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Quantification of E.coli 0157:H7 Virulence Factors In Vivo using Real-Time RT-PCR and In Situ RT-PCR. - OZ0704

Description
Various virulence determinants have been identified by culturing Escherichia coli O157:H7 in vitro and in studies using mutated organisms. However, whilst the role of some of these determinants in pathogenicity has been established, the precise molecular events associated with colonisation, persistence of infection/carriage and clearance of carriage have not been determined.

In this project technology of real-time, conventional PCR and RT-PCR will be applied to quantitate the number of organisms and the expression of mRNAs for various virulence factors produced by Escherichia coli O157:H7 organisms in vivo. This will be combined with in situ RT-PCR techniques to obtain a detailed understanding of microbial gene expression during colonisation/attachment/carriage in the bovine alimentary tract.

Thus modern molecular techniques will be used to obtain a clearer understanding of the distribution of infection and pathogenesis of Escherichia coli O157:H7 in cattle. This data would underpin future studies investigating in detail host responses in the carrier animal, relating these to microbial virulence factors and host-pathogen interactions. Development of these techniques for Escherichia coli O157:H7 could also form the basis of investigating host-pathogen interactions of other food borne zoonoses at the molecular level.
Objective
1. Develop and validate a RT-PCR technique to quantiitate expression of E. coli 0157:H7 virulence determinants in vivo.
i) design and synthesis Taq Man probes for putative virulence factors and other relevant sequences derived from the E. coli 0157:H7 genome.
ii) validate the probes using organisms grown in vitro.
iii) validate the probes using organsims grown in vitro
iii) quantitative expression of mRNA for these virulence factors in E. coli 0157:H7 shed in faeces.
iv) quantitative factor mRNAs of E.coli 0157:H7 organisms from washed, tissue homogenates of defined regions of the alimentary tract.

2. Develop and validate an in situ RT-PCR techique for E. coli 0157:H7 virulence deteminants
i) design and synthesisi the require PCR primers
ii) examine virulence factor expression in paraformaldehyde fixed, wax embedded tissue sections known from immunocytochemistry studies to contain E. coli 0157:H7 organisms.
iii) examine virulence factor expression in tissue sections in neighbouring regions of the alimentary tract
iv) develop a quantitative methodology for in situ RT-PCR
The above objectives will be addressed in: 1)known carrier adult cattle
2) Calves born by carrier cows to examine age-related factors in the extent of colonisation of the alimentary tract and any varience in virulence factor expression in these different categories of animal.
Project Documents
• Final Report : Quantification of E.Coli 0157:H7 virulence factors in vivo using real-time RT-PCR   (49k)
Time-Scale and Cost
From: 1999

To: 2002

Cost: £200,764
Contractor / Funded Organisations
University - Glasgow
Keywords
Animal Health              
Biotechnology              
E.coli O157              
Epidemiology              
GM Non-Food              
Plants and Animals              
Zoonoses              
Fields of Study
Animal Health