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

The forest fly <i>(Hippobosca equina</i>) 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 <i>Anaplasma phagocytophilum</i>, <i>Borrelia burgdorferi</i> s.l., <i>Babesia caballi</i>, and <i>Theileria equi</i>, 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 <i>H. equina</i>, 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 <i>A. phagocytophilum</i>, whereas one fly was positive for <i>B. burgdorferi</i> s.l. (0.6%). In contrast, <i>T. equi</i> and <i>B. caballi</i> DNA were detected in 11.2% and 1.2% of flies, respectively, and all positive flies were collected from horses positive for equine piroplasmosis (<i>T. equi</i>/<i>B. caballi</i> 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 <i>H. equina</i> and support further investigation into the epidemiological importance of forest flies in equine pathogen surveillance.