriology. 2:162-174.

Nisbet, D.J., Edrington, T.S., Farrow, R.L., Genovese, K.J., Callaway, T.R., Anderson, R.C., Krueger, N.A. 2012. Lack of effect of feeding lactoferrin on intestinal populations and fecal shedding of Salmonella typhimurium in experimentally-infected weaned pigs. Agriculture, Food and Analytical Bacteriology. 2:280-290.

Krueger, N.A., Edrington, T.S., Farrow, R.L., Hagevoort, R., Anderson, R.C., Loneragan, G.H., Callaway, T.R., Nisbet, D.J. 2012. Evaluation of an experimental sodium chlorate product, with and without nitroethane, on Salmonella in cull dairy cattle. Agriculture, Food and Analytical Bacteriology. 2:82-87.

Ramlachan, N., Anderson, R.C., Andrews, K., Harvey, R.B., Nisbet, D.J. 2012. Transfer of tylosin resistance between Enterococcus spp. during continuous-flow culture of feral or domestic porcine gut microbes. Agriculture, Food and Analytical Bacteriology. 2:111-120.

Edrington, T.S., Dowd, S.E., Farrow, R.F., Hagevoort, G.R., Callaway, T.R., Anderson, R.C., Nisbet, D.J. 2012. Development of colonic microflora as assessed by pyrosequencing in dairy calves fed waste milk. Journal of Dairy Science. 95:4519-4525.

Edwards, H.D., Anderson, R.C., Miller, R.K., Taylor, T.M., Hardin, M.D., Smith, S.B., Krueger, N.A., Nisbet, D.J. 2012. Glycerol inhibition of ruminal lipolysis in vitro. Journal of Dairy Science. 95:5176-5181.

Poole, T.L., Callaway, T.R., Bischoff, K.M., Longeragan, G.H., Anderson, R.C., Nisbet, D.J. 2012. Competitive effect of commensal faecal bacteria from growing swine fed chlortetracycline-supplemented feed on beta-haemolytic Escherichia coli strains with multiple antimicrobial resistance plasmids. Journal of Applied Microbiology. 113:659-668.

Luo, P.J., Jiang, W.X., Beier, R.C., Shen, J.Z., Jiang, H.Y., Miao, H., Zhao, Y.F., Chen, X., Wu, Y.N. 2012. Development of an enzyme-linked immunosorbent assay for determination of the furaltadone metabolite, 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) in animal tissues. Biomedical and Environmental Sciences. 25:449-457.

Dunkley, K.D., Callaway, T.R., O Bryan, C.A., Kundinger, M.M., Dunkley, C.S., Anderson, R.C., Nisbet, D.J., Crandall, P.G., Ricke, S.C. 2012. Comparison of real time polymerase chain reaction quantification of changes in hilA and rpoS gene expression of a Salmonella typhimurium poultry isolate grown at fast versus slow dilution rates in an anaerobic continuous culture system. Food Biotechnology. 26:239-251.

Callaway, T.R., Edrington, T.S., Harvey, R.B., Anderson, R.C., Nisbet, D.J. 2012. Prebiotics in food animals, a potential to reduce foodborne pathogens and disease. Romanian Biotechnological Letters. 17:7808-7816.

Free, A.L., Duoss, H.A., Bergeron, L.V., Shields-Menard, S.A., Ward, E., Callaway, T.R., Carroll, J.A., Schmidt, T.B., Donaldson, J.R. 2012. Survival of O157:H7 and non-O157 serogroups of Escherichia coli in bovine rumen fluid and bile salts. Foodborne Pathogens and Disease. 9:1010-1014.

Duoss, H.A., Donaldson, J.R., Callaway, T.R., Carroll, J.A., Broadway, P.R., Martin, J.M., Shields-Menard, S., Schmidt, T.B. 2013. Survival of Escherichia coli O157:H7 transformed with either the pAK1-lux or pXEN-13 plasmids in in vitro bovine ruminal and fecal microbial fermentations. Foodborne Pathogens and Disease. 10:1-5. doi: 10.1089/fpd.2012.1234.

Gragg, S.E., Loneragan, G.H., Brashears, M.M., Arthur, T.M., Bosilevac, J.M., Kalchayanand, N., Wang, R., Schmidt, J.W., Brooks, J.C., Shackelford, S.D., Wheeler, T.L., Brown, T.R., Edrington, T.S., Brichta-Harhay, D.M. 2013. Cross-sectional study examining Salmonella enterica carriage in subiliac lymph nodes of cull and feedlot cattle at harvest. Foodborne Pathogens and Disease. 10:368-374.

Beier, R.C., Poole, T.L., Brichta-Harhay, D.M., Anderson, R.C., Bischoff, K.M., Hernandez, C.A., Bono, J.L., Arthur, T.M., Nagaraja, T.G., Crippen, T.L., Sheffield, C.L., Nisbet, D.J. 2013. Disinfectant and antibiotic susceptibility profiles of Escherichia coli O157:H7 strains from cattle carcasses, feces, and hides and ground beef from the United States. Journal of Food Protection. 76:6-17.

Wang, Z., Beier, R.C., Sheng, Y., Zhang, S., Jiang, W., Wang, Z., Wang, J., Shen, J. 2013. Monoclonal antibodies with group specificity toward sulfonamides: Selection of hapten and antibody selectivity. Analytical and Bioanalytical Chemistry. 405:4027-4037.

Edrington, T.S., Loneragan, G.H., Hill, J.E., Genovese, K.J., He, L.H., Callaway, T.R., Anderson, R.C., Brichta-Harhay, D.M., Nisbet, D.J. 2013. Development of a transdermal Salmonella challenge model in calves. Journal of Food Protection. 76:1255-1258.

Edrington, T.S., Loneragan, G.H., Hill, J.E., Genovese, K.J., Brichta-Harhay, D.M., Farrow, R.L., Krueger, N.A., Callaway, T.R., Anderson, R.C., Nisbet, D.J. 2013. Development of challenge models to evaluate the efficacy of a vaccine to reduce carriage of Salmonella in peripheral lymph nodes of cattle. Journal of Food Protection. 76:1259-1263.

Duoss-Jennings, H.A., Schmidt, T.B., Callaway, T.R., Carroll, J.A., Martin, J.M., Shields-Menard, S.A., Broadway, P.R., Donaldson, J.R. 2013. Effect of citrus byproducts on survival of O157:H7 and non-O157 Escherichia coli serogroups within in vitro bovine ruminal microbial fermentations. International Journal of Microbiology. 2013:Article 398320. doi: 10.1155/2013/398320.

Edwards, H.D., Anderson, R.C., Taylor, T.M., Miller, R.K., Hardin, M.D., Nisbet, D.J., Krueger, N.A., Smith, S.B. 2013. Interactions between oil substrates and glucose on pure cultures of ruminal lipase-producing bacteria. Lipids. 48:749-755.

Schuster, G.L., Donaldson, J.R., Buntyn, J.O., Duoss, H.A., Callaway, T.R., Carroll, J.A., Falkenberg, S.M., Schmidt, T.B. 2013. Use of bioluminescent Escherichia coli to determine retention during the life cycle of the housefly, Musca domestica (Diptera: Muscidae, L). Foodborne Pathogens and Disease. 10:442-447.

Petrujkic, B.T., Sedej, I., Beier, R.C., Anderson, R.C., Harvey, R.B., Epps, S.V., Stipanovic, R.D., Krueger, N.A., Nisbet, D.J. 2013. Ex vivo absorption of thymol and thymol-beta-D-glucopyranoside in piglet everted jejunal segments. Journal of Agricultural and Food Chemistry. 61:3757-3762.

Min, B.R., Pinchak, W.E., Hernandez Jr, C.A., Hume, M.E. 2013. Grazing activity and ruminal bacterial population associated with frothy bloat in steers grazing winter wheat. Professional Animal Scientist. 29:179-187.

Callaway, T.R., Edrington, T.S., Loneragan, G.H., Carr, M.A., Nisbet, D.J. 2013. Shiga toxin-producing Escherichia coli (STEC) ecology in cattle and management based options for reducing fecal shedding. Agriculture, Food and Analytical Bacteriology. 3:39-69.

Callaway, T.R., Edrington, T.S., Loneragan, G.H., Carr, M.A., Nisbet, D.J. 2013. Current and near-market intervention strategies for reducing Shiga Toxin-producing Escherichia coli (STEC) shedding in cattle. Agriculture, Food and Analytical Bacteriology. 3:103-120.

Last Modified: 04/29/2017
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