Background
Hippoboscid flies are obligate hematophagous Diptera that infest mammals and birds worldwide. Hippobosca equina, commonly known as the forest fly, primarily affects horses and is considered both a mechanical and/or biological vector of various bacteria, viruses, and protozoa. Equine piroplasmosis (EP) is a tick-borne disease caused by three intra-erythrocytic hemoprotozoans: Theileria equi, Babesia caballi, and the recently identified species Theileria haneyi. To date, no studies have reported the detection of any Piroplasmida in H. equina. This study provides the first evidence of the presence of T. equi and B. caballi in forest flies infesting horses in endemic areas, identified using molecular techniques.
Methods
A total of 170 forest flies were collected from 35 Equidae across five locations in Spain (Segovia, Madrid, Toledo, and Menorca Island). Blood samples for equine piroplasmosis (EP) testing were drawn from 27 examined horses. All insects were identified both morphologically and molecularly, and genomic DNA from each fly and their equine host blood samples was screened for EP using real-time and nested PCR. Phylogenetic analyses were performed on all obtained T. equi and B. caballi sequences.
Results
All collected hippoboscids were identified as H. equina using a stereomicroscope and taxonomic keys, as well as by sequencing the mitochondrial cytochrome oxidase subunit 1 (COI) gene. Molecular screening detected T. equi DNA in 19 of 170 (11.2%) collected forest flies and B. caballi DNA in 2 of 170 (1.2%). All EP-positive flies were collected from EP-positive horses, with sequences from the flies and their equine hosts being identical. Two distinct T. equi and two B. caballi sequences were identified.
Conclusions
This study is the first to report the detection of T. equi and B. caballi in H. equina collected from horses. Given that approximately one-quarter of the flies sampled from EP-positive horses tested positive for the disease, sampling forest flies could be a practical method for detecting equine piroplasmosis in feral horse populations. Experimental transmission studies are required to assess the vector competence of H. equina for EP and other equine diseases.