Source: KANSAS STATE UNIV submitted to
PRE-HARVEST FOOD SAFETY AND FOOD ANIMAL PRODUCTIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0217044
Grant No.
(N/A)
Project No.
KS419
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2008
Project End Date
Sep 30, 2014
Grant Year
(N/A)
Project Director
Nagaraja, TI, G.
Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506
Performing Department
Diagnostic Medicine & Pathobiology
Non Technical Summary
The food supply in the US is one of the safest in the world; however, foodborne illnesses do occur and frequently are associated with foods derived from animal agriculture. The livestock and meat industries have continuously sought improvements in the safety of their products and have demanded efforts to eliminate or reduce pathogen load, focusing on both pre- and post-harvest intervention strategies. The two foodborne pathogens of interest are Shiga-toxin-producing E. coli and Salmonella. Cattle feces are a major source of contamination of beef products, produce, and recreational and drinking water. Despite rigorous surveillance of meat processors by the USDA Food Safety and Inspection Service, the number of confirmed human illnesses caused by E. coli O157:H7 and Salmonella continues at unacceptable levels. Control strategies aimed at reducing the prevalence of E. coli O157:H7 and Salmonella in cattle and swine, thus reducing the overall number of bacteria entering both the food and environmental pathways, may be the most effective approach for reducing the risk of human infections. Antimicrobial resistance is a naturally occurring biological phenomenon amplified because of misuse and overuse of antimicrobial drugs. The proposed research would greatly enhance our understanding of the ecology of E. coli O157:H7, Salmonella, and antimicrobial resistant bacteria in cattle and swine, including contamination of carcasses at slaughter, and provides an opportunity to evaluate existing and novel intervention strategies to mitigate risks associated with foodborne pathogens.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7123310104060%
7123310110040%
Goals / Objectives
The research will focus on prevalence, ecology and on-farm interventions of Shiga-toxin-producing Escherichia coli and Salmonella and prevalence, amplification and dissemination of antimicrobial resistance in cattle and swine. The specific objectives are: 1. Develop and improve methods to detect and characterize E. coli O157 and Salmonella serotypes in cattle and swine. 2. Study the prevalence and factors affecting gut persistence and fecal shedding of E. coli O157 and Salmonella serotypes. 3. Identify management strategies and production practices that might be manipulated to reduce fecal shedding. 4. Study the ecology (prevalence, amplification, and dissemination) of antimicrobial resistance in fecal bacteria in cattle and swine.
Project Methods
In the first objective, methods to detect E. coli O157 and Salmonella serotypes will be based on cultural procedures. We will evaluate the potential use of PCR-based (single or multiplex) procedure to initially screen samples. In the second objective, factors affecting gut persistence and fecal shedding will be determined in one more of the following ways: a. Natural prevalence field studies: Samples (feces, hide, or carcass) will be collected from feedlot cattle during the finishing phase or at a slaughter plant after cattle are slaughtered. b. Natural prevalence experimental studies: A large population of cattle will be prescreened and a subset of cattle confirmed as persistent shedders of E. coli O157 or Salmonella will be assigned to treatments. Experimental challenge studies: Cattle or swine in this study will be prescreened to assess the negative status for E. coli O157 or Salmonella and then orally inoculated with a cocktail of nalidixic acid-resistant E. coli O157 and Salmonella strains to determine the influence of various factors of interest. In vitro Fermentation studies: Batch-culture fermentation systems with ruminal microbial or fecal microbial inoculum will be used to test the effects of various factors (diets and dietary ingredients, feed additives, etc) on survival or growth of E. coli O157:H7 or other pathogens. For the third objective, control strategies aimed at reducing the prevalence 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 risk of human infections. This is a multi-step effort; potential management factors and production practices are initially identified in our studies on the ecology of E. coli O157 and Salmonella, and then these factors and practices will be structured into formal hypotheses for evaluation. In the objective 4, the effect of antibiotics and metals on the antimicrobial susceptibilities of foodborne pathogens (E. coli O157, Salmonella and Campylobacter) and commensal (E. coli and Enterococcus spp.,) bacteria in feces of cattle and swine will be studied. Antimicrobial susceptibility patterns of all isolates will be determined by microbroth dilution method manually or using automated commercial systems. Real-time PCR will be used to quantify specific antimicrobial resistance genes in feces.

Progress 10/01/08 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 a number of students to obtain graduate degrees in Pathobiology, specifically in Microbiology and Epidemiology. In the past 5 years, the following students have gotten their MS or PhDs and have moved on to have employment in academia, federal research labs, and or industry: Getahun Agga (PhD), Raghavendra Amachawadi (MS and PhD), Charles Dodd (PhD), Trent Fox (PhD), Megan Jacob (MS and PhD), Zac Paddock (PhD), and Greg Peterson (PhD). Currently, the following students are enrolled in graduate program and are working in the area of preharvest food safety: 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? Nagaraja, T. G. 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. Diet, Hindgut Ecosystem, and Foodborne Pathogens in Cattle. Invited presentation at the Glance 2014-Global Animal Nutrition Conference in Bangalore, India (Apr 20-23, 2014). What do you plan to do during the next reporting period to accomplish the goals? 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). Shridhar, P., L. W. Noll, B. An, X. Shi, T. G. Nagaraja, and J. Bai. Development of multiplex real time PCR assays for the detection and quantification of the top six non-O157 Escherichia coli serogroups. Phi Zeta Research Day, College of Veterinary Medicine, Kansas State University. March 5, 2013. Manhattan, KS.

Impacts
What was accomplished under these goals? Shiga toxin-producing Escherichia coli. Shiga toxin-producing Escherichia coli (STEC), particularly O157, are major food borne pathogens. In recent years, six non-O157 STEC, O26, O45, O103, O111, O121, and O145, have also been recognized as a major public health concern. A PCR assay was developed to detect six genes of E. coli O157:H7. An 11-plex PCR assay was developed to identify seven serogroups and four virulence genes (stx1, stx2, eae, and ehxA). The hindgut was the major site of prevalence of E. coli O157:H7 in cattle, a majority of the isolates within the same animal were clonally similar and acid tolerance of hindgut isolates were not different from that of ruminal isolates. Availability of mucous constituents, particularly gluconic acid, may explain the higher prevalence of E. coli O157 in the large intestine compared to the rumen of the digestive tract. Human clinical strains of E. coli O157:H7 produce more Shiga toxins (Stx2) than strains isolated from cattle. The presence of Q933 gene is a better indicator of high Stx2 production and may be a useful marker to assess potential of cattle strains to cause human disease. Distillers grains (DG), a co-product of ethanol production used as protein and energy supplements in cattle diets, was shown to increase fecal shedding of E 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. A study was conducted in experimentally inoculated steers 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 lack of effect of starch addition to the DG diet on shedding of E. coli O157 may be because either the decreased starch content in the DG-supplemented diet is not a factor in the increased shedding of E. coli O157 or inclusion of pure starch in the diet may not have achieved our intended goal to have starch flow into the hindgut similar to that of corn grain. Cattle feces pose a potential risk for E. coli O157 contamination of carcasses. Truckload may be an important factor in the potential transmission of E. coli O157, but isolates from carcasses also may be similar to those from feces of cattle on different truckloads and harvest days. The E. coli O157:H7 SRP® vaccine may serve as a preharvest intervention to reduce the burden of E. coli O157:H7 on cattle presented for slaughter. A study was conducted to determine whether fecal shedding of non-O157 STEC in feedlot cattle was affected by the use of an E. coli O157 vaccine and a direct-fed microbial, and to determine whether the shedding of a particular non-O157 STEC serogroups within feces was associated with O157 or other non-O157 serogroups. The vaccine, DFM or both had no significant effect on fecal prevalence of the six non-O157 STEC in feedlot cattle. Fecal shedding of O157 was associated with an increased probability of shedding of O26, O45, O103 and O121. A unique serotype of STEC, O104:H4, was the cause of a large food-borne outbreak of hemorrhagic colitis and hemolytic uremic syndrome in Germany in 201. Because cattle are known reservoirs of STEC, fecal carriage of E. coli O104 in feedlot cattle was investigated. A multiplex PCR was designed and validated to detect the genes characteristic of O104:H4. A total of 248 fecal samples from 8 feedlots were tested. Overall, 20.6% of fecal samples were positive for the serogroup-specific gene. None of the fecal samples was positive for the gene characteristic of the EAEC. The results suggest that E. coli O104 is present in cattle feces, but the strains did not carry genes characteristic of the hybrid strain responsible for the 2011German outbreak. Salmonella: We showed that vaccination of feeder cattle with the Salmonella Newport SRP® vaccine had no significant effect on Salmonella fecal prevalence, cattle health, or cattle performance. In a feedlot study, fecal prevalence of Salmonella within a cohort at feedlot entry was not associated with preharvest prevalence. Preharvest prevalence was positively associated with the number of days in the feedlot (P = 0.02), cumulative morbidity (P = 0.01), and cumulative mortality (P = 0.03). We recovered Salmonella isolates with identical PFGE profiles both at feedlot entry and preharvest and PFGE subtypes of Salmonella appear to persist within and among feedlot cohorts. We developed and validated two molecular methods for identification of Salmonella serovars. A 70mer oligonucleotide spotted microarray was developed that consisted of probes which detected genes responsible for genetic variation between isolates of Salmonella that can be used for serotyping. A multiplex PCR assay was also developed that are capable of identifying 42 serovars and provided a valuable prediction of the pathogenicity of the isolates by detecting virulence genes sseL, invA, and spvC. The gene spvC was the best predictor of pathogenicity. Antimicrobial resistance: Our study is the first report on the occurrence of the transferable copper resistance (tcrB) gene in enterococci of swine in the U. S. Supplementation of copper at elevated level appears to select for resistance. The predominant tcrB-positive species were E. faecium and E. faecalis. All tcrB-positive isolates contained macrolide resistance (erm[B]) gene but none had vancomycin resistance (vanA) gene. The significance of this finding is the potential association between copper resistance and resistance to other antibiotics and the propensity of enterococci to transfer tcrB and antibiotic resistance genes to other strains within the same species, other enterococcal species, and possibly to species of other genera. Antibiotic resistant enterococci are opportunistic and nosocomial pathogens in humans. Because of the widespread occurrence of resistant enterococci in animals, it has been suggested that these enterococci may serve as a reservoir of potential resistant genes capable of transferring from animal to human bacteria. Zinc (Zn) is also supplemented at elevated concentrations in swine diets to promote growth and to prevent enteric infections. Studies from Denmark have suggested a genetic linkage and an association between Zn resistance, encoded by czrC, and methicillin-resistance, encoded by mecA, in Staphylococcus aureus. Such an association has not been reported in the U.S. swine. Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a potentially opportunistic zoonotic pathogen with swine as a primary reservoir around the world. A preliminary study was conducted to investigate the effects of Zn on the MRSA prevalence in nursery (n=40) and finisher pigs (n=40). Nasal swabs, collected from nursery and finisher pigs, were inoculated on to a chromogenic medium to isolate MRSA. The prevalence of MRSA was 10% and 20% among nursery and finisher pigs, respectively. The presence of MRSA was positively associated with higher levels of Zn supplementation. The finding of strong associations between copper and zinc resistance and other antibiotic resistance determinants is significant and potentially of public health concern. A critical issue is the co-selection of metal and antibiotic resistance on the same genetic determinant, often on a plasmid or a transposon. These plasmids or transposons are of concern because they harbor antibiotic resistance genes and have the potential to spread between species. The conjugative transposons have a broad bacterial host range and are capable of being transferred horizontally to a variety of gram positive and gram negative bacteria in the gut bacterial community. The existence of a metal-associated co-selection mechanism could potentially be a major issue relative to public health and further studies are needed to determine the magnitude of such an association.

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


Progress 01/01/13 to 09/30/13

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? Nothing Reported How have the results been disseminated to communities of interest? Antimicrobial resistance: The use of in-feed antimicrobials for growth promotion and disease prevention is an integral part of swine and cattle production and management. Because of concerns that bacteria, including pathogens, could develop resistance to antibiotics and get transfered to humans via food chains, alternatives to antibiotics, such as heavy metals (copper and zinc), are increasingly employed in swine production systems. Copper is often used at pharmacological levels for growth promotion in cattle and swine diets. Gut bacteria, particularly enterococci, exposed to copper acquire resistance, which is conferred by a transferable copper resistance (tcrB) gene carried on a plasmid. The plasmid also carries macrolide resistance gene, erm(B), suggesting that copper supplementation may co-select for macrolide resistance. A study was conducted to determine the relationship between copper and tylosin supplementations on the prevalence of copper and macrolide resistance genes. Copper and tylosin supplementations increased the prevalence of tcrB and erm(B) in fecal enterococci. Cattle fed both copper and tylosin had higher prevalence of tcrB- and erm(B)-positive enterococci compared to the control. Results provide evidence that copper supplementation has the potential to co-select for macrolide resistance and raises possibility for horizontal gene transfer to other gut bacteria. Zinc (Zn) is also supplemented at elevated concentrations in swine diets to promote growth and to prevent enteric infections. Studies from Denmark have suggested a genetic linkage and an association between Zn resistance, encoded by czrC, and methicillin-resistance, encoded by mecA, in Staphylococcus aureus. Such an association has not been reported in the U.S. swine. Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a potentially opportunistic zoonotic pathogen with swine as a primary reservoir around the world. A preliminary study was conducted to investigate the effects of Zn on the MRSA prevalence in nursery (n=40) and finisher pigs (n=40). Nasal swabs, collected from nursery and finisher pigs, were inoculated on to a chromogenic medium to isolate MRSA. The prevalence of MRSA was 10% and 20% among nursery and finisher pigs, respectively. The presence of MRSA was positively associated with higher levels of Zn supplementation. The finding of strong associations between copper and zinc resistance and other antibiotic resistance determinants is significant and potentially of public health concern. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The Research Team has focused on two major areas of preharvest food safety. Shiga toxin-producing Escherichia coli (STEC) and antimicrobial resistance in gut bacteria. STEC: Shiga toxin-producing Escherichia coli (STEC), particularly O157, are major food borne pathogens. In recent years, Six non-O157 STEC, O26, O45, O103, O111, O121, and O145, have also been recognized as a major public health concern. The prevalence of STEC and associated virulence genes in feces of feedlot cattle were determined. Fecal samples were collected from individual cattle (n = 960) and subjected to two detection protocols: 1. an 11-gene multiplex PCR that identified seven O serogroups (O26, O45, O103, O111, O121, O145, and O157) and four virulence genes applied to extracted total DNA (“direct PCR”); and 2. cultural procedures that involved immunomagnetic separation with O26, O103 and O111 beads, plating on a non-differential medium, followed by the multiplex PCR (mPCR) of pooled colonies (“culture-based method”). Based on direct PCR detection, O157 (49.9%) was the most prevalent serogroup followed by O26 (20.3%), O103 (11.8%), O121 (10.7%), O45 (10.4%), O145 (2.8%) and O111 (0.8%). Prevalence estimates were 22.3, 24.6 and 0.01% for O26, O103 and O111, respectively, based on culture-based methods. A substantial proportion of serogroup-positive samples did not harbor Shiga toxin genes. A study was conducted to determine whether fecal shedding of non-O157 STEC in feedlot cattle was affected by the use of an E. coli O157 vaccine and a direct-fed microbial, and to determine whether the shedding of a particular non-O157 STEC serogroups within feces was associated with O157 or other non-O157 serogroups. Cattle (n=17,148) in 40 pens were randomized to receive one, both or neither (control) of the two interventions: a siderophore receptor and porin proteins-based vaccine and a Lactobacillus-based direct-fed microbial product (DFM). Fecal samples (30/pen) were collected weekly for four consecutive weeks beginning 56 days after study allocation. Extracted DNA were tested for O157 and non-O157 serogroups O26, O45, O103, O111, O121 and O145, and four major virulence genes using an 11-gene mPCR. Results indicated that O157 (14.6%), O26 (10.5%) and O103 (10.3%) were the most prevalent serogroups. The vaccine, DFM or both had no significant effect on fecal prevalence of the six non-O157 STEC in feedlot cattle. Fecal shedding of O157 was associated with an increased probability of shedding of O26, O45, O103 and O121. The beef industry has implemented many intervention strategies in harvest facilities to reduce the likelihood of carcass contamination with STEC and other potential food-borne pathogens. In addition, it is recognized that STEC beef contamination can originate from the live animal production environment. Thus, reduction of pathogen load through the use of pre-harvest strategies, in the gastrointestinal tracts, systemically or on the hides of animals presented for harvest could improve beef safety. A unique serotype of STEC, O104:H4, was the cause of a large food-borne outbreak of hemorrhagic colitis and hemolytic uremic syndrome in Germany in 2011. The serotype was a hybrid of STEC and enteroaggregative E. coli (EAEC) pathotypes. Because cattle are known reservoirs of STEC, fecal carriage of E. coli O104 in feedlot cattle was investigated. A mPCR was designed and validated to detect the genes characteristic of O104:H4. A total of 248 fecal samples from 8 feedlots were tested. Overall, 20.6% of fecal samples were positive for the serogroup-specific gene. None of the fecal samples was positive for the gene characteristic of the EAEC. Only 7 samples yielded pure cultures of O104 and all 7 were negative for genes characteristics of STEC and EAEC. The results suggest that E. coli O104 is present in cattle feces, but the strains did not carry genes characteristic of the hybrid strain responsible for the 2011German outbreak. 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 E 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. A study was conducted in experimentally inoculated steers 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. Steers, housed individually in a biosafety level 2 facility, were randomly allocated to treatment diets and orally inoculated with a five-strain mixture of E. coli O157. Fecal samples were collected, and on d 35, steers were euthanized and necropsied to collect gut content samples. Steers fed DG or DG supplemented with starch shed E. coli O157 more often and at higher concentrations than those fed control diet. The lack of effect of starch addition to the DG diet on shedding of E. coli O157 may be because either the decreased starch content in the DG-supplemented diet is not a factor in the increased shedding of E. coli O157 or inclusion of pure starch in the diet may not have achieved our intended goal to have starch flow into the hindgut similar to that of corn grain.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: 1. Paddock, Z. D, D. G. Renter, X. Shi, C. R. Krehbiel, B. DeBey, and T. G. Nagaraja. 2013. Effects of feeding dried distillers grains with supplemental starch on fecal shedding of Escherichia coli O157:H7 in experimentally inoculated steers. J. Anim. Sci. 91:1362-1370. 2. 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. 3. Jacob M. E., J. Bai, D. G. Renter, A. T. Rogers, X. Shi and T. G. Nagaraja. 2013. 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. 4. Cernicchiaro N, C. A. Cull, Z. D. Paddock, X. Shi, J. Bai, T. G. Nagaraja, and D. G. Renter. 2013. Prevalence of Shiga toxin-producing Escherichia coli and associated virulence genes in feces of commercial feedlot cattle. Foodborne Pathog. Dis. 10:835-841. 5. Jacob M. E., D. M. Foster, A. T. Rogers, C. C. Balcomb, X. Shi and T. G. Nagaraja. 2013. Evidence of non-O157 Shiga toxin-producing Escherichia coli O157 in the feces of meat goats at a U. S. slaughter plant. J Food Prot. 76: 1626-1629. 6. Amachawadi, R. G., H. M. Scott, C. A. Alvarado, T. R. Mainini, J. Vinasco , J. S. Drouillard, and T. G. Nagaraja. 2013. Occurrence of the transferable copper resistance gene, tcrB, among fecal enterococci of U.S. feedlot cattle fed copper-supplemented diets. Appl. Environ. Microbiol. 79:4369-4375. 7. Paddock, Z. D., J. Bai, X. Shi, D. G. Renter, and T. G. Nagaraja. 2013. Detection of Escherichia coli O104 in the feces of feedlot cattle by a multiplex PCR assay designed to target major genetic traits of the virulent hybrid strain responsible for the 2011 German outbreak. Appl. Environ. Microbiol. 79:3522-3525.


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: The Research Team has focused on two major areas of preharvest food safety. food borne pathogens and antimicrobial resistance in gut bacteria of swine and cattle. The food borne pathogens of interest are Shiga toxin-producing Escherichia coli (STEC), Salmonella, and Campylobacter. STEC: Shiga toxin-producing Escherichia coli (STEC), particularly O157, are major food borne pathogens. Six non-O157 STEC, O26, O45, O103, O111, O121, and O145, have also been recognized as a major public health concern. Unlike O157, detection procedures for non-O157 have not been fully developed. We developed a multiplex PCR to distinguish O157 and the top six non-O157 serogroups. The assay was later modified to include 4 major virulence genes. The modified assay, an 11-gene multiplex PCR, was developed based on genes that code for serogroup-specific O-antigens and four major virulence factors (intimin, hemolysin, and Shiga toxins [Stx] 1 and 2), to detect O157 and six non-O157. The assay specificity was validated with pure cultures of STEC and non-STEC. Sensitivity of the assay with cattle fecal sample spiked with pooled cultures of seven major STEC was determined. The applicability of the assay to detect STEC in fecal samples, before and after enrichment, was evaluated by comparing with culture-based methods. Campylobacter and Salmonella: Both are leading causes of bacterial food borne illnesses in the US. Cattle are also reservoirs of both pathogens. Not much is known about prevalence of Campylobacter in cattle feces. A study was conducted to determine the prevalence and fluoroquinolone-susceptibilities of Campylobacter and Salmonella in cattle feces from feedlots that used a fluoroquinolone to treat respiratory disease. Pen-floor fecal samples were collected from each of ten pens of near-harvest cattle within five commercial feedlots. Cattle demographic and antimicrobial use data were collected from feedlot records. Fecal samples were cultured for Salmonella and Campylobacter using selective enrichment methods; isolates were confirmed using latex agglutination and PCR. Susceptibility to ciprofloxacin and naladixic acid was evaluated using broth micro-dilution methods. Antimicrobial resistance: Copper, an essential micronutrient, is supplemented at elevated levels in the diet to reduce morbidity and mortality and to promote growth in feedlot cattle. Gut bacteria exposed to copper can acquire resistance, which among enterococci is conferred by a transferable copper resistance (tcrB) gene borne on a plasmid. A study was undertaken to investigate whether the feeding of copper at levels sufficient to promote growth increases the prevalence of the tcrB gene among the fecal enterococci of feedlot cattle. The study was performed with 261 crossbred yearling heifers housed in 24 pens, with pens assigned randomly to a 2 by 2 factorial arrangement of treatments consisting of dietary copper (10 or 100 mg/kg of the diet) and a commercial linseed meal-based energy protein supplement (0 or 10 percent of the diet). Eight group pen floor fecal samples were collected on days 116 and 132 of the study, and two enterococcal isolates were obtained from each sample. PARTICIPANTS: Team Leaders: T. G. Nagaraja and Dave Renter T. G. Nagaraja: Microbiologist; Principal investigator; involved in all planning and decision making processes, and overseeing laboratory analysis and reporting of results. David Renter: Epidemiologist; 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. Action Team Members: M. M. Chengappa, Steve Dritz, Dick Oberst, and Dan Thomson Laboratory technicians: Xiaorong Shi, 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: Zac Paddock, Ragu Amachawadi, Charley Cull M. S. 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. TARGET AUDIENCES: Scientists, Veterinarians, Food Safety Inspection Agency, Cattlemen, Packers, Students PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
STEC: The primers designed to detect 11 genes formed distinct bands on agarose gels and were specific for the seven serogroups. A minimum concentration of 6.5 x 105 CFU/g was needed to detect all 11 genes. The sensitivity of the assay was improved to 6.5 x 102 CFU/g with the inclusion of a 6-h enrichment step in EC broth. An obvious limitation of the multiplex PCR is that the concurrent detection of virulence genes and the seven serogroups in a sample does not necessarily mean that the virulence genes are associated with the seven serogroups in the same sample. Campylobacter and Salmonella: Overall proportions of positive samples were 38.0 percent for Salmonella and 26.8 percent for Campylobacter. Salmonella prevalence varied from 1.0 to 76.5 percent among feedlots and from 0 to 100.0 percent among pens. Prevalence of Campylobacter ranged from 16.0 to 41.5 percent among feedlot and from 0 to 60.0 percent among pens. The number of fluoroquinolone treatments per pen ranged from 1 to 103. Most Salmonella isolates (99.7 percent) were susceptible to ciprofloxacin and naladixic acid. Susceptibility testing of Campylobacter and multivariable data analyses are currently underway. Preliminary conclusions are that Salmonella and Campylobacter prevalence is highly variable across feedlots and pens. In addition, it appears that most Salmonella in feces of pre-harvest feedlot cattle are susceptible to fluoroquinolones. Prevalence estimates and results from analyses of antimicrobial use and susceptibility data will assist in further assessments of potential impacts of fluoroquinolone use in feedlot production systems. Antimicrobial resistance: In the US, cattle diets are supplemented with in-feed antimicrobials for growth promotion, to increase feed efficiency, or to decrease infections. There is concern that gut bacteria in food-producing animals develop and propagate resistance to antibiotics and thereafter contaminate food products, providing a direct pathway for transmission to humans. Therefore, alternatives to antibiotics, including feeding of heavy metals and particularly copper and zinc, have been employed as feed additives in food animal production. In cattle fed a higher concentration of copper, only a small proportion (6.9 percent) of fecal enterococcal isolates harbored the tcrB, a gene that confers copper resistance. In contrast, Danish studies have reported a prevalence of 76 percent of copper resistant enterococci in pig feces obtained at the time of slaughter, perhaps because of higher levels of copper and supplementation for a longer period of time during their production cycle. The finding of strong associations between copper resistance and other antibiotic resistance determinants may be significant because of the propensity of enterococci to transfer resistance genes to other bacteria in the gut when placed under selection pressure. Further studies are needed to investigate the effects of feeding antibiotics and their association with copper resistance gene in enterococci to better understand the potential co-selection and molecular epidemiology of multidrug resistant enterococci in cattle.

Publications

  • Jacob, M. E., J. T. Fox, and T. G. Nagaraja. 2012. Prevalence of food-borne pathogens in organic beef. p 287-300. In: Organic Meat Production and Processing. S. C. Ricke, E. J. Van Loo, M. G. Johnson, and C. A. OBryan (eds.) Wiley-Blackwell, Ames, IA.
  • Jacob, M. E. and T. G. Nagaraja. 2012. Use of direct-fed microbials as a preharvest food safety intervention in cattle. p 189-202. In: Direct-Fed Microbials and Probiotics for Animals: Science and Mechanisms of Action. T. R. Callaway and S. C. Ricke (eds.) Springer Publ., NY.
  • Paddock, Z. D, D. G. Renter, X. Shi, C. R. Krehbiel, B. DeBey, and T. G. Nagaraja. 2013. Effects of feeding dried distillers grains with supplemental starch on fecal shedding of Escherichia coli O157:H7 in experimentally inoculated steers. J. Anim. Sci. In Press.
  • 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.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: The Research Team has focused on two major food borne pathogens, Escherichia coli O157:H7 and Salmonella, and on prevalence of antimicrobial resistance in gut bacteria of swine and cattle. E. coli O157:H7: Cattle harbor E. coli O157 in the hindgut and serve as a primary reservoir. The organism is shed in the feces, which serves as a major vehicle for food borne infections. The genetic diversity of E. coli O157 within an individual bovine fecal sample based was determined. Fecal samples were collected from cattle and E. coli O157:H7 was isolated. From each positive sample, up to 10 putative colonies were tested and isolates from samples with at least 7 positive colonies were subtyped using PFGE. The results of this study suggest that determining the PFGE pattern of a single isolate from a bovine sample may not be sufficient when comparing to other isolates as multiple PFGE subtypes are present. A study was conducted to determine the reason why feeding distiller's grains (DG) increases fecal shedding of E. coli O157:H7. We hypothesized that decreased starch content of the DG-supplemented diet may be the reason for increased fecal shedding of E. coli O157:H7. We designed a study to determine whether addition of starch to the DG-supplemented diet will negate the increased fecal shedding of E. coli O157:H7. Our results indicated that starch in the DG diet had no effect fecal shedding of E. coli O157:H7. Salmonella: The effects of a Salmonella Newport siderophore receptor and porin protein (SRP) vaccine on cattle health and performance and on prevalence of fecal shedding of Salmonella in feedlot cattle were evaluated. Cattle were randomly allocated within a replicate (n = 10 replicates), administered 2 mL of SRP vaccine or a placebo, and revaccinated after 21 days. Health and performance data were recorded by trained feedlot personnel who were blinded to treatment. Significant differences in fecal prevalence of Salmonella or health and performance variables were not detected between vaccinated and control cattle. In this setting, administration of the Salmonella SRP vaccine in feedlot cattle had no effect on fecal prevalence of Salmonella or cattle health and performance. Antimicrobial resistance: Copper, as copper sulfate, is used as an alternative to in-feed antibiotics for growth-promotion in weaned piglets. Acquired copper resistance, conferred by a plasmid-borne, transferable copper resistance (tcrB) gene has been reported in Enterococcuss. A study was undertaken to determine the relationship between copper supplementation and prevalence of tcrB-positive enterococci in piglets. The study consisted of weaned piglets. Overall prevalence of tcrB-positive enterococci was 21.1% in piglets fed elevated copper and 2.8% in the control. Among the 43 tcrB-positive isolates, 35 were E. faecium and 8 were E. faecalis. The mean MIC of copper for tcrB-negative and tcrB-positive enterococci was 6.2 and 22.2 mM, respectively. The higher prevalence of tcrB-positive enterococci in piglets fed elevated copper compared to normal copper suggests that supplementation of copper in swine diets selected for resistance. PARTICIPANTS: T. G. Nagaraja: Microbiologist; Principal investigator; involved in all planning and decision making processes, and overseeing laboratory analysis and reporting of results. David Renter: Epidemiologist; 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. Action Team Members: M. M. Chengappa, Steve Dritz, Dick Oberst, and Dan Thomson Laboratory technicians: Xiaorong Shi, 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 Post Doc: Megan Jacob Ph. D. students: Zac Paddock, Ragu Amachawadi, Charley Cull M. S. Students: Elena Gart 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. TARGET AUDIENCES: Scientists, Veterinarians, Food Safety Inspection Agency, Cattlemen, Packers, Students PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
E. coli O157:H7: The results of this study suggest that determining the PFGE pattern of a single isolate from a bovine sample may not be sufficient because multiple E. coli O157:H7 subtypes can co-exist in cattle feces. Additionally, since some variability in PFGE patterns is found within a bovine fecal sample, the same is likely true when analyzing other possible sources (such as meat or water) and human cases. The common practice of determining the PFGE typing of only one isolate from a human patient or the suspected source of infection may result in an incomplete description of the genotypic diversity of E. coli O157:H7 within that sample. Inclusion of starch in the DG diet had no effect fecal shedding of E. coli O157:H7 suggesting that the increase in fecal shedding E. coli O157 was not because of reduced starch content in the feed. Further research is needed to understand whether other components of DG, protein, lipid, or fiber, may be responsible for the positive association with E. coli O157:H7. Salmonella: Although no effects of vaccination with the Salmonella Newport SRP vaccine on the fecal prevalence of Salmonella bacteria or cattle health and performance were detected, further investigation of the use of this vaccine in other cattle production settings could provide evidence of vaccine efficacy. The lower than expected prevalence of Salmonella bacteria in feces combined with the extreme variability in prevalence among replicates and within replicates over time may have adversely affected the ability to detect significant vaccine effects in the study. Furthermore, the present study was conducted in only one commercial feedlot, and prevalence and serotypes of Salmonella bacteria vary among feedlots and regions. Because the control of Salmonella bacteria in commercial feedlot production systems may enhance food safety and potentially cattle health and performance, further studies are necessary to validate control methods. Antimicrobial resistance: Antibiotic resistant enterococci are opportunistic and nosocomial pathogens in humans. Because of the widespread occurrence of resistant enterococci in animals, it has been suggested that these enterococci may serve as a reservoir of potential resistant genes capable of transferring from animal to human bacteria. A critical issue is the co-selection of metal and antibiotic resistance on the same genetic determinant, often on a plasmid or a transposon. These plasmids or transposons are of concern because they harbor antibiotic resistance genes and have the potential to spread between species. The conjugative transposons have a broad bacterial host range and are capable of being transferred horizontally to a variety of gram positive and gram negative bacteria in the gut bacterial community. The existence of a metal-associated co-selection mechanism could potentially be a major issue relative to public health and further studies are needed to determine the magnitude of such an association.

Publications

  • Dodd C., M. W. Sanderson, M. E. Jacob, and D. G. Renter. 2011. Modeling preharvest and harvest interventions for Escherichia coli O157 contamination on beef cattle carcasses. J Food Prot. 74:1422-1433.
  • Dodd, C., D. G. Renter, X. Shi, M. Sanderson, M. Alam, and T. G. Nagaraja. 2011. Prevalence and Persistence of Salmonella in Cohorts of Feedlot Cattle. Foodborne Path. Dis. 8:781-789.
  • Taylor, E., X. Shi, J. Alam, G. Peterson, S. K. Narayanan, D. G. Renter and T. G. Nagaraja. 2011. Genetic variation and Shiga toxin producing abilities of bovine and human Escherichia coli O157:H7. Zoonoses Publ. Hlth. 58:185-191.
  • Dodd C., D. G. Renter, D. U. Thomson, and T. G. Nagaraja. 2011. Evaluation of the effects of a commercially available Salmonella Newport siderophore receptor and porin protein vaccine on fecal shedding of Salmonella bacteria and health and performance of feedlot cattle Amer. J. Vet. Res. 72:239-247.
  • Jacob, M. E., K. M. Almes, X. Shi., J. M. Sargeant, and T. G. Nagaraja. 2011. Escherichia coli O157:H7 genetic diversity in bovine fecal samples. J. Food Prot. 74:1186-1188.
  • Amachawadi, R. G., N. W. Shelton, X. Shi, J. Vinasco, S. S. Dritz, M. D. Tokach, J. L. Nelssen, H. M. Scott, and T. G. Nagaraja*. 2011. Selection of fecal enterococci exhibiting tcrB-mediated copper resistance in pigs fed diets supplemented with copper. Appl. Environ. Microbiol., 77:5597-5603.
  • Paddock, Z. D., C. E. Walker, J. S. Drouillard, and T. G. Nagaraja*. 2011. Dietary monensin level, supplemental urea, and ractopamine on fecal shedding of Escherichia coli O157:H7 in feedlot cattle. J. Anim. Sci., 89:2829-2835.


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: The Research Team has focused on two areas of food safety research: Prevalence, ecology and on-farm interventions of two major food borne pathogens, Escherichia coli O157:H7 and Salmonella, and prevalence, amplification and dissemination of antimicrobial resistance in cattle. E. coli O157:H7: Fecal shedding of Escherichia coli O157:H7 in cattle is due to the organism's ability to persist in the gut. The site of prevalence in the gut is important to understand mechanisms, factors affecting fecal shedding and the site could be targeted for intervention. The prevalence of E. coli O157:H7 in the rumen, cecum, colon, and rectum were determined with contents collected from slaughtered cattle at an abattoir. Isolation and identification of E. coli O157:H7 were by selective enrichment, immunomagnetic separation, plating on selective medium, agglutination for O157 antigen and presence of virulence genes. We used pulsed-field gel electrophoresis to genetically compare isolates from different gut locations. Acid tolerance for cattle with positive rumen (pre-gastric) isolates and at least one other positive hindgut (post-gastric) isolate within the same animal was determined. Salmonella: We determined factors associated with fecal prevalence of Salmonella at feedlot entry and prior to harvest, and to assess potential persistence of Salmonella strains within cattle populations. This repeated cross-sectional study followed 5,559 beef cattle within 30 feedlot cohorts. Samples (n = 30) of fresh feces were collected from the pen floor of each cohort at feedlot entry and again within 24 hrs of harvest. Samples were subjected to a selective Salmonella isolation protocol and serotypes were determined for all Salmonella isolates. Genetic relatedness of a subset of isolates was determined using pulsed-field gel electrophoresis (PFGE). Cattle health and performance data were recorded electronically by feedlot personnel. Cohort-level generalized linear mixed models were used to assess bivariable associations. Antimicrobial resistance: Copper, as copper sulfate, is increasingly used as an alternative to in-feed antibiotics for growth-promotion in weaned piglets. Acquired copper resistance, conferred by a plasmid-borne, transferable copper resistance (tcrB) gene has been reported in Enterocoocus faecium and E. faecalis. We conducted a study to determine the relationship between copper supplementation and prevalence of tcrB-positive enterococci in piglets. The study consisted of 60 weaned piglets, housed in 10 pens with 6 piglets per pen, fed normal or elevated level of copper. Fecal samples were collected from three piglets per pen on days 0, 14, 28, and 42, and three enterococcal isolates were obtained from each sample. We developed a spotted microarray for rapid identification and characterization of bacterial pathogens and their antimicrobial resistance genes. Our spotted microarray consisted of 489 70mer probes that detect 40 bacterial pathogens of medical, veterinary and zoonotic importance and associated genes that encode resistance for antimicrobial and metal resistance, and are important for horizontal gene transfer among bacteria. PARTICIPANTS: Team Leaders: T. G. Nagaraja and Dave Renter T. G. Nagaraja: Microbiologist; Principal investigator; involved in all planning and decision making processes, and overseeing laboratory analysis and reporting of results. David Renter: Epidemiologist; 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. Action Team Members: M. M. Chengappa, Steve Dritz, Dick Oberst, and Dan Thomson Laboratory technicians: Xiaorong Shi, Neil Wallace, Greg Peterson 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: Megan Jacob, Zac Paddock, Ragu Amachawadi, Greg Peterson M. S. Students: Elena Gart 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 significant outcomes of the various studies conducted in the previous year are outlined below: E. coli O157:H7: The prevalence in the rumen, cecum, colon, and rectum was 4.9, 9.9, 7.6, and 11.1%, respectively. Overall prevalence of E. coli O157:H7 in cattle sampled, based on being positive in any one gut location, was 20.3. The hindgut was the major site of prevalence of E. coli O157:H7 in cattle, a majority of the isolates within the same animal were clonally similar and acid tolerance of hindgut isolates were not different from that of ruminal isolates. Salmonella: In the Salmonella study, fecal prevalence within a cohort at feedlot entry was not associated with preharvest prevalence. Preharvest prevalence was positively associated with the number of days in the feedlot (P = 0.02), cumulative morbidity (P = 0.01), and cumulative mortality (P = 0.03). We recovered Salmonella isolates with identical PFGE profiles both at feedlot entry and preharvest and PFGE subytpes of Salmonella appear to persist within and among feedlot cohorts. We developed and validated two molecular methods for identification of Salmonella serovars. A 70mer oligonucleotide spotted microarray was developed that consisted of probes which detected genes responsible for genetic variation between isolates of Salmonella that can be used for serotyping. A multiplex PCR assay was also developed that are capable of identifying 42 serovars and provided a valuable prediction of the pathogenicity of the isolates by detecting virulence genes sseL, invA, and spvC. The gene spvC was the best predictor of pathogenicity. Antimicrobial resistance: A total of 45 enterococcal isolates were obtained per treatment group at each sampling time and tested for tcrB gene by PCR. Overall prevalence of tcrB-positive enterococci were 28.1% in piglets fed elevated copper and in the control 2.1%. Among the 43 tcrB-positive isolates, 35 were E. faecium and 8 were E. faecalis. All tcrB-positive isolates contained erm(B) gene but none had vanA gene. The mean minimum inhibitory concentrations of copper for tcrB-negative and tcrB-positive enterococci were 6.2 and 22.2 mM, respectively. Supplementation of copper at elevated level appears to select for resistance. In the spotted microarray method, high specificity and reliability were achieved for bacterial pathogens by validating multi drug resistant pathogenic bacteria as pure cultures or by following their inoculation in complex and highly organic sample matrices, such as soil and manure. The food supply in the US is one of the safest in the world; however, food-borne illnesses do occur and frequently are associated with foods derived from animal agriculture. Control strategies aimed at reducing the prevalence and concentration of E. coli O157:H7 and Salmonella 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. In recent decades, there has been a significant rise in the prevalence of multi-drug resistant strains of bacteria in the U.S. and in countries worldwide.

Publications

  • Jacob M. E., Z. D. Paddock, D. G. Renter, K. F. Lechtenberg, and T. G. Nagaraja. 2010. Inclusion of dried or wet distillers' grains at different levels in diets of feedlot cattle affects fecal shedding of Escherichia coli O157:H7. Appl. Environ. Microbiol. 76:7238-7242.
  • Bai J., X. Shi and T. G. Nagaraja. 2010. A Multiplex PCR Procedure for detection of six major virulence genes in Escherichia coli O157:H7. J. Microbiol. Meth. 82:85-89.
  • Jacob, M. E., J. T. Fox, T. G. Nagaraja, J. S. Drouillard, R. G. Amachavadi, S. K. Narayanan. 2010. Effects of feeding elevated concentrations of supplemental copper and zinc on antimicrobial susceptibilities of fecal bacteria in feedlot cattle. Foodborne Path. Dis. 7:643-648.
  • Peterson, G., B. Gerdes, Jami Brodges, T. G. Nagaraja, J. G. frye, D. S. Boyle, and S. K. Narayanan. 2010. Development of microarray and multiplex polymerase chain reaction assays for identification of serovars and virulence genes in Salmonella enterica of human and or animal origin. J. Vet. Diagn. Inest. 22:559-569.
  • Dodd, C., D. G. Renter*, T. Fox, X. Shi, M. Sanderson, A. Nutsch, and T. G. Nagaraja. 2009. Genetic relatedness of Escherichia coli O157 isolates from cattle feces and pre-intervention beef carcasses. Foodborne Path. Dis.7:357-365.
  • Jacob M. E., D. G. Renter, and T. G. Nagaraja. 2010. Animal- and truckload-level associations between E. coli O157:H7 in feces and on hides at harvest and contamination of pre-evisceration beef carcasses. J. Food Prot. 6:1030-1037.
  • Dodd C., D. G. Renter, D. U. Thomson, and T. G. Nagaraja. 2010. A randomized field trial to determine the effects of a commercially available vaccine against Salmonella in feedlot cattle. Amer. J. Vet. Res.
  • Peterson, G., J. Bai, T. G. Nagaraja, and S. K. Narayanan. 2010. Diagnostic microarray for human and animal bacterial diseases. J. Microbiol. Meth. 80:223-230.
  • Taylor, E., X. Shi, J. Alam, G. Peterson, S. K. Narayanan, D. G. Renter and T. G. Nagaraja. 2010. Genetic variation and Shiga toxin producing abilities of bovine and human Escherichia coli O157:H7. Zoonoses Publ. Hlth.
  • Amachawadi, R. G., N. W. Shelton, M. E. Jacob, X. Shi, S. K. Narayanan, L. Zurek, S. S. Dritz, J. L. Nelssen, M. D. Tokach, and T. G. Nagaraja. 2010. Occurrence of tcrB, a transferable copper resistance gene, in fecal enterococci of swine. Foodborne Path. Dis. 9:1089-1097
  • Walker, C. X. Shi, M. J. Sanderson, J. S. Sargeant, and T. G. Nagaraja. 2010. Prevalence of Escherichia coli O157:H7 in gut contents of beef cattle at slaughter. Foodborne Path. Dis. 7:249-255.


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: The Research Team has focused on two areas of food safety research: Prevalence, ecology and on-farm interventions of Escherichia coli O157:H7 and Salmonella, and prevalence, amplification and dissemination of antimicrobial resistance in cattle. A multiplex PCR procedure that detected six major virulence genes, fliC, stx1, stx2, eae, rfbE, and hlyA, in E. coli O157:H7 was developed. The procedure was validated with a total of 222 strains that included cattle and human strains. We defined and compared pulsed-field gel electrophoresis profiles of E. coli O157 isolated from cattle feces and carcass samples to evaluate relationships between beef carcass contamination and fecal shedding of E. coli O157 at harvest. Within truckload, the percentages of carcass isolates that were identical to high-shedder or low-shedder fecal isolates were 69.2 and 46.0 percent, respectively. We conducted a study on the effects of mucin and its carbohydrate constituents on E. coli O157 growth in batch culture fermentations with ruminal or fecal microbial inoculums. Gluconic acid was the most stimulatory substrate increasing E. coli O157 by more than 1.0 log in ruminal fermentations and 2.0 log in fecal fermentations. We conducted a cross-sectional study to evaluate associations among fecal, hide and pre-evisceration carcass prevalence of E. coli O157:H7 and assess factors affecting carcass contamination. Fecal, hide and carcass prevalence of E. coli O157:H7 within truckload were significantly (P less than 0.05) correlated with each other. We evaluated the efficacy of an anti-E. coli O157 SRP-based vaccine in feedlot cattle naturally shedding the organism. The SRP vaccine at the 3 ml dose reduced the prevalence of E. coli O157 compared to the control (17.7 percent vs 33.7 percent). We determined the effects of a commercially available Salmonella Newport SRP vaccine on cattle health and performance and on fecal shedding of Salmonella in commercial feedlot cattle. Cumulative prevalence of fecal shedding was 10.2 and 10.9 percent for SRP vaccinated and control cattle, respectively. Enteric pathogens respond to catecholamines by increasing growth, motility and virulence. We have determined that catecholamines increased the rate of conjugative transfer of genetic material between Salmonella Typhimurium and laboratory strain of E. coli. An in vitro pharmacodynamic model suggested that oxytetracycline concentrations exceeding the MIC of the recipient bacteria suppressed plasmid transfer. We determined the occurrence of a plasmid-borne transferable copper resistance (tcrB) gene that confers copper resistance in fecal enterococci from weaned piglets fed diets with normal or elevated supplemental level of copper. The mean MIC of copper for tcrB-positive was higher compared to tcrB-negative isolates. The transferability of the tcrB gene from tcrB-positive strains to tcrB-negative strains was demonstrated by conjugation. The potential link between tcrB and antibiotic resistance genes and the propensity of enterococci to transfer tcrB to other strains raises the possibility that copper supplementation may exert selection pressure for antibiotic resistant enterococci in pigs. PARTICIPANTS: Team Leaders: T. G. Nagaraja and Dave Renter T. G. Nagaraja: Microbiologist; Principal investigator; involved in all planning and decision making processes, and overseeing laboratory analysis and reporting of results. David Renter: Epidemiologist; 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. Action Team Members: M. M. Chengappa, Steve Dritz, Dick Oberst, and Dan Thomson Laboratory technicians: Xiaorong Shi, Neil Wallace, Greg Peterson 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, Zac Paddock; MPH students: Ethel Taylor; M. S. Students: Shelby Reinstein, Callie Walker, Raghavendra Amachawadi, Elena Gart 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 US 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 Salmonella are important food-borne pathogens that cause hemorrhagic colitis, hemolytic uremic syndrome and thrombocytopenic purpura in humans. Control strategies aimed at reducing the prevalence and concentration of E. coli O157:H7 and Salmonella 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. In recent decades, there has been a significant rise in the prevalence of multi-drug resistant strains of bacteria in the U.S. and in countries worldwide. The economic impact of antibiotic resistance has been estimated to extend healthcare costs to over $5 billion annually in the U.S. alone. The significant outcomes of the various studies conducted in the previous year are outlined below. E. coli 157: The new procedure we developed achieves amplification of six genes in a single reaction, which is a significant improvement. Cattle feces pose a potential risk for E. coli O157 contamination of carcasses. Truckload may be an important factor in the potential transmission of E. coli O157, but isolates from carcasses also may be similar to those from feces of cattle on different truckloads and harvest days. Our results confirm that human clinical strains of E. coli O157:H7 produce more Stx 2 than strains isolated from cattle. The presence of Q933 gene is a better indicator of high Stx2 production and may be a useful marker to assess potential of cattle strains to cause human disease. Availability of mucous constituents, particularly gluconic acid, may explain the higher prevalence of E. coli O157 in the large intestine compared to the rumen of the digestive tract. Gluconic acid may serve as a unique substrate available to promote growth of E. coli O157 in the large intestine of beef cattle. The E. coli O157:H7 SRP vaccine may serve as a preharvest intervention to reduce the burden of E. coli O157:H7 on cattle presented for slaughter. Salmonella: We showed that vaccination of feeder cattle with the Salmonella Newport SRP vaccine had no significant effect on Salmonella fecal prevalence, cattle health, or cattle performance. Antimicrobial resistance: We have identified a mechanism by which host factors may drive the development of highly virulent and multidrug resistant strains of enteric bacterial pathogens. We have demonstrated that plasmid transfer rates were not correlated with the pharmacokinetic parameters of oxytetracycline. Finally, our study is the first report on the occurrence of the tcrB gene in enterococci of swine in the U. S. The significance of this finding is the potential association between copper resistance and resistance to other antibiotics and the propensity of enterococci to transfer tcrB and antibiotic resistance genes to other strains within the same species, other enterococcal species, and possibly to species of other genera.

Publications

  • Dodd C., Renter D.G., Fox J.T., Shi X., Sanderson M.W., Nagaraja T.G. 2009. Genetic relatedness of Escherichia coli O157 isolates from cattle feces and pre-intervention beef carcasses. Foodborne Pathogens and Disease, in press.
  • Taylor E.V., Kastner J.J., Renter D.G. 2009. Challenges involved in the Salmonella Saintpaul outbreak and lessons learned. Journal of Public Health Management and Practice, in press.
  • Walker, C., Shi X., Sanderson M. J., Sargeant J. S., Nagaraja T. G. 2009. Prevalence of Escherichia coli O157:H7 in gut contents of beef cattle at slaughter. Foodborne Pathogens and Disease, in press.
  • Reinstein S., Fox J.T., Shi X., Alam M.J., Renter D.G., Nagaraja T.G. 2009. Prevalence of Escherichia coli O157:H7 in organically and naturally raised beef cattle. Applied and Environmental Microbiology 75(16): 5421-5423.
  • Alam M.J., Renter D.G., Ives S.E., Thomson D.U., Hollis L.C., Sanderson M.W., Nagaraja T.G. 2009. Potential associations between fecal shedding of Salmonella in feedlot cattle treated for apparent respiratory disease and subsequent adverse health outcomes. Veterinary Research 40:02.
  • Jacob M.E., Fox J.T., Drouillard J.S., Renter D.G., Nagaraja T.G. 2009. Evaluation of feeding dried distillers grains with solubles and dry-rolled corn on the fecal prevalence of Escherichia coli O157:H7 and Salmonella spp. in cattle. Foodborne Pathogens and Disease 6(2): 145-153.
  • Fox, J. T., Drouillard, J. S., and Nagaraja T. G. 2009. Competitive exclusion Escherichia coli cultures on E. coli O157 batch culture ruminal or fecal microbial fermentation. Foodborne Pathogens and Disease 6:193-199.
  • Thomson D. U., Loneragan, G. H., Thornton A. B., Lechtenberg K. F., Emery D. A., Burkhardt D. T., Nagaraja T. G. 2009. Use of a siderophore receptor and porin proteins-based vaccine to control the burden of Escherichia coli O157:H7 in feedlot cattle. Foodborne Pathogens and Disease 6:871-877.
  • Fox, J. T., Thomson, D. U., Drouillard J. S., Thornton, A. B., Burkhardt D. T., Emery D. A., Nagaraja T. G. 2009. Efficacy of Escherichia coli O157:H7 siderophore receptor/porin proteins-based vaccine in feedlot cattle naturally shedding E. coli O157. Foodborne Pathogens and Disease 6:893-899.
  • Fox, J. T., Drouillard J. S., Nagaraja T. G. 2009. Effects of mucin and its carbohydrate constituents on Escherichia coli O157 in batch culture fermentations with ruminal or fecal microbial inoculums. Journal of Animal Science 87:1304-1313.
  • Thornton, A. B., Thomson D. U., Fox J. T., Loneragan G. H., Burkhardt D. T., Emery D. A., Nagaraja T G. 2009. Siderophore receptor/porin proteins-based vaccination reduces prevalence and shedding of Escherichia coli O157:H7 in experimentally inoculated cattle. Journal of Food Protection 72:866-869.
  • Jacob, M. E., Callaway T. R., Nagaraja T. G. 2009. Dietary interactions and interventions affecting Escherichia coli O157 colonization and shedding in cattle. Foodborne Pathogens and Disease 6:785-792.