Rapid Detection of Virulence-Associated Genes in Avian Pathogenic Escherichia coli by Multiplex Polymerase Chain Reaction

Abstract

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.

Molecular Characterization and Antimicrobial Resistance of Uropathogenic Escherichia coli

“…List of primers used for detection of the virulence genes, and antibiotic resistance genes are shown in Table 1 (4,5,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). Table 2 presents PCR conditions.…”

Genotypic Analyses of Escherichia coli Isolated from Chickens with Colibacillosis and Apparently Healthy Chickens in Japan

“…Some of these traits are encoded chromosomally (11,15): fimC and papC responsible for adhesion factors F1 and P fimbriae, respectively, irp2 and fyuA for iron uptake systems, astA and vat for heat-stable toxin and vacuolating autotransporter toxin, respectively (7). Others are encoded in a plasmid termed "ColV plasmid" (12,20): colV for colicin V production, iss implicated in increased serum survival, tsh encoding temperature-sensitive hemagglutinin, and iucD encoding aerobactin secretions.…”

“…Recent genetic approaches have suggested that a single or combination of these genes is prevalent in APEC. For instance, Dozois et al (5) suggested that tsh contributes most to APCE pathogenicity in the early stages of infection, whereas Ewers et al (6,7) reported that some virulence genes, especially fimC, fyuA, irp2, iss, and iucD are highly prevalent in APEC strains. Most recently, Johnson et al (12) claimed that a region of the ColV plasmid including iss and tsh is highly prevalent among APEC.…”

Similaridade genética entre APEC e cepas de Escherichia coli isoladas de Guaruba guarouba em um estudo com psitacídeos hígidos de cativeiro

“…Two strains, G12 and G13, possessed four or more virulence genes and therefore are considered APEC (Figure 3). G13 had the highly pathogenic phenotype containing iuc, iss is not well understood 13,12,20 The prevalence of the papC gene, which encodes for P fimbriae, may vary largely among APEC isolates, but it is very common in human uropathogenic E.…”