2011 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.
This report documents progress for Project 5438-42000-014-00D "Pathogen Mitigation in Livestock and Red Meat Production" which started February 2011 and continues research from Project 5438-42000-013-00D "Control of Pathogenic and Spoilage Bacteria on Red Meat." In 2011, we conducted extensive research projects focused on methodology for detecting E. coli O157:H7 in beef samples. Initial experiments were conducted to determine the ability of commercial detection systems to detect very low levels of E. coli O157:H7 and Salmonella. This research will identify the enrichment conditions that allow reliable detection of < 5 E. coli O157:H7 and Salmonella organisms per sample and are suitable for use with the following sample types relevant to industrial testing: ground beef, beef trim, beef lymph nodes, fat reduced beef, cattle fecal samples, beef hide sponge samples, and beef carcass sponge samples. We also studied the effects of changing sample size, enrichment media volume, and sample matrix on detection of low levels of E. coli O157:H7 using culture, molecular, and antibody mediated methods. Testing schemes for E. coli O157:H7 are constantly evolving with improvements made to the test protocols. Unfortunately, there is a tendency to implement changes while not recognizing potential problems that may result later. Investigation is ongoing to determine if changes that reduce the volume of enrichment media in order to concentrate the E. coli, or changes that increase the size of the sample to provide greater sensitivity, must be revalidated by users instead of relying on test manufacturer provided documentation or AOAC validation documents.
To understand a potential avenue by which Salmonella evades beef carcass decontamination steps, we have completed a variety of studies on bovine lymph nodes. Pathogens such as Salmonella have the ability to survive within bovine lymph nodes. When Salmonella are present in lymph nodes, they are protected from chemical and thermal antimicrobial interventions used in packing plants. We collaborated with industry groups and university scientists in isolating Salmonella from peripheral lymph nodes of healthy cattle presented for harvest. A necessary component of this study was validation of the microbiological analysis methods used to detect Salmonella harbored within lymph nodes. The results of these validation studies verified that the pathogens recovered originate from within lymph node tissues and are not the result of cross contamination of pathogens present on the surrounding adipose tissue.
We completed work on multiple intervention studies. Most antimicrobial interventions in use in commercial beef processing plants were validated for reducing E. coli O157:H7. We determined the efficacy of these interventions against other Shiga toxin-producing E. coli and Salmonella. In addition, we evaluated the effectiveness and parameters of UV and UV-ozone as a non-thermal intervention of red meat.
Detection of low levels of Escherichia coli O157:H7 in ground beef samples. Production of ground beef is on a scale of thousands of pounds per day while the amount of E. coli O157:H7 bacterial cells required to cause human disease has been determined to be very low, around 10 to 100 cells. In this scenario, it is imperative that the method used to detect E. coli O157:H7 in the finished product be very sensitive. ARS researchers in Clay Center, NE, determined that commercial detection methods were able to detect very low concentrations (< 5 organisms) of E. coli O157:H7 in ground beef under conditions typically used by industrial testing laboratories. They also determined that when ground beef was treated improperly, such as being held at too high of temperature, background bacteria levels could become elevated, causing commercial rapid detection methods to fail. This research demonstrated that current methods employed by the beef industry for detecting E. coli O157:H7 will detect very low levels of E. coli O157:H7 contamination, but proper handling of the product is necessary to ensure optimal test performance for ensured food safety.
Antimicrobial interventions on Shiga toxin-producing E. coli. Although the bacterium E. coli O157:H7 is currently most widely recognized, other E. coli types producing the same toxins have been implicated in cases of human diseases and are the subject of pending regulatory policy. ARS scientists in Clay Center, NE, investigated whether antimicrobial compounds currently used by the meat industry to control E. coli O157:H7 are effective against these non-O157 Shiga toxin-producing groups (O26, O103, O111, and O145). They determined that six antimicrobial compounds were equally effective against E. coli O157:H7 and non-O157 on fresh beef. These results will assist the meat industry in developing effective antimicrobial intervention programs against newly recognized pathogens.
Non-O157 Shiga toxin-producing E. coli in commercial ground beef. The bacteria called non-O157 Shiga toxin-producing E. coli are a collection of E. coli strains that produce Shiga toxins. There are over 200 types of these E. coli strains and their ability to cause human foodborne illness ranges from those that are harmless to those that can cause severe disease. Recently, these strains have become an increasing concern to the beef industry, regulatory officials, and the public. ARS researchers at Clay Center, NE, determined the prevalence and characterized non-O157 Shiga toxin-producing E. coli from over 4,000 commercial ground beef samples obtained from numerous manufacturers across the United States over a period of 24 months. Markers of the bacteria were present in approximately one quarter of ground beef samples. However, characterization of the specific bacterial strains obtained from the samples identified very few organisms that should be considered significant food safety threats. The project provided the first large scale analysis of non-O157 in ground beef, and the results have been used by the beef industry and FSIS to determine the best measures to take in regards to eliminating these pathogens from the beef supply.