1972

Journal of Food Protection Vol 66 No 11 2003 Pages 1972ndash1977Copyright q International Association for Food Protection

Incidence Duration and Prevalence of Escherichia coli O157H7Fecal Shedding by Feedlot Cattle during the Finishing Period

M L KHAITSA1 D R SMITH1 J A STONER2 A M PARKHURST3 S HINKLEY1 T J KLOPFENSTEIN4 AND

R A MOXLEY1

1Department of Veterinary and Biomedical Sciences Institute of Agriculture and Natural Resources University of NebraskandashLincoln PO Box830905 Lincoln Nebraska 68583-0905 2Biostatistics Section Preventive and Societal Medicine University of Nebraska Medical Center PO Box984350 Omaha Nebraska 68198-4350 3Department of Biometry Institute of Agriculture and Natural Resources University of NebraskandashLincoln

PO Box 830905 Lincoln Nebraska 68583-0712 and 4Department of Animal Science Institute of Agriculture and Natural Resources University ofNebraskandashLincoln PO Box 830908 Lincoln Nebraska 68583-0908 USA

MS 03-72 Received 14 February 2003Accepted 25 May 2003

ABSTRACT

The objective was to describe variability in prevalence incidence and duration of fecal shedding of naturally occurringE coli O157H7 by a group of feedlot cattle over time One hundred steers randomly assigned to 10 pens were fed a high-concentrate nishing diet for 136 days (19 weeks) Rectal feces from each animal were tested for E coli O157H7 every weekfor 19 weeks E coli O157H7 was recovered from each animal that completed the study and was detected from at least oneanimal every week Average pen prevalence of cattle shedding E coli O157H7 varied signi cantly over time (P 00001)and across pens (P 00001) ranging from 1 to 80 Pairwise comparisons of mean pen prevalence of E coli O157H7between weeks and estimation of the predicted probability of an incident case of E coli O157H7 over time allowed thede nition of three distinct phasesmdashnamely the preepidemic epidemic and postepidemic periods Average pen prevalencevaried signi cantly over time (P 001) and across pens (P 0001) for all time periods The odds of an incident case weresigni cantly greater during epidemic and postepidemic periods relative to the preepidemic period (P 5 00002 and P 5 003respectively) Duration of infection was signi cantly longer for rst or second infections that began during epidemic orpostepidemic periods relative to the preepidemic period (P 0001) Both incidence and duration of shedding peaked duringthe epidemic period Pen-level prevalence of cattle shedding E coli O157H7 was affected by both incidence and duration ofshedding and could be explained by time- or pen-dependent risk factors or both

E coli O157H7 has been identi ed as a potential haz-ard associated with consumption of beef products (1 1328) This organism is widely distributed among groups offeedlot cattle (6 10 27) Determining critical control pointsfor E coli O157H7 however requires a greater under-standing of the ecology of this organism Unfortunately itsepidemiology in cattle production systems is still poorlyunderstood (25)

Fecal and hide prevalence of E coli O157H7 fromcattle presented for slaughter at meat processing plants havebeen signi cantly correlated with carcass contamination(6) indicating a role for control of E coli O157H7 in livecattle It is important that studies of the ecology of E coliO157H7 identify the factors that in uence shedding of thisorganism in cattle so as to minimize the level of sheddingparticularly in those cattle going to slaughter

In studies conducted in commercial feed yards we foundthat the percentage of cattle shedding E coli O157H7 in apen ranged from 1 to 80 (27) Also the percentage of cattleshedding E coli O157H7 varied greatly within pens in afeedlot but not between feedlots (27) Because each pen ofcattle in that study was tested only once we were unable tomonitor changes in prevalence over time The objective ofthis study was to describe the prevalence incidence and du-

Author for correspondence Tel 402-472-2362 E-mail dsmithunledu

ration of fecal excretion of E coli O157H7 by feedlot cattleover the course of the feeding period and to determine wheth-er factors that contributed to E coli O157H7 shedding wererelated to time or place

MATERIALS AND METHODS

Study design The design was a longitudinal observationalstudy to monitor individual cattle for the presence of E coliO157H7 in rectal feces during the feedlot nishing period

Animals One hundred steers were randomly assigned to 10pens (10 animals each) on arrival to the feed yard at the Agri-culture Research and Development Center University of Nebras-kandashLincoln Ithaca Nebr The pens were arranged in a row witha water trough shared between adjacent pens and a separate feedbunk for each pen The steers were fed a high-concentrate nish-ing diet for 136 days starting in June 2000 One animal was re-moved from the study during the seventh week because of itsaggressive behavior The cattle were tested once each week for 19weeks Previously these calves had been purchased from Nebras-ka ranchers during October and November 1999 They were thenfed cornstalks corn gluten feed and hay until the observationalstudy started The calves were turned onto harvested corn eldson arrival then raised in con nement from 10 March until the startof the study

Laboratory methods Feces were collected from the rectumof each animal in each pen each week of the study The fecal

J Food Prot Vol 66 No 11 PATTERNS OF E COLI O157H7 SHEDDING OVER TIME BY FEEDLOT CATTLE 1973

FIGURE 1 The proportion of cattle detected shedding Escherich-ia coli O157H7 each week of the 19-week feeding period (Juneto October 2000) Statistical analysis revealed three distinct prev-alence periods termed preepidemic epidemic and postepidemic(double arrows)

samples were transported to the laboratory and within 3 to 4 hafter collection from the animal subsamples were put into en-richment culture Culture methods optimized for the detection ofE coli O157H7 in fecal specimens were used (27) A multiplexPCR assay was modi ed from existing protocols using primers todetect genes for O157 antigen synthesis (rfbE (22)) intimin(eaeO157 (9)) Shigatoxin-1 (stx1 (21)) Shigatoxin-2 (stx2 (23))and H7 ( iC (8)) Isolates were initially identi ed as E coli bybiochemical tests and API 20E (bioMerieux Hazelwood Mo) aspreviously described (27) Isolates that we nally designated asE coli O157H7 were positive for the O157 antigen by latex ag-glutination (Remel Lenexa Kans) and further tested by multiplexPCR Isolates with a positive PCR reaction for both rfbEO157H7

and iCH7 and at least one of the virulence factor genes (ie stx1stx2 or eaeO157) were designated enterohemorrhagicE coli O157H7 (8) These criteria allow for detection of E coli O157H7strains that have lost stx or eae genes and also circumvent theambiguity associated with the nonmotile designation (8)

Data imputation During weeks 11 12 and 17 a portion ofthe samples were cultured in selective enrichment media that con-tained a tenfold increase in the concentrationof ce xime resultingin no growth on the respective isolation plates The presence ofE coli O157H7 was imputed for all no-growth observations (84of 1888) during these weeks as well as for four additional ob-servations in which a sample was not collected Logistic regres-sion (SAS Proc Logistic) was used to model the probability ofgrowth given the animalrsquos culture results prior to and after theweek in question There was one missing observation each inweeks 6 8 13 and 19 There were 59 17 and 9 missing obser-vations in weeks 11 12 and 17 respectively We generated aresponse for the observations that were in question with the pre-dicted probabilities of E coli O157H7 presence for periods thatwere similar to the weeks with missing values Information fromweeks 1 through 8 was used to impute E coli O157H7 valuesfor the missing observations between weeks 1 and 8 informationfrom weeks 9 through 15 was used to impute E coli O157H7values for the missing observations between weeks 9 and 15 andinformation from weeks 1 through 18 was used to impute E coliO157H7 values for the missing observations between weeks 16through 19 Missing observations from the animal removed fromthe study were not imputed because of insuf cient follow-up in-formation

Statistical methods E coli O157H7 pen prevalence wascomputed from the E coli O157H7 status of individual animalswithin the pen and was de ned as the proportion of cattle in apen shedding a detectable level of E coli O157H7 on any givensampling date To describe the sources of variability in the pen-level prevalence of E coli O157H7 a mixed model analysis ofvariance (SAS Proc Mixed) was used with an AR(1) correlationstructure and the variable pen as the repeated measure Place(pen) time (week) and water tank sharing were investigated assources of variability in the E coli O157H7 prevalencedata Pensand the sharing of water tanks were modeled as random effectsand week was modeled as a xed effect Each week correspondedto a particular time point along the feeding period of the cattleso week-speci c average responses were of interest Pairwisecomparisons of the mean pen prevalence of E coli O157H7 be-tween weeks were made using Tukeyrsquos honestly signi cant dif-ference test As an exploratory analysis the effect of time wasinvestigated both as a continuous and categorical effect on thebasis of the E coli O157H7 shedding pattern observed and themixed model was t within each time period to determine whetherpen and time were sources of variability in these time periods

This analysis enabled us to informally test for an interaction be-tween time and pen effects on E coli O157H7 prevalence

Incidence of E coli O157H7 shedding for a particular weekwas de ned as the proportion of cattle whose feces were culture-positive that week but were negative the previous week (at risk)Each animal could have more than one incidence of shedding overthe course of the feeding period each with its own duration Alogistic regression model was used to model the probability of anincident case of E coli O157H7 as a function of time Time (thefeeding period) was split into three periods (weeks 1 to 8 9 to15 and 16 to 19) according to the observed patterns of E coliO157H7 shedding The logistic regression models were t byGeneralized Estimating Equations to account for the likely cor-relation of responses within animals over time and within pens(14 16) Computations were made using GenMod and GEEPLUSSAS Macro (15)

The duration of E coli O157H7 shedding was de ned asthe number of consecutive weeks an animal shed a detectable levelof the organism Data on duration of E coli O157H7 sheddingwas complicated because at week 19 some of the infection du-rations were censored so that only the minimum length of theinfections that were still present at week 19 could be determinedTherefore a summary of the median time to infection estimatedfrom survival curves for the animals in each pen was analyzedA survival regression modeling procedure was used to determinewhether the duration distribution differed depending on when theinfection started or the infection number ( rst infection secondinfection etc) among recurrent infections The method whichwas proposed and described earlier (24) is appropriate for ana-lyzing the duration of recurrent infections within the same animal

RESULTS

Prevalence of E coli O157H7 shedding During thecourse of the study 697 E coli O157H7 isolates were re-covered PCR detected stx genes in all isolates and theeaeO157 gene in 696 of 697 isolates The proportion of cattledetected shedding E coli O157H7 each week of the feed-ing period is shown in Figure 1 E coli O157H7 was re-covered at least once during the feeding period from eachanimal that completed the study and the organism was de-tected from at least one animal every week

At the pen level the percentage of cattle shedding a

J Food Prot Vol 66 No 111974 KHAITSA ET AL

FIGURE 2 The prevalence of Escherichia coli O157H7 fecal shedding by pen and week for the 19 weeks of the feeding period (Juneto October 2000) illustrates the distribution of the agent by time and place

FIGURE 3 Incidence density and duration of Escherichia coliO157H7 shedding by cattle for the 19 weeks of the feeding period(June to October 2000)

detectable level of E coli O157H7 within a given pen ofsteers in the feed yard over the feeding period ranged from0 to 100 (Fig 2) A pattern of E coli O157H7 sheddingcould be seen whereby the rst 8 weeks were characterizedby low pen prevalence (range 0 to 70 median 0)followed by 7 weeks of high pen prevalence (range 0 to100 median 79) and nally 4 weeks of medium penprevalence (range 0 to 100 median 30) On the lastsampling date 30 of 99 steers that completed the study(303) were excreting a detectable level of E coli O157H7 in their feces and 9 of 10 pens had at least one steershedding E coli O157H7 in its feces Ninety percent ofthe animals in one pen 40 of the animals in three pens30 of the animals in one pen 20 of the animals in two

pens and 10 to 11 of the animals in two pens were cul-ture-positive for E coli O157H7 on the last sampling date

Both pen (P 00001) and week (P 00001) weresigni cant sources of variability in E coli O157H7 prev-alence Pairwise comparisons of mean pen prevalence of Ecoli O157H7 between weeks allowed the de nition ofthree distinct phases within the feeding period We there-after named the three periods preepidemic (weeks 1 to 8)epidemic (weeks 9 to 15) and postepidemic (weeks 16 to19) (Fig 1) When the mixed model analysis of variancewas t onto data for the three prevalence periods observedE coli O157H7 prevalence differed signi cantly over time(P 001) and across pens (P 0001) for the three pe-riods Results of a mixed model analysis of variance indi-cated no evidence of the water tank acting as a signi cantsource of variability in E coli O157H7 prevalence (P 507)

Incidence of E coli O157H7 shedding The rst 8weeks of the feeding period were characterized by low in-cidence (001 to 010 new cases per animal per week) offecal excretion of E coli O157H7 The incidence increaseddramatically in week 9 (05 new cases per animal perweek) reached the highest rate in week 12 (07 new casesper animal per week) and decreased during the last 5 weeksof the study (Fig 3) Because new cases of E coli O157H7 for the proportion of samples affected by the delay inculture in weeks 11 12 and 17 as well as the four timepoints for which no sample was drawn were based on im-puted data incidence values for those weeks could havebeen under- or even overestimated

The logistic model investigating the time period effecton the odds of an incident case included an intercept andtwo terms representing the time period of the observationduring either the epidemic period (weeks 9 to 15) or the

J Food Prot Vol 66 No 11 PATTERNS OF E COLI O157H7 SHEDDING OVER TIME BY FEEDLOT CATTLE 1975

TABLE 1 Median duration in weeks that E coli O157H7 wasrecovered from individual cattle according to pen and infectionnumber (the rank of each sequential new infection)

Pen

Infection numbera

1 2 3 4 5

309310311312313314315316317318

2313352422

1323312315

132241231ndash

ndash24

1

ndash1ndash

ndash

ndash

a An empty cell implies that no animals experienced the higherorder infection a dash implies that the median could not beestimated from the data

postepidemic period (weeks 16 to 19) The odds of an in-cident case were signi cantly greater during the epidemicperiod than the preepidemic period (P 5 00002) Speci -cally the odds of an incident case during the epidemic pe-riod were 211 times the odds of an incident case duringthe preepidemic period (95 CI 75 597) The odds ofan incident case were signi cantly greater during thepostepidemic period than the preepidemic period (P 5003)mdashspeci cally 42 times the odds of an incident caseduring the preepidemic period (95 CI 12 153) Thepredicted probabilities of an incident case during the preepi-demic epidemic and postepidemic periods were 006 059and 022 respectively

Duration of E coli O157H7 shedding The rst 7weeks of the feeding period were characterized by esti-mated short mean duration (25 weeks) of shedding ofthe organism The duration of shedding increased dramat-ically in week 8 peaked in week 9 and gradually decreasedduring the last 10 weeks of the study (Fig 3) Note thatthe estimate of duration could have been underestimatedbecause some animals were still shedding at week 19 whenthe study ended and the duration of their infections werecensored The mean duration of fecal shedding was longestduring the epidemic period with cases starting in weeks 8and 9 and lasting on average 39 and 45 weeks respec-tively On the last sampling date (week 19) 30 of thesteers were excreting a detectable level of E coli O157H7in their feces and these animals had been shedding theorganism for a mean duration of 31 weeks (range 1 to 10median 2) The average number of times each steer shedE coli O157H7 in the feces during the course of the studywas 25 times (range 1 to 5 median 2)

The median duration (weeks) of E coli O157H7 in-fection is summarized for cattle within each pen accordingto infection number (Table 1) From the descriptive sum-mary of the median duration times it did not appear thatthe duration differed depending on infection sequence num-ber ( rst infection second infection etc) Also from the

regression model the duration of infection did not appearto differ signi cantly between rst and second infections or rst and third infections (P 04 for both comparisonsrelative risk for infection ending was 11 and 12 for secondand third relative to rst infections respectively)

The results of survival regression modeling proceduresto determine whether the duration distribution differed de-pending on when the infection started were based only oninformation from the rst and second infections experi-enced by each animal The rst and second infections com-prised about 75 of the observed infections When lookingfor preepidemicepidemicpostepidemic time period effectswe did not see third infections in the preepidemic periodand we did not see rst infections in the postepidemic pe-riod The analysis compared the duration of infections thatbegan in the preepidemic period to the duration of infec-tions that began during either the epidemic period or thepostepidemic period The regression model results suggest-ed that the duration of infection was signi cantly longer for rst or second infections that began during the epidemic orpostepidemic periods relative to the preepidemic period (P 0001) Speci cally the risk of the rst or second infec-tion ending when it began in the epidemic or postepidemicperiod was 036 relative to the risk of the infection endingwhen it began in the preepidemic period (95 CI 020066)

DISCUSSION

The range of E coli O157H7 prevalence in pens offeedlot cattle in this longitudinal study was nearly identicalto that we previously reported in a cross-sectional study ofcommercial feedlot cattle (27) Other researchers havereported similar (3 6 18 29) or lower (4 7 11 12)prevalence values than reported herein The lower E coliO157H7 prevalence estimates were supposedly attributedto lower sensitivity methods and differences in methodol-ogy (17)

In an earlier study (27) E coli O157H7 prevalencevaried between pens within a feedlot but not between feed-lots and we were unable to test the role of time on vari-ability In this study we describe the variation in E coliO157H7 shedding between pens and across time duringthe feeding period for this group of cattle Other longitu-dinal studies of feedlot cattle have been reported (18 19)This pattern of change over time might suggest the exis-tence of time-dependent risk factors for E coli O157H7shedding which underscores the importance of longitudinalstudy designs in our effort to understand the epidemiologyof this organism in feedlot cattle

The pattern of E coli O157H7 prevalence observedin this feedlot allowed us to de ne three phases within thefeeding period Further investigation showed a differencein the odds of E coli O157H7 incidence among the threephases and this was the basis of de ning the three periodsas preepidemic epidemic and postepidemic The limita-tions in our estimation of the odds of an incident case ofE coli O157H7 stemmed from some imputed values andfrom the assumption that the odds of E coli O157H7 in-cidence were similar across all pens It is possible this was

J Food Prot Vol 66 No 111974 KHAITSA ET AL

FIGURE 2 The prevalence of Escherichia coli O157H7 fecal shedding by pen and week for the 19 weeks of the feeding period (Juneto October 2000) illustrates the distribution of the agent by time and place

FIGURE 3 Incidence density and duration of Escherichia coliO157H7 shedding by cattle for the 19 weeks of the feeding period(June to October 2000)

detectable level of E coli O157H7 within a given pen ofsteers in the feed yard over the feeding period ranged from0 to 100 (Fig 2) A pattern of E coli O157H7 sheddingcould be seen whereby the rst 8 weeks were characterizedby low pen prevalence (range 0 to 70 median 0)followed by 7 weeks of high pen prevalence (range 0 to100 median 79) and nally 4 weeks of medium penprevalence (range 0 to 100 median 30) On the lastsampling date 30 of 99 steers that completed the study(303) were excreting a detectable level of E coli O157H7 in their feces and 9 of 10 pens had at least one steershedding E coli O157H7 in its feces Ninety percent ofthe animals in one pen 40 of the animals in three pens30 of the animals in one pen 20 of the animals in two

pens and 10 to 11 of the animals in two pens were cul-ture-positive for E coli O157H7 on the last sampling date

Both pen (P 00001) and week (P 00001) weresigni cant sources of variability in E coli O157H7 prev-alence Pairwise comparisons of mean pen prevalence of Ecoli O157H7 between weeks allowed the de nition ofthree distinct phases within the feeding period We there-after named the three periods preepidemic (weeks 1 to 8)epidemic (weeks 9 to 15) and postepidemic (weeks 16 to19) (Fig 1) When the mixed model analysis of variancewas t onto data for the three prevalence periods observedE coli O157H7 prevalence differed signi cantly over time(P 001) and across pens (P 0001) for the three pe-riods Results of a mixed model analysis of variance indi-cated no evidence of the water tank acting as a signi cantsource of variability in E coli O157H7 prevalence (P 507)

Incidence of E coli O157H7 shedding The rst 8weeks of the feeding period were characterized by low in-cidence (001 to 010 new cases per animal per week) offecal excretion of E coli O157H7 The incidence increaseddramatically in week 9 (05 new cases per animal perweek) reached the highest rate in week 12 (07 new casesper animal per week) and decreased during the last 5 weeksof the study (Fig 3) Because new cases of E coli O157H7 for the proportion of samples affected by the delay inculture in weeks 11 12 and 17 as well as the four timepoints for which no sample was drawn were based on im-puted data incidence values for those weeks could havebeen under- or even overestimated

The logistic model investigating the time period effecton the odds of an incident case included an intercept andtwo terms representing the time period of the observationduring either the epidemic period (weeks 9 to 15) or the

J Food Prot Vol 66 No 11 PATTERNS OF E COLI O157H7 SHEDDING OVER TIME BY FEEDLOT CATTLE 1975

TABLE 1 Median duration in weeks that E coli O157H7 wasrecovered from individual cattle according to pen and infectionnumber (the rank of each sequential new infection)

Pen

Infection numbera

1 2 3 4 5

309310311312313314315316317318

2313352422

1323312315

132241231ndash

ndash24

1

ndash1ndash

ndash

ndash

a An empty cell implies that no animals experienced the higherorder infection a dash implies that the median could not beestimated from the data

postepidemic period (weeks 16 to 19) The odds of an in-cident case were signi cantly greater during the epidemicperiod than the preepidemic period (P 5 00002) Speci -cally the odds of an incident case during the epidemic pe-riod were 211 times the odds of an incident case duringthe preepidemic period (95 CI 75 597) The odds ofan incident case were signi cantly greater during thepostepidemic period than the preepidemic period (P 5003)mdashspeci cally 42 times the odds of an incident caseduring the preepidemic period (95 CI 12 153) Thepredicted probabilities of an incident case during the preepi-demic epidemic and postepidemic periods were 006 059and 022 respectively

Duration of E coli O157H7 shedding The rst 7weeks of the feeding period were characterized by esti-mated short mean duration (25 weeks) of shedding ofthe organism The duration of shedding increased dramat-ically in week 8 peaked in week 9 and gradually decreasedduring the last 10 weeks of the study (Fig 3) Note thatthe estimate of duration could have been underestimatedbecause some animals were still shedding at week 19 whenthe study ended and the duration of their infections werecensored The mean duration of fecal shedding was longestduring the epidemic period with cases starting in weeks 8and 9 and lasting on average 39 and 45 weeks respec-tively On the last sampling date (week 19) 30 of thesteers were excreting a detectable level of E coli O157H7in their feces and these animals had been shedding theorganism for a mean duration of 31 weeks (range 1 to 10median 2) The average number of times each steer shedE coli O157H7 in the feces during the course of the studywas 25 times (range 1 to 5 median 2)

The median duration (weeks) of E coli O157H7 in-fection is summarized for cattle within each pen accordingto infection number (Table 1) From the descriptive sum-mary of the median duration times it did not appear thatthe duration differed depending on infection sequence num-ber ( rst infection second infection etc) Also from the

regression model the duration of infection did not appearto differ signi cantly between rst and second infections or rst and third infections (P 04 for both comparisonsrelative risk for infection ending was 11 and 12 for secondand third relative to rst infections respectively)

The results of survival regression modeling proceduresto determine whether the duration distribution differed de-pending on when the infection started were based only oninformation from the rst and second infections experi-enced by each animal The rst and second infections com-prised about 75 of the observed infections When lookingfor preepidemicepidemicpostepidemic time period effectswe did not see third infections in the preepidemic periodand we did not see rst infections in the postepidemic pe-riod The analysis compared the duration of infections thatbegan in the preepidemic period to the duration of infec-tions that began during either the epidemic period or thepostepidemic period The regression model results suggest-ed that the duration of infection was signi cantly longer for rst or second infections that began during the epidemic orpostepidemic periods relative to the preepidemic period (P 0001) Speci cally the risk of the rst or second infec-tion ending when it began in the epidemic or postepidemicperiod was 036 relative to the risk of the infection endingwhen it began in the preepidemic period (95 CI 020066)

DISCUSSION

The range of E coli O157H7 prevalence in pens offeedlot cattle in this longitudinal study was nearly identicalto that we previously reported in a cross-sectional study ofcommercial feedlot cattle (27) Other researchers havereported similar (3 6 18 29) or lower (4 7 11 12)prevalence values than reported herein The lower E coliO157H7 prevalence estimates were supposedly attributedto lower sensitivity methods and differences in methodol-ogy (17)

In an earlier study (27) E coli O157H7 prevalencevaried between pens within a feedlot but not between feed-lots and we were unable to test the role of time on vari-ability In this study we describe the variation in E coliO157H7 shedding between pens and across time duringthe feeding period for this group of cattle Other longitu-dinal studies of feedlot cattle have been reported (18 19)This pattern of change over time might suggest the exis-tence of time-dependent risk factors for E coli O157H7shedding which underscores the importance of longitudinalstudy designs in our effort to understand the epidemiologyof this organism in feedlot cattle

The pattern of E coli O157H7 prevalence observedin this feedlot allowed us to de ne three phases within thefeeding period Further investigation showed a differencein the odds of E coli O157H7 incidence among the threephases and this was the basis of de ning the three periodsas preepidemic epidemic and postepidemic The limita-tions in our estimation of the odds of an incident case ofE coli O157H7 stemmed from some imputed values andfrom the assumption that the odds of E coli O157H7 in-cidence were similar across all pens It is possible this was

J Food Prot Vol 66 No 11 PATTERNS OF E COLI O157H7 SHEDDING OVER TIME BY FEEDLOT CATTLE 1975

TABLE 1 Median duration in weeks that E coli O157H7 wasrecovered from individual cattle according to pen and infectionnumber (the rank of each sequential new infection)

Pen

Infection numbera

1 2 3 4 5

309310311312313314315316317318

2313352422

1323312315

132241231ndash

ndash24

1

ndash1ndash

ndash

ndash

a An empty cell implies that no animals experienced the higherorder infection a dash implies that the median could not beestimated from the data

postepidemic period (weeks 16 to 19) The odds of an in-cident case were signi cantly greater during the epidemicperiod than the preepidemic period (P 5 00002) Speci -cally the odds of an incident case during the epidemic pe-riod were 211 times the odds of an incident case duringthe preepidemic period (95 CI 75 597) The odds ofan incident case were signi cantly greater during thepostepidemic period than the preepidemic period (P 5003)mdashspeci cally 42 times the odds of an incident caseduring the preepidemic period (95 CI 12 153) Thepredicted probabilities of an incident case during the preepi-demic epidemic and postepidemic periods were 006 059and 022 respectively

Duration of E coli O157H7 shedding The rst 7weeks of the feeding period were characterized by esti-mated short mean duration (25 weeks) of shedding ofthe organism The duration of shedding increased dramat-ically in week 8 peaked in week 9 and gradually decreasedduring the last 10 weeks of the study (Fig 3) Note thatthe estimate of duration could have been underestimatedbecause some animals were still shedding at week 19 whenthe study ended and the duration of their infections werecensored The mean duration of fecal shedding was longestduring the epidemic period with cases starting in weeks 8and 9 and lasting on average 39 and 45 weeks respec-tively On the last sampling date (week 19) 30 of thesteers were excreting a detectable level of E coli O157H7in their feces and these animals had been shedding theorganism for a mean duration of 31 weeks (range 1 to 10median 2) The average number of times each steer shedE coli O157H7 in the feces during the course of the studywas 25 times (range 1 to 5 median 2)

The median duration (weeks) of E coli O157H7 in-fection is summarized for cattle within each pen accordingto infection number (Table 1) From the descriptive sum-mary of the median duration times it did not appear thatthe duration differed depending on infection sequence num-ber ( rst infection second infection etc) Also from the

regression model the duration of infection did not appearto differ signi cantly between rst and second infections or rst and third infections (P 04 for both comparisonsrelative risk for infection ending was 11 and 12 for secondand third relative to rst infections respectively)

The results of survival regression modeling proceduresto determine whether the duration distribution differed de-pending on when the infection started were based only oninformation from the rst and second infections experi-enced by each animal The rst and second infections com-prised about 75 of the observed infections When lookingfor preepidemicepidemicpostepidemic time period effectswe did not see third infections in the preepidemic periodand we did not see rst infections in the postepidemic pe-riod The analysis compared the duration of infections thatbegan in the preepidemic period to the duration of infec-tions that began during either the epidemic period or thepostepidemic period The regression model results suggest-ed that the duration of infection was signi cantly longer for rst or second infections that began during the epidemic orpostepidemic periods relative to the preepidemic period (P 0001) Speci cally the risk of the rst or second infec-tion ending when it began in the epidemic or postepidemicperiod was 036 relative to the risk of the infection endingwhen it began in the preepidemic period (95 CI 020066)

DISCUSSION

The range of E coli O157H7 prevalence in pens offeedlot cattle in this longitudinal study was nearly identicalto that we previously reported in a cross-sectional study ofcommercial feedlot cattle (27) Other researchers havereported similar (3 6 18 29) or lower (4 7 11 12)prevalence values than reported herein The lower E coliO157H7 prevalence estimates were supposedly attributedto lower sensitivity methods and differences in methodol-ogy (17)

In an earlier study (27) E coli O157H7 prevalencevaried between pens within a feedlot but not between feed-lots and we were unable to test the role of time on vari-ability In this study we describe the variation in E coliO157H7 shedding between pens and across time duringthe feeding period for this group of cattle Other longitu-dinal studies of feedlot cattle have been reported (18 19)This pattern of change over time might suggest the exis-tence of time-dependent risk factors for E coli O157H7shedding which underscores the importance of longitudinalstudy designs in our effort to understand the epidemiologyof this organism in feedlot cattle

The pattern of E coli O157H7 prevalence observedin this feedlot allowed us to de ne three phases within thefeeding period Further investigation showed a differencein the odds of E coli O157H7 incidence among the threephases and this was the basis of de ning the three periodsas preepidemic epidemic and postepidemic The limita-tions in our estimation of the odds of an incident case ofE coli O157H7 stemmed from some imputed values andfrom the assumption that the odds of E coli O157H7 in-cidence were similar across all pens It is possible this was

J Food Prot Vol 66 No 111976 KHAITSA ET AL

not the case Also because of sample size limitation (10pens only) we were unable to test for space-time interac-tion on these data Nevertheless it was possible to discernan epidemic curve-like pattern of E coli O157H7 sheddingin this feedlot

In this study the mean duration of E coli O157H7fecal shedding according to the week in which the infectionstarted was longest (45 weeks) during the epidemic periodAnother study reported a typical duration of fecal sheddingin cattle of approximately 1 month (2) Also duration offecal shedding of E coli O157H7 did not differ signi -cantly between rst and second infections or rst and thirdinfections This might be an indication that rst infectionsof cattle with E coli O157H7 neither confer signi cantlong-term immunity to the cattle to further infections norreduce duration of shedding of the organism Another pos-sible explanation of this observation is that cattle are ex-posed to different challenge levels or clonal types of E coliO157H7 during the different phases of an epidemic Thereis a report (5) that a relatively high challenge dose (104

and probably $107 CFU) of E coli O157H7 grown invitro is required to infect adult cattle compared to pre-weaned calves Information concerning the immune re-sponses and their potential roles in protection of cattleagainst E coli O157H7 infection is minimal Experimentalinfection studies have shown that 1-week-old calves (26)8- to 10-month-old ruminating calves (5) and adult (1year old) cattle (20) have reduced shedding of E coliO157H7 following repeat challenge with the homologousstrain compared to the rst challenge suggesting some lev-el of protective immunity results from infection A limita-tion of this study was not knowing whether the infectionsat the different time sequences were caused by the same ordifferent E coli O157H7 strains This analysis is currentlyin progress Furthermore no information is currently avail-able to suggest whether strain homology or heterologyplays a signi cant role in increased susceptibility to re-peated infection

Prevalence of a disease is a function of both its inci-dence and duration In this study we investigated whetherchanges in E coli O157H7 prevalence were caused bychanges in incidence or duration Our results suggest thatvariation in E coli O157H7 pen-level prevalence was afunction of changes in both incidence and duration indi-cating a need to identify factors and subsequently controlstrategies that will reduce both incidence and duration ofE coli O157H7 fecal shedding Also pens and time(weeks) were signi cant sources of variability in E coliO157H7 prevalence indicating a need to study both pen-level and time-dependent factors Therefore longitudinalstudy designs are important for understanding the epide-miology and methods needed to control this organism

ACKNOWLEDGMENTS

The authors thank Doreen Bailey Karen Hansen Chris Riggert An-gela Schlueter Spring Younts Jeff Folmer and Sharon Clowser for tech-nical assistance This study was supported in part by grants from theNebraska State Legislative Appropriation Bill LB 1206 the Nebraska BeefCouncil and the US Department of Agriculture (NR1CGP 2000-02501)

Journal series contribution no 13968 of the University of Nebraska Ag-ricultural Research Division Lincoln NE 68583

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2 Besser T E D D Hancock L C Pritchard E M McRae D HRice and P I Tarr 1997 Duration of detection of fecal excretionof Escherichia coli O157H7 in cattle J Infect Dis 175726ndash729

3 Blanco M J E Blanco J Blanco et al 1997 Distribution andcharacterization of faecal verotoxin-producing Escherichia coli(VTEC) isolated from healthy cattle Vet Microbiol 54309ndash319

4 Chapman P A C A Siddons A T Gerdan Malo and M AHarkin 1997 A 1-year study of Escherichia coli O157H7 in cattlesheep pigs and poultry Epidemiol Infect 119245ndash250

5 Cray W C Jr and H W Moon 1995 Experimental infection ofcalves and adult cattle with Escherichia coli O157H7 Appl Envi-ron Microbiol 611586ndash1590

6 Elder R O J E Keen G R Siragusa G A Barkocy-GallagherM Koohmaraie and W W Laegreid 2000 Correlation of entero-hemorrhagic Escherichia coli O157H7 prevalence in feces hidesand carcasses of beef cattle during processing Proc Natl Acad SciUSA 972999ndash3003

7 Galland J C 2001 Prevalence antibiotic susceptibility and diver-sity of Escherichia coli O157H7 isolates from a longitudinal studyof beef cattle feedlots Appl Environ Microbiol 671619ndash1627

8 Gannon V P J S DrsquoSouza T Graham R K King K Rahn andS Read 1997 Use of the agellar H7 gene as a target in multiplexPCR assays and improved speci city in identi cation of enterohem-orrhagic Escherichia coli strains J Clin Microbiol 35656ndash662

9 Gannon V P J M Rashed R K King and E J Golsteyn Thomas1993 Detection and characterization of the eae gene of Shiga-liketoxin-producing Escherichia coli using polymerase chain reactionJ Clin Microbiol 311268ndash1274

10 Gansheroff L and A D OrsquoBrien 2000 Escherichia coli O157H7in beef cattle presented for slaughter in the US higher prevalencerates than previously estimated Proc Natl Acad Sci USA 972959ndash2961

11 Hancock D T E Besser M L Kinsel P I Tarr D H Rice andM G Paros 1994 The prevalence of Escherichia coli O157 in dairyand beef cattle in Washington state Epidemiol Infect 113199ndash207

12 Hancock D D D H Rice L Thomas D A Dargatz and T EBesser 1997 Epidemiology of Escherichia coli O157 in feedlot cat-tle J Food Prot 60462ndash465

13 Hogue A T L W Patricia and J A Heminover 1998 Pathogenreduction and hazard analysis and critical control point (HACCP)systems for meat and poultry Vet Clin N Am Food Anim Pract14151ndash164

14 Liang K-Y and S L Zeger 1986 Longitudinal data analysis usinggeneralized linear models Biometrika 7313ndash22

15 Mancl L A 2001 GEEPLUS SAS macro for regression modelingof correlated and longitudinal data Technical Report 9503 (Version2) Biometry Core Regional Clinical Dental Research Center Uni-versity of Washington Technical Report Series University of Wash-ington Seattle Wash

16 Mancl L A and T A DeRouen 2001 A covariance estimator forGEE with improved small-sample properties Biometrics 57126ndash134

17 Meyer-Broseta S S N Bastian P D Arne O Cerf and M Sanaa2001 Review of epidemiological surveys on the prevalence of con-tamination of healthy cattle with Escherichia coli serogroup O157H7 Int J Hyg Environ Health 203347ndash361

18 Midgley J and P Desmarchelier 2001 Pre-slaughter handling ofcattle and Shiga toxin-producing Escherichia coli (STEC) LettAppl Microbiol 32307ndash311

19 Midgley J N Fegan and P Desmarchelier 1999 Dynamics ofShiga toxin-producing Escherichia coli (STEC) in feedlot cattleLett Appl Microbiol 2985ndash89

J Food Prot Vol 66 No 11 PATTERNS OF E COLI O157H7 SHEDDING OVER TIME BY FEEDLOT CATTLE 1977

20 Moxley R A 2002 Personal communication21 Paton A W L Beutin and J C Paton 1995 Heterogeneity of the

amino acid sequences of Escherichia coli Shiga-like toxin type-Ioperons Gene 15371ndash74

22 Paton A W and J C Paton 1998 Detection and characterizationof Shiga toxigenic Escherichia coli by using multiplex PCR assaysfor stx1 stx2 eaeA enterohemorrhagic E coli hlyA rfbO111 andrfbO157 J Clin Microbiol 36598ndash602

23 Paton A W J C Paton and P A Manning 1993 Polymerasechain reaction ampli cation cloning and sequencing of variant Esch-erichia coli Shiga-like toxin type II operons Microb Pathog 1577ndash82

24 Prentice R L B J Williams and A V Peterson 1981 On theregression analysis of multivariate failure time data Biometrika 68373ndash379

25 Rasmussen M A and T A Casey 2001 Environmental and foodsafety aspects of Escherichia coli O157H7 infections in cattle CritRev Microbiol 2757ndash73

26 Sanderson M W T E Besser J M Gay C C Gay and D DHancock 1999 Fecal Escherichia coli O157H7 shedding patternsof orally inoculated calves Vet Microbiol 69199ndash205

27 Smith D R M Blackford S M Younts et al 2001 Ecologicalrelationships between the prevalence of cattle shedding Escherichiacoli O157H7 and characteristics of the cattle or conditions of thefeedlot pen J Food Prot 641899ndash1903

28 Smith R A D D Grif n and D A Dargatz 1997 The risks andprevention of contamination of beef feedlot cattle the perspectiveof the United States of America Rev Sci Technol 16359ndash368

29 Van Donkersgoed J T Graham and V Gannon 1999 The prev-alence of verotoxins Escherichia coli and Salmonella in the fecesand rumen of cattle at processing Can Vet J 40332ndash338