Escherichia coli O 157 : H 7 Populations in Ruminants Can Be Reduced by Orange Peel Product Feeding 3

Foodborne pathogenic bacteria such as Escherichia coli O157:H7 are threats to the safety of beef. Citrus peel and dried orange pulp are by-products from citrus juice production that have natural antimicrobial effects and are often incorporated into least-cost ration formulations for beef and dairy cattle. This study was designed to determine if orange peel and pulp affected E. coli O157:H7 populations in vivo. Sheep (n ~ 24) were fed a cracked corn grain–based diet that was supplemented with a 50-50 mixture of dried orange pellet and fresh orange peel to achieve a final concentration (dry matter basis, wt/wt) of 0, 5, or 10% pelleted orange peel (OP) for 10 days. Sheep were artificially inoculated with 10 CFU of E. coli O157:H7 by oral dosing. Fecal shedding of E. coli O157:H7 was measured daily for 5 days after inoculation, after which all animals were humanely euthanized. At 96 h postinoculation, E. coli O157:H7 shedding was reduced (P , 0.05) in sheep fed 10% OP. Populations of inoculated E. coli O157:H7 were reduced by OP treatment throughout the gastrointestinal tract; however, this reduction reached significant levels in the rumen (P , 0.05) of sheep fed 10% OP diets. Cecal and rectal populations of E. coli O157:H7 were reduced (P , 0.05) by inclusion of both 5 and 10% OP diets. Our results demonstrate that orange peel products can be used as a preharvest intervention strategy as part of an integrated pathogen reduction scheme. In spite of the numerous hurdles that have been implemented throughout the food production chain to ensure food safety, foodborne illnesses still affect more than 48 million Americans each year (27). One of the most significant foodborne pathogenic bacteria is Escherichia coli O157:H7, which is typically associated with beef due to its natural reservoir in the gastrointestinal tract of cattle and other ruminant animals (6, 11). Each year, E. coli O157:H7 and other related enterohemorrhagic E. coli strains cause 93,000 human illnesses at a cost to the U.S. economy of more than US$1 billion per year (27, 28). Since E. coli O157:H7 is an asymptomatic resident of the gastrointestinal tract of cattle, reducing E. coli O157:H7 populations in the live animal can have a significant impact with regard to improving human health and safety (7, 24, 26). Phytochemicals, such as essential oils, are naturally occurring antimicrobials found in many plants that show promise in a variety of applications by decreasing bacterial growth and survival (15, 16, 30). The addition of . 1% orange peel and pulp to mixed ruminal fluid fermentations reduced populations of E. coli O157:H7 and Salmonella Typhimurium (9, 22). Further studies have demonstrated that feeding orange peel and pulp reduced intestinal populations of diarrheagenic E. coli in weaned swine (12) and of Salmonella in experimentally infected sheep (8). Orange peel and pulp are by-product feedstuffs commonly fed to cattle and have a good nutritive value for ruminants (2). Therefore, the present study was designed to determine if using fresh and preserved orange peel and pulp as a dietary component could reduce gastrointestinal populations of E. coli O157:H7 in an experimentally inoculated sheep model. MATERIALS AND METHODS Bacterial cultures. E. coli O157:H7 strain 43895, taken from the Food and Feed Safety Research Unit culture collection, was repeatedly grown by 10% (vol/vol) transfer in anoxic (85% N2, 10% CO2, 5% H2 atmosphere) tryptic soy broth (TSB) medium at 37uC. This strain was selected for resistance to novobiocin and nalidixic acid (20 and 25 mg/ml, respectively) by repeated transfer and selection in the presence of sublethal concentrations of each antibiotic. This resistant phenotype was stable through multiple unselected transfers in batch culture and through repeated culture * Author for correspondence. Tel: 979-260-9374; Fax: 979-260-9332; E-mail: todd.callaway@ars.usda. gov. { Proprietary or brand names are necessary to report factually on available data; however, the U.S. Department of Agriculture neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies neither approval of the product, nor exclusion of others that may be suitable. USDA is an equal opportunity provider and employer. 1917 Journal of Food Protection, Vol. 74, No. 11, 2011, Pages 1917–1921 doi:10.4315/0362-028X.JFP-11-234 vessel turnovers in continuous culture (data not shown). Overnight cultures (1 liter) were harvested by centrifugation (7,500 | g, 10 min), and cell pellets were resuspended in TSB medium (total volume, 150 ml). Populations of E. coli O157:H7 in these cell suspensions were determined to be approximately 3 | 10 CFU/ ml, by serial dilution and plating as described below. Sheep, rations, and experimental design. All procedures in this study were approved by the Institutional Animal Care and Use Committee (IACUC protocol 05-001). Rambouillet/Suffolk sheep (average body weight, 60 kg) were purchased from a commercial feedlot and were transported to the laboratory. Sheep were fed a commercial high-grain ration composed of (on a dry matter [DM] basis) cracked corn (74.4%), soybean meal (9.2%), urea (0.7%), and trace mineral salts (0.4%) and supplemented with coastal Bermuda grass hay (15.3%). The diet was formulated according to National Research Council recommendations, and sheep were allowed ad libitum access to water. Sheep were housed in environmentally controlled facilities, and feces from each sheep were sampled on arrival and each subsequent day (n ~ 7) during the dietary and facility adaptation period to verify that no organisms capable of growth on MacConkey’s agar plates supplemented with novobiocin (20 mg/ ml) and nalidixic acid (25 mg/ml) (MacNN) were present in the sheep. During this period no colonies grew on any of the MacNN plates. Sheep (n ~ 24) were randomly assigned to one of three treatment groups (n ~ 8 in each group) that were fed diets supplemented with a 50-50 (DM basis, wt/wt) mixture of dried orange pellet and fresh orange peel to obtain final concentrations (DM basis, wt/wt replacement of concentrate portion of the ration) of 0, 5, or 10% orange product (OP; Texas Citrus Exchange, Mission, TX). Sheep were housed in enclosed facilities and were maintained on their respective diets for 10 days prior to the initiation of the study and throughout the entire study. Sheep were artificially inoculated with E. coli O157:H7 (3 | 10 CFU per sheep) via oral gavage (total volume per sheep, 10 ml) at 0 h. Every 24 h for 96 h after inoculation, fecal samples were collected via rectal grab from individual sheep, and populations of inoculated E. coli O157:H7 were determined. Gastrointestinal sample collection. Sheep were humanely euthanized at 96 h after the E. coli O157:H7 inoculation.