Development of DNA probes for the ovine Eimeria species E. crandallis and E. ovinoidalis

As an alternative to morphological speciation, DNA probes have been developed to identify Eimeria crandallis and E. ovinoidalis, the species associated with ovine coccidiosis. Coccidia-free lambs were infected with pure cultures of E. crandallis and E. ovinoidalis. Oocysts separated from faeces were sporulated and treated to release sporozoites from which DNA was purified. Repetitive DNA sequences were identified by hybridisation and cloned. The specificity of selected clones was tested by hybridisation with dot blots of four other ovine Eimeria species, a mixture of caprine Eimeria species and E. acervulina of the chicken. The probes allowed the detection of DNA equivalent to that present in approximately 1,500 oocysts. To increase the sensitivity of the test, a polymerase chain reaction (PCR) approach was adopted, using oligonucleotide primers specific for the cloned repeats. By this method, less than 10 oocysts of E. ovinoidalis could be reliably detected. In addition to the species-specific probes, other highly repetitive DNA sequences from E. crandallis and E. ovinoidalis were cloned and sequenced; these repeats hybridised to all ovine, caprine and avian Eimeria species tested and contained tandem CGA and putative telomeric repeats.