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Pathogens (Basel, Switzerland)2026; 15(1); 94; doi: 10.3390/pathogens15010094

Molecular Detection of Theileria equi, Babesia caballi, and Borrelia burgdorferi Sensu Lato in Hippobosca equina from Horses in Spain.

Abstract: The forest fly ) is an obligate haematophagous dipteran insect (order Diptera) that primarily infests horses and may contribute to the circulation of vector-borne pathogens. This study aimed to investigate the presence of , s.l., , and , important vector-borne pathogens of equids, in forest flies collected from horses in endemic areas of Spain. A total of 170 forest flies were collected from 39 equids across four geographical regions in Spain (Segovia, Madrid, Toledo, and Menorca) and blood samples were collected from 27 of these horses. All flies were morphologically and molecularly identified as , and DNA extracted from flies and equine blood was screened using multiplex real-time and nested PCR, followed by sequencing and phylogenetic analysis. Neither flies nor horses tested positive for , whereas one fly was positive for s.l. (0.6%). In contrast, and DNA were detected in 11.2% and 1.2% of flies, respectively, and all positive flies were collected from horses positive for equine piroplasmosis (/ infection), with identical 18S rRNA sequences between hosts and flies. Nested PCR showed a higher detection rate than real-time PCR for the detection of these piroplasms in flies and blood samples. These findings provide the first molecular evidence of EP pathogens in and support further investigation into the epidemiological importance of forest flies in equine pathogen surveillance.
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Publication Date: 2026-01-15 PubMed ID: 41599078PubMed Central: PMC12844906DOI: 10.3390/pathogens15010094Google Scholar: Lookup
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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 examined the presence of certain bloodborne pathogens in forest flies (Hippobosca equina) collected from horses in Spain, identifying infection rates and exploring the potential role of these flies in transmitting diseases to horses.

Background

  • Hippobosca equina: Also known as the forest fly, it is an obligate blood-feeding insect that mainly infests horses.
  • These flies are considered potential vectors (carriers) for several pathogens affecting horses, including Theileria equi, Babesia caballi (both causing equine piroplasmosis), and bacteria from the Borrelia burgdorferi sensu lato group (associated with Lyme disease).
  • Understanding the presence of these pathogens in forest flies can help clarify their role in disease transmission and aid in epidemiological surveillance.

Aim of the Study

  • To detect the presence of Theileria equi, Babesia caballi, and Borrelia burgdorferi s.l. in forest flies collected from horses in Spain.
  • To compare the infection status of flies with that of their equine hosts.
  • To assess the potential epidemiological importance of forest flies in spreading these pathogens.

Methods

  • A total of 170 forest flies were collected from 39 horses located across four regions in Spain: Segovia, Madrid, Toledo, and Menorca.
  • Blood samples were taken from 27 of these horses to test for infections.
  • All flies were both morphologically and molecularly identified as Hippobosca equina.
  • DNA was extracted from both the flies and the horse blood samples.
  • Molecular screening involved multiplex real-time PCR and nested PCR techniques targeting the pathogens’ DNA, followed by sequencing and phylogenetic analyses to confirm pathogen identity.

Key Findings

  • No samples from flies or horses tested positive for Borrelia burgdorferi s.l., the bacterium associated with Lyme disease.
  • One fly (0.6%) was positive for Borrelia burgdorferi s.l., indicating a very low detection rate.
  • Theileria equi and Babesia caballi DNA were detected in forest flies at rates of 11.2% and 1.2%, respectively.
  • All flies found positive for these protozoan parasites were collected from horses that were also confirmed to be infected with equine piroplasmosis, showing a direct correlation.
  • Identical 18S rRNA gene sequences were obtained from both the infected flies and their horse hosts, suggesting possible transmission or overlap of infection.
  • Nested PCR was more sensitive than real-time PCR in detecting these pathogens in both flies and horses.

Significance and Implications

  • This study provides the first molecular evidence documenting the presence of equine piroplasm pathogens in Hippobosca equina flies.
  • The detection of pathogen DNA in flies associated with infected horses suggests that forest flies could play a role in maintaining or spreading equine piroplasmosis infections.
  • The low detection rate of Borrelia burgdorferi s.l. suggests that this bacterium is less likely to be transmitted by forest flies in the studied regions.
  • The findings encourage further research into the epidemiological role of forest flies in disease surveillance and control strategies for vector-borne equine diseases.
  • Improved diagnostic approaches, particularly using nested PCR, may enhance the detection of these pathogens in both insect vectors and horses.

Conclusion

  • Forest flies (Hippobosca equina) collected from horses in Spain harbor DNA of important piroplasm pathogens (Theileria equi and Babesia caballi), but not significantly of the Lyme disease group (Borrelia burgdorferi s.l.).
  • This supports the hypothesis that these flies potentially contribute to the epidemiology of equine piroplasmosis, although further studies are needed to confirm their vector role.

Cite This Article

APA
Dorrego A, Olvera-Maneu S, Jose-Cunilleras E, Gago P, Raez A, Rivera B, Oporto A, Gonzalez S, Cruz-Lopez F. (2026). Molecular Detection of Theileria equi, Babesia caballi, and Borrelia burgdorferi Sensu Lato in Hippobosca equina from Horses in Spain. Pathogens, 15(1), 94. https://doi.org/10.3390/pathogens15010094

Publication

Pathogens (Basel, Switzerland)
ISSN: 2076-0817
NlmUniqueID: 101596317
Country: Switzerland
Language: English
Volume: 15
Issue: 1
PII: 94

Researcher Affiliations

Dorrego, Abel
  • VISAVET Health Surveillance Centre, Universidad Complutense, 28040 Madrid, Spain.
Olvera-Maneu, Sergi
  • Department of Veterinary Medicine, School of Veterinary Medicine, University of Nicosia, 2414 Nicosia, Cyprus.
Jose-Cunilleras, Eduard
  • Department of Animal Medicine and Surgery, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain.
Gago, Paloma
  • VISAVET Health Surveillance Centre, Universidad Complutense, 28040 Madrid, Spain.
  • Animal Health Department, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain.
Raez, Alejandra
  • VISAVET Health Surveillance Centre, Universidad Complutense, 28040 Madrid, Spain.
Rivera, Belen
  • VISAVET Health Surveillance Centre, Universidad Complutense, 28040 Madrid, Spain.
Oporto, Ariana
  • VISAVET Health Surveillance Centre, Universidad Complutense, 28040 Madrid, Spain.
Gonzalez, Sergio
  • VISAVET Health Surveillance Centre, Universidad Complutense, 28040 Madrid, Spain.
Cruz-Lopez, Fatima
  • VISAVET Health Surveillance Centre, Universidad Complutense, 28040 Madrid, Spain.

MeSH Terms

  • Animals
  • Horses
  • Spain / epidemiology
  • Babesia / genetics
  • Babesia / isolation & purification
  • Babesia / classification
  • Theileria / genetics
  • Theileria / isolation & purification
  • Theileria / classification
  • Horse Diseases / microbiology
  • Horse Diseases / parasitology
  • Horse Diseases / epidemiology
  • Diptera / parasitology
  • Diptera / microbiology
  • Babesiosis / parasitology
  • Babesiosis / epidemiology
  • Phylogeny
  • Borrelia burgdorferi Group / genetics
  • Borrelia burgdorferi Group / isolation & purification
  • Lyme Disease / veterinary
  • Lyme Disease / microbiology
  • Theileriasis / parasitology
  • Theileriasis / epidemiology
  • Borrelia burgdorferi / genetics
  • Borrelia burgdorferi / isolation & purification
  • Anaplasma phagocytophilum / isolation & purification
  • Anaplasma phagocytophilum / genetics
  • DNA, Protozoan / genetics

Grant Funding

  • CT82/20-CT83/20 / Grants for Predoctoral Researcher Training Contracts. Funding Program of the Complutense University of Madrid-Banco Santander
  • I0015-2023-000004 / Research grants from the Institut Menorquí d'Estudis 2023

Conflict of Interest Statement

The authors declare no conflicts of interest in the subject, matter or materials discussed in this manuscript.

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