BACKGROUND
attaching and effacing escherichia coli  are characterized by their ability to cause attaching-and-effacing  lesions in the gut mucosa of human and animal hosts leading to diarrhoea.
the main mechanism of aeec pathogenesis is the destruction of the gastric microvillus brush border through restructuring of the underlying cytoskeleton by signal transduction between bacterial and host cells, intimate adherence of strains to the intestinal epithelium, pedestal formation and aggregation of polymerized actin at the sites of bacterial attachment  <cit> .
the adherence of bacteria to the enterocytes is mediated by intimin, an outer membrane protein encoded by the eae  gene  <cit> .
intimin genes are present in enteropathogenic e. coli  and some shigatoxin-producing e. coli .
epec strains are defined as eae harbouring diarrhoeagenic e. coli that possess the ability to form a/e lesions on intestinal cells and that do not possess shigatoxin encoding genes  <cit> .
according to their virulence markers, epec strains are subdivided into typical and atypical epec.
typical epec harbour the eaf  plasmid, which carries genes for regulation of lee functions and for production of bundle-forming pili , which interconnect bacteria within microcolonies and lead to a characteristic localized adherence pattern.
atypical epec strains are negative for both, the eaf plasmid and bfp and show diffuse, aggregative or localized-like adherence patterns  <cit> .
moreover, typical and atypical epec usually belong to certain serotype cluster and differ in their geographic distribution and their natural reservoir.
typical epec are still a major cause of infantile diarrhoea in developing countries and are rarely found in animals.
atypical epec strains predominate in industrialized countries and can be isolated from both humans and animals  <cit> .
atypical epec appear to be more closely related to stec and as such are considered emerging pathogens  <cit> .
their role in human infections is probably underestimated.
the second group of aeec is formed by stec strains, which produce shigatoxins.
stec are responsible for a number of human gastrointestinal diseases, including diarrhoea and hemorrhagic colitis .
in a proportion of individuals, particularly in children, these conditions may be complicated by neurological and renal sequelae, including haemolytic-uremic syndrome   <cit> .
although a number of studies have looked for the eae gene in stec strains isolated from sheep and pigs, only a limited number of studies have been undertaken to screen pigs and sheep for aeec and to further characterize such strains  <cit> .
the aim of this study was to isolate eae positive e. coli strains carried by healthy pigs and sheep at slaughterhouse level and to provide further characterization data for such strains.
RESULTS
a total of  <dig> faecal samples from pigs and  <dig> faecal samples from sheep collected at slaughter were tested for the presence of eae positive e. coli.
aeec were shed by  <dig>  and  <dig>  of pigs and sheep, respectively.
using colony dot-blot hybridization,  <dig> porcine and  <dig> ovine aeec strains were identified and isolated.
twenty-seven of the  <dig> eae positive porcine e. coli strains were typeable with o antisera .
they belonged to ten o serogroups, whereas  <dig> strains were of three serogroups, namely o <dig> , o <dig>  and o <dig> .
eighteen strains were not typeable for the o-antigen  and five had a rough lipopolysaccharide .
strains of the serogroup o <dig> showed different h types and the two strains of serotype o88:h <dig> carried two different intimin variants .
thirty-six of the  <dig> eae positive ovine e. coli strains were typeable with o antisera .
they belonged to  <dig> o serogroups, whereas  <dig> strains were of three serogroups, namely o <dig> , o <dig>  and o <dig> .
sixteen strains were ont and one had a rough lps type.
strains of the serogroup o <dig> and o <dig> showed different h types.
overall, only five o26:h <dig> strains  belonged to a known human epec serotype.
besides, this serotype  has also been reported in association with human enterohaemorrhagic e. coli  strains causing severe disease.
y-p: epec-type translocated intimin receptor
s: stec-type translocated intimin receptor
eae: gene encoding intimin
asta: gene encoding east1
tir: gene encoding translocated intimin receptor
stx: gene encoding shigatoxin
y-p: epec-type translocated intimin receptor
s: stec-type translocated intimin receptor
eae: gene encoding intimin
asta: gene encoding east1
tir: gene encoding translocated intimin receptor
stx: gene encoding shigatoxin
in total, a variety of  <dig> different intimin types and subtypes were found.
the  <dig> isolated porcine aeec  harboured  <dig> variants, and β <dig> , γ <dig> , ε , γ2/θ , ι , κ  were the frequently found.
the  <dig> isolated ovine eae positive e. coli strains  harboured  <dig> different intimin variants, and β <dig> , β2/δ ,ε , γ2/θ  were the most frequently found.
one strain was not typeable.
for this strain no pcr product was obtained with sk <dig> as the forward primer and all used reverse primers.
all but two ovine aeec strains  tested negative for genes encoding shigatoxins.
all strains tested negative for bfpa and the eaf plasmid.
east <dig>  was present in  <dig> strains, four porcine and  <dig> ovine strains.
forty-one porcine and  <dig> ovine strains tested positive for tir-y-p .
for one porcine strain there were no pcr products present in the tir specific pcrs.
this strain was therefore not typable for this genotype.
remarkably, all strains encoding intimin γ <dig> were associated with tir-s .
discussion
the high percentages of animals shedding aeec are remarkable, especially in view of frequencies of sheep and pigs colonized with aeec found in these animal species in previous works  <cit> .
indeed, de la fuente et al.
<cit>  and krause et al.
<cit>  detected aeec in less than 20% of the sheep and pigs studied, while in our work these percentages were 89% for pigs and 55% for sheep.
aktan et al.
<cit>  found  <dig> % and  <dig> % aeec strains from sheep and pigs, respectively.
in our study, no strains showing characteristics of typical epec were found.
these results are consistent with earlier reports that most aeec strains from sheep and pigs are negative for stx genes and bfpa, and are therefore considered as atypical epec  <cit> .
pigs and sheep are reservoirs of serologically and genetically diverse intimin harbouring e. coli.
in our study, only strains of the serotypes o2:h <dig>  o3:h <dig> and o26:h <dig> were commonly isolated from both pigs and sheep.
all other serotypes were different.
this could indicate that atypical epec are adapted to their particular animal host.
in pigs, serogroups o <dig>  o <dig> and o <dig> and serotypes o2:h <dig>  o2:h <dig>  o108:h <dig>  o145:h <dig> were most frequently found.
intimin types showed a wide diversity with θ, β <dig> and γ <dig> as the most frequent types.
these results partly agree with other studies performed on healthy pigs in germany and hungary  <cit> , considering that the latter study was performed on weaned pigs.
our results show more intimin types and slightly different serotype patterns with more untypeable o serogroups and o <dig> as the most frequent one.
in sheep, serogroups o <dig>  o26; o <dig> and o <dig> and serotypes o2:h <dig>  o26:h <dig>  o70:h <dig>  o146:h <dig> were most frequently found.
intimin types showed a wide diversity with γ2/θ and β <dig> as the most frequent types.
compared to studies performed on healthy sheep in england, wales, and australia  <cit> , the ovine strains isolated in switzerland harboured more frequently the intimin γ2/θ type.
but a high prevalence  of this intimin type in healthy sheep was also shown in a spanish study  <cit> .
ovine isolates reported in the literature show more genetic similarity to bovine than porcine strains.
namely o <dig>  o <dig>  o <dig>  o <dig>  o <dig>  o <dig> and o <dig> were found in studies on healthy cattle in germany, england and wales, spain, switzerland and brazil  <cit> .
apart from intimin γ2/θ, which was found much more often in ovine strains than bovine, the distribution of intimin types is rather similar in the present study.
the only classical human epec serotype found in our study was o26:h <dig> , indicating that classical epec serotypes might not be very common in these animal species.
moreover, no serotypes known for causing large outbreaks such as o39:h-, o88:h <dig>  o91:h <dig> and o111:h <dig> were found.
however, 17% of all strains carried east <dig>  the percentage  of ovine aeec strains that harboured this gene was similar to that found by aktan et al.
<cit> , but much higher than that found by yuste et al.
<cit> .
even the significance of this low-molecular-weight, plasmid-mediated, heat-stable enterotoxin in aeec pathogenesis is still unknown, large epec outbreaks in adults have been associated with east <dig> positive strains and recent studies found east <dig> to be associated significantly with diarrhoea  <cit> .
hence most of the strains showed characteristics of atypical epec.
nevertheless, there are seven stx negative aeec strains belonging to serotypes and eae types that are associated with typical ehec strains .
the reason that these strains are stx negative might be due to the fact that stec strains can undergo ephemeral interconversions via loss and gain of stx-encoding phages, which leads to different pathotypes.
this was already shown for e. coli o <dig> of the h <dig> clone  <cit>  and for e. coli o <dig>  <cit> .
ongoing studies in our laboratories aim to further characterize the o <dig> and o <dig> strains in view of phage integration sites as well as to perform transduction experiments on these strains.
CONCLUSIONS
our data show that pigs and sheep are a source of serologically and genetically diverse intimin harbouring e. coli strains.
most of the strains show characteristics of atypical epec.
nevertheless, there are stx negative aeec belonging to serotypes and intimin types that are associated with typical ehec strains .

 attaching and effacing escherichia coli are characterized by their ability to cause attaching-and-effacing lesions in the gut mucosa of human and animal hosts leading to diarrhoea. the main mechanism of aeec pathogenesis is the destruction of the gastric microvillus brush border through restructuring of the underlying cytoskeleton by signal transduction between bacterial and host cells, intimate adherence of strains to the intestinal epithelium, pedestal formation and aggregation of polymerized actin at the sites of bacterial attachment. the adherence of bacteria to the enterocytes is mediated by intimin, an outer membrane protein encoded by the eae gene. intimin genes are present in enteropathogenic e. coli and some shigatoxin-producing e. coli. epec strains are defined as eae harbouring diarrhoeagenic e. coli that possess the ability to form a/e lesions on intestinal cells and that do not possess shigatoxin encoding genes. according to their virulence markers, epec strains are subdivided into typical and atypical epec. typical epec harbour the eaf plasmid, which carries genes for regulation of lee functions and for production of bundle-forming pili, which interconnect bacteria within microcolonies and lead to a characteristic localized adherence pattern. atypical epec strains are negative for both, the eaf plasmid and bfp and show diffuse, aggregative or localized-like adherence patterns. moreover, typical and atypical epec usually belong to certain serotype cluster and differ in their geographic distribution and their natural reservoir. typical epec are still a major cause of infantile diarrhoea in developing countries and are rarely found in animals. atypical epec strains predominate in industrialized countries and can be isolated from both humans and animals. atypical epec appear to be more closely related to stec and as such are considered emerging pathogens. their role in human infections is probably underestimated. the second group of aeec is formed by stec strains, which produce shigatoxins. stec are responsible for a number of human gastrointestinal diseases, including diarrhoea and hemorrhagic colitis. in a proportion of individuals, particularly in children, these conditions may be complicated by neurological and renal sequelae, including haemolytic-uremic syndrome. although a number of studies have looked for the eae gene in stec strains isolated from sheep and pigs, only a limited number of studies have been undertaken to screen pigs and sheep for aeec and to further characterize such strains. the aim of this study was to isolate eae positive e. coli strains carried by healthy pigs and sheep at slaughterhouse level and to provide further characterization data for such strains.  a total of faecal samples from pigs and faecal samples from sheep collected at slaughter were tested for the presence of eae positive e. coli. aeec were shed by and of pigs and sheep, respectively. using colony dot-blot hybridization, porcine and ovine aeec strains were identified and isolated. twenty-seven of the eae positive porcine e. coli strains were typeable with o antisera. they belonged to ten o serogroups, whereas strains were of three serogroups, namely o, o and o. eighteen strains were not typeable for the o-antigen and five had a rough lipopolysaccharide. strains of the serogroup o showed different h types and the two strains of serotype o88:h carried two different intimin variants. thirty-six of the eae positive ovine e. coli strains were typeable with o antisera. they belonged to o serogroups, whereas strains were of three serogroups, namely o, o and o. sixteen strains were ont and one had a rough lps type. strains of the serogroup o and o showed different h types. overall, only five o26:h strains belonged to a known human epec serotype. besides, this serotype has also been reported in association with human enterohaemorrhagic e. coli strains causing severe disease. y-p: epec-type translocated intimin receptor s: stec-type translocated intimin receptor eae: gene encoding intimin asta: gene encoding east1 tir: gene encoding translocated intimin receptor stx: gene encoding shigatoxin y-p: epec-type translocated intimin receptor s: stec-type translocated intimin receptor eae: gene encoding intimin asta: gene encoding east1 tir: gene encoding translocated intimin receptor stx: gene encoding shigatoxin in total, a variety of different intimin types and subtypes were found. the isolated porcine aeec harboured variants, and β, γ, ε, γ2/θ, ι, κ were the frequently found. the isolated ovine eae positive e. coli strains harboured different intimin variants, and β, β2/δ,ε, γ2/θ were the most frequently found. one strain was not typeable. for this strain no pcr product was obtained with sk as the forward primer and all used reverse primers. all but two ovine aeec strains tested negative for genes encoding shigatoxins. all strains tested negative for bfpa and the eaf plasmid. east was present in strains, four porcine and ovine strains. forty-one porcine and ovine strains tested positive for tir-y-p. for one porcine strain there were no pcr products present in the tir specific pcrs. this strain was therefore not typable for this genotype. remarkably, all strains encoding intimin γ were associated with tir-s. discussion the high percentages of animals shedding aeec are remarkable, especially in view of frequencies of sheep and pigs colonized with aeec found in these animal species in previous works. indeed, de la fuente et al. and krause et al. detected aeec in less than 20% of the sheep and pigs studied, while in our work these percentages were 89% for pigs and 55% for sheep. aktan et al. found % and % aeec strains from sheep and pigs, respectively. in our study, no strains showing characteristics of typical epec were found. these results are consistent with earlier reports that most aeec strains from sheep and pigs are negative for stx genes and bfpa, and are therefore considered as atypical epec. pigs and sheep are reservoirs of serologically and genetically diverse intimin harbouring e. coli. in our study, only strains of the serotypes o2:h o3:h and o26:h were commonly isolated from both pigs and sheep. all other serotypes were different. this could indicate that atypical epec are adapted to their particular animal host. in pigs, serogroups o o and o and serotypes o2:h o2:h o108:h o145:h were most frequently found. intimin types showed a wide diversity with θ, β and γ as the most frequent types. these results partly agree with other studies performed on healthy pigs in germany and hungary, considering that the latter study was performed on weaned pigs. our results show more intimin types and slightly different serotype patterns with more untypeable o serogroups and o as the most frequent one. in sheep, serogroups o o26; o and o and serotypes o2:h o26:h o70:h o146:h were most frequently found. intimin types showed a wide diversity with γ2/θ and β as the most frequent types. compared to studies performed on healthy sheep in england, wales, and australia, the ovine strains isolated in switzerland harboured more frequently the intimin γ2/θ type. but a high prevalence of this intimin type in healthy sheep was also shown in a spanish study. ovine isolates reported in the literature show more genetic similarity to bovine than porcine strains. namely o o o o o o and o were found in studies on healthy cattle in germany, england and wales, spain, switzerland and brazil. apart from intimin γ2/θ, which was found much more often in ovine strains than bovine, the distribution of intimin types is rather similar in the present study. the only classical human epec serotype found in our study was o26:h, indicating that classical epec serotypes might not be very common in these animal species. moreover, no serotypes known for causing large outbreaks such as o39:h-, o88:h o91:h and o111:h were found. however, 17% of all strains carried east the percentage of ovine aeec strains that harboured this gene was similar to that found by aktan et al., but much higher than that found by yuste et al.. even the significance of this low-molecular-weight, plasmid-mediated, heat-stable enterotoxin in aeec pathogenesis is still unknown, large epec outbreaks in adults have been associated with east positive strains and recent studies found east to be associated significantly with diarrhoea. hence most of the strains showed characteristics of atypical epec. nevertheless, there are seven stx negative aeec strains belonging to serotypes and eae types that are associated with typical ehec strains. the reason that these strains are stx negative might be due to the fact that stec strains can undergo ephemeral interconversions via loss and gain of stx-encoding phages, which leads to different pathotypes. this was already shown for e. coli o of the h clone and for e. coli o. ongoing studies in our laboratories aim to further characterize the o and o strains in view of phage integration sites as well as to perform transduction experiments on these strains.  our data show that pigs and sheep are a source of serologically and genetically diverse intimin harbouring e. coli strains. most of the strains show characteristics of atypical epec. nevertheless, there are stx negative aeec belonging to serotypes and intimin types that are associated with typical ehec strains. 
our data show that pigs and sheep are a source of serologically and genetically diverse intimin-harbouring e. coli strains. nevertheless, there are stx-negative aeec strains belonging to serotypes and intimin types that are associated with classical enterohaemorrhagic e. coli strains. strains of the serotypes o2:h o3:h and o26:h were found in both pigs and sheep. this study reports data on the occurrence of eae positive e. coli carried by healthy pigs and sheep at the point of slaughter, and on serotypes, intimin variants, and further virulence factors of isolated aeec strains. the genetic determinants for the production of a/e lesions are located on the locus of enterocyte effacement, a pathogenicity island that also contains the genes encoding intimin. eleven different intimin types were detected, whereas γ2/θ was the most frequent, followed by β ε and γ all but two ovine strains tested negative for the genes encoding shiga toxins. by colony dot-blot hybridization, aeec were isolated from and randomly selected porcine and ovine samples and further characterized. attaching and effacing escherichia coli are characterized by their ability to cause attaching-and-effacing lesions in the gut mucosa of human and animal hosts leading to diarrhoea. all strains tested negative for the bfpa gene and the eaf plasmid.
 attaching and effacing escherichia coli are characterized by their ability to cause attaching-and-effacing lesions in the gut mucosa of human and animal hosts leading to diarrhoea. the genetic determinants for the production of a/e lesions are located on the locus of enterocyte effacement, a pathogenicity island that also contains the genes encoding intimin. this study reports data on the occurrence of eae positive e. coli carried by healthy pigs and sheep at the point of slaughter, and on serotypes, intimin variants, and further virulence factors of isolated aeec strains.  faecal samples from finished pigs and sheep were examined at slaughter. the proportion of eae positive samples was 89% for pigs and 55% for sheep. by colony dot-blot hybridization, aeec were isolated from and randomly selected porcine and ovine samples and further characterized. strains of the serotypes o2:h o3:h and o26:h were found in both pigs and sheep. in pigs o2:h o2:h o108:h o145:h and in sheep o2:h o26:h o70:h o146:h were the most prevalent serotypes among typable strains. eleven different intimin types were detected, whereas γ2/θ was the most frequent, followed by β ε and γ all but two ovine strains tested negative for the genes encoding shiga toxins. all strains tested negative for the bfpa gene and the eaf plasmid. east was present in of the isolated strains.  our data show that pigs and sheep are a source of serologically and genetically diverse intimin-harbouring e. coli strains. most of the strains show characteristics of atypical enteropathogenic e. coli. nevertheless, there are stx-negative aeec strains belonging to serotypes and intimin types that are associated with classical enterohaemorrhagic e. coli strains. 