BACKGROUND
in the last  <dig> years, escherichia coli serogroup o <dig>  has become an important cause of severe gastrointestinal illness in westernised countries, warranting substantial public health concern.
clinical signs range from mild diarrhoea to haemorrhagic colitis and haemolytic uraemic syndrome  which may result in death  <cit> .
hus usually occurs in young children and is the major cause of acute renal failure in children in western countries  <cit> .
clinical surveillance in scotland has shown that over 90% of hus cases are associated with e. coli o <dig> infection  <cit> ; similar observations have been made in other countries  <cit> .
cattle are the main reservoir for e. coli o <dig>  <cit> , and play a major role in the epidemiology of human infections  <cit> .
visits to farms, contact with animal excreta and recreational use of animal pasture have all been identified as significant risk factors for sporadic human infections  <cit> .
spatial analyses suggest that human incidence is positively associated with indicators such as livestock density and the ratio of cattle to human population, although the relationship appears complex  <cit> .
considerable effort has been made to determine the prevalence of e. coli o <dig> in cattle worldwide .
estimates of prevalence range from  <dig> to 71% of animals and  <dig> to 100% of herds  <cit> .
two of the world's largest surveys of animal e. coli o <dig> prevalence were conducted in the past decade in scotland.
the first  <cit>  estimated herd-level and animal-level prevalence for  <dig> farms throughout scotland in a study funded by the scottish executive environment and rural affairs department  conducted from march  <dig> to may  <dig>  since then a second survey, funded by the wellcome foundation international partnership research award in veterinary epidemiology  was conducted on a subsample of the  <dig> seerad farms, from february  <dig> to february  <dig>  data from the seerad and iprave studies are presented in this paper.
in scotland, the first reported cases of human e. coli o <dig> infection were identified in  <dig>  currently, health protection scotland  conducts active, population based enhanced surveillance in close collaboration with the scottish e. coli o157/vtec reference laboratory   <cit> .
over the  <dig> year period 1998- <dig>  an annual average of  <dig> culture positive cases has been reported to hps, which is an average annual rate of  <dig>  cases per  <dig>  population  <cit> .
rates in scotland are generally higher than in most other united kingdom, european and north american countries  <cit> .
a recent publication proposed a specific mechanism for the link between human infection and livestock carriage of e. coli o <dig>  <cit>  which involved a subset of shedding animals known as super-shedders.
super-shedders are individuals who for a period yield more infectious organisms  than typical individuals of the same host species  <cit> .
shedding high concentrations of e. coli o <dig> has been proposed as a major contributor to cattle-to-cattle transmission  <cit>  and possibly cattle-to-human transmission.
although little is known about super-shedders it has been shown that they have been associated with the presence of phage type  21/ <dig> whereas non super-shedders are more likely to be associated with pt <dig>  <cit> .
recent evidence has shown pt21/ <dig> to be associated with higher transmission in livestock when compared to pt <dig>  <cit> .
pt21/ <dig> is the most predominant phage type in both cattle  <cit>  and human cases  <cit>  whereas pt <dig> is a common phage type in cattle only  <cit> .
in humans, pt21/ <dig> is of particular concern because of its association with more severe morbidity.
in the uk and ireland , the mean risk of developing diarrhoea-associated hus was significantly higher in children in scotland infected with pt21/ <dig> compared with other phage types  <cit> .
phage type 21/ <dig> strains possess the vtx <dig> gene alone, which appears to be associated with more serious health outcomes  <cit> .
the objectives of this study were three-fold.
first, to calculate the mean prevalence of e. coli o <dig> in cattle using the data from both the seerad  and iprave  surveys.
second, to examine temporal patterns in the overall as well as regional, seasonal and phage type specific prevalence of bovine shedding.
third, to examine the incidence levels and relative proportions of common phage types associated with human cases over the same periods and the proportion of phage types pt21/ <dig> and pt <dig> in bovine isolates and human cases, for evidence of any epidemiological link between the two.
methods
animal prevalence studies
livestock sampling design
two surveys of scottish store and finishing cattle were conducted: the first from march  <dig> to may  <dig>  the second from february  <dig> to february  <dig>  the first study was funded by the scottish executive environment and rural affairs department ; the second by a wellcome foundation international partnership research award in veterinary epidemiology .
details on the methodology of both surveys have been published elsewhere  <cit> , however, a brief outline is given below.
in  <dig>  seerad provided the scottish agricultural college  with a list comprising  <dig>  farms with cattle, randomly selected from  <dig> scottish agricultural and horticultural census data.
for the seerad survey,  <dig> farms across the  <dig> state animal health divisions    were randomly selected and surveyed  <cit> .
owners or managers of  <dig> of these  <dig> farms consented to an additional sampling visit and these  <dig> farms were used as the sampling frame for the second survey .
within the sampling frame for the iprave survey there were insufficient farms to adequately represent two state animal health divisions: highland and islands.
additional farms  for these two ahds were recruited by random selection from the remainder of  <dig>  farms not sampled in the seerad survey.
in total,  <dig> farms were sampled for the iprave survey,  <dig> of which had been previously sampled in the seerad survey.
instead of randomly sampling farms within each ahd, the iprave study used a stratified sampling plan to select farms to sample  <cit> .
this was done to ensure that similar numbers were included from each region and that regions were sampled evenly over time.
both surveys preferentially sampled cattle groups composed only of store  or finishing cattle closest to sale or slaughter.
if such groups did not exist, one or more mixed groups with store or finishing cattle closest to sale or slaughter were sampled.
from each group fresh faecal pats were sampled.
the number of faecal pats tested in each group was determined from the number of cattle in the group using a prescribed sampling schedule.
for the seerad survey, sufficient numbers of faecal pats were tested to ensure prospectively an 80% chance of sampling at least one positive pat if there was a shedding prevalence of at least 2% within the group  <cit> .
based on results from the seerad survey, in the iprave survey, it was assumed that, on average, 8% of the animals in positive groups would be shedding, with shedding distributed as seen in the seerad survey  <cit> .
for each iprave group, sufficient fresh pat samples were taken to ensure prospectively a mean 90% probability of detecting shedding of e. coli o <dig> if at least one shedding animal was indeed present.
samples were collected from freshly voided faecal pats, refrigerated at 5°c as soon as possible and processed within  <dig> hours of collection.
no direct animal sampling was involved and the study was not regulated by the animals  act  <dig>  at present the seerad and iprave data are not available on open-access databases, however, requests for data can be made though the corresponding author.
immunomagnetic separation  and phage typing of livestock samples
within  <dig> hours of sampling, one gram of faeces from each sample was tested for the presence of e. coli o <dig> as previously described  <cit> .
following ims, one e. coli o <dig> isolate from each faecal sample was submitted to the scottish e. coli o157/vtec reference laboratory  for phage typing  <cit> , and tested for the presence of genes encoding the virulence factors verocytotoxin  <dig> , verocytotoxin  <dig>  and intimin  using multiplex pcr  <cit> .
human case identification, data collection and phage typing
health protection scotland  receives reports of human cases of e. coli o <dig> infection from serl and from diagnostic laboratories throughout scotland.
diagnostic laboratories submit samples  to serl for further testing in line with scottish guidance  <cit> .
using a series of phenotypic and genotypic tests, serl confirms the identity of submitted isolates of e. coli o <dig>  or identifies and isolates e. coli o <dig> from submitted faecal samples  <cit> .
serl also types all isolated organisms using a hierarchical array of methods including phage typing, polymerase chain reaction  and pulse-field gel electrophoresis .
the results of phage and verotoxin typing undertaken by serl are also reported to hps.
since establishing enhanced surveillance in  <dig>  hps has compiled a standard dataset for every case, in which microbiological data shared by serl is a crucial component  <cit> .
a case is defined as one person-infection-episode, with microbiological confirmation of infection, defined as culture positive i.e.
isolates of e. coli o <dig> cultured from faeces.
although hps enhanced surveillance also includes cases identified by serl by detection of antibodies to e. coli o <dig> in serum, these serum positive cases were excluded from data entered into this study as they by definition had no available phage type results.
hps integrates laboratory data including serl typing results, with epidemiological details from local investigators .
these include clinical and exposure details, including travel.
prior to  <dig>  the number of cases that might potentially have acquired infection outside the uk could only be estimated according to whether non-uk countries were noted on laboratory forms; details of whether travel occurred before, during or after the incubation period were not available to hps.
since  <dig>  enhanced surveillance at hps has enabled more accurate differentiation of imported cases defined as likely to have acquired infection outside the uk, based on examination of travel, clinical and exposure histories provided by local investigators  <cit> .
data on culture-positive, indigenous human cases with known phage type results identified by serl, for the period march  <dig> to may  <dig>  and february  <dig> to february  <dig> , were therefore entered into this study by hps, in collaboration with serl.
statistical analysis
animal studies - prevalence of e. coli o157
the sas v <dig> .
<dig> package  was used to fit generalised linear mixed models , to generate bootstrap-based estimates of key parameters and to carry out non-parametric statistical testing.
the excel  <dig> package  was used to implement a latin hypercube sampling algorithm to convert results from the glmms into prevalence, taking into account the influence of random effects  <cit>  and to assess the group sensitivity of the two sampling regimens.
seasons were defined as: winter, december, january and february; spring, march, april, and may; summer, june, july and august; and winter, september, october and november.
statistical significance of pairwise comparisons was determined using students t-test.
farm-level data analysis
the mean percentage of farms with shedding cattle was estimated using glmms  <cit> , fitting models with bernoulli response terms and a logit link function.
a farm was classed as positive if at least one animal was identified as shedding.
farm cluster and/or farm were fitted as random effects depending on the sampling design of the program.
including ahd and season as fixed effects, glmms were used to determine the impact of ahd and season on the proportion of farms with shedding cattle in each ahd and season.
confidence intervals for means were derived by reweighting output from the appropriate glmm, taking into account the influence of random effects  <cit> .
latin hypercube sampling of the observed non-zero prevalences and sample sizes was used to provide inputs to a simple probabilistic calculation, assuming sampling with replacement, of mean estimates of the sensitivity of the sampling procedures in identifying positive groups.
pat-level data analysis
for both the seerad and iprave surveys, sampling distributions of the overall mean prevalence of shedding, overall mean shedding prevalence by specific phage type, and mean shedding prevalence within ahd or seasonal subsets were generated using bootstrap sampling with  <dig>  iterations.
in each iteration, farms and pats from each farm were sampled from the overall data or respective ahd or seasonal subsets arising from the original surveys.
the same number of pats sampled in the original surveys was sampled using the sampling procedure used in the original surveys, but with replacement both at the farm and pat strata.
the mean and upper and lower confidence limits of the mean shedding prevalence were derived from the respective bootstrap distributions.
these calculations make no adjustment for the sensitivity and specificity of the assay.
human data analysis--incidence of common phage types
the number of human cases entered into the study and the duration of the surveys were used to calculate the comparative incidence of human cases.
this was then expressed as an equivalent annual figure.
incidence was calculated as the number of human cases with each of the more common phage types  and 'other' pts  reported to hps over the time periods equivalent to the seerad and iprave surveys.
comparison of phage types from cattle and human cases
the overall temporal pattern of the most common phage types ie pt <dig>  pt21/ <dig>  pt <dig>  pt <dig>  pt <dig> and 'other' pts  were examined for human cases and cattle isolates using the cochran mantel haenzel  test  .
temporal patterns of human cases and bovine shedding were then examined separately using the exact chi-square test .
further analysis was conducted on pt21/ <dig> and pt <dig> to compare the relative ratio of the two phage types in bovine isolates and human cases.
if pt21/ <dig> is associated with super-shedders  we should see high proportions in both cattle and humans whereas pt <dig>  should be relatively over-represented in cattle.
RESULTS
animal studies
<dig>  faecal pats across  <dig> farms were sampled in the seerad study and  <dig>  pats across  <dig> farms in the iprave study.
a total of  <dig>  e. coli o <dig> strains were isolated from the seerad study  and  <dig> strains in the iprave study .
the spatial distribution of positive farms in the seerad and iprave study are shown in figure  <dig>
among strains isolated during the seerad study,  <dig> % ,  <dig> %  and  <dig> %  possessed genes encoding the virulence factors vtx <dig> only, vtx <dig> only and vtx1vtx <dig> respectively.
among strains isolated during the iprave study,  <dig> % ,  <dig> %  and  <dig> %  possessed genes encoding vtx <dig> only, vtx <dig> only and vtx1vtx <dig> respectively.
all strains isolated from both studies possessed eae, the gene encoding the virulence factor intimin.
farm and pat-level mean prevalence estimates for the two surveys are given in tables  <dig> and  <dig> respectively.
the point-estimate and confidence interval of group prevalence are both slightly higher than the raw estimates given earlier  <cit>  as the figures now average over unbalanced random effects from the studies.
mean overall farm-level mean prevalence decreased slightly from  <dig>  to  <dig>  but this was not statistically significant .
similarly, there was no significant change in temporal, seasonal or phage specific shedding at the farm-level.
mean overall pat-level mean prevalence of e. coli o <dig> more than halved from  <dig>  to  <dig>   .
the farm-level sensitivity of the iprave study was only marginally smaller, at  <dig> %, than that of the seerad study , the effect of larger mean sample sizes being outweighed by the lower pat-level prevalences seen in the iprave study.
over the same period, there were statistically significant decreases in the mean prevalence of shedding in all seasons.
the mean pat-level prevalence decline was highly statistically significant  in the north east and central ahds.
statistically significant decreases were also observed in the highland and south east ahds .
among the major most common phage types, there was a substantial decrease in the mean pat-level prevalence of pt21/ <dig> shedding from  <dig>  to  <dig>  .
pt21/ <dig> was the dominant phage type isolated in both studies, representing 56% of strains in the seerad study and 51% of strains in the iprave study.
a statistically significant decrease in mean pat-level prevalence was also observed for pt <dig> .
changes in the mean pat-level prevalence of pts  <dig> and  <dig> were not statistically significant.
in the majority of farms sampled in both surveys, no shedding animals were detected.
the distribution of the prevalence on e. coli o <dig> positive farms is shown in figure  <dig> for both the seerad and iprave surveys.
the distribution of prevalence for the two studies was different .
the median prevalence of shedding animals was statistically significantly lower  in the iprave compared with the seerad survey ; iprave:  <dig>  .
results from human data
acomparative incidence is equivalent to the number of cases per year.
bincludes pt <dig>  pt <dig>  pt <dig>  pt <dig>  pt <dig>  rdnc and untypeable
comparison of phage types for animal and human cases
the proportion of human cases and cattle isolates identified with e. coli o <dig> pt21/ <dig> was much higher than any other phage type .
overall there was a statistically significant association between time  and pt for human cases and cattle isolates .
when human cases and cattle isolates were examined separately there were significant associations between time and pt although the associations for cattle isolates  were stronger than human cases .
these results suggest that there was more temporal change in cattle isolates than in human cases.
aincludes pt <dig>  pt <dig>  pt <dig>  pt <dig>  pt <dig>  rdnc and untypeable
discussion
the surveys examined in this study represent the only reported systematic national surveys of bovine e. coli o <dig> shedding and present a valuable opportunity to examine changes in patterns of shedding and strain characteristics.
knowledge of bovine shedding is important as cattle represent a major risk factor both for human e. coli o <dig> infection, whether from contamination of food or water by bovine faeces, or from direct contact with cattle or their environments, and for transmission to other animals.
this is of particular concern in scotland which has consistently higher rates of human e. coli o <dig> cases than the rest of the united kingdom, and other european and north american countries  <cit> .
in most instances it is difficult to compare results from different prevalence studies as different study designs, sampling procedures and microbiological methods have been used.
the use of similar sampling and identical laboratory methods in the seerad and iprave studies allowed direct comparison of e. coli o <dig> prevalence estimates.
estimates of the prevalence of e. coli o <dig> from the seerad study have been published, but in this study the estimates were recalculated to accommodate differences in sampling design and changes in statistical methodology.
the farm-level and pat-level mean prevalence calculated for the seerad survey was  <dig>   and  <dig>   respectively  <cit> .
in this study the same quantities were recalculated to be  <dig>   and  <dig>  .
these minor differences are the result of using different statistical models.
pat-level mean prevalence estimates for the iprave study were generated using a bootstrapping technique given the clustered nature of data collection and the zero-inflated nature of the resulting data.
for comparability the seerad pat-level prevalence was recalculated using the same methodology.
an overall comparison of the mean prevalence of e. coli o <dig> shedding for the seerad and iprave surveys indicated a statistically significant decline in the mean prevalence of e. coli o <dig> at the pat-level but no statistically significant change at the farm-level.
over the 4-year period between the surveys there was a substantial decrease in the mean proportion of cattle shedding e. coli o <dig> on farms.
the mean pat-level prevalence of e. coli o <dig> more than halved from  <dig>  to  <dig>  between the two surveys.
this result possibly reflects a change in on-farm transmission rate between the two surveys, although the effect of environmental conditions or survival outside the host cannot be eliminated as possible causes of the differences observed.
in two separate publications  <cit> , the r <dig>  of the seerad and iprave surveys were reported as  <dig>   <cit>  and  <dig>   <cit>  respectively.
a difference in transmission dynamics could explain the different distribution of prevalences observed in figure  <dig>  higher transmission on a farm has been linked to the presence of super-shedding or high-level shedding animals  <cit> .
as part of the iprave survey, counts of e. coli o <dig> in pat samples were estimated.
unfortunately there is no data from the seerad survey on the density of e. coli o <dig> in farm pat samples.
therefore, no direct comparison between the numbers of super-shedders can be made between the two surveys.
research has shown that there is an association between the presence of a super-shedder and the presence of pt21/ <dig> on a farm  <cit> .
therefore, we might hypothesise that there were fewer super-shedders on farms in the iprave survey as opposed to the seerad survey as there were significantly fewer pt21/ <dig> strains isolated in the iprave survey.
assuming an association between shedding rates and transmission rates   <cit> , fewer super-shedders may explain lower transmission rates on farms in the iprave study and hence the lower mean on-farm prevalence.
unfortunately, in the absence of enumeration data from the seerad study this supposition cannot be tested.
mean prevalence was calculated for different seasons, animal health districts  and phage types .
as observed with the overall prevalence results, statistically significant declines in mean prevalence of e. coli o <dig> were observed at the pat-level only.
marginal changes were observed at the farm-level but these were not statistically significant.
the decline in the mean prevalence of pat-level shedding appears to have been driven by statistically significant reductions in the mean prevalence of pt21/ <dig> as well as specific seasonal  and regional  decreases.
despite the statistically significant pairwise reductions in mean pat-level prevalences there was no equivalent change in overall mean prevalence at the farm-level.
the mean farm-level prevalence between surveys did decline but it was not statistically significant.
changes in sampling strategy between the two surveys had a negligible effect on the power to identify positive farms, with the only potential effect of these changes being to reduce further the absolute size of the change in mean farm-level prevalences across the surveys.
dissociation between the mean prevalence at the pat and farm-level has been described for non-o <dig> strains of e. coli  <cit> .
it is possible that at farm-level, e. coli o <dig> shedding may stop or remain undetectable in many cattle but still remain on the farm, and there are reports of extended e. coli o <dig> activity on individual farms  <cit> .
this point has important implications for control programmes and assessment of their efficacy.
is it reasonable to conjecture that reductions in farm-level prevalence lag behind pat-level prevalence?
do we need to see more significant reductions in pat shedding over longer time periods before we might see a significant impact at the farm-level?
is this the result of bacteria maintained within the environment re-infecting cattle, or of a few persistently shedding cattle that are shedding at detectable levels but not transmitting to the rest of the group?
low-level shedders may have different risk factors but could have an important role in the maintenance of e. coli o <dig> populations on farms.
sustained farm-level prevalence indicates persistence of e. coli o <dig> on farms, but decreases at the pat-level imply a lower environmental load which would expect to lower the force of infection to both cattle and humans.
concurrent declines in the total number and comparative annual incidence of human cases in this survey may reflect a link between human infection and the level of bovine shedding on a farm.
however, the drivers of e. coli o <dig> infection are likely to be multifactorial, and as the infectious dose for e. coli o <dig> is low  <cit> , a substantial reduction in environmental load may therefore be required to significantly reduce the risk of infection for humans.
pt21/ <dig> is of particular concern because of its association with more severe human disease  <cit> .
analysis of human e. coli o <dig> cases over the same period as this study show that although it remains the dominant phage type, the incidence of phage type pt21/ <dig> e. coli cases in humans declined  <cit>  as did the prevalence of bovine shedding, providing circumstantial evidence of a link between bovine shedding and human infection.
our findings show that the relative ratio of pt32:pt21/ <dig> in cattle pats compared with pt32:pt21/ <dig> in human e. coli o <dig> cases was  <dig>  during the course of the seerad study and  <dig>  during the iprave study.
this supports the contention that phage type pt21/ <dig> is more transmissible from cattle to humans than phage type pt <dig>  the relative proportion of pt32:pt21/ <dig> in cattle pats compared to human cases was much higher for the iprave survey period, a reflection of the significant temporal change in the proportion of pt <dig> and pt21/ <dig> in cattle between the two survey periods.
the decrease in the proportion of pt21/ <dig> and increase in pt <dig> were not mirrored by data on the human cases.
such results may be a reflection of the proposed heterogeneity in transmission  <cit> .
in addition, pt <dig> may either be less stable in the environment than pt21/ <dig> and/or less virulent to humans  <cit> .
in this paper we have highlighted the importance of cattle as the primary source of human e. coli o <dig> infection.
cattle are the major reservoirs of e. coli o <dig>  <cit> , they carry it asymptomatically in their intestines and excrete it in their faeces.
excretion rates for some animals  can be high  per gram of faeces)  <cit> .
the potentially high excretion rate, longevity of e. coli o <dig> in pasture and soil  <cit>  and the low infectious dose for human infection  <cit>  mean that the environment is an important source of infection for humans.
comparison of  <dig> published e. coli o <dig> outbreaks meeting certain criteria  from  <dig> countries  <cit>  has identified exposure to contaminated food  and environmental sources   as the most frequently reported primary modes of transmission  <cit> .
analysis of general outbreaks  of e. coli o <dig> infection in scotland associated with either meat or dairy foods, or with environmental transmission  showed that approximately 40% of these outbreaks were associated with foodborne transmission, 54% with environmental transmission and 6% with both modes of transmission  <cit> .
however, most infections in scotland are sporadic or single household cases, and not part of general outbreaks.
contact with livestock faeces was the risk factor most strongly associated with sporadic infection  <cit> .
this further highlights the cattle and the environment as an important sources of e. coli o <dig> infections in humans.
it remains to be seen whether the decline in the mean prevalence of e. coli o <dig> cattle shedding observed between the seerad and iprave surveys continues, but there are precedents among other members of the enterobacteriaceae family e.g.
salmonella  <cit>  to suggest that this is possible.
despite observing declines in the number of human e. coli o <dig> cases over the time periods equivalent to the two cattle surveys, incidence rates, at least from  <dig>  do not seem to suggest a downward trend .
although these data were not generated by our study, examination of the reported rate of e. coli o <dig> infection per  <dig>  population in scotland shows that from  <dig> to  <dig> there was no change in the reported national rate of human cases  .
between  <dig> and  <dig> the average annual rate  was  <dig>    <cit> .
the iprave survey included the year  <dig>  a year which had the lowest reported rate of human cases in scotland since the early 1990s  <cit> , suggesting that  <dig> may have been an unusual year.
in some regions of scotland,  <dig> was characterised by the highest temperatures and lowest rain fall since  <dig>  <cit> , and in the islands, highlands, and north east ahds, the mean prevalence of e. coli o <dig> shedding in cattle was much lower in  <dig> compared with  <dig>  without linked data on the prevalence of bovine e. coli o <dig> shedding and the incidence of human cases over a longer time period, and more detailed linkage of geographical, temporal and meteorological data, the possible effects of climate must remain as conjecture.
CONCLUSIONS
the objectives of this study were to assess the prevalence of bovine e. coli o <dig> shedding in scotland; determine changes in the temporal, spatial and phage patterns of bovine shedding between the periods 1998- <dig> and 2002-2004; and compare the phage types of e. coli o <dig> associated with human infections with those shed by cattle.
between the two survey periods, farm-level prevalence of shedding changed little, yet pat-level prevalence of shedding halved.
this study also demonstrated that season, location and phage type are linked to pat-level prevalence of shedding.
between the two survey periods, human e. coli o <dig> case numbers also declined and the pattern of phage types shed by cattle were comparable to those isolated from human patients suggesting a link between bovine shedding and human infection.
our findings reinforce the need to reduce the prevalence and virulence of e. coli o <dig> shed by cattle in scotland and the health risk posed by this organism  <cit> .
authors' contributions
mcp collected farm data, analysed and interpreted data and prepared the manuscript.
mect analysed data and prepared the manuscript.
ijm specified analyses and interpreted data.
djm and het collected the farm data and interpreted data.
la, hik and aws conducted the laboratory analysis.
mel collected, applied inclusion criteria to, and provided the human data and contributed to the manuscript; wr authorised use of the human data.
lm interpreted data and prepared the manuscript.
mejw, swjr, bas, jcl and gg supervised the study and interpreted data.
all authors read and approved the final manuscript.

 in the last years, escherichia coli serogroup o has become an important cause of severe gastrointestinal illness in westernised countries, warranting substantial public health concern. clinical signs range from mild diarrhoea to haemorrhagic colitis and haemolytic uraemic syndrome which may result in death. hus usually occurs in young children and is the major cause of acute renal failure in children in western countries. clinical surveillance in scotland has shown that over 90% of hus cases are associated with e. coli o infection; similar observations have been made in other countries. cattle are the main reservoir for e. coli o, and play a major role in the epidemiology of human infections. visits to farms, contact with animal excreta and recreational use of animal pasture have all been identified as significant risk factors for sporadic human infections. spatial analyses suggest that human incidence is positively associated with indicators such as livestock density and the ratio of cattle to human population, although the relationship appears complex. considerable effort has been made to determine the prevalence of e. coli o in cattle worldwide. estimates of prevalence range from to 71% of animals and to 100% of herds. two of the world's largest surveys of animal e. coli o prevalence were conducted in the past decade in scotland. the first estimated herd-level and animal-level prevalence for farms throughout scotland in a study funded by the scottish executive environment and rural affairs department conducted from march to may since then a second survey, funded by the wellcome foundation international partnership research award in veterinary epidemiology was conducted on a subsample of the seerad farms, from february to february data from the seerad and iprave studies are presented in this paper. in scotland, the first reported cases of human e. coli o infection were identified in currently, health protection scotland conducts active, population based enhanced surveillance in close collaboration with the scottish e. coli o157/vtec reference laboratory. over the year period 1998- an annual average of culture positive cases has been reported to hps, which is an average annual rate of cases per population. rates in scotland are generally higher than in most other united kingdom, european and north american countries. a recent publication proposed a specific mechanism for the link between human infection and livestock carriage of e. coli o which involved a subset of shedding animals known as super-shedders. super-shedders are individuals who for a period yield more infectious organisms than typical individuals of the same host species. shedding high concentrations of e. coli o has been proposed as a major contributor to cattle-to-cattle transmission and possibly cattle-to-human transmission. although little is known about super-shedders it has been shown that they have been associated with the presence of phage type 21/ whereas non super-shedders are more likely to be associated with pt. recent evidence has shown pt21/ to be associated with higher transmission in livestock when compared to pt. pt21/ is the most predominant phage type in both cattle and human cases whereas pt is a common phage type in cattle only. in humans, pt21/ is of particular concern because of its association with more severe morbidity. in the uk and ireland, the mean risk of developing diarrhoea-associated hus was significantly higher in children in scotland infected with pt21/ compared with other phage types. phage type 21/ strains possess the vtx gene alone, which appears to be associated with more serious health outcomes. the objectives of this study were three-fold. first, to calculate the mean prevalence of e. coli o in cattle using the data from both the seerad and iprave surveys. second, to examine temporal patterns in the overall as well as regional, seasonal and phage type specific prevalence of bovine shedding. third, to examine the incidence levels and relative proportions of common phage types associated with human cases over the same periods and the proportion of phage types pt21/ and pt in bovine isolates and human cases, for evidence of any epidemiological link between the two. methods animal prevalence studies livestock sampling design two surveys of scottish store and finishing cattle were conducted: the first from march to may the second from february to february the first study was funded by the scottish executive environment and rural affairs department; the second by a wellcome foundation international partnership research award in veterinary epidemiology. details on the methodology of both surveys have been published elsewhere, however, a brief outline is given below. in seerad provided the scottish agricultural college with a list comprising farms with cattle, randomly selected from scottish agricultural and horticultural census data. for the seerad survey, farms across the state animal health divisions were randomly selected and surveyed. owners or managers of of these farms consented to an additional sampling visit and these farms were used as the sampling frame for the second survey. within the sampling frame for the iprave survey there were insufficient farms to adequately represent two state animal health divisions: highland and islands. additional farms for these two ahds were recruited by random selection from the remainder of farms not sampled in the seerad survey. in total, farms were sampled for the iprave survey, of which had been previously sampled in the seerad survey. instead of randomly sampling farms within each ahd, the iprave study used a stratified sampling plan to select farms to sample. this was done to ensure that similar numbers were included from each region and that regions were sampled evenly over time. both surveys preferentially sampled cattle groups composed only of store or finishing cattle closest to sale or slaughter. if such groups did not exist, one or more mixed groups with store or finishing cattle closest to sale or slaughter were sampled. from each group fresh faecal pats were sampled. the number of faecal pats tested in each group was determined from the number of cattle in the group using a prescribed sampling schedule. for the seerad survey, sufficient numbers of faecal pats were tested to ensure prospectively an 80% chance of sampling at least one positive pat if there was a shedding prevalence of at least 2% within the group. based on results from the seerad survey, in the iprave survey, it was assumed that, on average, 8% of the animals in positive groups would be shedding, with shedding distributed as seen in the seerad survey. for each iprave group, sufficient fresh pat samples were taken to ensure prospectively a mean 90% probability of detecting shedding of e. coli o if at least one shedding animal was indeed present. samples were collected from freshly voided faecal pats, refrigerated at 5°c as soon as possible and processed within hours of collection. no direct animal sampling was involved and the study was not regulated by the animals act at present the seerad and iprave data are not available on open-access databases, however, requests for data can be made though the corresponding author. immunomagnetic separation and phage typing of livestock samples within hours of sampling, one gram of faeces from each sample was tested for the presence of e. coli o as previously described. following ims, one e. coli o isolate from each faecal sample was submitted to the scottish e. coli o157/vtec reference laboratory for phage typing, and tested for the presence of genes encoding the virulence factors verocytotoxin, verocytotoxin and intimin using multiplex pcr. human case identification, data collection and phage typing health protection scotland receives reports of human cases of e. coli o infection from serl and from diagnostic laboratories throughout scotland. diagnostic laboratories submit samples to serl for further testing in line with scottish guidance. using a series of phenotypic and genotypic tests, serl confirms the identity of submitted isolates of e. coli o or identifies and isolates e. coli o from submitted faecal samples. serl also types all isolated organisms using a hierarchical array of methods including phage typing, polymerase chain reaction and pulse-field gel electrophoresis. the results of phage and verotoxin typing undertaken by serl are also reported to hps. since establishing enhanced surveillance in hps has compiled a standard dataset for every case, in which microbiological data shared by serl is a crucial component. a case is defined as one person-infection-episode, with microbiological confirmation of infection, defined as culture positive i.e. isolates of e. coli o cultured from faeces. although hps enhanced surveillance also includes cases identified by serl by detection of antibodies to e. coli o in serum, these serum positive cases were excluded from data entered into this study as they by definition had no available phage type results. hps integrates laboratory data including serl typing results, with epidemiological details from local investigators. these include clinical and exposure details, including travel. prior to the number of cases that might potentially have acquired infection outside the uk could only be estimated according to whether non-uk countries were noted on laboratory forms; details of whether travel occurred before, during or after the incubation period were not available to hps. since enhanced surveillance at hps has enabled more accurate differentiation of imported cases defined as likely to have acquired infection outside the uk, based on examination of travel, clinical and exposure histories provided by local investigators. data on culture-positive, indigenous human cases with known phage type results identified by serl, for the period march to may and february to february, were therefore entered into this study by hps, in collaboration with serl. statistical analysis animal studies - prevalence of e. coli o157 the sas v. package was used to fit generalised linear mixed models, to generate bootstrap-based estimates of key parameters and to carry out non-parametric statistical testing. the excel package was used to implement a latin hypercube sampling algorithm to convert results from the glmms into prevalence, taking into account the influence of random effects and to assess the group sensitivity of the two sampling regimens. seasons were defined as: winter, december, january and february; spring, march, april, and may; summer, june, july and august; and winter, september, october and november. statistical significance of pairwise comparisons was determined using students t-test. farm-level data analysis the mean percentage of farms with shedding cattle was estimated using glmms, fitting models with bernoulli response terms and a logit link function. a farm was classed as positive if at least one animal was identified as shedding. farm cluster and/or farm were fitted as random effects depending on the sampling design of the program. including ahd and season as fixed effects, glmms were used to determine the impact of ahd and season on the proportion of farms with shedding cattle in each ahd and season. confidence intervals for means were derived by reweighting output from the appropriate glmm, taking into account the influence of random effects. latin hypercube sampling of the observed non-zero prevalences and sample sizes was used to provide inputs to a simple probabilistic calculation, assuming sampling with replacement, of mean estimates of the sensitivity of the sampling procedures in identifying positive groups. pat-level data analysis for both the seerad and iprave surveys, sampling distributions of the overall mean prevalence of shedding, overall mean shedding prevalence by specific phage type, and mean shedding prevalence within ahd or seasonal subsets were generated using bootstrap sampling with iterations. in each iteration, farms and pats from each farm were sampled from the overall data or respective ahd or seasonal subsets arising from the original surveys. the same number of pats sampled in the original surveys was sampled using the sampling procedure used in the original surveys, but with replacement both at the farm and pat strata. the mean and upper and lower confidence limits of the mean shedding prevalence were derived from the respective bootstrap distributions. these calculations make no adjustment for the sensitivity and specificity of the assay. human data analysis--incidence of common phage types the number of human cases entered into the study and the duration of the surveys were used to calculate the comparative incidence of human cases. this was then expressed as an equivalent annual figure. incidence was calculated as the number of human cases with each of the more common phage types and'other' pts reported to hps over the time periods equivalent to the seerad and iprave surveys. comparison of phage types from cattle and human cases the overall temporal pattern of the most common phage types ie pt pt21/ pt pt pt and'other' pts were examined for human cases and cattle isolates using the cochran mantel haenzel test. temporal patterns of human cases and bovine shedding were then examined separately using the exact chi-square test. further analysis was conducted on pt21/ and pt to compare the relative ratio of the two phage types in bovine isolates and human cases. if pt21/ is associated with super-shedders we should see high proportions in both cattle and humans whereas pt should be relatively over-represented in cattle.  animal studies faecal pats across farms were sampled in the seerad study and pats across farms in the iprave study. a total of e. coli o strains were isolated from the seerad study and strains in the iprave study. the spatial distribution of positive farms in the seerad and iprave study are shown in figure among strains isolated during the seerad study, %, % and % possessed genes encoding the virulence factors vtx only, vtx only and vtx1vtx respectively. among strains isolated during the iprave study, %, % and % possessed genes encoding vtx only, vtx only and vtx1vtx respectively. all strains isolated from both studies possessed eae, the gene encoding the virulence factor intimin. farm and pat-level mean prevalence estimates for the two surveys are given in tables and respectively. the point-estimate and confidence interval of group prevalence are both slightly higher than the raw estimates given earlier as the figures now average over unbalanced random effects from the studies. mean overall farm-level mean prevalence decreased slightly from to but this was not statistically significant. similarly, there was no significant change in temporal, seasonal or phage specific shedding at the farm-level. mean overall pat-level mean prevalence of e. coli o more than halved from to. the farm-level sensitivity of the iprave study was only marginally smaller, at %, than that of the seerad study, the effect of larger mean sample sizes being outweighed by the lower pat-level prevalences seen in the iprave study. over the same period, there were statistically significant decreases in the mean prevalence of shedding in all seasons. the mean pat-level prevalence decline was highly statistically significant in the north east and central ahds. statistically significant decreases were also observed in the highland and south east ahds. among the major most common phage types, there was a substantial decrease in the mean pat-level prevalence of pt21/ shedding from to. pt21/ was the dominant phage type isolated in both studies, representing 56% of strains in the seerad study and 51% of strains in the iprave study. a statistically significant decrease in mean pat-level prevalence was also observed for pt. changes in the mean pat-level prevalence of pts and were not statistically significant. in the majority of farms sampled in both surveys, no shedding animals were detected. the distribution of the prevalence on e. coli o positive farms is shown in figure for both the seerad and iprave surveys. the distribution of prevalence for the two studies was different. the median prevalence of shedding animals was statistically significantly lower in the iprave compared with the seerad survey; iprave:. results from human data acomparative incidence is equivalent to the number of cases per year. bincludes pt pt pt pt pt rdnc and untypeable comparison of phage types for animal and human cases the proportion of human cases and cattle isolates identified with e. coli o pt21/ was much higher than any other phage type. overall there was a statistically significant association between time and pt for human cases and cattle isolates. when human cases and cattle isolates were examined separately there were significant associations between time and pt although the associations for cattle isolates were stronger than human cases. these results suggest that there was more temporal change in cattle isolates than in human cases. aincludes pt pt pt pt pt rdnc and untypeable discussion the surveys examined in this study represent the only reported systematic national surveys of bovine e. coli o shedding and present a valuable opportunity to examine changes in patterns of shedding and strain characteristics. knowledge of bovine shedding is important as cattle represent a major risk factor both for human e. coli o infection, whether from contamination of food or water by bovine faeces, or from direct contact with cattle or their environments, and for transmission to other animals. this is of particular concern in scotland which has consistently higher rates of human e. coli o cases than the rest of the united kingdom, and other european and north american countries. in most instances it is difficult to compare results from different prevalence studies as different study designs, sampling procedures and microbiological methods have been used. the use of similar sampling and identical laboratory methods in the seerad and iprave studies allowed direct comparison of e. coli o prevalence estimates. estimates of the prevalence of e. coli o from the seerad study have been published, but in this study the estimates were recalculated to accommodate differences in sampling design and changes in statistical methodology. the farm-level and pat-level mean prevalence calculated for the seerad survey was and respectively. in this study the same quantities were recalculated to be and. these minor differences are the result of using different statistical models. pat-level mean prevalence estimates for the iprave study were generated using a bootstrapping technique given the clustered nature of data collection and the zero-inflated nature of the resulting data. for comparability the seerad pat-level prevalence was recalculated using the same methodology. an overall comparison of the mean prevalence of e. coli o shedding for the seerad and iprave surveys indicated a statistically significant decline in the mean prevalence of e. coli o at the pat-level but no statistically significant change at the farm-level. over the 4-year period between the surveys there was a substantial decrease in the mean proportion of cattle shedding e. coli o on farms. the mean pat-level prevalence of e. coli o more than halved from to between the two surveys. this result possibly reflects a change in on-farm transmission rate between the two surveys, although the effect of environmental conditions or survival outside the host cannot be eliminated as possible causes of the differences observed. in two separate publications, the r of the seerad and iprave surveys were reported as and respectively. a difference in transmission dynamics could explain the different distribution of prevalences observed in figure higher transmission on a farm has been linked to the presence of super-shedding or high-level shedding animals. as part of the iprave survey, counts of e. coli o in pat samples were estimated. unfortunately there is no data from the seerad survey on the density of e. coli o in farm pat samples. therefore, no direct comparison between the numbers of super-shedders can be made between the two surveys. research has shown that there is an association between the presence of a super-shedder and the presence of pt21/ on a farm. therefore, we might hypothesise that there were fewer super-shedders on farms in the iprave survey as opposed to the seerad survey as there were significantly fewer pt21/ strains isolated in the iprave survey. assuming an association between shedding rates and transmission rates, fewer super-shedders may explain lower transmission rates on farms in the iprave study and hence the lower mean on-farm prevalence. unfortunately, in the absence of enumeration data from the seerad study this supposition cannot be tested. mean prevalence was calculated for different seasons, animal health districts and phage types. as observed with the overall prevalence results, statistically significant declines in mean prevalence of e. coli o were observed at the pat-level only. marginal changes were observed at the farm-level but these were not statistically significant. the decline in the mean prevalence of pat-level shedding appears to have been driven by statistically significant reductions in the mean prevalence of pt21/ as well as specific seasonal and regional decreases. despite the statistically significant pairwise reductions in mean pat-level prevalences there was no equivalent change in overall mean prevalence at the farm-level. the mean farm-level prevalence between surveys did decline but it was not statistically significant. changes in sampling strategy between the two surveys had a negligible effect on the power to identify positive farms, with the only potential effect of these changes being to reduce further the absolute size of the change in mean farm-level prevalences across the surveys. dissociation between the mean prevalence at the pat and farm-level has been described for non-o strains of e. coli. it is possible that at farm-level, e. coli o shedding may stop or remain undetectable in many cattle but still remain on the farm, and there are reports of extended e. coli o activity on individual farms. this point has important implications for control programmes and assessment of their efficacy. is it reasonable to conjecture that reductions in farm-level prevalence lag behind pat-level prevalence? do we need to see more significant reductions in pat shedding over longer time periods before we might see a significant impact at the farm-level? is this the result of bacteria maintained within the environment re-infecting cattle, or of a few persistently shedding cattle that are shedding at detectable levels but not transmitting to the rest of the group? low-level shedders may have different risk factors but could have an important role in the maintenance of e. coli o populations on farms. sustained farm-level prevalence indicates persistence of e. coli o on farms, but decreases at the pat-level imply a lower environmental load which would expect to lower the force of infection to both cattle and humans. concurrent declines in the total number and comparative annual incidence of human cases in this survey may reflect a link between human infection and the level of bovine shedding on a farm. however, the drivers of e. coli o infection are likely to be multifactorial, and as the infectious dose for e. coli o is low, a substantial reduction in environmental load may therefore be required to significantly reduce the risk of infection for humans. pt21/ is of particular concern because of its association with more severe human disease. analysis of human e. coli o cases over the same period as this study show that although it remains the dominant phage type, the incidence of phage type pt21/ e. coli cases in humans declined as did the prevalence of bovine shedding, providing circumstantial evidence of a link between bovine shedding and human infection. our findings show that the relative ratio of pt32:pt21/ in cattle pats compared with pt32:pt21/ in human e. coli o cases was during the course of the seerad study and during the iprave study. this supports the contention that phage type pt21/ is more transmissible from cattle to humans than phage type pt the relative proportion of pt32:pt21/ in cattle pats compared to human cases was much higher for the iprave survey period, a reflection of the significant temporal change in the proportion of pt and pt21/ in cattle between the two survey periods. the decrease in the proportion of pt21/ and increase in pt were not mirrored by data on the human cases. such results may be a reflection of the proposed heterogeneity in transmission. in addition, pt may either be less stable in the environment than pt21/ and/or less virulent to humans. in this paper we have highlighted the importance of cattle as the primary source of human e. coli o infection. cattle are the major reservoirs of e. coli o, they carry it asymptomatically in their intestines and excrete it in their faeces. excretion rates for some animals can be high per gram of faeces). the potentially high excretion rate, longevity of e. coli o in pasture and soil and the low infectious dose for human infection mean that the environment is an important source of infection for humans. comparison of published e. coli o outbreaks meeting certain criteria from countries has identified exposure to contaminated food and environmental sources as the most frequently reported primary modes of transmission. analysis of general outbreaks of e. coli o infection in scotland associated with either meat or dairy foods, or with environmental transmission showed that approximately 40% of these outbreaks were associated with foodborne transmission, 54% with environmental transmission and 6% with both modes of transmission. however, most infections in scotland are sporadic or single household cases, and not part of general outbreaks. contact with livestock faeces was the risk factor most strongly associated with sporadic infection. this further highlights the cattle and the environment as an important sources of e. coli o infections in humans. it remains to be seen whether the decline in the mean prevalence of e. coli o cattle shedding observed between the seerad and iprave surveys continues, but there are precedents among other members of the enterobacteriaceae family e.g. salmonella to suggest that this is possible. despite observing declines in the number of human e. coli o cases over the time periods equivalent to the two cattle surveys, incidence rates, at least from do not seem to suggest a downward trend. although these data were not generated by our study, examination of the reported rate of e. coli o infection per population in scotland shows that from to there was no change in the reported national rate of human cases. between and the average annual rate was. the iprave survey included the year a year which had the lowest reported rate of human cases in scotland since the early 1990s, suggesting that may have been an unusual year. in some regions of scotland, was characterised by the highest temperatures and lowest rain fall since, and in the islands, highlands, and north east ahds, the mean prevalence of e. coli o shedding in cattle was much lower in compared with without linked data on the prevalence of bovine e. coli o shedding and the incidence of human cases over a longer time period, and more detailed linkage of geographical, temporal and meteorological data, the possible effects of climate must remain as conjecture.  the objectives of this study were to assess the prevalence of bovine e. coli o shedding in scotland; determine changes in the temporal, spatial and phage patterns of bovine shedding between the periods 1998- and 2002-2004; and compare the phage types of e. coli o associated with human infections with those shed by cattle. between the two survey periods, farm-level prevalence of shedding changed little, yet pat-level prevalence of shedding halved. this study also demonstrated that season, location and phage type are linked to pat-level prevalence of shedding. between the two survey periods, human e. coli o case numbers also declined and the pattern of phage types shed by cattle were comparable to those isolated from human patients suggesting a link between bovine shedding and human infection. our findings reinforce the need to reduce the prevalence and virulence of e. coli o shed by cattle in scotland and the health risk posed by this organism. authors' contributions mcp collected farm data, analysed and interpreted data and prepared the manuscript. mect analysed data and prepared the manuscript. ijm specified analyses and interpreted data. djm and het collected the farm data and interpreted data. la, hik and aws conducted the laboratory analysis. mel collected, applied inclusion criteria to, and provided the human data and contributed to the manuscript; wr authorised use of the human data. lm interpreted data and prepared the manuscript. mejw, swjr, bas, jcl and gg supervised the study and interpreted data. all authors read and approved the final manuscript. 
for both cattle and humans, the predominant phage type between and was pt21/ comprising over 50% of the positive cattle isolates and reported human cases respectively. the proportion of pt however, was represented by few of reported human cases despite comprising over 10% of cattle isolates. across the two studies there were differences in the proportion of pts 21/ and'other' pts in both cattle isolates and reported human cases; however, only differences in the cattle isolates were statistically significant. however, the mean pat-level prevalence decreased from to between the seerad and iprave studies respectively. highly significant reductions in mean pat-level prevalence were also observed in the spring, in the north east and central scotland, and in the shedding of phage type 21/ human cases: contrasting the same time periods, there was a decline in the overall comparative annual reported incidence of human cases as well as in all the major pt groups except'other' pts. there was no significant decrease in the mean farm-level prevalence of e. coli o between and in scotland, despite significant declines in mean pat-level prevalence. incidence rates for human e. coli o infection in scotland are higher than most other united kingdom, european and north american countries. although there were declines in the number of human cases between the two study periods, there is no statistically significant evidence that the overall rate of human e. coli o infections in scotland over the last years has altered. escherichia coli o is an important cause of acute diarrhoea, haemorrhagic colitis and, especially in children, haemolytic uraemic syndrome. cattle are considered the main reservoir for e. coli o significant associations between livestock related exposures and human infection have been identified in a number of studies. comparable patterns in the distribution of pts 21/ and between cattle and humans support a hypothesized link between the bovine reservoir and human infections. animal studies: there were no statistically significant differences in the mean farm-level prevalence between the two studies; iprave: ).
 escherichia coli o is an important cause of acute diarrhoea, haemorrhagic colitis and, especially in children, haemolytic uraemic syndrome. incidence rates for human e. coli o infection in scotland are higher than most other united kingdom, european and north american countries. cattle are considered the main reservoir for e. coli o significant associations between livestock related exposures and human infection have been identified in a number of studies.  animal studies: there were no statistically significant differences in the mean farm-level prevalence between the two studies; iprave: ). however, the mean pat-level prevalence decreased from to between the seerad and iprave studies respectively. highly significant reductions in mean pat-level prevalence were also observed in the spring, in the north east and central scotland, and in the shedding of phage type 21/ human cases: contrasting the same time periods, there was a decline in the overall comparative annual reported incidence of human cases as well as in all the major pt groups except'other' pts. for both cattle and humans, the predominant phage type between and was pt21/ comprising over 50% of the positive cattle isolates and reported human cases respectively. the proportion of pt however, was represented by few of reported human cases despite comprising over 10% of cattle isolates. across the two studies there were differences in the proportion of pts 21/ and'other' pts in both cattle isolates and reported human cases; however, only differences in the cattle isolates were statistically significant.  there was no significant decrease in the mean farm-level prevalence of e. coli o between and in scotland, despite significant declines in mean pat-level prevalence. although there were declines in the number of human cases between the two study periods, there is no statistically significant evidence that the overall rate of human e. coli o infections in scotland over the last years has altered. comparable patterns in the distribution of pts 21/ and between cattle and humans support a hypothesized link between the bovine reservoir and human infections. this emphasizes the need to apply and improve methods to reduce bovine shedding of e. coli o in scotland where rates appear higher in both cattle and human populations, than in other countries. 