Zoonotic infections infections in livestock and the risk to public health - Poster Abstract

A quantitative PCR assay for Escherichia coli O157:H7 in bovine gut contents

Dr N Parham et al

Beef products, from carcasses contaminated with faeces, appear to be one of the main sources for human infection with Escherichia coli serotype O157:H7. This organism is prevalent in cattle, with asymptomatic bovines shedding the bacterium intermittently throughout their life. However, clinical disease in cattle is rare and the precise molecular events associated with colonisation, persistence of infection/carriage and clearance of carriage have not been determined. Hence, it is not known whether true colonisation actually occurs and why bovine gut contents should represent such a significant risk for infection.

To address the question of colonisation, we are examining the numbers of O157 organisms throughout the gastro-intestinal tract, in the lumen and attached to the mucosa, of carrier animals. For this purpose, a specific and sensitive quantitative test is required; hence, a PCR-based approach seemed to be most appropriate.

Real-time PCR, using the TaqMan system (PE Applied Biosystems, Warrington, UK), is a highly specific, sensitive, rapid and reproducible technique for quantification of target sequences. PCR amplification of the target sequence is continuously monitored throughout thermal cycling by detection of fluorescent light. A probe, which contains a 5'-fluorescent reporter dye, anneals to the target sequence but only a very low level of emission occurs due to the presence of a 3'-quencher dye. As PCR progresses annealed probe is digested due to the 5'-exonuclease activity of the Taq DNA polymerase. Hence, reporter dye becomes separated from the quencher with concomitant increase in light emission. The point at which light intensity exceeds a predetermined threshold (CT) is proportional to the initial level of target sequence in the sample. The CT is determined and compared to a calibration graph for calculation of starting copy number in the test sample.

TaqMan primers and probe were designed for a specific assay against the rfbE gene (encoding perosamine synthetase) of E. coli O157:H7. This was based on the work of Paton and Paton (1998), who developed a conventional PCR assay to this target and validated against a range of VT-positive and VT-negative O157 strains, other STEC and EPEC. A BLAST search against the rfbE gene sequence (accession S83460) also indicated very low levels of conservation among bacterial species. Our TaqMan assay was further validated against a VT-negative O157:H7 (strain 6252, kindly provided by Dr. F. Thomson-Carter), a range of other E. coli serotypes (including O55:H7 and H-) and bacterial species that are known to bind to immunomagnetic beads, both by specific cross-reaction and by non-specific attachment to the bead surface (e.g. Escherichia, Pseudomonas and Proteus species).

O157-negative bovine faeces was spiked with strain 6252 to give a range of concentrations from approximately 108 to 1 CFU g-1. Target cells were separated from faecal suspensions using anti-E. coli O157:H7 immunomagnetic beads (Dynal, Bromborough, UK) and DNA collected by phenol:chloroform extraction. Initial results were promising with detection levels of about 102 CFU g-1, however, we aim to improve sensitivity of the assay.

However, two E. coli isolates (one bovine and one feline) recovered during diagnostic bacteriological examination were tested by the TaqMan assay. These isolates were found to be positive; they were not O157 but novel serotypes. Hence other potential PCR targets are now being investigated.

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