First molecular detection of Theileria haneyi infection in horses in Southern Spain

Background Equine piroplasmosis (EP) is a tick-borne disease of equids caused by the hemoparasites Theileria equi, Babesia caballi , and Theileria haneyi . T. haneyi , a close relative of T. equi that causes mild clinical signs under experimental conditions, has been reported in several countries, adding complexity to EP ecology, diagnosis, and treatment. However, its presence in Spain remains unknown, which is concerning given the country's importance in equine genetics and international trade. Hereby, we investigated the presence of T. haneyi in blood samples from equids in Spain. Methods Blood samples from 222 equids (218 horses, two donkeys, two mules) suspected of EP across nineteen Spanish provinces were analyzed. DNA was extracted using standard protocols. T. haneyi was detected by nested PCR targeting a hypothetical protein gene (GenBank MT896770.1), and T. equi by nested PCR targeting the EMA1 gene. For T. haneyi -positive samples, the EMA11 gene was amplified and sequenced, and nucleotide alignments were generated using ClustalW. Results Three horses out of 222 (3/222; 1.35%) were positive for T. haneyi . These three positive horses were located in Southern Spain in the provinces of Huelva, Cadiz, and Malaga. Additionally, 76 (76/222; 34.2%) were positive for T. equi . All three T. haneyi -positive horses were also found PCR-positive for T. equi . Alignment analysis of a 209-nucleotide sequence from the EMA11 gene revealed a high level of identity between the Spanish and the T. haneyi reference isolate U.S. Eagle Pass sequences. One nucleotide change, representing a silent mutation, was detected in one of the T. haneyi -positive samples, while the other two showed 100% nucleotide identity. Conclusion The results indicate the first detection of T. haneyi in horses in Spain. These findings have important implications for equine clinicians, regulatory agencies, and animal health surveillance programs not only in Spain but also in other countries with significant equine industries, particularly in those European countries within the Schengen area. A systematic epidemiological study should be now undertaken to determine the prevalence and geographic distribution of T. haneyi infections in Spain.