Colonization of the respiratory tract by a virulent strain of avian Escherichia coli requires carriage of a conjugative plasmid

Abstract

The E3 strain of E. coli was isolated in an outbreak of respiratory disease in broiler chickens, and experimental aerosol exposure of chickens to this strain induced disease similar to that seen in the field. In order to establish whether the virulent phenotype of this strain was associated with carriage of particular plasmids, four plasmid-cured derivatives, each lacking two or more of the plasmids carried by the wild-type strain, were assessed for virulence. Virulence was found to be associated with one large plasmid, pVM01. Plasmid pVM01 was marked by introduction of the transposon TnphoA, carrying kanamycin resistance, and was then cloned by transformation of E. coli strain DH5alpha. The cloned plasmid was then reintroduced by conjugation into an avirulent plasmid-cured derivative of strain E3 which lacked pVM01. The conjugant was shown to be as virulent as the wild-type strain E3, establishing that this plasmid is required for virulence following aerosol exposure. This virulence plasmid conferred expression of a hydroxamate siderophore, but not colicins, on both strain E3 and strain DH5alpha. Carriage of this plasmid was required for strain E3 to colonize the respiratory tracts of chickens but was not necessary for colonization of the gastrointestinal tract. However, the virulence plasmid did not confer virulence, or the capacity to colonize the respiratory tract, on strain DH5alpha. Thus, these studies have established that infection of chickens with E. coli strain E3 by the respiratory route is dependent on carriage of a conjugative virulence plasmid, which confers the capacity to colonize specifically the respiratory tract and which also carries genes for expression of a hydroxymate siderophore. These findings will facilitate identification of the specific genes required for virulence in these pathogens.

FIG. 1

Comparison of the plasmid profiles of avian E. coli strain E3 and its plasmid-cured derivatives. Positions of plasmids pVM01 (01), pVM02 (02), pVM03 (03), and pVM04 (04) are indicated. Other bands of greater mobility are either smaller plasmids or different forms of the higher-molecular-weight plasmids. The positions of fragments of HindIII-digested phage lambda DNA, which were used as molecular size markers, are shown (M). This image was obtained using a Nikon Scantouch scanner and Adobe Photoshop.

FIG. 2

Plasmid profiles of selected E. coli strain E3/2.4 progeny following conjugation with DH5α/4 or DH5α/5 and selection in the presence of ampicillin and kanamycin. Positions of plasmids pVM01 (01), pVM02 (02), pVM03 (03), and pVM04 (04) are indicated in the parental E3 strain. Strains E3/2.4/1 and E3/2.4/6 (derived from DH5α/4) and strains E3/2.4/9 and E3/2.4/10 (derived from DH5α/5), all of which carried pVM01, pVM02, and pVM03, were chosen for further characterization. This image was obtained using a Nikon Scantouch scanner and Adobe Photoshop.