Comparison of 18S rRNA gene fragments and reference databases for assessing bovine Eimeria diversity using the Illumina platform.

This study compared the effects of different 18S rRNA target regions and reference databases on assessing the diversity of bovine Eimeria spp. using metabarcoding. A total of 32 diarrheic bovine fecal samples, confirmed positive for Eimeria through microscopy and PCR, were collected in Korea. The DNA from each sample was pooled for DNA metabarcoding, which targeted the V4 and V9 regions of the 18S rRNA gene, as well as the genus-specific Eimeria 18S rRNA gene fragment (Pan-Eim-18S), using the Illumina MiSeq platform. Additionally, taxonomic assignments were compared using two reference databases: one from the NCBI and one from the SILVA. The results showed that Pan-Eim-18S analysis identified more Eimeria species than analysis of the V4 and V9 regions. Furthermore, taxonomic assignments based on the NCBI database identified more Eimeria species than those based on the SILVA database. The 18S rRNA gene metabarcoding results were validated using conventional PCR and cloning. The identical DNA pool used in metabarcoding was also used in these subsequent analyses, which were followed by phylogenetic analysis. The phylogenetic analysis revealed that the obtained sequences clustered with the E. bovis/E. ellipsoidalis/E. zuernii group, as well as with E. auburnensis and Eimeria sp. In conclusion, the target region and the reference database critically influence the identification of Eimeria species, especially among closely related species. The results of this study suggest that longer amplicon analysis and use of the NCBI database enable high-resolution identification of diverse Eimeria species.