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Based on recently published prevalence data of virulence-associated factors in avian pathogenic Escherichia coli (APEC) and their roles in the pathogenesis of colibacillosis, we developed a multiplex polymerase chain reaction (PCR) as a molecular tool supplementing current diagnostic schemes that mainly rely on serological examination of strains isolated from diseased birds. Multiple isolates of E. coli from clinical cases of colibacillosis known to possess different combinations of eight genes were used as sources of template DNA to develop the multiplex PCR protocol, targeting genes for P-fimbriae (papC), aerobactin (iucD), iron-repressible protein (irp2), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), enteroaggregative toxin (astA), increased serum survival protein (iss), and colicin V plasmid operon genes (cva/cvi). In order to verify the usefulness of this diagnostic tool, E. coli strains isolated from fecal samples of clinically healthy chickens were also included in this study, as were uropathogenic (UPEC), necrotoxigenic, and diarrhegenic E. coli strains. The application of the multiplex PCR protocol to 14 E. coli strains isolated from septicemic poultry showed that these strains harbored four to eight of the genes mentioned above. In contrast, those isolates that have been shown to be nonpathogenic for 5-wk-old chickens possessed either none or, at most, three of these genes. We found only one enterohemorrhagic (EHEC), one enteropathogenic (EPEC), and two enterotoxic (ETEC) E. coli strains positive for irp2, and another two ETEC strains positive for astA. As expected, UPEC isolates yielded different combinations of the genes iss, papC, iucD, irp2, and a sequence similar to vat. However, neither the colicin V operon genes cva/cvi nor tsh were amplified in UPEC isolates. The multiplex PCR results were compared with those obtained by DNA-DNA-hybridization analyses to validate the specificity of oligonucleotide primers, and the protocol was concluded to be a useful, sensitive, and rapid assay system to detect avian pathogenic E. coli and differentiate them from nonpathogenic strains and those belonging to other pathotypes.

Long-lasting allergen treatment is the most efficient therapy in alopecia areata (AA). The underlying mechanism is unknown. We here asked whether treatment with a contact sensitizer influences leukocyte migration such that dendritic cell (DC) migration or the recruitment of activated T-cells towards the skin become hampered. Allergen treatment of AA mice was not accompanied by a decrease in skin-infiltrating leukocytes or draining lymph node cells (LNC). However, the distribution of leukocyte subsets was changed with a dominance of monocytes in the skin and a reduced percentage of DCs in draining nodes. Chemokine and chemokine receptor expression in skin and draining nodes was strikingly increased and LNC from untreated and allergen-treated AA mice showed high migratory activity in vitro and readily homed in draining nodes and skin after intravenous injection. However, FITC labelling of the skin and subcutaneous transfer of dye-labelled DC revealed that allergen treatment created a chemokine milieu severely hampering DC migration from the skin towards the draining node. An allergic eczema-induced reduction in DC migration and antigen transfer could well contribute to insufficient T-cell activation and the recovery of hair follicle in AA and possibly be of relevance for other skin-related autoimmune diseases.

We describe a locus of enterocyte effacement (LEE) which is part of a new pathogenicity island (PAI) detected in the bovine Shiga toxin-producing Escherichia coli strain RW1374 (O103:H2). This PAI is at least 80 kb in size and inserted in the vicinity of the pheV tRNA gene at 67 min of the E. coli chromosome. Furthermore, the PAI differs from the previously described LEEs by unique flanking regions at both sides, which harbor one copy each of an insertion element in an inverted orientation that is 96% identical to insertion site (IS)629. In addition, a 5-kb PAI-specific sequence downstream of the LEE core region and adjacent to the E. coli K12 region is duplicated upstream of the LEE core region as well. The duplicated sequences are more than 80% identical to each other and consist partially of prophage sequences.