ntroduction: Tick-borne diseases caused by Anaplasma, Babesia, and Theileria species are prevalent in tropical and subtropical regions, resulting in substantial economic losses. The present study aimed to microscopically and molecularly detect Anaplasma, Babesia, and Theileria spp. in sheep in Ilam province, Iran. This evaluation is of great importance given the crucial role of these diseases in livestock health and their associated economic losses, as well as the contribution of this evaluation to selecting the most appropriate diagnostic method for the region. Materials & Methods: Blood samples (n=73) were collected from clinically symptomatic sheep between June and September 2024. Giemsa-stained blood smears were examined under light microscopy and DNA was extracted for polymerase chain reaction (PCR) targeting Theileria spp. 18S rRNA, Babesia ovis 18S rRNA, and Anaplasma ovis msp4 genes. The PCR products were sequenced, and phylogenetic relationships were analyzed using MEGA software. Statistical tests were used to evaluate the associations between infection rates, age, and gender. Results: Microscopy detected Theileria spp. in all 73 samples and Anaplasma spp. in 12, while PCR identified Theileria ovis in 60 (82.2%) samples, A. ovis in 21 (28.76%), and mixed infections in 13 (17.80%). No B. ovis was found. PCR proved significantly more sensitive than microscopy. Sequence analysis revealed over 99% identity to reference sequences for T. ovis and A. ovis, indicating limited genetic diversity. There was no significant association between age or gender and infection with Theileria or Anaplasma. Conclusion: The results provide important epidemiological data, highlighting the need for improved tick control and molecular diagnostics to reduce economic losses in Iran’s livestock industry.
