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Study on Prevalence and Characterization of Shiga Toxin-producing E. Coli (stec) from Cattle Farms in the Arkansas Delta Region


<p>This study proposes to determine the prevalence of Shiga-toxin producing E. coli (STEC) in cattle farms located in the Arkansas Delta region, and to develop a sensitive detection assay for STEC in farm environments. The proposed study offers a novel approach in assessing STEC prevalence</p>
<p>(1) by focusing on only small-sized farms (30 to 40 mature cows on average) in the Arkansas Delta region, primarily managed by family members, and</p>
<p>(2) by focusing on mature cows, commonly used for the production of ground beef, the main food source of human STEC infection. Despite the adoption of in-plant intervention, human STEC infection is still continuously being reported. Additionally, most human infection cases are linked with cattle farms or manure-contaminated fruits and vegetables. Therefore, implementing pre-harvest intervention strategies at the farm level will have the largest impact on improving food safety. This study will lead to development of proactive measures against pre-harvest STEC contamination in small-sized farms by identifying potential risk factors associated with STEC prevalence and establishing STEC awareness and education programming for cattle farmers. </p>
<p>The long-term goals of this study are</p>
<p>(1) to develop pre-harvest risk management strategies to effectively reduce STEC colonization in beef cattle,</p>
<p>(2) to develop an on-farm STEC monitoring system employing a rapid detection assay for farm environmental samples, and</p>
<p>(3) to characterize STEC strains isolated from pre-harvest environments through further genotyping and serotyping. This long-term study will significantly reduce STEC contamination at the pre-harvest level and thus improve food safety and public health by reducing beef-associated human infections. </p>
<p>Specific objectives of this project are to:</p>
<p>1. Collect farm environmental samples (rectal swabs, soil, water and feeds) from 25 farms in Arkansas Delta region.</p>
<p>2. Compare three different STEC detection techniques for sensitivity and specificity to develop a rapid and sensitive detection method specific to farm environments.</p>
<p>3. Perform a statistical analysis on the data and analyze risk factors associated with STEC contamination in small-sized cattle farms.</p>
<p>4. Organize educational workshops to help regional cattle farmers develop control measures on identified risk factors.</p>

More information

<p>NON-TECHNICAL SUMMARY:<br/> This proposal addressed the FY2010 priority identified in Research Area A4101 -Prevention, Detection and Control of Shiga-toxin producing E. coli (STEC) from Pre-Harvest through Consumption of Beef Products, specifically focusing on identification of risk factors involved in STEC contamination in the pre-harvest environments and development of sensitive detection methods for environmental samples. STEC is a major concern for food industry because of its deleterious effects on public health and economy. Since cattle are at the top of integrated beef production, developing pre-harvest intervention strategies would provide the most cost-effective control with the biggest impact in beef safety. Here we propose a novel approach of studying STEC prevalence in beef cattle by specifically focusing on mature cows in small-sized farms in the historically
underserved Arkansas Delta region. This approach will help recognize STEC-associated risk factors in small farms, and thus help develop specific management practices for small farms in the underprivileged regions. In addition, we propose to develop a rapid and sensitive STEC detection assay for environmental samples by comparing sensitivity and specificity of new antibody-based and DNA-based methods, which have been successfully applied to complex food samples, to a standard culture method. Knowledge obtained from the proposed study will lead to development of on-farm STEC monitoring system and pre-harvest risk management strategies for small-sized farms in the Arkansas Delta region, which is a long-term goal for this study. This study will ultimately improve food safety by significantly reducing beef-associated human STEC infections.

<br/>We will collect the samples including rectal swabs, feces, soil and water, from 25 beef cow-calf farms in the Arkansas Delta region based on their location and size. All samples will be immediately packed on ice, transported to the labs at ASU, and analyzed within 3 days after arrival. Using collected samples, we will compare three different techniques (culture method, immunoassay and PCR-based method) for their sensitivity and specificity in STEC detection. STEC will be identified by culture method of selective media and enrichment. Along with culture methods, immunoassay combined with magnetic separation and tyramide signal amplification and PCR-based methods of two different approaches, duplex TaqMan real-time PCR and Bioplex bead-suspencion array, will be employed to detect and characterize STEC from the collected samples, and their results will be
analyzed for statistical validity and compared from those of microbiological tests for their sensitivity and specificity. Based on the prevalence of STEC from detection assays, associated risk factors will be analyzed. At the end of the proposed study, we will organize workshop to share research data with participating farmers and to help them develop control measures for risk factors. For the workshop organization, we will work closely with Arkansas Cattlemen Association and Center for Food Safety at University of Arkansas - Fayetteville.</p>

<br/>2012/07 TO 2013/06
<br/>Target Audience: Small beef cattle (30- 40 cows in herd) producers in the Mississipi Delta in Arkansas and cooperative extension agents.
<br/>Changes/Problems: We asked and were granted and 6-month extenstion period to complete project goals. I have included the request: Please consider the request for a 6-month, one-time, no-cost extension of time for USDA-NIFA Project, �Study of Prevalence and Characterization of Shiga-toxin producing (STEC) E. coli from Cattle Farms in the Arkansas Delta� (NIFA Award # 2011-68003-30417). I am asking for an extension for the three reasons stated below. Reason #1: The original project PD/PI, Dr. Soohyoun Ahn, left Arkansas State University (ASU) for a new position at University of Florida (UF) in December 2011. Submission of documentation and approval process for PD/PI change (from and Dr. Ahn
to Dr. Donald Kennedy) and UF subaward creation delayed the start of the project significantly. Delayed transfer of subaward fund, which became available for spending in August of 2012, and the new laboratory set-up after the relocation prevented Ahn from following the original timeline proposed in the original proposal. While Ahn has made major progress in her assigned task of comparing multiple techniques for the detection of shiga toxin-producing E. coli (STEC) (see Progress Report for details), she could not test sufficient samples due to the delayed project start and the results still need to be analyzed using statistical method (which will be performed by Choi at ASU). Reason #2: ASU had to seek approval from NIFA for a new PI, Dr. Donald Kennedy, the also caused a delay. Reason #3: Because Dr. Ahn�s expertise is in food microbiology, we also had to recruit Dr. David
Gilmore from the ASU Department of Biology to provide the expertise necessary to conduct microbiological assays at ASU. Because Dr. Gilmore was new to the project and food microbiological assays, the project was delayed further while he supplied and equipped his lab to perform the particular assays acquired for this project.
<br/>Extension Request: Award # 2011-68003-30417 Page 2 Use of extended time: 1) Extended time will be used for Ahn and her research group to test more environmental samples to compare the sensitivity and specificity of each method. Additionally, blind tests using artificially contaminated samples will be performed during the extended period. 2) Kennedy and the ASU group will have ample time to assess risk factors (Project Objective 3), and plan and conduct workshops for producers (Project Objective 4). What opportunities for training and professional development
has the project provided? Nothing Reported How have the results been disseminated to communities of interest? We have reported results to beef producers that have particpated in the study. During the 6-month extension period (July 1, 2013 - December 31, 2013) we will continue outreach to disseminat this information to cattel producers in Mississippi Delta Region of Arkansas. The results from this study were presented in International Association of Food Protection annual meeting in July 2013 (Charlotte, NC) through 2 oral presentations. Additionally, manuscripts are currently being prepared for the publication in refereed journals. What do you plan to do during the next reporting period to accomplish the goals? Goals 1 and 4. Further analyze data to determine risk factors. Conduct meeting with producer to discuss risk factors and best management practices to reduce prevalence of E. coli
STEC on beef farms.
<br/>2011/07/01 TO 2012/06/30
<br/>Activities: Methodology developed: Study design and sample collection. Samples were collected in the months of February-April from 20 different beef cattle farms in NE Arkansas (Craighead, St. Francis, Poinsett, Clay, and Green counties). Research has shown that prevalence rates of STEC are highest in summer , so a second round of samples will be collected in August. Each farmer was given information about the details of this study and familiarized with the sampling protocol. Four types of samples are collected from each farm - rectal swabs, feed, soil and water samples. Rectal swab samples were collected from 10 randomly chosen animals per farm. These were stored, and transported individually by using transport culture swabs (Amies Clear culturettes). Feed samples were also collected. Soil samples (approximately 20-80 g)
were collected from areas in which the animal lived or migrated such as at grazing pastures. Water samples were collected from ponds, creeks, or troughs at each site. Feed, soil, and water samples were collected in Whirl-pak bags and kept on ice until processed in the lab. The rectal swab samples were transferred to 5 ml mTSB for enrichment. The soil and feed samples (5 g) were combined with 45 ml sterile buffered Peptone water (BPW) in sterile, filter-lined stomacher bags and homogenized for 1 min in a stomacher. An aliquot (1 ml) of this homogenate was combined with 5 ml mTSB. Similarly, 1 ml of each water sample was combined with 5 ml mTSB. All samples in mTSB were incubated at 37C for 24 h. After incubation, samples were serially diluted in BPW to a 1:25 dilution. Aliquots (10 microliters of diluents) were spread via spreader onto CT-SMAC plates and incubated at 37C for 18-20 h.
Plates were then observed for different types of colonies. E.coli O157:H7 appears as colorless colonies, sometimes with an orange halo, on CT-SMAC plates. The screening for STEC was done on O157 Rainbow agar plates (Biolog). The plates were divided into 16 sections. Different types of four colonies were picked and patched onto a spot on the rainbow agar. The plates were incubated at 37C for 24 h then observed for different colors of the patches. E.coli O157:H7 appears black-gray on Rainbow agar. Confirmation of E.coli O157:H7. A latex agglutination test (Hardy Diagnostics) detecting the O157 antigen was used as a confirmation test for E.coli O157:H7. The colonies tested were those appearing black-gray color on the Rainbow agar; appropriate controls were also performed. Cultures: Because the initial phase of the project requires identification of E. coli strains by their appearance on
differential and selective media, numerous isolates were obtained. These included E.coli O157:H7, O111:H , O103:H11, O121:H19, and O126:H11. Project Second Half Culturettes used to collect samples consist of duplicate swabs. In the next round of sampling, one swab will be sampled as before, and the duplicate will be sent to Dr. Sooyoun Ahn for research on the identification of STEC using molecular approaches. The current protocol described above will be examined for weaknesses, and solutions for the inconsistencies in the identification data will be sought.
<br/>PARTICIPANTS: Donald Kennedy, Ph.D. (PI/PD). - primary role is coordinator of field sampling and to oversee the project. Donald is the supervisor for a graduate student, Monica Yarbrough. David Gilmore, Ph.D. (PI) - microbiologist; primary role is to oversee and conduct microbiological analysis of samples. David is the supervisor for
a graduate student, Harneet Kaur. Soohyoun Ahn, Ph.D. (PI) - food microbiologist; primary role is to compare three different STEC detection techniques for sensitivity and specificity to develop a rapid an sensitive detection method specific to farm environments. Seo-eun Choi, Ph.D. (PI) - statistician Jennifer Bouldin, Ph.D. - ecotoxicologist; consultant for soil and water sampling and analyses Monica Yarbrough - graduate student; in charge field samples and assists with microbiological assays Harneet Kaur - graduate student; in charge of microbiological assays.
<br/>TARGET AUDIENCES: Nothing significant to report during this reporting period.
<br/>PROJECT MODIFICATIONS: Dr. Sooyhoun Ahn was the original PI/PD for this project. Dr. Ahn left Arkansas State University in December 2011 to take a job at the University of Florida (UF). Because she left, Dr. Gilmore was recruited to serve as the
project's microbiologists. Because food micro is not his expertise, Dr. Gilmore had to spend a considerable amount of time working out lab protocols. Also, working out the sub award agreement with the UF and Dr. Ahn took a significant amount of time. Consequently, we were delayed approximately 2 months. Also, because of unexpected results from the first set up samples, modifications to lab protocol have been made. Therefore, our second round of sampling has been delayed until we are confident that the improved lab protocol is reliable. Because of problems, we may only be able to use the second round of samples four analyses. There is a possibility that we could collect a third round in the spring of 2013. However, this will depend on whether or not we can complete the collection and analysis of samples before the training phase of the project begins.</p>

Kennedy, Donald; Choi, Seo-eun; Bouldin, Jennifer
Arkansas State University
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