Progress 07/01/11 to 12/31/13
Outputs
Target Audience: Small beef producers in eastern Arkansas. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Provided graduates and undergraduates training in microbiological assays. Allowed faculty members to atttend professional meeting to present their research. How have the results been disseminated to communities of interest? Goal 1: All particapting producers were provided a report about the prevalence of STEC on their farm as compared to other producers. Three beef producer meetings were held in three different counties during December 2013 to disseminate the results of this project. Donald Kennedy presented a ppt presentation and discussed why STEC is a food safety concern in th US. Furthermore, Kennedy discussed management strategies with the goal of reducing STEC on beef farms. A total of 75 people attended the 3 meetings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Goals 1, 2 and 3. Out of 681 samples tested (534 rectal swabs, 39 feed samples, 54 soil samples, and 54 water samples) from 20 farms, 61% of the tested samples (417 samples) were STEC-positive and 25% and 45% of the samples were positive for O157:H7 and non-O157 STEC, respectively. When three detection methods were compared for their sensitivity and specificity, culture method, PCR, and IFA showed 68%, 99%, and 92% of sensitivity, and 77%, 91% and 90% of specificity, respectively. It was notable that culture method showed higher false-positive rates (23%) than other three methods tested. Rainbow agar-based culture method showed lower sensitivity and specificity of 64% and 72% for non-O157 STEC (compared to 71% and 80% of sensitivity and specificity for O157:H7), which suggests Rainbow agar might not be an appropriate method to identify STEC in environmental samples. Even though IFA shows high sensitivity and specificity, its high sensitivity and specificity were achieved only when antibodies to shiga toxin were used with antibodies to O157:H7 and big 6 STEC together. The results from this study suggest PCR-based assay has the great potential to be used as a routine STEC monitoring system due to its high sensitivity and specificity. Additionally, PCR had advantages over other methods that it can identify serotypes of STEC and/or virulence factors depending on the primers employed for PCR reactions. In this study, the samples were tested for the presence of shiga toxin genes (stx1 and stx 2) and O157:H7 (rfbE), and if they are shiga toxin-positive but not O157:H7, they were further tested with big 6 STEC primers to identify their serotypes. And approximately 25% of the STEC positive samples were from unidentified serogroups (neither O157:H7 nor big 6 STEC groups). Results Dissemination: 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 The objective of this study was to determine the prevalence of E. coli STEC on small (30- 40 head) cattle farms in the Arkansas Delta. Results indicated that the prevalence E. coli O157 and non-O157 STEC was highly variable among the farms studed. The proportion of positives for O157 STEC in fecal samples was highly variable and ranged from 0 to 75%. Farm 8 (Greene County) and Farm 7 (St. Francis County) were significantly higher in the proportion of positive samples for O157. Three farms (6, 9, and 12) had fewer than 10% positive samples. Positive samples ranged from 6.7 to 100%, and 100% of the farms tested positive for non-0157 STEC. Farm 13 (Poinsett County) was significantly lower in the proportion of positive samples for non-O157 STEC. Over half of the farms had zero positive environmental samples for O157 STEC and values ranged from 0 to 50%. Among the 18 farms, significant differences were observed for the proportion of environmental samples that tested positive for non- O157 STEC. Proportion of positive samples ranged from 0 to 100% and 83% of the farms tested positive for non-O157. In this study, the prevalence rates of E. coli STEC on small cattle farms in the Arkansas Delta were relatively high and widespread. Further work is needed to determine factors that may be associated with high prevalence rates for both O157 and non-O157 STEC. 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? Goal 4. Three producer meeting were held in three counties during December 2013. Producers were presented the results of the project. The food safety concern STEC was emphasized and produers were made aware how they might manage theiri cattle with the goal of reducing STEC on their farms. A total of 95 people attended 1 of 3 meetings.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Ahn, S., M. Yarbrough, H. Kaur, S-E. Choi, D. Gilmore, and D. Kennedy. 2013. Prevalence of shiga toxin-producing Escherichia coli (STEC) in beef cattle and cattle farms in Arkansas Delta region. International Association for Food Protection (IAFP) Annual Meeting. Book of Abstract, #4750.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Ahn, S., T. Austin, S. Wu, S-E, Choi, D. Gilmore and D. Kennedy. 2013. Comparison of four different methods for detection of shiga toxin-producing Escherichia coli (STEC) in environmental samples. International Association for Food Protection (IAFP) Annual Meeting. Book of Abstract, #4803.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
David Gilmore, Harneet Kaur, Monica Yarbrough, and Donald Kennedy. 2013. Prevalence of shiga-toxin producing Escherichia coli on cattle farms in Northeast Arkansas. South central branch of the American society for microbiology annual meeting. Oct 31-Nov 1. New Orleans, LA.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2013
Citation:
Kaur, Harneet. 2013. Detection of Shiga-toxin producing E. Coli Prevalence of Northeast Arkansas using cultural methods. Thesis. Arkansas State University.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2013
Citation:
Yarbrough, Monica. 2013. Prevalence of Shiga-toxin Producing E. coli on Cattle Farms Located in the Arkansas Delta Region. Thesis. Arkansas State University.
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Progress 07/01/12 to 06/30/13
Outputs
Target Audience: Small beef cattle (30- 40 cows in herd) producers in the Mississipi Delta in Arkansas and cooperative extension agents. 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. 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.
Impacts
What was accomplished under these goals?
Goal 2 and 3. Out of 681 samples tested (534 rectal swabs, 39 feed samples, 54 soil samples, and 54 water samples) from 20 farms, 61% of the tested samples (417 samples) were STEC-positive and 25% and 45% of the samples were positive for O157:H7 and non-O157 STEC, respectively. When three detection methods were compared for their sensitivity and specificity, culture method, PCR, and IFA showed 68%, 99%, and 92% of sensitivity, and 77%, 91% and 90% of specificity, respectively. It was notable that culture method showed higher false-positive rates (23%) than other three methods tested. Rainbow agar-based culture method showed lower sensitivity and specificity of 64% and 72% for non-O157 STEC (compared to 71% and 80% of sensitivity and specificity for O157:H7), which suggests Rainbow agar might not be an appropriate method to identify STEC in environmental samples. Even though IFA shows high sensitivity and specificity, its high sensitivity and specificity were achieved only when antibodies to shiga toxin were used with antibodies to O157:H7 and big 6 STEC together. The results from this study suggest PCR-based assay has the great potential to be used as a routine STEC monitoring system due to its high sensitivity and specificity. Additionally, PCR had advantages over other methods that it can identify serotypes of STEC and/or virulence factors depending on the primers employed for PCR reactions. In this study, the samples were tested for the presence of shiga toxin genes (stx1 and stx 2) and O157:H7 (rfbE), and if they are shiga toxin-positive but not O157:H7, they were further tested with big 6 STEC primers to identify their serotypes. And approximately 25% of the STEC positive samples were from unidentified serogroups (neither O157:H7 nor big 6 STEC groups). Results Dissemination: 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 Goal 3. The objective of this study was to determine the prevalence of E. coli STEC on small (30- 40 head) cattle farms in the Arkansas Delta. Results indicated that the prevalence E. coli O157 and non-O157 STEC was highly variable among the farms studed. The proportion of positives for O157 STEC in fecal samples was highly variable and ranged from 0 to 75%. Farm 8 (Greene County) and Farm 7 (St. Francis County) were significantly higher in the proportion of positive samples for O157. Three farms (6, 9, and 12) had fewer than 10% positive samples. Positive samples ranged from 6.7 to 100%, and 100% of the farms tested positive for non-0157 STEC. Farm 13 (Poinsett County) was significantly lower in the proportion of positive samples for non-O157 STEC. Over half of the farms had zero positive environmental samples for O157 STEC and values ranged from 0 to 50%. Among the 18 farms, significant differences were observed for the proportion of environmental samples that tested positive for non-O157 STEC. Proportion of positive samples ranged from 0 to 100% and 83% of the farms tested positive for non-O157. In this study, the prevalence rates of E. coli STEC on small cattle farms in the Arkansas Delta were relatively high and widespread. Further work is needed to determine factors that may be associated with high prevalence rates for both O157 and non-O157 STEC.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2013
Citation:
Yarbrough, Moncia. 2013. Prevalence of Shiga-toxin Producing E. coli on Cattle Farms Located in the Arkansas Delta Region. Thesis. Arkansas State University.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2012
Citation:
Kuar, Harneet. 2012. Identification of Shiga toxin-producing Escherichia coli in Beef Cattle of Northeast-Arkansas using Cultural Methods. Thesis. Arkansas State University.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
S Ahn, M Yarbrough, H Kaur, S-E Choi, D Gilmore and D Kennedy, "Prevalence of Shiga toxin-producing Escherichia coli (STEC) in beef cattle and cattle farms in Arkansas Delta region", International Association for Food Protection Annual Meeting, Jul 29, 2013, Charlotte, NC.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
S Ahn, T Austin, S Wu, D Gilmore and D Kennedy, "Comparison of Four Different Methods for Detection of Shiga Toxin-producing Escherichia coli (STEC) in Environmental Samples", International Association for Food Protection Annual Meeting, Jul 30, 2013, Charlotte, NC.
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Progress 07/01/11 to 06/30/12
Outputs
OUTPUTS: 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. 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 TARGET AUDIENCES: Nothing significant to report during this reporting period. 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.
Impacts
As result of our first sample analysis, we determined that modifications in detection of microorganisms were needed. Below is a description of the modified approach. Summary 1. Four types of samples will be collected using sterile culturette swabs from different farms: rectal swabs, feed, soil and water samples. Also, the samples will be collected in triplicate. These will be stored in an ice box while at the field. Each culturette contains two swabs; one will be used for classical identification and the other sent for molecular identification. For rectal swabs: Rectal swabs will be transferred to 5 ml sterile mTSB (containing novobiocin) tubes and incubated for 42C for 18-20 h. For feed and soil samples: Portions (5 g) of feed and soil samples will be weighed on a balance using weighing boats and spatula. These 5 g samples will be transferred to sterile stomacher bags containing 45 ml of BPW and homogenized for 1 min. Aliquots (1 ml) of each soil and feed samples will be transferred to 5 ml sterile mTSB tubes and incubated for 42C for 18-20 h. For water samples: Aliquot (1 ml) of each water sample will be transferred to the 5 ml sterile mTSB tubes and incubated for 42C for 18-20 h. 2. After incubation, the swabs will be agitated using vortex mixer to remove the adherent bacteria and then will be disposed of. The remaining samples in the tubes will be used for selective plating. 3. A loopful of culture from each mTSB tubes (3 samples per cow) will be streaked on a CT-SMAC and a CT- Rainbow O157 agar plate. Plates will be incubated at 35C for 18-20h. 4. Each plate will be scored for the presence of non-STEC E. coli, E. coli O157, and non-O157 STEC by colony appearance. Non-O157 STEC colonies will be confirmed as E. coli by routine biochemical tests. O157 colonies will be confirmed by Latex Agglutination assays for the O157 antigen. 5. Cultures of interest that cannot be immediately tested will be maintained on Tryptic Soy Agar slants until tests can be completed.
Publications
- No publications reported this period
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