Genetic diversity and phylogenetic relationships of Theileria equi inferred from heat shock protein 70 kDa gene sequences in Brazilian horses

Equine piroplasmosis, a tick-borne disease caused by hemoparasites of the Babesia and Theileria genera, has significant clinical and economic impacts worldwide. This study aims to characterize the heat shock protein 70 kDa (hsp70) gene sequences of Theileria equi from naturally infected horses across the five geographic regions in Brazil, and to analyze the phylogenetic relationships between T. equi and other parasites within the order Piroplasmida. Primers specific to T. equi were designed through in silico analysis of hsp70 gene sequences available in GenBank. Forty samples of equine whole blood were subjected to DNA extraction and conventional PCR targeting the hsp70 gene. The resulting amplicons were purified and sequenced. The Hsp70 sequences generated were aligned using the ClustalW method, and phylogenetic analyses were conducted using Bayesian Inference (BI) and Maximum Likelihood (ML) methods. The evolutionary distance within and between species of parasites from the order Piroplasmida was evaluated, as was the entropy of the hsp70 gene sequence among Theileria species. Phylogenetic analysis of the forty sequences identified in this study revealed two circulating genotypes of T. equi in Brazil. One genotype comprised the American sequence along with thirty-six Brazilian sequences, while the other grouped a sequence from Mongolia with four Brazilian sequences. The Equus clade was distinctly placed with strong support, indicating a different evolutionary ancestor compared to Theileria spp. and Babesia spp. groups. The results confirmed hsp70 as an effective molecular marker for phylogeny, yielding topologies similar to those observed with 18 S rDNA while distinguishing closely related protozoan species such as T. haneyi and T. equi. This study provides valuable evolutionary insights into the phylogenetic relationships within the order Piroplasmida.