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United States Department of Agriculture

Agricultural Research Service

2011 Annual Report

1a.Objectives (from AD-416)
1) Develop phylogenetic and phenotypic markers for E. coli O157:H7, non-O157 STEC/EHEC, and Salmonella spp. based on genomic and proteomic strain comparisons, expression analysis, and multi-drug resistance profiles for use in molecular strain typing, intervention method development, and design of multiple pathogen detection schemes. 2) Determine prevalence of unrecognized foodborne pathogens such as Shiga toxigenic E. coli on fresh imported beef to be used for ground beef and establish necessary profiling to insure imported beef products meet the same levels of safety as domestic products. 3) Identify sources of spoilage bacteria and pathogen contamination during beef transport/processing/slaughter (i.e., transport vehicles, lairage pens, air, hides, feces) and develop novel antimicrobial intervention strategies. 4) Determine the microbiological safety of lamb processed in the United States and determine the efficacy of currently used intervention technologies during various stages of lamb processing.

1b.Approach (from AD-416)
Strain specific markers will be identified and will be used for tracking, typing, virulence, and detection assays. Identification of the strain specific markers will lead to a more complete understanding of bovine-related foodborne pathogen ecology in which the pathogens of concern are not the same as those of the United States. The microbial profile of ground beef imported from countries will be determined in order to establish the most effective testing guidelines. Employing traditional methodologies as well as implementing new strategies developed in Objective 1, researchers in this unit will continue longstanding efforts in tracking pathogen contamination. To better understand the contribution of feedlot settings, livestock transport, and husbandry equipment to pathogen contamination, focus will be placed on identifying surface and airborne bacterial populations associated with transport vehicles, lairage pens, and slaughter facilities. Efforts will continue in evaluating pathogen carriage on hides and in feces. New efforts will be initiated to identify sources of other pathogen and spoilage bacterial contaminants including, but not limited to, non-O157 Shiga toxin-producing E. coli (STEC), Salmonella spp. and Clostridium spp. As new sources of pathogen contamination are identified, research will be undertaken to develop and evaluate novel antimicrobial strategies. Projects will be performed to determine the prevalence of foodborne pathogens (i.e., Escherichia coli O157:H7, Salmonella, and non-O157 STEC) and the level of aerobic bacteria on lamb carcasses processed in the United States. Understanding sources of carcass contamination will identify critical control points where antimicrobial intervention technologies need to be used to reduce or eliminate carcass contamination and to ensure wholesome meat. These results will be useful for the lamb industry and the USDA Food Safety and Inspection Service (FSIS).

3.Progress Report
This is the final report for the 5 year project 5438-42000-013-00D, which was terminated in 2011, and replaced with project 5438-42000-014-00D "Pathogen Mitigation in Livestock and Red Meat Production." Over the lifespan of this research project, we have collected a great deal of data regarding foodborne pathogens and their association with beef, pork, and lamb supplies in the United States. Studies were conducted in live animal settings during production and on carcasses and the finished product during processing. Through these efforts, our knowledge of pathogen ecology has grown, giving insights into new intervention development. Many of these experiments could only be realized by utilizing detection methodology also developed in this project. We have compiled evidence that cattle shedding high amounts of E. coli O157:H7 are of particular concern in a feedlot setting, as they serve as the largest source of the bacterial pathogen and lead to high rates of hide contamination among the other animals in the pen. We have determined the contamination present on the carcass during processing is more likely to originate from the processing plant holding pen environment than from the feedlot. A multitude of novel antimicrobial interventions have been evaluated for efficacy including 1, 3-Dibromo-5, 5-Dimethylhydantoin (DBDMH) or commercially called Bovibrome, electrolyzed water, Fresh FX, ozone, and UV light.

Prior to initiating much of this work, methodologies for the assessment of microbial pathogen load, at various steps in the beef production process, were lacking. We developed two methods for the direct enumeration of E. coli O157:H7 and Salmonella spp. Also, work was conducted to enhance detection of foodborne pathogens in the finished product. The beef industry has used a process called test-and-hold to sample beef trim and ground beef to test for the presence of E. coli O157:H7 and only release product that tests negative. We determined the optimal medium and growth conditions for improving the speed at which results can be obtained. Further work determined that the volume of media could be reduced by 2/3, thus, creating a significant cost savings for the end user. The cost reduction for these changes has reduced the cost from ~$10 to ~$4 per test. These results demonstrate that beef processors can use a low cost medium and reduce the amount of medium used to enrich for possible contaminating E. coli O157:H7 while maintaining a high level of accuracy in the testing of beef trim and ground beef. Experiments performed under this research project also evaluated the potential contamination risk presented by novel sources. Airborne pathogens in slaughter facilities have the potential to contaminate beef carcasses after interventions have been performed. Our work determined that airborne contamination in areas immediately adjacent to the hide puller equipment could be a source for spreading E. coli O157:H7 and Salmonella.

1. Bacteria colonization sites in cattle excreting disproportionately high levels of E. coli O157:H7. E. coli O157:H7 contamination of beef products has resulted in human disease outbreaks and large financial losses to the beef industry. This pathogen utilizes the intestinal tract of cattle as a primary habitat. Some cattle have been shown to shed E. coli O157:H7 in their feces at disproportionately high levels. It is thought that if the excretion of disproportionately high levels of E. coli O157:H7 by cattle could be prevented, the transmission of E. coli O157:H7 could be controlled. ARS scientists in Clay Center, NE, identified cattle shedding disproportionately high levels of E. coli O157:H7 and determined that the distal colon was the intestinal tract location most frequently found to harbor E. coli O157:H7. In addition, it was shown that at times of peak shedding E. coli O157:H7 could be isolated from sites encompassing the entire bovine gastrointestinal tract from mouth to rectum. At no time was E. coli O157:H7 ever detected from samples of lymph nodes or gall bladders. This study emphasized the contamination potential of cattle that shed disproportionately high levels of E. coli O157:H7 in beef processing. A better understanding of E. coli O157:H7 shedding will aid in reducing the spread of this pathogen among cattle and throughout the beef production chain and thereby prevent economic loss to farmers, ranchers, and United States and foreign consumers of American beef.

2. Method revalidation for test-and-hold programs. Method revalidation is required when changes are made in sample size and/or enrichment media (volume or type). Testing schemes are constantly evolving with improvements to the test protocols. There is a tendency across the beef processing industry to implement changes while not recognizing potential problems that may result later. ARS scientists at Clay Center, NE, demonstrated that changes that reduced the volume of enrichment media in order to concentrate the E. coli, or changes that increased the size of the sample to provide greater sensitivity, must be revalidated by all users in their own systems. In addition, the scientists noted that rapid detection methods can be used on numerous sample types, except those that might alter the pH of the enrichment media during incubation. These results identified situations when a revalidation is needed and when it is not. The results can also be used by in-house laboratories of beef processors, ground beef manufacturers, and others, such as third party laboratories, as supporting documents in their testing programs.

3. Salmonella prevalence is low in commercial ground beef. Salmonella is a major foodborne pathogen in the United States. Data has been lacking regarding the presence of Salmonella in ground beef, especially for those Salmonella that are resistant to multiple antibiotics. ARS scientists in Clay Center, NE, analyzed several thousand ground beef samples from commercial establishments and determined that the prevalence and level of Salmonella were very low (4.2%). Only 0.6% of samples harbored Salmonella that were resistant to antibiotics. From these results, it can be concluded that Salmonella and specifically antibiotic resistant Salmonella present a very low risk to the safety of the United States beef supply.

4. Prevalence and characterization of multidrug resistant (MDR) Salmonella Newport. Each year in the United States an estimated 1.4 million people contract foodborne illnesses due to Salmonella. Most cases of salmonellosis are not serious; however, some infections result in invasive salmonellosis, a more severe form of illness requiring antibiotic therapy. The emergence of multidrug resistant Salmonella is concerning. Salmonella Newport is frequently a cause of salmonellosis in both humans and cattle, and also frequently MDR. Cattle have been identified as a potential reservoir for Salmonella and this association presents a food safety risk for Salmonella contamination of ground beef. ARS scientists (Clay Center, NE) examined the prevalence of multidrug resistant Newport associated with cattle at slaughter in the United States. Regional differences in prevalence were observed across the United States. Drug resistance Newport isolates were resistance to ceftriaxone, a key antibiotic for treatment of human illness cases of salmonellosis. The research provided key information for hazard analysis and critical control point analysis and cattle processing risk assessment modeling. The results from these studies will aid cattle producers in implementing farm management practices that will reduce the dissemination of multidrug resistant pathogens and protect American and international consumers of United States beef.

5. Beef carcass contamination linked to cattle transportation and processing plant holding pen environment. Cattle hide has been established as the main source of carcass contamination during beef processing; therefore, it is crucial to minimize the amount of Escherichia coli O157:H7 on hides prior to slaughter. There are several potential sources of E. coli O157:H7 encountered in the transportation to and in the holding pen environment at beef processing facilities that could increase the prevalence and levels of E. coli O157:H7 on the hides of cattle. ARS scientists in Clay Center, NE, investigated the extent to which E. coli O157:H7 is transferred to cattle hides during transport and while in holding at beef processing plants and, subsequently, transferred to beef carcasses. They determined that transport to and holding at processing plants leads to increased prevalence and levels of E. coli O157:H7 contamination on hides and carcasses. Thus, intervention efforts by beef producers are negated by pathogen transfer in the beef processing facility.

6. Small and inexpensive beef cattle hide wash cabinet to manage bacterial contamination. Harborage of Escherichia coli O157:H7 and Salmonella on animal hides at slaughter is the main source of beef carcass contamination during processing. Interventions have been designed and implemented to target the hides of cattle following entry into beef processing plants. Previous interventions targeting hides have not been suitable for all beef processing plants to implement due to cost and space restrictions. ARS scientists in Clay Center, NE, evaluated a hide wash cabinet design that was smaller and more economical and, therefore, might be more amenable to widespread use in the beef processing industry. They discovered large reductions in E. coli O157:H7 and Salmonella levels after hide washing in the test cabinet. The hide wash cabinet was effective and should provide beef processors, especially small and medium-sized processing plants, with an affordable hide wash intervention strategy.

7. Microbiological characterization of imported and domestic boneless beef trim used for ground beef. The United States imports lean boneless beef trim from Australia (AUS), New Zealand (NZL), and Uruguay (URY) to meet demand for ground beef production. The reported incidence of, and the bacteria responsible for, foodborne disease differs between these countries and the United States. ARS scientists in Clay Center, NE, investigated if current United States microbiological profiling adequately addresses the potential differences in the foreign and domestic beef trim. Additionally, they compared the prevalence of pathogens between imported and domestic samples. Researchers determined that current pathogen monitoring procedures in the United States do not need to be changed for imported beef trim.

8. Determination of E. coli O157:H7 concentration for ground beef and hides, carcasses, and feces of cattle. Methodologies for the assessment of microbial pathogen load, at various steps in the beef production process, are lacking. In order to quantify the risks associated with the slaughter of animals that may harbor or shed E. coli O157:H7 or Salmonella spp., accurate estimates of the prevalence and frequency of distribution of these pathogens and their relative concentration on hides and in feces is needed to assess the food safety risk associated with bacterial pathogens found at various process steps in ground beef production. ARS scientists in Clay Center, NE, developed two methods for the direct enumeration of E. coli O157:H7 and Salmonella spp. One method enumerated E. coli O157:H7 and Salmonella spp. from samples with high bacterial loads such as cattle hide and fecal samples. The second enumerated E. coli O157:H7 and Salmonella spp. from low bacterial count samples such as beef carcass and ground beef samples. Analysis of pathogen enumeration data, in combination with pathogen prevalence, should provide processors with greater control of potential pathogen contamination and further improve the safety of beef for consumers. In addition, these methods will be very useful to researchers studying sources of pathogen contamination and its prevention.

Review Publications
Bosilevac, J.M., Kalchayanand, N., Schmidt, J.W., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M. 2010. Inoculation of beef with low concentrations of Escherichia coli O157:H7 and examination of factors that interfere with its detection by culture isolation and rapid methods. Journal of Food Protection. 73:2180-2188.

Harhay, D.M., Arthur, T.M., Bosilevac, J.M., Kalchayanand, N., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M. 2011. Diversity of multidrug-resistant Salmonella enterica strains associated with cattle at harvest in the United States. Applied and Environmental Microbiology. 77(5):1783-1796.

Arthur, T.M., Nou, X., Kalchayanand, N., Bosilevac, J.M., Wheeler, T.L., Koohmaraie, M. 2011. Survival of Escherichia coli O157:H7 on cattle hides. Applied and Environmental Microbiology. 77(9):3002-3008.

Bosilevac, J.M., Koohmaraie, M. 2011. Prevalence and characterization of non-O157 Shiga toxin producing Escherichia coli isolates from commercial ground beef in the United States. Applied and Environmental Microbiology. 77(6):2103-2112.

Last Modified: 11/29/2015
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