2013 Annual Report
1a.Objectives (from AD-416):
Objective 1: Develop and validate intervention strategies that reduce or eliminate foodborne pathogens at the animal and processing levels.
Objective 2: Determine and validate detection methods for foodborne pathogen colonization and contamination at various stages in the production of red meat.
Objective 3: Examine host pathogen interactions with an emphasis on host-specific determinants of pathogen colonization.
1b.Approach (from AD-416):
The research to be conducted in this project will focus on Shiga-toxin producing E. coli (STEC) and Salmonella at multiple stages of the beef production continuum and contains both basic and applied aspects. The research objectives have been divided into classifications of antimicrobial intervention, detection methodology, and host-pathogen interaction. Antimicrobial interventions to be investigated include applications for the live animal and for carcasses during harvest. Feed supplements will be studied as a means of reducing E. coli O157:H7 in feedlot cattle. Feed supplements are more easily administered than other potential preharvest interventions, such as vaccines, and may provide cross protection against a variety of pathogens. As the hide has been shown to be the source of carcass contamination at processing, any reduction in hide pathogen load should result in lower carcass contamination rates. Thermal dehairing will be investigated as a means to sanitize the cattle hide prior to hide removal. Work will be done to evaluate application of bacteriophage to the hide of the live animal just prior to entrance into the processing plant as an additional step to reduce carriage of E. coli O157:H7 on the animal’s hide.
Basic research to model the colonization of E. coli O157:H7 at the bovine recto-anal junction will allow for in vitro assay development to identify direct methods of colonization disruption and mitigation to reduce or eliminate the pathogen from the gastrointestinal tract of cattle. Non-O157 STEC are becoming an increasing burden on beef production with potential regulatory policy based on these organisms. The project described herein will endeavor to develop and validate methodologies for the detection of non-O157 STEC that will provide the beef industry with more sensitive and specific tools to combat these pathogens.
While STEC contamination of beef carcasses occurs predominantly through transfer of the pathogens from the hide of the animal to the carcass as the hide is removed, Salmonella contamination has been shown to reside within the tissues of the animal. Previous work has shown that Salmonella can be isolated from lymph nodes located within meat cuts destined for human consumption. Experiments have been designed to study the dissemination of Salmonella in bovine lymph nodes throughout the animal during an active infection and after clinical symptoms have subsided.
Progress was made on all three objectives. Under Objective 1, intervention strategies aimed at reducing contamination of veal calf hides and beef cheek meat were evaluated. Pre- and post-intervention veal hide samples were collected from three veal processing plants to investigate the levels and prevalence of Salmonella and various Enterohemorrhagic Escherichia coli (EHECs), including E. coli O157:H7 and the non-O157 EHECs (O26, O45, O103, O111 O121 and O145). These studies will identify the most useful hide interventions to reduce contamination during veal production. We also evaluated the immersion of beef cheek meat in seven antimicrobial solutions as an intervention against EHEC and Salmonella. Our results indicated that immersion in 5% lactic acid for 1 minute or 80 degree C water for 10 seconds were the most effective interventions to apply in order to reduce EHEC and Salmonella.
Under Objective 2, FSIS and ARS are continuing their collaboration on determining the sources of positive EHEC screening tests of beef trim and ground beef constituents. We have processed FSIS broths and isolated strains of E. coli that are not adulterants and which are responsible for many of the false positive screening tests. We are characterizing these isolates in order to identify methods that will keep them from interfering with the EHEC screening assays.
Experiments under Objective 3 have continued examining bovine immune responses before, during, and after E. coli O157:H7 super shedding and found an ARS collaborator to work with characterizing E. coli O157:H7 in in vitro binding assays. Serum, fecal, and nasal swab samples were collected from USMARC feedlot cattle identified as super shedders, normal shedders, and non-shedders of E .coli O157:H7 in their feces. Bacterial adherence to cells of bovine origin by strains of O157 and non-O157 EHEC from the USMARC strain collection was characterized in collaboration with ARS scientists in Ames, Iowa. Our collaboration in this area is focused on the binding and colonization in the bovine intestinal tract by STEC using a novel adherence assay they developed that uses cattle rectal-anal squamous epithelial cells.
Electrolyzed oxidizing (EO) water shown to reduce levels of various Shiga toxin-producing Escherichia coli. EO water has emerged as a potential intervention in the beef processing industry but its efficacy against E. coli O157:H7 and other non-O157 EHEC has not been clearly established. ARS scientists at Clay Center, Nebraska working in collaboration with scientists at the University of Georgia measured the resistance of Escherichia coli O157:H7, the major serotypes of non-O157 EHEC, and the German outbreak E. coli O104:H4 to EO water. The results demonstrated that EO water treatment can reduce E. coli O157:H7 and other non-O157 STEC and therefore holds promise as a novel antimicrobial intervention technology.
Molecular tests for multiple virulence factors aid in predicting the presence of non-O157 Shiga toxin-producing Escherichia coli in ground beef. The USDA Food Safety and Inspection Service announced it would consider six non-O157 Shiga toxin- producing Escherichia coli (STEC) O26, O45, O103, O111, O121 and O145 adulterants in certain beef products. Most of the current methods for the detection of these organisms are based on molecular detection of Shiga toxin, intimin and O group specific genes in an enriched beef sample. ARS scientists at Clay Center, Nebraska applied this detection scheme to a set of 3,972 samples containing 6 known positives and showed that 183 were identified as potentially positive, and that 4 of the known positives were not identified. The results reveal the weaknesses of the current testing approaches and demonstrate the need for more specific molecular targets for STEC detection.
Characterization of E. coli 0157:H7 strains isolated from super shedding cattle. A small proportion of cattle shedding high levels (super shedding) of E coli O157:H7 are the main source for the transmission of this pathogen. ARS scientists at Clay Center, Nebraska found that no particular E. coli O157:H7 strain is responsible for super-shedding. Characterization of these supershedder strains identified multiple strain-specific trait combinations that were previously associated with strains causing human illness. These results indicate that strain-specific targeting will not reduce supershedding and that other avenues need to be explored to restrict the E. coli O157:H7 bovine-colonization cycle.
Physiologic state of E. coli O157:H7 effects detection in ground beef when using molecular-based methods. There is disagreement on the use of real-time molecular-based tests to predict pre-enrichment concentrations of E. coli O157:H7, as well as disagreement pertaining to the influence of nonviable cells causing false positive results. ARS scientists at Clay Center, Nebraska investigated the effects of bacterial physiologic states (injured or healthy) and the presence of nonviable cells on the ability of real-time molecular tests to accurately measure contamination levels of E. coli O157:H7 in ground beef. This study provides scientific advice regarding how to use these assays properly and accurately for food safety control in the meat processing industry.
Wang, R., Schmidt, J.W., Arthur, T.M., Bosilevac, J.M. 2013. The physiologic state of Escherichia coli O157:H7 does not affect its detection in two commercial real-time PCR-based tests. Food Microbiology. 33: 205-212.
Jadeja, R., Hung, Y., Bosilevac, J.M. 2013. Resistance of various shiga toxin-producing Escherichia coli to electrolyzed oxidizing water. Food Control. 30(2):580-584.
Kalchayanand, N., Arthur, T.M., Bosilevac, J.M., Wells, J., Wheeler, T.L. 2013. Chromogenic agar medium for detection and isolation of Escherichia coli serogroups O26,O45,O103,O111,O121, and O145 from fresh beef and cattle feces. Journal of Food Protection. 76(2):192-199.
Bosilevac, J.M., Koohmaraie, M. 2012. Predicting the presence of non-O157 Shiga toxin-producing Escherichia coli in ground beef by using molecular tests for Shiga toxins, intimin, and O serogroups. Applied and Environmental Microbiology. 78(19):7152-7155.
Schmidt, J.W., Arthur, T.M., Bosilevac, J.M., Kalchayanand, N., Wheeler, T.L. 2012. Detection of Escherichia coli O157:H7 and Salmonella enterica in air and droplets at three United States commercial beef processing plants. Journal of Food Protection. 75(12):2213-2218.
Kalchayanand, N., Arthur, T.M., Bosilevac, J.M., Harhay, D.M., Shackelford, S.D., Wells, J., Wheeler, T.L., Koohmaraie, M. 2013. Isolation and characterization of Clostridium difficile associated with beef cattle and commercially produced ground beef. Journal of Food Protection. 76(2):256-264.
Arthur, T.M., Ahmed, R., Chase-Topping, M., Kalchayanand, N., Schmidt, J.W., Bono, J.L. 2013. Characterization of Escherichia coli 0157:H7 strains isolated from supershedding cattle. Applied and Environmental Microbiology. 79(14):4294-4303.