Development and Validation of a Most-Probable-Number–Immunomagnetic Separation Methodology of Enumerating Escherichia coli O157 in Cattle Feces

2007 ◽  
Vol 70 (5) ◽  
pp. 1072-1075 ◽  
Author(s):  
T. P. STEPHENS ◽  
G. H. LONERAGAN ◽  
W. E. CHANEY ◽  
L. A. BRANHAM ◽  
M. M. BRASHEARS
Keyword(s):  
Escherichia Coli ◽  
Immunomagnetic Separation ◽  
Feedlot Cattle ◽  
Most Probable Number ◽  
Escherichia Coli O157 ◽  
Probable Number ◽  
Direct Plating ◽  
Fecal Samples ◽  
E Coli ◽  
The Difference

A method to validate enumeration of Escherichia coli O157 in fecal samples from feedlot cattle was developed in these studies. Due to background flora, bovine fecal sample enumeration cannot be performed by simple direct plating techniques. Known quantities of E. coli O157:H7 were inoculated into feces, and populations were determined by direct plating of the cocktail (studies 1, 2, and 3) and manure and cocktail (studies 4 and 5) mixtures and compared with a most-probable-number (MPN)–immunomagnetic separation (IMS) method. The three-tube MPN combined preenrichment in gram-negative broth with confirmation using IMS. Five separate enumeration studies (study 1, sterile feces inoculated with 102 E. coli O157:H7 per g; study 2, nonsterile feces inoculated with 103 E. coli O157:H7 per g; study 3, nonsterile feces inoculated with 101 E. coli O157:H7 per g; study 4, sterile feces inoculated with 104 streptomycin-resistant E. coli O157:H7 per g; and study 5, sterile feces inoculated with 102 streptomycin-resistant E. coli O157:H7 per g) were conducted. These studies were performed to determine the precision, accuracy, and specificity at low and high levels of pathogen contamination in feces, using direct plating compared with the MPN-IMS methodology tested. There was an overall difference (P < 0.01) between direct plating and MPN-IMS methodologies, but this difference was biologically negligible due to the difference in least-squares means (0.29 ± 0.10) being so low. The direct plating and MPN-IMS methods were correlated (r = 0.93). These results suggest that using the MPN-IMS procedures is an effective method of estimating E. coli O157 populations in naturally infected bovine fecal samples.

An Investigation of Escherichia coli O157 Contamination of Cattle during Slaughter at an Abattoir

2005 ◽  
Vol 68 (3) ◽  
pp. 451-457 ◽  
Author(s):  
NARELLE FEGAN ◽  
GLEN HIGGS ◽  
PAUL VANDERLINDE ◽  
PATRICIA DESMARCHELIER
Keyword(s):  
Escherichia Coli ◽  
Oral Cavity ◽  
Immunomagnetic Separation ◽  
Most Probable Number ◽  
Escherichia Coli O157 ◽  
Probable Number ◽  
Fecal Samples ◽  
E Coli ◽  
Cell Counts ◽  
Carcass Contamination

The extent of contamination with Escherichia coli O157 was determined for 100 cattle during slaughter. Samples from 25 consecutively slaughtered cattle from four unrelated groups were collected from the oral cavity, hide, rumen, feces after evisceration, and pre- and postchill carcass. Ten random fecal samples were collected from the pen where each group of animals was held at the abattoir. E. coli O157 was detected using automated immunomagnetic separation (AIMS), and cell counts were determined using a combination of most probable number (MPN) and AIMS. E. coli O157 was isolated from 87 (14%) of the 606 samples collected, including 24% of 99 oral cavity samples, 44% of 100 hides, 10% of 68 fecal samples collected postevisceration, 6% of 100 prechill carcass swabs, and 15% of 40 fecal samples collected from holding pens. E. coli O157 was not isolated from rumen or postchill carcass samples. E. coli O157 was isolated from at least one sample from each group of cattle tested, and the prevalence in different groups ranged from less than 1 to 41%. The numbers of E. coli O157 differed among the animals groups. The group which contained the highest fecal (7.5 × 105 MPN/g) and hide (22 MPN/cm2) counts in any individual animal was the only group in which E. coli O157 was isolated from carcasses, suggesting a link between the numbers of E. coli O157 present and the risk of carcass contamination. Processing practices at this abattoir were adequate for minimizing contamination of carcasses, even when animals were heavily contaminated with E. coli O157.

Prevalence and Enumeration of Escherichia coli O157 in Steers Receiving Various Strains of Lactobacillus-Based Direct-Fed Microbials

2007 ◽  
Vol 70 (5) ◽  
pp. 1252-1255 ◽  
Author(s):  
T. P. STEPHENS ◽  
G. H. LONERAGAN ◽  
L. M. CHICHESTER ◽  
M. M. BRASHEARS
Keyword(s):  
Escherichia Coli ◽  
Lactobacillus Acidophilus ◽  
Immunomagnetic Separation ◽  
Feedlot Cattle ◽  
Most Probable Number ◽  
Escherichia Coli O157 ◽  
Feeding Period ◽  
Probable Number ◽  
Selective Enrichment ◽  
E Coli

The objective of this research was to evaluate the effect of daily dietary inclusion of specific strains of Lactobacillus acidophilus on prevalence and concentration of Escherichia coli O157 in harvest-ready feedlot cattle. Five hundred yearling steers were housed in pens of 10 animals each. At arrival, steers were randomly allocated to one of five cohorts. Four of the cohorts were fed various strains and dosages of Lactobacillus-based direct-fed microbials throughout the feeding period. Fecal samples were collected from the rectum of each animal immediately prior to shipment to the abattoir. E. coli O157 was detected using selective enrichment and immunomagnetic separation techniques. For positive samples, E. coli O157 concentration was estimated using a most-probable-number (MPN) technique that included immunomagnetic separation. Prevalence varied among the cohorts (P < 0.01). The prevalence in the controls (26.3%) was greater (P < 0.05) than that in cattle supplemented with L. acidophilus strains NP51, NP28, or NP51-NP35 (13.0, 11.0, and 11.0%, respectively). The greatest E. coli O157 concentration was also observed in the controls (3.2 log MPN/g of feces); this concentration was greater (P < 0.05) than that observed in positive animals receiving NP51, NP28, or NP51-NP35 (0.9, 1.1, 1.7 log MPN/g of feces, respectively). Specific strains of Lactobacillus-based direct-fed microbials effectively reduced the prevalence and concentration of E. coli O157 in harvest-ready cattle, whereas others did not. When using direct-fed microbials to reduce carriage of E. coli O157 in cattle, it is important to select appropriately validated products.

A Direct Plating Method for Estimating Populations of Escherichia coli O157 in Bovine Manure and Manure-Based Materials†

2008 ◽  
Vol 71 (11) ◽  
pp. 2233-2238 ◽  
Author(s):  
ELAINE D. BERRY ◽  
JAMES E. WELLS
Keyword(s):  
Escherichia Coli ◽  
Soil Amendments ◽  
Most Probable Number ◽  
Escherichia Coli O157 ◽  
Surface Material ◽  
Potassium Tellurite ◽  
Probable Number ◽  
Direct Plating ◽  
E Coli ◽  
Plating Method

Escherichia coli O157:H7 outbreaks associated with produce consumption have brought attention to livestock manures and manure-based soil amendments as potential sources of pathogens for the contamination of these crops. Procedures for enumeration of E. coli O157:H7 are needed to assess the risks of transmission from these manures and their by-products. A direct plating method employing spiral plating onto CHROMagar O157 was investigated for enumeration of E. coli O157:H7 in feedlot surface material, aged bovine manure, bovine manure compost, and manure-amended soil. In studies utilizing samples spiked with a five-strain cocktail of E. coli O157:H7 at levels ranging from 102 to 105 CFU/g of sample, there were strong correlations between the observed and predicted levels of this pathogen. Although the addition of 2.5 mg/liter potassium tellurite and 5 mg/liter novobiocin made the medium more restrictive, these amendments enhanced the ability to identify and enumerate E. coli O157:H7 in feedlot surface material, which contained a higher proportion of fresh feces than did the other three sample types and therefore higher levels of interfering bacterial microflora. The spiral plating method was further assessed to determine its ability to enumerate E. coli O157:H7 in naturally contaminated feedlot surface material. Comparison of E. coli O157:H7 counts in feedlot surface material obtained by the spiral plating method and a most probable number technique were well correlated. We conclude that direct spiral plating onto CHROMagar O157 is effective for estimating E. coli O157: H7 levels in a variety of manures and manure-containing sample types to a lower detection limit of 200 CFU/g. The method has application for determining E. coli O157:H7 concentrations in manures and composts before their sale and use as soil amendments and for measuring the effectiveness of manure treatment processes to reduce or inactivate this pathogen.

scholarly journals Escherichia coli O157:H7 in Drin Prevalence of Escherichia coli O157:H7 in Drink from Different Food Premises in Kota Samarahan Sarawak

2019 ◽  
Vol 2 (2) ◽  
pp. a13-19
Author(s):  
ELEXSON NILLIAN ◽  
AMIZA NUR ◽  
DIYANA NUR ◽  
AMIRAH ZAKIRAH ◽  
GRACE BEBEY
Keyword(s):  
Escherichia Coli ◽  
Drinking Water ◽  
Coliform Bacteria ◽  
Most Probable Number ◽  
Plain Water ◽  
Escherichia Coli O157 ◽  
Probable Number ◽  
E Coli ◽  
Preventive Actions ◽  
Stx1 Gene

Contamination of drinks with E. coli O157:H7 served in food premises such as restaurants can cause haemorrhagic colitis and haemolytic uremic syndrome to humans. The presence or absence of faecal pathogen was demonstrated using coliform group as indicator microorganisms. Therefore, this study was conducted to detect the presence of E. coli O157:H7 in drinking water from food restaurant premise in Kota Samarahan and Kuching to ensure safe and potable drinking water is served to the consumer. A total of thirty (n=30) drink samples including six types of each of the samples are cold plain water, iced tea, iced milo, syrup and iced milk tea. Most Probable Number (MPN) procedure was used in this study to enumerate the MPN values of coliform bacteria in each drink collected. A total of 53.33% (16/30) of the drink samples showed positive E. coli detection. Then, the PCR assay showed 6.25% (one out of 16 isolates) samples were positive and carried stx1 gene produced by E. coli O157:H7 in iced milo sample types. This study showed the drinks collected from food premises was contaminated with faecal contamination, which was not safe to drink by the consumer. Therefore, preventive actions should be taken to prevent foodborne illness outbreak in future

Comparison of Rapid Enzyme-Linked Immunosorbent Assay and Immunomagnetic Separation Methods for Detection of Escherichia coli O157 in Fecal, Hide, Carcass, and Ground Beef Samples

2007 ◽  
Vol 70 (10) ◽  
pp. 2230-2234 ◽  
Author(s):  
T. W. THOMPSON ◽  
T. P. STEPHENS ◽  
G. H. LONERAGAN ◽  
M. F. MILLER ◽  
M. M. BRASHEARS
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Separation Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Escherichia Coli O157 ◽  
Perfect Agreement ◽  
Fecal Samples ◽  
E Coli ◽  
Beef Products

Rapid enzyme-linked immunosorbent assays (ELISAs) are approved for detection of Escherichia coli O157 in beef products. However, these kits have also been used in the industry to detect this pathogen on hides or in feces of cattle, although this use has not been validated. The objective of this study was to compare commercially available ELISAs (E. coli Now, Reveal, and VIP) with immunomagnetic separation along with selective media to detect E. coli O157 on hides, in feces, and in medium- and low-level-inoculated ground beef and carcasses (simulated by using briskets) samples. Naturally infected hide and fecal samples were subjected to both the immunomagnetic separation method and ELISAs for the detection of E. coli O157. Additionally, E. coli O157 inoculated and noninoculated ground beef and beef briskets were used to simulate meat and carcass samples. When comparing the detection results from the ELISAs (E. coli Now, Reveal, and VIP) to the immunomagnetic separation method, poor agreement was observed for fecal samples (kappa = 0.10, 0.02, and 0.03 for E. coli Now, Reveal, and VIP, respectively), and fair-to-moderate agreement was observed for hide samples (kappa = 0.30, 0.51, and 0.29 for E. coli Now, Reveal, and VIP, respectively). However, there was near-perfect agreement between the immunomagnetic separation method and ELISAs for ground beef (kappa = 1, 1, and 0.80 for E. coli Now, Reveal, and VIP, respectively) and brisket (kappa = 1, 1, and 1 for E. coli Now, Reveal, and VIP, respectively) samples. Assuming immunomagnetic separation is the best available method, these data suggest that the ELISAs are not useful in detecting E. coli O157 from hide or fecal samples. However, when ELISAs are used on ground beef and beef brisket samples they can be used with a high degree of confidence.

Epidemiology of Escherichia coli O157 in Feedlot Cattle

1997 ◽  
Vol 60 (5) ◽  
pp. 462-465 ◽  
Author(s):  
DALE D. HANCOCK ◽  
DANIEL H. RICE ◽  
LEE ANN THOMAS ◽  
DAVID A. DARGATZ ◽  
THOMAS E. BESSER
Keyword(s):  
United States ◽  
Escherichia Coli ◽  
The United States ◽  
Feedlot Cattle ◽  
Escherichia Coli O157 ◽  
Fecal Samples ◽  
E Coli ◽  
Geographical Regions ◽  
Flagellar Antigen ◽  
Low Prevalence

Fecal samples from cattle in 100 feedlots in 13 states were bacteriologically cultured for Escherichia coli O157 that did not ferment sorbitol, lacked beta-glucuronidase, and possessed genes coding for Shiga-like toxin. In each feedlot 30 fresh fecal-pat samples were collected from each of four pens: with the cattle shortest on feed, with cattle longest on feed, and with cattle in two randomly selected pens. E. coli O157 was isolated from 210 (1.8%) of 11,881 fecal samples. One or more samples were positive for E. coli O157 in 63 of the 100 feedlots tested. E. coli O157 was found at roughly equal prevalence in all the geographical regions sampled. The prevalence of E. coli O157 in the pens with cattle shortest on feed was approximately threefold higher than for randomly selected and longest on feed pens. Of the E. coli O157 isolates found in this study, 89.52% expressed the H7 flagellar antigen. E. coli O157 was found to be widely distributed among feedlot cattle, but at a low prevalence, in the United States.

Relative Sensitivity of Escherichia coli O157 Detection from Bovine Feces and Rectoanal Mucosal Swabs

2014 ◽  
Vol 77 (6) ◽  
pp. 972-976 ◽  
Author(s):  
K. J. WILLIAMS ◽  
M. P. WARD ◽  
O. DHUNGYEL ◽  
L. VAN BREDA
Keyword(s):  
Escherichia Coli ◽  
Confidence Interval ◽  
Human Health Risk ◽  
Relative Sensitivity ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Escherichia Coli O157 ◽  
Prevalence Studies ◽  
E Coli ◽  
The Difference

The need to quantify the potential human health risk posed by the bovine reservoir of Escherichia coli O157 has led to a wealth of prevalence studies and improvements in detection methods over the last two decades. Rectoanal mucosal swabs have been used for the detection of E. coli O157 fecal shedding, colonized animals, and those predisposed to super shedding. We conducted a longitudinal study to compare the detection of E. coli O157 from feces and rectoanal mucosal swabs (RAMS) from a cohort of dairy heifers. We collected 820 samples that were tested by immunomagnetic separation of both feces and RAMS. Of these, 132 were detected as positive for E. coli O157 from both samples, 66 were detected as positive from RAMS only, and 117 were detected as positive from feces only. The difference in results between the two sample types was statistically significant (P < 0.001). The relative sensitivities of detection by immunomagnetic separation were 53% (confidence interval, 46.6 to 59.3) from RAMS and 67% (confidence interval, 59.6 to 73.1) from fecal samples. No association between long-term shedding (P = 0.685) or super shedding (P = 0.526) and detection by RAMS only was observed.

scholarly journals Evaluation of Culture Methods To Identify Bovine Feces with High Concentrations of Escherichia coli O157

2007 ◽  
Vol 73 (16) ◽  
pp. 5253-5260 ◽  
Author(s):  
J. Trent Fox ◽  
David G. Renter ◽  
Michael W. Sanderson ◽  
Daniel U. Thomson ◽  
Kelly F. Lechtenberg ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sensitivity And Specificity ◽  
Immunomagnetic Separation ◽  
Most Probable Number ◽  
Culture Methods ◽  
Potassium Tellurite ◽  
Probable Number ◽  
E Coli ◽  
Concentration Sensitivity ◽  
High Concentrations

ABSTRACT Our objective was to evaluate methods for identifying cattle with high concentrations of Escherichia coli O157 in their feces. In two experiments, feces were collected from cattle orally inoculated with nalidixic acid (Nal)-resistant E. coli O157, and direct plating of diluted feces on sorbitol MacConkey agar with cefixime and potassium tellurite (CT-SMAC) containing Nal was considered the gold standard (GS) method. In experiment 1, methods evaluated were preenrichment direct streak, immunomagnetic separation with most probable number (MPN), and postenrichment direct streak with MPN, all using CT-SMAC. The mean concentration of Nal-resistant E. coli O157 in samples (n = 59) by use of the GS was 3.6 log10 CFU/g. The preenrichment streak detected >3.0 log10 CFU/g samples with a 74.4% sensitivity and 68.8% specificity. Postenrichment direct streak-MPN and immunomagnetic separation-MPN concentrations were correlated significantly with GS concentrations (r = 0.53 and r = 0.39, respectively). In experiment 2 (480 samples), pre- and postenrichment direct streaking performed in triplicate and spiral plating on CT-SMAC were evaluated. For preenrichment streaks, sensitivity was 79.7% and specificity was 96.7% for detecting >3.0 log10 CFU/g when the criterion was positive cultures on at least two plates. For spiral plating at that concentration, sensitivity and specificity were 83.9% and 56.3%, respectively. Postenrichment streaking performed relatively poorly. Triplicate preenrichment streaks of 1:10-diluted feces on CT-SMAC may be useful for identifying cattle shedding high concentrations of E. coli O157. Estimates of sensitivity and specificity enable appropriate application of methods and interpretation of results and may enhance applied research, surveillance, and risk assessments.

Enumeration of Escherichia coli O157 in Outbreak-Associated Gouda Cheese Made with Raw Milk

2015 ◽  
Vol 78 (9) ◽  
pp. 1733-1737 ◽  
Author(s):  
ALEXANDER GILL ◽  
DENISE OUDIT
Keyword(s):  
Escherichia Coli ◽  
Raw Milk ◽  
Most Probable Number ◽  
Prolonged Storage ◽  
Escherichia Coli O157 ◽  
Probable Number ◽  
E Coli ◽  
Aging Period ◽  
Gouda Cheese ◽  
Cheese Products

In this article, we discuss the enumerative analysis for Escherichia coli O157 in two raw milk Gouda cheese products (A and B), implicated in an outbreak of 29 cases of E. coli O157:H7 illness that occurred across Canada in 2013. Samples were enumerated for E. coli O157 by most probable number (MPN) over a period of 30 to 60 days after the end of the outbreak. Samples (55.55 g) of product A (n = 14) were analyzed at 146 to 180 days postproduction. E. coli O157 was isolated from six samples at 19.9 to 44.6 MPN/kg. The E. coli O157 concentration of product A estimated from the results of all 14 samples was 9.5 MPN/kg. Samples (55.55 g) of product B (n = 20) were analyzed at 133 to 149 days postproduction. E. coli O157 was isolated from four samples at 19.9 MPN/kg. The E. coli O157 concentration of product B estimated from the results of all 20 samples was 3.7 MPN/kg. Analysis of a 305-g sample of product A (n = 1) stored at 4°C until 306 days postproduction revealed that the E. coli O157 concentration had declined to 3.6 MPN/kg. E. coli O157 could not be isolated from 555-g samples of product B (n = 5) after 280 days postproduction. The physicochemical parameters (pH, water activity, percent moisture, and percent salt) of both cheese products were found to be in the normal range for this type of product. The results of this study demonstrate that E. coli O157 could not replicate during storage at 4°C in the products tested but was capable of survival following aging and prolonged storage. This indicates that, if contaminated, the minimum 60-day aging period, which is required for raw milk Gouda cheeses, is not sufficient in all cases to ensure that the product does not contain viable cells of E. coli O157. The results also indicate that samples sizes greater than 100 g may be required to reliably detect E. coli O157 in cheese products associated with outbreaks.

Prevalence of Escherichia coli O157:H7 and Salmonella on Inshell California Walnuts

2015 ◽  
Vol 78 (8) ◽  
pp. 1547-1553 ◽  
Author(s):  
GORDON R. DAVIDSON ◽  
JOHN C. FRELKA ◽  
MAI YANG ◽  
THOMAS M. JONES ◽  
LINDA J. HARRIS
Keyword(s):  
Escherichia Coli ◽  
Confidence Interval ◽  
Most Probable Number ◽  
Escherichia Coli O157 ◽  
Probable Number ◽  
E Coli ◽  
Annual Prevalence

Inshell walnuts collected from California walnut handlers over four harvests were evaluated for the presence of Escherichia coli O157:H7 and Salmonella. E. coli O157:H7 was not detected in any of 2,903 375-g samples evaluated in 2011, 2012, and 2013 (<0.034% prevalence; 95% confidence interval [CI], 0 to 0.13%). Salmonella was not isolated from any of the 935 samples in 2010 (100 g evaluated; <0.11% prevalence; 95% CI, 0 to 0.41%) but was isolated from 2 of 905 (375 g; 0.22% prevalence; 95% CI, 0.061 to 0.80%), 1 of 998 (375 g; 0.10% prevalence; 95% CI, 0.018 to 0.56%), and 1 of 1,000 (375 g; 0.10% prevalence; 95% CI, 0.018 to 0.56%) samples in 2011, 2012, and 2013, respectively, for an average annual prevalence of 0.14% (375 g; 95% CI, 0.054 to 0.35%). The levels of Salmonella in positive samples determined by a modified most-probable-number (MPN) method were estimated to be 0.32 to 0.42 MPN/100 g (95% CI, 0.045 to 3.6 MPN/100 g).

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