Individual oocysts of Isospora (Apicomplexa: Coccidia) parasites from avian feces: from photo to sequence.

Numerous microscopic studies of coccidian oocysts from avian feces have become the basis for species identification. In contrast, molecular studies of wild birds' Coccidia are still in their infancy and are mostly based on DNA extracted from the blood stages of these parasites. Linking microscopic and molecular data requires a method that reliably extracts DNA from single oocysts with parallel detailed morphological examination of the same cell. We offer a thorough manual of isolating, photographing, and trapping single oocysts from avian feces, followed by extraction of parasite DNA and amplification of mitochondrial DNA from the same cells. In 39 single oocysts from 6 wild blackcaps, we combined microscopic studies of individual cells with studies on their mitochondrial haplotype. In 72% of the single oocysts sampled, we detected unambiguous sequences. From feces and blood of investigated birds, we obtained 6 different haplotypes of Isospora sp. (iSAT1-iSAT 6), finding both the same haplotype in different host individuals and various haplotypes in the same host individual. Our described methodology enables linking the huge amount of morphological data with innovative gene analysis. This method expands the scope of genetic studies conducted on Isospora species, including routine molecular analysis of single oocysts isolated from fecal samples.