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Applied and Environmental Microbiology, April 2008, p. 2492-2504, Vol. 74, No. 8
0099-2240/08/$08.00+0 doi:10.1128/AEM.01242-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Real-Time PCR Approach for Detection of Environmental Sources of Campylobacter Strains Colonizing Broiler Flocks
Anne M. Ridley,1*
Vivien M. Allen,2
Meenaxi Sharma,1
Jill A. Harris,2 and
Diane G. Newell1
Veterinary Laboratories Agency, Addlestone, Surrey KT15 3NB, United Kingdom,1 Division of Food Animal Science, Department of Clinical Veterinary Science, University of Bristol, Langford, Bristol BS40 5DU, United Kingdom2
Received 5 June 2007/ Accepted 7 January 2008
Reducing colonization of poultry flocks by Campylobacter spp. is a key strategy in the control and prevention human campylobacteriosis. Horizontal transmission of campylobacters, from in and around the farm, is the presumed route of flock colonization. However, the identification and prioritization of sources are confounded by the ubiquitous nature of these organisms in the environment, their poor rates of recovery by standard culture methods, and the need for cost-effective and timely methods for strain-specific comparison. A real-time PCR screening test for the strain-specific detection of campylobacters in environmental samples has been developed to address this issue. To enable this approach, fluorescently labeled PCR oligonucleotide probes suitable for a LightCycler-based assay were designed to match a highly variable DNA segment within the flaA short variable region (SVR) of Campylobacter jejuni or C. coli. The capacity of such probes to provide strain-specific tools was investigated by using bacterial cultures and spiked and naturally contaminated poultry fecal and environmental samples. The sensitivity of two representative probes was estimated, by using two different C. jejuni strains, to be 1.3 x 102 to 3.7 x 102 CFU/ml in bacterial cultures and 6.6 x 102 CFU/ml in spiked fecal samples. The specificity of the SVR for C. jejuni and C. coli was confirmed by using a panel of strains comprising other Campylobacter species and naturally contaminated samples. The approach was field tested by sampling the environment and feces of chickens of two adjacently located poultry houses on a conventional broiler farm throughout the life of one flock. All environmental samples were enriched for 2 days, and then DNA was prepared and stored. Where feasible, campylobacter isolates were also recovered and stored for subsequent testing. A strain-specific probe based on the SVR of the strain isolated from the first positive chicken fecal sample was developed. This probe was then used to screen the stored environmental samples by real-time PCR. Pulsed-field gel electrophoresis was used to compare recovered environmental and fecal isolates to assess the specificity of the method. The results established the proof of principle that strain-specific probes, based on the SVR of flaA, can identify a flock-colonizing strain in DNA preparations from enriched environmental cultures. Such a novel strategy provides the opportunity to investigate the epidemiology of campylobacters in poultry flocks and allows targeted biosecurity interventions to be developed. The strategy may also have wider applications for the tracking of specific campylobacter strains in heavily contaminated environments.
* Corresponding author. Mailing address: Veterinary Laboratories Agency, Woodham Lane, Addlestone, Surrey KT15 3NB, United Kingdom. Phone: 44 1932 357489. Fax: 44 1932 357268. E-mail: a.ridley{at}vla.defra.gsi.gov.uk
Published ahead of print on 18 January 2008.
Applied and Environmental Microbiology, April 2008, p. 2492-2504, Vol. 74, No. 8
0099-2240/08/$08.00+0 doi:10.1128/AEM.01242-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
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