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Background  Piroplasms are tick-borne hemoprotozoans with a major impact on extensive management systems. Detection of sub-clinical low-level carriers, which can act as source of infection for vector ticks, is key to protect livestock trade and facilitate preventive control programs. The purpose of this study was to develop a method for the detection of ovine piroplasms and to use it in a field study aimed at investigating piroplasms infection in semi-extensive production systems in the Basque Country (northern Spain).    Methods  A DNA bead-based suspension array using the Luminex® xMAP technology that included a generic  Theileria - Babesia  control probe, 6 species-specific probes, and an internal control probe was developed to detect and identify piroplasms that infect sheep. To monitor piroplasm infection in clinically healthy sheep from 4 flocks that share communal mountain pastures, blood samples were collected during 2 grazing seasons.    Results  Piroplasms were detected in 48% (214/446) of blood samples, nearly half of them (49.1%, 105/214) as mixed infections. Five different piroplasms were identified:  Theileria  sp. OT3 in 34.8% of the samples,  Theileria ovis  in 20.9%, and at lower prevalences  Babesia motasi  (12.3%),  Theileria luwenshuni /OT1 (10.5%) and  Babesia ovis  (6.3%). Despite differences among flocks associated to differences in management, an increasing trend in the incidence of piroplasm infection with increasing age of animals after increased tick exposure was observed. This increment could be attributed to continued re-infection associated with re-exposure to ticks at grazing. Ticks were collected from animals (4 species) and vegetation (8 species), and associations between tick abundance seasonality and risk of infection with the different piroplasms were established.    Conclusion  The multiplex Luminex® xMAP procedure is a rapid and high throughput technique that provided highly specific and sensitive identification of single and mixed piroplasm infections in blood of sheep carriers. This study confirmed a situation of endemic stability for piroplasm infection in the region, where infection is present in the absence of clinical signs, and mountain grazing allows for sufficient inoculation rates to maintain such situation.

Assessment of exposure to piroplasms in sheep grazing in communal mountain pastures by using a multiplex DNA bead-based suspension array