Identification of Avian Pathogenic Escherichia Coli Genes Required for Carriage and Virulence in Poultry

Avian pathogenic E. coli (APEC) cause colibacillosis, a severe systemic disease of profound economic and welfare importance for the poultry industry world-wide. Birds may be prediposed to colibacillosis by viral infection and other factors, however APEC are widely agreed to be a key control point and a pressing need exists for cross-protective vaccines. However, the bacterial factors mediating carriage and virulence of APEC in poultry are poorly characterised. Furthermore, colibacillosis is caused by diverse APEC serogroups, often O1, O2 and O78. To date, few genetic traits have been identified that delineate virulent and avirulent APEC and the role of such genes in the pathogenesis in food-producing animals has rarely been tested.<P>
We propose to employ signature-tagged mutagenesis to identify APEC genes mediating respiratory tract colonisation and systemic virulence in turkeys. A bank of tagged-mini-Tn5 APEC O78 mutants will be screened by intra-tracheal inoculation of turkeys predisposed by prior inoculation with avian pneumovirus. The composition of output pools from the respiratory tract and systemic sites will be compared to the input and attenuated mutants identified by negative selection. The location of Tn-insertion sites will be determined by subcloning and sequencing. <P>
We will then survey the prevalence of APEC virulence factors among a collection of diverse APEC strains available at IAH. The role of selected conserved virulence factors, in particular those predicted to be surface-exposed, will be confirmed by analysis of non-polar deletion mutants. The basis of attenuation will be assessed in vitro by quantifying the ability of deletion and/or Tn-mutants to resist serum killing and adhere to and invade epithelial cells, relative to parent and trans-complemented strains.<P>
It is envisaged that selected conserved virulence factors will then be cloned, expressed and purified and their ability to induce cross-protective immunity against colibacillosis tested.