Automated fluorescence-based approach for the specific diagnosis of chicken coccidiosis

We have established a fluorescence-based electrophoretic approach for the s pecific identification of all seven currently recognised species of Eimeria infecting chickens. The second internal transcribed spacer (ITS-2) of ribo somal DNA is amplified by polymerase chain reaction (PCR) from any of the s even species using a single set of oligonucleotide primers (of which the re verse one is fluorescently labelled). The amplicons are heat-denatured, sub jected to denaturing polyacrylamide gel electrophoresis in a 377 DNA sequen cer (ABI). The chromatograms produced are stored electronically and then an alysed using GeneScan 3.1 software. Using control DNA samples representing monospecific lines of Emeria regions in the chromatograms have been defined for the specific identification of each of the seven species, although som e variation in the chromatograms (reflecting population variation) was dete ctable within two species. Electrophoretic reading and analysis is carried out automatically using a computer imaging system, thus making it a time- a nd cost-effective approach. It is well suited for high-throughput diagnosti c screening of oocyst samples and should find applicability as a tool for p revalence studies, monitoring of coccidiosis outbreaks and the quality cont rol of vaccines.