Research/Improving control of colibacillosis using genomic information
From Poultry Hub
Source: Rocky Mountain Laboratories, NIAID, NIH
Title: Improving control of colibacillosis using genomic information
Project Leader: Marc Marenda - at University of Melbourne funded by Poultry CRC (Project 07-08)
Duration of Project: 1 July 2007 to 31 December 2009
Contents |
Project overview
Colibacillosis is a common disease problem in broiler flocks and in recent years has been increasingly seen in broiler breeders, as well as layers and layers breeders. Only specific strains of E. coli can cause disease and these strains carry a large virulence plasmid enabling them to colonise and persist in the respiratory tract of birds. We have identified the regions of these plasmids that play a significant role in virulence, and have developed and applied a diagnostic tool for confirming that isolates of E. coli are likely to be virulent. However this tool is not suitable for large scale screening. Some of the proteins involved in virulence are located on the external surface of the bacterial cell (generally in the outer membrane), and thus are likely to induce protective antibody, while others are located inside the bacterial cell (particularly in the cytoplasmic membrane), and thus are unlikely to induce protective immunity, even though they contribute to virulence. This information can be used to develop novel vaccine strains of APEC that have increased immunogenicity but are still attenuated.
Ultimately, the benefits would be a reduced risk of serious outbreaks of colibacillosis and reduced need for antimicrobial therapy to control outbreaks.
Project objectives
- This project aims to aid development of novel vaccines to control colibacillosis in broilers and broiler breeders. Researchers are also seeking the development of a novel method for screening poultry sheds for high concentrations of avian pathogenic Escherichia coli, thereby identifying farms at risk and determining factors that may increase risks on farms.
Project progress
A live attenuated vaccine is developed by inactivating iron uptake systems as the ability for a bacterium to source iron is essential for survival within the host environment. A diagnostic test designed to assess APEC levels in air samples by detecting specific virulence genes is planned.
Progress to date: Vaccine development 1. Targets for mutation in APEC include: tonB, fur, ftn, and feoB. Single, double and triple mutant combinations are designed. The tonB mutant will be impaired in iron uptake mediated by Salmochelin (iro), and Aerobactin (iut) siderophores. The ftn mutant will be impaired in iron storage capacity. The fur mutant will over-express the iron siderophore receptors at the surface of the cells. 2. The cloning and inactivation strategy for generating each mutant has been designed. 3. PCR primers were designed for initial amplification and subsequent cloning of each of the target genes. 4. PCR protocols have been optimized for tonB, fur, ftn and feoB. The PCR products of tonB and fur have been successfully cloned into vector pGEM-T (Promega) and confirmed by RFLP and DNA sequence characterization. The cloning of ftn, and feoB is ongoing. 5. A temperature sensitive vector (pKD46) to be used to deliver inactivated target genes to facilitate selection of chromosomal knock-out mutants is under finalisation. An alternative mutagenesis strategy (Lambda Red) is to be run in parallel and the specific primers have been designed.
Detection system development 1. Target genes have been selected based on identification of virulence associated genes described in the literature. The four target genes are iroB, iss, iucA and sitA. All genes are located on the pVM01 virulence plasmid of APEC strain E3 and are common to other APEC virulence plasmids 2. PCR primers were designed for amplification of the four target genes and the PCRs are currently being optimised.
