Progress 09/01/10 to 08/31/12
Outputs
OUTPUTS: Escherichia coli O157:H7, a food borne pathogen, is of major public health concern. Cattle are asymptomatic reservoirs for Escherichia coli O157. The organism generally colonizes the hindgut of cattle and is shed in the feces at low concentrations, which serves as a major source of contamination of human food and water. The goals of our research program are to develop improved methods to detect and quantify E. coli O157, improve our understanding of the ecology of E. coli O157 in cattle operations, and to identify and test on-farm intervention strategies for control of E. coli O157:H7. A subset of the cattle population, called "super shedders", shed high concentrations of E. coli O157 (~103-4 CFU/g) in their feces. Cattle shedding E. coli O157 at higher concentrations transmit the organism to a greater number of animals and significantly increase the risk of carcass contamination compared to animals shedding the organism at low levels. Therefore, it is important to identify high shedding animals when evaluating intervention strategies. The culture-based methods are time consuming and expensive, and are generally logistically cumbersome for a large number of samples. Therefore, a multiplex, quantitative PCR assay, based on rfbE, stx1, and stx2, was developed and validated to detect and quantify E. coli O157 in cattle feces. The primers and probes chosen were based on unique sequences in the stx1 subunit A (stx1A), the stx2 subunit A (stx2A), and the rfbE gene from the E. coli O157 EDL 933 strain. Primer efficiency and analytical sensitivity of the assay were evaluated with a single or pooled (five strain) culture of E. coli O157. Diagnostic sensitivity was analyzed using DNA extracted from cattle feces spiked with E. coli O157. The assay was also evaluated with feces from cattle experimentally inoculated with E. coli O157 by comparing the results to a culture-based method. Distillers grains (DG), a co-product of ethanol production used as protein and energy supplements in cattle diets, have been shown to increase fecal shedding of Escherichia coli O157. The reason for the positive association is not known. Because DG often replaces grain in the diet, decreased starch content and flow to the hindgut may create a favorable environment for E. coli O157. Therefore, a study was to determine whether the addition of starch to a corn DG-supplemented diet negates the effects of DG on fecal shedding of E. coli O157. The study included 21 steers fed one of three diets: a corn grain-based basal diet (CON), basal diet supplemented with 25% corn dried DG (DDG), and basal diet supplemented with 25% DDG with corn starch (DDG+S) added at a level intended to raise starch concentration to that of the CON diet. Steers, housed individually in a biosafety level 2 animal facilities, were randomly allocated to treatment diets and orally inoculated with a 5-strain mixture of nalidixic acid-resistant (NalR) E. coli O157. Fecal samples were collected for 5 wk, and on d 35, steers were euthanized and necropsied to collect gut content samples. Fecal or gut samples were cultured to determine prevalence and concentrations of NalR E. coli O157. PARTICIPANTS: Investigators: Nagaraja, T. G., Dave Renter, Richard Oberst, Mike Sanderson, and Ludek Zurek T. G. Nagaraja: Microbiologist; Principal investigator; involved in all planning and decision making processes, managing funds, and overseeing laboratory analysis and reporting of results. David Renter: Epidemiologist; Co-Principal investigator; Responsible for the study design and statistical analysis; Also involved in all planning and decision making processes and overseeing laboratory analysis and reporting of results. Mike Sanderson: Epidemiologist; Involved in study design and statistical analysis Ludek Zurek: Medical Entomologist; Involved in studies on flies and E. coli O157:H7 Laboratory technicians: Xiaorong Shi and Neil Wallace Xiaorong Shi: Responsible for bacteriological procedures and molecular analyses (PCR and PFGE typing of isolates). Neil Wallace: Responsible for bacteriological procedures, ordering supplies, maintaining inventory and supervising undergraduate students. Graduate students Ph. D. students: Charles Dodd, Megan Jacob, Zac Paddock MPH students: Ethel Taylor MS students: Lance Noll Graduate students are responsible for sample collection and processing in laboratory, recording of data, analysis of results, conference presentations and manuscript writing. Undergraduate students: A number of graduate students work part time in the laboratory. The students are trained in laboratory safety and analytical skills. The students presently working in the laboratory are: Blair Wyrick, Samantha Smith, Cody Strodtman, Katie Flock, Kristina Wert, Nick Reams, and Eric Haney. TARGET AUDIENCES: Scientists, Veterinarians, Food Safety Inspection Agency, Cattlemen, Packers, Students PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
(N/A)
Publications
- Beier R. C., T. L. Poole, D. M. Brichta-Harhay, R. C. Anderson, K. M. Bischoff, C. A. Hernandez, J. L. Bono, T. M. Arthur, T. G. Nagaraja, T. L. Crippen, C. L. Sheffield and D. J. Nisbet. 2013. Disinfectant and antibiotic susceptibility profiles of Escherichia coli O157:H7 strains from cattle carcasses, feces, hides, and ground beef from the United States. J. Food Prot.76:6-17.
- Cull, C. A., Z. D. Paddock, T. G. Nagaraja, N. M. Bello, A. H. Babcock, and D. G. Renter. 2012. Efficacy of a vaccine and a direct-fed microbial against fecal shedding of Escherichia coli O157:H7 in a randomized pen-level field trial of commercial feedlot cattle. Vacine 30:6210-6215.
- Bai, J., Z. D. Paddock, X. Shi, S. Li, B. An, and T. G. Nagaraja.. 2012. Applicability of a Multiplex PCR to detect the seven major Shiga toxin-producing Escherichia coli based on genes that code for serogroup-specific O-antigens and major virulence factors in cattle feces. Foodborne Path Dis. 9:541-548.
- Paddock, Z., X. Shi, J. Bai, and T.G. Nagaraja. 2012. Applicability of a multiplex PCR to detect O26, O45, O103, O111, O121, O145, and O157 serogroups of Escherichia coli in cattle feces. Vet. Microbiol. 156:381-388.
- Jacob, M. E., X. Shi, B. An, T. G. Nagaraja, and J. Bai. 2012. Evaluation of a multiplex real-time PCR for the quantification of Escherichia coli O157 in cattle feces. Foodborne Path. Dis. 9:79-85.
- Paddock, Z. D., X. Shi, T. G. Nagaraja, and J. Bai. 2011. Detection of major serotypes of Shiga-toxin producing E. coli in bovine feces by multiplex PCR. J. Anim. Sci. 89 (E suppl.).
- Schaefer, L., Z. Paddock, C. Cull, X. Shi, S. Li, J. Bai, T. G. Nagaraja, and D. Renter. 2010. Prevalence of Shiga toxin-producing Escherichia coli serogroup O26 in feces of feedlot cattle. (Abstract # 038). Poster presented in the Conference for Research Workers in Animal Diseases, Chicago, IL (Dec 4-6, 2011).
- Paddock, Z, D. G. Renter, C. A. Cull, L. A. Schaffer, X. Shi, J. Bai, and T. G. Nagaraja. 2011. Fecal shedding of Escherichia coli O26 in feedlot cattle from a field trial evaluating Escherichia coliO157:H7 vaccine and a direct-fed microbial (Abstract # 087). Paper presented in the Conference for Research Workers in Animal Diseases, Chicago, IL (Dec 4-6, 2011).
- Cull, C. A., D. G. Renter, Z. Paddock, N. M. Bello, A. H. Babcock, and T. G. Nagaraja. 2011. Effects of a vaccine and a direct-fed microbial on fecal shedding of Escherichia coli O157:H7 in pens of commercial feedlot cattle fed a diet supplemented with distiller's grains (Abstract # 087). Paper presented in the Conference for Research Workers in Animal Diseases, Chicago, IL (Dec 4-6, 2011).
- Jacob, M. E., K. F. Lechtenberg, L. L. Burnham, D. Haverkamp, and T. G. Nagaraja. 2010. Feeding MSE direct-fed microbials in finishing cattle decreases the fecal shedding of Escherichia coli O157:H7. Paper presented at the Midwest Animal Science and Dairy Science Associations Meeting, Des Moines, IA (Mar 15-17, 2010).
- Jacob, M. E., D. G. Renter, Z. D. Paddock, K. F. Lechtenberg, and T. G. Nagaraja. 2010. Feeding dried or wet distillers grains at varying inclusion levels to feedlot cattle affects the fecal prevalence of Escherichia coli O157:H7. Paper presented at the Midwest Animal Science and Dairy Science Associations Meeting, Des Moines, IA (Mar 15-17, 2010).
- Paddock, Z. D., C. E. Walker, J. S. Drouillard, D. G. Renter, and T. G. Nagaraja. 2011. Monensin level, supplemental urea, and administration of ractopamine on fecal shedding of Escherichia coli O157:H7 in feedlot cattle (Abstarct # 1086). J. Anim. Sci 88 (E-Suppl.):854.
- Wilson, B. K., B. P. Holland, T. G. Nagaraja, and C. R. Krehbiel, 2011. Feeding Lactobacillus acidophilus combined with Propionibacterium freudenreichii to determine performance and carcass characteristics in feedlot heifers fed with or without wet distiller's grains plus solubles. (Abstarct # 206). J. Anim. Sci 88 (E-Suppl.):232.
- Jacob, M. E., B. An, X. Shi, T. G. Nagaraja, and J. Bai. 2010. A multiplex, real-time PCR assay based on stx1, stx2, and rfbE for the quantification of Escherichia coli O157 in cattle feces (Abstract # 089). Paper presentation in the Conference for Research Workers in Animal Diseases, Chicago, IL (Dec 7-9, 2010).
- Paddock, Z, X. Shi, T. G. Nagaraja, and J. Bai. 2010. Development of a multiplex for the detection of major serotypes of Shiga toxin producing E. coli in bovine feces (Abstract # 088). Paper presentation in the Conference for Research Workers in Animal Diseases, Chicago, IL (Dec 7-9, 2010).
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: Escherichia coli O157:H7, a food borne pathogen, is of major public health concern. Cattle are natural reservoirs of this organism and the organism inhabits the hindgut and is then shed in the feces, which serves as a major source of contamination of human food and water. The goals of our research program are to develop improved methods to detect and quantify E. coli O157:H7, improve our understanding of the ecology of E. coli O157:H7 in cattle operations, and to identify and test on-farm intervention strategies for control of E. coli O157:H7. Inclusion of distiller's grains (DG) in cattle diets has been shown to increase fecal shedding of E. coli O157:H7. It is hypothesized that altered gut fermentation by DG may be responsible for the positive association. Therefore, feed additives affecting ruminal or hindgut fermentation of DG also may impact fecal shedding of E. coli O157:H7. A study was conducted to evaluate effects of monensin (33 or 44 mg/kg DM), supplemental urea (0, 0.35, or 0.70% of DM), and ractopamine (0 or 200 mg/steer daily administered during the last 42 d of finishing) in a steam-flaked corn grain-based diet containing 30% wet sorghum DG on fecal shedding of E. coli O157:H7. Seven-hundred and twenty steers, housed in 48 pens (15 steers/pen), were assigned to dietary treatments. Pen floor fecal samples (10 per/pen) were collected every 2 wk for 14 wk and cultured for E. coli O157:H7. Mean fecal prevalence of E. coli O157:H7 was 7.6%, and ranged from 1.6 to 23.6%. Cattle fed monensin at 44 mg/kg of feed had lower (P = 0.05) fecal E. coli O157:H7 prevalence than cattle fed 33 mg/kg (4.3 vs 6.8%). Supplemental urea at 0.35 or 0.70% had no effect (P = 0.87) on fecal shedding of E. coli O157:H7. Fecal prevalence of E. coli O157:H7 were 5.3, 5.7, and 5.9 % for groups fed 0, 0.35 and 0.7% urea, respectively. The inclusion of ractopamine at 0 or 200 mg/head/d had no effect (P = 0.89) on fecal prevalence of E. coli O157:H7 (4.4 vs 4.0 %). Another study was conducted to determine the genetic diversity of E. coli O157:H7 within a single individual bovine fecal sample based on PFGE typing. Fecal samples (n=601) were collected from dairy and beef cattle at three separate facilities. The prevalence of E. coli O157:H7 was 7.7% (46/601). From each positive fecal sample, up to 10 putative colonies were tested and isolates from samples with at least 7 positive colonies were subtyped using PFGE and tested for six major virulence genes by multiplex PCR. A total of 254 E. coli O157:H7 isolates from 27 samples met these criteria and were included in PFGE analysis. Fifteen PFGE subtypes (< 100% Dice similarity) were detected among the 254 isolates and there were no common subtypes between the three locations. Seven of 27 (26%) fecal samples had E. coli O157:H7 isolates with different PFGE subtypes (mean = 2.1) within the same sample. The virulence gene profiles of different isolates from the same sample were always identical, regardless of the number of PFGE types. PARTICIPANTS: T. G. Nagaraja: Microbiologist; Principal investigator; involved in all planning and decision making processes, managing funds, and overseeing laboratory analysis and reporting of results. David Renter: Epidemiologist; Co-Principal investigator; Responsible for the study design and statistical analysis; Also involved in all planning and decision making processes and overseeing laboratory analysis and reporting of results. Richard Oberst: Molecular Biologist; Involved in PCR and PFGE analyses Mike Sanderson: Epidemiologist; Involved in study design and statistical analysis Ludek Zurek: Medical Entomologist; Involved in studies on flies and E. coli O157:H7 Laboratory technicians: Xiaorong Shi and Neil Wallace Xiaorong Shi: Responsible for bacteriological procedures and molecular analyses (PCR and PFGE typing of isolates). Neil Wallace: Responsible for bacteriological procedures, ordering supplies, maintaining inventory and supervising undergraduate students. Graduate students Ph. D. students: Charles Dodd, Trent Fox, Megan Jacob, Zac Paddock MPH students: Ethel Taylor Graduate students are responsible for sample collection and processing in laboratory, recording of data, analysis of results, conference presentations and manuscript writing. Undergraduate students: A number of graduate students work part time in the laboratory. The students are trained in laboratory safety and analytical skills. The students presently working in the laboratory are: Blair Wyrick, Samantha Smith, Cody Strodtman, Katie Flock, Kristina Wert, Nick Reams, and Eric Haney. TARGET AUDIENCES: Scientists, Veterinarians, Food Safety Inspection Agency, Cattlemen, Packers, Students PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The food supply in the United States is one of the safest in the world; however, food-borne illnesses do occur and frequently are associated with foods derived from animal agriculture. Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (non-O157 serotypes) are important food-borne pathogens that cause hemorrhagic colitis, hemolytic uremic syndrome and thrombocytopenic purpura in humans. Besides food, there are other modes of transmission such as direct contact with cattle (farm workers, farm visitors, children visiting fairs and petting zoos), exposure to contaminated drinking and recreational water, and consumption of contaminated vegetables and fruits. Despite rigorous surveillance of meat processors by the USDA Food Safety and Inspection Service following the 1994 regulatory declaration of E. coli O157:H7 as an adulterant in raw ground beef, the number of confirmed human illnesses caused by E. coli O157:H7 continues at unacceptable levels. Control strategies aimed at reducing the prevalence and concentration of E. coli O157:H7 in cattle feces, thus reducing the overall number of bacteria entering both the food and environmental pathways, may be the most effective approach for reducing the overall risk of human infections. Furthermore, effective control of E. coli O157:H7 in the beef food supply chain will require interventions at multiple levels and by multiple means. The study to evaluate dietary feed additives on fecal shedding of E. coli O157:H7 suggests that inclusion of supplemental urea had no effect. Our premise in testing the effect of supplemental urea was the potential effects of altered ruminal and possibly hindgut fermentation on E. coli O157:H7. Because we did not monitor ruminal or hindgut fermentation changes, we have no evidence that fermentation was altered by feeding supplemental urea. Similarly, the low prevalence of E. coli O157:H7 during ractopamine feeding precluded us from assessing the impact of ractopamine. The reduction in fecal shedding of E. coli O157:H7 associated with feeding the high dose of monensin is interesting and somewhat surprising considering that prior studies (although with lower doses) had shown no effect. Therefore, additional research is needed to confirm the reduction in fecal shedding of E. coli O157:H7 in cattle fed 44 mg/kg monensin. The results of the study on genetic diversity of E. coli O157:H7 suggest that multiple PFGE subtypes are present in cattle feces, and determining the PFGE pattern of a single isolate from a bovine sample may not be sufficient when comparing to other isolates from feces, hides or carcasses. The long term goal of our studies is to reduce the E. coli O157 load in cattle presented for slaughter. Control strategies aimed at reducing the prevalence and concentration of E. coli O157:H7 in cattle feces, thus reducing the overall number of bacteria entering both the food and environmental pathways, may be the most effective approach for reducing the overall risk of human infections. Furthermore, effective control of E. coli O157:H7 in the beef food supply chain will require interventions at multiple levels and by multiple means.
Publications
- Jacob, M. E., Callaway, T. R., and Nagaraja, T. G. (2009). Dietary interactions and interventions affecting Escherichia coli o157 colonization and shedding in cattle, Foodborne Pathogens and Diseases, 6:785-792.
- Jacob, M. E., Fox, J. T., Reinstein, S., and Nagaraja, T. G. (2008). Antimicrobial susceptibility of foodborne pathogens in organic or natural production systems: an overview, Foodborne Pathogens and Diseases, 5:720-730.
- Fox, J. T., Reinstein, J., Jacob, M. E., and Nagaraja, T. G. (2008). Niche marketing production practices for beef cattle in the United States and prevalence of foodborne pathogens, Foodborne Pathogens and Diseases, 5:1-12.
- Paddock, Z. D., Walker, C. E., Drouillard, J. S., and Nagaraja, T. G. (2011). Dietary monensin level, supplemental urea, and ractopamine on fecal shedding of Escherichia coli O157:H7 in feedlot cattle. Journal of Animal Sciences, 89:2829-2835.
- Jacob, M. E., Almes, K. M., Shi, X., Sargeant, J. M., and Nagaraja, T. G. (2011). Escherichia coli O157 diversity in bovine fecal samples. J. Food Prot. 74:1186-1188.
- Bai, J., Shi, X and Nagaraja, T. G. (2008). An improved multiplex PCR method to detect eae, stx1, stx2, fliC, and hlyA in Escherichia coli O157:H7. Poster presented at the 108 th General Meeting of the American Society for Microbiology, Boston, MA., June 1-5.
- Paddock, Z. D., Walker, C. E., Drouillard, J. S., Renter, D. G., and Nagaraja, T. G. (2010). Monensin level, supplemental urea, and administration of ractopamine on fecal shedding of Escherichia coli O157:H7 in feedlot cattle (Abstract # 1086). J. Anim. Sci. 88 (E-Suppl.):854.
- Paddock, Z, Shi, X., Nagaraja, T. G., and Bai, J. (2010). Development of a multiplex for the detection of major serotypes of Shiga toxin producing E. coli in bovine feces (Abstract # 088). Paper presentation in the Conference for Research Workers in Animal Diseases, Chicago, IL (Dec 7-9).
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