Geographical distribution and genotypic diversity of Theileria orientalis with emphasis on Indian isolates

Globally, Theileria orientalis leads to considerable economic losses in cattle. This study carried out an in-depth analysis of T. orientalis MPSP sequences to explore their phylogeny, genetic diversity, and population structure. We analyzed 800 blood samples from northern India between 2023 and 2025 using microscopy. The results showed a higher prevalence of Theileria in cattle at 29.25%, compared to 2.75% in buffaloes. PCR amplification targeting the 18 S rRNA gene confirmed the presence of the genus in 39.5% of cattle and 6.0% of buffaloes. Additionally, T. orientalis-specific PCR detected a prevalence of 4.5% in cattle and 3.0% in buffaloes. The phylogenetic analysis included 981 MPSP sequences from diverse geographical regions with 15 new isolates from India. This revealed 12 genotypes, classified as Types 1-5, 7, 8, and N1-N5. This study also supports recent updates in the taxonomic interpretation of these genotypes, suggesting that type 6 aligns closely with T. sinensis, while type 9 appears to group within the type 4 lineage. Furthermore, we found inaccuracies in GenBank typing, like the clustering of type 8 isolates with type 4 and vice versa. In India, we identified eight genotypes, with types 2, N1, and N5 being reported for the first time. Geographic analyses indicated that genotypes 1 and 3 have a cosmopolitan distribution. The population showed high diversity with 552 haplotypes, a nucleotide diversity (π) of 0.14, and haplotype diversity (Hd) of 0.988. The consistently high haplotype diversities across all 12 genotypes, along with significant negative values for Tajima's D and Fu's Fs statistics, suggest that the parasite population is expanding. Different T. orientalis genotypes showed higher Fst and lower Nm values between them, which indicates significant genetic differentiation and limited gene flow.