Frontiers in veterinary science2026; 13; 1841333; doi: 10.3389/fvets.2026.1841333

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

Mendoza, FJ
·
Pérez-Écija, A
·
Suarez, CE
·
Martínez, E
·
Navarro, A
·
Kappmeyer, LS
·
Bastos, RG

Abstract: Equine piroplasmosis (EP) is a tick-borne disease of equids caused by the hemoparasites , and . , a close relative of 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 in blood samples from equids in Spain. Unassigned: 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. was detected by nested PCR targeting a hypothetical protein gene (GenBank MT896770.1), and by nested PCR targeting the EMA1 gene. For -positive samples, the EMA11 gene was amplified and sequenced, and nucleotide alignments were generated using ClustalW. Unassigned: Three horses out of 222 (3/222; 1.35%) were positive for . 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 . All three -positive horses were also found PCR-positive for . Alignment analysis of a 209-nucleotide sequence from the EMA11 gene revealed a high level of identity between the Spanish and the reference isolate U.S. Eagle Pass sequences. One nucleotide change, representing a silent mutation, was detected in one of the -positive samples, while the other two showed 100% nucleotide identity. Unassigned: The results indicate the first detection of 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 infections in Spain.

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Publication Date: 2026-06-04 PubMed ID: 42328053PubMed Central: PMC13275341DOI: 10.3389/fvets.2026.1841333Google Scholar: Lookup

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Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

This study reports the first detection of Theileria haneyi, a tick-borne parasite, in horses from Southern Spain, highlighting its presence alongside Theileria equi, another causative agent of equine piroplasmosis (EP).
The findings have key implications for disease diagnosis, management, and control in equine populations in Spain and potentially across Europe.

Background

Equine piroplasmosis (EP) is a disease transmitted by ticks affecting horses, donkeys, and mules.
It is caused by hemoparasites, mainly Theileria equi and Babesia caballi.
Theileria haneyi is a recently identified close relative of T. equi causing milder clinical symptoms in experimental settings.
The presence of T. haneyi is known in several countries but had not been previously documented in Spain.
Understanding T. haneyi’s presence is important due to Spain’s vital role in equine genetics and international horse trade.

Objectives

To investigate the presence of Theileria haneyi in equids from Spain, particularly in animals suspected of having equine piroplasmosis.
To assess co-infection rates with Theileria equi in the tested animals.
To provide genetic characterization of the identified T. haneyi strains in Spain.

Methods

Blood samples were collected from 222 equids (218 horses, two donkeys, two mules) across nineteen provinces in Spain which were suspected of having EP.
DNA extraction was performed on these blood samples using standard laboratory protocols.
Nested PCR techniques were used to detect:
- Theileria haneyi by targeting a hypothetical protein gene (GenBank accession MT896770.1).
- Theileria equi by targeting the EMA1 gene.
For samples positive for T. haneyi, a further PCR amplification and sequencing of the EMA11 gene was carried out to analyze genetic similarities.
Sequence alignments were performed using ClustalW software to compare Spanish isolates with known reference sequences.

Results

Three out of 222 horses (1.35%) tested positive for Theileria haneyi.
These horses were located in southern provinces of Spain: Huelva, Cadiz, and Malaga.
Theileria equi was found in 76 of the 222 equids tested (34.2%).
All three T. haneyi-positive horses were also positive for T. equi, indicating co-infection.
Sequence analysis of a 209-nucleotide region of the EMA11 gene showed:
- High identity between Spanish T. haneyi sequences and the U.S. Eagle Pass reference isolate.
- One silent mutation (a nucleotide substitution that does not alter the amino acid sequence) was found in one Spanish sample, while two others had complete sequence identity.

Conclusions and Implications

This research documents the first molecular evidence of T. haneyi in horses in Spain.
Presence of T. haneyi complicates the epidemiology and diagnosis of equine piroplasmosis since it shares similarities with T. equi but may produce different clinical outcomes.
Co-infection with both T. haneyi and T. equi suggests the need for diagnostic tests that can differentiate between these parasites.
The findings have significant implications for veterinary clinicians managing equine health in Spain and neighboring European countries, especially those within the Schengen area where horse movement is frequent.
It calls for expanded epidemiological surveillance and systematic studies to:
- Determine the distribution and prevalence of T. haneyi in Spain.
- Understand the impact of co-infections on horse health and disease control programs.
- Evaluate implications for international trade and regulatory policies concerning equine infectious diseases.

Cite This Article

APA

Mendoza FJ, Pérez-Écija A, Suarez CE, Martínez E, Navarro A, Kappmeyer LS, Bastos RG. (2026). First molecular detection of Theileria haneyi infection in horses in Southern Spain. Front Vet Sci, 13, 1841333. https://doi.org/10.3389/fvets.2026.1841333

Publication

Frontiers in veterinary science

ISSN: 2297-1769

NlmUniqueID: 101666658

Country: Switzerland

Language: English

Volume: 13

Pages: 1841333

PII: 1841333

Researcher Affiliations

Mendoza, Francisco J

Department of Animal Medicine and Surgery, College of Veterinary Medicine, University of Cordoba, Cordoba, Spain.

Pérez-Écija, Alejandro

Department of Animal Medicine and Surgery, College of Veterinary Medicine, University of Cordoba, Cordoba, Spain.

Suarez, Carlos E

Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA, United States.

Martínez, Esther

Gasset Laboratory, DAV Salud Group SL, Granada, Spain.

Navarro, Ana

Gasset Laboratory, DAV Salud Group SL, Granada, Spain.

Kappmeyer, Lowell S

Animal Disease Research Unit, Agricultural Research Service, United States Department of Agriculture (USDA), Pullman, WA, United States.

Bastos, Reginaldo G

Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA, United States.
Animal Disease Research Unit, Agricultural Research Service, United States Department of Agriculture (USDA), Pullman, WA, United States.

Conflict of Interest Statement

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Theauthors RB and CS declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

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