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Identification of colonisation factors of bovine EHEC strains using intestinal explants

The first step in the pathogenesis of most intestinal bacterial pathogens is the adherence to the cytoplasmic membrane of enterocytes via specific bacterial structures and proteins (= primary colonisation factors). This step is also often the basis of the host specificity of the bacterial pathogens. The identification of such primary colonisation factors could greatly help in the comparison of bovine and human strains identical by other properties. The general objectives of WP14 were to identify such adhesions present in bovine EHEC strains, to confirm their role in intestinal colonisation and in enteropathogenicity in calves and to look for the expression of these new adhesin(s) in human EHEC.

Major finding during the project:
Ex vivo and in vitro adherence assays.
- Intestinal explants experiments with bovine EPEC/EHEC strains. Intestinal explants experiments were performed using mucosal tissues from the colon obtained from 18-month-old cattle at the slaughterhouse. Explants were inoculated with two O26 EHEC strains and with an in vivo pathogenic O118 EHEC strain as positive control. This part of the work-package suffered a lot of problems regarding the short survival time of the intestinal explants (less than 5 hours), despite testing several variations in the culture conditions and parameters of the explants. No further work was therefore performed with explants and efforts were re-directed towards the development of an adherence assay on cells in culture.

- Adherence assay on MDBK (Madin Darby Bovine Kidney) cells with bovine EPEC/EHEC strains. The tests were performed with 64 strains of bovine origin belonging to the O5, O26, O103, O111, O118, O145 and O157 serogroups. With the exception of the O145 serogroup, the other strains presented a phenotype with various intensity of adherence: localised-like (LAL), diffuse (DA) and aggregative (AA) adherence patterns were observed irrespectively of the serotype or the pathotype of the strain. A quantitative assay on the MDBK cell line was also derived, by comparing adherent bovine EPEC/EHEC strains and negative control strains. In spite of some variations during the assay and of a significant level of non-specific adherence by the negative control strains, a one-log difference in the numbers of adherent bacteria was consistently observed between EPEC/EHEC and negative control strains. This assay is therefore suitable to test mutant strains.

- Adherence assay on primary intestinal cell cultures with bovine EPEC/EHEC strains. The adherence assay with the 64 strains of bovine origin belonging to the O5, O26, O103, O111, O118, O145 and O157 serogroups was also adapted to primary culture of bovine jejunocytes and colonocytes. The adherence patterns of most strains were similar to the ones on MDBK cells: localised-like (LAL), diffuse (DA) and aggregative (AA) adherence patterns.

Production and testing of mutants:
i) Presence of adhesin-encoding genes in bovine EPEC/EHEC strains. Following sequence alignments and gene probe derivation, the collection of bovine EPEC/EHEC strains was screened for the presence of putative adhesin-encoding genes (afa8, f17, afr2, clp, bfp, lpfA, lifA/efa1, iha). The positive hybridisation results can be summarised as follows: bfp-like gene was detected in human EPEC strains, but not in the other strains; lpfA-like gene was detected in all O157 and O145 EHEC, but not in the other serogroups; whereas lifA/efa1-like gene was detected in most non-O157 non-O145 EHEC; iha-like gene was present in several EPEC/EHEC too, irrespectively of the serotypes; and clpE-like gene (but not clpG) was present in O26 EPEC strains only.

- Production of mutants in putative adhesin-encoding genes of bovine EPEC/EHEC strains. In order to evaluate the relative implication of the putative adhesins encoded by the lifA/efa1, clpE and iha gene-like sequences in the adherence on MDBK cells of bovine EPEC/EHEC stains, a mutagenesis strategy by allelic exchange was followed. Two mutants in the lifA/efa1 and clpE genes were obtained from an O26 EPEC strain which show a rise (one log difference) in their adherence properties on MDBK cells and primary enterocyte cultures, but a decrease of adherence in the porcine and rabbit ligated loop assays (see also WP11). They are being further studied for the exact localisation and genetic consequence of the mutations.

- Experimental challenge of calves with mutants in putative adhesin-encoding genes of bovine EPEC/EHEC strains. Six calves were experimentally orally infected with deletion mutant strains of lifA/efa1, clpE or cif genes (see WP05 for cif gene), and two calves per strains. Preliminary results indicate a less clinically severe enteritis in those calves in comparison with those infected with the wild O26 strains.

Reported by

Faculté de Médecine Vétérinaire, Sart Tilman, Bât B43a
4000 LIEGE
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