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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). <P>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. <P>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.
Non-Technical Summary:<br/>
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.
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Approach:<br/>
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.
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Progress:<br/>
2012/01 TO 2012/12<br/>
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.
<br/>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.
<br/>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 <br/>MODIFICATIONS: Nothing significant to report during this reporting period.
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IMPACT: 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.