We describe a polymerase chain reaction (PCR)-based assay for the detection, identification and differentiation of pathogenic species of Eimeria in poultry. The internal transcribed spacer 1 (ITS1) regions of ribosomal DNA (rDNA) from Eimeria acervulina, E. brunetti, E. necatrix and E. tenella were sequenced and regions of unique sequences identified. Four pairs of oligonucleotide primers, each designed to amplify the ITS1 region of a single Eimeria species, were synthesised for use in the PCR assay. In tests on purified genomic DNA from all seven species of Eimeria that infect the chicken, each of the four primer pairs amplified the ITS1 region from only their respective target species. The robustness of the approach was further demonstrated by the amplification of specific DNA fragments from tissues of experimentally infected animals and from oocysts recovered from field samples. We conclude that the ITS1 regions of Eimeria species contain sufficient inter-specific sequence variation to enable the selection of primers that can be applied in PCR analyses to detect and differentiate between species. In future work they may provide excellent markers for epidemiological studies.

A polymerase chain reaction (PCR) assay, based on the amplification of internal transcribed spacer 1 (ITS1) regions of ribosomal DNA, was developed for the chicken coccidian species Eimeria maxima, E. mitis and E. praecox. Thus, taking into account our previous work, a complete set of ITS1-based, species-specific primers for the detection and discrimination of all seven Eimeria species that infect the domestic fowl is now available. ITS1 primers for each of these seven species of Eimeria were also used as capture probes in a paper chromatography assay (PACHA). The addition of PACHA to the PCR assay provided a faster, more simplified read-out compared to staining of amplified bands in an agarose gel with ethidium bromide.

The phylogenetic relationships and taxonomic affinities of coccidia with isosporan-type oocysts have been unclear as overlapping characters, recently discovered life cycle features, and even recently discovered taxa, continue to be incorporated into biological classifications of the group. We determined the full or partial 18S ribosomal RNA gene sequences of three mammalian Isospora spp., Isospora felis, Isospora ohioensis and Isospora suis, and a Sarcocystis sp. of a rattlesnake, and used these sequences for a phylogenetic analysis of the genus Isospora and the cyst-forming coccidia. Various alveolate 18S rDNA sequences were aligned and analyzed using maximum parsimony to obtain a phylogenetic hypothesis for the group. The three Isospora spp. were found to be most closely related to Toxoplasma gondii and Neospora caninum. This clade in turn formed the sister group to the Sarcocystis spp. included in the analysis. The results confirm that the genus Isospora does not belong to the family Eimeriidae, but should be classified together with the cyst-forming coccidia in the family Sarcocystidae. Furthermore, there appear to be two lineages within the Sarcocystidae. One lineage comprises Isospora and the Toxoplasma/Neospora clade which share the characters of having a proliferative phase of development preceding gamogony in the definitive host and an exogenous phase of sporogony. The other lineage comprises the Sarcocystis spp. which have no proliferative phase in the definitive host and an endogenous phase of sporogony.