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Pathogenicity and Medical Microbiology |
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
Attaching and effacing Escherichia coli (AEEC) strains isolated from diarrhoeic lambs and goat kids were characterized for intimin (eae) and EspB (espB) gene subtypes by PCR and sequencing, and for genetic relatedness by PFGE. Fifty (23 ovine and 27 caprine) AEEC strains of 398 (246 ovine and 152 caprine) analysed were detected by colony blot hybridization. These strains were epidemiologically unrelated since they were isolated from different outbreaks of neonatal diarrhoea over a long period. Ovine AEEC strains belonged to serogroups O2, O4, O26, O80, O91 or were untypable, and caprine strains belonged to serogroups O3, O153 and O163. Two intimin subtypes were detected among the ovine and caprine strains studied. Most of the strains (43/50) had the ß type intimin gene, but seven ovine strains possessed a variant 
type intimin gene (V). Analysis of deduced amino acid sequences of the eae gene revealed that the sequences of ß intimin of ovine and caprine strains were virtually identical to those of ß intimin of rabbit EPEC, human EPEC clone 2 and swine AEEC, whereas the V intimin present in seven ovine strains had 75–76% identity with intimin of human EHEC clone 1 strains, and 96% of identity with intimin of the human EHEC strain 95NR1 of serotype O111:H–. A PCR test was developed to identify the three different espB gene subtypes, espB of human EPEC clone 1 (espB
), espB of human EHEC clone 1 (espB) and espB of rabbit EPEC and human EPEC clone 2 (espBß). There was close correlation between the intimin ß type and the espBß gene subtype in the ovine and caprine AEEC strains. The seven ovine strains possessing the V intimin gene possessed the espB gene subtype. None of the strains studied possessed the espB gene found in human O157:H7 EHEC strains. PFGE analysis of genomic DNA of selected strains showed a great diversity among strains. Cluster analysis of PFGE patterns showed greater divergence between strains with the V intimin gene than between strains with the ß intimin gene. This study showed that most of the AEEC strains isolated from diarrhoeic lambs and goat kids possessed ß intimin and espB genes identical to those of rabbit EPEC, and they may be associated with enteric disease in small ruminants.
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)
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