Applied and Environmental Microbiology, September 2003, p . 5690-5692, Vol . 69, No . 9

We used PCR to investigate the prevalence of eae-positive E . coli in cattle feces and genetically characterized the intimin types found, as well as various virulence genes seen in the isolated strains .

E . coli O157:H7 strain ATCC 35150 (American Type Culture Collection, Manassas, Va.) was used as the positive control for stx₁, stx₂, eae, and intimin type (intimin ) . The E . coli strains JS144, 166, VR299-2, and EPEC108, which were used as positive-control strains for intimins , ß, and and bundle-forming pilus (bfp), respectively, were derived from the stock culture collection of the National Institute of Animal Health, Tsukuba, Japan . A total of 444 rectal stool grab samples were collected from healthy dairy cattle (35 calves under 1 month old, 107 calves more than 1 to 3 months old, 88 heifers more than 3 to 6 months old, 214 heifers more than 6 months old, and cows) on 95 farms located in the western and central parts of Japan between May and November 2001 . All rectal stool samples were sampled by veterinarians at regional governmental animal hygiene centers . The samples were placed in cool boxes (4 to 8°C) and taken to the laboratory for immediate processing (usually within 24 h) . Each sample of 1 g of rectal stool was enriched in 19 ml of Trypticase soy broth (Eiken, Tokyo, Japan) at 37°C for 18 h . Ten microliters of the Trypticase soy broth culture was inoculated onto MacConkey agar (MAC; Eiken) . The MAC plates were incubated at 37°C for 18 h, and a loopful of colonies from an area of confluent growth was tested using eae, stx₁, and stx₂ PCR (5, 12) . From eae-PCR-positive samples we isolated eae-positive colonies by colony hybridization with an eae DNA probe or by eae PCR of randomly isolated individual colonies . The eae probes were prepared by labeling eae-PCR amplicons from E . coli strain ATCC 35150 with a DIG High Prime kit (Roche Diagnostics GmbH, Mannheim, Germany) . The pair of primers for eae PCR were located at the 5' end of the eae gene, a common region for intimin subtypes (5) . For colony hybridization or individual colony PCR, up to 24 typical E . coli isolates were taken from the MAC plate . All eae-positive isolates were confirmed to be E . coli by conventional biochemical tests and, if needed, by an API 20E system . Up to three eae-positive colonies per sample were randomly chosen and subjected to O-serogroup typing and classification of intimin types , ß, (1), and (13) by PCR . If the intimin types of isolates could not be classified by the PCR technique described above, the isolates were analyzed by DNA sequencing of 800 bp at the 3' end of the eae gene (1) . One strain per farm was selected for further study from strains showing the same O serogroup and intimin type profile at each farm . The eae-positive strains selected were characterized by PCR to determine the presence of stx₁, stx₂, bfp, EHEC hlyA (14), and irp2 located in the high-pathogenicity island (HPI) (6) .

eae and stx were detected in 74 (16.7%) and 138 (31.1%) of 444 animals, respectively (Table 1) . There were no regional variations seen in eae detection rates . The eae gene was detected more often in neonatal animals (1 month old) than in older animals . The stx genes were detected more often in older animals but least often in animals over 6 months old . Interestingly, animals aged >1 to 3 months showed the least frequent presence of both eae and stx; in contrast, those >3 to 6 months showed a generally higher prevalence . Intimin is known to be a strong immunogen (18), and it is classified into 10 types (21) . Although several intimin type groups might generate immunological cross-reactions, E . coli strains possessing other intimin types can still infect animals . Therefore, we believe that neonates, which do not have an immunological history of contact with any intimin-expressing organisms, are likely to be the most easily infected by intimin-producing E . coli . According to this hypothesis, once an animal has acquired immunity against one intimin type, E . coli strains expressing the same type of intimin are eliminated around 2 months after the onset of infection . Consequently, other types of intimin-producing E . coli from neighboring or imported animals are likely to infect the animal at heifer age . If this hypothesis is true, intimin types ß and , especially the intimin seen in O157:H7 strains, should act as an effective vaccine for eliminating the human EHEC strain from cattle .

 
Forty-five of the 51 eae-positive strains selected belonged to 20 different O serogroups, with 6 strains being nontypeable . Two isolates were members of serogroup O157, and 26 of the typeable strains could be classified into five serogroups (O26, O70, O103, O145, and O156) (Table 2) . The O26, O103, O111, O145, and O157 serogroups, which are frequently detected as causal agents of hemorrhagic colitis and hemolytic-uremic syndrome, together accounted for more than 40% of the eae-positive E . coli strains examined (19 of 45 strains) . These major human potential EHEC strains were isolated from various locations on farms . Almost all of these strains, except strains determined as O145, possessed one or more stx genes . On the other hand, when stx was used for screening STEC in cattle in 1998, only 20% of STEC strains could be classified into these potential EHEC serogroups (9) . The O113 and O116 STEC strains, which have only rarely been isolated from humans, were the most predominant STEC strains in cattle in Japan (9) . Moreover, except for two O157 strains, no isolates tested possessed stx₂ . This suggests that most E . coli strains with stx₂ in cattle in Japan do not possess eae . This fact was confirmed in a previous study (9), in which only 3 of 22 STEC strains with stx₂ from cattle possessed eae . The three strains were classified as either O111 or O157 (9) . Therefore, when potential EHEC strains are screened in cattle fecal samples by PCR, eae is likely to be a more useful target than the stx gene . However, O26 strains from humans are the most common O serogroup of E . coli strains possessing the HPI locus (6); irp2 in the HPI was also detected in three strains belonging to serogroups O35, O49, and O70 . In a recent study, the gene was found in O39 strains isolated from broiler chickens in Finland (8) . The HPI locus might be prevalent in many types of E . coli in various kinds of animals . In contrast, bfp was not found in any of the strains . Organisms with bfp seem to be isolated very rarely from cattle, since there are no reports of E . coli isolates with bfp from cattle samples . In contrast, EHEC-hlyA was detected at a high rate of more than 75% (39 of 51 animals) . This virulent gene was also detected at a higher rate in STEC in 71.7% (66 of 92) of cattle tested in Japan in 1998 (9) . In this study, which confirmed the prevalence in cattle of the EHEC-hlyA gene in E . coli strains, regarded as gut flora, 23 strains from 23 cattle fecal samples at different farms, which were identified as neither STEC nor AEEC, were examined by PCR . Fourteen of these 23 strains (60.9%) possessed the gene . Therefore, the EHEC-hlyA gene is believed to have spread into gut flora E . coli in cattle in Japan . It may therefore no longer be an effective marker for identifying pathogenic E . coli strains in cattle .

 
Concerning the typing of intimin, 39 of 51 strains could be identified by established PCR (1, 13) . In particular, any one of the strains typed as ß, , or intimin could be amplified by PCR . However, of nine strains identified as intimin, two strains could not be amplified but were instead identified by DNA sequence analysis . It appears that the DNA sequences of -intimin-specific regions show greater variation than those of other intimins . This fact has also been recognized in E . coli strains with avian intimin (8) . According to one report, the DNA homology among -intimin-specific regions in strains was calculated to be 86 to 100% . On the other hand, the remaining 10 strains were determined as either not typeable (strains which could not be subcloned) or having recently reported intimin types (21) . Intimins , , and are newly categorized intimin types that have been reported in E . coli strains from patients . In this study, we also detected these new types not only from bovine E . coli strains but also from different O serogroups (O49, O145, and O153) of human origin (21) . For example, Zhang et al . reported that intimin was detected in O2, O12, and O125 and intimin was present in O118 and O157:H42 (21) . On the other hand, in a previous report, 6 of 15 eae-positive E . coli strains isolated from wild birds could not be intimin typed by PCR (8) . Since the nontypeable strains are of types that are either newly established or hitherto unknown, if some have newly established types, then the newly categorized intimin types are likely to have been more prevalent in various O serogroups than previously realized, since these strains were serotyped as O54, O72, O103, and O144, which are very rare types in mammals, except O103 (8) . We conclude that E . coli strains with newly categorized intimin types have different serotypes according to the animal species from which they are derived .

This work was supported by grants from the National Agricultural Research Organization, Japan .

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