Source: MICHIGAN STATE UNIV submitted to
HOST, GENETIC, MICROBIAL, AND ENVIRONMENTAL FACTORS ASSOCIATED WITH SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) SHEDDING
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0224218
Grant No.
2011-67005-30004
Project No.
MICL08452
Proposal No.
2010-04451
Multistate No.
(N/A)
Program Code
A4111
Project Start Date
Jan 15, 2011
Project End Date
Jan 14, 2017
Grant Year
2011
Project Director
Manning, S. D.
Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
Microbiology & Molecular Genetics
Non Technical Summary
STEC is a leading cause of foodborne and waterborne infections. Cattle are the primary reservoir of STEC, but little is known about those factors that impact shedding levels. While some STEC genotypes have been suggested to more effectively colonize cattle, the type and distribution of the gastrointestinal microbiota, cattle immune responses, and the composition of the rumen may also impact shedding. To comprehensively study the ecology of STEC shedding, we will: 1) Identify bacterial genotypes and epidemiological factors important for shedding in multiple herds over time; 2) Compare the composition, diversity and function of the microbial communities within the rectoanal junction and ruminal fluid of STEC shedders and non-shedders; 3) Determine how STEC affects the bovine immune response; and 4) Identify STEC inhibitors from the ruminal fluid of non-shedders and test control strategies aimed at decreasing shedding levels. Our long term goal is to assess how bacterial, epidemiological, immunological, and host factors work together to impact shedding. Multidisciplinary studies of this scope are required to better understand shedding in cattle and aid in the development of enhanced detection methods and control strategies.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7124010104050%
7123310117025%
7123410117025%
Goals / Objectives
To comprehensively study the ecology of STEC shedding, we will pursue the following objectives: 1) Identify bacterial genotypes and epidemiological factors important for shedding in multiple herds by genotyping >750 strains to examine genotype distributions, genetic diversity and changes in shedding status over time; 2) Use 454 pyrosequencing to compare the composition, diversity and function of the microbial communities within the rectoanal junction and ruminal fluid of super-shedders and non-shedders; 3) Determine how STEC affects the bovine immune response by comparing antibody levels, and the expression of cytokine and bovine genes among super-shedders and non-shedders; and 4) Identify STEC inhibitors from the ruminal fluid of non-shedders, and test control strategies aimed at decreasing shedding levels. The long-term goal of our collaborative research effort is to determine the impact of bacterial, epidemiological, immunological, and ecological factors on STEC shedding in cattle. Through this work, we expect to develop new ideas for direct-fed microbials, vaccines, therapies and other control strategies that can reduce the frequency and level of STEC shedding, an end result that will likely translate to a reduction in food contamination, transmission to humans, and STEC-associated morbidity and mortality.
Project Methods
To enhance our understanding of STEC colonization of cattle, we will evaluate temporal changes in bacterial genotype distributions as well as the microbial communities and chemical composition of ruminal fluid from shedders, super-shedders and non-shedders. To do this, STEC-positive cattle will be identified from multiple herds with varying management practices in Michigan and Texas. STEC shedders and a subset of non-shedders will be followed over a 6 month period to examine colonization and transmission dynamics by herd and identify epidemiological associations with shedding. Rectoanal junction (RAJ) swabs and tissue, blood, and ruminal fluid will be collected from all super-shedders, matched nonshedders and a subset of low-level shedders to identify differences in the i) genotypic and phenotypic characteristics of STEC; ii) microbiome of the RAJ and rumen via 454 pyrosequencing of 16S rRNA genes and metagenomics; iii) immunological response by examining antibody levels and cytokine expression profiles; iv) composition of the ruminal fluid; and v) genetic makeup of cattle. In vitro studies of super-shedder and nonshedder ruminal fluid will also enable the identification of STEC inhibitors. Based on these results, we will develop control strategies for reducing shedding in vitro, while alternative strategies will be evaluated in the field with naturally infected shedders. These control methods could potentially result in long-range improvements to the agricultural and food industries by reducing STEC colonization in cattle, the subsequent contamination of the food supply, and morbidity and mortality in humans.

Progress 01/15/11 to 01/14/17

Outputs
Target Audience:Target audiences: Farm owners in mid-Michigan serve as a target audience for one aspect of this study as well as people with enteric infections living in the state of Michigan. We also aim to disseminate our findings to members of the scientific research community including researchers, health professionals in human and veterinary medicine, public health officials, and food safety and consumer groups. Efforts: Educating residents in areas with the highest risk of disease or greatest exposure levels to STEC can potentially result in better food handling and consumption practices and decrease disease rates. Research findings have been and will continue to be disseminated to members of the research community with similar interests via publications, presentations at conferences and symposia as well as interviews. Changes/Problems:Our goal to identify STEC inhibitors from the ruminal fluid of non-shedders and test these inhibitors in cattle could not be performed. Indeed, inhibitors could not be identified for use. In an effort to identify other prevention strategies aimed at controlling STEC shedding, we did pursue "protective" bacteriophage populations isolated from feces as well as antibiofilm compounds. Data generated from both of these alternative projects, however, were too preliminary to warrant use in live cattle populations. What opportunities for training and professional development has the project provided?Many of the students and postdoctoral researchers have had the opportunity to present their data at a national conference and/or attend workshops that had educated them on various analytical methods including R, genome assemblies, RNA sequencing, metagenomics, etc. Many new research projects have also resulted from the work, providing new opportunities for learning different techniques and tools in the laboratory and pursuing different hypotheses. How have the results been disseminated to communities of interest?We have disseminated our findings to members of the scientific research community including researchers, health professionals in human and veterinary medicine, public health officials, and food safety and consumer groups via scientific papers, presentations and attendance at national meetings. Farm owners have been briefed on study findings, particularly when associations between STEC shedding and certain factors were identified. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In the final year of our study, we have continued to analyze data and prepare and submit manuscripts for publication. Our papers on the epidemiology of STEC and Campylobacter jejuni in humans and cattle have been published, with the cattle paper having a big impact in that it was selected for a press release by ASM. Importantly, we found that the number of cattle shedding Shiga toxin-producing E. coli (STEC) in their feces varied considerably across herds in Michigan and fecal shedding frequencies of STEC serotypes other than O157 (non-O157) far exceeded the frequencies of STEC O157. Dairy cattle sampled at higher temperatures, in their first lactation, and early in the milk production stage were significantly more likely to shed STEC, which could be due to stress or a negative energy balance. Future interventions could therefore focus on the isolation of high-risk animals to decrease herd shedding levels and the potential for contamination of the food supply. We have also conducted an in-depth screen of our isolates (C. jejuni, STEC, and non-typhoidal Salmonella) for resistance to commonly used antibiotics, and an abstract on the STEC data was presented ASM Microbe 2016. Additionally, we are continuing our microbiota profile analyses that aim to compare gut intestinal communities among cattle belonging to different herds and between deer and cattle by 16S rDNA sequencing. There appears to be no difference in community profiles by STEC status, though other variables (e.g., temperature, diet, age, season, etc) appear to be important. We have also conducted a similar analysis in humans to determine the impact that age and sex have on enteric infections caused by STEC and other bacterial pathogens. With regard to our collaboration with Texas Tech University and collegues in Argentina, we have completed an analysis of O157 strains collected from cattle in diverse locations (e.g., Texas, Argentina, and Michigan) to assess the distribution of O157 STEC lineages (clades) by region. The preliminary data were presented at ASM Microbe 2016, one manuscript was published, and another manuscript is in preparation. It is interesting to note that the cattle population sampled in Michigan had a very low prevalence of O157 and a much higher prevalence of non-O157 STEC. Four additional abstracts were submitted by different undergraduate researchers to the Michigan State University (MSU) undergraduate research conference (UURAF), which gave them the opportunity to present posters highlighting their work last April. Finally, we have been to explore the identification of alternative therapeutics aimed at disrupting non-O157 biofilms, which we have identified to be important for persistence and transmission in cattle belonging to the same herd. The data associated with biofilm production are almost complete and will be incorporated into a manuscript.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Mukherjee, S., P. Singh, R. Mosci, J.T. Rudrik, and S.D. Manning. Antimicrobial Resistance in Shiga Toxin-Producing Escherichia coli (STEC) and Non-Typhoidal Salmonella in Michigan. ASM Microbe Boston 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Nohomovich, B., S.L. Wengert, J.T. Rudrik, and S.D. Manning. Impact of Intestinal Bacteriophage Communities on Growth of Enteric Pathogens. Molecular Genetics of Bacteria & Phages Meeting, Madison, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Singh, P., R.E. Mosci, C. Venegas Vargas, K. Hussnaetter, H. Selheimer, K. Jernigan, J.T. Rudrik, G. Loneragan, J. Funk, B. Norby, S. Rust, P. Bartlett, D. Grooms, and S.D. Manning. Characteristics of STEC isolates recovered from dairy and beef cattle in Michigan. Annual USDA NIFA PI Meeting, St. Louis 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Cha, W., P.M. Fratamico, L. Ruth, G.M. Baranzoni, G-H. Kim, A.S. Bowman, J. Nolting, S.D. Manning, and J.A. Funk. Prevalence and characteristics of Shiga toxin-producing E. coli in finishing pigs: the implication on public health. Annual USDA NIFA PI Meeting, St. Louis 2016.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Cha, W., R. Mosci, S. Wengert, P. Singh, D. Newton, H. Salimnia, P. Lephart, W. Khalife, L.S. Mansfield, J.T. Rudrik, and S.D. Manning. Antimicrobial susceptibility profiles of human Campylobacter jejuni isolates in Michigan and the association with phylogenetic lineage and disease severity. Front Microbiol 2016;7:589.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Venegas Vargas, M.C., S. Henderson, A. Khare, R. Mosci, J.D. Lehnert, P. Singh, L. Ouellette, B, Norby, J.A. Funk, S. Rust, P. Bartlett, D. Grooms, and S.D. Manning. Factors associated with Shiga toxin-producing Escherichia coli shedding in dairy and beef cattle. Appl Environ Microbiol. 2016: doi: 10.1128/AEM.00829-16
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Cha, W., J. Collins, T. Henderson, and S.D. Manning. Factors associated with increasing campylobacteriosis incidence in Michigan, 2004-2013. Epidemiol Infect. 2016:1-10.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Galli, L, V. Brusa, P. Singh, A.A. Cataldi, S. Manning, P.P. Garc�a, and G.A. Leotta. High prevalence of Escherichia coli O157:H7 clade 8 isolated from retail meat and butcher shop environment. Infect Genet Evol. 2016;45:1-5.
  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Venegas Vargas, M.C., S.D. Manning, P. Coussens, J. Roussey, P. Bartlett, and D. Grooms. Association of bovine leukemia virus and Mycobacterium avium subsp. paratuberculosis with shedding of Shiga toxin-producing Escherichia coli. J Food Protect. In press.
  • Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Delgado, M.L, P. Singh, J. Funk, J. Moore, E. Cannell, J. Kanesfsky, S.D. Manning, and K.T. Scribner. Intestinal microbial community dynamics of white-tailed deer (Odocoileus virginianus) in an agroecosystem. Microbial Ecology. July 2016. In revision.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Moore, M., R. Mosci, and S.D. Manning. Correlation between Shiga-toxin Producing Escherichia coli Serotypes and Disease Severity in Michigan Patients. MSU Undergraduate Research and Arts Forum (UURAF), 2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Peters, M., R. Mosci, and S.D. Manning. Epidemiological analysis of confirmed Shiga toxin-producing E. coli (STEC) O157:H7 cases in four Michigan hospitals, 2007-2014. MSU Undergraduate Research and Arts Forum (UURAF), 2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Bednarczyk, J., J. Rodrigues, W. Cha, R. Mosci, and S.D. Manning. Antibiotic Resistance of Campylobacter jejuni in Michigan. MSU Undergraduate Research and Arts Forum (UURAF), 2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Singh, P., R. Mosci, M. Moore, K. Jernigan, J.T. Rudrik, G. Loneragan, and S.D. Manning. Variation in the distribution of Shiga toxin-producing Escherichia coli O157 genotypes from humans and cattle. ASM Microbe Boston 2016.


Progress 01/15/15 to 01/14/16

Outputs
Target Audience:The target of our efforts during the past year has been the research community and news sources that promote the dissemination of our findings. Changes/Problems:Our intervention study originally planned for the summer of 2015 was delayed and consequently, a no-cost extension was requested and approved. What opportunities for training and professional development has the project provided?Throughout the project, a total of 28 students, technicians and postdoctoral researchers have been learning the tools needed to recover STEC from different matrices, isolate the bacterial DNA andcharacterize each isolate for the O-antigen and H-antigen type as well as key virulence factor genes. A subset of individuals have been trained to extract phage communities and bacterialcommunities from feces and to perform 16s rRNA sequencing. This same subset of individuals has learnedvarioussequence analysis pipelines for whole genomes, microbial communities and RNA sequencing via the High Perrformance Computing Cluster (HPCC) at MSU. Several students have had the opportunity to take workshops on sequence analyses and 7 students have presented posters or given oral presentations at internal and external conferences. How have the results been disseminated to communities of interest?Dissemination of findings has occurred via journal articles, attendance and presentations at MSU and national conferences as well as via news sources highlighting the work. We have also conducted various outreach activities at local elementary schools to enhance knowledge of foodborne pathogens and proper food handling techniques. What do you plan to do during the next reporting period to accomplish the goals?The remainder of the manuscripts will be submitted in the upcoming year and data analysis will continue on the long term shedding dynamics of STEC, the immunological changeswith colonizationas well as the microbiome analyses. In addition, we are assessing the feasibility of conducting an intervention study to utilize novel therapeutics developed in the lab.

Impacts
What was accomplished under these goals? We have completed the analysis of STEC, EHEC and enteropathogenic E. coli (EPEC) recovered from deer and cattle sharing an agroecosystem, which was published in Frontiers in Cellular and Infection Microbiology under the "Ecology and population biology of shiga toxin-producing E. coli" Research Topic. The data presented in this manuscript highlight the diversity of pathogenic E. coli in ruminant reservoirs and demonstrate that white-tailed deer play a role in STEC shedding dynamics and persistence in cattle herds. We have also examined the microbiota profiles between deer and cattle fecal samples using 16S rDNA sequencing. The intestinal microbiota profiles differ in deer and cattle feces; STEC positivity does not appear to influence microbiota composition in deer or cattle from one dairy herd. Ongoing analyses focus on examining intestinal microbiota differences across beef and dairy cattle from four herds in an effort to identify profiles that may be linked to STEC status.We have alsosubmitted and published several manuscripts that focus on the epidemiology of both STEC and Campylobacter jejuni in humans and cattle. Amanuscript was submitted to Applied and Environmental Microbiology highlighting results from the STEC cross-sectional study of 1,096 cattle in five dairy herds (n=718 animals) and six beef herds (n=378 animals). The prevalence of STEC was higher in beef (21%) versus dairy (13%) cattle (odds ratio (OR): 1.76; 95% confidence interval (CI): 1.25, 2.47) with considerable variation across herds (range: 6% to 54%). In addition, we found that dairy cattle were significantly more likely to shed STEC when the average temperature was >28.9°C 1-5 days prior to sampling (OR: 2.5; 95% CI: 1.25, 4.91), during their first lactation (OR: 1.8; 95% CI: 1.1, 2.8), and when they were <31 days in milk (OR: 3.9; 95% CI: 2.1, 7.2). These data suggest that the stress associated with lactation may result in increased STEC shedding frequencies in Michigan during the warm summer months. A manuscript associated with STEC and bovine leukemia virus is also in preparation, while two papers were submitted on the microbiota composition and abundance of humans with STEC and other bacterial enteric infections. Finally, we presented a poster on the distribution of O157 STEC lineages (clades) in Michigan patients relative to O157 isolates recovered from cattle and gave two presentations at the VTEC meeting in Boston.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Singh, P., Q. Sha, Q., D.W. Lacher, R. Mosci, J. Del Valle, J. Moore, K. Scribner, and S.D. Manning. Characterization of enteropathogenic and Shiga toxin-producing Escherichia coli in cattle and deer in a shared agroecosystem. Front Cell Infect. Microbiol. 2015; 5:29.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Cataldi, A.A., Amigo, N., E. Mercado, P. Singh, D. Vilte, E. Gerhardt, E. Zotta, C. Ibarra, S.D. Manning, and M. Larzabal. Clade 8 and clade 6 strains of Escherichia coli O157:H7 from cattle in Argentina have hypervirulent-like phenotypes. PLoS One. 2015; 10(6): e0127710.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Corbett, E.M., B. Norby, L.W. Halbert, S.T. Henderson, D.L. Grooms, S.D. Manning, and J.B. Kaneene. Effect of feeding a direct-fed microbial on total and antimicrobial-resistant fecal coliform counts in preweaned dairy calves. Am J Vet Res. 2015; 76:780-788
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Habing, G.G., S. Manning, C. Bolin, Y. Cui, J. Rudrik, S. Dietrich, J.B. Kaneene. Within-farm changes in dairy farm-associated Salmonella subtypes and comparison to human clinical isolates in Michigan, 2000-2001and 2009. Appl Environ Microbiol. 2015; 81(17):5724-35.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Singh, P., T. Teal, T. Marsh, J. Tiedje, K.Jernigan, R. Mosci, A. Zell, D. Newton, H. Salimnia, P. Lephart, H. Salimnia, W. Khalife, D. Sundin, R.A. Britton, J.T. Rudrik, and S.D. Manning. Intestinal microbial communities associated with acute enteric infections and disease recovery. Microbiome. 2015; 3:45.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Tseng, M., J.T. Rudrik, J. Collins, T. Henderson, J.A. Funk, and S.D. Manning. Increasing incidence of non-O157 Shiga toxin-producing Escherichia coli (STEC) in Michigan and association with disease. Epidemiol Infect. 2015: 1-12.
  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Hayden, H.S., S. Matamouros; K.R. Hager, M.J. Brittnacher, L. Rohmer, M.C. Radey, E.J. Weiss, K.B. Kim, M.A. Jacobs, E.H. Sims-Day, M. Yue, M.B. Zaidi, D.M. Schifferli, S.D. Manning, J.I. Walson, and S.I. Miller. Genomic analysis of Salmonella Typhimurium characterizes strain diversity for recent U.S. Salmonellosis cases and identifies mutations linked to loss of fitness under nitrosative and oxidative stress. BMC Genomics.
  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Cha, W., R. Mosci, S. Wengert, P. Singh, D. Newton, H. Salimnia, P. Lephart, W. Khalife, L.S. Mansfield, J.T. Rudrik, and S.D. Manning. Antimicrobial susceptibility profiles of human Campylobacter jejuni isolates in Michigan and the association with phylogenetic lineage and disease severity. Antimicrob Agents Chemother.
  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Cha, W., J. Collins, T. Henderson, and S.D. Manning. Factors associated with increasing campylobacteriosis incidence in Michigan, 2004-2013. Submitted to Emerg Infect Dis.
  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Venegas-Vargus, C.M., S. Henderson, A. Khare, R. Mosci, J.D. Lehnert, P. Singh, L. Ouellette, B, Norby, J.A. Funk, S. Rust, P. Bartlett, D. Grooms, and S.D. Manning. Factors associated with Shiga toxin-producing Escherichia coli shedding in dairy cattle. Appl Environ Microbiol. October 2015.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Moore, M., R.E. Mosci, J.T. Rudrik, and S.D. Manning. Virulence characteristics of Shiga toxin-producing Escherichia coli recovered in Michigan between 2007-2014. MSU Undergraduate Research and Arts Forum (UURAF). April 2015.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Mukherjee, S., P. Singh, R.E. Mosci, J.T. Rudrik, and S.D. Manning. Prevalence of antibiotic resistant Shiga toxin-producing Escherichia coli in Michigan. Midwest Microbial Pathogenesis Conference. Indianapolis, IN. August 2015.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Singh, P., R.E. Mosci, M. Moore, K. Jernigan, J.T. Rudrik, and S.D. Manning. Distribution of Shiga toxin-producing Escherichia coli O157 clades among patients and cattle in Michigan. Midwest Microbial Pathogenesis Conferences. Indianapolis, IN. August 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Amigo, N., E. Mercado, P. Singh, D. Vilte, E. Gerhardt, E. Zotta, C. Ibarra, S.D. Manning, M. Larzabal, and A.A. Cataldi. Clade 8 and clade 6 strains of Escherichia coli O157:H7 from cattle in Argentina have hypervirulent-like phenotypes. VTEC. Boston, MA; 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Ison, S. Genetic Diversity and pathogenic potential of attaching and effacing E. coli O26:H11 strains recovered from bovine feces. Oral presentation at VTEC. Boston, MA; 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: S.D. Manning. Ecology of STEC in cattle- A summary of findings from Michigan, Washington, and Nebraska. Presented at the VTEC conference. Boston, MA. September 13, 2015.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Ison, S.A., S. Delannoy, M. Bugarel, K.K. Nightingale, H.E. Webb, David G. Renter, T.G. Nagaraja, G.H. Loneragan, P. Fach. Genetic diversity and pathogenic potential of attaching and effacing Escherichia coli O26:H11 recovered from bovine feces in the United States. Appl Environ Microbiol. 2015; 81: 3671-8.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Mukherjee, S., P. Singh, R.E. Mosci, J.T. Rudrik, and S.D. Manning. Prevalence of antibiotic resistant Shiga toxin- producing Escherichia coli in Michigan. Michigan branch of the American Society for Microbiology, Bacterial Pathogenicity. Ann Arbor, MI. October 2015.


Progress 01/15/14 to 01/14/15

Outputs
Target Audience: The research community represents the main audience during this phase of the study. Dissemination of research findings occurs via publications, presentations at national conferences, and interviews. Changes/Problems: Given the diversity of STEC identified among the herds sampled, we are less sure about our ability to prevent STEC shedding and are in the process of developing therapeutic tools. It is therefore not clear whether these tools will be able to be tested in the challenge studies initially proposed. Discussions regarding these and similar types of studies are ongoing. What opportunities for training and professional development has the project provided? Dr. Manning has mentored 25 students, technicians and/or postdoctoral fellows on study design, epidemiological analyses and the use of multiple molecular techniques since the project began. Individuals, who worked on the project over the current reporting period, include Dr. Pallavi Singh, Lindsey Ouellette, Wonhee Cha, Yi-An Yang, Rebekah Mosci, Katherine Jernigan, Jonathon Lehnert, Dr. Qiong Sha, Dr. Joshua Herr, Marion Tseng, Lisette Delgado, Brian Nohomovich, Megan Shiroda, Lillian Jensen, Kai Hussnaetter, and Moriah Moore. Drs. Daniel Grooms, Julie Funk and Paul Bartlett have mentored Dr. Cristina Venegas, who is completing her thesis work on the epidemiology of STEC in Michigan farms. During the reporting period, seven students or postdoctoral fellows submitted abstracts and presented either posters or gave oral presenations at several national conferences or local symposia. Three students have participated in workshops to better understand genome analysis, programming, 16s rDNA analyses, and RNA sequencing. One student was given the opportunity to participate in the University of Michigan graduate summer program on geographic information systems (GIS) mapping. How have the results been disseminated to communities of interest? Dissemination of research findings is underway through classes, publications, and presentations at conferences including the American Society for Microbiology, the Governor's conference on STEC at the University of Nebraska, and local meetings at MSU and the University of Michigan. Dr. Manning has also been interviewed for several news articles. What do you plan to do during the next reporting period to accomplish the goals? Complete the analysis of deer and cattle-derived STEC, EPEC, and EHEC as well as the assessment of differences within the microbiota profiles among cattle from different herds and deer and cattle sharing the same environment. Our main focus will be to continue to publish the findings in the primary scientific literature and present the data at various scientific meetings, while generating additional data on other aspects of the study that we have yet to work on. We are also in the process of determining whether a challenge study can be done in summer 2015 to potentially limit STEC shedding in cattle.

Impacts
What was accomplished under these goals? We have completed the analysis of STEC, EHEC and enteropathogenic E. coli (EPEC) recovered from deer and cattle sharing an agroecosystem, which is currently in press at Frontiers in Cellular and Infection Microbiology under the "Ecology and population biology of shiga toxin-producing E. coli" Research Topic. The data presented in this manuscript highlight the diversity of pathogenic E. coli in ruminant reservoirs and demonstrate that white-tailed deer play a role in STEC shedding dynamics and persistence in cattle herds. In this study we sought to investigate transmission of STEC, enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) between cattle and white-tailed deer in a shared agroecosystem. Cattle feces were collected from 100 animals in a Michigan dairy farm in July 2012, while 163 deer fecal samples were collected during two sampling periods (March and June). The locations of deer fecal pellets were recorded via geographic information system mapping and microsatellite multi-locus genotyping was used to link the fecal samples to individual deer at both time points. STEC and EHEC were cultured from 12% and 16% of cattle, respectively, and EPEC was found in 36%. Deer were significantly less likely to have a pathogen in March versus June where the frequency of STEC, EHEC, and EPEC was 1%, 6% and 22%, respectively. PCR fingerprinting and MLST clustered the cattle- and deer-derived strains together in a phylogenetic tree. Two STEC strains recovered from both animal species shared MLST and fingerprinting profiles, thereby providing evidence of interspecies transmission and highlighting the importance of wildlife species in pathogen shedding dynamics and persistence in the environment and cattle herds. We have also begun to examine the microbiota profiles between deer and cattle fecal samples using 16S rDNA sequencing. The intestinal microbiota profiles differ in deer and cattle feces; STEC positivity does not appear to influence microbiota composition in deer or cattle from one dairy herd. Ongoing analyses focus on examining intestinal microbiota differences across beef and dairy cattle from four herds in an effort to identify profiles that may be linked to STEC status. Manuscripts are in preparation for epidemiological factors associated with STEC shedding as well as the association between STEC and chronic infections. For the first paper, a multivariable model, with herd included as a random effect, was used to evaluate both herd-level and cow-level risk factors for dairy cattle. Dairy cattle were more likely to shed STEC when the average temperature was > 84°F 1-5 days before sampling (OR: 2.5; 95% CI: 1.25- 4.91). Dairy cows were more likely to shed STEC in their first lactation (OR: 1.8; 95% CI: 1.1-2.8) and when they were < 31 days in milk (OR: 3.9; 95% CI: 2.1-7.2). The second paper involves better understanding the relationship between bovine leukemia virus (BLV) and STEC across all 11 herds. Blood samples were collected from a subsample (n=497) to quantify the percentage of lymphocytes, monocytes and neutrophils using flow cytometry. Of the animals sampled, 35% were BLV positive while 3% were MAP positive and 16% were shedding STEC. Dairy herds had a higher frequency of BLV and MAP than did beef herds, but beef herds had more STEC. Neither BLV nor MAP was associated with STEC shedding. We also observed no association between percentage of white blood cells and STEC status. Although controlling both BLV and MAP is important for overall herd health and productivity, controlling BLV and MAP will not likely have an impact on STEC shedding in cattle. We have also begun an analysis of shedding dynamics in the149 cattle sampled longitudinally at four consecutive times separated by an average of 19 days. STEC shedding was intermittent with only five animals shedding STEC continuously throughout the study. Twenty-seven cattle shed STEC for at least two consecutives sample times and the average duration of shedding was 24 days. Notably, a large number (n=28) of cattle were STEC negative throughout the entire study period. The rate of STEC loss was higher than the rate of STEC acquisition in all visits, and although these rates did not vary between visits, there was variation in acquisition frequencies across herds. In addition, we have developed a novel Stx-bacteriophage genotyping tool that allows us to characterize the Stx-phage as it is incorporated into different STEC strains. The diversity of Stx-phages within herds is low relative to the strain diversity. Finally, we have developed a method to extract bacteriophage from cattle feces.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Iyoda, S. S.D. Manning, K. Seto, K. Kimata, J. Isobe, Y. Etoh, S. Ichihara, Y. Migita, et. al. and the EHEC working group in Japan. Phylogenetic clades 6 and 8 of enterohemorrhagic Escherichia coli O157:H7 with particular stx subtypes are more frequently found in isolates from hemolytic uremic syndrome patients than from asymptomatic carriers. Open Forum Infectious Diseases (OFID). 2014; 10: 1093
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Franz, F., P. Delaquis, S. Morabito, L. Beutin, K. Gobius, D. Rasko, J. Bono, N. French, J. Osek, B. Lindstedt, M. Muniesa, S.D. Manning, J. LeJeune, T. Callaway, S. Beatson, M. Eppinger, T. Dallman, K. Forbes, H. Aarts, D. Pearl and N. Strachan. Exploiting the explosion of information associated with whole genome sequencing to tackle Shiga toxin-producing Escherichia coli (STEC) in global food production systems. Int J Food Microbiol. 2014; 187:57-72.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Tseng, M., P. Fratamico, L. Bagi, S. Delannoy, P. Fach, S.D. Manning, and J. Funk. Diverse virulence gene content of Shiga toxin-producing Escherichia coli from finishing swine. Appl Environ Microbiol. 2014; 80(20):6395.
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Singh, P., Q. Sha, Q., D.W. Lacher, R. Mosci, J. Del Valle, J. Moore, K. Scribner, and S.D. Manning. Characterization of enteropathogenic and Shiga toxin-producing Escherichia coli in cattle and deer in a shared agroecosystem. Frontiers in Cellular and Infect Microbiol.
  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Habing, G.G., S.D. Manning, C. Bolin, Y. Cui, J.T. Rudrik, S. Dietrich, J.B. Kaneene. Changes in human and dairy farm associated Salmonella subtypes in Michigan, USA, 2000-2009. Appl Environ Microbiol.
  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Cataldi, A.A., Amigo, N., E. Mercado, P. Singh, D. Vilte, E. Gerhardt, E. Zotta, C. Ibarra, S.D. Manning, and M. Larzabal. Clade 8 and novel genotypes of Escherichia coli O157:H7 from cattle in Argentina have hypervirulent-like phenotypes. PLoS One.
  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Hayden, H.S., S. Matamouros; K.R. Hager, M.J. Brittnacher, L. Rohmer, M.C. Radey, E.J. Weiss, K.B. Kim, M.A. Jacobs, E.H. Sims-Day, M.B. Zaidi, D.M. Schifferli, S.D. Manning, and S.I. Miller. Genomic analysis of recent U.S. isolates of Salmonella typhimurium reveals three lineages with distinct evolutionary signatures. BMC Genomics
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Singh, P. and S.D. Manning. Molecular characterization of Shiga toxin-producing Escherichia coli. In: DNA Methods in Food Safety: Molecular Typing of Foodborne and Waterborne Bacterial Pathogens. Wiley-Blackwell
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Hussnaetter, K., P. Singh, L. Ouellette, J.D. Lehnert, L. Pena Cortes, and S.D. Manning. Variation in biofilm formation among Shiga toxin-producing Escherichia coli isolated from beef cattle in Michigan. MSU UURAF
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Cha, W., R. Mosci, S. Wengert, J.T. Rudrik, and S.D. Manning. Antimicrobial susceptibility profiles of human Campylobacter jejuni isolates from Michigan and association with phylogenetic lineage and disease severity. American Society for Microbiology Annual Meeting; Boston, MA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Yang, Y-A., P. Singh, L. Ouellette, S.D. Manning. Genetic Diversity of Shiga Toxin (Stx)-Producing Bacteriophages in Escherichia coli Isolated from Cattle in Multiple Herds. American Society for Microbiology Annual Meeting; Boston, MA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Herr, J.R., D.W. Lacher, R. Al Safadi, C.T. Brown, J.T. Rudrik, and S.D. Manning. Comparative genomics and epidemiology of European Escherichia coli O104:H4 outbreak strains and novel counterparts isolated from the United States. Oral presentation, American Society for Microbiology Annual Meeting; Boston, MA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Habing, G.G., S.D. Manning, C. Bolin, J.T. Rudrik, J.B. Kaneene. Changes in the frequency of reported human illnesses associated with Salmonella subtypes recovered from Michigan dairy farms in either 2000-2001 or 2009. CRWAD
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Cha, W., R. Mosci, S. Wengert, C. Venegas, P.Bartlett, D. Grooms, S.D. Manning. Campylobacter jejuni isolated from cattle in Michigan: The impact on public health. . Oral presentation. Phi Zeta Research Day, MSU College of Veterinary Medicine. Michigan State University, E. Lansing, MI
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Cha, W., R. Mosci, S. Wengert, C. Venegas, P.Bartlett, D. Grooms, S.D. Manning. Campylobacter jejuni isolated from cattle in Michigan; antimicrobial resistance, genetic diversity, and the impact on public health. CRWAD (oral presentation). Chicago, IL
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: S.D. Manning. STEC in Michigan- from shedding in cattle to human disease. Presented at the Governors Conference on Ensuring Food Safety. Lincoln, NE. May 29, 2014
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: S.D. Manning. The importance of intestinal microbial communities and pathogen characteristics for the development of enteric infections. Presented at the Department of Microbiology and Molecular Genetics, MSU. E. Lansing, MI. August 12, 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: S.D. Manning. Impact of intestinal community composition and pathogen characteristics on enteric disease. Presented at the Department of Food Science and Technology, University of Nebraska. Lincoln, NE. August 18, 2014
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: S.D. Manning. Impact of intestinal community composition and pathogen characteristics on enteric disease. Distinguished Lectures in Microbiology series, Department of Bacteriology, University of Wisconsin-Madison. October 23, 2014.
  • Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Genetic Diversity of Shiga Toxin-Producing Prophages Among Escherichia coli Isolated from Cattle in Multiple Herds


Progress 01/15/13 to 01/14/14

Outputs
Target Audience: Farm owners in mid-Michigan and Texasrepresentedonetarget audience for the field portion of the study, while scientists studying STEC, genomics,and pre-harvest food safety represent another audience via publications, presentations, and attendance at national conferences. Changes/Problems: One unexpected outcome is the high frequency of non-O157 STEC and non-typeable (NT) STEC that have been recovered from cattle in the study. Because these strains are difficult to characterize, we have had to modify our genotyping strategy. For example, it is difficult to determine whether 2 NT strains with a similar toxin profile are identical and represent duplicate colony picks. Consequently, we have used PCR-based fingerprinting tools to omit duplicates and have sequenced the genomes of the distinct STEC strains. This approach has allowed us to decrease the number of isolates to be sequenced, while the genomic information provides the opportunity to better understand diversity and evolution of STEC within an animal and herd where transmission is common. What opportunities for training and professional development has the project provided? Dr. Manning has mentored 17 students, technicians and/or postdoctoral fellows on study design, epidemiological analyses and the use of multiple molecular techniques. Individuals include Dr. Pallavi Singh, Lindsey Ouellette, Wonhee Cha, Yi-An Yang, Rebekah Mosci, Akanksha Khare, Katherine Jernigan, Scott Henderson, Jonathon Lehnert, Aaron Balogh, Davis Thomas, A. Cody Springman, Dr. Qiong Sha, Marion Tseng, and Dr. Rim Al Safadi. Drs. Daniel Grooms and Paul Bartlett have mentored Dr. Cristina Venegas, who is completing her thesis work on the epidemiology of STEC in Michigan farms. Student support was also obtained through the Merial Veterinary Scholars program and several students/postdoctoral fellowshave been given the opportunity to present their findings at national conferences (n=4), MSU conferences (n=2)and attend workshops including genome assembly and epidemiological studies (n=3). How have the results been disseminated to communities of interest? Dissemination of research findings is underway through classes and presentations at conferences including the International Association of Food Protection meeting, the American Society for Microbiology, the Merial NIH Veterinary Scholars Symposium, the STEC Genomics workshop, and the American Association of Bovine Practitioners. Dr. Manning has also been interviewed for several newspapers and radio shows including a program on pathogen evolution, which aired on WKAR on 3/21/2013. She also presented her research findings in lay terms to members of the MSU alumni group affiliated with the College of Natural Sciences. What do you plan to do during the next reporting period to accomplish the goals? Complete the genome analysis of STEC strains isolated from cattle as well as the epidemiological and microbiome studies in cattle and humans. Our main focus will be to publish the findings in the primary scientific literature and present the data at various international conferences, while generating additional data on other aspects of the study that we have yet to work on.

Impacts
What was accomplished under these goals? The processing of over 5,000 bacterial isolates recovered from cattle in 11 herds in mid-Michigan between 2011 and 2012 is nearly complete. Specific factors including days in milk, lactation status and temperature have been found to be associated with STEC shedding in cattle. The epidemiological analysis of risk factors for shedding is nearly complete as is the qPCR assessment to identify the frequency of super shedders across all STEC-positive animals per herd. The longitudinal data have demonstrated considerable changes in STEC shedding dynamics in animals per herd over time and have uncovered a highly diverse population of STEC. Although many animals are positive for STEC, there is a high frequency of animals that acquire and lose specific genotypes over time. The distribution of genotypes varies across herds with most of the isolates representing non-O157 STEC strains and not O157. Genome sequencing has been performed on a subset of 40 STEC strains from one dairy herd that varied by DNA fingerprint profiling to better understand the diversity of STEC in a given herd. 16S rRNA sequencing has also been performed on STEC-positive and STEC-negative cattle from 4 herds (2 dairy and 2 beef) to examined microbiome composition differences across animals in relation to STEC shedding. A comparative strain study has also been performed in humans and cattle infected with Campylobacter jejuni. MLST was performed on all ~250 strains and genome sequencing has been performed on a subset to better understand the genetic differences in strains from each host.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Tseng, M., P.M. Fratamico, S.D. Manning, and J.A. Funk. Shiga toxin-producing Escherichia coli in swine: the public health perspective. Animal Health Research Reviews. In press
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Hamilton, E.A, Kruger, K., Schall, Beal, M., S.D. Manning, and J. Kaneene. Acquisition and persistence of antimicrobial resistant bacteria isolated from companion animals admitted to a veterinary teaching hospital 2007-2009. J Am Vet Med Assoc. 2013;7(1):990-1000.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Manning, S.D. Evolution, epidemiology and ecology of Shiga toxin-producing E. coli. Presented at the MSU Microbiology Club. E. Lansing, MI. February 6, 2013.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Manning, S.D. Pathogen Evolution: The E. coli are coming. Presented at the 4th annual College of Natural Science Classes without Quizzes. E. Lansing, MI. April 13, 2013.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Manning, S.D. Relevance of Current Molecular Typing Methodology to Epidemiological Investigations and Food Safety. Presented at the International Association of Food Protection. Charlotte, NC. July 29, 2013.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Venegas Vargas, C., P. Bartlett, S.D. Manning, and D. Grooms. Do cattle with immunocompromising infections shed Shiga toxin-producing E. coli more frequently than otherwise healthy animals? American Association of Bovine Practitioners. Milwaukee, WI. September 2013.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Amigo, Larz�bal, Vilte, E. Mercado, S.D. Manning, and A. Cataldi. Molecular markers (SNPS and Shiga Toxin types) and Shiga Toxin expression in potentially hypervirulent Escherichia coli O157:H7 strains isolated from cattle in Argentina. Sociedad Argentina de Microbiologia General; Nacional de Rosario, Rosario, Santa Fe, Argentina; August 2013.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Yang, Y-A, P. Singh, L. Ouellette, and S.D. Manning. Genetic diversity of Shiga toxin-producing bacteriophages in Escherichia coli isolated from cattle in Multiple Herds. 2013. Phi Zeta Research Day, MSU College of Veterinary Medicine. Michigan State University, E. Lansing, MI.


Progress 01/15/12 to 01/14/13

Outputs
OUTPUTS: Through this study, the prevalence of STEC has been estimated in twelve Michigan cattle herds sampled in 2011 and 2012 by culturing STEC from 1,195 animals; microbial communities have been examined in several herds. In all, the herd prevalence varied considerably (range: 10%-53 %), while the average herd prevalence for dairy and beef cattle was 14% and 27%, respectively. Spatial variation in the prevalence of human STEC infections reported by the Michigan Department of Community Health (MDCH) was correlated with STEC herd prevalence in cattle in the same county. A subset of animals was also sampled at four different time points and culture data for 153 animals in 10 herds demonstrated significant variation in STEC prevalence over time, with shedding levels peaking at different times in different farms. In addition to STEC, we have also cultured 6 herds for Campylobacter jejuni and the prevalence ranged from 8% to 87% across herds. A comparative study of the genetic characterization of both STEC and C. jejuni is underway that aims to examine the frequency and type of genotypes, as determined by multilocus sequence typing (MLST), from humans with clinical infections and cattle. A modified single nucleotide polymorphism (SNP) genotyping scheme has been developed for the characterization of STEC O157, while serotyping, RAPD PCR and other fingerprinting methods have been used to confirm transmission of specific STEC among herd members. A protocol has also been developed to measure the level of STEC shedding from the corresponding fecal grab specimen via quantitative PCR (qPCR) protocol targeting the stx1 and stx2 genes. Twelve students have been mentored on field methods and collecting samples from cattle, and an additional 10 students on study design, epidemiological analyses and the use of multiple molecular techniques. To date, we have recovered over 300 bacterial isolates from the MDCH, including two E. coli O104:H4 isolates from the 2011 German outbreak, which were characterized. The results were published in PLoS One and summarized in science blogs, newspaper articles and radio interviews in July 2012. Dr. Manning gave an oral presentation on the E. coli O104 study at VTEC 2012 in Amsterdam in May, 2012 and 7 additional posters/presentations have been given by individuals working on several related projects at multiple conferences. The research team holds lab meetings weekly and the extended research team has met several times per year. In addition, Dr. Manning has worked with BioRad, Corp. to validate a STEC detection qPCR method and curates the STEC Center, a bacterial repository comprising over 16,000 strains. The STEC Center website is active with ~400 hits per week and the Center ships and receives an average of 1,390 and 1,176 strains, respectively, each year. PARTICIPANTS: Dr. Manning has mentored 12 students, technicians and/or postdoctoral fellows on study design, epidemiological analyses and the use of multiple molecular techniques. Individuals include Dr. Pallavi Singh, Lindsey Ouellette, Rebekah Mosci, Akanksha Khare, Katherine Jernigan, Jonathon Lehnert, Davis Thomas, A. Cody Springman, Dr. Qiong Sha, and Dr. Rim Al Safadi. Drs. Daniel Grooms, Paul Bartlett and Steven Rust, the primary MSU collaborators associated with the cattle study, oversaw the farm visits and have mentored up to 12 students and/or postdoctoral fellows on field methods and collecting samples from cattle. Individuals supported on the project include Dr. Cristina Venegas, Scott Henderson, and Lei Zhang. Cristina Venegas (Ph.D. student) will complete her thesis work on the epidemiology of STEC in Michigan farms. Jacquelyn Del Valle, a D.V.M student, was funded through the Merial Veterinary Scholars program, to sample cattle and deer sharing pasture in the summer of 2012. Dr. Paul Coussens oversaw the immunology portion of the project and trained 3 individuals in the use of flow cytometry and blood sample preparation. Individuals include Jon Roussey, Christopher Colvin and Aaron Balogh. Dr. Britton has worked to refine the microbial ecology protocols and analyses and Dr. Mansfield has worked on the Campylobacter jejuni research projects. Partner organizations include the Michigan Dept. of Community Health and 4 participating clinical laboratories (Detroit Medical Center, Sparrow Health System, Spectrum Health, and the University of Michigan Hospital), who provide bacterial isolates for characterization. Collaborators include Dr. Kathryn Eaton of the University of Michigan, Drs. Christopher Waters, John Kaneene, and Kim Scribner of Michigan State University, and Drs. Guy Loneragan and Mindy Brashears of Texas Tech University. Farm managers at each of the farms have also been actively involved in the sampling. TARGET AUDIENCES: Farm owners in mid-Michigan serve as the target audience for one aspect of this study as well as people with enteric infections as determined by the Michigan Department of Community Health. In addition to educating them about STEC and other pathogens, cattle sampling, and food safety, participating farms and patients have been appropriately compensated for enrolling in the studies. Dr. Manning has given several presentations at multiple venues aimed at disseminating knowledge gained from the studies, while Drs. Manning, Bartlett, and Grooms have incorporated study information into their courses: VM 817 - Issues in Pre-Harvest Food Safety (Grooms); VM 831 - Foodborne Disease Epidemiology, VM 832 - Food Safety Disease Control (Bartlett); and VM 811 - Evolution of Foodborne Pathogens, MMG 461 - Molecular Pathogenesis, MMG 861 - Advanced Microbial Pathogenesis (Manning). Similarly, Dr. Funk is routinely incorporating study findings into her curriculum, as she is the Director of the MSU Online Professional Masters of Science in Food Safety. PROJECT MODIFICATIONS: Because each farm was positive for STEC, we did not feel that it was necessary to sample 40 herds as we had originally proposed. It is now clear that 40 herds would be difficult and not cost effective. Consequently, we have decided to more extensively sample 12 herds with diverse landscapes and management practices to better understand factors that are associated with high versus low STEC prevalence.

Impacts
We have refined our methodology for sampling and culturing thousands of samples from cattle and humans with clinical infections caused by multiple enteric pathogens as well as our community DNA and 454 pyrosequencing methods for fecal grab, rumen and tonsil samples. We observed that a many animals are shedding more than one type of STEC strain, as determined by stx profiling. Most prior studies have selected the predominant STEC isolate from a given fecal sample, which may have underestimated the genetic diversity and frequency of STEC in cattle. The majority of the strains isolated from our study are non-O157, which is consistent with data from the MDCH highlighting the high frequency of non-O157 STEC in humans with clinical illness. Although serotyping data is not yet available for all strains recovered, we have found that most of the STEC do not represent one of the top seven serotypes in circulation in human populations. A comparison of these strain types to those recovered from patients in Michigan during the same time period will enhance our knowledge of strains and strain characteristics that are most important for clinical disease. Although we have previously reported a high frequency of false positivity by PCR, we suspect that the prevalence of the Stx phage is extremely high in cattle feces, though it is likely that the bacterial host is not E. coli. This hypothesis is based on our inability to culture STEC from conventional PCR- and qPCR-positive samples. Alternatively, it is possible that some STEC are present in low densities within the cattle reservoir and current microbiological practices are not useful for isolation. Through our study presented at VTEC 2012 and the subsequent PLoS One publication on the E. coli O104:H4 outbreak strain from Germany, we enhanced our understanding of how this organism may have caused high frequencies of HUS. Specifically, we demonstrated that the outbreak strains has an enhanced ability to form biofilms in vivo, which is correlated with enhanced expression of Stx and other important virulence genes. These data suggest that inhibition of biofilms in E. coli O104:H4 may represent an important future disease prevention strategy. Because of the impact that this outbreak had on human health, we have changed our serotyping protocols to screen for the presence of E. coli O104:H4 and other enteroaggregative E. coli (EAEC) in all of our cattle- and human-derived isolates.

Publications

  • St. Charles, J.L., R. Mosci, J.T. Rudrik, S.D. Manning, L.S. Mansfield. 2012. Campylobacter jejuni isolates from calves have A, B and C lipooligosaccharide (LOS) biosynthetic locus classes similar to human Guillain Barre syndrome associated strains. CRWAD. Chicago, IL.
  • Habing, G.G., C. Bolin, S.D. Manning, J.B. Kaneene. 2012. Prevalence, distribution, and diversity of Salmonella subtypes on Michigan dairy farms in 2000 to 2001 and 2009. CRWAD. Chicago, IL.
  • PAPERS IN PROCEEDINGS Manning, S.D. 2012. Evolution and pathogenesis of Escherichia coli O104:H4 infections. Zoonoses Pub Health. 59 (s1);i-1. VTEC 2012. Amsterdam, Netherlands.
  • Hazen, T.H., Sahl, J.W., Redman, J.C., Morris, C.R., Daugherty, S.C., Chibucos, M.C., Sengamalay, N.A., Fraser-Liggett, C.M., Steinsland, H., Whittam, T.S., Whittam, B., Manning, S.D., and Rasko, D.A. 2012. Draft Genome Sequences of the Diarrheagenic Escherichia coli Collection. J Bacteriol. ;194:3026-7.
  • ABSTRACTS Singh, P. K. Jernigan, C. Radek, R. Britton, C. Venegas, S. Rust, P. Bartlett, D. Grooms, S.D. Manning. 2012. Variation in Microbiota Diversity between Beef and Dairy Cattle. American Society for Microbiology Annual Meeting, San Francisco, CA.
  • Al Safadi, R., G. Abu-Ali, R.E. Sloup, J.T. Rudrik, C.M. Waters, K.A. Eaton, and S.D. Manning. 2012. Biofilm-Associated Pathogenesis of Escherichia coli O104:H4. American Society for Microbiology Annual Meeting, San Francisco, CA.
  • PUBLICATIONS Al Safadi, R., G.S. Abu-Ali, R.E. Sloup, J.T. Rudrik, C.M. Waters, K.A Eaton, and S.D. Manning. 2012. Correlation between in vivo biofilm formation and virulence gene expression in Escherichia coli O104:H4. PLoS One;7(7): e41628.
  • Eaton K.A., C. Fontaine, S. Poe, R. Al Safadi., J.T. Rudrik, S.D. Manning. 2012. Acute colitis and hemolytic uremic syndrome in germ free mice colonized by shigatoxigenic Escherichia coli O104:H4. American College of Veterinary Pathologists. Seattle, WA.
  • Del Valle, J.M., Venegas-Vargas, C., Henderson, S., Khare, A., Ouellette, L.M., Lehnert, J.D., Thomas, D., Grooms, D.L., Bartlett, P.C., Moore, J., Scribner, K.T., Manning, S.D. 2012. Evidence for interspecies transmission of Shiga toxin-producing Escherichia coli (STEC) between wild deer and dairy cattle. Phi Zeta Research Day, MSU College of Veterinary Medicine. Michigan State University, E. Lansing, MI.
  • Del Valle, J.M., Venegas-Vargas, C., Henderson, S., Khare, A., Ouellette, L.M., Lehnert, J.D., Thomas, D., Grooms, D.L., Bartlett, P.C., Moore, J., Scribner, K.T., Manning, S.D. 2012. Evidence for interspecies transmission of Shiga toxin-producing Escherichia coli STEC) between wild deer and dairy cattle. Merial-NIH National Veterinary Scholars Symposium. Colorado State University, CO.


Progress 01/15/11 to 01/14/12

Outputs
OUTPUTS: We have developed a sampling protocol that allows us to screen all animals from multiple herds in Michigan for STEC. Phase I involves collecting and culturing environmental samples and interviewing farm managers for epidemiological information. Phase II involves sampling each animal at a given farm by collecting fecal grab (FG) specimens, rectoanal junction (RAJ) swabs, and blood for STEC quantification, culture and community DNA isolation for 454 pyrosequencing. Blood samples are tested for immunosuppressive infections. Phase III samplings target a subset of STEC-positive and -negative animals followed over time to evaluate changes in shedding and colonization status and better understand STEC transmission dynamics. At each visit, one FG, two RAJ swabs, one rumen fluid sample, and two pharyngeal swabs are collected per animal for culture, community DNA isolation and 454 pyrosequencing. RAJ biopsies and blood are collected to evaluate the bovine immune response to STEC. Following collection, specimens are stored on ice until transport to MSU. Each specimen is inoculated in modified gram-negative broth and incubated overnight at 37oC. The inoculum is resuspended in TE buffer and heated to 94oC for 10 mins to isolate crude DNA. PCR using primers specific for stx1, stx2, and eae is performed to identify STEC-positive samples. To date, 702 samples have been screened for by multiplex PCR. Approximately 140 of the 702 were tested using three different multiplex PCR assays to ensure that we are using the most sensitive system. For stx1 and/or stx2 positive samples, the STEC are cultured by plating the broth on SMAC and rainbow agar. Immunomagnetic separation has been used for O157 detection. Crude DNA is then isolated from suspect colonies and multiplex PCR is performed to determine which colonies harbor stx genes. Isolates are frozen for future genotyping studies. A protocol has also been developed to measure the level of STEC shedding from the corresponding fecal grab specimen via quantitative PCR (qPCR) protocol targeting the stx1 and stx2 genes. Dr. Grooms, Bartlett and Rust use the farm visits as a way to teach D.V.M. and other graduate students how to sample animals. Dr. Manning teaches students epidemiological analyses and molecular techniques needed to answer the research questions. Seven students have been instructed thus far. All instructors have incorporated material from the study into undergraduate and graduate level courses. Two half day workshops, which included presentations from all aspects of the study, were well attended. Dr. Manning outlined the study in a presentation to the MSU Board of Trustees, MSU Bad Bugs Club, University of Michigan Molecular and Clinical Epidemiology Microbial Ecology Symposium, Neogen Corp., the MSU Global Water Initiative and a poster presentation at the USDA PI annual meeting. New collaborations have been initiated with Drs. Andy Benson and Tom Besser, who received similar awards, as well as Dr. Bo Norby of MSU. Dr. Benson will receive blood cards for his bovine genetics component and sequencing data to allow comparisons across states. PARTICIPANTS: Dr. Shannon Manning (PD) has spent 8 person months (pms) on the study since January 2011. She has overseen the design, survey development, cattle sampling, specimen processing, STEC detection and methods development. She has presented data and jointly trained all students and postdoctoral fellows with the Co-PDs, Drs. Grooms, Bartlett and Rust, who have each spent 5 pms on the study. They have assisted with study design, farm identification, cattle sampling, survey design and implementation, and epidemiological analyses. New personnel (role, pms) include Akanksha Khare (technician, 10 pms), A. Cody Springman (technician, 5 pms), Katherine Jernigan (technician, 10 pms), Pallavi Singh (postdoctoral fellow, 10 pms), and Christopher Colvin (technician, 2 pms). Ms. Khare and Ms. Springman are responsible for specimen processing (i.e., subculturing, IMS, serotyping, PCR, STEC quantification and genotyping). Dr. Singh and Ms. Jernigan are responsible for isolating and purifying DNA for sequencing. Dr. Singh conducts the 454 pyrosequencing analysis to characterize microbial communities and examines DNA for other bacterial pathogens. Mr. Colvin processes blood specimens and conducts the immunology analyses under the supervision of Dr. Paul Coussens (Co-PD, 1 pm). Additional Co-investigators have committed some time as well. Dr. Inge Langhor (1 pm) has examined the histology of bovine RAJ tissue, while Dr. Funk (0.5 pms) has assisted with the study design. The Center for Microbial Pathogenesis has funded a complementary study that aims to culture and sequence DNA isolated from the pharyngeal area and to identify factors important for STEC transmission. Drs. Ned Walker and Roy Kirkwood were brought in as collaborators. The Michigan Department of Community Health (MDCH) provides STEC isolates obtained from humans with clinical infections for comparison, while Neogen Corp. offers free diagnostic testing services. Drs. Guy Loneragan and Mindy Brashears of Texas Tech University (TTU) have assisted with the study design. The TTU study will begin in year 2 as will the collaboration with Drs. Britton and Hashsham, who will assist with the sequencing analyses. This project has been used to train several individuals in the fields of molecular biology, epidemiology, animal science, and veterinary medicine. Cristina Venegas (Ph.D. student) will complete her thesis work on the epidemiology of STEC in Michigan farms. Rebekah Mosci (M.P.H. student), Lindsey Ouellette (M.P.H. student), Marion Tseng (Ph.D. student), and Scott Henderson (M.S. student) are learning how to conduct epidemiological and laboratory-based studies through his project. Several D.V.M. students, Justine Zingsheim, Tonya Neuman, and Lee Ann Peasley, who are funded through the Merial Veterinary Scholars program, and 3 undergraduates (Erin Jagodzinski, Cassandra Martin, and Samantha Wengert) have enhanced their understanding of cattle and farm studies. Farm managers at each of the 6 farms have also been actively involved in the sampling. TARGET AUDIENCES: Farm owners in mid-Michigan serve as the target audience for this study. In addition to educating them about STEC, cattle sampling, and food safety, all owners have been appropriately compensated for their participation. Dr. Manning has participated in several workshops (e.g., MSU Global Water Initiative, Neogen Corp. summit) aimed at disseminating knowledge gained from the study, while Drs. Manning, Bartlett, and Grooms have incorporated study information into their courses: VM 817 - Issues in Pre-Harvest Food Safety (Grooms); VM 831 - Foodborne Disease Epidemiology, VM 832 - Food Safety Disease Control (Bartlett); and VM 811 - Evolution of Foodborne Pathogens, MMG 461 - Molecular Pathogenesis, MMG 861 - Advanced Microbial Pathogenesis (Manning). Similarly, Dr. Funk is routinely incorporating study findings into her curriculum, as she is the Director of the MSU Online Professional Masters of Science in Food Safety. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Multiple new methods have been developed since the study began. These methods are different than the older, more commonly used methods, which were not useful for our purposes. For example, STEC quantification is time consuming via bacterial plating and cell counting; therefore, we have developed a new method based on qPCR targeting the Stx genes. Similarly, previously published protocols were limited in that they mostly focus on the identification and culture of STEC O157. Very little information is available on the prevalence of non-O157 STEC. This represents a change in our current knowledge, as it is possible that those STEC genotypes present in low frequencies are cattle-specific and more difficult to get into the food supply. If so, then these may not represent useful intervention targets regardless of serotype. Another change in current knowledge is with regard to the site of STEC colonization in cattle and the high frequency of false positivity by PCR. Prior studies have reported that STEC colonization occurs primarily at the RAJ. However, we have identified STEC in rumen and feces in RAJ-negative animals. RAJ samples have been sent to Neogen Corp. to be tested with their novel MALDI-TOF assay that detects STEC and distinguishes the serotype; these data will be used to validate the test for more widespread use. The high rate of PCR positivity does not correlate with a high rate of STEC recovery; therefore, it is not clear whether STEC is often present in low quantities or is transient in that only DNA can be recovered in a large percentage of animals. While many researchers have published the isolation of high numbers of STEC, in actuality, these organisms are difficult to culture and may only represent a small proportion of the total microbial communities of many animals; these communities likely vary across animals. Indeed, preliminary sequencing data from two animals has demonstrated that the microbial communities are similar in STEC-negative sites, while STEC-positivity appears to throw off the balance of the healthy community. Examining this relationship in a larger subset of animals is important to better understand how microbial communities of the gastrointestinal tract impact STEC colonization and shedding. These findings will facilitate the development of future intervention strategies that will eventually result in a safer food supply. Through Dr. Manning's presentations to the MSU Board of Trustees and at the MSU Global Water Initiative, she was able to provide information with regard to STEC contamination of the environment and food supply that may improve the existing infrastructure. For example, the MSU Global Water Initiative is interested in protecting our waterways and drinking water; STEC has become an area where MSU now has considerable expertise. The Trustees meeting involved a discussion of how MSU can help support and provide resources that are important for the STEC ecology study. The presentations given to Neogen Corp and the University of Michigan aimed to provide information that will enhance current technology.

Publications

  • Neuman, T., C. Venegas, J. Zingsheim, E. Jagodzinski, L. Ouellette, R. Mosci, S.D. Manning, P. Bartlett and D. Grooms. Relationship between chronic infection with Bovine Viral Diarrhea Virus, Bovine Leukosis Virus, or Mycobacterium avium ssp. paratuberculosis and fecal shedding of Shiga toxin-producing Escherichia coli (STEC) in adult cattle on Michigan farms. Phi Zeta Research Day, MSU College of Veterinary Medicine. Michigan State University, E. Lansing, MI. October 7, 2011.
  • Neuman, T., C. Venegas, J. Zingsheim, E. Jagodzinski, L. Ouellette, R. Mosci, S.D. Manning, P. Bartlett and D. Grooms. Relationship between chronic infection with Bovine Viral Diarrhea Virus, Bovine Leukosis Virus, or Mycobacterium avium ssp. paratuberculosis and fecal shedding of Shiga toxin-producing Escherichia coli (STEC) in adult cattle on Michigan farms. Merial Veterinary Scholars meeting. Orlando, FL. July, 2011.
  • Zingsheim J., Neuman, T., C. Venegas, E. Jagodzinski, L. Ouellette, R. Mosci, S.D. Manning, P. Bartlett and D. Grooms. Comparison of Environmental Sampling Strategies for Shiga toxin-producing E. coli on Michigan Farms. Phi Zeta Research Day, MSU College of Veterinary Medicine. Michigan State University, E. Lansing, MI. October 7, 2011.
  • Abstracts/Proceedings: Venegas, C., R. Mosci, L. Ouellete, A. Khare, P. Singh, S. Rust, P. Bartlett, D. Grooms, S.D. Manning. Prevalence of Shiga toxin-producing Escherichia coli (STEC) genes by multiplex PCR in cattle and their environment, Michigan 2011. CRWAD. Chicago, IL. December 2011.