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April 2017 | Volume 5, Issue 1

STEC CAP News CONTROLLING SHIGA TOXIN-PRODUCING E. coli TO IMPROVE BEEF SAFETY

Director’s Update

This morning, I read back through several of the STEC CAP newsletters that the EMT has issued during the course of our grand project, and it sort of blows me away how many truly remarkable accomplishments have been made by our team. We have improved detection methods for STECs, we better understand where these non-O157 STECs hide out in production units and how prevalent they are across different cattle populations, we continue to investigate the virulence profiles of various STEC serogroups isolated from cattle and how they might

Randy Phebus

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appy spring to all of you, our STEC CAP family and friends. I sit today in my beloved mancave enjoying the gorgeous Kansas sunshine and thinking about the myriad of accomplishments we have made related to the STEC CAP grant from back in 2010 when a small group of us began the torturous endeavor of writing the NIFA AFRI grant and pulling the team together, to today where we are embarking on our 6th year of focused translational research, education and outreach to improve the safety of beef products, and ultimately to improve public health.

impact food safety and public health, our team of economists are involved to gauge cost-benefit relationships of different STEC aspects in the production to consumption chain, we have validated many intervention technologies in lab studies and in fullscale production facility scenarios, we have addressed many consumerlevel food safety risks and gained a better understanding of human behavior related to food safety, and our robust Quantitative Microbial Risk Assessment model continues to evolve. We have trained a huge number of graduate students who will go on to accomplish great things in food safety. We are nearing our 100th STEC-STEP internship being filled, which was the achievement benchmark set in our original grant proposal. These interns have done tremendous work to add to the overall output and outcomes of all five STEC CAP grant objectives. ...continued page 4 STEC CAP Team 126D VBS Lincoln, NE 68905 PHONE: 402-472-8564 FAX: 402-472-9690

stecbeefsafety.org

Rapid-testing research targets O serogroups

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or the past three years, rapid testing of Shiga toxin-producing Escherichia coli (STEC), one of the causes of foodborne illnesses, has been one focus of the Coordinated Agricultural Program (CAP) grant from the U.S. Department of Agriculture (USDA). CAP grant researchers especially are developing and testing detection methods for the several STEC pathogens, including O26, O45, O103, O104, O111, O121, O145 and O157, in the beef industry. One such DNA-based detection method is multiplex oligonucleotide ligation-PCR (MOL-PCR), of which CAP grant researchers Travis Wood, a graduate student at the University of New Mexico, and his advisor, Alina Deshpande, a scientist at Los Alamos National Laboratory, led the development and validation. The assay is now in the process of independent testing by another CAP grant researcher. MOL-PCR can rapidly screen samples concurrently for several STEC O serogroups and three major virulence factors (genes for two Shiga toxins and intimin). Inside this issue

Director’s Update .......................................1 O Serogroups ..............................................1 Student Perspective....................................3 IAFP 2017.......................................................4 Dodge City ....................................................5


STEC CAP News In turn, the CAP grant is using a waveguide-based optical biosensor, with development led by Loreen Stromberg, currently a postdoctoral research associate at Iowa State University, and her advisor, Harshini Mukundan, a scientist at the Los Alamos National Laboratory, to detect the binding of a key surface molecule, lipopolysaccharide, which is the outer part of the membrane of the E. coli. CAP grant researchers use antibodies to detect the lipopolysaccharide in a very sensitive method that eliminates a lot of background.

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Rapid-testing research targets O An isolate would have to be positive for one of the targeted O serogroups, as well as a Shiga toxin and intimidating, to be classified as an adulterant.

According to the USDA Food Safety and Inspection Service (FSIS), to be classified as an adulterant, an isolate would have to be positive for one of the targeted O serogroups as well as a Shiga toxin and intimin. The MOL-PCR STEC assay that has been developed is a screening test, and CAP grant researchers have developed it to run on DNA from culture. Beginning with the culture material, MOL-PCR produces results in the same day. Beginning with a ground beef sample, results are possible in two days. Besides independent lab testing of the MOL-PCR screening test, CAP grant researchers are working on a MOL-PCR characterization assay. This assay detects the seven non-O157 STEC that are adulterants, but it is looking at a different gene than the first MOL-PCR. In essence, the characterization assay is testing for individual genes representing each of the seven types of adulterants that would be further confirmation that particular sample has the adulterant in it, explains Rod Moxley, a professor at the School of Veterinary Medicine and Biomedical Sciences at the University of Nebraska– Lincoln and project director of the CAP grant. CAP grant researchers are looking at a variation of one gene, called a single nucleotide polymorphism (SNP). One individual nucleic acid substitution in that gene makes it unique, and the types of E. coli that are adulterants would have that kind of substitution. “It’s targeting a sequence that’s very typical for a particular, let’s say O1O3, O111 or O26, but it would be a second type of MOL-PCR assay that would be called a characterization assay,” Moxley explains. “We could do it on that same sample that’s been screened, and then come up with more additional confirmatory evidence that the adulterant is there.” While several PCR assays that can search for multiple genes at a time exist, MOL-PCR is rapid and high

STEC CAP News

throughput, translating to a lot more samples being run through, in comparison to end-point PCR and quantitative real-time PCR (qPCR). MOL-PCR also is relatively inexpensive. In addition, while MOL-PCR has been developed to test for 11 markers, it is adaptable and can potentially go up to 50 at a time. “What that means is that you can get a tremendous amount of information about a sample because you are looking at many, many different targets at once,” Moxley explains. “The more evidence you have, the greater the likelihood of something being positive. You also can easily add more genetic markers to this assay that you already developed. Instead of starting all over again, you can add things into the mix, which is a big advantage.” Outside of the CAP grant research, another rapid technology researchers are studying involves droplet digital PCR (ddPCR). Like qPCR, this method allows for quantification of STEC target genes; however, its researchers say ddPCR has a couple of advantages. First, ddPCR yields accurate absolute quantification without the need to rely on a standard curve. This is important because the construction of a standard curve requires accurately quantified template DNA, which might be difficult to obtain. Second, ddPCR is less prone to inhibitors of DNA polymerase, e.g., bile salts, than qPCR.

Detecting biomarkers

CAP grant researchers also have been working to address the USDA request for development of biosensors, which in essence would detect biological phenomenon. For example, this could be a binding of part of a pathogen to a surface or to a receptor that would normally be present on a cell or body. “The way pathogens infect you is by binding to something that allows them to get a foothold on the surface and then establish a connection that allows them to grow,” Moxley says.

“It works by detecting key molecules in a pathogen by an ability to bind to the surface,” Moxley explains. “Then antibodies are used that are specific for that molecule that have a fluorescent label on them, and that florescent light can be detected. Then another key part of the way this system works is that light fades out a short distance above the surface where the pathogen is bound. The further you get, and we are talking in an extremely minute space here above the membrane, that light completely disappears. That really eliminates

a lot of artificial signal, which is important in the system.” This waveguide biosensor assay is very different from the MOL-PCR or any of the DNA-based methods. “This is detecting an actual part of the bacteria, an outer membrane, as opposed to its DNA,” Moxley says. “There aren’t many places around the country that are developing methods for detecting non-DNA targets.”

From Student to Scientist

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riginally from the Gem State of Idaho, my journey to work with the best food safety professionals in the country brought me to the Midwest. When I think about the first time I walked into the Food Safety and Defense Laboratory at Kansas State University, I had no idea the level or the number of meaningful research projects I would be exposed to. With the support of Dr. Randall Phebus and the STEC CAP grant, I began working towards a Master’s degree in August 2014. My thesis project consisted of a two-part evaluation of a novel chlorinated nanobubble antimicrobial technology for use in proprietary beef processing developments. This was especially exciting as nanobubble technology is relatively new to the food industry, but proved to be equally challenging due to its minimally-researched state. Fortunately, this served as an excellent opportunity to team up with industry partners to evaluate the antimicrobial properties of chlorinated nanobubble waters against the foodborne pathogens STEC and Salmonella, and non-pathogenic E. coli surrogates, in pure and organically-loaded solution scenarios on a benchtop level. The second stage of this research project consisted of conducting a month-long in-plant validation of a recirculating chlorinated nanobubble solution within a beef trim processing plant—an enormous task that could not have been completed without expert help (specifically Drs. John Luchansky and Anna Porto-Fett and graduate student Nick Sevart for sacrificing a month of their lives to help me) and extensive planning. One huge benefit of the STEC CAP grant is the ability to work with and bounce ideas off of seasoned researchers from other institutions—my own research could not have been successful without many pairs of eyes and thoughtful input. Learning to collaborate with industry members,

academic researchers, and accounting for regulatory requirements was difficult, but rewarding once finished with the project. Before working with STEC CAP, I felt my understanding of the beef industry to be fairly extensive due to my previous internship experiences at Washington Beef, LLC in Toppenish, WA and my personal background of raising livestock on a family farm; but, I had no idea the level of scientific depth the industry utilized to mitigate foodborne pathogens and evaluate antimicrobial interventions. Working with Dr. Phebus and my lab mates on several of the STEC CAP’s Objective 3 projects really broadened my depth of understanding and deepened my appreciation for academic research. I can honestly say that the experiences I gained working with STEC CAP associated projects, specifically my own thesis project, developed my professional and personal skills tremendously. The grant provided real-life multidisciplinary collaborative research opportunities, something that has helped me transition smoothly into a career as a scientist in industry. My ability to now confidently conduct research and critically evaluate and solve problems will forever benefit my future endeavors, a feeling I hope other STEC CAP students will experience in their time working with grant-assisted projects. In December 2016, I walked away from my STEC CAP supported project with a Master’s of Science in Food Science from Kansas State University and a job as an R&D Concept Developer with Boar’s Head Brand in Holland, MI.


STEC CAP News

STEC CAP News

...Directors Update ...continued from page 1 STEC CAP NATION … YOU HAVE PERFORMED WELL … thus far! A significant amount of work remains to be completed by every person affiliated with the STEC CAP grant, as we get closer to the end. We will characterize this work as 1) finishing/expanding current projects, of which there are many; 2) starting and completing several new projects; 3) reporting and documenting past, current, and future projects and grant-related achievements in a timely manner; 4) comprehensively summarizing the accomplishments of our work towards our originally stated goals; and 5) efficiently reassessing all outstanding budgets where dollars remain unspent to ensure the best and complete use of our remaining resources. The EMT, along with our grant’s financial/business advisors at UNL, will be devoting a huge effort to make sure that all of the above happen smoothly and completely. We ask all of you to assess the status of your own STEC CAP obligations over the next couple of weeks to align your unspent budgets with your

research, education and outreach activities through this no-cost extension period ending November 30, 2017. Objective team leaders… we ask you to help us across your respective teams to accomplish this evaluation and propose ideas for best achieving remaining goals with any unspent funds. By the end of May 2017, the EMT will be making significant programmatic and budgetary decisions related to closing out the grant. We would like to hear from all co-PIs before these decisions are made. Now… an announcement that makes the EMT a little nervous to make. Through recent discussions with our Program Leader at NIFA, it may be possible that we can make the case for a 7th year nocost extension to finish off key elements of the grant’s deliverables. However, there has to be very good justification for this extension and NIFA has to approve it. The EMT is encouraging co-PIs to target completion of projects by our 6th year deadline of November 30, 2017, but we want you to almost

Mark your calendars now for the STEC CAP IAFP Breakfast!

The annual STEC CAP IAFP breakfast will be held Tuesday, July 11th at the Downtown Embassy Suites Tampa, FL. Continental breakfast at 7:00 AM with meeting to follow.

immediately inform us if you feel you have a justifiable need (and remaining funding) to extend your STEC CAP work into 2018. Dr. Moxley will put together a formal 7th year no-cost extension request with detailed justification and submit it to NIFA shortly after our summer STEC CAP annual conference (Dr. Walls has asked for it to be submitted in August 2017). As you evaluate your needs in this regards, please understand that if we are awarded the 7th year nocost extension, UNL will require all subcontractors to have completed their projects and any expenditures (including salaries/student stipends) by August ?? (JILL), 2018 (no extensions or exceptions). The most obvious indication of our STEC CAP efforts is our output of refereed journal manuscripts. In case you haven’t noticed, we have a beautiful and valuable website that is one of our best informational avenues to the outside world. Under a tab called Researchers in Action (http://stecbeefsafety. org/researchers-action), you will see a comprehensive listing of published and in-press manuscripts from our grant activities, with links to most of these articles. Currently, we sit at 80 peer-reviewed manuscripts. A significant number of projects have been completed and manuscripts are in various stages of development. The EMT and our NIFA program leader are sending out this request, or we will call it a challenge…LET’S HAVE A MINIMUM OF 100 MANUSCRIPTS SUBMITTED TO JOURNALS BEFORE OUR JUNE 13-15, 2017 ANNUAL CONFERENCE! Please prioritize getting these manuscripts submitted so that we can show our great productivity as we request the 7th year no-cost

EARLY REGISTRATION PRICING UNTIL APRIL 30TH Students can register for FREE See complete agenda online

http://stecbeefsafety.org/2017-steccap-annual-conference extension paperwork. Don’t forget to fill out and submit the short Manuscript Documentation Form (http://stecbeefsafety.org/points-board) to the EMT immediately before you send these manuscripts to journals. While you’re at this area of our website, click on the Points-on-the-Board link and record all of the great things you’re group has done so we can get these into our agency reporting. The STEC CAP grant continues to receive frequent and positive media coverage, and our team has been busy on many fronts spreading the word of our contributions towards beef/food safety. Dr. Moxley visited K-State last month and presented an outstanding keynote seminar on virulence characteristics of STEC to our College of Veterinary Medicine. I gave a keynote address at Oklahoma State University in February that focused on validation of interventions (http://fapc.biz/workshops/2017research-symposium/researchsymposium) to control pathogens in foods (skip to the 47 min mark for STEC CAP piece), and was fortunate to be invited to Texas A&M also to speak to food/meat science students and faculty regarding our STEC CAP accomplishments. Just this week, the faculty retirees social club from the K-State Colleges of Ag and Vet Med invited me to speak to them about our STEC CAP’s educational and outreach efforts. What an energetic group they were! Please, let the EMT know when you make presentations or have significant outreach communications (and report it in the POBs) so we can thank you and promote your effort. You may want to check out the additional national coverage we’ve received through these links: http://www.stopfoodborneillness.org/candcrodney-moxley-december2016/

http://www.hpj.com/ag_news/dodge-city-sharesin-usda-grant/article_562cf5c0-a29b-11e6-8a9193b6545b0706.html http://www.leavenworthtimes.com/news/20161025/ schools-to-benefit-from-grant http://www.provisioneronline.com/articles/104052rapid-testing-research-targets-o-serogroups http://news.unl.edu/newsrooms/today/article/e-coliproject-generates-new-detection-control-methods/ It’s exciting to think about our upcoming STEC CAP annual conference that will occur June 13-15, 2017 in Lincoln, NE, again at the Embassy Suites. This is our fifth one! This time the conference will be open to the public with a proper registration. We will have some very high-profile speakers from both within and outside of the STEC CAP family. Encourage your students and colleagues to log in and register soon (http://stecbeefsafety.org/2017-stec-capannual-conference). On behalf of my EMT comrades, thank you for your continued professionalism, great science, and passion for addressing a significant challenge to our food system… Shiga toxigenic E. coli in beef products. Please re-energize yourselves to help us sprint towards the finish line with this landmark grant, and let’s look for ways that our 6-7 year group effort can synergize to go after future support opportunities. The EMT is at your service, so feel free at anytime to contact us with ideas, wishes, concerns, and usable information. OK…I’m now back to enjoying my Spring Break (not really, we have an EMT teleconference in 15 min. Ughhhh! by Randy Phebus (phebus@ksu.edu)


STEC CAP News

STEC CAP News industry. Local employers understand what type of employees they need and Dodge City schools can help guide students to those careers. Employers and educators can serve as mentors, she said. Through mentoring experience a student may find the career path is not what he or she wanted to do, which can also be a productive experience. Kyle Longacre, an educator from Souderton, said the award and program is designed for the entire community. “It will help our children be better prepared for the world,” said Dodge City Superintendent Alan Cunningham.

Jason Ellis, an associate professor in the College of Agriculture at K-State, said the program represents an opportunity to build economic value and development from within a community. Regardless of where he travels in the Midwest, the idea of providing jobs and opportunities for youth, is a common theme in which community leaders acknowledge lost opportunities when young people leave. Development and redevelopment are important and growing future employees and businesses from within a community is a successful and sustaining formula. Business leaders commended the school district for pursing the grant and for looking for ways to partner with them.

Dodge City Receives STEC Grant

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odge City High School hosted representatives from Pennsylvania’s Souderton High School, Kansas State University, as well as the University of Nebraska-Lincoln on Oct. 27 and 28 for the purpose of exploring ways to more effectively educate and prepare students for future careers. The U.S. Department of Agriculture’s National Institute of Food and Agriculture funded the STEC Coordinated Agriculture Project goal is to reduce the public health risk and number of cases of Shiga toxin-producing Escherichia-coli infections in the population. As part of the event on Oct. 28, Dodge City High School was awarded a $100,000 from USDA. Dodge City was one of 18 institutions sharing in a $2.5 million grant program. “The STEC Cap partnered with Pathway 360 Degrees to increase awareness about the potential dangers of Shiga toxin-producing Escherichia-coli in beef while giving students a powerful venue to expose them to career opportunities and new possibilities,” according to a news release from Dodge City schools. Finding ways to develop careers in food science and related fields can occur with proper preparation that can start at the secondary level before a student even reaches college, representatives said. The Pathway 360 program was developed by Souderton High School.

“Through the program, students critically think about potential career choices, which allows them to make better decisions about their professional paths,” stated Rod Moxley, STEC Cap grant director. “Pathway 360 can help educate students on not just food safety, but all types of agricultural careers that are continually increasing, offering students real world experience,” according to Jill Hochstein, STEC CAP project manager. “We are hopeful that the program will continue to grow and expand to more areas across the country. Souderton’s Pathway 360 program is an inspiration to the growing career needs of our young people and the STEC CAP Grant is proud to be a partner in this initiative.” Dodge City High School is working in conjunction with the STEC Cap Project. “We’re currently in the research and planning phase to determine how we might implement the key components of the Pathway 360 program to enhance the educational experiences of our students,” Dodge City High School Principal Jacque Feist said. “This fits nicely with the individual plans of study all our students are currently building as part of the New Vision for Education in Kansas.” During a public presentation, members of the business community, school board members and local government officials, as well as educators from the schools and local community college. Feist said Dodge City was a natural place for such a project because agriculture and livestock production is the region’s top

Check us out on the Web! Visit us at: www.stecbeefsafety.org Subscribe to the listserv. Send an email to: listserv@unl.edu In the message field: subscribe stecbeefsafety This project was supported by Agriculture and Food Research Initiative Grant No. 2012-68003-30155 from the USDA National Institute of Food and Agriculture, Prevention, Detection and Control of Shiga Toxin Producing Escherichia coli (STEC) from Pre-Harvest Through Consumption of Beef Products Program –A4101.

STEC CAP Spring 2017 Newsletter  
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