Molecular evidence of co-infections with Anaplasma and Theileria species in domestic sheep and goats from Alentejo, Portugal.

Anaplasma spp. and piroplasmids are tick-borne pathogens with worldwide distribution, affecting domestic animals, wildlife, and humans. Although several tick-borne species have relevance in animal health and can cause economic losses in livestock farms, little is known about the epidemiological situation in small ruminants in Portugal. We aimed to assess the infection prevalence, molecular characterization, co-infection patterns and risk factors of Anaplasma spp. and piroplasmids in sheep and goat farms from the Alentejo region (south-central Portugal). A total of 290 blood samples collected in 2019 were analyzed using conventional PCR assays targeting partial fragments of the 16S rRNA and RpoB genes for Anaplasma spp., and 18S rRNA gene for piroplasmids. The results confirmed a wide distribution and a high prevalence of Anaplasma spp. (34.5%, 100/290) and piroplasmids (25.2%, 73/290). The following species were identified: Anaplasma ovis (n = 31), "Candidatus Anaplasma corsicanum" (n = 1), Theileria ovis (n = 21), and Theileria capreoli (n = 1). Co-infections occurred in 11.0% (32/290) of animals, and the most common association was A. ovis/T. ovis (n = 20), followed by "Candidatus A. corsicanum"/T. ovis (n = 1) and A. ovis/T. capreoli (n = 1). The remaining 10 co-infected individuals harbored A. ovis and an uncharacterized piroplasmid. This study represents the first molecular detection of "Candidatus A. corsicanum" in Portugal. Risk factors positively associated with Anaplasma infection were age and breed, while piroplasmid prevalence was significantly higher in sheep and crossbred animals. The presence of mixed infections may influence the pathogenicity of existing pathogens and complicate both diagnosis and treatment, potentially leading to considerable economic impacts on the livestock sector. These findings highlight the need for an effective monitoring system to enhance disease surveillance.