Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Scientists, Veterinarians, Food Safety Inspection Agency, Cattlemen, Packers, Students Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The research project has provided opportunities for the following students to obtain graduate degrees in Pathobiology, specifically in Microbiology and Epidemiology: Charley Cull: PhD Diana Dewsbury: MS Pius Ekong: PhD Lance Noll: MS Pragathi Shridhar: PhD The graduate students get trained and become proficient in designing the study, collection of samples, processing of samples, microbiological and molecular analyses of samples, data collection and management, statistical analyses, and data interpretation. Students have opportunities to present their findings as poster or oral presentations in a number of scientific conferences. Also, students receive the training in writing manuscripts for submission to peer-reviewed journals for publications. Additionally, a number of undergraduate students, including freshman and sophomore DVM students, have worked in the laboratory and received training in culture and PCR-based methods to detect foodborne pathogens. How have the results been disseminated to communities of interest? The following presentations were made in a variety of conferences and meetings: Nagaraja T.G. (invited): Diet, Hindgut Ecosystem, and Foodborne Pathogens in Cattle. Glance 2014-Global Animal Nutrition Conference in Bangalore, India (Apr 20-23, 2014). Nagaraja T.G. PCR- and culture-based methods of detection of STEC-7 in cattle feces. Shiga toxin-Producing Escherichia coli-Coordinated Agricultural Project Annual Conference, Lincoln, NE (May 28-29, 2014). Nagaraja T.G. (invited): Detection methods for STEC-7. Shiga toxin-Producing Escherichia coli-Coordinated Agricultural Project Annual Meeting, Indianapolis, IN (Aug 4-6, 2014). Nagaraja T.G. (invited): Future use of antimicrobial additives and alternatives to prevent acidosis. Ruminant Nutrition Conference, Universidad Autónomo de Nuevo León, Monterrey, Mexico (Oct 7-8, 2014). Nagaraja T.G. (invited): Shiga toxin-producing Escherichia coli. Meeting of the NC 1202 Regional Research Technical Committee on Enteric Diseases of Food Animals: Enhanced Prevention, Control and Food Safety, Chicago, IL (Dec 6-7, 2014) Renter D. G. (invited): Ecology and Epidemiology of STEC in the Feedlot Through Harvest. In a Symposium entitled "Update on the STEC CAP Grant", at the Annual Meeting of the International Association of Food Protection, Indianapolis, IN, August 4, 2014. Renter D. G. (invited): Recent Research on the Epidemiology of STEC in Beef Production Systems. 2014 Governor's Conference on Ensuring Food Safety and the Annual Meeting of the Shiga toxin-producing Escherichia coli Coordinated Agriculture Project (STEC CAP), Lincoln, NE. 2014. Renter D. G. (invited): Salmonella Research 2013 - results of SRP® vaccine efficacy trial and related studies. Zoetis sponsored Salmonella Research Cluster meeting; in conjunction with the Beef Industry Safety Summit. Dallas, TX. 2014. Renter D. G. (invited): Shiga toxin-producing E. coli CAP Grant: preharvest progress and plans. Beef Industry Safety Summit, Dallas, TX. 2014. Noll, L. W., Belagola, P. B. Shridhar, W. C. Baumgartner, X. Shi, T. G. Nagaraja. Evaluation of chromID EHEC agar for detection of seven major serogroups of Shiga toxin-producing Escherichia coli from cattle feces. Annual International Association for Food Protection Conference, Indianapolis, Indiana, August 3-6, 2014 (Poster presentation). Noll, L. W., P. B. Shridhar, D. Dewsbury, X. Shi, N. Cernicchiaro, D. G. Renter, and T. G. Nagaraja. Prevalence of seven major serogroups of Shiga toxin-producing Escherichia coli in cattle feces from a large commercial feedlot. Governor's E. coli Conference/Shiga toxin-producing E. coli Coordinated Agricultural Project Annual Conference, Lincoln, NE., May 27-29, 2014 (Poster presentation). Noll, L. W., P. B. Shridhar, X. Shi, B. An, T. G. Nagaraja, and J. Bai. A four-plex real-time PCR assay for the detection and quantification of Escherichia coli O157 in cattle feces. BioKansas One Health Summit Annual Conference, Kansas City, Kansas, Mar 5, 2014 (Poster presentation). Shridhar, P. B., L. Noll, X. Shi, N. Cernicchiaro, D. G. Renter, J. Bai and T. G. Nagaraja, Prevalence, isolation and characterization of E. coli O104 in cattle feces. Annual American Association of Veterinary Laboratory Diagnosticians Meeting, Kansas City, October 16-22, 2014 (Oral presentation). Shridhar, P. B., L. Noll, B. An, X. Shi, T. G. Nagaraja and J. Bai. TaqMan-based multiplex real time PCR assays for the detection and quantification of the six major non-O157 Shiga toxin-producing Escherichia coli in cattle feces. Annual International Association for Food Protection Conference, Indianapolis, Indiana, August 3-6, 2014 (Oral presentation). Shridhar, P. B., L. Noll, X. Shi, N. Cernicchiaro, J. Bai and T. G. Nagaraja. Prevalence and characterization of E. coli O104 in cattle feces. Annual International Association for Food Protection Conference, Indianapolis, Indiana, August 3-6, 2014 (Oral presentation). Shridhar, P. B., L. Noll, B. An, X. Shi, N. Cernicchiaro, D. G. Renter, J. Bai and T. G. Nagaraja. Spiral plating method to quantify six major non-O157 E. coli serogroups in cattle feces. Governor's E. coli Conference/Shiga toxin-producing E. coli Coordinated Agricultural Project Annual Conference, Lincoln, NE., May 27-29, 2014 (Poster presentation). Shridhar, P. B., L. Noll, B. An, X. Shi, N. Cernicchiaro, J. Bai, and T. G. Nagaraja. Prevalence and characterization of E. coli O104 in cattle feces, Annual Phi-Zeta Research Day, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, March 4, 2014 (Oral presentation). Shridhar, P. B., L. Noll, B. An, X. Shi, N. Cernicchiaro, J. Bai, and T. G. Nagaraja. Development of multiplex real time PCR assays for the detection and quantification of the six major non-O157 Escherichia coli serogroups. Capitol Graduate Research Summit (CGRS), Topeka, Kansas, Feb 12, 2014 (Poster presentation). Shridhar, P. B., Noll L.W., Kim E., Cull C. A., Dewsbury D. M., Shi X., Cernicchiaro N., Renter D. G., Bai J., Nagaraja T.G. Quantification of six non-O157 E. coli serogroups in cattle feces by spiral plating method. Conference of Research Workers in Animal Disease, Chicago, IL, 014. 048 (Oral presentation).. Cull, C.A., Renter D.G., Dewsbury D.M., Noll L.W., Shridhar P. B., Shi X., Nagaraja T.G., Cernicchiaro N. Feedlot- and pen-level prevalence of Shiga toxin-producing Escherichia coli in feces of commercial feedlot cattle. Conference of Research Workers in Animal Disease, Chicago, IL, 2014. 046 (Oral presentation).. Dewsbury, D.M., Noll L.W., Shridhar P.B., Shi X., Renter D.G., Nagaraja T.G., Cernicchiaro N. Summer and winter prevalence of O26, O45, O103, O111, O121, O145 and O157 Shiga toxin-producing Escherichia coli (STEC) in feces of feedlot cattle. Conference of Research Workers in Animal Disease, Chicago, IL, 2014. 045 (Oral presentation).. Noll, L.W., Baumgartner W.C., Shridhar P. B., Cull C.A., Dewsbury D.M., Shi X., Cernicchiaro N., Renter D. G., Nagaraja T.G. Pooling of immunomagnetic separation beads does not affect sensitivity of detection of seven serogroups of Shiga toxin-producing Escherichia coli in cattle feces. Conference of Research Workers in Animal Disease, Chicago, IL, 2014. 129 (Oral presentation). Moxley, R. (invited): Renter, D., Luchansky, J., Chapman, B., Ekong, P., Sanderson, M., Gallagher, D., and G. Acuff. 2014. Update on the Shiga toxin-producing Escherichia coli Coordinated Agricultural Project (STEC-CAP). J. Food Prot. Supplement A, 77:6 (Abstract S7). Gallagher DL, (invited): Sanderson M, Ekong, P. Quantitative Risk Assessment Model for STEC in the Beef Continuum. Int. Assoc Food Protection Annual Meeting, Indianapolis, IN. August 3-6, 2014. Sanderson, M.W. (invited): Ekong, PS. Gallagher. D. L. Quantitative Microbial Risk Assessment: An Update on Objective 4 of the STEC-CAP Grant. Proceedings 4th Governor's Conference on Ensuring Food Safety, 2nd STEC CAP Annual Conference. Lincoln, NE. May 27-29, 2014. Ekong, PS. Sieg, J. Sanderson, M.W. A systematic review of harvest intervention for the reduction of shiga-toxin producing Escherichia coli on beef carcass surfaces. Proceedings 4th Governor's Conference on Ensuring Food Safety, 2nd STEC CAP Annual Conference. Lincoln, NE. May 27-29, 2014. Sieg, J. Ekong PS. Sanderson, M.W. A systematic review of peri-harvest intervention for the reduction of shiga-toxin producing Escherichia coli on cattle hides. Proceedings 4th Governor's Conference on Ensuring Food Safety, 2nd STEC CAP Annual Conference. Lincoln, NE. May 27-29, 2014. What do you plan to do during the next reporting period to accomplish the goals? Evaluation of the applicability of Real-Time PCR assays to detect and quantify seven serogroups of Shiga-toxin producing E. coli in cattle feces. Evaluation of the applicability of the spiral plate method to quantify Shiga-toxin producing E. coli in cattle feces, hide and carcass samples. Development, validation and applicability of Real-Time PCR assay to detect and quantify Salmonella in cattle feces and lymph nodes. Study on bacterial probiotics in cattle and swine to determine whether they carry antimicrobial resistance genes.
Impacts
What was accomplished under these goals?
The Research Team has focused on Shiga toxin-producing Escherichia coli (STEC) and antimicrobial resistance in gut bacteria. STEC: Shiga toxin-producing E. coli (STEC) are major foodborne pathogens that cause illnesses in humans with symptoms ranging from diarrhea, with or without blood, to hemolytic uremic syndrome, and even death. Among STEC, the serotype O157:H7 has caused a greater number of foodborne outbreaks than any other serotype. Recent epidemiological data have shown that six more serogroups of STEC (O26, O45, O103, O111, O121, and O145) represent a major portion of foodborne STEC infections. A study was conducted to determine fecal prevalence and characteristics of E. coli O26, which second only to O157 in causing STEC infections in commercial feedlot cattle. Pen floor fecal samples (n=4,800) were enriched in E. coli broth and subjected to a multiplex PCR designed to detect O26-specific wzx gene and four major virulence genes (stx1, stx2, eae, and ehxA) and to a culture-based procedure that involved immunomagnetic separation and plating on MacConkey agar. The overall prevalence E. coli O26 was higher (P < 0.001) by the culture-based method compared to the PCR assay (22.7 vs.10.5%). Of the 260 isolates of O26 obtained in the study, only seven were positive for stx gene and a majority of them possessed an eaeβ gene that codes for intimin subtype β, but not the bfpA gene, which codes for bundle-forming pilus. Therefore, the majority of the O26 recovered from feedlot cattle feces was atypical enteropathogenic E. coli, and not STEC. Culture-based methods to detect and isolate all six major non-O157 STEC are not well established. We have developed a culture-based method and compared with PCR method for the detection of six non-O157 STEC serogroups in cattle feces. Fecal samples (n=576) were collected in a commercial feedlot and enriched in E. coli broth at 40 C for 6 h. Enriched samples were subjected to immunomagnetic separation (IMS), spread-plated onto selective media and initially pooled colonies, and subsequently, single colonies were tested by 7-plex (seven serogroups only) and 11-plex PCR (seven serogroups plus four virulence genes, stx1, stx2, eae, and ehxA), respectively. Fecal suspensions, before and after enrichment, were tested by an 11-plex PCR targeting seven serogroups and four virulence genes. There was no difference in the proportions of fecal samples that tested positive (84.0 vs. 83.5%) for one or more of the seven serogroups by either PCR or the culture method. Both culture method and PCR indicated that O26, O45, O103, and O157 were the dominant serogroups. None of the fecal samples contained more than five serogroups by either method. Higher proportions (P > 0.05) of fecal samples were positive for O26 (44.5 vs.22.7%), O121 (22.9 vs.2.3%) and O157 (54.7 vs. 42.9%) serogroups by PCR than by the culture method; however, each method detected O26, O45, O103 and O157 in samples that were negative by the other method. Also, 99/576 (17.2%) samples harbored STEC not associated with one of the seven serogroups. Our data indicate that neither culture nor PCR method was better than the other in detecting the seven serogroups in cattle feces, and only a small proportion of the non-O157 serogroups (23/640; 3.6%) isolated carried Shiga toxin genes. Another study was conducted to estimate the prevalence of E. coli O104 serogroup in feedlot cattle feces and characterize the isolated strains. The reason for the study was that a unique serotype of O104:H4 was reported in a 2011 foodborne outbreak in Germany, which possessed characteristics of two pathotypes, STEC and enteroaggregative E. coli. Fecal samples (n=757) were enriched in E. coli broth for 6 h at 400C. DNA extracted from pre and post enriched fecal samples were tested by a multiplex PCR to detect serogroup O104 and associated virulence genes of the hybrid strain. Post-enriched fecal samples were also subjected to culture-based method of detection that involved serogroup-specific immunomagnetic separation. Of the 757 fecal samples, 38 (5%) were positive before enrichment and 349 were positive (46%) after enrichment for O104 serogroup specific gene. A total of 143 O104 isolates was obtained in pure culture, 92 of them were positive for O8/O9 serogroup antigen genes. Of the 51 O104 isolates, only 16 (31.4%) carried stx1, none of them carried eae. The stx1 was of the stubtype 1c in all 16 isolates. The O104 isolates harbored diverse flagellar (H) antigens with 37 isolates containing H7, 4 H2, 1 H21 and 1 H1. Cattle shed serogroup O104 in feces, but only a few strains (31.4 %) carried stx1 gene and none of the isolated strains carried genes characteristic of the hybrid pathotype. Antimicrobial resistance: Feed grade antimicrobials are supplemented in cattle and s wine diets to promote growth, improve feed efficiency, and to reduce the incidence of respiratory or gut infections. The use of antimicrobial feed additives is controversial because of the emergence and potential dissemination of antimicrobial resistance and subsequent risk to human health. Therefore, alternatives to conventional antimicrobials, such as heavy metals, are being used to achieve improved animal performance. Copper, an essential micronutrient, is supplemented in feedlot cattle diets at elevated levels to promote growth and improve feed efficiency. Resistance to copper among gut bacteria is common and in enterococci, a transferable copper resistance gene, tcrB, is linked to erm(B) and tet(M) genes on a plasmid. The potential genetic link between copper and antibiotic resistance is of interest because tylosin, a macrolide, and tetracyclines are widely used as feed additives in feedlot cattle. A study was conducted to investigate whether feed supplementation of copper, at elevated level, co-selects for macrolide resistance in fecal enterococci in the absence of tylosin. A longitudinal study was conducted in cattle (n=80) with 2×2 factorial arrangements of copper (10 or 100 mg/kg of feed) and tylosin (0 or 10 mg/kg of feed). Thirty-seven isolates (4.6%; 37/800) were positive for the tcrB and all were E. faecium. The prevalence was higher among cattle fed diets supplemented with copper and tylosin (8.5%) compared to control (2.0%), copper (4.5%), and tylosin (3.5%) alone. All tcrB-positive isolates were positive for erm(B) and tet(M) genes. Median copper MICs for tcrB-positive and tcrB-negative enterococci were 20 and 4 mM, respectively. The overall prevalence of the erm(B) and tet(M) genes among enterococcal isolates was 46.8%and 57.5% , respectively. Feeding of elevated dietary copper and tylosin alone or in combination resulted in an increased prevalence of tcrB and erm(B) mediated copper and tylosin resistant fecal enterococci in feedlot cattle. A longitudinal study was conducted to investigate the effects of in-feed copper, chlortetracycline, and tylosin alone or in combination on the selection and co-selection of antimicrobial resistant enterococci. The study included 240 weaned piglets assigned randomly to six dietary treatment groups; control, copper, chlortetracycline, tylosin, copper and chlortetracycline, and copper and tylosin. Feces was collected before, during, and after initiating treatment, and enterococcal isolates were tested for genotypic and phenotypic resistance to copper and antibiotics. The overall prevalence of tcrB-positive enterococci was 14.3% (372/2,592). Among the tcrB-positive isolates, 331 were E. faecium and 41 were E. faecalis. All tcrB-positive isolates contained both erm(B) and tet(M) genes. The median MIC of copper for tcrB-negative and tcrB-positive enterococci was 6 and 18 mM, respectively. In conclusion, supplementing copper or antibiotics alone did not increase copper resistant enterococci; however, supplementing antibiotics with copper increased the prevalence of tcrB gene among fecal enterococci of piglets.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Jacob M. E., J. Bai J, D. G. Renter, A. T. Rogers, X. Shi and T. G. Nagaraja. 2014. Comparing real-time and conventional PCR to culture-based methods for detecting and quantifying Escherichia coli O157 in cattle feces. J Food Prot. 77:314-319.
Paddock Z. D, D. G. Renter, C. A. Cull, J. Bai, and T. G. Nagaraja*. 2014. Escherichia coli O26 in feedlot cattle: Fecal prevalence, isolation, characterization and effects of an E. coli O157 vaccine and a direct-fed microbial. Foodborne Pathog Dis. 11: 186-193.
Cernicchiaro, N., D. G. Renter, C. A. Cull, Z. D. Paddock, X. Shi, and T. G. Nagaraja. 2014. Fecal shedding of non-O157 serogroups of Shiga toxin-producing Escherichia coli in feedlot cattle vaccinated with an Escherichia coli O157:H7 SRP vaccine or fed a Lactobacillus-based direct-fed microbial. J. Food Prot. 77:732-737.
Agga, G. E., H. M. Scott, R. G. Amachawadi, T. G. Nagaraja, J. Vinasco, J. Bai, B. Norby, D. G. Renter, S. S. Dritz, J. L. Nelssen, and M. D. Tokach. 2014. Effects of chlortetracycline and copper supplementation on antimicrobial resistance of Escherichia coli isolates from nursery pigs: Phenotypic and Genotypic analysis. Prev.Vet. Med. 114:231-246
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