Development of methods for the recovery of Escherichia coil O157:H7 and Salmonella from beef carcass sponge samples and bovine fecal and hide samples

Culture methods were developed for the concurrent recovery of Escherichia coli O157:H7 and Salmonella from bovine carcass, hide, and fecal samples. Several enrichment conditions were tested for the overall growth of pure cultures; tryptic soy broth for 2 h at 25 degrees C and then for 6 h at 42 degrees C was the protocol selected for use. Immunomagnetic separation (IMS) was incorporated for sensitivity and selectivity, along with a post-IMS enrichment for the recovery of Salmonella as recommended by the manufacturer. Selective agars for plating after IMS were chosen on the basis of ease of target colony identification. Sorbitol MacConkey agar supplemented with cefixime and potassium tellurite and Rainbow agar supplemented with novobiocin and potassium tellurite were chosen for the recovery of E. coli O157:H7. Brilliant green agar with sulfadiazine and Hektoen enteric agar supplemented with novobiocin were selected for the recovery of Salmonella. The resulting methods were evaluated along with standard or previously used methods for the recovery of E. coli O157:H7 and Salmonella from bovine hide and fecal samples and carcass sponge samples. The Meats Research Unit (MRU) methods performed at least as well as the established methods, except that a secondary enrichment in tetrathionate (TT) broth prior to IMS was required for the optimal recovery of Salmonella from feces. Thus, the MRU and MRU-TT methods are effective in the recovery of both E. coli O157:H7 and Salmonella from a single bovine carcass, hide, or fecal sample.     

Use of steam condensing at subatmospheric pressures to reduce Escherichia coli O157:H7 numbers on bovine hide.

This study used a laboratory-scale apparatus to apply subatmospheric steam to bovine hide pieces inoculated with Escherichia coli O157:H7 in maximum recovery diluent (MRD) and in high-liquid content and low-liquid content fecal suspensions (HLC fecal and LLC fecal, respectively). The survival of the organism in fecal clods, which were stored for 24 days in a desiccated state, was assessed. Inoculated fecal clods were also treated with subatmospheric steam. Steam treatment at 80 +/- 2 degrees C for 20 s reduced E. coli O157:H7 concentrations on hide inoculated to initial concentrations of approximately 7 log10 CFU/g by 5.46 (MRD inoculum), 4.17 (HLC fecal inoculum), and 5.99 (LLC fecal inoculum) log10 CFU/g. The reductions achieved in samples inoculated with LLC feces were larger than in samples inoculated with HLC feces (P < 0.05). Treatment at 80 +/- 2 degrees C for 10 s resulted in significantly smaller reductions (P < 0.05) on hide pieces of 2.54 (MRD), 1.94 (HLC fecal), and 2.15 (LLC fecal) log10 CFU/g. There were no significant differences among the reductions observed in all inoculum types in samples treated for 10 s. E. coli O157:H7 inoculated in fecal clods to 7.78 log10 CFU/g and stored at 4 or 15 degrees C survived for at least 24 days. Steam treatment (20 s) of 3-day-old clods reduced surviving E. coli O157:H7 numbers from 4.20 log10 CFU/g to below the limit of detection of the assay used (1.20 log10 CFU/g). This study shows that steam condensing at or below 80 +/- 2 degrees C can reduce E. coli O157:H7 when present on bovine hide, reducing the risk of cross contamination to the carcass during slaughter and dressing.     

Development of an experimental model to assess the ability of Escherichia coli O157:H7-inoculated fecal pats to mimic a super shedder within a feedlot environment

This study was conducted to develop an experimental model that could assess the ability of Escherichia coli O157:H7-inoculated fecal pats to mimic a super shedder (>10(4) CFU/g of feces) within a feedlot environment. The day before the study began, 48 steers that had been negative for E. coli O157:H7 in feces for three consecutive weeks were sorted into three treatment groups, with two replicate pens per treatment and 8 steers per pen. Steers within the pens (20.50 by 10.75 m) were exposed to control feces or feces inoculated with two levels of a mixture of five strains of nalidixic acid-resistant E. coli O157:H7 (low level, 10(2) CFU/g; high level, 10(5) CFU/g). Five 300-g fecal pats were introduced into the pens twice daily (10:00 a.m. and 2:30 p.m.) on days 0 through 6 and days 14 through 20. Pats were placed in the pen at random locations to mimic defecation of a steer within the pen. Fecal grab samples, hide swab samples (500-cm2 area of the rump), natural fecal pat samples (freshly voided), and rope samples (1.22-m-long manila rope) where obtained at multiple times during the 49-day trial to evaluate the spread of nalidixic acid-resistant E. coli O157:H7 throughout the feedlot environment and among penmates. Immunomagnetic separation and selective media were used to detect E. coli O157:H7. Nalidixic acid-resistant E. coli O157:H7 was detected in 13 high-level treatment fecal grab samples, 7 high-level treatment hide swab samples, 1 low-level hide swab sample, and 2 high-level rope samples. For both fecal grab and hide swab samples, the overall prevalence of E. coli O157:H7 in the high-level group was greater (P < 0.01) than that for the pooled low-level and control groups. Addition of inoculated fecal pats to pens increased transmission of E. coli O157:H7 among penmates, but cattle that acquired E. coli O157:H7 shed the bacterium for only a short time at low levels. Transmission of E. coli O157:H7 from the feces of super shedders to naive penmates may contribute to the observed transient nature of shedding of E. coli O157:H7 among feedlot cattle.     

Effects of recovery, plating, and inoculation methods on quantification of Escherichia coli O157:H7 and Listeria monocytogenes from strawberries

Effects of different recovery and inoculation methods on quantification of Escherichia coli O157:H7 and Listeria monocytogenes from strawberries were studied. Strawberries were spot or dip inoculated with 7 to 8 log CFU per strawberry of each pathogen, air dried for 2 h, and stored for 1, 3, and 7 days at 4 degrees C. The inoculated samples were stomached or washed with phosphate-buffered saline (PBS; pH 7.2) or with modified PBS (pH 8.4). Bacterial levels were determined using a direct selective plating, thin agar layer plating, or membrane-transferring plating (MTP) with tryptic soy agar and sorbital MacConkey agar (E. coli O157:H7) or modified Oxford agar (L. monocytogenes). Under most test conditions, washing with PBS followed by MTP had significantly higher (P < 0.05) recovery for both bacteria compared with other tested methods. Within a 7-day storage period for spot-inoculated strawberries, a stomaching step resulted in an injury of 0.9 to 1.4 log CFU for E. coli O157: H7 and 1.4 to 1.7 log CFU for L. monocytogenes. When a washing step was used instead, this resulted in an injury of only 0.2 to 0.6 log CFU for E. coli O157:H7 and 0.2 to 0.7 log CFU for L. monocytogenes. Both bacteria could survive on strawberry surfaces, but their recovered levels decreased with the increase of storage time at 4 degrees C for both spot and dip inoculation methods. Dip inoculation generally had a lower recovery than spot inoculation. An ideal protocol to recover and enumerate E. coli O157:H7 and L. monocytogenes from strawberries involved shaking and washing samples with 100 ml of PBS for 15 min at 22 degrees C coupled with a MTP enumeration method.     

Seasonal prevalence of Shiga toxin-producing Escherichia coli, including O157:H7 and non-O157 serotypes, and Salmonella in commercial beef processing plants

The seasonal prevalence of Escherichia coli O157:H7, Salmonella, non-O157 E. coli (STEC), and stx-harboring cells was monitored at three Midwestern fed-beef processing plants. Overall, E. coli O157:H7 was recovered from 5.9% of fecal samples, 60.6% of hide samples, and 26.7% of carcasses sampled before the preevisceration wash. This pathogen also was recovered from 1.2% (15 of 1,232) of carcasses sampled at chilling (postintervention) at approximate levels of <3.0 cells per 100 cm2. In one case, the E. coli O157:H7 concentration dropped from ca. 1,100 cells per 320 cm2 at the preevisceration stage to a level that was undetectable on ca. 2,500 cm2 at the postintervention stage. The prevalence of E. coli O157:H7 in feces peaked in the summer, whereas its prevalence on hide was high from the spring through the fall. Overall, Salmonella was recovered from 4.4, 71.0, and 12.7% of fecal, hide, and preevisceration carcass samples, respectively. Salmonella was recovered from one postintervention carcass (of 1,016 sampled). Salmonella prevalence peaked in feces in the summer and was highest on hide and preevisceration carcasses in the summer and the fall. Non-O157 STEC prevalence also appeared to vary by season, but the efficiency in the recovery of isolates from stx-positive samples ranged from 37.5 to 83.8% and could have influenced these results. Cells harboring stx genes were detected by PCR in 34.3, 92.0, 96.6, and 16.2% of fecal, hide, preevisceration carcass, and postintervention carcass samples, respectively. The approximate level of non-O157 STEC and stx-harboring cells on postintervention carcasses was > or = 3.0 cells per 100 cm2 for only 8 of 199 carcasses (4.0%). Overall, the prevalence of E. coli O157:H7, Salmonella, and non-O157 STEC varied by season, was higher on hides than in feces, and decreased dramatically, along with pathogen levels, during processing and during the application of antimicrobial interventions. These results demonstrate the effectiveness of the current interventions used by the industry and highlight the significance of hides as a major source of pathogens on beef carcasses.     

Rapid detection of Escherichia coli O157:H7 inoculated in ground beef,chicken carcass,and lettuce samples with an immunomagnetic chemiluminescence fiber-optic biosensor

A biosensor was evaluated with regard to its usefulness in the rapid detection of Escherichia coli O157:H7 inoculated in ground beef, chicken carcass, and romaine lettuce samples. The biosensor consisted of a chemiluminescence reaction cell, a fiber-optic light guide, and a luminometer linked to a personal computer in conjunction with immunomagnetic separation. The samples inoculated with E. coli O157:H7 were first centrifuged and suspended in buffered peptone water and then incubated with anti-E. coli O157 antibody-coated magnetic beads and horseradish peroxidase (HRP)-labeled anti-E. coli O157 antibodies to form antibody-coated bead-bacterium-HRP-labeled antibody sandwich complexes. Finally, the sandwich complexes were separated from the samples in a magnetic field and reacted with luminol in the reaction cell. The number of E. coli O157:H7 cells was determined by collecting the HRP-catalyzed chemiluminescence signal from the bead surface through a fiber-optic light guide and measuring the signal with a luminometer. The chemiluminescence biosensor was specific for E. coli O157:H7 in samples containing other bacteria, including Salmonella Typhimurium, Campylobacter jejuni, and Listeria monocytogenes. The chemiluminescence signal was linear on a log scale from 10(2) to 10(5) CFU of E. coli O157:H7 per ml in samples. Detection could be completed within 1.5 h without any enrichment. The detection limits for ground beef, chicken carcass, and lettuce samples were 3.2 x 10(2), 4.4 x 10(2), and 5.5 x 10(2) CFU of E. coli O157:H7 per ml, respectively.     

Comparison of fecal versus rectoanal mucosal swab sampling for detecting Escherichia coli O157:H7 in experimentally inoculated cattle used in assessing bacteriophage as a mitigation strategy

This study was conducted to compare fecal grab (FEC) and rectoanal mucosal swab (RAMS) techniques as sampling methods for surveillance of Escherichia coli O157:H7 in conjunction with administration of a mitigation therapy. The study was nested within a larger experiment that investigated bacteriophage as a preharvest strategy for controlling E. coli O157:H7 in feedlot steers. Samples (FEC and RAMS) were collected from 16 of the 32 feedlot steers (control and oral bacteriophage treatment; n = 8) involved in the mitigation study. All steers had been inoculated on day 0 with 10(10) CFU of nalidixic acid-resistant E. coli O157:H7, and samples were collected on 16 occasions over the next 83 days. FEC samples were assessed by direct plating of serial dilutions in PBS, plus a 6-h enrichment and immunomagnetic separation when E. coli O157:H7 concentrations were below limits detectable by direct plating (i.e., <1 log CFU/g). All RAMS samples were assessed by enrichment and immunomagnetic separation. E. coli O157:H7 was detected more frequently (P < 0.01) by FEC than by RAMS. Overall, 213 of 256 samples were positive either by FEC or RAMS. Discrepancies between sampling techniques were observed in 63 of the 213 positive samples; FEC missed 11 samples that were positive by RAMS, and RAMS missed 52 of those positive by FEC (miss rates of 5.16 and 24.41%, respectively). Kappa values (0.36 to 0.45) indicated only fair to moderate agreement between FEC and RAMS results, but this agreement was higher at lower levels of E. coli O157:H7 shedding (later in the experimental period). Selection of sampling procedure could significantly influence the assessed merit during testing of potential strategies for controlling E. coli O157:H7 on the farm.     

Evaluation of peroxyacetic acid as a post-chilling intervention for control of Escherichia coli O157:H7 and Salmonella Typhimurium on beef carcass surfaces

Four experiments were conducted to test the efficacy of peroxyacetic acid as a microbial intervention on beef carcass surfaces. In these experiments, beef carcass surfaces were inoculated with fecal material (no pathogens) or fecal material containing rifampicin-resistant Escherichia coli O157:H7 and Salmonella Typhimurium. Inoculated surfaces were subjected to a simulated carcass wash with and without 2% l-lactic acid treatment before chilling. In Experiments 1 and 2, the chilled carcass surfaces were sprayed with peroxyacetic acid (200 ppm; 43°) for 15 s. Peroxyacetic acid had no effect on microbial counts of any organism measured on these carcass surfaces. However, lactic acid reduced counts of E. coli Type I (1.9log(10) CFU/cm(2)), coliforms (3.0log(10) CFU/cm(2)), E. coli O157:H7 (2.7log(10) CFU/cm(2)), and S. Typhimurium (2.8log(10) CFU/cm(2)) entering the chilling cooler and prevented growth during the chilling period. In Experiment 3, peroxyacetic acid at different concentrations (200, 600, and 1000 ppm) and application temperatures (45 and 55 °C) were used to investigate its effectiveness in killing E. coli O157:H7 and S. Typhimurium compared to 4% l-lactic acid (55 °C). Application temperature did not affect the counts of either microorganism. Peroxyacetic acid concentrations up to 600 ppm had no effect on these microorganisms. Concentrations of 1000 ppm reduced E. coli O157:H7 and S. Typhimurium by up to 1.7 and 1.3log(10) CFU/cm(2), respectively. However, 4% lactic acid reduced these organisms by 2.7 and 3.4log(10) CFU/cm(2), respectively. In Experiment 4, peroxyacetic acid (200 ppm; 43 °C) was applied to hot carcass surfaces. This treatment caused a 0.7log(10) CFU/cm(2) reduction in both E. coli O157:H7 and S. Typhimurium. The collective results from these experiments indicate that peroxyacetic acid was not an effective intervention when applied to chilled inoculated carcass piece surfaces.     

Inoculation of beef with low concentrations of Escherichia coli O157:H7 and examination of factors that interfere with its detection by culture isolation and rapid methods

Currently used industry testing programs require the ability to detect Escherichia coli O157:H7 in samples of beef trim or ground beef at levels as low as 1 CFU/375 g. We present a reliable protocol for generating a control inoculum for verification testing at this low concentration and evaluate its use. Results show that half of all samples received no cells when 1 CFU was the target concentration and that targets greater than 3 CFU were much more reliable. Detection by culture isolation and two commercial assays, Qualicon BAX-MP and BioControl GDS, detected 94% ± 11%, 92% ± 10%, and 92% ± 7% of samples inoculated with 5.4 CFU (range 1 to 9 CFU), respectively. We also examined the effect of background aerobic plate count (APC) bacteria and fat content effects on the detection of E. coli O157:H7. At APC concentrations below 6 log CFU/g, the rapid methods detected all beef trim samples inoculated with 26 CFU of E. coli O157:H7 per 65 g. At an APC of 6.7 log CFU/g, culture, BAX-MP, and GDS detected 100, 75, and 13%, respectively, of inoculated samples. Neither commercial method detected E. coli O157:H7 in the samples when APC was 7.7 log CFU/g, whereas culture was able to detect 63% of E. coli O157:H7 in the samples when APC was at this concentration. Increased fat content correlated with decreasing recovery of immunomagnetic separation beads, but this was not observed to interfere with detection of E. coli O157:H7.     

Horizontal transmission of Escherichia coli O157:H7 during cattle housing

Ruminant livestock, particularly cattle, is considered the primary reservoir of Escherichia coli O157:H7. This study examines the transmission of E. coli O157:H7 within groups of cattle during winter housing. Holstein Friesian steers were grouped in six pens of five animals. An animal inoculated with and proven to be shedding a marked strain of E. coli O157: H7 was introduced into each pen. Fecal (rectal swabs) and hide samples (900 cm2 from the right rump) were taken from the 36 animals throughout the study. Water, feed, and gate or partition samples from each pen were also examined. Within 24 h of introducing the inoculated animals into the pens, samples collected from the drinking water, pen barriers, and animal hides were positive for the pathogen. Within 48 h, the hides of 20 (66%) of 30 cohort animals from the six pens were contaminated with E. coli O157:H7. The first positive fecal samples from the noninoculated cohort animals were detected 3 days after the introduction of the inoculated steers. During the 23 days of the study, 15 of 30 cohort animals shed the marked E. coli O157:H7 strain in their feces on at least one occasion. Animal behavior in the pens was monitored during a 12-h period using closed circuit television cameras. The camera footage showed an average of 13 instances of animal grooming in each pen per hour. The study suggests that transmission of E. coli O157:H7 between animals may occur following ingestion of the pathogen at low levels and that animal hide may be an important source of transmission.     

Transfer of Escherichia coli O157:H7 to romaine lettuce due to contact water from melting ice

Ice can be used to chill romaine lettuce and maintain relative humidity during transportation. Escherichia coli O157:H7 may contaminate water used for ice. The objective of this study was to determine the potential for E. coli O157:H7 contamination of romaine lettuce from either ice contaminated with the pathogen or by transfer from lettuce surfaces via melting ice. In experiment 1, lettuce was spot inoculated with E. coli O157:H7 and chilled with ice prepared from uncontaminated tap water. In experiment 2, water inoculated with this pathogen was frozen and used to ice lettuce. Three heads of lettuce were stacked in each container and stored at 4 or 20 degrees C. After the ice melted, E. coli O157:H7 attachment to and recovery from the lettuce leaves were determined. For experiment 1, the population of E. coli O157:H7 attached to inoculated sites averaged 3.8 and 5.5 CFU/cm2 at 4 and 20 degrees C, respectively. Most of the uninoculated sites became contaminated with the pathogen due to ice melt. For experiment 2, 3.5 to 3.8 log CFU E. coli O157:H7 per cm2 was attached to the top leaf on the first head. After rinsing with chlorinated water (200 microg/ml), E. coli O157:H7 remained on the surface of the top head (1.8 to 2.0 log CFU/cm2). There was no difference in numbers of E. coli O157:H7 recovered from each sampling site at 4 and 20 degrees C. Results show that E. coli O157:H7 can be transferred onto other produce layers in shipping containers from melted ice made of contaminated water and from contaminated to uncontaminated leaf surfaces.     

Molecular characterization of Escherichia coli O157:H7 hide contamination routes: feedlot to harvest

This study was conducted to identify the origin of Escherichia coli O157:H7 contamination on steer hides at the time of harvest. Samples were collected from the feedlot, transport trailers, and packing plant holding pens and from the colons and hides of feedlot steers. A total of 50 hide samples were positive for E. coli O157:H7 in two geographical locations: the Midwest (25 positive hides) and Southwest (25 positive hides). Hide samples were screened, and the presence of E. coli O157: H7 was confirmed. E. coli O157:H7 isolates were fingerprinted by pulsed-field gel electrophoresis and subjected to multiplex PCR procedures for amplification of E. coli O157:H7 genes stx1, stx2, eaeA, fliC, rfbEO157, and hlyA. Feedlot water trough, pen floor, feed bunk, loading chute, truck trailer side wall and floor, packing plant holding pen floor and side rail, and packing plant cattle drinking water samples were positive for E. coli O157:H7. Pulsed-field gel electrophoresis banding patterns were analyzed after classifying isolates according to the marker genes present and according to packing plant. In this study, hide samples positive for E. coli O157:H7 were traced to other E. coli O157:H7-positive hide, colon, feedlot pen floor fecal, packing plant holding pen drinking water, and transport trailer side wall samples. Links were found between packing plant side rails, feedlot loading chutes, and feedlot pens and between truck trailer, different feedlots, and colons of multiple cattle. This study is the first in which genotypic matches have been made between E. coli O157:H7 isolates obtained from transport trailer side walls and those from cattle hide samples within the packing plant.     

Effects of a minimal hide wash cabinet on the levels and prevalence of Escherichia coli O157:H7 and Salmonella on the hides of beef cattle at slaughter

Harborage of Escherichia coli O157:H7 and Salmonella on animal hides at slaughter is the main source of beef carcass contamination during processing. Given this finding, interventions have been designed and implemented to target the hides of cattle following entry into beef processing plants. Previous interventions targeting hides have not been suitable for all beef processing plants because of cost and space restrictions. In this study, a hide wash cabinet was evaluated to determine whether it was more amenable to widespread use in the beef processing industry, especially for small and medium-size plants. Overall, 101 (35.1%) of 288 beef cattle hides sampled before entry into the hide wash cabinet harbored E. coli O157:H7 at or above the limit of detection (40 CFU/100 cm2). After passage through the hide wash cabinet, only 38 (13.2%) of 288 hides had E. coli O157:H7 levels > or =40 CFU/100 cm2. Before the hide wash cabinet, 50 (17%) of 288 hides harbored E. coli O157:H7 at levels above 100 CFU/100 cm2, with one sample as high as 20,000 CFU/100 cm2. In contrast, only 14 (5%) of 288 hides had E. coli O157:H7 levels above 100 CFU/100 cm2 after hide washing, with the highest being 2000 CFU/100 cm2. These same trends also were found for Salmonella before and after hide washing. These results indicate that the hide wash cabinet described in this study was effective and should provide small and medium-size processing plants with an affordable hide wash intervention strategy.     

Evaluation of a polymerase chain reaction-based system for detection of Salmonella enteritidis, Escherichia coli O157:H7, Listeria spp., and Listeria monocytogenes on fresh fruits and vegetables

A polymerase chain reaction (PCR)-based detection system, BAX, was evaluated for its sensitivity in detecting Salmonella Enteritidis, Escherichia coli O157:H7, Listeria sp., and Listeria monocytogenes on fresh produce. Fifteen different types of produce (alfalfa sprouts, green peppers, parsley, white cabbage, radishes, onions, carrots, mushrooms, leaf lettuce, tomatoes, strawberries, cantaloupe, mango, apples, and oranges) were inoculated, in separate studies, with Salmonella Enteritidis, E. coli O157:H7, and L. monocytogenes down to the predicted level of 1 CFU per 25-g sample. Detection by BAX was compared to recovery of the inoculated bacteria by culture methods according to the Food and Drug Administration's (FDA) Bacteriological Analytical Manual (BAM). BAX was essentially as sensitive as the culture-based method in detecting Salmonella Enteritidis and L. monocytogenes and more sensitive than the culture-based method for the detection of E. coli O157:H7 on green pepper, carrot, radish, and sprout samples. Detection of the pathogenic bacteria in samples spiked with a predicted number of less than 10 CFU was possible for most produce samples, but both methods failed to detect L. monocytogenes on carrot samples and one of two mushroom and onion samples spiked with less than 100 CFU. Both BAX and the culture method were also unable to consistently recover low numbers of E. coli O157:H7 from alfalfa sprouts. The PCR method allowed detection of Salmonella Enteritidis, E. coli O157:H7, and L. monocytogenes at least 2 days earlier than the conventional culture methods.     

Evaluation of Lactic Acid as an Initial and Secondary Subprimal Intervention for Escherichia coli O157:H7, Non-O157 Shiga Toxin-Producing E. coli, and a Nonpathogenic E. coli Surrogate for E. coli O157:H7

Lactic acid can reduce microbial contamination on beef carcass surfaces when used as a food safety intervention, but effectiveness when applied to the surface of chilled beef subprimal sections is not well documented. Studies characterizing bacterial reduction on subprimals after lactic acid treatment would be useful for validations of hazard analysis critical control point (HACCP) systems. The objective of this study was to validate initial use of lactic acid as a subprimal intervention during beef fabrication followed by a secondary application to vacuum-packaged product that was applied at industry operating parameters. Chilled beef subprimal sections (100 cm(2)) were either left uninoculated or were inoculated with 6 log CFU/cm(2) of a 5-strain mixture of Escherichia coli O157:H7, a 12-strain mixture of non-O157 Shiga toxin-producing E. coli (STEC), or a 5-strain mixture of nonpathogenic (biotype I) E. coli that are considered surrogates for E. coli O157:H7. Uninoculated and inoculated subprimal sections received only an initial or an initial and a second "rework" application of lactic acid in a custombuilt spray cabinet at 1 of 16 application parameters. After the initial spray, total inoculum counts were reduced from 6.0 log CFU/cm(2) to 3.6, 4.4, and 4.4 log CFU/cm(2) for the E. coli surrogates, E. coli O157:H7, and non-O157 STEC inoculation groups, respectively. After the second (rework) application, total inoculum counts were 2.6, 3.2, and 3.6 log CFU/cm(2) for the E. coli surrogates, E. coli O157:H7, and non-O157 STEC inoculation groups, respectively. Both the initial and secondary lactic acid treatments effectively reduced counts of pathogenic and nonpathogenic strains of E. coli and natural microflora on beef subprimals. These data will be useful to the meat industry as part of the HACCP validation process.     

Shedding of escherichia coli O157:H7 by cattle fed diets containing monensin or tylosin

Monensin and tylosin have activity against gram-positive bacteria, and it has been theorized that their effects on the intestinal environment may promote proliferation of gram-negative bacteria such as Escherichia coli. Effects of these antibiotics on the shedding of E. coli O157:H7 were studied in a feedlot environment, using 32 finishing steers. A diet containing 85% barley grain, 10% barley silage, and 5% supplement was amended with 33 ppm monensin, 11 ppm tylosin, both of these additives, or no additives (control). All steers were orally inoculated with 10(10) CFU of a mixture of four strains of nalidixic acid-resistant E. coli O157:H7. Fecal (grab), oral (mouth swab) and water, water-water bowl interface, feed, and pen floor fecal pat samples were collected weekly for 12 weeks. Prevalence of E. coli O157:H7-positive fecal grab samples did not differ (P = 0.26) among treatments, nor did the rate (P = 0.81) or duration (P = 0.85) of shedding of the organism. Fecal grab samples were positive for E. coli O157:H7 more frequently (P < 0.001) than were oral swabs. More (P = 0.02) E. coli O157:H7-positive oral swabs were recovered from the tylosin group than from controls. E. coli O157:H7 was not detected in any of 47 water samples, but was present in 1 of 47 water bowl swabs, 7 of 48 feed samples, and 36 of 48 fecal pats. Pulsed-field gel electrophoresis suggested that differences existed among inoculated strains in their ability to persist in animals and in the environment. However, this study revealed no evidence that dietary inclusion of monensin or tylosin, alone or in combination, increased fecal shedding of E. coli O157:H7 or its persistence in the environment.     

Comparison of sampling methods for microbiological testing of beef animal rectal/colonal feces, hides, and carcasses

This study compared sampling methods for detecting Escherichia coli O157:H7 and Salmonella in beef cattle feces and on hides and carcasses and for enumerating E. coli biotype I counts (ECC) on carcasses. Fecal samples were collected by rectal/colonal palpation and colonal sponge swabbing. Hides were sampled by sponge swabbing three sites, hair clipping, excision, rinsing, and gauze swabbing, whereas carcasses were sampled by three-site thoracic and pattern-mark sponge swabbing and tissue excision. Overall, irrespective of sampling method, 36.7, 13.3, and 0.0% of lots contained at least one E. coli O157:H7-positive hide, fecal, and carcass sample, respectively, while the corresponding prevalence of Salmonella was 70.0, 16.7, and 6.7%, respectively. For hide sampling, excision and gauze swabbing yielded the fewest (13.3%) E. coli O157:H7-positive samples, while hair clipping and sponge swabbing yielded the most (23.3%). None of the carcass-sampling methods detected E. coli O157:H7 or differed (P > 0.05) in their ability to enumerate ECC. Colonal swabbing was the most effective (10.0%) method for detecting E. coli O157:H7 in feces. No differences (P > 0.05) in Salmonella prevalence were observed between carcass-sampling methods, although three-site sponge swabbing and tissue excision detected the most (3.3%). Hide rinsing was the most effective (P < 0.05) Salmonella detection method (63.3%), but dangers associated with its application may preclude its use by industry; there were no differences (P > 0.05) among other hide-sampling methods. No differences (P > 0.05) in Salmonella detection were observed between fecal-sampling methods. Overall, three-site sponge swabbing was the most feasible and effective sampling method for the detection of E. coli O157:H7 and Salmonella on hides and carcasses.     

Monitoring Escherichia coli O157:H7 in inoculated and naturally colonized feedlot cattle and their environment

On-farm methods of monitoring Escherichia coli O157:H7 were assessed in 30 experimentally inoculated steers housed in four pens over a 12-week period and in 202,878 naturally colonized feedlot cattle housed in 1,160 pens on four commercial Alberta feedlots over a 1-year period. In the challenge study, yearling steers were experimentally inoculated with 10(10) CFU of a four-strain mixture of nalidixic acid-resistant E. coli O157:H7. After inoculation, shedding of E. coli O157:H7 was monitored weekly by collecting rectal fecal samples (FEC), oral swabs (ORL), pooled fecal pats (PAT), manila ropes (ROP) orally accessed for 4 h, feed samples, water, and water bowl interface. Collection of FEC from all animals per pen provided superior isolation (P < 0.01) of E. coli O157:H7 compared with other methods, although labor and animal restraint requirements for fecal sample collection were high. When one sample was collected per pen of animals, E. coli O157:H7 was more likely to be detected from the ROP than from the FEC, PAT, or ORL (P < 0.001). In the commercial feedlot study, samples were limited to ROP and PAT, and E. coli O157:H7 was isolated in 18.8% of PAT and 6.8% of ROP samples. However, for animals that had been resident in the feedlot pen for at least 1 month, isolation of E. coli O157:H7 from ROP was not different from that from PAT (P = 0.35). Pens of animals on feed for <30 days were six times more likely to shed E. coli O157:H7 than were animals on feed for >30 days. However, change in diet did not affect shedding of the organism (P > 0.23) provided that animals had acclimated to the feedlot for 1 month or longer. Findings from this study indicate the importance of introduction of mitigation strategies early in the feeding period to reduce transference and the degree to which E. coli O157:H7 is shed into the environment.     

Efficacy of a peroxyacetic acid formulation as an antimicrobial intervention to reduce levels of inoculated Escherichia coli O157:H7 on external carcass surfaces of hot-boned beef and veal

The efficacy of a peroxyacetic acid formulation (POAA) at reducing Escherichia coli O157:H7 contamination on external carcass surfaces of hot-boned beef and veal with a commercial spray apparatus was determined. Hot-boned external carcass surfaces were inoculated with either a high dose (10(6) CFU/cm2) in fresh bovine feces or with a low dose (10(3) CFU/cm2) in diluent of laboratory-cultured E. coli O157:H7. Treatments included a water wash, a POAA (180 ppm) wash, or a water plus POAA wash. Samples were extracted from the external carcass surface with a cork borer to determine the numbers of viable E. coli O157:H7 remaining on the carcass surface after treatment. Although a water wash alone resulted in a 1.25 (94.4%) and a 1.31 (95.1%) mean log reduction on veal and beef inoculated with a high dose of E. coli O157:H7, the POAA treatment resulted in a substantially greater mean log reduction of 3.56 and 3.59 (>99.9%). The water wash only resulted in a 33.9% reduction on veal and 62.8% on beef inoculated with a low dose of E. coli O157:H7, whereas POAA treatment greatly improved pathogen reduction to 98.9 and 97.4% on veal and beef, respectively. The combination of a water wash followed by a POAA treatment resulted in a similar E. coli O157:H7 reduction to that achieved by POAA treatment alone. In conclusion, POAA treatment significantly reduced viable E. coli O157:H7 numbers on experimentally contaminated beef and veal carcasses, which justifies its use as a chemical intervention for the removal of this human pathogen.     

Collaborative evaluation of detection methods for Escherichia coli O157:H7 from radish sprouts and ground beef

For the evaluation of plating and immunological methods applicable to the detection of Escherichia coli O157:H7 from ground beef and radish sprouts, a collaborative study was conducted. It focused on a comparison of the efficiency of the plating and immunological methods using various plating agars and immuno-kits in combination with enrichment in modified E. coli broth supplemented with novobiocin (mEC + n), and using immunomagnetic separation. The plating media tested were sorbitol MacConkey agar (SMAC), SMAC supplemented with cefixime (0.05 mg/l) and potassium tellurite (2.5 mg/l) (CT-SMAC), and agars containing beta-glucuronidase substrates such as BCM O157 and CHROMagar O157. The immuno-kits used were Now E. coli, Path-Stick O157, VIP, EHEC-Tek ELISA System and Rapiblot E. coli O157. The 20 participating laboratories attempted to detect E. coli O157:H7 in 25 g chilled and frozen samples of ground beef uninoculated and inoculated with E. coli O157:H7 at levels of 138.9 and 23.9 cfu/25 g, and in 25 g chilled and frozen samples of radish sprouts uninoculated and inoculated at levels of 20.4 and 1.7 cfu/25 g. E. coli O157:H7 was recovered well from ground beef by all of the methods except direct plating with SMAC. For radish sprouts, the IMS-plating methods with CT-SMAC, BCM O157 and CHROMagar O157 were most efficient at detecting E. coli O157:H7 in more than 90% of the chilled samples inoculated at the level of 20.4 cfu/25 g. All the methods were less sensitive when applied to similar levels of E. coli O157:H7 in radish sprouts (20.4 cfu/25 g) compared with ground beef (23.9 cfu/25 g) especially if the sprouts were frozen. The sensitivity of the immuno-kits appeared to be similar to the IMS-plating methods, but the specificity was lower. Based on the results, we recommend the IMS-plating method using CT-SMAC and agars containing beta-glucuronidase substrate in combination with static enrichment incubation in mEC + n at 42 degrees C.