2013 Beef Industry Safety Summit

Transcription

1 2013 Beef Industry Safety Summit Research Update Category: Methodology Improvements Project Title: Comparison of non O157 Shiga Toxin producing E. coli Detection Systems Mick Bosilevac, USDA, ARS, US Meat Animal Research Center Objective: To identify strengths and weaknesses of Shiga toxin producing E coli detection systems and kits in a side by side fashion Three commercial Shiga toxin producing E. coli (STEC) detection tests and two third party laboratories were compared to the MLG (5B) and USMARC molecular methods for ability to detect top 6 STEC (aka enterohemorrhagic E. coli [EHEC]). FSIS MLG 5B STEC Detection DuPont Qualicon BAX System STEC Suite commercial detection test BioControl Systems Assurance GDS Top STEC commercial detection test Pall GeneDisc System for non O157 STEC commercial detection test IEH Laboratories third party lab Neogen NeoSEEK third party lab US Meat Animal Research Center STEC detection and isolation protocols The study design was a three part evaluation in two phases: Two inoculation studies Each method/system used according to its protocol (medias, incubation temperatures & times, material). Each method/system used to test the same TSB enrichments (ground beef varying weight/volume/time). One set of Real World samples Each method/system used on 500 enrichments from a regional service lab. The results indicated all methods, used as directed, can detect low levels of inoculated top 6 EHEC though results varied among methods (Table 1). One hundred sixty of 500 samples were found to be potential positive by one or more methods, but only 2 samples were positive by all methods. This information allows users of STEC detection tests to draw comparisons and critically consider the value of a potential positive when multiple organisms may be present. The results also provide a baseline of what number of potential positives different methods may give.

2 Project Title: Escherichia coli O104 in the feces of feedlot cattle do not harbor genetic traits of the strain responsible for the 2011 German outbreak T. G. Nagaraja, Kansas State University Objective: To test feedlot cattle feces with a multiplex PCR (mpcr) designed to detect serogroup O104 and genes characteristic of the STEC and EAEC pathotypes and isolate and characterize E. coli O104 from PCRpositive fecal samples. Escherichia coli O104:H4 was the cause of a large foodborne outbreak of hemorrhagic colitis and hemolytic uremic syndrome in Germany and other European countries in The serotype was a hybrid of Shiga toxinproducing E. coli (STEC) and enteroaggregative E. coli (EAEC) pathotypes. Because cattle are known reservoirs of STEC, the researchers designed and validated a mpcr to detect E. coli O104 with STEC and or EAEC traits by targeting the following 8 genes: stx1, stx2, terd (tellurite resistance), eae, wzx O104 (O104 specific O antigen flippase), flic H4 (H4 specific flagella), ehxa and agga (pilin subunit of aggregative adherence fimbriae). The mpcr assay of 248 fecal samples, collected from 8 different feedlots, showed only three samples (1.1%) were positive before enrichment and 51 samples (20.6%) positive after enrichment in EC broth for the wzx O104 gene specific for the O104 serogroup. None of the 248 fecal samples contained the agga, a gene that is characteristic of the EAEC pathotype (Table 2). No significant association between the presence of wzx O104 and either of the Shiga toxin genes was detected. Only seven (13.7%) samples yielded pure cultures of serogroup O104 and all 7 isolates were negative for flic H4 and genes characteristics of STEC and EAEC. These results show that cattle are not a likely reservoir for E. coli O104:H4 with characteristics of STEC and EAEC (Table 2). Table 2. Presence of genes that encode for Escherichia coli O104 serogroup specific traits in cattle fecal samples (n=248) before and after enrichment in Escherichia coli broth Genes (encoded protein or function) E. coli O104:H4 (German strain) No. of samples positive (%) Before enrichment 1 After enrichment Amplicon size, Kb wzx O104 (O104 antigen flippase) (1.2) 51 (20.6) flic H4 (H4 flagellar antigen) (41.5) 214 (86.3) stx1 (Shiga toxin 1) (14.) 144 (58.1) stx2 (Shiga toxin 2) (40.3) 188 (75.8) eae (Intimin) (37.1) 204 (82.3) ehxa (enterohemolysin) (84.5) 243 (97.8) terd (tellurite resistance) (48.0) 233 (94.0) agga (aggregative fimbrial adhesin) wzx O104 + flic H4 + 2 (0.8) 47 (20.0) wzx O104 + flic H4 + stx1 or stx2 + 2 (0.8) 41(16.5) Category: Pre harvest Pathogen Reduction Project Title: Direct Fed Microbials as an Aid in the Control of Foodborne Pathogens in Cattle Lymph Nodes and Fecal Samples Mindy Brashears, Texas Tech University Objective: To determine if supplementing diets with a high dose (10 9 /animal/day) of Lactobacillus acidophilus NP51 (HNP51) will reduce Salmonella in lymph nodes at slaughter and determine reductions in fecal pathogens for cattle supplemented with HNP51.

3 This project consisted of both commercial and research feedlot studies. The treatments for both studies were controls (i.e., not fed HNP51) and animals whose diets were supplemented with 10 9 /head/day HNP51. In the commercial study, approximately 1,800 cattle were randomized into two treatments with 12 pens/treatment and 75 head/pen. In the research feedlot study, 112 cattle were randomized to 14 pens/treatment and 4 head/pen. For the commercial and research feedlot studies, Salmonella prevalence in the lymph nodes (Figure 1) was 25% (p < 0.01) and 88% (p < 0.05) less, respectively, for cattle fed HNP51. The concentration of Salmonella on a CFU/g LN and CFU/LN basis was also less for animals given HNP51 in the commercial feedlot. A 45% (p = 0.02) and 60% (p = 0.10) reduction in E. coli O157 prevalence in fecal samples was observed among cattle administered HNP51 in the commercial and research feedlots, respectively. Fewer fecal samples collected in the commercial feedlot from animals administered HNP51 were positive for the genes that encode non O157 serogroups O26 (53% reduction relative to control; p = 0.02), O45 (41%; p = 0.02), O103 (35%; p = 0.03), and O121 (47%; p = 0.02). No differences were observed in the research feedlot study. The results of both the commercial and research feedlot studies indicate that the pre harvest administration of the direct fed microbial, HNP51, is an effective tool that aids in the control of Salmonella within lymph nodes and various pathogens in the feces of feedlot cattle. Project Title: Characterization of the Baseline Fecal Prevalence of O26, O103 and O111 Shiga Toxin producing Escherichia coli in Commercial Feedlot Cattle Cohorts Natalia Cernicchiaro, Kansas State University Objective: To characterize the baseline prevalence of O26, O103, and O111 Shiga toxin producing Escherichia coli (STEC) serogroups and associated virulence genes (stx1, stx2, eae and ehxa), based on culture and multiplex PCR, in feces of cattle in a commercial feedlot. A total of 960 fecal samples from 10 cattle cohorts (96 per cohort) representative of a population of more than 17,000 cattle that originated from six states (CO, IA, MO, ND, NE, and SD) were collected rectally prior to the animals being allocated to pens in a commercial feedlot. Cattle cohorts were defined as a group of animals that were purchased and managed similarly and were eligible for sampling based on allocation date at the feedlot. Ten presumptive E. coli colonies per sample were randomly picked, pooled and then tested by a multiplex PCR assay targeted to detect O specific serogroups (O26, O103, and O111) and four major virulence genes (stx1, stx2, eae and ehxa; Bai et al., 2012). The results showed (Table 3): 1. O26 (30.5%) and O103 (29.7%) were more prevalent than O111 (10.1%). 2. Large variability from cohort to cohort (week to week) was seen. 3. Only 12 to 24% of O serogroup positive samples had genes to be considered adulterants by FSIS.

4 Table 3. Model adjusted, animal level, fecal prevalence estimates for STEC O serogroups and virulence genes (percentages and 95% confidence intervals) by type of IMS bead. Bold type emphasizes serogroup prevalence estimates for the corresponding IMS bead type. Serogroup specific and virulence genes O26 IMS beads O103 IMS beads O111 IMS beads O ( ) 3.0 ( ) 2.3 ( ) O ( ) 24.6 ( ) 17.7 ( ) O ( ) 0.01 ( ) 0.01 ( ) stx ( ) 8.8 ( ) 7.2 ( ) stx ( ) 18.4 ( ) 14.1 ( ) eae 8.8 ( ) 10.9 ( ) 9.2 ( ) ehxa 39.1 ( ) 45.7 ( ) 45.0 ( ) The baseline prevalence estimates presented in this study provide initial point in time measures of STEC frequency at the feedlot level. In addition, these data contribute to a better understanding of the potential performance and limitations of STEC diagnostic assays and the distribution of genes indicating the potential virulence of important serogroups. Category: Pre and Post Harvest Pathogen Reduction Salmonella Project Title: Do Pre Harvest Interventions Applied for Controlling E. coli O157:H7 in Feedlot Cattle Affect Fecal Shedding of Non O157 E. coli or Salmonella? Natalia Cernicchiaro, Kansas State University Objective: To determine the effects of commercially available pre harvest interventions (vaccine and a directfed microbial) intended to reduce fecal shedding of E. coli O157 in feedlot cattle on fecal prevalence of six non O157 Shiga toxin producing E. coli (STEC) serogroups and Salmonella, and whether the presence of non O157 STEC and Salmonella are associated with the presence of O157 within feedlot cattle fecal samples. More than 17,000 cattle fed a high concentrate finishing diet with > 25% distiller s grains in 40 pens were randomized to receive one, both or neither (control) of the two interventions: a siderophore receptor and porin proteins based vaccine (E. coli SRP vaccine, Pfizer, New York, NY) and a direct fed microbial product (DFM; Bovamine, Nutrition Physiology Co., Guymon, OK). Cattle in the vaccinated groups received one dose at enrollment and again three weeks later. The DFM was fed (10 6 CFU/animal/day) throughout the study period (mean = 87 days). DNA extracted from fecal samples was tested for detection of O157 and six non O157 STEC serogroups (O26, O45, O103, O111, O121, and O145) and four major STEC virulence genes (stx1, stx2, eae, and ehxa) by an 11 gene multiplex PCR (Bai et al., 2012). In addition, a duplex real time PCR assay, based on inva and pagc genes was used to detect Salmonella. The results showed (Table 4): The vaccine, the direct fed microbial or both had no statistically significant effect on fecal prevalence of non O157 STEC or Salmonella. E. coli O157 was associated (P < 0.05)* with increased shedding of O26, O45, O103, and O121 STEC in feces; but no significant associations with Salmonella. O157, O26 and O103 were the most prevalent STEC O serogroups based on direct PCR results. Salmonella fecal prevalence was 3.3%. Two pre harvest interventions did not reduce fecal shedding of non O157 or Salmonella.

5 This information in this study showed that two commercially available pre harvest interventions did not reduce fecal shedding of non O157 or salmonella. Project Title: Methods for Controlling Escherichia coli O157:H7 and Salmonella Surrogates during the Production of Non Intact Beef Products Lindsey Mehall, Texas A&M University Objective: To evaluate the reduction and internalization of E. coli O157:H7 and Salmonella surrogate microorganisms by applying five different antimicrobial treatments to subprimal surfaces before marination. Strip loins were inoculated with one of two levels (High [~10 7 ] and low [~10 4 ]) of non pathogenic, rifampicinresistant E. coli organisms. After aging, each strip was assigned to one of five antimicrobial treatments or control: 2.5% L lactic acid and 5.0% L lactic acid applied at ~53 C, and 1,050 ppm acidified sodium chlorite, 205 ppm peroxyacetic acid, and tap water applied at room temperature (~25 C). Treated and control loins were vacuum tumbled with a commercial marinade, chilled for 24 hours, vacuum packaged, and aged for 7 to 24 days. Samples were collected throughout the experiment. Results: 24 h chill had minimal impact on surface surrogate microorganisms for both high and low inoculated strips. The log reductions following aging ranged from 0.6 to 1.8 log 10 CFU/cm 2. For both high and low inoculated strips, the 5% lactic acid treatment had the greatest log reduction of surface surrogate microorganisms, whereas water had the least. Following antimicrobial treatments, the log reductions of surface surrogate microorganisms ranged from 0.5 to 2.6 log 10 CFU/cm 2. Strips treated with peroxyacetic acid had the greatest reduction of surface surrogate organisms in the finished, marinated product. After marination, a greater than one log reduction of surface surrogate microorganisms was seen for all treatments with the exception of 2.5% lactic acid. Strips receiving water treatment resulted in greater internalization of surrogate microorganisms when compared to the control. The results indicate that lactic acid, acidified sodium chlorite, and peroxyacetic acid solutions used as treatments can minimize the number of surface pathogens capable of translocating or internalizing during the production process.

6 Project Title: Variation of Salmonella Prevalence in Bovine Lymph Nodes among South Texas Feedyards Ashley N. Haneklaus, Texas A&M University Objective: To determine the impact of cattle source on Salmonella prevalence in bovine lymph nodes located in beef products destined for further processing. A total of 307 bovine lymph nodes were collected in four collection trips over a three month span from seven feedyards selected based on predicted contribution to the harvest facility s Salmonella positive test results(table 5). Salmonella prevalence in lymph nodes was found to be 0% in cattle sourced from one of the seven feed yards. Lymph nodes from cattle sourced from the other feedyards yielded positive samples with varying prevalence. One feedyard yielded 88.2% prevalence of Salmonella in the lymph nodes, which was significantly higher than all other feedyards (42.9, 40.0, 40.0, 24.0, and 4.0%). The reason for the complete absence of Salmonella positive lymph nodes in one feedyard is unknown. Subsequent research has been initiated to help understand what environmental factors and management practices contribute to the differing results. Understanding the route of entry and duration of Salmonella infection is necessary before optimal preventive measures for managing the prevalence of Salmonella in the lymph nodes of feedyard cattle can be identified and implemented. Category: Post Harvest Pathogen Reduction Project Title: Comparison of Current and Novel Chemical Treatments to Control Escherichia coli O157:H7 and O26 on Beef Trims and Ground Beef Patties Ravirajsinh Jadeja, University of Georgia Objective: To evaluate effectiveness of various current and novel intervention treatments to reduce E. coli O157:H7 and E. coli O26:H11 from spiked beef trims and ground beef patties. For each treatment planned, beef trim samples were inoculated to achieve one of each of the following: E. coli O157:H7 at 6 (high) log CFU/cm 2 concentration, E. coli O157:H7 at 2 (low) log CFU/cm 2 concentration, E. coli O26:H11 at 6 (high) log CFU/cm 2 concentration, and E. coli O26:H11 at 2 (low) log CFU/cm 2 concentration. Inoculated trims were spray treated with one of eight single or combined antimicrobial treatments: Electrolyzed oxidizing water (EO water) 50 mg/l available chlorine for 30 s 3% Levulinic acid+ 0.5% sodium dodecyl sufate (LA SDS) for 30 s Lactic acid (LA) 5% for 30 s Blitz TM 0.02% for 30 s HB2 TM 0.03% for 30 s Deionized water (W) for 30 s Blitz for 15 s followed by lactic acid for 15 s EO water for 15 s followed by LA SDS for 15 s At the end of each treatment, two samples were used for microbial analysis while the remaining two were ground, made into four beef patties and stored at 20⁰C followed by analysis for pathogen presence on days 0, 1, 7, and 14 after storage.

7 All chemical treatments yielded significant reductions of both E. coli O157:H7 and O26 from trim and ground beef at high inoculation levels. Peroxyacetic acid (Blitz) treatment was most effective and reduced E. coli O157 and O26 by 1.70 and 1.76 logs respectively on trims. Plate counts of pathogens from frozen beef patties after 0, 1, 7 and 14 days of storage showed a declining trend with additional reductions up to 1.30 and 1.58 log cycles for E. coli O157:H7 and O26 respectively after 14 days of frozen storage. The efficacy of the treatments for reducing E. coli O157:H7 was equally if not more effective in reduction of E. coli O26:H11 from beef trims and beef patties: Blitz HB2 (Blits+La) LA SDS La (EO+LA SDS) EO>W. Project Title: Inactivation of the Emerging Pathogen Escherichia albertii in Ground Beef following Cooking T. Matt Taylor, Texas A&M University Objective: To determine reduction of E. albertii in vitro following exposure to 3.0% lactic acid as a function of acid solution ph and verify E. albertii inactivation through cooking of ground beef to USDA recommended inhome cooking guidelines The Gram negative enteric pathogen E. albertii represents an emerging food safety challenge. It has been recently associated with the onset of a localized outbreak of foodborne disease in an industrialized nation. While the U.S. disease risk has not been estimated, researchers have detailed the ability of this pathogen to survive on ground beef during refrigerated storage and to resist several classes of therapeutic antibiotics. Lactic acid interventions, provided sufficient contact with the pathogen and proper application (temperature of solution, concentration, ph of 3.0), are expected to be effective for a reduction in all strains of E. albertii on meat carcasses or products. Lactic acid at a ph of 4.0 significantly reduced pathogen levels but not as significantly for all strains. Cooking the inoculated ground beef to 71⁰C afforded at least a 4.5 log reduction in E. albertii. Medium rare preparation produced a minimum 3.6 log reduction in pathogen loads on average. Should high levels of contamination be observed on trim prior to grinding, such cooking procedures are not necessarily effective at fully eradicating the pathogen, though at levels typical of sanitary processing facilities, such reductions would be expected to produce a product safe for consumption. Figure 2 illustrates the log reduction as a result of cooking to three different temperatures for E. albertii and its Rif mutant, E. albertii 9194 and its Rif mutant, and E. albertii and its Rif mutant. Figure 2

8 Project Title: Escherichia coli O157:H7 as an Indicator for Efficacy of Antimicrobial Interventions to Control non O157 Shiga Toxin producing E. coli (STEC) Alejandro Echeverry, Texas Tech University Objective: To validate and provide scientific support for the assumption that currently implemented interventions will efficaciously reduce the non O157 STECs (O121, O45, O145, O26, O111 and O103) as effectively as O157:H7. The study characterized reductions of E. coli O157 and six non O157 STEC, in parallel, following application of hot water (HW, 180 F), lactic acid (LA, 5% ± 0.1) and acidified sodium chlorite (ASC, 1200 ppm), individually, utilizing replicate hot brisket and chilled subprimal models. The results varied among intervention methods and STEC strains as follows: 5% lactic acid efficacy on hot brisket o Significant reductions ranging from 0.3 to over 1 log 10 to all STEC serogroups o Most effective against STEC O121 and least effective against O111 o STEC O111 was the only serogroup that differed significantly from O157 5% lactic acid efficacy on chilled subprimal o Efficacy ranged from 0.34 to 0.66 log 10 on all STECs except O103 which was not effectively reduced o Most effective against O157 on chilled tissue 180⁰F hot water efficacy on hot brisket o Less effective against STEC serogroups and more variable o Serogroups O157, O26, O121, O145 and O45 exhibited reduction while O103 and O111 were not reduced 180⁰F hot water efficacy on chilled subprimal o Variable reduction efficacy across serogroups o Reduction ranged from 0.07 for O103 to 0.62 log 10 for O157, while all non O157 STECs differed significantly when compared to O157 with the exception of O ppm acidified sodium chlorite efficacy on hot brisket o Not effective against O103 and O111 (similarly to hot water) o Most effective against O ppm acidified sodium chlorite efficacy on chilled subprimal o Reductions across all STEC serogroups o Most effective against O45 and least effective against O111 o All non O157s were reduced similarly to, or more than O157. Project Title: Simulation of Contamination through the Post Harvest Environment Using Fluorescent Surrogate Organisms Mariana Villarreal Silva, Texas A&M University Objective: The purpose of this study was to determine the effect of the post harvest environment on the direct and indirect transmission of pathogens. Researchers traced the cross contamination from carcasses inoculated with non virulent Escherichia coli used as surrogates for E. coli O157:H7 and Salmonella to adjacent carcasses, equipment and plant environment in three different abattoirs. Inoculated carcasses and carcasses immediately following each of the inoculated carcasses were sampled at different stages of harvesting. Samples were also collected from floors, walls, and air, personnel gear including gloves, boots and aprons, and equipment and utensils including hand knives, air knives, meat hooks, hide puller and split saw.

9 The results indicated that cross contamination occurred between inoculated and immediately adjacent carcasses, and from carcasses to walls, floor, aprons, boots and hide pullers. Generally, the microorganisms remained on the carcasses until the final carcass intervention though the counts differed significantly between abattoirs. The lowest counts and prevalence of positive samples were found in the smallest abattoir (25 head/hour) and with a plant layout following a straight line. The fact that none of the carcasses showed detectable counts after the carcass intervention step underscores the importance of interventions in reducing pathogens on beef carcasses. In general, inoculated bacteria were detected in 15% of the samples from equipment and in 10% of the environmental samples. Project Title: Evaluation of Beef Carcass Washing Technologies: Inorganic Acid vs. Organic Acid Jason Sawyer, Tarleton State University Objective: To evaluate carcass washing technologies of organic acid (lactic acid) and an inorganic acid (buffered sulfuric acid AFTEC 3000) for effectiveness at reducing microbial loads during beef carcass processing within a commercial processing facility. Researchers sampled sixty beef carcasses (N = 30/treatment) at three intervals (prior to entering the wash cabinet; following a 180 second drip period after entering the chilling coolers; four hours after carcass washing) during a normal production shift at a small commercial slaughter facility. Results suggested AFTEC as a carcass washing intervention for beef carcasses is as effective as lactic acid in reducing surface organisms (Figure 3). The benefits of using AFTEC include its more stable shelf life and its usage rate is 50% less than recommended Lactic Acid levels for comparable microbial reductions.

10 Project Title: Validation of the Commercial Use of Thermal Pasteurization Combined with a Bromine Solution as Whole Carcass Antimicrobial Intervention when Tested at Operational Parameters Xiang Yang, Colorado State University Objective: To validate the use of a bromine solution independently or combined with hot water pasteurization cabinets, to reduce bacterial pathogen surrogates inoculated onto beef carcasses in a large scale plant (Experiment 1) and a small scale plant (Experiment 2). On forty eight carcasses, researchers designated three sites for non inoculated sampling to numerate natural flora and three sites for inoculated areas using a 5 strain mixture of non pathogenic Escherichia coli, previously found to be surrogates for E. coli O157:H7 and Salmonella Typhimurium. Each carcass side was conveyed automatically through a bone dust cabinet spraying a bromine chemical solution (sodium bromide [NaBr] with bleach) followed by a cabinet applying hot water at 94.6⁰C. Overall, while the bromine solutions statistically lowered the bacterial populations, their impact was not of biological significance. The reductions were small in contrast to the impact of thermal treatments using hot water (Figure 6). Project Title: Validation of Commercial Hot Water Thermal Pasteurization for use as a Whole Carcass Antimicrobial Intervention at Operational Parameters Carlie C. Perham, Colorado State University Objective: To validate the use of a hot water pasteurization cabinet to reduce bacterial populations on beef carcasses at operational parameters at a large and small beef plant. On forty eight carcasses at each of two plants, researchers designated two sites for non inoculated sampling to numerate natural flora and two sites for inoculated areas using a 5 strain mixture of non pathogenic Escherichia coli, previously found to be surrogates for E. coli O157:H7 and Salmonella Typhimurium. Samples were collected directly prior to and after pasteurization.

11 Hot water pasteurization reduced populations of natural and surrogate bacteria on all beef carcasses (Figure 4). With high percentages of samples being below the detectable limit after treatment, the effectiveness of thermal pasteurization in both plant systems was achieved Cattlemen s Beef Board and National Cattlemen s Beef Association May 29, 2013