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Implementation of targeted interventions to control Escherichia coli O157:H7 in a commercial abattoir
Meat Science 87 (2011) 361–365
Contents lists available at ScienceDirect
Meat Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m e a t s c i
Implementation of targeted interventions to control Escherichia coli O157:H7 in a commercial abattoir Corri L. Rekow, Mindy M. Brashears ⁎, J. Chance Brooks, Guy H. Loneragan, Sara E. Gragg, Mark F. Miller Texas Tech University, P.O. Box 42141, Lubbock, TX 80631, USA
a r t i c l e
i n f o
Article history: Received 20 May 2010 Received in revised form 8 October 2010 Accepted 12 November 2010 Keywords: Beef carcass Carcass swab Carcass mapping Targeted intervention Steam Escherichia coli O157:H7
a b s t r a c t The objective of this study was to define locations on the carcass with highest contamination of E. coli O157 throughout the harvest process and implement targeted interventions to reduce or eliminate contamination. To establish a pathogen baseline, samples were collected at the foreshank, hindshank, inside round, neck and midline area and evaluated for E. coli O157:H7 presence. Environmental samples were also collected in the harvest area and the fabrication area of the facility. E. coli O157:H7 prevalence was highest on the foreshank, hindshank and inside rounds in the baseline study and steam vacuums/cones were implemented as an intervention in these specific areas on the harvest floor. At pre-evisceration, foreshank prevalence of E. coli O157:H7 was significantly (P b 0.05) reduced from 21.7% to 3.1% after the application of steam interventions. At the final rail, foreshank prevalence in the baseline study was 4.2% while no E. coli O157:H7 was detected post-intervention implementation. E. coli O157:H7 on hindshanks and inside rounds was significantly reduced after intervention implementation from 24.2 to 11.5% and 37.5 to 16.7%, respectively at the final rail. Pathogen contamination of environmental samples collected in fabrication declined from 6.7% to 0.7% after slaughter interventions were implemented. Data indicate the identifying areas of contamination on the carcass and implementing interventions can significantly reduce E. coli O157 on the carcasses and in the fabrication environment. © 2010 The American Meat Science Association. Published by Elsevier Ltd. All rights reserved.
1. Introduction Escherichia coli O157:H7 is a foodborne pathogen and a primary cause of hemorrhagic colitis, severe bloody diarrhea, and hemorrhagic uremic syndrome (HUS) in humans (O'Brien & Kaper, 1998). Hemorrhagic colitis caused by E. coli O157:H7 was first recognized in 1982, when two outbreaks of severe bloody diarrhea were linked to the consumption of undercooked ground beef (Centers for Disease Control and Prevention, 1982) and the microorganism was subsequently designated as a food-borne pathogen. Due to past outbreaks and recent recalls in the beef industry and its status as an adulterant in raw, ground beef, new control measures must be investigated to reduce the incidence of this pathogen on beef carcasses and other raw beef products. The understanding of how E. coli O157:H7 is distributed on carcasses, as well as how to prevent and reduce contamination, can prevent it from entering the food supply. Cattle are the primary reservoir of E. coli O157:H7 and their hides are typically the site of origin for contamination during slaughter (BarkocyGallagher et al., 2003; Smith et al., 2001). Several studies have documented cross-contamination onto carcasses from the hides (Arthur et al., 2004; Bell, 1997; Smith et al., 2001). However, past studies have
⁎ Corresponding author. Tel.: +1 806 742 2805; fax: +1 806 742 4003. E-mail address: [email protected] (M.M. Brashears).
been unable to pinpoint the primary areas of carcass contamination. A study by Keen and Elder (2002) reported that different hide sampling areas such as the neck, ventrum, back, flank and hock contain different amounts of contamination. Another study (Barkocy-Gallagher et al., 2003) obtained samples from individually tagged carcasses from the hide, the carcass prior to the pre-evisceration wash, and from the carcass post-intervention. They isolated E. coli O157:H7 from 781 (60.6%) of 1288 hides and the prevalence of E. coli O157:H7 on pre-evisceration carcass samples was 26.7% and the post-intervention samples had a prevalence of 1.2%, which is similar to the prevalence found in other studies (Barkocy-Gallagher & Koohmaraie, 2002; Barkocy-Gallagher et al., 2003). While these data indicate that the prevalence was reduced after the application of interventions, different locations on the carcasses were sampled throughout the process and it is unclear where the primary sites on the carcasses are that are most frequently contaminated. Some research indicates that prevalence of E. coli O157:H7 on chilled beef carcasses is low. In 1992 and 1993 a nationwide beef microbiological baseline study was conducted by the U.S. Food Safety and Inspection Service (FSIS) where a 300 cm2 composite area of each carcass was sampled at the rump, brisket, and flank (Food Safety and Inspection Service, 1994). This study determined that only 0.2% (4 of 2081) of carcasses sampled after interventions were positive for E. coli O157:H7. A study (Ransom et al., 2002) reported the prevalence of E. coli O157:H7 in feces (3.3 and 10.0%, using two different isolation
0309-1740/$ – see front matter © 2010 The American Meat Science Association. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2010.11.012
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methods) and hides (13.3 to 23.3%, using two different isolation methods) but did not detect E. coli O157:H7 on any of the carcasses sampled immediately after an intervention was applied. While much information is available on the pathogen prevalence on the hides and carcasses, little data exist on the specific locations (mapping) of pathogen distribution on the carcass itself after dehiding. The ability to consistently identify patterns of contamination on the carcass in processing plants allows for the implementation of interventions that target high contamination areas and result in improved reduction of E. coli O157:H7 in the beef supply. From 2004 to 2006, the number of E. coli O157:H7 recalls in the beef industry had declined to an average of 6 recalls per year from up to 20 in previous years (Food Safety and Inspection Service, 2007). However, in 2007 there were 20 recalls due to E. coli O157:H7 contamination in ground beef with the nation's second largest beef recall in history being initiated by Topps Meat Company, who recalled 21.7 million pounds of frozen ground beef products and subsequently declared bankruptcy (Food Safety and Inspection Service, 2007). The outbreak resulted in 40 illnesses and 21 hospitalizations in eight different states (Center for Disease Control and Prevention, 2007). An outbreak and recall such as this has a major impact on the meat industry from both a consumer perception as well as an industry stability standpoint. Research is needed to determine how microorganisms contaminate final beef products and how to control them. The objectives of this study were to identify specific locations of carcass contamination with E. coli O157 at various points during harvest and to determine if targeted interventions will reduce the amount of contamination. 2. Materials and methods 2.1. Sample collection In this study, a pathogen baseline was established on various sites on the carcass and then targeted interventions were applied to control pathogens in the areas of highest contamination and sampling was conducted again in these areas alone at the final rail to validate pathogen control. To establish baseline contamination prevalence of E. coli O157 in the facility, samples were collected over a total of 5 days during the month of August at a Midwestern beef slaughter facility. Twenty-four carcass samples were collected from five sampling sites on the carcass (foreshank, hindshank, inside round, midline, and neck) on each day, for a total of 120 carcass samples per collection (Fig. 1). Environmental samples from the slaughter and fabrication environments, as well as twenty-four hide samples, were also collected per visit. For the baseline study, carcass samples were obtained by swabbing a 500 cm2 area of the foreshank immediately after hide removal, at pre-evisceration, on the final rail after FSIS inspection, both before application of the final hot water intervention and post-intervention. The hindshank, midline, neck and inside round were individually sampled by swabbing a 500 cm2 area at the final rail only (not at the other sampling areas) after FSIS inspection but prior to application of the final hot water intervention. The hide samples were taken before hind leg transfer was complete by swabbing a 500 cm2 area of the animals hide from the anus to the hindshank (Fig. 2). In order to validate the effectiveness of the interventions, the same carcass sampling sites and locations within the plant were used. Environmental samples were collected in the slaughter and the fabrication areas. A total of 225 slaughter environmental samples were collected throughout the study with 45 collected on each day during the establishment of baseline data. A 100 cm2 template was used to collect random samples from the floor, wall, rails, drains, and viscera table (8 each). Because of the low detection rate in the slaughter area, no samples were collected after the implementation of interventions. This same method was used to collect samples in the
Fig. 1. Carcass sampling locations for the carcass mapping study to determine E. coli O157 contamination patterns.
fabrication area with a total of 35–40 samples taken on each day before and after the implementation of interventions. Environmental fabrication samples were taken from the cutting tables where the high risk cuts were processed (inside round saw/table, shank saw/belt). Half of the samples were taken at the middle and half of the samples were taken at the end of the shift. All samples were obtained using a sterile sponge (HydraSponge™, Biotrace Int., Bridgend, UK), which was pre-moistened with 10 ml of sterile buffered peptone water (BPW) to hydrate the dehydrated, sterile sponge. All sample bags were individually labeled, collected using sterile disposable gloves and placed into sanitized, insulated coolers chilled with frozen gel-packs. These coolers were shipped overnight to the Texas Tech University Experimental Sciences Building. Upon arrival to the laboratory, samples were stored at 4 °C and analyzed on the day of arrival. Each sample bag received an additional 15 ml of BPW and was homogenized for 1 min (Seward Model 400, Bohemia, NY). Following the first 5 days of sampling and establishment of the baseline, two interventions were implemented in areas on the carcass with the highest pathogen contamination, the foreshank, hindshank and inside round areas. The first intervention, hot water (82.2 °C), was applied to the hindshank and foreshank just after the hide was removed from those areas. The hot water was not circulated and used only once. Steam vacuum was the second intervention and was applied to the inside round just after the final hide removal. Following implementation of the targeted interventions, an additional four sample collection days occurred as previously described. For all nine sampling times, foreshanks were sampled at pre-evisceration (prior
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2.3. Statistical analysis In order to determine the influence of the interventions, while controlling for hide prevalence, add data were entered into an Excel spreadsheet for analyses. The average pathogen prevalence in the baseline study was analyzed in comparison to the data generated in after interventions are applied to determine the effectiveness of the interventions. The data were analyzed using generalized and general linear mixed models in Statistical Analysis Software (SAS Release 9.1.3., SAS Inc., Cary, NC), a commercially available software package. Statistical significance was measured at the P b 0.05 level of significance.
3. Results and discussion 3.1. Baseline for E. coli O157:H7
Fig. 2. Hide sampling location for the carcass mapping study to determine E. coli O157 contamination patterns.
The foreshanks were selected to establish the baseline data for this study because plant personnel had identified this section of the carcass as being associated with E. coli O157:H7 positive samples in the trim area of the plant. At the pre-evisceration sampling location, E. coli O157:H7 prevalence was detected to be 21.7% on the foreshanks. After the application of interventions, a significant reduction of the contamination (P b 0.05) occurred with only 3.1% of the foreshanks testing positive. At the final rail, in the baseline study, foreshanks had a prevalence of 4.2%; however no E. coli O157:H7 was detected after interventions were applied in any location (Table 1). Samples were also collected from the hindshank, inside round, midline and neck area immediately prior to receiving interventions at the final rail because these are areas that had been reported to be contaminated with the pathogen in previous studies. The hindshank and inside round samples resulted in the highest prevalence at 24.2% and 27.5%, respectively (Fig. 3). Only 1.4% of midline and 0.5% of the neck samples tested positive for E. coli O157:H7 (data not illustrated) and therefore, no interventions were applied to these areas and samples were not collected on these sites for the validation study.
3.2. Microbial profiles after the implementation of interventions to pre-intervention application), immediately prior to the application of interventions (final rail) and immediately following the application of interventions (post-interventions). The hindshanks, inside rounds, midlines and necks were sampled at the final rail only; immediately before the carcasses entered the cooler and after final FSIS inspection. A pre-evisceration wash cabinet was the first intervention utilized by the plant throughout the study and comprised a combination of hot (82.2 °C) water and 5% lactic acid in a single use (not re-circulating) system. Following the pre-evisceration cabinet, carcasses were eviscerated, washed with a cool water wash and were finally treated with two washes, the first being a 5% lactic acid wash and the second a 82.2 °C hot water wash following the final rail inspection. All targeted interventions were implemented in addition to these two interventions already employed by the plant.
2.2. Microbial analyses Carcass samples were processed for detection of E. coli O157:H7 by enriching 1 ml of the homogenized sample in 9 ml of Tryptic Soy Broth (TSB). TSB sample tubes were vortexed thoroughly and incubated for 24 h at room temperature. Following the enrichment period, E. coli O157:H7 was detected using the AOAC approved and USDA-FSIS approved BAX® detection system according to published USDA protocols (DuPont Qualicon, Wilmington, DE). Isolates were recovered from positive BAX samples.
The prevalence of E. coli O157:H7 was significantly (P b 0.05) reduced from 21.7% to 3.1% on foreshank samples after the initial application of the steam treatment immediately after the preevisceration wash when comparing the baseline study data to the data collected in the validation study (Table 1). At the final rail in the baseline study, 4.2% of the samples were positive while none tested positive after interventions had been applied. A similar trend was observed on the hindshanks and inside rounds with E. coli O157:H7 prevalence significantly reduced (P b 0.05) after the intervention was implemented from 24.2 to 11.5% and 37.5 to 16.7%, respectively at the final rail (Fig. 3). Data indicate that the prevalence of the pathogen can be reduced on the harvest floor with the implementation of interventions in areas of highest pathogen contamination. Currently, a whole carcass intervention cannot be applied until after the final rail inspection, but interventions can be applied in certain locations to control pathogen contamination.
Table 1 Prevalence (%) of Escherichia coli O157:H7 on foreshank samples during the baseline study and after implementation of the targeted interventions.
Baseline study After interventions
Pre-evisceration
Final rail
21.7% 3.1%⁎
4.2% 0
⁎ Samples collected after the application of steam to the foreshank area.
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40
Precentage Positive
35 30 25 20 15 10 5 0 Fore Shank
Hind Shank
Inside Round Before
Environment
Hide
After
Fig. 3. Prevalence of E. coli O157:H7 detected on selected areas of a carcass at the final rail prior to the implementation of carcass interventions and after the implementation of targeted interventions in these areas.
3.3. Pathogen prevalence on hides Previous studies have reported that the hides contribute a significant amount to the total bacterial load on the carcass (Arthur et al., 2004; Bell, 1997; Smith et al., 2001) and decontamination methods should be considered to reduce the amount of cross-contamination that occurs from the hide to the carcass (Nou et al., 2003). The hide prevalence in this study was 37% before the implementation of interventions and 35% after (Fig. 3). There were no interventions applied to the hides. This indicates that baseline contamination levels arriving into the plant did not change before and after implementation of the targeted interventions; however, the level of contamination on the carcasses did. 3.4. E. coli O157:H7 in the environment When establishing the initial baseline for pathogen prevalence, 14 of 225 (6.2%) samples obtained from the fabrication environment were positive, indicating that some pathogens are transported through the cooler and into the fabrication area. Following the implementation of targeted interventions, only 1 of 145 (0.7%) of samples from the fabrication environment were positive (Table 2). This reduction in the number of positive samples indicates that the implemented interventions were effective in controlling the pathogens not only on the carcasses themselves, but also in the fabrication area. Utilizing multiple interventions is beneficial and improves the microbial quality of the carcass. Other studies have shown that the use of steam vacuuming, pre-evisceration and post-evisceration water washes, organic acids and hot water washes are effective in decreasing carcass contamination (Bacon et al., 2000; Castillo, Lucia, Goodson, Savell, & Acuff, 1999; Hardin, Acuff, Lucia, Oman, & Savell, 1995). The whole carcass application of interventions can only occur at pre-evisceration or after the final rail. Data in this study indicate that utilizing proven interventions in targeted locations on the
Table 2 Prevalence (%) of Escherichia coli O157:H7 in environmental samples collected from the fabrication area during the baseline study and after implementation of the targeted interventions.
Before implementation of interventions After implementation of interventions Total positive
Fabrication
Positive sampling locations
14/225 (6.2%)
Inside round table (5) shank saw (3)
1/145 (0.70%) 15/370 (4.06)
Round saw table (6) Inside round saw (1)
harvest floor can add a measure of security in reducing contamination on the final carcasses and in the fabrication area. An understanding of E. coli O157:H7 distribution on beef cattle carcasses during slaughter can assist the industry with implementation of targeted interventions to control this pathogen. However, pathogens are still transferred into the fabrication environment and must be monitored even if interventions are used during slaughter. Within the fabrication environment, cross-contamination from the carcass to equipment occurred most commonly in areas that come into contact with the inside round (round saw table and inside round table). The shank saw also tested positive prior to the implementation of interventions (Table 2). Sampling sites on the carcass with the highest prevalence for E. coli O157:H7 include the inside round, foreshank and the hindshank were the most contaminated in this plant. Because these carcass sites had the highest prevalence of E. coli O157:H7, higher contamination levels for the corresponding fabrication locations was also observed. After fabrication, the majority of foreshank and hindshank product is processed into trimmings, which are frequently used to make ground beef. Therefore, this illustrates how trim or ground beef may become contaminated and enter the food supply. Results indicate that E. coli O157:H7 can be transferred from the carcass to the fabrication environment and result in contaminated trim if proper precautions are not taken. Without the proper implementation of interventions, sanitation and dressing procedures, food safety hazards will enter the food supply. 4. Conclusion In the present study, the foreshank, hindshank and inside round samples contained the highest prevalence of E. coli O157:H7. The implementation of targeted interventions resulted in significant reductions in pathogen loads in these samples. Another benefit is the fact that these interventions can be applied prior to the final rail, in contrast to whole carcass interventions, which cannot be applied until after the final rail after evisceration has occurred. These data also indicate that E. coli O157:H7 is transferred from contaminated carcasses to the fabrication area and can potentially cross-contaminate meat that is pathogen free. The targeted interventions successfully reduced the pathogen loads in the slaughter process as well as microbial loads in the fabrication environment. Therefore, the entire beef production process benefited from these targeted interventions. Acknowledgements Funding for this research was provided by The Beef Checkoff program. References Arthur, T. M., Bosilevac, J. M., Nou, X., Shackelford, S. D., Wheeler, T. L., Kent, M. P., et al. (2004). Escherichia coli O157 prevalence and enumeration of aerobic bacteria, Enterobacteriaceae, and Escherichia coli O157 at various steps in commercial beef processing plants. Journal of Food Protection, 67, 658−665. Bacon, R. T., Belk, K. E., Sofos, J. N., Clayton, R. P., Reagan, J. O., & Smith, G. C. (2000). Microbial populations on animal hides and beef carcasses at different stages of slaughter in plants employing multiple-sequential interventions for decontamination. Journal of Food Protection, 63, 1080−1086. Barkocy-Gallagher, G. A., Arthur, T. M., Rivera-Bentancourt, M., Nou, X., Shackelford, S. D., Wheeler, T. L., et al. (2003). Seasonal prevalence of shiga toxin-producing Escherichia coli, including O157:H7 and non-O157 serotypes, and Salmonella in commercial beef processing plants. Journal of Food Protection, 66, 1978−1986. Barkocy-Gallagher, G. A., & Koohmaraie, M. (2002). Prevalence of Escherichia coli O157: H7 in cattle and during processing. Proceedings of the 55th Reciprocal Meat Conference, 55, 15−20. Bell, R. G. (1997). Distribution and sources of microbial contamination on beef carcasses. Journal of Applied Microbiology, 82, 292−300. Castillo, A., Lucia, L. M., Goodson, K. J., Savell, J. W., & Acuff, G. R. (1999). Decontamination of beef carcass surface tissue by steam vacuuming alone and
C.L. Rekow et al. / Meat Science 87 (2011) 361–365 combined with hot water and lactic acid sprays. Journal of Food Protection, 62, 146−151. Center for Disease Control and Prevention (2007). Multistate outbreak of E. coli O157 infections linked to Topp's brand ground beef patties.: Department of Health and Human Services, Centers for Disease Control and Prevention Available at:. http:// www.cdc.gov/ecoli/2007/october/100207.html Accessed July 21, 2008. Centers for Disease Control and Prevention (1982). Isolation of E. coli O157:H7 from sporadic cases of hemorrhagic colitis—United States. Morbidity and Mortality Weekly Report, 31, 580−585. Food Safety and Inspection Service (1994). Nationwide beef microbiological baseline data collection program: steers and heifers, October 1992–September 1993.Washington, D.C: U.S. Department of Agriculture, Food Safety and Inspection Service, Science and Technology, Microbiology Division Available at:. http://www.fsis.usda.gov/Science/ Baseline_Data/index.asp Accessed July 27, 2008. Food Safety and Inspection Service (2007). Recall case archive.Washington, D.C: U.S. Department of Agriculture, Food Safety and Inspection Service Available at:. http:// www.fsis.usda.gov/fsis_recalls/Recall_Case_Archive_2007/index.asp Accessed July 21, 2008. Hardin, M. D., Acuff, G. R., Lucia, L. M., Oman, J. S., & Savell, J. W. (1995). Comparison of methods for decontamination from beef carcass surfaces. Journal of Food Protection, 58, 368−374.
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Keen, J. E., & Elder, R. O. (2002). Isolation of shiga-toxigenic Escherichia coli O157 from hide surfaces and the oral cavity of finished beef feedlot cattle. Journal of the American Veterinary Medical Association, 220, 756−763. Nou, X., Rivera-Betancourt, M., Bosilevac, J. M., Wheeler, T. L., Shackelford, S. D., Gwartney, B. L., et al. (2003). Effect of chemical dehairing on the prevalence of Escherichia coli O157:H7 and the levels of aerobic bacteria and Enterobacteriaceae on carcasses in a commercial beef processing plant. Journal of Food Protection, 66, 2005−2009. O'Brien, A. D., & Kaper, J. B. (1998). Shiga toxin-producing Escherichia coli: yesterday, today, and tomorrow. In J. B., & A. D. (Eds.), Escherichia coli O157:H7 and other shiga toxin-producing E. coli strains (pp. 3−5). Washington, D.C: ASM Press. Ransom, J. R., Belk, K. E., Bacon, R. T., Sofos, J. N., Scanga, J. A., & Smith, G. C. (2002). Comparison of sampling methods for microbiological testing of beef animal rectal/ colonal feces, hides, and carcasses. Journal of Food Protection, 65, 621−626. Smith, D., Blackford, M., Younts, S., Moxley, R., Gray, J., Hungerford, L., et al. (2001). Ecological relationships between the prevalence of cattle shedding Escherichia coli O157:H7 and characteristics of the cattle or conditions of the feedlot pen. Journal of Food Protection, 64, 1899−1903.
Contents lists available at ScienceDirect
Meat Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m e a t s c i
Implementation of targeted interventions to control Escherichia coli O157:H7 in a commercial abattoir Corri L. Rekow, Mindy M. Brashears ⁎, J. Chance Brooks, Guy H. Loneragan, Sara E. Gragg, Mark F. Miller Texas Tech University, P.O. Box 42141, Lubbock, TX 80631, USA
a r t i c l e
i n f o
Article history: Received 20 May 2010 Received in revised form 8 October 2010 Accepted 12 November 2010 Keywords: Beef carcass Carcass swab Carcass mapping Targeted intervention Steam Escherichia coli O157:H7
a b s t r a c t The objective of this study was to define locations on the carcass with highest contamination of E. coli O157 throughout the harvest process and implement targeted interventions to reduce or eliminate contamination. To establish a pathogen baseline, samples were collected at the foreshank, hindshank, inside round, neck and midline area and evaluated for E. coli O157:H7 presence. Environmental samples were also collected in the harvest area and the fabrication area of the facility. E. coli O157:H7 prevalence was highest on the foreshank, hindshank and inside rounds in the baseline study and steam vacuums/cones were implemented as an intervention in these specific areas on the harvest floor. At pre-evisceration, foreshank prevalence of E. coli O157:H7 was significantly (P b 0.05) reduced from 21.7% to 3.1% after the application of steam interventions. At the final rail, foreshank prevalence in the baseline study was 4.2% while no E. coli O157:H7 was detected post-intervention implementation. E. coli O157:H7 on hindshanks and inside rounds was significantly reduced after intervention implementation from 24.2 to 11.5% and 37.5 to 16.7%, respectively at the final rail. Pathogen contamination of environmental samples collected in fabrication declined from 6.7% to 0.7% after slaughter interventions were implemented. Data indicate the identifying areas of contamination on the carcass and implementing interventions can significantly reduce E. coli O157 on the carcasses and in the fabrication environment. © 2010 The American Meat Science Association. Published by Elsevier Ltd. All rights reserved.
1. Introduction Escherichia coli O157:H7 is a foodborne pathogen and a primary cause of hemorrhagic colitis, severe bloody diarrhea, and hemorrhagic uremic syndrome (HUS) in humans (O'Brien & Kaper, 1998). Hemorrhagic colitis caused by E. coli O157:H7 was first recognized in 1982, when two outbreaks of severe bloody diarrhea were linked to the consumption of undercooked ground beef (Centers for Disease Control and Prevention, 1982) and the microorganism was subsequently designated as a food-borne pathogen. Due to past outbreaks and recent recalls in the beef industry and its status as an adulterant in raw, ground beef, new control measures must be investigated to reduce the incidence of this pathogen on beef carcasses and other raw beef products. The understanding of how E. coli O157:H7 is distributed on carcasses, as well as how to prevent and reduce contamination, can prevent it from entering the food supply. Cattle are the primary reservoir of E. coli O157:H7 and their hides are typically the site of origin for contamination during slaughter (BarkocyGallagher et al., 2003; Smith et al., 2001). Several studies have documented cross-contamination onto carcasses from the hides (Arthur et al., 2004; Bell, 1997; Smith et al., 2001). However, past studies have
⁎ Corresponding author. Tel.: +1 806 742 2805; fax: +1 806 742 4003. E-mail address: [email protected] (M.M. Brashears).
been unable to pinpoint the primary areas of carcass contamination. A study by Keen and Elder (2002) reported that different hide sampling areas such as the neck, ventrum, back, flank and hock contain different amounts of contamination. Another study (Barkocy-Gallagher et al., 2003) obtained samples from individually tagged carcasses from the hide, the carcass prior to the pre-evisceration wash, and from the carcass post-intervention. They isolated E. coli O157:H7 from 781 (60.6%) of 1288 hides and the prevalence of E. coli O157:H7 on pre-evisceration carcass samples was 26.7% and the post-intervention samples had a prevalence of 1.2%, which is similar to the prevalence found in other studies (Barkocy-Gallagher & Koohmaraie, 2002; Barkocy-Gallagher et al., 2003). While these data indicate that the prevalence was reduced after the application of interventions, different locations on the carcasses were sampled throughout the process and it is unclear where the primary sites on the carcasses are that are most frequently contaminated. Some research indicates that prevalence of E. coli O157:H7 on chilled beef carcasses is low. In 1992 and 1993 a nationwide beef microbiological baseline study was conducted by the U.S. Food Safety and Inspection Service (FSIS) where a 300 cm2 composite area of each carcass was sampled at the rump, brisket, and flank (Food Safety and Inspection Service, 1994). This study determined that only 0.2% (4 of 2081) of carcasses sampled after interventions were positive for E. coli O157:H7. A study (Ransom et al., 2002) reported the prevalence of E. coli O157:H7 in feces (3.3 and 10.0%, using two different isolation
0309-1740/$ – see front matter © 2010 The American Meat Science Association. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2010.11.012
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methods) and hides (13.3 to 23.3%, using two different isolation methods) but did not detect E. coli O157:H7 on any of the carcasses sampled immediately after an intervention was applied. While much information is available on the pathogen prevalence on the hides and carcasses, little data exist on the specific locations (mapping) of pathogen distribution on the carcass itself after dehiding. The ability to consistently identify patterns of contamination on the carcass in processing plants allows for the implementation of interventions that target high contamination areas and result in improved reduction of E. coli O157:H7 in the beef supply. From 2004 to 2006, the number of E. coli O157:H7 recalls in the beef industry had declined to an average of 6 recalls per year from up to 20 in previous years (Food Safety and Inspection Service, 2007). However, in 2007 there were 20 recalls due to E. coli O157:H7 contamination in ground beef with the nation's second largest beef recall in history being initiated by Topps Meat Company, who recalled 21.7 million pounds of frozen ground beef products and subsequently declared bankruptcy (Food Safety and Inspection Service, 2007). The outbreak resulted in 40 illnesses and 21 hospitalizations in eight different states (Center for Disease Control and Prevention, 2007). An outbreak and recall such as this has a major impact on the meat industry from both a consumer perception as well as an industry stability standpoint. Research is needed to determine how microorganisms contaminate final beef products and how to control them. The objectives of this study were to identify specific locations of carcass contamination with E. coli O157 at various points during harvest and to determine if targeted interventions will reduce the amount of contamination. 2. Materials and methods 2.1. Sample collection In this study, a pathogen baseline was established on various sites on the carcass and then targeted interventions were applied to control pathogens in the areas of highest contamination and sampling was conducted again in these areas alone at the final rail to validate pathogen control. To establish baseline contamination prevalence of E. coli O157 in the facility, samples were collected over a total of 5 days during the month of August at a Midwestern beef slaughter facility. Twenty-four carcass samples were collected from five sampling sites on the carcass (foreshank, hindshank, inside round, midline, and neck) on each day, for a total of 120 carcass samples per collection (Fig. 1). Environmental samples from the slaughter and fabrication environments, as well as twenty-four hide samples, were also collected per visit. For the baseline study, carcass samples were obtained by swabbing a 500 cm2 area of the foreshank immediately after hide removal, at pre-evisceration, on the final rail after FSIS inspection, both before application of the final hot water intervention and post-intervention. The hindshank, midline, neck and inside round were individually sampled by swabbing a 500 cm2 area at the final rail only (not at the other sampling areas) after FSIS inspection but prior to application of the final hot water intervention. The hide samples were taken before hind leg transfer was complete by swabbing a 500 cm2 area of the animals hide from the anus to the hindshank (Fig. 2). In order to validate the effectiveness of the interventions, the same carcass sampling sites and locations within the plant were used. Environmental samples were collected in the slaughter and the fabrication areas. A total of 225 slaughter environmental samples were collected throughout the study with 45 collected on each day during the establishment of baseline data. A 100 cm2 template was used to collect random samples from the floor, wall, rails, drains, and viscera table (8 each). Because of the low detection rate in the slaughter area, no samples were collected after the implementation of interventions. This same method was used to collect samples in the
Fig. 1. Carcass sampling locations for the carcass mapping study to determine E. coli O157 contamination patterns.
fabrication area with a total of 35–40 samples taken on each day before and after the implementation of interventions. Environmental fabrication samples were taken from the cutting tables where the high risk cuts were processed (inside round saw/table, shank saw/belt). Half of the samples were taken at the middle and half of the samples were taken at the end of the shift. All samples were obtained using a sterile sponge (HydraSponge™, Biotrace Int., Bridgend, UK), which was pre-moistened with 10 ml of sterile buffered peptone water (BPW) to hydrate the dehydrated, sterile sponge. All sample bags were individually labeled, collected using sterile disposable gloves and placed into sanitized, insulated coolers chilled with frozen gel-packs. These coolers were shipped overnight to the Texas Tech University Experimental Sciences Building. Upon arrival to the laboratory, samples were stored at 4 °C and analyzed on the day of arrival. Each sample bag received an additional 15 ml of BPW and was homogenized for 1 min (Seward Model 400, Bohemia, NY). Following the first 5 days of sampling and establishment of the baseline, two interventions were implemented in areas on the carcass with the highest pathogen contamination, the foreshank, hindshank and inside round areas. The first intervention, hot water (82.2 °C), was applied to the hindshank and foreshank just after the hide was removed from those areas. The hot water was not circulated and used only once. Steam vacuum was the second intervention and was applied to the inside round just after the final hide removal. Following implementation of the targeted interventions, an additional four sample collection days occurred as previously described. For all nine sampling times, foreshanks were sampled at pre-evisceration (prior
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2.3. Statistical analysis In order to determine the influence of the interventions, while controlling for hide prevalence, add data were entered into an Excel spreadsheet for analyses. The average pathogen prevalence in the baseline study was analyzed in comparison to the data generated in after interventions are applied to determine the effectiveness of the interventions. The data were analyzed using generalized and general linear mixed models in Statistical Analysis Software (SAS Release 9.1.3., SAS Inc., Cary, NC), a commercially available software package. Statistical significance was measured at the P b 0.05 level of significance.
3. Results and discussion 3.1. Baseline for E. coli O157:H7
Fig. 2. Hide sampling location for the carcass mapping study to determine E. coli O157 contamination patterns.
The foreshanks were selected to establish the baseline data for this study because plant personnel had identified this section of the carcass as being associated with E. coli O157:H7 positive samples in the trim area of the plant. At the pre-evisceration sampling location, E. coli O157:H7 prevalence was detected to be 21.7% on the foreshanks. After the application of interventions, a significant reduction of the contamination (P b 0.05) occurred with only 3.1% of the foreshanks testing positive. At the final rail, in the baseline study, foreshanks had a prevalence of 4.2%; however no E. coli O157:H7 was detected after interventions were applied in any location (Table 1). Samples were also collected from the hindshank, inside round, midline and neck area immediately prior to receiving interventions at the final rail because these are areas that had been reported to be contaminated with the pathogen in previous studies. The hindshank and inside round samples resulted in the highest prevalence at 24.2% and 27.5%, respectively (Fig. 3). Only 1.4% of midline and 0.5% of the neck samples tested positive for E. coli O157:H7 (data not illustrated) and therefore, no interventions were applied to these areas and samples were not collected on these sites for the validation study.
3.2. Microbial profiles after the implementation of interventions to pre-intervention application), immediately prior to the application of interventions (final rail) and immediately following the application of interventions (post-interventions). The hindshanks, inside rounds, midlines and necks were sampled at the final rail only; immediately before the carcasses entered the cooler and after final FSIS inspection. A pre-evisceration wash cabinet was the first intervention utilized by the plant throughout the study and comprised a combination of hot (82.2 °C) water and 5% lactic acid in a single use (not re-circulating) system. Following the pre-evisceration cabinet, carcasses were eviscerated, washed with a cool water wash and were finally treated with two washes, the first being a 5% lactic acid wash and the second a 82.2 °C hot water wash following the final rail inspection. All targeted interventions were implemented in addition to these two interventions already employed by the plant.
2.2. Microbial analyses Carcass samples were processed for detection of E. coli O157:H7 by enriching 1 ml of the homogenized sample in 9 ml of Tryptic Soy Broth (TSB). TSB sample tubes were vortexed thoroughly and incubated for 24 h at room temperature. Following the enrichment period, E. coli O157:H7 was detected using the AOAC approved and USDA-FSIS approved BAX® detection system according to published USDA protocols (DuPont Qualicon, Wilmington, DE). Isolates were recovered from positive BAX samples.
The prevalence of E. coli O157:H7 was significantly (P b 0.05) reduced from 21.7% to 3.1% on foreshank samples after the initial application of the steam treatment immediately after the preevisceration wash when comparing the baseline study data to the data collected in the validation study (Table 1). At the final rail in the baseline study, 4.2% of the samples were positive while none tested positive after interventions had been applied. A similar trend was observed on the hindshanks and inside rounds with E. coli O157:H7 prevalence significantly reduced (P b 0.05) after the intervention was implemented from 24.2 to 11.5% and 37.5 to 16.7%, respectively at the final rail (Fig. 3). Data indicate that the prevalence of the pathogen can be reduced on the harvest floor with the implementation of interventions in areas of highest pathogen contamination. Currently, a whole carcass intervention cannot be applied until after the final rail inspection, but interventions can be applied in certain locations to control pathogen contamination.
Table 1 Prevalence (%) of Escherichia coli O157:H7 on foreshank samples during the baseline study and after implementation of the targeted interventions.
Baseline study After interventions
Pre-evisceration
Final rail
21.7% 3.1%⁎
4.2% 0
⁎ Samples collected after the application of steam to the foreshank area.
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40
Precentage Positive
35 30 25 20 15 10 5 0 Fore Shank
Hind Shank
Inside Round Before
Environment
Hide
After
Fig. 3. Prevalence of E. coli O157:H7 detected on selected areas of a carcass at the final rail prior to the implementation of carcass interventions and after the implementation of targeted interventions in these areas.
3.3. Pathogen prevalence on hides Previous studies have reported that the hides contribute a significant amount to the total bacterial load on the carcass (Arthur et al., 2004; Bell, 1997; Smith et al., 2001) and decontamination methods should be considered to reduce the amount of cross-contamination that occurs from the hide to the carcass (Nou et al., 2003). The hide prevalence in this study was 37% before the implementation of interventions and 35% after (Fig. 3). There were no interventions applied to the hides. This indicates that baseline contamination levels arriving into the plant did not change before and after implementation of the targeted interventions; however, the level of contamination on the carcasses did. 3.4. E. coli O157:H7 in the environment When establishing the initial baseline for pathogen prevalence, 14 of 225 (6.2%) samples obtained from the fabrication environment were positive, indicating that some pathogens are transported through the cooler and into the fabrication area. Following the implementation of targeted interventions, only 1 of 145 (0.7%) of samples from the fabrication environment were positive (Table 2). This reduction in the number of positive samples indicates that the implemented interventions were effective in controlling the pathogens not only on the carcasses themselves, but also in the fabrication area. Utilizing multiple interventions is beneficial and improves the microbial quality of the carcass. Other studies have shown that the use of steam vacuuming, pre-evisceration and post-evisceration water washes, organic acids and hot water washes are effective in decreasing carcass contamination (Bacon et al., 2000; Castillo, Lucia, Goodson, Savell, & Acuff, 1999; Hardin, Acuff, Lucia, Oman, & Savell, 1995). The whole carcass application of interventions can only occur at pre-evisceration or after the final rail. Data in this study indicate that utilizing proven interventions in targeted locations on the
Table 2 Prevalence (%) of Escherichia coli O157:H7 in environmental samples collected from the fabrication area during the baseline study and after implementation of the targeted interventions.
Before implementation of interventions After implementation of interventions Total positive
Fabrication
Positive sampling locations
14/225 (6.2%)
Inside round table (5) shank saw (3)
1/145 (0.70%) 15/370 (4.06)
Round saw table (6) Inside round saw (1)
harvest floor can add a measure of security in reducing contamination on the final carcasses and in the fabrication area. An understanding of E. coli O157:H7 distribution on beef cattle carcasses during slaughter can assist the industry with implementation of targeted interventions to control this pathogen. However, pathogens are still transferred into the fabrication environment and must be monitored even if interventions are used during slaughter. Within the fabrication environment, cross-contamination from the carcass to equipment occurred most commonly in areas that come into contact with the inside round (round saw table and inside round table). The shank saw also tested positive prior to the implementation of interventions (Table 2). Sampling sites on the carcass with the highest prevalence for E. coli O157:H7 include the inside round, foreshank and the hindshank were the most contaminated in this plant. Because these carcass sites had the highest prevalence of E. coli O157:H7, higher contamination levels for the corresponding fabrication locations was also observed. After fabrication, the majority of foreshank and hindshank product is processed into trimmings, which are frequently used to make ground beef. Therefore, this illustrates how trim or ground beef may become contaminated and enter the food supply. Results indicate that E. coli O157:H7 can be transferred from the carcass to the fabrication environment and result in contaminated trim if proper precautions are not taken. Without the proper implementation of interventions, sanitation and dressing procedures, food safety hazards will enter the food supply. 4. Conclusion In the present study, the foreshank, hindshank and inside round samples contained the highest prevalence of E. coli O157:H7. The implementation of targeted interventions resulted in significant reductions in pathogen loads in these samples. Another benefit is the fact that these interventions can be applied prior to the final rail, in contrast to whole carcass interventions, which cannot be applied until after the final rail after evisceration has occurred. These data also indicate that E. coli O157:H7 is transferred from contaminated carcasses to the fabrication area and can potentially cross-contaminate meat that is pathogen free. The targeted interventions successfully reduced the pathogen loads in the slaughter process as well as microbial loads in the fabrication environment. Therefore, the entire beef production process benefited from these targeted interventions. Acknowledgements Funding for this research was provided by The Beef Checkoff program. References Arthur, T. M., Bosilevac, J. M., Nou, X., Shackelford, S. D., Wheeler, T. L., Kent, M. P., et al. (2004). Escherichia coli O157 prevalence and enumeration of aerobic bacteria, Enterobacteriaceae, and Escherichia coli O157 at various steps in commercial beef processing plants. Journal of Food Protection, 67, 658−665. Bacon, R. T., Belk, K. E., Sofos, J. N., Clayton, R. P., Reagan, J. O., & Smith, G. C. (2000). Microbial populations on animal hides and beef carcasses at different stages of slaughter in plants employing multiple-sequential interventions for decontamination. Journal of Food Protection, 63, 1080−1086. Barkocy-Gallagher, G. A., Arthur, T. M., Rivera-Bentancourt, M., Nou, X., Shackelford, S. D., Wheeler, T. L., et al. (2003). Seasonal prevalence of shiga toxin-producing Escherichia coli, including O157:H7 and non-O157 serotypes, and Salmonella in commercial beef processing plants. Journal of Food Protection, 66, 1978−1986. Barkocy-Gallagher, G. A., & Koohmaraie, M. (2002). Prevalence of Escherichia coli O157: H7 in cattle and during processing. Proceedings of the 55th Reciprocal Meat Conference, 55, 15−20. Bell, R. G. (1997). Distribution and sources of microbial contamination on beef carcasses. Journal of Applied Microbiology, 82, 292−300. Castillo, A., Lucia, L. M., Goodson, K. J., Savell, J. W., & Acuff, G. R. (1999). Decontamination of beef carcass surface tissue by steam vacuuming alone and
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