2012 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, we investigated Salmonella mitigation in ground beef through Lymph node removal. This work builds on previous work from our Unit that determined Salmonella-harboring lymph nodes could circumvent conventional antimicrobial interventions that are applied to the carcass surface. We are testing the hypothesis that if Salmonella-containing lymph nodes are removed by beef processors, the resulting ground beef will have reduced levels and prevalence of Salmonella. Under the same objective, we evaluated the utility of UV light in removing carcass surface contamination. Ultraviolet light (UV) creates non-ionizing radiation and has been used extensively in pharmaceutical and medical device companies to control microbial contamination. Lately, UV treatment has received attention from the beef processing industry because it is a non-thermal processing technology that does not leave any chemical residues on products. We evaluated the ability of UV and a UV-ozone combination to inactivate Shiga toxin-producing E. coli (STEC), Salmonella, and L. monocytogenes on surfaces of fresh beef. We also evaluated the effects of UV and a UV-ozone combination on the meat quality. The preliminary findings indicated that use of UV or UV-ozone has the potential as an intervention to reduce foodborne pathogens on surfaces of fresh beef and had no effect on meat quality.
Under Objective 2, experiments were conducted to improve the detection of STEC. Recently, Food Safety Inspection Service (FSIS) declared six serogroups of non-O157 STEC to be adulterants in raw beef. This decision had been delayed due to a lack of validated detection methodologies. Experiments were performed to determine the efficacy of available and pre-market non-O157 STEC detection assays to meet the needs of beef processors and others who have begun testing for these Top-6 STEC. In another project under this objective, FSIS and ARS are collaborating on determining the sources of positive screening test results that are not confirmed by culture analysis according to FSIS methods. We processed enrichments from FSIS to isolate and characterize the organisms responsible for the false positive screening tests.
Experiments under Objective 3 have been focused on identifying host factors that affect O157:H7 colonization and fecal shedding levels by detecting and characterizing the bovine immune response to super-shedding of E. coli O157:H7. Serum, fecal, and nasal swab samples were collected from feedlot cattle. The animals were identified as super-shedders, normal shedders, and non-shedders based on the enumeration and prevalence results of O157:H7 in fecal samples. Total IgG and IgA were measured as indicators of general immune response from animals shedding E. coli O157:H7 at different levels. In addition, whole cell lysates of E. coli O157:H7 were prepared to test the serum, fecal, and nasal swab samples in order to compare specific serological and mucosal immune responses to O157:H7 antigens between the super-shedders and other control animals over the time course of before, during, and after O157:H7 shedding.
Airborne pathogen transport during cattle hide removal. For a long time, it has been speculated that bacteria present in airborne droplets released during hide removal were a source of beef carcass contamination but no data had been collected to confirm or refute this idea. ARS scientists in Clay Center, Nebraska, demonstrated the contribution of liquid splatter during hide removal as a means of transferring bacterial pathogens to carcasses. Recognition of the contamination risk posed by pathogen-containing airborne droplets should lead to the implementation of strategies to protect adjacent carcasses.
Use of hypobromous acid as an antimicrobial intervention. Beef processing companies continually seek more effective and/or less expensive antimicrobial interventions to prevent carcass contamination by bacterial pathogens. ARS scientists in Clay Center, Nebraska, demonstrated that application of hypobromous acid to cattle hides significantly reduced the prevalence of E. coli O157:H7 and S. enterica on hides. These results demonstrate that adoption of a hypobromous acid hide wash by beef processors would be effective in reducing carcass contamination.
Novel growth medium for detection of E. coli pathogens based on color. Some strains of E. coli produce Shiga toxins(STEC)and are clinically significant foodborne pathogens. The Centers for Disease Control (CDC) reported that the top six non-0157 STEC strains accounted for 71% of the human cases in the U.S. Unlike the most notorious E. coli strain (O157:H7), however, these foodborne pathogens have no unique characteristics to readily distinguish them from other E. coli. ARS scientists in Clay Center, Nebraska, developed a novel agar medium that indicates different bacterial types by producing different colors. The medium allowed successful detection and isolation of the naturally occurring top six non-O157 STEC present in bovine feces. This is the first bacterial agar medium validated for isolation of all of the top-6 non-O157 STEC and should significantly improve their detection.
Schmidt, J.W., Harhay, D.M., Kalchayanand, N., Bosilevac, J.M., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M. 2012. Prevalence, enumeration, serotypes, and antimicrobial resistance phenotypes of Salmonella enterica isolates from carcasses at two large United States pork processing plants. Applied and Environmental Microbiology. 78(8):2716-2726.
Schmidt, J.W., Wang, R., Kalchayanand, N., Wheeler, T.L., Koohmaraie, M. 2012. Efficacy of hypobromous acid as a hide-on carcass antimicrobial intervention. Journal of Food Protection. 75 (5): 955-958.
Harhay, D.M., Arthur, T.M., Bosilevac, J.M., Kalchayanand, N., Schmidt, J.W., Wang, R., Shackelford, S.D., Loneragan, G.H., Wheeler, T.L. 2012. Microbiological analysis of bovine lymph nodes for the detection of Salmonella enterica. Journal of Food Protection. 75(5):854-858.