Theileria equi sensu lato genetic diversity and phylogeny: new insights from mitochondrion, apicoplast and nucleus multilocus gene analysis

Equine piroplasmosis is a significant tick-borne disease with a global distribution. Among the two causative agents, Theileria equi has undergone numerous taxonomic reclassifications over time, reflecting advances in our understanding of its biology, molecular characteristics, and phylogenetic relationships. Formerly named Babesia equi, now classified in the Equus group apart from Babesia and Theileria species, its genus name will again certainly change. The genetic diversity also within T. equi lead to the renaming of one lineage as Theileria haneyi. Theileria equi should better now be considered as T. equi sensu lato (including T. haneyi) with a set of different cryptic species awaiting for formal taxonomical changes to be taken. A better knowledge within this group is therefore essential as this diversity has an impact on the diagnostic of this disease which is submitted to strict regulations governing the international movement of horses. In the present study, we designed primers to study the same five genes from the nucleus (hsp70, clamp and ama-1), the mitochondria (cox1) and the apicoplast (tufA), on a set of 36 mono-infected samples of the lineages A, C (T. haneyi) and E of T. equi sensu lato. The phylogenetic separation in lineages A, C and E based on 18S rRNA sequencing was confirmed by this multi-locus analysis. Genetic diversity and phylogenetic separation within lineage A into two sub-lineages A1 and A2 were demonstrated on the basis of all five genes, with an additional A3 sub-lineage on the basis of nuclear (hsp70) and mitochondrial (cox1) markers. All these sub-lineages would deserve the rank of species based on cox1 divergence confirmed by other markers. Phylogenetic and haplotype networks analyses were also conducted on genes used in molecular or serological diagnostic, ema-1 and ema-11, to evaluate the impact of this diversity on currently used tests. We confirmed the absence of molecular detection of the lineage E DNA with any of these two gene targets. We linked the sub-lineages A1 and A3 to the ema-1 clades A and B, with a worldwide distribution. We confirmed the link of the rare A2 sub-lineage from France to a new ema-1 clade D, with additional Eurasian sequences from Croatia, Mongolia and China. A fifth ema-1 clade E was also confirmed. Separation of lineage C (T. haneyi) into at least two sub-lineages was also confirmed with the few ema-11 sequences available, based on their genetic diversity, and their phylogenetic and haplotype network analyses. From this study, it becomes clear that the division of T. equi sensu lato into species corresponding either to the lineage (5 species) or to the sub-lineage (at least 10) should await a more comprehensive knowledge of its genetic diversity worldwide, as it may impact the diagnostic tools to be developed.