SHIGA TOXIN-PRODUCING E. COLI (STEC) IN MICHIGAN: FROM COLONIZATION OF CATTLE TO HUMAN DISEASE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0227730
• Project Start Date: Dec 1, 2011
• Project End Date: Nov 30, 2016
• Program Code: [(N/A)]- (N/A)

Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824

Performing Department
Microbiology & Molecular Genetics

Non Technical Summary
Our preliminary studies indicate a high level of genetic diversity among cattle- and human-derived STEC strains, though there is significant variation in the ability of different genotypes to colonize and cause severe disease. Additionally, some cattle-derived genotypes upregulate acid resistance and stress fitness genes and survive better following passage through a model stomach. Therefore, determining STEC genotype and serotype distributions as well as phenotypic variation in strains from cattle with a range of shedding phenotypes is a logical and important future direction. Making comparisons to strains from infected humans will enhance our understanding of those strain types that most frequently cause disease. While most of our preliminary data focuses on STEC variation, we hypothesize that shedding in cattle is a multifactorial process. Additional factors, including the immune response and microbial ecology of the GI tract, are also important to address. If, for example we identify a specific O157:H7 genotype to be associated with persistent or super-shedding, then genotype-specific intervention protocols can be developed. Examples of such protocols include creating immunoglobulins or vaccines that target a virulence gene allele unique to the genotype. In addition, a predominance of more virulent strain types in cattle could lead to the development of rapid diagnostic tools to prevent entry into the food supply. Alternatively, if an inadequate immune response appears to be the most important factor for super-shedding, then control methods can be developed accordingly. It is possible, for instance, that some STEC strains lack the ability to effectively stimulate the immune system, thereby preventing bacterial clearance. In this scenario, compounds known to effectively elicit a similar response could be incorporated into vaccines. Moreover, for the proposed microbiome experiments, we expect to identify specific microbes and microbial by-products that are negatively associated with STEC. If cultivable, multiple microbes could be administered as direct-fed microbial cocktails. Finally, with the exception of epidemiological factors, we would expect that some host and bacterial factors identified to be important for shedding in cattle can be applied to different animal species. This becomes more important as new sources and reservoirs for STEC are identified. In short, these and similar 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.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	3310	1040	10%
712	3310	1100	10%
712	3310	1170	10%
712	3410	1040	10%
712	3410	1100	10%
712	3410	1170	10%
722	4010	1040	10%
722	4010	1100	10%
722	6010	1040	10%
722	6010	1100	10%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 722 - Zoonotic Diseases and Parasites Affecting Humans;

Subject Of Investigation
3310 - Beef cattle, live animal; 4010 - Bacteria; 6010 - Individuals; 3410 - Dairy cattle, live animal;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology; 1170 - Epidemiology;

Keywords

shiga toxin

escherichia coli

shedding

virulence

microbiome

immune response

Goals / Objectives
Foodborne infections are a major public health concern in the U.S. and are estimated to cause >76 million illnesses, 300,000 hospitalizations and 5,000 deaths each year, with annual costs of ~$23 billion. In the U.S. in 2007, Salmonella, Campylobacter, Shigella, and Shiga toxin-producing Escherichia coli (STEC) caused the majority (91%) of foodborne cases, with most cases reporting severe diarrhea or hemorrhagic colitis. While prior studies have demonstrated that there is variation in virulence among different enteric bacterial genotypes, it is unclear how these genotypes affect the host and the host microbiota. In addition to the morbidity, mortality and substantial costs associated with sporadic and outbreak-associated human STEC infections, some STEC serotypes also cause clinical illness in calves, thereby increasing the disease burden. While our prior studies have detected variation in virulence, acid resistance, stress response, and survivability among STEC O157:H7 lineages, the distribution of lineages in the bovine reservoir and their relationship with shedding and human disease is not known. Similarly, little is known about non-O157 distributions in cattle or associations with shedding and non-bacterial factors, including the bovine immune response, the microbial communities of the GI tract, and epidemiological factors (e.g., diet, stocking density, herd type). The goals of this project are to: 1) Identify bacterial genotypes, phenotypes, and epidemiological factors important for STEC shedding and temporal changes in shedding status in cattle and make comparisons to strains isolated from patients with STEC infections and meat; 2) Examine how the composition, diversity and function of various microbial communities impacts shedding in cattle and disease in humans with STEC infections; and 3) Determine how STEC affects antibody and blood leukocyte levels, and cytokine and leukocyte expression profiles of STEC-positive and STEC-negative cattle. One 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 aim to develop new ideas for direct-fed microbials, vaccines, therapies and other control strategies that can reduce the frequency and level of STEC shedding. Another goal is to identify beneficial microbial communities, microbes and microbial products that are important for preventing STEC infections in humans. Together, these studies could facilitate the development of prevention and treatment strategies used to restore beneficial microbial communities, thereby resulting in decreased contamination of beef products, transmission to humans, and infections in humans. Furthermore, a comprehensive evaluation of STEC genotypic and phenotypic variation in cattle and humans and knowledge of additional factors important for bovine colonization and human disease will facilitate development of effective prevention and intervention methods useful for the agricultural, food, and health care industries.

Project Methods
In April-May of year 1, we have begun sampling MI dairy and beef herds that vary with regard to maintenance, forage diets and environmental conditions. At each farm, pen bedding, manure storage areas and water troughs will be tested, and personnel will complete a short questionnaire focusing on the farm environment and cattle. At each STEC+ herd, fresh fecal grab and recto anal junction (RAJ) samples will be collected from all animals. Detection methods specific for O157:H7 and non-O157 STEC (culture, multiplex PCR) will be used. Suspect STEC isolates will be confirmed using multiplex PCR for stx, and serotypes will be confirmed by latex slide agglutination. In addition to conducting a risk factor analysis to identify epidemiological factors associated with shedding status changes in cattle, additional sampling (blood, RAJ biopsy, RAJ swabs, and ruminal fluid) will be conducted on a carefully selected subset of animals followed over time. We are most interested in sampling animals with an observed change in shedding status, namely 1) super-shedders that convert to non-shedders, and 2) non-shedders that convert to super-shedders. ELISAs will also be used to screen cattle for infections with common immunosuppressive agents and blood samples will be used to quantify total leukocytes. At the same time, stools from patients with diarrhea in the state of Michigan will be cultured for STEC. Strains from both cattle and humans will be characterized using MLST and SNP genotyping, while total community DNA will be extracted and sequenced from stools and RAJ swabs to characterize the microbial communities. A combination of ANOVA, regression, and multivariate approaches will be used to visualize relationships between variables and community structure. Although there is likely to be considerable variation in the microbiome of different animals and people, we expect to identify commonalities, which will allow us to create community profiles for epidemiological analyses. The overall frequency of profiles will be determined in humans as well as bovine super-shedders and non-shedders, and profile frequencies will be stratified by host, clinical, epidemiological, and bacterial factors obtained from questionnaires and additional molecular data.

Progress 12/01/11 to 11/30/16

Outputs
Target Audience:Target audiences: Public health professionals, health practicioners and farm owners in mid-Michigan serve as a target audience for different aspects of this study as well as patients with enteric infections living in the state of Michigan. We also aim to disseminate our findings to members of the scientific community including researchers and food safety and consumer groups.Dr. Manning has attended an elementary school to demonstrate to students how to culture bacteria and inform them about proper food safety practices as well. Efforts: Educating residents in areas with the highest risk of disease or greatest exposure levels to foodborne pathogens 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 and press releases. Changes/Problems: Nothing Reported 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, 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, and Dr. Manning has been to elementary schools to teach kids about STEC and food safety. 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. In addition to examining the epidemiology of STEC in cattle and making comparisons to human isolates and cases, we have also included a comparative study of Campylobacter jejuni and an examination of antibiotic resistance rates in both the cattle- and human-derived STEC and C. jejuni isolates. A subset of these analyses have been published. The STEC epidemiology paper published in Applied and Environmental Epidemiology was selected for a press release by ASM. Key findings of the latter study were: 1) the number of cattle shedding Shiga toxin-producing E. coli (STEC) in their feces varied considerably across herds in Michigan; and 2) 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 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 to highlight the findings.Our analysis of strains collected longitudinally is ongoing. For aim 2, 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. A paper on the deer samples is under review in the journal "Microbial Ecology". There appears to be no difference in community profiles by STEC status in either deer or cattle, though other variables (e.g., temperature, diet, age, season, etc) appear to be important. We have also conducted a follow-up analysis in humans to determine the impact that age and sex have on individuals with and without enteric infections, which was published in Annals of Epidemiology. Overall, we observed an increase in Bacteroides abundance and decrease in Escherichia abundance with age, though these differences were most apparent for patients with enteric infections. Genus Bacteroides was also higher in female communities while Escherichia predominated in males. Because Escherichia abundance was previously linked to symptom severity in our prior study (Singh et al, Microbiome. 2015; 3:45), children with enteric infections may be most susceptible to severe disease outcomes due to high and low abundance of Escherichia and Bacteroides, respectively. For the last aim, we have examined the role of immune function in cattle and STEC shedding. Our paper on the association between STEC shedding and infection with the bovine leukemia virus (BLV) and Mycobacterium avium subsp. paratuberculosis (MAP) is currently in press in the Journal of Food Protection.Because both infections are chronic in nature, we tested the hypothesis that cattle infected with BLV and/or MAP are more likely to shed STEC. Of the animals sampled in our study, 34.9% were BLV positive while 2.7% 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 and no association was observed between STEC status and percentage of neutrophil, lymphocytes. Although controlling both BLV and MAP is important for overall herd health and productivity, we found no evidence that controlling BLV and MAP would have an impact on STEC shedding in cattle.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Singh, P. R.Mosci, J.T. Rudrik, and S.D. Manning. Draft genome sequence of a diarrheagenic Morganella morganii isolate. Genome Announc. 2015: e01165-15.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Singh, P. and S.D. Manning. Impact of age and sex on the composition and abundance of the intestinal microbiota in individuals with and without enteric infections. Annals Epidemiol. 2016; 26:380-85.
• Type: Journal Articles Status: Published Year Published: 2016 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. mBio. 2016; 7(2):e00154-16.
• 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: Published 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: 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. 1st place poster award.
• 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.
• 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: Moore, M., R. Mosci, and S.D. Manning. Correlation between Shiga-toxin Producing Escherichia coli Serotypes and Disease Severity in Michigan Patients. MSU 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 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 UURAF, 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.

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

Outputs
Target Audience: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. In addition, multiple manuscripts have been submitted and published to disseminate information to other scientists in the field, and Dr. Manning has attended an elementary school to demonstrate to students how to culture bacteria and inform them about proper food safety practices. Changes/Problems: Nothing Reported 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 working on the project over the reporting period included: Dr. Pallavi Singh, Rebekah Mosci, Katherine Jernigan, Dr. Qiong Sha, Dr. Marion Tseng, Brian Nohomovich, Matt Peters, and Moriah Moore. Dr. Wonhee Cha completed her Ph.D. on Campylobacter jejuni in cattle versus humans in July, 2015 under Dr. Manning's guidance, and a new graduate student, Sanjana Mukherjee, joined the team. Drs. Daniel Grooms and Paul Bartlett have mentored Dr. Cristina Venegas, who completed her thesis work on the epidemiology of STEC in Michigan farms in spring, 2015, while Drs. Kim Scribner and Manning co-advised, Lisette Delgado, who earned her masters degree in spring 2015. Dr. Mansfield continued to collaborate on the C. jejuni research projects; both have multiple students involved in these analyses. 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. Several undergraduates attended the undergraduate research conference and graduate students presented their work at the Midwest Microbial Pathogenesis Conference (MMPC) in August 2015. Dr. Cha presented her work at CRWAD in December 2014. How have the results been disseminated to communities of interest?Dissemination of research findings is underway through classes, manuscripts, and presentations at conferences including the CWRAD, MMPC and VTEC meetings. Dr. Manning has also presented her work at different universities and institutes within MSU. What do you plan to do during the next reporting period to accomplish the goals?For the final reporting period, we aim to continute to disseminate our research findings via classes, manuscripts and conferences. We are also planning to complete one additional study in cattle in the spring of 2016 in an effort to test novel therapeutics that can decrease the likelihood of STEC shedding. Additional sequencing analyses will also be conducted to compare intestinal microbial communties from cattle of different herds and of humans with and without enteric infections.

Impacts
What was accomplished under these goals? We have been working hard to get manuscripts prepared, submitted and published and in disseminating our research findings. For the cattle studies of STEC, we have completed the analyses that identified risk factors for shedding in 11 herds, and have assessed associations between immunocompromising conditions and STEC. Two papers are almost ready for submission. Another paper highlighting the likelihood of transmission between dairy cattle and deer was published in 2015 as well as a paper on Salmonella and antibiotic resistant E. coli. We did a comparative analysis of cattle from Argentina as well. In addition, we have completed the analyses of the Campylobacter jejuni population from Michigan cattle and humans with infections and three papers are ready for submission. With regard to the human infection projects, a paper is under review that highlights the number of STEC infections in Michigan over time and illustrates an increasing frequency of non-O157 STEC. We have also completed the analysis of 275 intestinal microbial communities and a paper is in press in Microbiome.

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: Accepted 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; In press.
• Type: Journal Articles Status: Submitted 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. Submitted to BMC Genomics. 2015.
• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Tseng, M., J.T. Rudrik, J. Collins, T. Henderson, J. Funk, and S.D. Manning. Increasing incidence of non-O157 Shiga toxin-producing Escherichia coli (STEC) in Michigan and association with disease. Epidemiol Infect. July 2015
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 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. 2014.
• 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; December 2014.
• Type: Conference Papers and Presentations Status: Published 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 UURAF. April 2015.
• Type: Conference Papers and Presentations Status: Published 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 Conferences. Indianapolis, IN. August 2015.
• Type: Conference Papers and Presentations Status: Published 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: Other Status: Published Year Published: 2015 Citation: Manning, S.D. Oral presentation. 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: Other Status: Published Year Published: 2015 Citation: Manning, S.D. Oral presentation. Efforts to understand bacterial pathogens and discover novel methods to kill them. Presented at the MSU Foundation Board of Directors meeting. E. Lansing, MI. September 18, 2015.
• Type: Other Status: Published Year Published: 2014 Citation: Manning, S.D. Oral presentation. 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: Other Status: Published Year Published: 2015 Citation: Manning, S.D. Oral presentation Transitioning from Wolverine to Spartan. Distinguished alumni seminar. University of Michigan School of Public Health. Jan. 30, 2015.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: For these studies, we have been trying to disseminate our research findings to members of the research community with similar interests via publications, presentations at national conferences, and interviews. Changes/Problems: One unexpected result was that we observed a high frequency of non-O157 STEC and non-typeable (NT) STEC from cattle in the study. Interestingly, an increase in non-O157 STEC was also observed in Michigan patients over time. Because these strains are difficult to characterize, we have modified our genotyping strategy and have relied on SNP-based array profiling in collaboration with the FDA and genome sequencing. Developing sequencing analytical pipelines and tools is an important current focus. What opportunities for training and professional development has the project provided? Dr. Manning has mentored 22 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, Marion Tseng, Brian Nohomovich, Lillian Jensen, Kai Hussnaetter, and Moriah Moore. 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, while Drs. Kim Scribner and Manning both advise a new masters student, Lisette Delgado, who is comparing the microbiome of STEC-positive deer and cattle. Dr. Britton has worked to refine the microbial ecology protocols and analyses and Dr. Mansfield has worked on the Campylobacter jejuni research projects; both have multiple students involved in these analyses. 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. Students interested in attending national conferences are encouraged to submit abstracts to attend and several students have participated in genome processing and metagenomics workshops in order to enhance sequence analysis skills. During the reporting period, seven students reported their findings at national conferences and two students participated in sequencing workshops. 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 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 scientific meetings, while generating additional data on other aspects of the study that we have yet to work on and maintaining collaborations.

Impacts
What was accomplished under these goals? We have continued our analysis of over 2,000 STEC isolates recovered from Michigan herds (6 dairy and 5 beef) as well as a subset of herds in Texas. The STEC prevalence in Michigan was 16% with significant variation across herds. We have developed models that identify epidemiological predictors of STEC shedding in dairy cattle and have evaluated the level of STEC diversity across herds and animals over time. In 5 herds (n=913 isolates), for example, we identified 3-6 distinct STEC strains circulating per herd by stx/eae profiling and serotyping; the majority of strains were nontypeable (negative for the top 7 serotypes). We have also characterized 87 STEC isolates from 13 animals in one feedlot over an 8 week period to better understand how the specific strain types change over time. Even in this small subset of animals we identified the emergence of new strain types and transmission to other animals; a separate project also identified transmission between dairy cattle and deer sharing a pasture. For the human enteric disease project, we have received a total of 802 stool samples from 741 participants with enteric infections to date. A total of 615 (83%) of the participants are cases and 128 (17%) are controls, while 60 (6%) were cases who had been re-examined up to a month after their infection was cleared. Pyrosequencing of the V3-V5 16S rRNA gene region was performed on a subset of 275 stools obtained during the first two years of the study. A total of 201 stools were from cases infected with Campylobacter (n=72), STEC (n=29), Salmonella (n=66) or Shigella (n=34) prior to antibiotic use and 74 were from healthy controls. Based on rarefaction by both chao1 and Shannon index the CB treated samples had a slight decrease in diversity and species richness; this difference is also seen weighted NJ tree. ANOVA revealed significant differences in 25 OTUs (p<0.05) between the 2 groups, of which 13 belonged to family Ruminococcaceae. In cases versus controls, construction of a rarefaction curve revealed lower species richness and evenness. Notably, controls had the highest number of observed species relative to cases; however, no differences in community diversity or abundance were observed among the cases infected with different types of pathogens. Additional support for this finding came from the principal component analysis, which resulted in close clustering of the control communities but not cases even after stratifying by age, gender, race, and type of pathogen.

Publications

• 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: 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. August 2014
• 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. April 2014.
• 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; June 2014.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; June 2014.
• 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; June 2014.
• 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: 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; June 2014.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Manning, SD. 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: Other Year Published: 2014 Citation: Manning, SD. 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: Other Year Published: 2014 Citation: Manning, SD. 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

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

Outputs
Target Audience: Farm owners in mid-Michigan and Texas serve as one of the target audiences for the field portion of the study, while patients with enteric infections and their family members represent another audience for the human health aspects of the study. For both studies, we also aim to disseminate the research findings to members of the research community with similar interests in the form of 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. Dr. Britton has worked to refine the microbial ecology protocols and analyses and Dr. Mansfield has worked on the Campylobacter jejuni research projects; both have multiple students involved in these analyses. 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. 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 study has also been conducted in humans, as the microbial communities from stool samples from patients with and without enteric infections have been examined. Interestingly, patients with enteric infections had a higher frequency of Proteobacteria than healthy controls. 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: 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: 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: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Zell, A., R. Mosci, D.W. Newton, H. Salimnia, P. Lephart, D. Sundin, W. Khalife, J. Collins, J.T. Rudrik and S.D. Manning. Overview of the Michigan State University Enterics Research Investigative Network (ERIN) Microbial Diversity Study. Michigan Public Health Association Annual Meeting. Ann Arbor, MI. April 2013
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Singh, P. K. Jernigan, R. Mosci, A. Zell, D. Newton, H. Salimnia, P. Lephart, W. Khalife, J.T. Rudrik, S.D. Manning. Intestinal Microbial Communities in Patients with Enteric Infections. American Society for Microbiology Annual Meeting; Denver, CO; May 2013.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Wengert, S., R. Mosci, J. St. Charles, W. Cha, D. Newton, H. Salimnia, P. Lephart, W. Khalife, J. Bell, L.S. Mansfield, J.T. Rudrik, S.D. Manning. Genetic Diversity of Campylobacter jejuni Isolated from Patients in Michigan. American Society for Microbiology Annual Meeting; Denver, CO; May 2013.
• Type: Conference Papers and Presentations Status: Published 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: 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: 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.
• 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: Other 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: Other Year Published: 2013 Citation: Manning, S.D. Pathogen Evolution and Implications for Food Safety. Presented at the annual Merial NIH Veterinary Scholars Symposium. E. Lansing, MI. August 3, 2013

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

Outputs
OUTPUTS: We have completed our sampling of 1,195 cattle from 12 herds located in different geographic locations in the state of Michigan; samplings took place between May and October of both 2011 and 2012. STEC isolation methods such as immunomagnetic separation and culture using multiple media types were optimized for use on fecal grab, rumen fluid, and tonsil samples. Over 3,000 samples have been processed to date. The herd prevalence varied considerably across 8 of the 12 herds with data available. The prevalence ranged between 10% and 53 %, while the average herd prevalence for dairy and beef cattle was 14% and 27%, respectively. Identifying factors important for the variation in pathogen prevalence across herds represents an important future goal. 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. 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. Dr. James Rudrik of the Michigan Department of Community Health (MDCH) continues to work with Dr. Manning to oversee the enteric disease surveillance system and ensure that STEC, C. jejuni, Shigella and Salmonella isolates are delivered to MSU for characterization. To date, we have recovered over 300 bacterial isolates. Notably, the surveillance system captured two E. coli O104:H4 isolates from two Michigan patients, which were characterized and examined in a pathogenesis study. Dr. Manning gave an oral presentation on E. coli O104 pathogenesis 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 closely with BioRad, Corp. to validate their new STEC detection qPCR method and continuously updates STEC genotyping and strain data in the STEC Center website (www.shigatox.net). The STEC Center is a bacterial repository comprising over 16,000 strains. The website allows users to search the database in a variety of ways and request bacterial cultures for research studies via the web page. The site is consistently active with roughly 400 hits to the homepage each week, while an average of 1,390 strains are shipped and 1,176 strains are received each year. PARTICIPANTS: Dr. Manning has mentored 10 students, technicians and/or postdoctoral fellows on study design, epidemiological analyses and the use of multiple molecular techniques. Individuals include Dr. Pallavi Singh, 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. 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). Collaborators include Dr. Kathryn Eaton of the University of Michigan, Drs. Christopher Waters, John Kaneene, Julie Funk and Kim Scribner of Michigan State University, and Drs. Guy Loneragan and Mindy Brashears of Texas Tech University. 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) learned how to conduct epidemiological and laboratory-based studies through his project. Jacquelyn Del Valle, a D.V.M student, was funded through the Merial Veterinary Scholars program, and 3 undergraduates (Jonathon Lehnert, Davis Thomas and Clare Laut) have enhanced their understanding of cattle and farm studies. Farm managers at each of the 12 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: Nothing significant to report during this reporting period.

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. Notably, we have observed that a high frequency of cattle is shedding of more than one type of STEC strain, as determined by Shiga toxin gene (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 pathogenesis. 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. The results of this study were reported on multiple websites, newspapers and radio stations including National Public Radio shortly after the manuscript was published. 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

• 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.
• 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.
• 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.
• 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.
• 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.