Departamento Patología Animal I, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain1
Centro de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain2
Author for correspondence: R. de la Fuente. Tel: +34 91 3943703. Fax: +34 91 3943908. e-mail: rifuente{at}eucmax.sim.ucm.es
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: pulsed field gel electrophoresis, attaching and effacing Escherichia coli strains, enteropathogenic E. coli, diarrhoea, adherence
Abbreviations: AE, attaching and effacing; AEEC, attaching and effacing E. coli; CNF, cytotoxic necrotizing factor; EHEC, enterohaemorrhagic E. coli; EPEC, enteropathogenic E. coli; Esp, EPEC secreted protein; HC, haemorrhagic colitis; HUS, haemolytic uraemic syndrome; LEE, locus for enterocyte effacement; Stx, Shiga toxin; VT, verotoxin
The GenBank accession numbers for the sequences reported in this paper are AF253560 (eae gene of strain CK379), AF253561 (eae gene of strain CL559), and AF254454, AF254455, AF254456 and AF254457 (espB nucleotide sequences of strains CL559, CL617, CK379 and CL398, respectively)
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
All the genes necessary for AE lesion formation by human EPEC are encoded in a pathogenicity island called the locus for enterocyte effacement (LEE) (McDaniel et al., 1995 ; McDaniel & Kaper, 1997
). The LEE is present in all the bacteria that induce AE lesions (McDaniel et al., 1995
; Wieler et al., 1998
). The complete sequences for the LEE of human EPEC strain E2348/69 and human EHEC O157:H7 strain EDL933 have been determined (Elliot et al., 1998
; Perna et al., 1998
). The LEE encodes a type III secretion system, a series of proteins secreted by this system called Esps (for EPEC secreted proteins), intimin, which mediates intimate bacterial adhesion to epithelial cells, and Tir, a receptor for intimin which is translocated into host cells (reviewed by Frankel et al., 1998
). The eae gene, encoding intimin, has been sequenced in a variety of AE bacteria including human EPEC and EHEC (Jerse et al., 1990
; Yu & Kaper, 1992
; McGraw et al., 1999
), AEEC strains isolated from a rabbit, a calf, a dog and a pig (Agin et al., 1996
; An et al., 1997
; China et al., 1999b
), and C. rodentium and H. alvei (Frankel et al., 1994
; Schauer & Falkow, 1993
). Comparison of the deduced amino acid sequences has revealed that intimins are highly conserved proteins at the N-terminal region, but highly variable at their C-termini. The differences in amino acid sequences at the C-termini are correlated with antigenic variation (Frankel et al., 1994
). On the basis of antigenic variation, PCR analysis and sequencing, at least five subtypes of intimins have been identified:
, ß,
,
and
(Agin & Wolf, 1997
; Adu-Bobie et al., 1998
; Oswald et al., 2000
). It appears that specific intimin subtypes are associated with the distinct lineages of human EPEC and EHEC (Adu-Bobie et al., 1998
).
The second gene that was identified in EPEC as necessary for induction of AE lesions was the espB gene (Donnenberg et al., 1993 ). Secretion of the EspB protein is essential for signal transduction in host cells and AE lesion formation (Foubister et al., 1994
). However its function has not been elucidated. The espB gene has been sequenced in several strains, including human EPEC (Donnenberg et al., 1993
), human and bovine EHEC (Ebel et al., 1996
), and rabbit EPEC (Abe et al., 1997
). Comparison of the deduced amino acid sequences has revealed that, like intimins, EspB are highly variable proteins. The variability in genes encoding proteins that interact directly with the host, in contrast to other LEE-encoded proteins, which are highly conserved, suggests that these variable proteins are subject to selection for evasion of the host immune system (Frankel et al., 1998
).
In small ruminants, AEEC strains and their possible association with diarrhoea in neonatal animals have not been studied, although AE lesions have been observed in these animals (Janke et al., 1989 ; Drolet et al., 1994
; Duhamel et al., 1992
). The aim of this study was to investigate the variability of eae and espB, two genes of the LEE directly involved in interactions with the host, in AEEC isolates from diarrhoeic lambs and goat kids. In addition, genetic relatedness of selected strains was investigated by PFGE.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
PCR amplification and nucleotide sequencing of eae genes.
Bacterial DNA samples for amplification were obtained with cetyltrimethylammonium bromide as described by Ausubel et al. (1991) . PCR reactions were performed in a final volume of 100 µl containing 1xPCR buffer (Perkin Elmer), 0·2 mM deoxynucleoside triphosphates, 1·5 mM MgCl2, 0·5 µM of each primer and 2·5 U AmpliTaq Gold polymerase (Perkin Elmer). PCR conditions used for all primers were a first cycle of denaturation at 94 °C for 10 min, 30 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 1 min and extension at 72 °C for 3 min, and a final extension at 72 °C for 10 min.
PCR to amplify the 5' region of the eae gene from field strains was performed with the forward primer AE9 (5'-ACG TTG CAG CAT GGG TAA CTC-3') previously described by Gannon et al. (1993) and the reverse consensus primer rEAE5 (5'-CGA AGT CTT ATC AGC CGT AAG T-3') complementary to bases 16851707 of the gene of strain E2348/69 (Jerse et al., 1990
). This primer pair amplifies a 2·3 kbp fragment comprising the 5' end of the eae gene and 590 bp upstream of the gene.
PCR products were analysed by electrophoresis in 1% agarose gels, recovered with the Qiaquick gel extraction kit (Qiagen) and characterized by restriction analysis with PstI (New England Biolabs). Purified PCR products obtained from two independent PCR reactions were ligated into the pMOSBlue vector and the plasmids generated used to transform competent E. coli strains using the pMOSBlue T-vector kit (Amersham Life Science). Transformants were selected on LB agar containing ampicillin (100 µg ml-1) and the presence of the appropriate fragment confirmed by PCR. DNA sequences of cloned PCR fragments were determined by automated sequencing using primers based on the previously determined sequence and a 373A DNA sequencer (Applied Biosystems). The sequences obtained were compared with eae gene sequences in the databases using the BLASTX program. The information generated in this manner was used to select reverse primers to amplify the 3' region of eae genes. Reverse primers were based on the 3' region of published eae gene sequences. These primers were combined with the forward primer fEAE-5, which is complementary to the rEAE-5 primer. PCR products obtained from two independent reactions were cloned and sequenced as described above. Finally, the entire genes were amplified using appropriate primers selected from those used to sequence the genes and sequences were confirmed by automated sequencing directly on purified PCR products. Sequences were compared with sequence databases using the BLASTX program. Multiple alignments of the sequences obtained and published eae sequences were performed with CLUSTAL (Higgins & Sharp, 1988 ).
Detection of espB gene by Southern blot hybridization.
Samples of bacterial DNA were digested with PstI and EcoRI (New England Biolabs); the restriction fragments were resolved by electrophoresis in 1% agarose gels and transferred onto nylon membranes (Hybond-N+, Amersham Life Science).
The DNA probe for detection of espB was generated by PCR amplification from EPEC reference strain E2348/69 (Donnenberg et al., 1993 ). A 930 bp fragment of espB was amplified with the feaeB and reaeB primers previously described by Beaudry et al. (1996)
(Table 1
). Bacterial DNA from strain E2348/69 was released by boiling in 500 µl sterile water at 100 °C for 10 min. The boiled suspension was centrifuged and 10 µl of the supernatant used as template. The amplification reactions were performed in a final volume of 100 µl containing 1xPCR buffer II (Perkin Elmer), 0·2 mM of each deoxynucleoside triphosphate, 1·5 mM MgCl2, 0·5 µM of each primer, 2·5 U AmpliTaq Gold polymerase (Perkin Elmer) and 10 µl of the DNA template. PCR reactions were carried out using an initial incubation at 94 °C for 10 min followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and elongation at 72 °C for 1 min, and a final incubation at 72 °C for 5 min. PCR products were separated by electrophoresis in 1% agarose gels, purified with the Qiaquick gel extraction kit (Qiagen) and labelled by random-primed DNA synthesis in the presence of [
-32P]dCTP as described above. Southern blot hybridizations with the espB probe were performed as described above for colony hybridization with the eae probe.
|
The nucleotide sequences of selected espB PCR products were determined directly from purified PCR products. The sequences of both strands were determined using the amplification primers and an Applied Biosystems model 373 A automated DNA sequencer. Analysis and assembly of sequences were performed with the Chromas (1.43) program and searches for homologous sequences in the databases were done with the BLASTX program.
PFGE.
Genomic DNA for contour-clamped homogeneous electric field electrophoresis (CHEF) was prepared in agarose plugs as described by Smith & Cantor (1987) . Genomic DNA of selected strains was digested with restriction enzymes SfiI or NotI (New England Biolabs) according to the manufacturers instructions. Restriction fragments were separated in 1% agarose gels using a CHEF apparatus (LKB) with pulse time conditions varied depending on the size range of fragments to be resolved. A lambda ladder (New England Biolabs) was used as molecular size marker. The PFGE patterns were compared by the unweighted pair group method with arithmetic averages (UPGMA) clustering method using the Dice coefficient. Relationships between isolates were determined using the criteria of Tenover et al. (1996)
.
Rabbit ligated intestinal loop assay and demonstration of AE lesions.
The ability of bacterial strains to produce AE lesions was determined in the rabbit ligated intestinal loop assay (Moon et al., 1983 ). Bacteria were grown in Penassay broth (Difco) at 37 °C overnight. They were washed once and resuspended in 10 mM phosphate-buffered saline (PBS), pH 7·4, at a final concentration of approximately 1·5x108 c.f.u. ml-1. Three-month-old New Zealand White rabbits were used. One millilitre of bacterial suspension was inoculated into each ligated loop. After 18 h, rabbits were killed by intravenous injection of sodium pentobarbital and tissue samples were taken for light and electron microscopic examination. Each strain was tested in at least two different animals.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Analysis and sequencing of eae genes
Since eae genes of ovine and caprine E. coli strains have not been described previously, we decided to amplify the highly conserved 5' region of eae genes from field strains to analyse them further. All the strains that were positive by colony hybridization yielded products of 2·3 kbp (Table 3). The PCR products were digested with PstI and the restriction patterns obtained were compared with the PstI restriction map of the corresponding 5' region of eae nucleotide sequences of
intimin prototype strain E2348/69, ß intimin prototype strain RDEC-1 and
intimin prototype strain EDL933 (Fig. 1
). Two different patterns were observed among 5' eae regions of field strains. Pattern I, which was identical to the restriction pattern of the 5' eae region of ß intimin prototype strain RDEC-1, was obtained from PCR products from 43 strains, and pattern II, identical to the restriction pattern of the 5' eae region of
and
intimin prototype strains, was obtained from PCR products from seven of the ovine strains. A caprine strain, CK379, which yielded pattern I and an ovine strain, CL559, which yielded pattern II were selected for sequencing of the 5' eae region. The DNA sequences of the 5' regions of both strains were analysed and compared with published eae sequences. The DNA sequence of the 5' eae region of strain CK379 showed the highest identity (99·6%) with that of rabbit strain RDEC-1 (Agin et al., 1996
; GenBank accession no. ECU60002) and the DNA sequence of the 5' eae region of strain CL559 showed the highest identity (99%) with that of the human O111:NM EHEC strain 95NR1 (Voss et al., 1998
; GenBank accession no. AF025311). Analysis of the sequences also revealed the presence of an ORF of 471 bp encoding a predicted protein of 156 amino acids. Comparison with sequence databases indicated that these ORFs were identical to the highly conserved orfU gene of the LEE. The 471 bp ORF of strains CK379 and CL559 showed 100% identity with orfU of strains RDEC-1 (Agin et al., 1996
) and 95NR1 (Voss et al., 1998
; GenBank accession no. AF025311), respectively.
|
|
|
Identification of espB genes and sequencing
To identify espB genes of ovine and caprine eae-positive strains, genomic DNAs were analysed by Southern blot hybridization with an espB probe consisting of a 930 bp PCR fragment of the espB gene of EPEC prototype strain E2348/69, which comprised nearly the entire espB gene. Only seven strains, all of ovine origin, hybridized with this probe (Table 4) even under hybridization conditions of low stringency. The presence of an espB gene similar to the espB of strain E2348/69 correlated with the presence of an eae gene encoding intimin subtype
V. Strains with an eae gene encoding the ß intimin subtype were not detected by the probe (Table 4
). Two of the espB-positive strains (CL559 of serogroup O91 and CL617 of serogroup O2) were selected for determining the extent of the similarity of their espB with that of human EPEC strain E2348/69. The nucleotide sequences of the 930 bp fragments generated by PCR from strains CL559 and CL617 were identical to each other, and had 95% identity with the corresponding fragment of the espB gene of strain E2348/69 (Donnenberg et al., 1993
).
|
The results of the PCR assays showed that each primer pair amplified a specific PCR product from the corresponding prototype strain and did not amplify any PCR product from strains with a heterologous espB gene subtype. EPEC strain E2430/78 (serotype O111:NM), which has been shown to posses ß intimin (Agin & Wolf, 1997 ), and strains CL617 and CL559 (this study), which harbour espB genes homologous to that of strain E2348/69, were also included. Strain E2430/78 yielded the expected espBß product with primers B1+B3. Strains CL559 and CL617 yielded product exclusively with the B1+reaeB primer pair, corresponding to an espB
gene subtype. The remaining eae-positive ovine and caprine strains were also analysed for espB subtype by PCR in three independent assays with each primer pair. The PCR results (Table 4
) showed that all the strains that were negative by Southern hybridization with the espB probe produced a specific PCR product of the expected size with primer pair B1+B3, corresponding to the espBß gene subtype. The presence in these strains of an espB gene homologous to that of strain RDEC-1 was confirmed by sequencing the PCR products from strains CK379 and CL398. Nucleotide sequences of the espB genes of both strains had 100% identity with the corresponding DNA sequence of the espB gene of strain RDEC-1 (Abe et al., 1997
).
PFGE analysis of AEEC strains
Six ovine (one of each identified serogroup and one untypable) and six caprine (two of each identified serogroup) eae-positive strains were selected for PFGE analysis (Fig. 3). The selected strains were epidemiologically unrelated, as they were collected in different years from different animals and different farms. All these selected strains induced typical AE lesions in the rabbit ligated ileal loop assay. The ovine and caprine strains adhered closely to ileal enterocytes and this adhesion was characterized by microvillus effacement and the induction of cup-like structures underneath adhering bacteria (Fig. 4
).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The other intimin subtype detected in seven of the ovine AEEC strains studied was classified as a variant of intimin (termed
V) on the basis of the identity of the deduced amino acid sequence of its C-terminal domain with those of previously published
intimin sequences. Intimin
is present in EHEC strains of clone 1 (serotypes O157:H7 and O157:H) and the atypical EPEC clone (serotype O55:H) from which O157:H7 EHEC strains are believed to have evolved (Whitman et al., 1993
). The
intimins of these strains are virtually identical, supporting the hypothesis that they belong to a single clonal lineage (McGraw et al., 1999
). The C-terminus of the
V intimin of the ovine strain CL559 had 7576% sequence identity with
intimin of EHEC clone 1 (McGraw et al., 1999
), and 96% identity with intimin of the human O111:H EHEC strain 95NR1 isolated from a patient with HUS and belonging to EHEC clone 2 (Voss et al., 1998
). It is possible that these differences in amino acid sequence in the cell-binding domain represent antigenic variations, as Voss et al. (1998)
found that serum from a HUS patient infected only with a serotype O111:H EHEC strain reacted with intimin from an O111 EPEC strain, but not with that of an EHEC strain of clone 1. Recently, Oswald et al. (2000)
detected intimin
in human EHEC strains of clone 2 (serotypes O111:H and O111:H8), and in human EPEC strains of serotypes O127:H40, O128:H8 and O128:H using PCR analysis. On the basis of the PstI restriction patterns of PCR products derived from the intimin gene, these authors differentiated two subtypes of
intimin, the first (
1) shared by the EHEC strains of clone 1, and the second (
2) represented by EHEC O111 and EPEC O127:H40 and O128:H8 (Oswald et al., 2000
). The intimin
2 detected by Oswald et al. (2000)
in EHEC strains of clone 2 probably corresponds to the
V intimin identified in human EHEC strain 95NR1 by Voss et al. (1998)
and in the CL559 ovine strain in our study. The two subtypes of
intimin may correspond to distinct clonal lineages of E. coli.
In this study, we developed a PCR test to identify the three different espB gene subtypes detected by analysis of espB published sequences (espB, espBß and espB
). Two types of espB gene were found among ovine and caprine strains by PCR, espB
and espBß. Using a multiplex PCR assay, China et al. (1999a
) also detected these two espB gene subtypes in bovine AEEC strains. Sequencing of PCR products from two selected strains with espB
(CL559 and CL617) and two with espBß (CK379 and CL398) confirmed the espB gene classification using the PCR assay. Only the ovine strains with an espB
gene hybridized with the espB probe derived from the espB gene of the human EPEC strain E2348/69. Previous studies have found that most AEEC strains isolated from diarrhoeic calves did not hybridize with the same espB probe as used in our study (Wieler et al., 1998
; China et al., 1998
). The PCR test developed in our study was found to be highly specific, and it will be useful for identification of espB gene subtypes in AEEC strains isolated from animals and humans.
In the strains studied, we found a close correlation between the intimin ß type and the espBß gene subtype. It has been shown that the strain RDEC-1 and the bovine EHEC strain 413-89-1 possessed identical espB genes (Ebel et al., 1996 ; Abe et al., 1997
). Analysis by multiple sequence alignment of published espB gene sequences revealed that other rabbit EPEC strains, and the swine strain 1390, also possessed espB genes identical to that of RDEC-1, and all of these strains contain ß intimin genes. Therefore, there seems to be a correlation between ß intimin and espBß subtype in AEEC strains of different origins. In addition to the eae and espB genes, the orfU gene of strain CK379 was also characterized, and it was identical to that of the prototype rabbit EPEC strain RDEC-1 (Abe et al., 1997
). Since eae and espB are among the most variable genes of the LEE (Elliot et al., 1998
; Perna et al., 1998
), it is possible that the entire LEE of strain CK379 was identical to that of RDEC-1.
The ovine strains with V intimin possessed an espB gene subtype (espB
) different from the espB
detected in the EHEC clone 1 prototype strain EDL933 possessing
intimin. The espB genes of EHEC clone 2 or animal AEEC strains producing
intimin have not yet been sequenced, so it is uncertain whether EHEC clone 2 strains possess an espB similar to the espB
defined in this study. However, the results of China et al. (1999a)
suggest that the association between intimin and espB gene subtypes described in this study is also present in bovine AEEC and in human EHEC strains. Using a multiplex PCR assay, these authors detected three different combinations: ß intimin with espBß,
intimin with espB
, and
intimin with espB
. The last combination was only detected in O157:H7 EHEC strains of clone 1, whereas the combination
intimin/espB
was present in human EHEC strains of serogroup O111 and in bovine AEEC strains of different serogroups. It is possible that at least some of these strains with the espB
gene subtype possess the
V intimin rather than the
intimin since the primers used for amplification of
intimin by China et al. (1999a
) do not allow distinction between
and
v intimin.
The ovine and caprine AEEC strains belonged to eight different serogroups (O2, O4, O26, O80, O91, O3, O153 and O163) or were untypable. It has been established that many serotypes of E. coli are genetically heterogeneous and, thus, the classification of strains based solely on O:H serotyping is not always indicative of genetic relatedness (Whittman et al., 1993 ). PFGE typing combined with genetic analysis of virulence determinants has been shown to be useful for evaluation of genetic relatedness of pathogenic E. coli strains (Arbeit et al., 1990
; Rios et al., 1999
). In this study, PFGE typing showed that two strains with
V intimin and espB
genes were genetically more distant than the strains with ß intimin and espBß genes (Fig. 3
). Among the latter, although a high degree of diversity was also found, a group of six (four caprine and two ovine) strains formed a cluster with 71% or greater similarity and, according to the criteria of Tenover et al. (1996)
, three of these epidemiologically unrelated isolates (the ovine CL258 and the caprine CK379 and CK513 strains) are possibly related genetically. Moreover, caprine strains with the ß intimin and espBß genes belonging to the same serogroup were also closely related, suggesting that they evolved from a common ancestor containing the LEE.
In cattle, both EPEC and EHEC strains are frequently isolated from diarrhoeic calves (Mainil et al., 1993 ; China et al., 1998
; Wieler et al., 1998
) and both types of strains have been shown to be pathogenic for neonatal calves (Moxley & Francis, 1986
; Fischer et al., 1994
). In contrast, the pathogenicity of AEEC strains for small ruminants has not been investigated, although the occurrence of AE lesions in these animals has been reported (Janke et al., 1989
; Duhamel et al., 1992
; Drolet et al., 1994
). AEEC strains isolated from diarrhoeic lambs and kids seem to be different from those isolated from diarrhoeic calves. None of the ovine and caprine eae-positive E. coli strains analysed in this study produced Stxs, and we have previously found that production of Stxs is not a common characteristic of E. coli strains isolated from diarrhoeic lambs and goat kids (Blanco et al., 1996
; Cid et al., 1996
).
Most of the ovine and caprine AEEC strains characterized in this study possessed the ß intimin gene. This intimin subtype is the most frequent subtype among EPEC strains isolated from diarrhoeic calves (China et al., 1999b ), and it is also characteristic of rabbit EPEC (Agin & Wolf, 1997
; Adu-Bobie et al., 1998
). Ovine and caprine strains with ß intimin were able to induce AE lesions in the rabbit ligated ileal loop model and they did not produce Stxs. Thus, these ovine and caprine AEEC strains should be classified as EPEC. It is possible that some of these strains, especially those of serogroup O26, which has been shown to be associated with enteric disease in calves and humans (China et al., 1999b
; Mainil et al., 1993
; Nataro & Kaper, 1998
), are pathogenic for lambs and kids. The association of the ovine AEEC strains possessing intimin
V with neonatal diarrhoea appears less likely. The isolation rate from diarrhoeic lambs is low, and it has been shown that intimin
is more frequent in EPEC strains isolated from healthy cattle than in EPEC isolated from diarrhoeic calves (China et al., 1999b
). Intimin
is characteristic of human EHEC strains, which cause HC and HUS (Adu-Bobie et al. 1998
; MGraw et al., 1999)
, and adult sheep are reservoirs of strains pathogenic for humans (Beutin et al., 1996
; Kuvda et al., 1996
). Although lambs could also be a reservoir of strains pathogenic for humans, the AEEC with the
V intimin subtype did not produce Stxs. We found that these strains possessed a
V intimin, which differed from
intimin of the human O157:H7 EHEC. This
V intimin was present in the human O111:H EHEC strain 95NR1 isolated from patients with enteric disease or HUS in an outbreak of disease caused by contaminated fermented sausage (Voss et al., 1998
). It is possible that the ovine AEEC strains with
V intimin characterized in this study are normal inhabitants of the gut of sheep. Further studies to investigate the distribution of intimin subtypes among E. coli strains isolated from diarrhoeic and healthy small ruminants, as well as experimental studies of pathogenicity in lambs and kids, are needed to establish the possible role of AEEC strains in neonatal diarrhoea of small ruminants.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adu-Bobie, J., Frankel, G., Bain, C., Goncalves, A. G., Trabulsi, L. R., Douce, G., Knutton, S. & Dougan, G. (1998). Detection of intimins alpha, beta, gamma, and delta, four intimin derivatives expressed by attaching and effacing microbial pathogens. J Clin Microbiol 36, 662-668.
Agin, T. S. & Wolf, M. K. (1997). Identification of a family of intimins common to Escherichia coli causing attaching-effacing lesions in rabbits, humans, and swine. Infect Immun 65, 320-326.[Abstract]
Agin, T. S., Cantey, J. R., Boedeker, E. C. & Wolf, M. K. (1996). Characterization of the eaeA gene from rabbit enteropathogenic Escherichia coli strain RDEC-1 and comparison to other eaeA genes from bacteria that cause attaching-effacing lesions. FEMS Microbiol Lett 144, 249-258.[Medline]
Albert, M. J., Faruque, S. M., Ansaruzzaman, M., Islam, M. M., Haider, K., Alam, L., Kabir, I. & Robins-Browne, R. (1992). Sharing of virulence-associated properties at the phenotypic and genetic levels between enteropathogenic Escherichia coli and Hafnia alvei. J Med Microbiol 37, 310-314.[Abstract]
An, H., Fairbrother, J. M., Dubreuil, J. D. & Harel, J. (1997). Cloning and characterization of the eae gene from a dog attaching and effacing Escherichia coli strain 4221. FEMS Microbiol Lett 148, 239-245.[Medline]
Arbeit, R. D., Arthur, M., Dunn, R., Kim, C., Selander, R. K. & Goldstein, R. (1990). Resolution of recent evolutionary divergence among Escherichia coli from related lineages: the application of pulsed field electrophoresis to molecular epidemiology. J Infect Dis 161, 230-235.[Medline]
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1991). Current Protocols in Molecular Biology. New York: Wiley.
Beaudry, M., Zhu, C., Fairbrother, J. M. & Harel, J. (1996). Genotypic and phenotypic characterization of Escherichia coli isolates from dogs manifesting attaching and effacing lesions. J Clin Microbiol 34, 144-148.[Abstract]
Beutin, L., Geiger, D., Zimmermann, S. & Karch, H. (1996). Virulence markers of shiga-like toxin-producing Escherichia coli strains originating from healthy domestic animals of different species. J Clin Microbiol 33, 631-635.[Abstract]
Blanco, J., Cid, D., Blanco, J. E., Blanco, M., Ruiz Santa Quiteria, J. A. & De la Fuente, R. (1996). Serogroups, toxins and antibiotic resistance of Escherichia coli strains isolated from diarrhoeic lambs in Spain. Vet Microbiol 49, 209-217.[Medline]
Broes, A., Drolet, R., Jacques, M., Fairbrother, J. M. & Johnson, W. M. (1988). Natural infection with an attaching and effacing Escherichia coli in a diarrheic puppy. Can J Vet Res 52, 280-282.[Medline]
Cantey, J. R. & Blake, R. K. (1977). Diarrhoea due to Escherichia coli in the rabbit: a novel mechanism. J Infect Dis 135, 454-462.[Medline]
China, B., Pirson, V. & Mainil, J. (1998). Prevalence and molecular typing of attaching and effacing Escherichia coli among calf populations in Belgium. Vet Microbiol 63, 249-259.[Medline]
China, B., Goffaux, F., Pirson, V. & Mainil, J. (1999a). Comparison of eae, tir, espA, and espB genes of bovine and human attaching and effacing Escherichia coli by multiplex polymerase chain reaction. FEMS Microbiol Lett 178, 177-182.[Medline]
China, B., Jacquemin, E., Devrin, A. C., Pirson, V. & Mainil, J. (1999b). Heterogeneity of the eae genes in attaching/effacing Escherichia coli from cattle: comparison with human strains. Res Microbiol 150, 323-332.[Medline]
Cid, D., Blanco, M., Blanco, J. E., Ruiz Santa Quiteria, J. A., De la Fuente, R. & Blanco, J. (1996). Serogroups, toxins and antibiotic resistance of Escherichia coli strains isolated from diarrhoeic goat kids in Spain. Vet Microbiol 53, 349-354.[Medline]
Donnenberg, M. S., Yu, J. & Kaper, J. B. (1993). A second chromosomal gene necessary for intimate attachment of enteropathogenic Escherichia coli to epithelial cells. J Bacteriol 175, 4670-4680.[Abstract]
Drolet, R., Fairbrother, J. M. & Vaillancourt, D. (1994). Attaching and effacing Escherichia coli in a goat with diarrhoea. Can Vet J 35, 122-123.
Duhamel, G. E., Moxley, R. A., Maddox, C. W. & Erickson, E. D. (1992). Enteric infection of a goat with enterohemorrhagic Escherichia coli (O103:H2). J Vet Diagn Invest 4, 197-200.[Medline]
Ebel, F., Deibel, C., Kresse, A. U., Guzman, C. A. & Chakraborty, T. (1996). Temperature- and medium-dependent secretion of proteins by shiga toxin-producing Escherichia coli. Infect Immun 64, 4472-4479.[Abstract]
Elliot, S. J., Wainwright, L. A., McDaniel, T. K., Jarvis, K. G., Deng, Y. K., Lai, L. C., McNamara, B. P., Donnenberg, M. S. & Kaper, J. B. (1998). The complete sequence of the locus of enterocyte effacement (LEE) from enteropathogenic Escherichia coli E2348/69. Mol Microbiol 28, 1-4.[Medline]
Fischer, J., Maddox, C., Moxley, R., Kinden, D. & Miller, M. (1994). Pathogenicity of a bovine attaching effacing Escherichia coli isolated lacking Shiga-like toxins. Am J Vet Res 55, 991-999.[Medline]
Foubister, V., Rossenshine, I., Donnenberg, M. S. & Finlay, B. B. (1994). The eaeB of enteropathogenic Escherichia coli is necessary for signal transduction in epithelial cells. Infect Immun 62, 3038-3040.[Abstract]
Frankel, G., Candy, D. C. A., Everest, P. & Dougan, G. (1994). Characterization of the C-terminal domains of intimin-like proteins of enteropathogenic and enterohemorrhagic Escherichia coli, Citrobacter freundii, and Hafnia alvei. Infect Immun 62, 1835-1842.[Abstract]
Frankel, G., Philips, A. D., Rosenshine, I., Dougan, G., Kaper, J. B. & Knutton, S. (1998). Enteropathogenic and enterohaemorrhagic Escherichia coli: more subversive elements. Mol Microbiol 30, 911-921.[Medline]
Gannon, V. P. J., Rashed, M., King, R. K. & Thomas, E. J. G. (1993). Detection and characterization of the eae gene of shiga-like toxin producing Escherichia coli using polymerase chain reaction. J Clin Microbiol 31, 1268-1274.[Abstract]
Griffin, P. M. & Tauxe, R. V. (1991). The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol Rev 13, 60-98.[Medline]
Higgins, D. G. & Sharp, P. M. (1988). CLUSTAL: a package for performing multiple sequence alignments on a microcomputer. Gene 73, 237-244.[Medline]
Janke, B. H., Francis, D. H., Collins, J. E., Libal, M. C., Zeman, K. H. & Johnson, D. D. (1989). Attaching and effacing Escherichia coli infections in calves, pigs, lambs, and dogs. J Vet Diagn Invest 1, 6-11.[Medline]
Jerse, A. E., Yu, J., Tall, B. D. & Kaper, J. B (1990). A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc Natl Acad Sci USA 87, 7839-7843.[Abstract]
Kudva, I. T., Hatfield, P. G. & Hovde, C. J. (1996). Escherichia coli O157:H7 in microbial flora of sheep. J Clin Microbiol 34, 431-433.[Abstract]
McDaniel, T. K & Kaper, J. B. (1997). A cloned pathogenicity island from enteropathogenic Escherichia coli confers the attaching and effacing phenotype on E. coli K-12. Mol Microbiol 23, 399-407.[Medline]
McDaniel, T. K., Jarvis, K. G., Donnenberg, M. S. & Kaper, J. B. (1995). A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. Proc Natl Acad Sci USA 92, 1664-1668.[Abstract]
McGraw, E. A., Li, J., Selander, R. K. & Whittam, T. S. (1999). Molecular evolution and mosaic structure of , ß, and
intimins of pathogenic Escherichia coli. Mol Biol Evol 16, 12-22.[Abstract]
Mainil, J. G., Jacquemin, E. R., Kaeckenbeeck, A. E. & Pohl, P. H. (1993). Association between the effacing (eae) gene and the Shiga-like toxin encoding genes in Escherichia coli isolated from cattle. Am J Vet Res 54, 1064-1068.[Medline]
Milon, A., Oswald, E. & De Rycke, J. (1999). Rabbit EPEC: a model for the study of enteropathogenic Escherichia coli. Vet Res 30, 203-219.[Medline]
Moon, H. W., Whipp, S. C., Argenzio, R. A., Levine, M. M. & Gianella, R. A. (1983). Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infect Immun 39, 1340-1351.
Moxley, R. A. & Francis, D. H. (1986). Natural and experimental infection with an attaching and effacing strain of Escherichia coli in calves. Infect Immun 53, 339-346.[Medline]
Nataro, J. P. & Kaper, J. B. (1998). Diarrhoeagenic Escherichia coli. Clin Microbiol Rev 11, 142-201.
Oswald, E., Schmidt, H., Morabito, S., Karch, H., Marches, O. & Caprioli, A. (2000). Typing of intimin genes in human and animal enterohemorrhagic and enteropathogenic Escherichia coli: characterization of a new intimin variant. Infect Immun 68, 64-71.
Perna, N. T., Mayhew, G. F., Posfai, G., Elliott, S., Donnenberg, M. S., Kaper, J. B. & Blattner, F. R. (1998). Molecular evolution of a pathogenicicty island from enterohemorrhagic Escherichia coli O157:H7. Infect Immun 66, 3810-3817.
Rios, M., Prado, V., Trucksis, M., Arellano, C., Borie, C., Alexandre, M., Fica, A. & Levine, M. M. (1999). Clonal diversity of Chilean isolates of enterohemorrhagic Escherichia coli from patients with hemolytic-uremic syndrome, asymptomatic subjects, animal reservoirs, and food products. J Clin Microbiol 37, 778-780.
Schauer, D. B. & Falkow, S. (1993). Attaching and effacing locus of a Citrobacter freundii biotype 4280 that causes transmissible murine colonic hyperplasia. Infect Immun 61, 2486-2492.[Abstract]
Smith, C. L. & Cantor, C. R. (1987). Purification, specific fragmentation, and separation of large DNA molecules. Methods Enzymol 55, 449-467.
Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, P. A., Murray, B. E., Persing, D. H. & Swaminathan, B. (1996). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis, criteria for bacterial strain typing. J Clin Microbiol 33, 2233-2239.
Voss, E., Paton, A. W., Manning, P. A. & Paton, J. C. (1998). Molecular analysis of shiga toxigenic Escherichia coli O111:H proteins which react with sera from patients with hemolytic-uremic syndrome. Infect Immun 66, 1467-1472.
Wells, J. G., Shipman, L. D., Greene, K. D. & 10 other authors (1991). Isolation of Escherichia coli serotype O157:H7 and other Shiga-like-toxin-producing E. coli from dairy cattle. J Clin Microbiol 29, 985989.[Medline]
Whitman, T. S., Wolfe, M. L., Wachsmuth, I. K., Ørskov, F., Ørskov, I. & Wilson, R. A. (1993). Clonal relationships among Escherichia coli strains that cause hemorrhagic colitis and infantile diarrhoea. Infect Immun 61, 16-19.
Wieler, L. H., Schwanitz, A., Vieler, E., Busse, B., Steinruck, H., Kaper, J. B. & Baljer, G. (1998). Virulence properties of Shiga toxin-producing Escherichia coli (STEC) strains of serogroup O118, a major group of STEC pathogens in calves. J Clin Microbiol 36, 1604-1607.
Yu, J. & Kaper, J. B. (1992). Cloning and characterization of the eae gene of enterohaemorrhagic Escherichia coli O157:H7. Mol Microbiol 6, 411-417.[Medline]
Zhu, C., Harel, J., Jacques, M., Desautels, C., Donnenberg, M. S., Beaudry, M. & Fairbrother, J. M. (1994). Virulence properties of attaching-effacing activity of Escherichia coli O45 from swine postweaning diarrhoea. Infect Immun 62, 4153-4159.[Abstract]
Received 17 November 2000;
revised 6 April 2001;
accepted 12 April 2001.