Absence of Internalization of Escherichia coli O157:H7 into Germinating Tissue of Field-Grown Leafy Greens

2014 ◽  
Vol 77 (2) ◽  
pp. 189-196 ◽  
Author(s):  
MARILYN C. ERICKSON ◽  
CATHY C. WEBB ◽  
JUAN CARLOS DÍAZ-PÉREZ ◽  
LINDSEY E. DAVEY ◽  
ALISON S. PAYTON ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Contaminated Soil ◽  
Spinacia Oleracea ◽  
Field Studies ◽  
Growth Chamber ◽  
Escherichia Coli O157 ◽  
Apparent Lack ◽  
E Coli ◽  
Leafy Greens ◽  
Natural Isolates

Both growth chamber and field studies were conducted to investigate the potential for Escherichia coli O157:H7 to be internalized into leafy green tissue when seeds were germinated in contaminated soil. Internalized E. coli O157:H7 was detected by enrichment in both spinach (Spinacia oleracea L.) and lettuce (Lactuca sativa L.) seedlings when seeds were germinated within the growth chamber in autoclaved and nonautoclaved soil, respectively, contaminated with E. coli O157:H7 at 2.0 and 3.8 log CFU/g, respectively. Internalized E. coli O157:H7 populations could be detected by enumeration within leafy green tissues either by increasing the pathogen levels in the soil or by autoclaving the soil. Attempts to maximize the exposure of seed to E. coli O157:H7 by increasing the mobility of the microbe either through soil with a higher moisture content or through directly soaking the seeds in an E. coli O157:H7 inoculum did not increase the degree of internalization. Based on responses obtained in growth chamber studies, internalization of E. coli O157:H7 surrogates (natural isolates of Shiga toxin–negative E. coli O157:H7 or recombinant [stx- and eae-negative] outbreak strains of E. coli O157:H7) occurred to a slightly lesser degree than did internalization of the virulent outbreak strains of E. coli O157:H7. The apparent lack of internalized E. coli O157:H7 when spinach and lettuce were germinated from seed in contaminated soil (ca. 3 to 5 log CFU/g) in the field and the limited occurrence of surface contamination on the seedlings suggest that competition from indigenous soil bacteria and environmental stresses were greater in the field than in the growth chamber. On the rare occasion that soil contamination with E. coli O157:H7 exceeded 5 log CFU/g in a commercial field, this pathogen probably would not be internalized into germinating leafy greens and/or would not still be present at the time of harvest.

Infrequent Internalization of Escherichia coli O157:H7 into Field-Grown Leafy Greens

2010 ◽  
Vol 73 (3) ◽  
pp. 500-506 ◽  
Author(s):  
MARILYN C. ERICKSON ◽  
CATHY C. WEBB ◽  
JUAN CARLOS DIAZ-PEREZ ◽  
SHARAD C. PHATAK ◽  
JOHN J. SILVOY ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Irrigation Water ◽  
Field Studies ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Leafy Greens ◽  
Contaminated Irrigation Water ◽  
Green Fluorescent ◽  
Field Plots ◽  
Spinach Leaves

Several sources of contamination of fresh produce by Escherichia coli O157:H7 (O157) have been identified and include contaminated irrigation water and improperly composted animal waste; however, field studies evaluating the potential for internalization of O157 into leafy greens from these sources have not been conducted. Irrigation water inoculated with green fluorescent plasmid–labeled Shiga toxin–negative strains (50 ml of 102, 104, or 106 CFU of O157 per ml) was applied to soil at the base of spinach plants of different maturities in one field trial. In a second trial, contaminated compost (1.8 kg of 103 or 105 CFU of O157 per g) was applied to field plots (0.25 by 3.0 m) prior to transplantation of spinach, lettuce, or parsley plants. E. coli O157:H7 persisted in the soil up to harvest (day 76 posttransplantation) following application of contaminated irrigation water; however, internalized O157 was not detected in any spinach leaves or in roots exposed to O157 during the early or late growing season. Internalized O157 was detected in root samples collected 7 days after plants were contaminated in mid-season, with 5 of 30 samples testing positive for O157 by enrichment; however, O157 was not detected by enrichment in surface-disinfected roots on days 14 or 22. Roots and leaves from transplanted spinach, lettuce, and parsley did not internalize O157 for up to 50 days in the second trial. These results indicate that internalization of O157 via plant roots in the field is rare and when it does occur, O157 does not persist 7 days later.

Transfer of Escherichia coli O157:H7 from Soil, Water, and Manure Contaminated with Low Numbers of the Pathogen to Lettuce Plants

2009 ◽  
Vol 72 (11) ◽  
pp. 2308-2312 ◽  
Author(s):  
GABRIEL MOOTIAN ◽  
WEN-HSUAN WU ◽  
KARL R. MATTHEWS
Keyword(s):  
Escherichia Coli ◽  
Contaminated Soil ◽  
Human Health Risk ◽  
Contaminated Water ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Leafy Greens ◽  
Entire Plant ◽  
Sources Of Contamination ◽  
Amended Soil

The sources of contamination of leafy greens remain unclear, but it is evident that contaminated water, soil amendments, and wildlife likely contribute. The objective of the present study was to determine transfer of low numbers of Escherichia coli O157:H7 from soil, manure-amended soil, and water to growing lettuce plants. Lettuce plants, young (12 days of age) or mature (30 days of age), were grown in soil, manure-amended soil, or irrigated with water containing 101, 102, 103, or 104 CFU E. coli O157:H7 per g or ml. Harvested plants were processed to determine whether E. coli O157:H7 was associated with the entire plant or within internal locations. Young plants (12 days) were harvested at 1, 10, 20, and 30 days postexposure. No samples were positive for E. coli O157:H7 after direct plating of serial dilutions. Enrichment of all samples from young plants exposed to contaminated soil, manure-amended soil, and irrigation water demonstrated that approximately 21% (113 of 552) of plants were positive for E. coli O157:H7. Approximately 30% (36 of 120) of the mature plants initially irrigated with or grown in contaminated soil (including manure-amended soil) for 15 days were positive for E. coli O157:H7. Based on sterilization of surface tissue, E. coli O157:H7 was in protected locations of lettuce tissue. The results suggest that lettuce exposed to, and grown in the presence of, low numbers of E. coli O157:H7 may become contaminated and thus present a human health risk.

Surface and Internalized Escherichia coli O157:H7 on Field-Grown Spinach and Lettuce Treated with Spray-Contaminated Irrigation Water

2010 ◽  
Vol 73 (6) ◽  
pp. 1023-1029 ◽  
Author(s):  
MARILYN C. ERICKSON ◽  
CATHY C. WEBB ◽  
JUAN CARLOS DIAZ-PEREZ ◽  
SHARAD C. PHATAK ◽  
JOHN J. SILVOY ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Irrigation Water ◽  
Enrichment Culture ◽  
Field Studies ◽  
Plate Count ◽  
Escherichia Coli O157 ◽  
Adaxial Side ◽  
Abaxial Side ◽  
E Coli ◽  
Contaminated Irrigation Water

Numerous field studies have revealed that irrigation water can contaminate the surface of plants; however, the occurrence of pathogen internalization is unclear. This study was conducted to determine the sites of Escherichia coli O157:H7 contamination and its survival when the bacteria were applied through spray irrigation water to either field-grown spinach or lettuce. To differentiate internalized and surface populations, leaves were treated with a surface disinfectant wash before the tissue was ground for analysis of E. coli O157:H7 by direct plate count or enrichment culture. Irrigation water containing E. coli O157:H7 at 102, 104, or 106 CFU/ml was applied to spinach 48 and 69 days after transplantation of seedlings into fields. E. coli O157:H7 was initially detected after application on the surface of plants dosed at 104 CFU/ml (4 of 20 samples) and both on the surface (17 of 20 samples) and internally (5 of 20 samples) of plants dosed at 106 CFU/ml. Seven days postspraying, all spinach leaves tested negative for surface or internal contamination. In a subsequent study, irrigation water containing E. coli O157:H7 at 108 CFU/ml was sprayed onto either the abaxial (lower) or adaxial (upper) side of leaves of field-grown lettuce under sunny or shaded conditions. E. coli O157:H7 was detectable on the leaf surface 27 days postspraying, but survival was higher on leaves sprayed on the abaxial side than on leaves sprayed on the adaxial side. Internalization of E. coli O157:H7 into lettuce leaves also occurred with greater persistence in leaves sprayed on the abaxial side (up to 14 days) than in leaves sprayed on the adaxial side (2 days).

Tracking an Escherichia coli O157:H7–Contaminated Batch of Leafy Greens through a Pilot-Scale Fresh-Cut Processing Line

2014 ◽  
Vol 77 (9) ◽  
pp. 1487-1494 ◽  
Author(s):  
ANNEMARIE L. BUCHHOLZ ◽  
GORDON R. DAVIDSON ◽  
BRADLEY P. MARKS ◽  
EWEN C. D. TODD ◽  
ELLIOT T. RYSER
Keyword(s):  
Escherichia Coli ◽  
Membrane Filtration ◽  
Fluorescent Protein ◽  
Pilot Scale ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Leafy Greens ◽  
Iceberg Lettuce ◽  
Processing Line ◽  
Fresh Cut

Cross-contamination of fresh-cut leafy greens with residual Escherichia coli O157:H7–contaminated product during commercial processing was likely a contributing factor in several recent multistate outbreaks. Consequently, radicchio was used as a visual marker to track the spread of the contaminated product to iceberg lettuce in a pilot-scale processing line that included a commercial shredder, step conveyor, flume tank, shaker table, and centrifugal dryer. Uninoculated iceberg lettuce (45 kg) was processed, followed by 9.1 kg of radicchio (dip inoculated to contain a four-strain, green fluorescent protein–labeled nontoxigenic E. coli O157:H7 cocktail at 106 CFU/g) and 907 kg (2,000 lb) of uninoculated iceberg lettuce. After collecting the lettuce and radicchio in about 40 bags (~22.7 kg per bag) along with water and equipment surface samples, all visible shreds of radicchio were retrieved from the bags of shredded product, the equipment, and the floor. E. coli O157:H7 populations were quantified in the lettuce, water, and equipment samples by direct plating with or without prior membrane filtration on Trypticase soy agar containing 0.6% yeast extract and 100 ppm of ampicillin. Based on triplicate experiments, the weight of radicchio in the shredded lettuce averaged 614.9 g (93.6%), 6.9 g (1.3%), 5.0 g (0.8%), and 2.8 g (0.5%) for bags 1 to 10, 11 to 20, 21 to 30, and 31 to 40, respectively, with mean E. coli O157:H7 populations of 1.7, 1.2, 1.1, and 1.1 log CFU/g in radicchio-free lettuce. After processing, more radicchio remained on the conveyor (9.8 g; P < 0.05), compared with the shredder (8.3 g), flume tank (3.5 g), and shaker table (0.1 g), with similar E. coli O157:H7 populations (P > 0.05) recovered from all equipment surfaces after processing. These findings clearly demonstrate both the potential for the continuous spread of contaminated lettuce to multiple batches of product during processing and the need for improved equipment designs that minimize the buildup of residual product during processing.

Inactivation of Stressed Escherichia coli O157:H7 Cells on the Surfaces of Rocket Salad Leaves by Chlorine and Peroxyacetic Acid

2014 ◽  
Vol 77 (1) ◽  
pp. 32-39 ◽  
Author(s):  
ANAS A. AL-NABULSI ◽  
TAREQ M. OSAILI ◽  
HEBA M. OBAIDAT ◽  
REYAD R. SHAKER ◽  
SADDAM S. AWAISHEH ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Deionized Water ◽  
Escherichia Coli O157 ◽  
Apparent Difference ◽  
Peroxyacetic Acid ◽  
Surface Disinfection ◽  
E Coli ◽  
Leafy Greens ◽  
Foodborne Outbreaks ◽  
Stressed Cells

Because Escherichia coli O157:H7 has been frequently associated with many foodborne outbreaks caused by consumption of leafy greens (lettuce, spinach, and celery), this study investigated the ability of deionized water, chlorine, and peroxyacetic acid to detach or inactivate stressed and unstressed cells of E. coli O157:H7 contaminating the surfaces of rocket salad leaves. E. coli O157:H7 cells stressed by acid, cold, starvation, or NaCl exposure, as well as unstressed cells, were inoculated on the surfaces of rocket salad leaves at 4°C. The effectiveness of two sanitizers (200 ppm of chlorine and 80 ppm of peroxyacetic acid) and deionized water for decontaminating the leaves treated with stressed and unstressed E. coli O157:H7 were evaluated during storage at 10 or 25°C for 0.5, 1, 3, and 7 days. It was found that washing with 80 ppm of peroxyacetic acid was more effective and reduced unstressed and stressed cells of E. coli O157:H7 by about 1 log CFU per leaf on the leaves. There was no apparent difference in the ability of stressed and unstressed cells to survive surface disinfection with the tested agents. Treatments to reduce viable E. coli O157:H7 cells on rocket leaves stored at 25°C were more effective than when used on those stored at 10°C. Washing with peroxyacetic acid or chlorine solution did not ensure the safety of rocket leaves, but such treatments could reduce the likelihood of water-mediated transfer of E. coli O157:H7 during washing and subsequent processing.

Effect of Modified Atmosphere Packaging on the Persistence and Expression of Virulence Factors of Escherichia coli O157:H7 on Shredded Iceberg Lettuce†

2011 ◽  
Vol 74 (5) ◽  
pp. 718-726 ◽  
Author(s):  
MANAN SHARMA ◽  
SUDESNA LAKSHMAN ◽  
SEAN FERGUSON ◽  
DAVID T. INGRAM ◽  
YAGUANG LUO ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Virulence Factors ◽  
Modified Atmosphere Packaging ◽  
Ambient Air ◽  
Modified Atmosphere ◽  
Escherichia Coli O157 ◽  
Atmospheric Conditions ◽  
E Coli ◽  
Leafy Greens ◽  
Fresh Cut

Fresh-cut leafy greens contaminated with Escherichia coli O157:H7 have caused foodborne outbreaks. Packaging conditions, coupled with abusive storage temperatures of contaminated lettuce, were evaluated for their effect on the potential virulence of E. coli O157:H7. Shredded lettuce was inoculated with 5.58 and 3.98 log CFU E. coli O157:H7 per g and stored at 4 and 15°C, respectively, for up to 10 days. Lettuce was packaged under treatment A (modified atmosphere packaging conditions used for commercial fresh-cut produce, in gas-permeable film with N2), treatment B (near–ambient air atmospheric conditions in a gas-permeable film with microperforations), and treatment C (high-CO2 and low-O2 conditions in a gas-impermeable film). E. coli O157:H7 populations from each treatment were determined by enumeration of numbers on MacConkey agar containing nalidixic acid. RNA was extracted from packaged lettuce for analysis of expression of virulence factor genes stx2, eae, ehxA, iha, and rfbE. E. coli O157:H7 populations on lettuce at 4°C under all treatments decreased, but most considerably so under treatment B over 10 days. At 15°C, E. coli O157:H7 populations increased by at least 2.76 log CFU/g under all treatments. At 15°C, expression of eae and iha was significantly greater under treatment B than it was under treatments A and C on day 3. Similarly, treatment B promoted significantly higher expression of stx2, eae, ehxA, and rfbE genes on day 10, compared with treatments A and C at 15°C. Results indicate that storage under near–ambient air atmospheric conditions can promote higher expression levels of O157 virulence factors on lettuce, and could affect the severity of E. coli O157:H7 infections associated with leafy greens.

Growth of Escherichia coli O157:H7 and Listeria monocytogenes in Packaged Fresh-Cut Romaine Mix at Fluctuating Temperatures during Commercial Transport, Retail Storage, and Display

2014 ◽  
Vol 77 (2) ◽  
pp. 197-206 ◽  
Author(s):  
WENTING ZENG ◽  
KEITH VORST ◽  
WYATT BROWN ◽  
BRADLEY P. MARKS ◽  
SANGHYUP JEONG ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Large Scale ◽  
Microbial Growth ◽  
Escherichia Coli O157 ◽  
Temperature Profiles ◽  
E Coli ◽  
Leafy Greens ◽  
Retail Display ◽  
Fluctuating Temperatures

Temperature abuse during commercial transport and retail sale of leafy greens negatively impacts both microbial safety and product quality. Consequently, the effect of fluctuating temperatures on Escherichia coli O157:H7 and Listeria monocytogenes growth in commercially-bagged salad greens was assessed during transport, retail storage, and display. Over a 16-month period, a series of time-temperature profiles for bagged salads were obtained from five transportation routes covering four geographic regions (432 profiles), as well as during retail storage (4,867 profiles) and display (3,799 profiles). Five different time-temperature profiles collected during 2 to 3 days of transport, 1 and 3 days of retail storage, and 3 days of retail display were then duplicated in a programmable incubator to assess E. coli O157:H7 and L. monocytogenes growth in commercial bags of romaine lettuce mix. Microbial growth predictions using the Koseki-Isobe and McKellar-Delaquis models were validated by comparing the root mean square error (RMSE), bias, and the acceptable prediction zone between the laboratory growth data and model predictions. Monte Carlo simulations were performed to calculate the probability distribution of microbial growth from 8,122,127,472 scenarios during transport, cold room storage, and retail display. Using inoculated bags of retail salad, E. coli O157:H7 and L. monocytogenes populations increased a maximum of 3.1 and 3.0 log CFU/g at retail storage. Both models yielded acceptable RMSEs and biases within the acceptable prediction zone for E. coli O157:H7. Based on the simulation, both pathogens generally increased <2 log CFU/g during transport, storage, and display. However, retail storage duration can significantly impact pathogen growth. This large-scale U.S. study—the first using commercial time/temperature profiles to assess the microbial risk of leafy greens—should be useful in filling some of the data gaps in current risk assessments for leafy greens.

scholarly journals Outbreaks of Escherichia coli O157:H7 Infections Linked to Romaine Lettuce in Canada from 2008 to 2018: An Analysis of Food Safety Context

2020 ◽  
Vol 83 (8) ◽  
pp. 1444-1462 ◽  
Author(s):  
GENEVIÈVE COULOMBE ◽  
ANGELA CATFORD ◽  
AMALIA MARTINEZ-PEREZ ◽  
ENRICO BUENAVENTURA
Keyword(s):  
Escherichia Coli ◽  
The United States ◽  
Escherichia Coli O157 ◽  
Romaine Lettuce ◽  
E Coli ◽  
Leafy Greens ◽  
Iceberg Lettuce ◽  
Canadian Population ◽  
The Difference ◽  
Commercial Distribution

ABSTRACT Foodborne diseases are a major cause of illness in Canada. One of the main pathogens causing cases and outbreaks of foodborne illness in Canada is Escherichia coli O157:H7. From 2008 to 2018, 11 outbreaks of E. coli O157:H7 infection in Canada were linked to leafy greens, including 7 (63.6%) linked to romaine lettuce, 2 (18.2%) linked to iceberg lettuce, and 2 (18.2%) linked to other or unspecified types of leafy greens. The consumption of lettuce in Canada, the behavior of E. coli O157:H7 on lettuce leaves, and the production practices used for romaine and iceberg lettuce do not seem to explain why a higher number of outbreaks of E. coli O157:H7 infection were linked to romaine than to iceberg lettuce. However, the difference in the shape of iceberg and romaine lettuce heads could be an important factor. Among the seven outbreaks linked to romaine lettuce in Canada between 2008 and 2018, an eastern distribution of cases was observed. Cases from western provinces were reported only twice. The consumption of romaine and iceberg lettuce by the Canadian population does not seem to explain the eastern distribution of cases observed, but the commercial distribution, travel distances, and the storage practices used for lettuce may be important factors. In the past 10 years, the majority of the outbreaks of E. coli O157:H7 infection linked to romaine lettuce occurred during the spring (March to June) and fall (September to December). The timing of these outbreaks may be explained by the availability of lettuce in Canada, the growing region transition periods in the United States, and the seasonality in the prevalence of E. coli O157:H7. The consumption of romaine lettuce by the Canadian population does not explain the timing of the outbreaks observed. HIGHLIGHTS

Preharvest Internalization of Escherichia coli O157:H7 into Lettuce Leaves, as Affected by Insect and Physical Damage

2010 ◽  
Vol 73 (10) ◽  
pp. 1809-1816 ◽  
Author(s):  
MARILYN C. ERICKSON ◽  
JEAN LIAO ◽  
ALISON S. PAYTON ◽  
DAVID G. RILEY ◽  
CATHY C. WEBB ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Insect Pests ◽  
Enteric Pathogen ◽  
Escherichia Coli O157 ◽  
Physical Damage ◽  
E Coli ◽  
Leafy Greens ◽  
Different Populations ◽  
Pathogen Exposure ◽  
Spinach Leaves

Environmental pests may serve as reservoirs and vectors of zoonotic pathogens to leafy greens; however, it is unknown whether insect pests feeding on plant tissues could redistribute these pathogens present on the surface of leaves to internal sites. This study sought to differentiate the degree of tissue internalization of Escherichia coli O157:H7 when applied at different populations on the surface of lettuce and spinach leaves, and to ascertain whether lettuce-infesting insects or physical injury could influence the fate of either surface or internalized populations of this enteric pathogen. No internalization of E. coli O157:H7 occurred when lettuce leaves were inoculated with 4.4 log CFU per leaf, but it did occur when inoculated with 6.4 log CFU per leaf. Internalization was statistically greater when spinach leaves were inoculated on the abaxial (underside) than when inoculated on the adaxial (topside) side, and when the enteric pathogen was spread after surface inoculation. Brief exposure (~18 h) of lettuce leaves to insects (5 cabbage loopers, 10 thrips, or 10 aphids) prior to inoculation with E. coli O157:H7 resulted in significantly reduced internalized populations of the pathogen within these leaves after approximately 2 weeks, as compared with leaves not exposed to insects. Surface-contaminated leaves physically injured through file abrasions also had significantly reduced populations of both total and internalized E. coli O157:H7 as compared with nonabraded leaves 2 weeks after pathogen exposure.

Reduction of Escherichia coli O157:H7 and Salmonella on Baby Spinach, Using Electron Beam Radiation

2008 ◽  
Vol 71 (12) ◽  
pp. 2415-2420 ◽  
Author(s):  
JACK A. NEAL ◽  
ELISA CABRERA-DIAZ ◽  
MAYRA MÁRQUEZ-GONZÁLEZ ◽  
JOSEPH E. MAXIM ◽  
ALEJANDRO CASTILLO
Keyword(s):  
Escherichia Coli ◽  
Electron Beam ◽  
Low Dose ◽  
Spinacia Oleracea ◽  
Microbial Populations ◽  
Escherichia Coli O157 ◽  
Beam Radiation ◽  
E Coli ◽  
Electron Beam Radiation ◽  
Multiple Strains

The effect of low-dose electron beam (e-beam) radiation on the reduction of Escherichia coli O157:H7 and Salmonella in spinach was studied. Fresh baby spinach (Spinacia oleracea) was inoculated with a bacterial cocktail containing multiple strains of rifampin-resistant E. coli O157:H7 and rifampin-resistant Salmonella. Inoculated samples were exposed to e-beam radiation from a linear accelerator and tested for counts of both E. coli O157:H7 and Salmonella. Irradiated spinach was also stored for 8 days at 4°C, and counts were made at 2-day intervals to determine if there was any effect of radiation on the survival trend of both pathogens. When no pathogens were detected on plates, additional enrichment plating was conducted to verify total destruction. Respiration rates were measured on spinach samples exposed to e-beam radiation. Each dose of e-beam radiation significantly reduced the numbers of E. coli O157:H7 and Salmonella from initial levels of 7 log CFU/g. Treatment by e-beam radiation at a dose of 0.40 kGy resulted in a reduction in populations of E. coli O157:H7 and Salmonella of 3.7 and 3.4 log cycles, respectively. At 0.70 kGy, both pathogens were reduced by 4 log. All doses above 1.07 kGy showed reductions greater than 6 log and decreased to undetectable levels when stored for 8 days. The respiration rate of spinach showed no changes after irradiation up to 2.1 kGy. These results suggest that low-dose e-beam radiation may be a viable tool for reducing microbial populations or eliminating E. coli O157:H7 and Salmonella from spinach without product damage.

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