Seroprevalence of Borrelia burgdorferi sensu lato in horses in Spain

Borrelia burgdorferi sensu lato is a tick-borne zoonotic pathogen of increasing importance in Europe, responsible for Lyme borreliosis and affecting both humans and domestic animals. In horses, infection is often subclinical and difficult to diagnose due to variable clinical presentation and limitations in diagnostic methods. Epidemiological data remain limited in several countries, including Spain. This study aimed to estimate the first nationwide seroprevalence in horses in Spain and to identify associated risk factors. A total of 716 serum samples from clinically healthy horses across 17 autonomous communities were analysed using a sequential diagnostic approach (ELISA screening followed by Western blot confirmation). Seropositive horses were further tested by real-time PCR. To identify horse- and management-related variables associated with seropositivity, a mixed-effects logistic regression model was fitted, incorporating previous data on equine piroplasmosis serology. The overall crude seroprevalence was 9.6% (69/716), increasing to 11.5% after weighting by the national equine census. Marked geographic heterogeneity was observed, with higher seroprevalence in northern regions. Molecular detection in whole blood was low (3%), consistent with the limited sensitivity of PCR in this matrix. Horses used for sport, residing in northern regions, and those both tick-exposed and seropositive for Babesia caballi showed the highest probability of B. burgdorferi s.l. seropositivity. A substantial farm-level effect indicated clustering within holdings. These findings provide the first nationwide evidence of exposure in horses in Spain, highlighting a heterogeneous distribution driven by environmental and management factors, and supporting the role of shared vector exposure in infection risk.