Identification of avian pathogenic Escherichia coli genes required for carriage and virulence in poultry

Escherichia coli is typically a harmless inhabitant of intestines of animals and humans. However, some E. coli strains possess traits that equip them with the potential to cause disease. Pathogenic E. coli are a major cause of diarrhoeal disease world-wide, but some strains can cause extra-intestinal disorders such as urinary tract infections, septicaemia and meningitis. The bacterial factors that enable E. coli to cross mucosal barriers and persist at systemic sites are poorly understood. One such group of extra-intestinal pathogens comprises avian pathogenic E. coli (APEC), which cause severe systemic disease in birds. APEC are a major economic and welfare problem for the poultry industry world-wide. APEC affect birds of all ages and cause colibacillosis which can result in sudden death, reduced egg hatching and condemnation of carcasses at slaughter. Whilst birds may be prediposed to colibacillosis by viral infection, stress and environmental factors, it is widely agreed that the problem could be alleviated by targeting APEC. However, the bacterial factors that influence the carriage and virulence of APEC in poultry are poorly understood. Furthermore, APEC isolated from poultry with colibacillosis are often unrelated and few conserved traits have been found to exist among virulent isolates. Antibiotic treatment is complicated by the lack of signs of disease prior to death and concern about the evolution and spread of new antibiotic resistances. Whilst vaccines against APEC have been developed, these are of limited value since they protect birds only against the strain from which they were derived. There is a pressing need to develop broadly cross-protective APEC vaccines. We believe that by dissecting the mechanisms by which APEC colonise the respiratory tract of poultry and spread to systemic sites we can identify bacterial factors that may be suitable for inclusion into a vaccine. For such vaccines to be effective against all APEC, it will be necessary to confirm that the factors are present in a wide variety of field APEC isolates. We propose to employ a technique called signature-tagged transposon mutagenesis (STM) to identify APEC factors required for the induction of colibacillosis in turkeys. STM makes it possible to track the fate of uniquely tagged bacterial mutants during their interaction with the host. The composition of a pool of mutants recovered from inoculated birds can be compared to the composition of the inoculum and in this way, mutants unable to survive in the host identified (Part 1. Fig. 1). The factors disrupted in such weakened mutants can then be identified. We have successfully used STM to identify genes of Salmonella and E. coli O157 required for colonisation of farm animals. With the information gleaned from such studies, we have then sought to develop vaccines for the control of these agents. In recognition of the value of the proposed studies to the poultry industry the British Poultry Council and Aviagen Turkeys (formerly British United Turkeys Ltd) have agreed to support the project in cash and in kind, respectively. The aims of the project are: 1. To identify factors that enable APEC to colonise the respiratory tract of turkeys and spread to other organs. 2. To confirm that such factors are found in a wide variety of field APEC isolates. 3. To confirm that such factors play an important role in disease by disrupting them and characterising the mutant strains in turkeys. The reasons for the loss of virulence will then be assessed in a number of laboratory-based experiments. 4. To determine whether purified APEC virulence factors can induce protection against APEC infection.

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. 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. 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. 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.