Tick-borne diseases (TBDs), including piroplasmosis and anaplasmosis, are globally distributed and pose significant threats to cattle. This study investigated the prevalence, co-infections, and risk factors of bovine piroplasmosis and anaplasmosis in three Egyptian governorates, Beni-Suef, Al-Faiyum, and Al-Minya, over a one year period. A total of 601 cattle blood samples were screened for hemopathogens using microscopic examination, Polymerase Chain Reaction (PCR), and nucleotide sequences analysis. Risk factors were analyzed using the chi-square (χ2) test (P < 0.05). Hemopathogens were detected in 41.09 % and 75.04 % of cattle through microscopic and PCR methods, respectively. Babesia bigemina was the most prevalent pathogen (33.44 %, including both single and co-infections), followed by Anaplasma marginale (20.63 %), Theileria annulata (10.64 %), and Babesia bovis (3.49 %). Co-infections were observed in 19.8 % of cattle in all three studied governorates, with the most common combination being A. marginale and B. bigemina (63.86 %). Significant risk factors included seasonality (B. bigemina and T. annulata), breed (B. bigemina), and acaricide application (A. marginale). Phylogenetic analysis confirmed the presence of three piroplasm species and revealed two potentially distinct genotypes of A. marginale. This study is the first to report B. bovis in cattle from Al-Faiyum and Al-Minya, expanding its known distribution in Egypt. Multiplex PCR was successfully applied for simultaneous detection of multiple pathogens, supporting its utility for efficient, large-scale epidemiological studies. In conclusion, B. bigemina and A. marginale were the most prevalent pathogens and frequently co-occurred. The obtained results contribute to a better understanding of pathogen prevalence, distribution, and co-infection patterns and advocate for the design and implementation of region-specificcontrol strategies in endemic regions. |