Source: KANSAS STATE UNIV submitted to
PRE-HARVEST FOOD SAFETY IN FOOD ANIMALS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1004187
Grant No.
(N/A)
Project No.
KS00-0093-HA
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2014
Project End Date
Sep 30, 2019
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, food-borne illnesses do occur and frequently are associated with foods derived from animal agriculture, particularly cattle and swine. The three major foodborne pathogens associated with cattle and swine are Shiga toxin-producing Escherichia coli, Salmonella, and Campylobacter. These pathogens reside in the intestinal tract of cattle and swine and are shed in the feces, which serves as a source of food and water contamination. The human illnesses caused by these pathogens range from mild to severe diarrhea, which could lead to serious complications, including death, particularly in children and elderly. 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 other countries. The potential impact of antimicrobial resistance of food borne pathogens and normal bacteria in gut of cattle and swine is profound and requires multifaceted approach to understand the emergence and dissemination of genetic elements that confer antimicrobial resistance. Control strategies aimed at reducing the prevalence and concentration of pathogens and antimicrobial resistance elements in cattle and swine feces, thus reducing the overall number of bacteria entering both the food and environmental pathways, is the most effective approach for reducing the overall risk of human infections.
Animal Health Component
100%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7123310104060%
7123310110040%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology;
Goals / Objectives
The project has the following two broad objectives:Determine prevalence, study ecology and develop and evaluate on-farm interventions of major foodborne pathogens, Shiga toxin-producing E. coli, Salmonella, and Campylobacter, in cattle and swine.Determine prevalence, and study amplification and dissemination of antimicrobial resistance in food borne pathogens and major gut commensals in cattle and swine and develop and evaluate methods to mitigate.
Project Methods
Detection and characterization of Shiga toxin-producing E. coli, Salmonella, and Campylobacter. We will utilize culture-based and PCR-based methods to detect and in some instances quantify the pathogens in fecal samples. Other samples that will be included, depending on the objectives, will be hide swabs and carcass swabs, and lymph nodes in case of Salmonella. Methods to detect E. coli O157, Salmonella, and Campylobacter have been generally well established. However, methodology to detect the six major serogroups (O26, O45, O103, O111, O121, and O145) needs to be optimized. We have initiated studies on this as part of the USDA CAP grant, but further evaluation and validation, particularly of selective agar medium for isolation, are needed. Similarly, PCR-based procedures, both conventional and real time, have well developed and validated for E. coli O157. However, PCR-based procedures to detect and quantify Salmonella and Campylobacter in cattle feces and other samples need to be developed and validated. The validation of the assays will be done initially with pure cultures and subsequently with feces spiked with pure cultures. Once the assays are validated, the applicability of the assay will be determined with field samples. Initial development of the assays will be with pure cultures, followed by feces spiked with pure cultures, and finally with field samples.One of the objectives of the project is to assess factors that affect fecal shedding of food borne pathogens. Studies to address this objective will be conducted in one more of the following ways:Natural prevalence field studies: Fecal samples will be collected from cattle at the farm or at a slaughter plant after cattle are slaughtered. Fecal samples from live cattle will be collected from the rectum or freshly defecated feces on the floor of the pen. At the slaughter plant, rectal contents (before postharvest intervention) will be collected to determine prevalence of the pathogens.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, non-O157 STEC, Salmonella or Campylobacter will be assigned to treatments. Characterizing the effects of factors on prevalence generally requires large sample sizes due to the relatively low prevalence of the organisms in cattle populations. Consequently, prevalence studies are expensive to conduct, and also are grossly limited in terms of the range of treatments that can be investigated in a given experiment. Quantitative models, as opposed to the traditional qualitative approaches, are far more robust in terms of characterization of the effects of potential influencing factors.Experimental challenge studies: This will be conducted in a BL-2 facility. Cattle in this study will be prescreened to assess the culture status (negative for pathogens of interest) and then orally inoculated with a cocktail of antibiotic-adapted (nalidixic acid, rifampicin, streptomycin, etc.) organisms to determine the influence of various factors of interest. This approach will allow us quantify level of fecal shedding and provide more robust evidence for factors influencing fecal shedding.Studies on antimicrobial resistance will be conducted in pigs or cattle. The assessment of treatment effects on antimicrobial resistance will be with gut commensals (Enterococcus [Gram Positive bacterium] and E. coli [Gram negative bacterium] and major food borne pathogens (E. coli O157, Salmonella, and Campylobacter). The resistance will be assessed phenotypically and genotypically.

Progress 10/01/14 to 09/30/19

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: Graduate Students completed: Mori Atobatele (MS), Kaylen Capps, Dian Dewsbury, Justin Ludwig, Lance Noll and Sarah Remfry (MS) Current Graduate Students completed: Charley Cull, Pius Ekong, Lance Noll, Pragathi Shridhar, Ashley Smith The graduate students were trained 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 had opportunities to present their findings as poster or oral presentations in a number of scientific conferences. Also, students received 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?A number of presentations, both oral and poster, were made by faculty and graduate students in a variety of conferences and meetings. The meetings included the following: American Society for Animal Science Annual Meeting Beef Industry Food Safety Summit Council (Organized by National Cattlemen Beef Association) Cattlemen's Day in Manhattan, Kansas (Organized by the Department of Animal Sciences and Industry at K-State) Internal Association for Food protection International Verotoxigenic (Shiga toxigenic) E. coli in Florence, Italy in May, 2018 Midwest American Society for Animal Science Animal Science Meeting Swine Day in Manhattan, Kansas (Organized by the Department of Animal Sciences and Industry at K-State) What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Accomplishments Impact: The research focused on three major foodborne pathogens associated with cattle and swine: Shiga toxin-producing Escherichia coli, Salmonella, and Campylobacter. These pathogens reside in the intestinal tract of cattle and swine and are shed in the feces, which serves as a source of food and water contamination. The human illnesses caused by these pathogens range from mild to severe diarrhea, which could lead to serious complications, including death, particularly in children and elderly. A priority focus area of the research team was Shiga toxin- producing E. coli, a serotype of STEC, called O157:H7, is the most common and causes more serious infections. However, the Centers for Disease Control, identified six other serogroups of STEC called 'non-O157 STEC', which include O26, O45, O103, O111, O121, and O145, were reported to cause a majority of non-O157 STEC infections in people. The non-O157 STEC also reside in the hindgut of cattle and not much was known because detection and identification methods were not available. Therefore, studies were designed to develop and validate culture-based and molecular techniques to isolate, identify, and quantify the non-O157 STEC in cattle feces. The techniques were later applied to understand the ecology of non-O157 STEC in the hindgut of cattle, fecal prevalnce, factors affecting prevalence and fecal shedding and subsequent contamination of hide carcass of cattle. Major findings of the studies conducted are as follows: Objective 1. Determine prevalence, study ecology and develop and evaluate on-farm interventions of three major foodborne pathogens, Shiga toxin-producing E. coli, Salmonella, and Campylobacter, in cattle and swine. Culture-based methods was developed and validated to detect the presence of six serogroups of non-O157 STEC, O26, O45, O111, O103, O121, and O145 in cattle feces and hide samples. Molecular (PCR)-based techniques were developed and validated to detect the presence of six serogroups of non-O157 STEC, O26, O45, O111, O103, O121, and O145 in cattle feces and hide samples. Culture-based and molecular-based methods developed and validated to quantify the presence of six serogroups of non-O157 STEC, O26, O45, O111, O103, O121, and O145 in cattle feces and hide samples. All six serogroups of non-O157 were detected in cattle feces with serogroups O26 and O103 were the most predominant and serogroup O121 was the lowest. Similar to O157, fecal shedding of non-O157 was highly variable between feedlots and between pens within a feedlot. Although prevalence of the serogroups, particularly O26 and O103, in cattle were high, the prevalence serogroups carrying Shiga toxin genes that encode for Shiga toxins, which are major virulence factors responsible for human infections, was low. Similar to O157, fecal shedding of non-O157 was affected by the season. The prevalence was higher in summer months and low or completely absent in winter months. Similar to O157, a subset of cattle (approx. 15% of cattle) shed high concentrations (>1,000 bacteria per gram of feces) of non-O157 serogroups. These 'super shedders' are likely to be major source of spread within a pen and subsequent hide and carcass contaminations. Cattle do not harbor O104:H4, a serotype that was involved in a major outbreak in Germany in 2011. However, cattle do harbor and shed Shiga toxin-positive O104 with O104:H7 being the most predominant. Indicator organisms (generic E. coli, coliform, and aerobic bacterial counts) were used to assess hide and carcass contaminations. Hide removal was identified as an important source of carcass contamination. Based on quantification of indicator organisms on carcass surfaces, interventions applied by the processing plants were shown to be effective. In studies done with culled dairy cows, differences in hygiene and control measures in lairage areas and the slaughterhouse environment likely explained the differences observed in prevalence of O157 and non-O157 on hide samples across slaughter plants. Molecular techniques (whole genome sequencing and microarray) were used to identify genes in non-O157 STEC to assess virulence potential of cattle strains. The genetic analysis suggested the potential of cattle STEC non-O157 strains to cause human illnesses. In swine, the predominant serogroups detected were O121, O26 and O157. Additionally, swine shed a number of other serogroups (O8, O86, O100, and O91) that cause only sporadic infections. Objective 2. Determine prevalence, and study amplification and dissemination of antimicrobial resistance (AMR) in food borne pathogens and major gut commensals in cattle and swine and develop and evaluate methods to mitigate. Antibiotic alternatives, such as heavy metals, probiotics, etc. have replaced the use of antibiotics because of the concern associated with antimicrobial resistance and public health implications. The major findings are as follows: Probiotics, an antibiotic alternative, are widely used as feed additives for performance benefits in cattle and swine production systems. Among bacterial species contained in probiotics, Enterococcus faecium is common. Antimicrobial resistance, particularly multi-drug resistance, is a common trait among enterococci because of their propensity to acquire resistance and horizontally transfer AMR genes. The E. faecium strains contained in a few commercial probiotics exhibited AMR to medically-important antimicrobials. Studies have suggested a link between added feeding copper (Cu) and co-selection of AMR in bacteria, but data are inconsistent. A study aimed to assess the impact of added Cu, alone or with a feed-grade antimicrobial (chlortetracycline) on Cu resistance and AMR in fecal enterococci in swine. Results indicated Cu, with or without a selection pressure of CTC, did not increase Cu-resistant enterococci and did not co-select resistance to antibiotics.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: 1. Shridhar P. B., I. R. Patel, J. Gangiredla, L. W. Noll, X. Shi, J. Bai, T. G. Nagaraja. 2019. DNA microarray-based genomic characterization of the pathotypes of Escherichia coli O26, O45, O103, O111, and O145 isolated from feces of feedlot cattle. Journal of Food Protection, 82:395-404.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: 2. Cernicchiaro, N., A. R. S. Olivera, A. Hoehn, C. A. Cull, L. W. Noll, P. B. Shridhar, T. G. Nagaraja, S. E. Ives, D. R. Renter, and M. W. Sanderson. 2019. Quantification of bacteria indicative of fecal and environmental contamination from hides and carcasses. Foodborne Pathogens and Diseases, 12:844-855.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: 3. Garcia-Macarro, J. F., S. L. Ishaq, M. V. Rodrguez-Herrera, C. A. Garcoia-Hernandez, J. R. Kawas, and T. G. Nagaraja. 2019. Review: Are there indigenous Saccharomyces in the digestive tract of livestock animal species? Implications for health, nutrition and productivity traits. Animal, 14:22-30.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: 4. Wu, F., M. D. Tokach, J. M. DeRouchey, S. S. Dritz, J. C. Woodworth, R. D. Goodband, K. Chitakasempornkul, N. M. Bello, K. Capps, S. Remfry, H. M. Scott, T. G. Nagaraja, M. D. Apley, and R. G. Amachawadi. 2019. Effects of tylosin administration routes on the prevalence of antimicrobial resistance among fecal enterococci of finishing swine. Foodborne Pathogens and Disease. 16:309-316. doi:10.1089/fpd.2018.2551.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: 5. Shridhar P. B., J. N. Worley, X. Gao, X. Yang, L. W. Noll, X. Shi, J. Bai, J. Meng, and T. G. Nagaraja. 2019. Analysis of virulence potential of Escherichia coli O145 isolated from cattle feces and hide samples based on whole genome sequencing. PLoS One, 14 (11):e0225057.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: 6. Capps, K. M., R. G. Amachawadi, M. B. Menegat, J. C. Woodworth, K. Perryman, M. D. Tokach, S. S. Dritz, J. M. DeRouchey, R. D. Goodband, J. Bai, M. D. Apley, B. V. Lubbers, and T. G. Nagaraja. 2019. Impact of added copper, alone or in combination with chlortetracycline, on growth performance, antimicrobial resistance of fecal enterococci of weaned piglets. Journal of Animal Science. (In Press)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 1. Amachawadi, R. G,, S. E. Remfry, X. Shi, L. Feuerbacher, J. Bai, M. D. Tokach, S. S. Dritz, R. D. Goodband, J. M. DeRouchey, J. C. Woodworth, and T. G. Nagaraja. 2019. Fecal prevalence of the top-7 Shiga toxin-producing Escherichia coli in finisher pigs. 100th Annual Proceedings of Conference of Research Workers in Animal Diseases, November 2-5, Chicago, Illinois. Oral presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 2. Sarwar, F., J. S. Suchodolski, R. G. Amachawadi, J. S. Drouillard, J. Vinasco, T. G. Nagaraja, K. N. Norman, G. H. Loneragan, and H. M. Scott. 2019. Changes in fecal microbiota of cattle fed the beta-adrenergic agonist ractopamine hydrochloride and elevated zinc. 100th Annual Proceedings of Conference of Research Workers in Animal Diseases, November 2-5, Chicago, Illinois. Oral presentation.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 4. Atobatele, M., S. E. Remfry, Y. Romero, X. Shi, R. Phebus, R. G. Amachawadi, and T. G. Nagaraja. 2019. Detection, isolation, and antimicrobial susceptibility testing of Salmonella enterica from wheat grain samples. BugAPalooza, Public Health event hosted in Manhattan City Park, April 1, Manhattan, Kansas, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 3. Remfry, S. E., R. G. Amachawadi, X. Shi, L. Feuerbacher, J. Bai, M. D. Tokach, S. S. Dritz, R. D. Goodband, J. M. DeRouchey, J. C. Woodworth, and T. G. Nagaraja. 2019. Shiga toxin-producing Escherichia coli in Swine: Prevalence, Serogroups, and Public Health Implications. Texas A&M University Graduate Student Association Meeting, April 22, College of Veterinary Medicine, Texas A&M University, College Station, Texas, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 5. Remfry, S. E., R. G. Amachawadi, X. Shi, L. Feuerbacher, J. Bai, M. D. Tokach, S. S. Dritz, R. D. Goodband, J. M. DeRouchey, J. C. Woodworth, and T. G. Nagaraja. 2019. Shiga toxin-producing Escherichia coli in commercial finisher pig feces. Proceedings of the Annual Phi-Zeta Research Day of College of Veterinary Medicine, March 26, Kansas State University, Manhattan, Kansas, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 6. Hancock, S., R. G. Amachawadi, G. Baca, S. Sexton-Bowser, D. Smolensky, D. Rhodes, T. Herald, J. S. Drouillard, D. U. Thomson, and T. G. Nagaraja. 2019. Evaluation of the antimicrobial activities of sorghum phenolic compounds. Proceedings of the Annual Phi-Zeta Research Day of College of Veterinary Medicine, March 26, Kansas State University, Manhattan, Kansas, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 7. Atobatele, M., S. E. Remfry, Y. Romero, X. Shi, R. Phebus, R. G. Amachawadi, and T. G. Nagaraja. 2019. Detection, isolation, and antimicrobial susceptibility testing of Salmonella enterica from wheat grain samples. Proceedings of the Annual Phi-Zeta Research Day of College of Veterinary Medicine, March 26, Kansas State University, Manhattan, Kansas, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 8. Amachawadi, R. G., X. Shi, L. George, M. Theurer, T. Marston, V. H. Holder, and T. G. Nagaraja. 2019. Late-breaking: Impact of feeding a proprietary yeast-based symbiotic product on fecal shedding of top-7 Shiga toxin-producing Escherichia coli in feedlot cattle. Journal of Animal Science, 97 (Suppl. 3):141. American Society for Animal Science Annual Meeting.


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Fellow scientists, Livestock producers, Animal health companies, Cattle and swine veterinary and nutrition consultants, Commodity groups (Kansas Beef Council, National Cattlemen's Beef Association, National Pork Board, etc.), and Policy makers 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: Graduate Students completed: Ashley Smith, PhD; Kaylen Capps (MS), Justin Ludwig (MS). Current Graduate Students: Hannah Seger (PhD), Mori Atobatele (MS), and Sarah Remfry (MS) 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?Journal articles and presentations. What do you plan to do during the next reporting period to accomplish the goals?The following studies are planned: Development and validation of multiplex PCR assays to identify STEC isolates from cattle and swine feces that do not belong to the top-7 STEC. Determination of prevalence of STEC in swine feces Effect of metaphylactic treatment with antibiotics on antimicrobial resistance of gut bacteria, including foodborne pathogens. Effects of route of antibiotic administration on antimicrobial resistance of gut bacteria, including foodborne pathogens

Impacts
What was accomplished under these goals? The Research Team has focused on the following food borne pathogens and prehatvest food safety issues: Shiga toxin-producing Escherichia coli (STEC), Salmonella, Campylobacter, and antimicrobial resistance in gut bacteria of cattle and swine. 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), often referred to as 'non-O157 STEC, represent a major portion of foodborne STEC infections. Cattle are asymptomatic reservoirs for STEC; the organisms reside in the hindgut and are shed in the feces, which serves as the source of food product contaminations. Escherichia coli O104:H4, a Shiga toxin-producing hybrid pathotype, has not been detected in cattle. However, serotypes of O104, other than O104:H4, have been isolated from cattle feces, with O104:H7 being the most predominant. We have assessed, on a whole genome scale, a genetic basis for virulence of E. coli O104 strains isolated from cattle feces. The genomes of ten bovine E. coli O104 strains (six O104:H7, one O104:H8, one O104:H12, Because bovine O104:H7 strains carried virulence genes similar to human clinical strains and one of the human clinical strains was phylogenetically related to bovine strains, the serotype has the potential to be a diarrheagenic pathogen in humans. We have used FDA E. coli identification (FDA-ECID) DNA microarray to identify the serotype, analyze virulence genes to assess the virulence potential and determine phylogenetic relationships of the five of the top six non-O157 E. coli serogroups isolated from cattle feces. ay. The cattle strains harbored diverse flagellar genes (H2, H4, H7, H8, H9, H11, H16, H19, H25, H28 and H31). Bovine non-O157 E. coli strains possessed, in addition to genes encoding Shiga toxins, other major virulence genes, including adhesins, type III secretory system proteins, and plasmid-borne virulence genes, similar to human clinical strains. Because virulence factors encoded by these genes are involved in the pathogenesis of various pathotypes of E. coli, the bovine non-O157 strains have the potential to cause human illness and FDA-ECID microarray provides a rapid profile of the virulence genes for assessment of the virulence potential. Several real-time quantitative PCR (qPCR) assays have been developed for detection and quantification of E. coli O157:H7 in complex matrices by targeting genes for serogroup-specific O-antigen (rfbEO157), H7 antigen, and one or more major virulence factors (Shiga toxin and intimin). A major limitation of such assays is that co-amplification of H7 and virulence genes in a sample does not signal association of those genes with the O157 serogroup. Clusters of regularly interspaced short palindromic repeats (CRISPR) polymorphisms are highly correlated with certain enterohemorrhagic E. coli (EHEC) serotypes, including O157:H7, and presence of Shiga-toxins (stx1 and stx2) and intimin (eae) genes. Therefore, we developed and validated a qPCR assay, targeting the CRISPR array, for the detection and quantification of EHEC O157:H7 in cattle feces, and evaluated the applicability of the assay to detect and compare to a 4-plex qPCR assay targeting rfbEO157, stx1, stx2 and eae genes and a culture method. Detection of E. coli O157 in feedlot cattle fecal samples (n=576) was compared among the CRISPRO157:H7 qPCR assay, culture method and 4-plex qPCR assay. The CRISPRO157:H7 qPCR detected 42.2% as positive for E. coli O157:H7, compared to 30.4% by culture method. Nearly all samples (97.2%) were positive for rfbEO157 by 4-plex PCR, but 21.8% of these samples were negative for stx and/or eae genes, making it unlikely that EHEC O157:H7 was present in the sample. Cohen's Kappa statistic indicated a fair and poor agreement beyond that due to chance among the CRISPR assay and culture method and 4-plex assay, respectively. The qPCR assay is novel in that it can detect EHEC O157:H7 serotype, in cattle feces, by targeting CRISPR polymorphisms. Campylobacter: Campylobacter spp. can be pathogenic to humans and often harbor antimicrobial resistance genes. A cross-sectional study of preharvestcattle evaluated Campylobacter prevalence and susceptibility to nalidixic acid and ciprofloxacin in feedlots that previously administered a fluoroquinolone as primary treatment for bovine respiratory disease. Overall, sample-level prevalence of Campylobacter was 27.2% (272/1000) and differed significantly among feedlots ( p < 0.01). Campylobacter coli was the most common species (55.1%), followed by C. hyointestinalis (42.6%). Within-pen prevalence was not significantly associated with the number of fluoroquinolone treatments, sex, body weight, or metaphylaxis use, but was associated with the number of days cattle were in the feedlot (p = 0.03). The MICs for the majority of Campylobacter isolates were above the breakpoints for nalidixic acid (68.4%) and for ciprofloxacin (65.6%). Distributions of MICs for nalidixic acid (p < 0.01) and ciprofloxacin (p < 0.05) were significantly different among feedlots, and by Campylobacter species. However, fluoroquinolone treatments, sex, body weight, days on feed, and metaphylaxis were not significantly associated with MIC distributions within pens. We found no evidence that the number of fluoroquinolone treatments within feedlot pens significantly affected the within-pen fecal prevalence or quinolone susceptibilies of Campylobacter in feedlots that used a fluoroquinolone as primary treatment for bovine respiratory disease. Antimicrobial resistance: Antibiotics can be administered via various routes in swine production, which may influence gastrointestinal bacteria antimicrobial resistance development. A total of 40 barrows and 40 gilts were used in a 35-d trial to determine the effects of tylosin administration route on antimicrobial resistance in fecal enterococcus isolates. The antibiotic treatments followed US label directions and were: 1) no antibiotic (CON), 2) 110 ppm tylosin in feed for 21 d (FEED), 3) 8.82 mg tylosin per kg BW through intramuscular injection twice daily for the first 3 d of each wk during the 3-wk treatment period (IM), and 4) 66 mg tylosin per liter of drinking water for the first 3 d of each wk during treatment period (WATER). No evidence for route × sampling day interactions (P>0.23) were observed for bacterial resistance development to any antibiotics. Overall, enterococcal isolates collected from pigs receiving FEED or IM were more resistant (P < 0.05) to erythromycin and tylosin compared with CON and WATER groups. In summary, tylosin administration through injection and feed resulted in greater probability of enterococcal resistance to erythromycin and tylosin compared with in-water treatment, which is likely a combined effect of administration route and dosage.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: 1. Amachawadi. R. G., J. Soto, X. Shi, F. Giok, M. D. Tokach, S. K. Narayanan, J. Pluske, and T. G. Nagaraja. 2017. Antimicrobial resistance in Enterococcus faecium isolated from commercial probiotic products used in cattle and swine. ASM Conference on Innovative Microbial Ecology for Mitigation of Antibiotic Resistance and Bacterial Diseases, March 22-25, Crystal City, VA (Oral presentation).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: 2. Williams, H. E., M. D. Tokach, S. S. Dritz, J. C. Woodworth, J. M. DeRouchey, R. G. Amachawadi, T. G. Nagaraja, and R. D. Goodband. 2017. Effects of feeding probiotic, chlortetracycline or a combination on nursery pig growth performance. 2017 Midwest American Society of Animal Science meeting, March 13-15, Omaha, NE (Poster presentation).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: 3. Herrick, R. T., C. L. Rogers, T. J. McEvers, R. G. Amachawadi, T. G. Nagaraja, C. L. Maxwell, and T. E. Lawrence. 2017. Exploratory observational quantification of liver abscess prevalence, economic impact and bacterial flora, specific to region and cattle type. The Plains Nutrition Council-Spring Conference, April 13-14, 2017, San Antonio, Texas (Poster presentation).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: 4. Capps, K., J. Ludwig, X. Shi, J. Bai, and T. G. Nagaraja. 2017. Detection and prevalence of serogroups O2, O74, O109, O131, O168, and O171 of Shiga toxin-producing Escherichia coli (STEC) in cattle feces. International association for Food Protection, Tampa, FL. (Oral presentation).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: 5. Remfry, S. E., R. G. Amachawadi, X. Shi, H. E. Williams, M. D. Tokach, S. S. Dritz, J. R. Pluske, M. D. Apley, and T. G. Nagaraja. 2017. Fecal prevalence and antimicrobial susceptibilities of Salmonella enterica and Campylobacter spp. in piglets diets supplemented with probiotics. 98th Annual Proceedings of Conference of Research Workers in Animal Diseases, December 3-5, Chicago, Illinois. (Oral presentation).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: 6. Seger, H. L., M. W. Sanderson, B. W. White, and T. G. Nagaraja. 2017. Nonpathogenic E. coli fecal shedding and environmental survival in weaned Holstein calves and feedlot steers. 98th Annual Proceedings of Conference of Research Workers in Animal Diseases, December 3-5, Chicago, Illinois ((Oral presentation).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: 7. Capps, K., J. Ludwig, X. Shi, J. Bai, R. Phebus and T. G. Nagaraja. 2017. Development and validation of multiplex PCR assays to identify serogroups of non top-7 Shiga toxin-producing Escherichia coli and determine their prevalence in cattle feces. 98th Annual Proceedings of Conference of Research Workers in Animal Diseases, December 3-5, Chicago, Illinois (Oral presentation).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: 8. Liu, X., Y. Wang, X. Shi, R. Ransburgh, E. Poulsen, V. Pasulapatti, A. Karczmarek, P. Boleij, T. G. Nagaraja, D. Renter, Y. Fang, G. Anderson, and J. Bai. 2017. Multiplex molecular serotyping of Salmonella using Luminex xMAP assay and Check&Trace Salmonella kit and comparing with serological method. 98th Annual Proceedings of Conference of Research Workers in Animal Diseases, December 3-5, Chicago, Illinois. (Poster presentation).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: 9. Schneider, L. G., G. L. Lewis, T. G. Nagaraja, R. A. Moxley, and D. R. Smith. 2017. Comparison of three detection methods to identify enterohemorrhagic Escherichia coli in samples of bovine origin. 98th Annual Proceedings of Conference of Research Workers in Animal Diseases, December 3-5, Chicago, Illinois (Oral presentation).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: 10. Murray, S. A., K. N. Norman, S. D. Lawhon, R. G. Amachawadi, J. Vinasco, R. A. Pugh, J. S. Drouillard, C. L. Van Bibber-Krueger, T. G. Nagaraja, and H. M. Scott. 2017. Zinc and menthol as alternatives to antibiotics: impacts on Enterococcus spp. resistance in feeder cattle. 98th Annual Proceedings of Conference of Research Workers in Animal Diseases, December 3-5, Chicago, Illinois (Oral presentation).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: 11. Remfry, S. E., R. G. Amachawadi, X. Shi, H. E. Williams, M. D. Tokach, S. S. Dritz, J. R. Pluske, M. D. Apley, and T. G. Nagaraja. 2017. Fecal prevalence and antimicrobial susceptibilities of Salmonella enterica and Campylobacter spp. in piglets diets supplemented with probiotics. Kansas City One Health Day. November 1, Olathe, Kansas (Poster presentation).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 12. Remfry. S., M. Atobatele, R. G. Amachawadi, X. Shi, R. Phebus, and T. G. Nagaraja. 2018. Prevalence of Top-7 Shiga toxin producing Escherichia coli in wheat grains by culture method. Proceedings of the Annual Phi-Zeta Research Day of College of Veterinary Medicine, March 6, Kansas State University, Manhattan, Kansas, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 13. Cochrane, R. A., R. G. Amachawadi, S. Remfry, A. B. Lerner, J. T. Gebhardt, T. G. Nagaraja, J. R. Pluske, M. C. Niederwerder, J. C. Woodworth, S. S. Dritz, and C. K. Jones. 2018. Young Scholar Presentation: A Review of Medium Chain Fatty Acids and Their Recent Role in Feed Safety. Midwest Animal Science Meeting, March 12-14, Omaha, NE.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 14. Sylvester. E. W., R. A. Cochrane, R. G. Amachawadi, S. Remfry, A. B. Lerner, T. G. Nagaraja, J. Pluske, M. C. Niederwerder, C. B. Paulk, C. R. Stark, J. C. Woodworth, S. S. Dritz, M. D. Tokach, J. M. DeRouchey, R. D. Goodband, and C. K. Jones.2018. Defining the Minimum Inhibitory Concentration of Synthetic and Commercial Medium Chain Fatty Acid Based Products Against Salmonella Typhimurium. Midwest Animal Science Meeting, March 12-14, Omaha, NE.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 15. Swanson. A. J., R. A. Cochrane, R. G. Amachawadi, S. Remfry, A. B. Lerner, T. G. Nagaraja1, J. R. Pluske, M. C. Niederwerder, C. R. Stark, C. B. Paulk, J. C. Woodworth, S. S. Dritz, M. D. Tokach, J. M. DeRouchey, R. D. Goodband, and C. K. Jones. 2018. Determination of the Minimum Inhibitory Concentration of Various Medium Chain Fatty Acid Based Products in E. coli, Enterotoxigenic E. coli, and Campylobacter coli. Midwest Animal Science Meeting, March 12-14, Omaha, NE.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 16. Wu. F., M. D. Tokach, J. M. DeRouchey, S. S. Dritz, J. C. Woodworth, R. D. Goodband, K. Capps, S. Remfry, K. Chitakasempornkul, N. M. Bello, T. G. Nagaraja, and R. G. Amachawadi. 2018. Effects of tylosin administration route on the development of antimicrobial resistance in fecal enterococci of finishing swine. Midwest Animal Science Meeting, March 12-14, Omaha, NE.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 17. Menegat. M. B., J. C. Woodworth, S. S. Dritz, R. G. Amachawadi, T. G. Nagaraja, K. Capps, M. D. Tokach, J. M. DeRouchey, and R. D. Goodband. 2018. Impact of added copper and chlortetracycline on growth performance of nursery pigs. Midwest Animal Science Meeting, March 12-14, Omaha, NE.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 18. Amachawadi. R. G., S. A. Murray, C. L. Van Bibber-Krueger, K. N. Norman, J. Vinasco, S. D. Lawhon, R. A. Pugh, J. S. Drouillard, T. G. Nagaraja, and H. M. Scott. 2018. Effects of zinc and menthol supplementation on antimicrobial resistance among fecal enterococci in feedlot cattle. 5th International Conference on Enterococci, April 15-20, Chamonix, France.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: 1. Smith, A. B., D. G. Renter, N. Cernicchirao, X. Shi, J. S. Nickell, D. J. Keil, and T. G. Nagaraja. 2017. A randomized trial to assess the effect of fluoroquinol metaphylaxis on the fecal prevalence and quinolone susceptibilities of Salmonella and Campylobacter in feedlot cattle. Foodborne Pathog. Dis. 14: 600-607.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: 2. Ekong, P. S., M. W. Sanderson, N. M. Bello, L. W. Noll, N. Cernicchiaro, D. G. Renter, J. Bai, and T. G. Nagaraja.. 2017. Bayesian estimation of true prevalence, sensitivity and specificity of three diagnostic tests for detection of Escherichia coli O157 in cattle feces. Prev. Vet. Med. 148:21-27.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: 5. Noll, L. W., J. N. Worley, X. Yang, P. B. Shridhar, J. Bai, J. Meng, D. Caragea, and T. G. Nagaraja.. 2017. Draft genome sequences of enteropathogenic Escherichia coli O103 strains isolated from feces of feedlot cattle. Genome Announc. 5:e00387-17.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: 6. Noll, L. W., J. N. Worley, X. Yang, P. B. Shridhar, J. Bai, J. Meng, D. Caragea, and T. G. Nagaraja.. 2017. Draft genome sequences of enterohemorrhagic Escherichia coli O103:H2 strains isolated from feces of feedlot cattle. Genome Announc. 5:e00094-17.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: 7. Shridhar, P. B., L. W. Noll, C. A. Cull, X. Shi, X., N. Cernicchiaro, D. G. Renter, D.G., J. Bai, and T. G, Nagaraja. 2017. Spiral plating method to quantify the six major non-O157 E. coli serogroups in cattle feces. J. Food Prot. 80:848-856.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: 8. Shridhar, P. B., C. Siepker, L. W. Noll, X. Shi, X., T. G, Nagaraja and J. Bai. 2017. Shiga toxin subtypes of non-O157 E. coli serogroups isolated from cattle feces. Frontiers Cell Infect. Microbiol. 7:121
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: 3. Shridhar, P. B., I. R. Patel, J. Gangiredla, M. K. Mammel, L. W. Noll, X. Shi, J. Bai, C. A. Elkins, N. Strockbine, and T. G, Nagaraja. 2017. Draft genome sequences of Escherichia coli O104 strains of bovine and human origin. Genome Announc. 5:e00630-17.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: 4. Cull, C. A., D. G. Renter, D. M., Dewsbury, L. W. Noll, P. B. Shridhar, S. E. Ives, and T. G. Nagaraja, and N. Cernicchiaro. 2017.Feedlot- and pen-level prevalence of enterohemorrhagic Escherichia coli in feces of commercial feedlot cattle in two major U.S. cattle feeding areas. Foodborne Pathog. Dis. 6:309-317.


Progress 10/01/16 to 09/30/17

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: Graduate Students completed: Lance Noll, PhD; Pius Ekong, PhD; Pragathi Shridhar, PhD. Current Graduate Students: Mori Atobatele, Kaylen Capps, Justin Ludwig, and Sarah Remfry 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?Presentations and journal articles. What do you plan to do during the next reporting period to accomplish the goals? The following studies are planned: Development, validation and application of real-time PCR assays, based on the clustered regularly interspaced short palindromic repeat sequence polymorphisms (CRISPR), for the serotype specific detection and quantification of E. coli O157:H7 in cattle feces. Whole genome sequencing-based DNA microarray-based identification and comparative analysis of major and putative virulence genes of the "top six" non-O157 Shiga toxin producing Escherichia coli of cattle origin. Development and validation of multiplex PCR assays to identify STEC isolates from feedlot cattle feces that did not belong to the top-7 STEC.

Impacts
What was accomplished under these goals? The Research Team has focused on Shiga toxin-producing Escherichia coli (STEC), Salmonella, and antimicrobial resistance in gut bacteria of cattle ands wine. 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), often referred to as 'non-O157 STEC, represent a major portion of foodborne STEC infections. Cattle are asymptomatic reservoirs for STEC; the organisms reside in the hindgut and are shed in the feces, which serves as the source of food product contaminations. Culture-based detection of non-O157 STEC involves an immunomagnetic separation (IMS) step to capture the specific serogroups in complex matrices, such as feces. The IMS procedure is time-consuming and labor-intensive because of the need to subject each fecal sample to six individual beads. Therefore, our objective was to evaluate whether pooling of IMS beads affects sensitivity of non-O157 STEC detection compared with using individual IMS beads. The evaluation was done by comparing detection of serogroups in feces spiked with pure cultures and from feces of naturally-shedding cattle. The results showed that the pooling of IMS beads is a better option for detection of STEC serogroups in fecal samples compared to individual beads. Besides prevalence in feces, concentrations of STEC in cattle feces plays a major role in their transmission dynamics. A subset of cattle, referred to as super shedders, shed the E. coli O157 at high concentrations (≥ 4 log CFU/g of feces). It is not known whether a similar pattern of fecal shedding exists for non-O157. Our objectives were to initially validate the spiral plating method to quantify the six non-O157 E. coli serogroups, and then determine the applicability of the method and compare with multiplex quantitative PCR (mqPCR) assays for the quantification of the six non-O157 E. coli serogroups in cattle fecal samples collected from commercial feedlots. Of the 1,152 cattle fecal samples tested from eight commercial feedlots, 122 (10.6%) and 320 (27.8%) harbored concentrations ≥ 4 log CFU/g of one or more of the six serogroups of non-O157 by spiral plating and mqPCR methods, respectively. We have developed and validateed two multiplex quantitative PCR (mqPCR) assays, targeting O antigen genes of O26, O103, and O111 (mqPCR-1) and O45, O121, and O145 (mqPCR-2), 2) and utilized the two assays, together with a previously developed four-plex qPCR assay (mqPCR-3) targeting the O157 antigen and three virulence genes (stx1, stx2, and eae ) to quantify seven serogroups and three virulence genes in cattle feces. Of the 576 fecal samples subjected, before enrichment, to three mqPCR assays for quantification, 175 (30.4%) were quantifiable (≥ log 4 CFU/g) for at least one of the seven serogroups, with O157 being the most common serogroup. Escherichia coli O104:H4, an hybrid pathotype of Shiga toxigenic and enteroaggregative E. coli, involved in a major foodborne outbreak in Germany in 2011, has not been detected in cattle feces. Our objectives were to determine the prevalence of O104 in feces of feedlot cattle, by culture and PCR detection methods, and characterize the isolated strains. Fecal samples, enriched in E. coli broth, were subjected to culture and PCR methods of detection. Model-adjusted prevalence estimates of E. coli O104 at the feedlot level were 5.7% and 21.2%, and at the sample level were 0.5% and 25.9% by culture and PCR, respectively. A total of 143 isolates, positive for the wzxO104, were obtained in pure culture from 146 positive fecal samples. Ninety-two of the 143 isolates (64.3%) also tested positive for the wbdDO8/O9/O9a, indicating that only 51 (35.7%) isolates truly belonged to the O104 serogroup (positive for wzxO104 and negative for wbdDO8/O9/O9a). All 51 isolates tested negative for eae, and 16 tested positive for stx1 gene of the subtype 1c. The predominant serotype was O104:H7. Although cattle do not harbor the O104:H4 pathotype, they do harbor and shed Shiga toxigenic O104 in the feces and the predominant serotype was O104:H7. STEC O26 is the second most important STEC worldwide. O26 strains are characterized in two groups: EPEC O26 carrying LEE, mostly causing milder disease and watery diarrhea, and STEC O26 that carry stx gene, responsible for more severe outcomes. stx-negative O26 can further be split in two groups. One O26 group differs significantly from O26 EHEC, whereby the other O26 EHEC-like shows all characteristics of EHEC O26, except production of Stx. In order to determine the different populations of O26 E. coli present in US cattle we sequenced several O26:H11 strains from US cattle and compared them to O26:H11 genomes available at NCBI. We found as observed previously in Europe, we found that O26:H11/H- in US cattle are highly diverse, with at least 5 different populations. Most strains were sequence type 29 (ST29). Two clear lineages could be distinguished among ST29 strains isolated from cattle. Lineage 1 composed of O26:H11 EHEC-like (ST29) and O26:H11 EHEC (ST21), and Lineage 2 showing four diverse populations with different virulence content. Our results showed that whole genome sequence analysis is a robust and valid approach to identify, and genetically characterize E. coli O26:H11, which is of paramount importance for food supply safety and public health. Salmonella Salmonella is an important foodborne pathogen and antimicrobial resistance can be a human health concern. The objectives of this cross-sectional study were to (1) determine the prevalence and quinolone susceptibility of Salmonella in feces of preharvest commercial feedlot cattle and (2) determine if the prevalence and susceptibility of Salmonella isolates were associated with previous fluoroquinolone use within pens. Five feedlots in western Kansas and Texas were selected based on their use of a commercially licensed fluoroquinolone for initial treatment of bovine respiratory disease (BRD). Twenty pen floor fecal samples were collected from each of 10 pens from each feedlot. Salmonella isolation was performed and microbroth dilution was used to determine susceptibility of isolates to nalidixic acid and ciprofloxacin. Generalized linear mixed models were used to assess associations between. Overall, cumulative fecal prevalence of Salmonella was 38.0% (380/1000), but prevalence varied significantly (p < 0.01) among the five feedlots: 0.5% (1/200), 17.5% (35/200), 37.0% (74/200), 58.5% (117/ 200), and 76.5% (153/200). There was no significant association ( p = 0.52) between Salmonella prevalence and the frequency of fluoroquinolone treatments within a pen. All Salmonella isolates were susceptible to ciprofloxacin, while one isolate exceeded the human breakpoint (‡32 lg/mL) for nalidixic acid. Nearly all Salmonella isolates were susceptible to quinolones, despite the fact that a fluoroquinolone was used as the primary therapeutic antimicrobial to treat BRD in these feedlot populations.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Feldpausch, J. A., R. G. Amachawadi, M. D. Tokach, H. M. Scott, T. G. Nagaraja, S. S. Dritz, R. D. Goodband*, J. C. Woodworth, and J. M. DeRouchey. 2016. Effects of dietary copper, zinc, and ractopamine HCl on finishing pig growth performance, carcass characteristics, and antimicrobial susceptibility of enteric bacteria. J. Anim. Sci., 94:3278-3293.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Cernicchiaro, N., S. E. Ives, T. S. Edrington, T. G. Nagaraja, D. G. Renter. 2016. Efficacy of a Salmonella siderophore receptor protein vaccine on fecal shedding and lymph node carriage of Salmonella in commercial feedlot cattle. Foodborne Pathog. Dis., 13:517-525.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Smith, A., D. G. Renter*, N. Cernicchiaro, X. Shi, and T. G. Nagaraja. 2016. Prevalence and fluoroquinolone-susceptibilities of Salmonella isolated from the feces of pre-harvest cattle within feedlots that use a fluoroquinolone to treat bovine respiratory disease. Foodborne Pathog. Dis., 13:303-308.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Woods, T. A., H. M. Mendez, S. Ortega, X. Shi, D. Marx, J. Bai, R. A. Moxley, T. G. Nagaraja, S. W. Graves, and A. Deshpande. 2016. Development of 11-plex MOL-PCR assay for the rapid screening of samples for Shiga toxin-producing Escherichia coli. Front. Cell. Infect. Microbiol., 6:92.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: 9. Murray, S. A., A. C. Holbert, K. N. Norman, S. D. Lawhon, R. G. Amachawadi, J. Vinasco, R. A. Pugh, J. S. Drouillard, C. L. Van Bibber-Krueger, T. G. Nagaraja, and H. M. Scott. 2016. Heavy metals, essential oils, and ?-adrenergic agonists as alternatives to antibiotics and their impacts on bacterial resistance in beef cattle. The 2nd International Symposium on Alternatives to Antibiotics (ATA): Challenges and Solutions in Animal Production. World Organization for Animal Health, December 12-15, Paris, France (Poster presentation).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: 10. McKiearnan, A., L. W. Noll, P. B. Shridhar, X. Shi, T. G. Nagaraja, S. E. Ives, D. G. Renter, M. W. Sanderson, N. Cernicchiaro. 2016. Enterohemorrhagic Escherichia coli on cattle hides and bacterial transfer from hides to carcasses in Midwestern commercial beef slaughter operations. Joint Annual Meeting (JAM) of the American Society of Animal Science, the American Dairy Science Association, the Western Section of the American Society of Animal Science, and the Canadian Society of Animal Science. July 19-23, 2016, Salt Lake City, UT.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Gonzales-Escalana N., M. Toro, L. V. Rump, G. Cao, T. G. Nagaraja, and J. Meng. 2016. Virulence gene profiles and clonal relationships of Escherichia coli O26:H11 isolates from feedlot cattle by whole genome sequencing. Appl. Environ. Microbiol., 82:3900-3912.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Amachawadi, R. G., M. Thomas, T. G. Nagaraja, and J. Scaria. 2016. Genome sequences of Salmonella enterica subsp. enterica Serovar Lubbock isolated from liver abscesses of feedlot cattle. Genome Announc., 4:e00319-16.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Noll, L. W., W. C. Baumgartner, P. B. Shridhar, C. A. Cull, D. M. Dewsbury, X. Shi, B. An, N. Cernicchiaro, D. G. Renter, and T. G. Nagaraja. 2016. Pooling of immunomagnetic separation beads does not affect sensitivity of detection of six serogroups of Shiga toxin-producing Escherichia coli in cattle feces. J. Food Prot., 79:59-65.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Shridhar, P. B., L. W. Noll, X. Shi, B. An, N. Cernicchiaro, D. G. Renter, T. G. Nagaraja, and J. Bai. 2016. Multiplex real-time PCR assays for the detection and quantification of the six major non-O157 Escherichia coli serogroups in cattle feces. J. Food Prot., 79:66-74.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Shridhar, P. B., L. W. Noll, X. Shi, N. Cernicchiaro, D. G. Renter, J. Bai, and T. G. Nagaraja. 2016. Escherichia coli O104 in feedlot cattle feces: Prevalence, isolation and characterization. PLoS One. 11:e0152101.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Wilson*, K., B. P. Holland, D. L. Step, M. E. Jacob, D. L. VanOverbeke, C. J. Richards, T. G. Nagaraja, and C. R. Krehbiel. 2016. Feeding wet distillers grains plus solubles with and without a direct-fed microbial to determine performance, carcass characteristics, and fecal shedding of Escherichia coli 0157:H7 in feedlot heifers. J. Anim. Sci., 94:297-305.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: 1. Chall, R. C., L. W. Noll, J. Bai, and T. G. Nagaraja. 2016. Development and validation of a real-time PCR assay, based on the clustered regularly interspaced short palindromic repeat sequence polymorphisms (CRISPR), for serotype-specific detection and quantification of enterohemorrhagic Escherichia coli O157:H7 in cattle feces. Phi Zeta Research Day (March 2016) (Poster presentation).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: 2. Noll, L. W., P. B. Shridhar, S. D. Menon, X. Shi, S. E. Ives, T. E. Lawrence, E. Cha, T. G. Nagaraja, and D. G. Renter. 2016. Quantification of coliforms and Escherichia coli on beef carcasses immediately before and after evisceration during slaughter. Annual STEC-CAP Collaborators Meeting, Lincoln, NE (Poster presentation)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: 3. Noll, L. W., K. L. Samuelson, P. B. Shridhar, J. B. Ludwig, X. Shi, M. E. Hubbert, C. A. L�est, E. Cha, D. G. Renter, and T. G. Nagaraja. 2016. Effects of dietary protein concentrations with different proportions of ruminally-degradable and -undegradable protein on fecal shedding of Escherichia coli O157 in finishing cattle. Annual STEC-CAP Collaborators Meeting, Lincoln, NE (Poster presentation)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: 4. Noll, L. W., J. N. Worley, X. Yang, P. B. Shridhar, X. Shi, J. Meng, and T. G. Nagaraja. 2016. Whole genome sequencing-based identification and comparative analysis of major and putative virulence genes of Escherichia coli O103 of bovine fecal origin. Annual Meeting of International Association for Food Protection, St. Louis, MO (Oral presentation).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: 5. Noll, L. W., J. N. Worley, X. Yang, X. Shi, J. Meng, and T.G. Nagaraja. 2016. Differences in virulence and antimicrobial resistance gene profiles of bovine and human enterohemorrhagic Escherichia coli O157 and O103: A comparative genomics approach. Conference of Research Workers in Animal Diseases, Chicago, IL (Oral presentation).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: 6. Shridhar, P. B., Isha R. Patel, Jayanthi Gangiredla, Christopher A. Elkins, Lance Noll, Xiaorong Shi, Jianfa Bai, and T. G. Nagaraja. 2016. Characterization of Shiga toxin-producing non-O157 E. coli serogroups isolated from cattle feces by DNA-microarray Annual STEC-CAP Collaborators Meeting (June , Lincoln, NE (Poster presentation).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: 7. Amachawadi, R. G., J. Scaria, and T. G. Nagaraja. 2016. Salmonella enterica in liver abscesses of beef cattle: Does it have an etiologic role? International Symposium Salmonella and Salmonellosis (I3S), June 6-8, Saint-Malo, France.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: 8. Scott, H. M., K. Rozas, J. A. Feldpausch, K. N. Norman, R. G. Amachawadi, G. Chalmers, J. Vinasco, R. A. Pugh, S. S. Dritz, M. D. Tokach, R. D. Goodband, J. C. Woodworth, J. M. DeRouchey, T. G. Nagaraja, and P. Boerlin. 2016. Effects of zinc, copper, oregano oil and ractopamine as antibiotic alternatives on bacterial resistance and growth performance in swine. The 2nd International Symposium on Alternatives to Antibiotics (ATA): Challenges and Solutions in Animal Production. World Organization for Animal Health, December 12-15, Paris, France (Poster presentation).


Progress 10/01/15 to 09/30/16

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: Graduate students completed: Diana Dewsbury, MS: Lance Noll, MSCurrent Graduate students: Charley Cull, PhD; Pius Ekong, PhD; Allison McKiearnan, PhD; Lance Noll, PhD; 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?Conference presentations and journal articles. What do you plan to do during the next reporting period to accomplish the goals?Continue focus on Shiga toxin-producing Escherichia coli (STEC) and antimicrobial resistance in gut bacteria.

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. 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: 2015 Citation: 7. Amachawadi, R. G., H. M. Scott, S. Nitikanchana, J. Vinasco, M. D. Tokach, S. S. Dritz, J. L. Nelssen, R. D. Goodband and T. G. Nagaraja. 2015. Nasal carriage of mecA-positive methicillin-resistant Staphylococcus aureus in pigs exhibits dose-response to zinc supplementation. Foodborne Path. Dis. 12:159=163
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: 8. Agga, G.E., H. M. Scott*, J. Vinasco, T. G. Nagaraja, R. G. Amachawadi, J. Bai, B. Norby, D. G. Renter, S. S. Dritz, J. L. Nelssen, and M. D. Tokach. 2015. Effects of chlortetracycline and copper supplementation on the prevalence and quantity of antimicrobial resistance genes in the fecal metagenome of weaned pigs. Prev. Vet. Med. 119:179-189.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: 9. Stromberg ZR, Baumann N, Lewis GL, Cernicchiaro N, Renter DG, Marx DB, Phebus RK, Moxley RA 2015. Prevalence of enterohemorrhagic Escherichia coli O26, O45, O103, O111, O121, O145, and O157 on hides and carcass surfaces of beef feedlot cattle at harvest. Foodborne Pathog Dis. 1`2:631-638.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: 10. Cull CA, Renter DG*, Bello NB, Ives SE, Babcock AH. 2015. Performance and carcass characteristics of commercial feedlot cattle from a study of vaccine and direct-fed microbial effects on E. coli O157:H7 fecal shedding. J Anim Sci. 93:3144-3151.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: 6. Ison, S. A., S. Delannoy, M. Bugarel, K. K. Nightingale, H. E. Webb, D. G. Renter, T. G. Nagaraja, G. H. Lomeragan, and P. Fach. 2015. Genetic diversity and pathogenic potential of attaching and effacing Escherichia coli O26:H11 strains recovered from bovine feces in United States. Appl. Environ. Microbiol. 81:3671-3678.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 1. PCR- and culture-based methods to detect and quantify six major non-O157 serogroups of Shiga toxin-producing Escherichia coli in cattle feces. Beef Food Safety Summit, National Cattlemens Association, TX (Mar 3-5, 2015).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 3. Noll LW, Shridhar PB, Dewsbury D, Shi X, Cernicchiaro N, Renter D, Bai J, Nagaraja TG. A Four-plex Quantitative PCR for the Detection and Quantification of Escherichia coli O157 in Cattle Feces and Comparison with Culture and Conventional PCR Methods. In: Proceedings of the American Society for Microbiology General Meeting; 2015 May 30- June 2, New Orleans, LA (Poster presentation).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 4. Noll LW, Shridhar PB, Shi X, Cernicchiaro N, Renter D, Bai J, Nagaraja TG. Application of a Novel Four-Plex Quantitative PCR Assay for Quantification of Escherichia coli O157 on Cattle Hide and Carcass. In: Proceedings of the Shiga toxin-producing Escherichia coli Coordinated Agricultural Project Conference; 2015 June 3-5, Manhattan, KS (Poster presentation).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 5. Noll LW, Shridhar PB, Shi X, Cernicchiaro N, Renter D, Bai J, Nagaraja TG. Application of a Novel Four-Plex Quantitative PCR Assay for Quantification of Escherichia coli O157 on Cattle Hide and Carcass. In: Proceedings of the International Association for Food Protection Annual Meeting; 2015 Jul 25-28, Portland, OR (Oral presentation).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 6. Noll LW, Shridhar PB, Menon SD, Shi X, Ives SE, Lawrence TE, Cha E, Nagaraja TG, Renter DG. Quantification of coliforms and Escherichia coli on beef carcasses immediately before and after evisceration during slaughter. In: Proceedings of the Conference of Research Workers in Animal Diseases Conference; 2015 Dec 6-8, Chicago, IL (Oral Presentation).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 7. Rozas, K., R. G. Amachawadi, K. Norman, J. Vinasco, R. Pugh, F. L. Perez, A. Wakil, D. Manriquez, M. D. Tokach, T. G. Nagaraja, and H. M. Scott. 2015. Effect of copper, zinc, and essential oil supplementation on antimicrobial resistance of fecal Escherichia coli in nursery piglets. 96th Proceedings of Conference on Research Workers in Animal Diseases, December 6-8, Chicago, Illinois.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 8. Amachawadi, R. G., H. M. Scott, J. Vinasco, J. Feldpausch, M. D. Tokach, S.S. Dritz, J. L. Nelssen, R. D. Goodband, and T. G. Nagaraja. 2015. Effects of chlortetracycline and zinc supplementation on nasal, skin, and tonsillar carriage of methicillin-resistant and zinc-resistant Staphylococcus aureus in piglets. 4th ASM Conference on Antimicrobial Resistance in Zoonotic Bacteria and Foodborne Pathogens. May 8-11, 2015. Washington, DC.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 9. Shridhar, P. B., Lance Noll, Charley Cull, Allison Mckiearnan, Xiaorong Shi, Natalia Cernicchiaro, Michael W. Sanderson, David G. Renter, Jianfa Bai and T. G. Nagaraja, Quantification of Six Major Non-O157 Escherichia coli Serogroups in cattle hide samples by Spiral Plating and Multiplex Quantitative PCR Methods In: Proceedings of the Conference of Research Workers in Animal Diseases Conference; 2015 Dec 6-8, Chicago, IL (Oral Presentation).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 10. Shridhar, P. B., Lance Noll, Ellen Kim, Charley Cull, Diana Dewsbury, Xiaorong Shi, 2015. Natalia Cernicchiaro, David Renter, Jianfa Bai and T.G. Nagaraja, Quantification of Six Major Non-O157 Escherichia coli Serogroups in Feces of Feedlot Cattle by Spiral Plating and Quantitative Real-time PCR Methods. In: Proceedings of the International Association for Food Protection Annual Meeting; 2015 Jul 25-28, Portland, OR (Oral presentation).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 11. Shridhar, P. B., Lance Noll, Jianfa Bai, Xiaorong Shi, Isha R. Patel, Jayanthi Gangiredla, Christopher A. Elkins and T. G. Nagaraja. DNA microarray-based assessment of virulence potential of Shiga toxigenic Escherichia coli O104:H7 isolated from feedlot cattle fece (Poster presentation, STEC CAP Annual conference, Manhattan, Kansas, June, 2015).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 12. Shridhar, P. B., Ellen kim, Lance Noll, Charlie Cull, Diana Dewsbury, Xiaorong Shi, Natalia Cernicchiaro, David G. Renter, Jianfa Bai and T. G. Nagaraja. Application of Spiral Plating and Quantitative Real-Time PCR Methods for Quantification of Six Major Non-O157 Escherichia coli Serogroups in Feces of Feedlot Cattle(Poster presentation, STEC CAP Annual conference, Manhattan, Kansas, June, 2015).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 13. Shridhar, P. B., Chris Siepker, Lance Noll, Xiaorong Shi, T. G. Nagaraja and Jianfa Bai, Shiga toxin subtypes of non-O157 E. coli serogroups isolated from various source. (Poster presentation, STEC CAP Annual conference, Manhattan, Kansas, June, 2015)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 2. Detection Methods for Non-O157 Shiga toxin-producing Escherichia coli (STEC) in Cattle Feces. International Verotoxigenic Escherichia coli Conference (VTEC-2015), Boston, MA (Sep 13-16, 2015).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 17. Shridhar, P. B., Ellen kim, Lance Noll, Charlie Cull, Diana Dewsbury, Xiaorong Shi, Natalia Cernicchiaro, David G. Renter, Jianfa Bai and T. G. Nagaraja. Spiral plating and quantitative real-time PCR based quantification of non-O157 E. coli in cattle feces (Oral presentation, Phi Zeta Research Day, Manhattan, Kansas, March, 2015).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 18. Cull CA, Renter DG, Bello N.B., Ives S.E., Babcock A.H. Performance and carcass characteristics of commercial feedlot cattle from a study of vaccine and direct-fed microbial effects on E. coli O157:H7 fecal shedding. Conference of Research Workers in Animal Diseases (CRWAD), December 6-8, 2015, Chicago, Illinois, USA P49.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 19. Cull CA, Cernicchiaro N, Dewsbury DM, Noll LW, Shridhar PB, Ives SE, Nagaraja TG, Renter DG. Feedlot- and pen-level prevalence of Shiga toxin-producing Escherichia coli in feces of commercial feedlot cattle in two major cattle feeding areas. STEC CAP Annual Meeting 2015, June 3-5, 2015, Kansas State University, Manhattan, KS, pp. 11.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 20. Cull CA, Renter DG, Ives SE, Dewsbury DM., Noll LW, Shridhar PB, Nagaraja TG, Cernicchiaro, N. Feedlot- and pen-level prevalence of Shiga toxin-producing Escherichia coli in feces of commercial feedlot cattle in two major cattle feeding states. Phi Zeta Day 2015 Clinical & Applied Sciences: Food Animal Presentation. College of Veterinary Medicine, Kansas State University. (*2nd place oral presentation).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 21. Maldonado P, Shi X, Nagaraja TG, Renter DG, Cernicchiaro N. Characterization of Salmonella enterica isolates from feces of feedlot cattle using pulsed-field gel electrophoresis. Phi Zeta Day 2015 Clinical & Applied Sciences: Food Animal Presentation. College of Veterinary Medicine, Kansas State University.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Toro, M., G. Cao, L. Rump, T.G. Nagaraja, J. Meng, and N. G. Escalona. 2015. Draft genome sequences of 64 strains of non-O157:H7 Shiga toxin-producing Escherichia coli. Genome A, 3:e01067-15.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 14. Shridhar, P. B., Lance Noll, Xiaorong Shi, Natalia Cernicchiaro, David G. Renter, Jianfa Bai and T. G. Nagaraja. Escherichia coli O104 in Feedlot Cattle Feces: Prevalence, Isolation and Characterization (Poster presentation, STEC CAP Annual conference, Manhattan, Kansas, June, 2015).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 15. Shridhar, P. B., Lance Noll, Jianfa Bai, Xiaorong Shi, Isha R. Patel, Jayanthi Gangiredla, Christopher A. Elkins and T. G. Nagaraja. DNA microarray-based assessment of virulence potential of Shiga toxigenic Escherichia coli O104:H7 isolated from feedlot cattle feces (Poster presentation, American Society of Microbiology General meeting, New Orleans, May 30 - June 2, 2015).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: 16. Shridhar, P. B., Lance Noll, Xiaorong Shi, Natalia Cernicchiaro, David G. Renter, Jianfa Bai and T. G. Nagaraja. Application of multiplex real-time PCR for the detection and quantification of seven major Escherichia coli serogroups and three virulence genes in feces of feedlot cattle (Oral presentation, K-State Research Forum, Manhattan, Kansas, March, 2015).
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: 2. Noll, L. W., P. B. Shridhar, D. M. Dewsbury, X. Shi, N. Cernicchiaro, D. G. Renter and T. G. Nagaraja. 2015. A comparison of culture- and PCR-based methods to detect six non-O157 serogroups of Shiga toxin-producing Escherichia coli in cattle feces. PLoS One, 10:e0135446.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: 3. Dewsbury D.M., L. W. Noll, P. B. Shridhar, X. Shi, D. G. Renter, T. G. Nagaraja, and N. Cernicchiaro. 2015. Prevalence of Shiga toxin-producing Escherichia coli (STEC) serogroups and associated virulence genes in feces of commercial feedlot cattle. Foodborne Path. Dis. 12:726-732.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: 4. Noll, L. W., P. B. Shridhar, X. Shi, B. An, N. Cernicchiaro, D. G. Renter, T. G. Nagaraja*, and J. Bai. 2015. A four-plex real-time PCR assay, based on rfbE, eae, stx1 and stx2 genes, for the detection and quantification of Escherichia coli O157 in cattle feces. Foodborne Path. Dis. 12:787-794.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: 5. Amachawadi, R. G., H. M. Scott, C. Aperce, J. Vinasco, J. S. Drouillard, and T. G. Nagaraja. 2015. Effects of in-feed copper and tylosin supplementations on copper and antibiotic resistance in fecal enterococci of feedlot cattle. J. Appl. Microbiol. 118:1287-1297.


Progress 10/01/14 to 09/30/15

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?Research outcomes generated from this project have been presented in a variety of national and international meetings. What do you plan to do during the next reporting period to accomplish the goals?Continue work towards the goals.

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: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: 22. Nagaraja, T. G. Future use of antimicrobial additives and alternatives to prevent acidosis. Ruminant Nutrition Conference, Universidad Aut�nomo de Nuevo Le�n, Monterrey, Mexico (Oct 7-8, 2014).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: 4. 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).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: 10. 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).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: 11. 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).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: 12. 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).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: 13. 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).
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: 39. 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.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: 40. 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.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: 41. 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.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: 42. 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