2012 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.
Work under this project in FY 2012 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 the application of an array of novel feed additives to reduce pathogen colonization in swine and cattle, finding that 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 in FY 2012 continues ongoing efforts aimed at 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. New technology and protocols developed from this work will help U.S. farmers and ranchers produce safer and more wholesome meat products at less cost for the consumer.
Swine are not carriers of a hyper-virulent, multi-drug-resistant strain of Clostridium difficile. Clostridium difficile is a ubiquitous opportunistic bacterial pathogen that is usually associated with infections acquired by immune-compromised patients during hospital stays. Recently there have been an increasing number of "community-acquired" cases among otherwise healthy individuals with no readily explained origin of infection; however, infectious disease scientists suspect that food-producing animals such as swine may be a potential reservoir of Clostridium difficile. ARS scientists at College Station, Texas, demonstrated that while Clostridium difficile was isolated at low frequency from non-diseased human and swine populations, none of the isolates were the hyper-virulent, multi-drug-resistant strains common to the community-acquired infections. Also, there was no evidence of transfer of the different isolates between animals and humans. This work is important because it indicates that swine should not be implicated as carriers of the newer "community-acquired" Clostridium difficile strains.
An improved and more rapid Salmonella serotyping method. Various supply, production, and environmental sources can serve as critical contamination points for the important foodborne pathogen Salmonella during food animal production. Cost-effective and rapid identification of different Salmonella strains (serotypes) would facilitate the recall and elimination of contaminated products from the production chain; however, traditional diagnostic protocols based on commonly used methods are slow and tedious or are not discriminating enough to enable molecular differentiation of the more than 1500 different types of Salmonella. ARS scientists at College Station, Texas, have shown that incorporating a procedure known as Denaturing Gradient Gel Electrophoresis into the diagnostic protocol enables sufficient resolution of a highly variable gene sequence to reliably discriminate more than 50 different commonly encountered Salmonella types into their appropriate category. Work is now focusing on development of a digital molecular typing library that includes the most prevalent Salmonella serotypes isolated from human cases. This new molecular typing technology will help producers and public health officials in their food safety assurance missions by providing near real time differentiation and traceability of Salmonella recovered from various supply, production, environmental, and clinical sources.
Callaway, T.R., Carroll, J.A., Arthington, J.D., Edrington, T.S., Rossman, M.L., Carr, M.A., Krueger, N.A., Ricke, S.C., Crandall, P., Nisbet, D.J. 2011. Escherichia coli O157:H7 populations in ruminants can be reduced by orange peel product feeding. Journal of Food Protection. 74:1917-1921.
Harvey, R.B., Norman, K.N., Andrews, K., Hume, M.E., Scanlan, C.M., Callaway, T.R., Anderson, R.C., Nisbet, D.J. 2011. Clostridium difficile in poultry and poultry meat. Foodborne Pathogens and Disease. 8:1321-1323.
Stancic, I., Stancic, B., Bozic, A., Harvey, R.B., Anderson, R.C., Gvozdic, D. 2011. Ovarian activity and uterus organometry in delayed puberty gilts. Theriogenology. 76:1022-1026.
Anderson, R.C., Krueger, N.A., Genovese, K.J., Stanton, T.B., MacKinnon, K.M., Harvey, R.B., Edrington, T.S., Callaway, T.R., Nisbet, D.J. 2012. Effect of thymol or diphenyliodonium chloride on performance, gut fermentation characteristics, and Campylobacter colonization in growing swine. Journal of Food Protection. 75:758-761.
Bozic, A.K., Anderson, R.C., Ricke, S.C., Crandall, P.G., O'Bryan, C.A. 2012. Comparison of nitroethane, 2-nitro-1-propanol, lauric acid, Lauricidin and the Hawaiian marine algae, Chaetoceros, for potential broad-spectrum control of anaerobically grown lactic acid bacteria. Journal of Environmental Science and Health, Part B. 47:269-274.
Hughes, D.T., Terekhova, D.A., Liou, L., Hovde, C.J., Sahl, J.W., Patankar, A.V., Gonzalez, J.E., Edrington, T.S., Rasko, D.A., Sperandio, V. 2010. Chemical sensing in mammalian host-bacterial commensal associations. Proceedings of the National Academy of Sciences. 107:9831-9836.
Edrington, T.S., Nisbet, D.J. 2011. Melatonin and food safety: Investigating a possible role in the seasonality of the bacterial pathogen Escherichia coli 0157:H7 in cattle. In: Watson, R.R., editor. Melatonin in the Promotion of Health. Boca Raton, FL: CRC Press. p. 211-218.
Raya, R.R., Oot, R.A., Moore-Maley, B., Wieland, S., Callaway, T.R., Kutter, E.M., Brabban, A.D. 2011. Naturally resident and exogenously applied T4-like and T5-like bacteriophages can reduce Escherichia coli O157:H7 levels in sheep guts. Bacteriophage. 1:15-24.
Callaway, T.R., Carroll, J.A., Arthington, J.D., Edrington, T.S., Anderson, R.C., Rossman, M.L., Carr, M.A., Genovese, K.J., Ricke, S.C., Crandall, P., Nisbet, D.J. 2011. Orange peel products can reduce Salmonella populations in ruminants. Foodborne Pathogens and Disease. 8:1071-1075.
Callaway, T.R., Edrington, T.S., Poole, T.L., Nisbet, D.J. 2011. Current status of practical applications: Probiotics in dairy cattle. In: Callaway, T.R., Ricke, S.C., editors. Direct Fed Microbials/Prebiotics for Animals: Science and Mechanisms of Action. New York, NY: Springer Verlag Publishing. p. 121-136.
Norman, K.N., Scott, H.M., Harvey, R.B., Norby, B., Hume, M.E., Andrews, K. 2011. Prevalence and genotypic characteristics of Clostridium difficle in a closed and integrated human and swine population. Applied and Environmental Microbiology. 77:5755–5760.
Harvey, R.B., Norman, K.N., Andrews, K., Norby, B., Hume, M.E., Scanlan, C.M., Hardin, M.D., Scott, H.M. 2011. Clostridium difficile in retail meat and processing plants in Texas. Journal of Veterinary Diagnostic Investigation. 23:807-811.
Njongmeta, N.L., Benli, H., Dunkley, K.D., Dunkley, C.S., Miller, D.R., Anderson, R.C., O'Bryan, C.A., Keeton, J.T., Nisbet, D.J. Crandall, P.G., Ricke, S.C. 2011. Application of acidic calcium sulfate and e-polylysine to pre-rigor beef rounds for reduction of pathogens. Journal of Food Safety. 31:395-400.
Edrington, T.S., Farrow, R.L., MacKinnon, K.M., Callaway, T.R., Anderson, R.C., Nisbet, D.J. 2012. Influence of vitamin D on fecal shedding of Escherichia coli O157:H7 in naturally colonized cattle. Journal of Food Protection. 75:314-319.
Kutter, E.M., Skutt-Kakaria, K., Blasdel, B., El-Shibiny, A., Castano, A., Bryan, D., Kropinski, A.M., Villegas, A., Ackermann, H., Toribio, A.L., Pickard, D., Anany, H., Callaway, T.R., Brabban, A.D. 2011. Characterization of a ViI-like phage specific to Escherichia coli O157:H7. Bacteriophage. 8:430-445.
Callaway, T.R., Edrington, T.S. 2012. Introduction. In: Callaway, T.R., Edrington, T.S., editors. On-Farm Strategies to Control Foodborne Pathogens. New York, NY: Nova Science Publishers, Inc. p. 1-4.
Xu, Z., Zeng, D., Yang, J., Shen, Y., Beier, R.C., Lei, H., Wang, H., Sun, Y. 2011. Monoclonal antibody-based broad-specificity immunoassay for monitoring organophosphorus pesticides in environmental water samples. Journal of Environmental Monitoring. 13:3040-3048.
Farrow, R.L., Edrington, T.S., Krueger, N.A., Genovese, K.J., Callaway, T.R., Anderson, R.C., Nisbet, D.J. 2012. Lack of effect of feeding citrus by-products in reducing Salmonella in experimentally infected weanling pigs. Journal of Food Protection. 75:573-575.
Loneragan, G.H., Thomson, D.U., McCarthy, R.M., Webb, H.E., Daniels, A.E., Edrington, T.S., Nisbet, D.J., Trojan, S.J., Rankin, S.C., Brashears, M.M. 2012. Salmonella diversity and burden in cows on and culled from dairy farms in the Texas High Plains. Foodborne Pathogens and Disease. 9:549-555.
Wang, Z., Kai, Z., Beier, R.C., Shen, J., Yang, X. 2012. Investigation of antigen-antibody interactions of sulfonamides with a monoclonal antibody in a fluorescence polarization immunoassay using 3D-QSAR models. International Journal of Molecular Sciences. 13:6334-6351.
Anderson, R.C., Stanton, T.B. 2012. Genus V. Denitrobacterium. In: Goodfellow, M., Kampfer, P., Busse, H., Trujillo, M., Suzuki, K., Ludwig, W., Whitman, W., editors. Bergey's Manual of Systematic Bacteriology. 2nd edition. New York, NY: Springer. p. 1988-1990.
Farrow, R.L., Edrington, T.S., Carter, B.H., Friend, T.H., Callaway, T.R., Anderson, R.C., Nisbet, D.J. 2011. Influence of winter and summer hutch coverings on fecal shedding of pathogenic bacteria in dairy calves. Agriculture, Food and Analytical Bacteriology. 1:98-104.
Anderson, R.C., Vodovnik, M., Min, B.R., Pinchak, W.E., Krueger, N.A., Harvey, R.B., Nisbet, D.J. 2012. Bactericidal effect of hydrolysable and condensed tannin extracts on Campylobacter jejuni in vitro. Folia Microbiologica. 57:253-258.