Avian pathogenic Escherichia coli (APEC) is an economically important respiratory pathogen of chickens worldwide. Factors previously associated with the virulence of APEC include adhesins, iron-scavenging mechanisms, the production of colicin V (ColV), serum resistance, and temperature-sensitive hemagglutination, but virulence has generally been assessed by parenteral inoculation, which does not replicate the normal respiratory route of infection. A large plasmid, pVM01, is essential for virulence in APEC strain E3 in chickens after aerosol exposure. Here we establish the size of pVM01 to be approximately 160 kb and show that the putative virulence genes iss (increased serum survival) and tsh (temperature-sensitive hemagglutinin) and the aerobactin operon are on the plasmid. These genes were not clustered on pVM01 but, rather, were each located in quite distinct regions. Examination of APEC strains with defined levels of respiratory pathogenicity after aerosol exposure showed that both the aerobactin operon and iss were associated with high levels of virulence in APEC but that the possession of either gene was sufficient for intermediate levels of virulence. In constrast, the presence of tsh was not necessary for high levels of virulence. Thus, both the aerobactin operon and iss are associated with virulence in APEC after exposure by the natural route of infection. The similarities between APEC and extraintestinal E. coli infection in other species suggests that they may be useful models for definition of the role of these virulence genes and of other novel virulence genes that may be located on their virulence plasmids.

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 DH5␣. 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 DH5␣. 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 DH5␣. 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.Lower-respiratory-tract infections are the most common disease syndrome associated with Escherichia coli in poultry. Although in extreme cases mortality can be over 20%, it is the high morbidity and associated loss in productivity which is responsible for the greatest economic loss (31).There is evidence to suggest that virulent strains of avian E. coli belong to a limited number of clone complexes (60,61) and that particular clones may be specific to particular manifestations of E. coli infection (47). A number of characteristics have been associated with virulence in avian E. coli, including colicin V production (22, 23, 50, 59), adhesins (17, 18, 20, 32, 46, 67), serum resistance (21,37,47,48,59,66), and iron sequestering (37,41,43,47,59,63), but specific attempts to establish the requirements of these factors for virulence are limited. While initial studies of avian E. coli led to the conclusion that certain serogroups, O1, O2, and O78 in particular, were more commonly associated with colibacillosis (27,28,29,33,35,36), the most prevalent serotypes vary with geographic location and many isolates are untypeable (3,6,12,19,38,45).Although plasmid-encoded virulence genes have been well investigated and described fo...

We have developed a reliable aerosol challenge method for reproduction of avian colibacillosis. This method involves intranasal administration of infectious bronchitis virus (IBV) to 1-day-old birds followed by three aerosol administrations of E. coli at 3 to 4-day intervals. In four separate experiments there was no significant difference in virulence for a single isolate of E. coli (E3) whereas four other field isolates of E. coli ranged from highly virulent to avirulent. We also observed that the dominant E. coli population isolated from 1-day-old birds in a flock of broiler breeders was highly virulent but that the dominant E. coli population isolated from birds in the same flock 2 weeks later was avirulent. The dominant E. coli population isolated from 1-day-old birds in this flock was sensitive to all of the antimicrobial agents tested whereas the dominant E. coli population in the 2-week-old birds was multiresistant. A previous study has shown that it is likely that the changed resistance pattern of the dominant E. coli population in the flock was linked to the administration of antimicrobial agents.

Escherichia coli isolates were obtained from two flocks of broiler breeder chickens beginning at 1 day old. Antimicrobial sensitivities were determined and isolates were grouped on the basis of their antibiogram patterns. In both flocks there was an initial dramatic shift in the antimicrobial resistance patterns of the E. coli isolates which changed from sensitive to multiresistant. Both flocks were given spectinomycin in drinking water during the first 3 days on the rearer farm. Statistical analysis of the E. coli isolates in Flock 2 revealed that there was a significant difference between E. coli obtained from 1-day-old birds and those obtained from 1-week-old birds in terms of the proportion of isolates that were resistant to spectinomycin. It is possible that the use of spectinomycin selected for resistant E. coli isolates which became dominant in the flocks soon after treatment. There was a strong association between resistance to spectinomycin and resistance to other antimicrobial agents, in particular, sulphafurazole and chloramphenicol.