Influence of b-Agonists (Ractopamine HCl and Zilpaterol HCl) on Fecal Shedding of Escherichia coli O157:H7 in Feedlot Cattle3

Ractopamine HCl and zilpaterol HCl, b-agonists recently approved for use in feedlot cattle to improve performance traits and carcass leanness, were examined for their effects on fecal shedding of Escherichia coli O157:H7 in feedlot cattle. Fecal samples (n ~ 2,454) were obtained from four experiments (one ractopamine HCl, three zilpaterol HCl) over the course of a 3year period, either by rectal palpation (ractopamine HCl experiment) or from pen-floor fecal pats. Samples were cultured quantitatively and qualitatively for E. coli O157:H7. No significant treatment differences were detected for fecal prevalence of E. coli O157:H7 in the ractopamine HCl experiment. Zilpaterol HCl feeding had no effect (P . 0.20) on fecal shedding in the first or second experiments, with overall E. coli O157:H7 prevalence relatively low (,7%). In the third zilpaterol HCl experiment, the percentage of fecal samples that were E. coli O157:H7 positive following qualitative culture was higher (P , 0.05) in the zilpaterol HCl treatment (10.3%) than for the control (6.1%). The current research showed minimal effects of b-agonists on fecal shedding of E. coli O157:H7 and indicated that these compounds (fed immediately prior to slaughter) are not a cause for concern from a food safety standpoint. b-Agonists (clenbuterol, cimaterol, ractopamine, zilpaterol) are organic molecules that bind to b-adrenergic receptors and repartition nutrients to increase the lean/ adipose tissue ratio (8, 9). Ractopamine HCl was approved for use in feedlot cattle in 2003, and in 2006 the U.S. Food and Drug Administration approved zilpaterol HCl to be fed to beef cattle in the last 20 to 40 days of the finishing period to increase rate of gain, improve feed efficiency, and increase carcass leanness (1, 8, 9, 13). The ‘‘physiological counterparts’’ to synthetic b-agonists are the hormones norepinephrine and epinephrine (7), reported to be involved in the quorum-sensing system that bacteria and host cells use to communicate (12). This communication system has been called ‘‘a global regulatory mechanism for basic physiological functions of E. coli O157:H7’’ (11); it regulates virulence gene expression (12) and stimulates the growth of intestinal populations (5, 6). Earlier research by the authors examined the effect of ractopamine HCl supplementation on fecal shedding of Escherichia coli O157:H7 in beef cattle, sheep, and pigs (3, 4); it was our hypothesis that ractopamine HCl would act similarly to the catecholamines and thereby increase fecal shedding. However, in two experiments we observed just the opposite effect: a decrease in fecal shedding of E. coli O157:H7 in ractopamine HCl–treated animals. While the effects on fecal shedding (measured qualitatively as shedding or not shedding) were statistically significant, they were not 100% consistent among all pens of cattle, nor did we measure fecal shedding quantitatively. Therefore, the objectives of the current research were to examine (quantitatively and qualitatively) the effect of b-agonist (ractopamine HCl and zilpaterol HCl) supplementation on fecal shedding of E. coli O157:H7 in feedlot cattle. MATERIALS AND METHODS The sample collections described below were made from four experiments designed to evaluate the effects of various feeding strategies of Optaflexx (10% ractopamine HCl; Elanco Animal Health, Greenfield, IL) or Zilmax (4.8% zilpaterol HCl; Intervet/ Schering-Plough Animal Health, De Soto, KS) on performance and carcass characteristics of feedlot cattle (results presented elsewhere). Fecal samples were collected from each of these larger experiments, with the objective of determining if supplementing feedlot cattle with either of these b-agonists influenced shedding of E. coli O157:H7. * Author for correspondence. Tel: 979-260-3757; Fax: 979-260-9332; E-mail: [email protected]. { Mention of trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable. 2587 Journal of Food Protection, Vol. 72, No. 12, 2009, Pages 2587–2591 Ractopamine HCl experiment. The effect of ractopamine HCl supplementation on feedlot cattle in a commercial feedlot in the southwestern United States was examined in the fall of 2006 (October through November). Fifty-six pens of crossbred beef heifers (nine head per pen; seven pens per treatment) were used in a study using a 2 | 3 factorial design consisting of two ractopamine HCl levels (0 and 200 mg per head per day) and three ractopamine HCl feeding durations (14, 28, or 42 days). Ractopamine HCl was incorporated into the total mixed ration per manufacturer’s recommendations during the final phase of the finishing period, immediately prior to slaughter. Aside from ractopamine HCl feeding and duration, all heifers were managed in a manner typical for a commercial feedlot operation in this region of the United States. One block of pens were harvested on day 1 to establish a baseline for comparison of the serial harvest data, but they were not sampled for the prevalence of E. coli O157:H7. Heifers were restrained in a squeeze chute, and fecal samples (approximately 50 g) were collected via rectal palpation using sterile palpation sleeves. Fecal samples were collected on day 0 (immediately prior to ractopamine HCl supplementation) and on days 14, 28, and 42 immediately prior to shipping to the slaughter facility. Fecal samples were placed on ice and shipped overnight to our laboratory in College Station, TX, for bacterial culture (described below) on the following day. Zilpaterol HCl: experiment 1. The animals sampled in this experiment were part of a larger experiment (conducted during the summer of 2007 in the western United States) in which 320 Holstein steers were used in a feedlot trial to determine the influence and duration of zilpaterol HCl on carcass yield. Upon arrival to the feedlot, steers were weighed, eartagged, and sorted by body weight into eight groups. Within body weight groupings, steers were assigned to 10 blocks of four pens, eight steers per pen. Treatments were randomly assigned to blocks and consisted of feeding zilpaterol HCl for 0 (control), 20, 25, or 30 days. All cattle were fed the control diet for a 9-day acclimation period prior to initiation of the experiment. Zilpaterol HCl was fed at 7.56 g/ton of the total mixed ration (100% dry matter basis). Following a 3-day withdrawal period, 20 blocks of cattle were shipped to slaughter; the remaining 5 blocks were shipped the following day. Fresh fecal samples (five per pen) were collected from the pen floor using sterile palpation sleeves at the time of shipment to the slaughter facility. One pen per treatment was used for animals not performing well on the study (for various health and other issues) and was therefore excluded from sampling (n ~ 180 fecal samples collected). Samples were shipped as above for processing. Zilpaterol HCl: experiment 2. Fecal samples were collected from two groups of crossbred beef steers located on a commercial feedlot in the southwestern United States. Each group of cattle had two staggered slaughter dates (January to April 2007). Fecal samples (collected as above) were obtained from each group of cattle on the later slaughter date (February and April) based on the expected low seasonal prevalence of E. coli O157:H7. Cattle were housed in drylot pens (approximately 100 head per pen) and subjected to management and feeding as normal for this region of the United States. Within each group of cattle, zilpaterol HCl (7.56 g of zilpaterol HCl per ton of total mixed ration [100% dry matter basis]) was fed for 0 (control), 20, 30, or 40 days prior to slaughter. A 3-day withdrawal period immediately preceded slaughter. Three pens from each treatment (feeding duration), representing both groups of cattle, were sampled (30 samples per pen; 360 samples per group; n ~ 720 total). Treatments were the same for each group, but due to the different collection times, data were analyzed and are presented by group (date of collection). Zilpaterol HCl: experiment 3. Crossbred beef heifers located on a commercial feedlot in the southwestern United States were fed 0 (control) or 7.56 g of zilpaterol HCl per ton of total mixed ration (100% dry matter basis) for 20 days immediately prior to slaughter. There was a 3-day withdrawal period immediately prior to slaughter. Animals were housed in drylot pens (approximately 95 head per pen) and managed as typical for a feedlot in this region of the United States. Cattle harvest dates were staggered; therefore, two fecal sample collections were made in June and July 2008, within 4 days of slaughter. Thirty fecal samples per pen (nine pens per treatment) were collected prior to each harvest date, for a total of 360 samples per date or 720 total samples. Fecal samples (approximately 50 g) were collected from freshly voided fecal pats, placed in sterile specimen cups, and shipped to our laboratory as described previously. Bacterial culture, isolation, and enumeration methods. All fecal samples were processed the day following collection for qualitative and quantitative analysis of E. coli O157:H7 as described previously (10) and modified (2). For the ractopamine HCl study only, all fecal samples were kept refrigerated and all immunomagnetic separation–positive samples were further examined for quantitative analysis of E. coli O157:H7. One gram of feces was serially diluted in phosphate-buffered saline, and 0.1-ml aliquots were plated and confirmed as E. coli O157:H7 as above. Enumeration of E. coli O157:H7 for the remainder of the studies was performed as referenced above (2, 10). Statistical analysis. Data were analyzed using SAS version 8.02 (SAS Institute, Inc., Cary, NC). The percentage of cattle shedding E. coli O157:H7 and the percentage of pens positive (pen with at least one animal shedding) were subjected to chi-square analysis using the PROC FREQ procedure. The concentration of E. coli O157:H7 [CFU (log)/g of feces] was subjected to analysis of variance appropriate for a completely randomized design. A value of 1.0 was assigned to all culture-negative animals for analysis of the quantitative shedding data. When appropriate, data were analyzed by duration of b-agonist feeding. Differences in means were considered significant at P , 0.05.