title: Automated, fluorescence-based approach for the specific diagnosis of chicken coccidiosis.
abstract: We have established a fluorescence-based electrophoretic approach for the specific identification of all seven currently recognised species of Eimeria infecting chickens. The second internal transcribed spacer (ITS-2) of ribosomal DNA is amplified by polymerase chain reaction (PCR) from any of the seven species using a single set of oligonucleotide primers (of which the reverse one is fluorescently labelled). The amplicons are heat-denatured, subjected to denaturing polyacrylamide gel electrophoresis in a 377 DNA sequencer (ABI). The chromatograms produced are stored electronically and then analysed using GeneScan 3.1 software. Using control DNA samples representing monospecific lines of Eimeria, regions in the chromatograms have been defined for the specific identification of each of the seven species, although some variation in the chromatograms (reflecting population variation) was detectable within two species. Electrophoretic reading and analysis is carried out automatically using a computer imaging system, thus making it a time- and cost-effective approach. It is well suited for high-throughput diagnostic screening of oocyst samples and should find applicability as a tool for prevalence studies, monitoring of coccidiosis outbreaks and the quality control of vaccines.
authors.affiliation: Department of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia. robinbg@unimelb.edu.au
authors.forename: R B
authors.initials: RB
authors.lastname: Gasser
authors.forename: W G
authors.initials: WG
authors.lastname: Woods
authors.forename: J M
authors.initials: JM
authors.lastname: Wood
authors.forename: L
authors.initials: L
authors.lastname: Ashdown
authors.forename: G
authors.initials: G
authors.lastname: Richards
authors.forename: K G
authors.initials: KG
authors.lastname: Whithear
mesh_terms: D000818:Animals; D001331:Automation; D001483:Base Sequence; D003048:Coccidiosis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D016054:DNA, Protozoan / Q000032:analysis; D021903:DNA, Ribosomal Spacer / Q000032:analysis; D004539:Eimeria / Q000145:classification / Q000235:genetics*; D016786:Eimeria tenella / Q000145:classification / Q000235:genetics*; D005452:Fluoresceins; D005453:Fluorescence; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D011201:Poultry Diseases / Q000175:diagnosis* / Q000469:parasitology; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D016054:DNA, Protozoan; D021903:DNA, Ribosomal Spacer; D005452:Fluoresceins; D005456:Fluorescent Dyes