Molecular epidemiology of Theileria parva in Eastern Democratic Republic of the Congo: implications for the introduction of the Muguga cocktail vaccine against East Coast Fever

East Coast Fever (ECF) is one of the most economically important tick-borne diseases of cattle in Eastern, Central, and Southern Africa, caused by the intracellular protozoan parasite Theileria parva ( T. parva ). This study investigated the seroprevalence and genetic diversity of T. parva populations in the eastern Democratic Republic of the Congo (DRC) to inform immunization strategies against ECF. A cross-sectional study was conducted between September 2012 and January 2013 in three provinces of eastern DRC (North Kivu, South Kivu, and Ituri) to assess the seroprevalence and confirm the presence of T. parva in cattle. Blood samples from 31 villages across different agro-ecological zones were analyzed using the PIM ELISA, and ELISA-positive samples were further screened by p104 PCR. An immunization and field challenge trial using the Muguga cocktail vaccine was conducted with 100 seronegative cattle. Molecular characterization of samples collected from the sentinel, immunized, and control groups was performed by sequencing the p67 , Tp1 , and Tp2 genes and conducting microsatellite genotyping. Data were analyzed using MEGA, DnaSP, GenAIEx, FSTAT, and LIAN to determine genetic variation, population structure, and linkage equilibrium among T. parva isolates. Of the 756 serum samples analyzed, 27.5% tested positive for T. parva antibodies by ELISA, with Ituri showing the highest seroprevalence and North Kivu the lowest. PCR confirmed T. parva DNA in 87.5% of ELISA-positive samples. Sentinel cattle exposed in Kabasha village developed severe ECF, confirming high transmission pressure, and were used to establish the immunization and challenge site. Vaccination with the Muguga cocktail induced seroconversion in 94% of cattle, with 46.5% surviving field challenge. Sequence analysis of p67 , Tp1 , and Tp2 genes revealed limited clustering with Muguga strains and high polymorphism, particularly in Tp2 (13.2%). Microsatellite genotyping detected substantial allelic diversity and population sub-structuring, with unique alleles predominant across loci (Fst = 0.118). This study revealed high and variable T. parva seroprevalence across eastern DRC, with genetic analyses indicating the coexistence of Muguga-like and distinct local parasite populations. The low survival rate in immunized cattle highlights limitations of the Muguga cocktail against field strains. These findings highlight the presence of diverse T. parva populations and emphasize the need for continuous genetic monitoring and tailored vaccine strategies to enhance ECF control in the region.