Comprehensive molecular characterization of Theileria equi: Detection, genetic diversity, and risk factors assessment

Equine piroplasmosis, caused by Theileria equi, has been widely studied globally, but genetic diversity research in India remains limited. Analysis of 306 horse blood samples revealed T. equi infection in 10.78% by microscopy and 21.24% by PCR. Universal primers targeting 18S rRNA of T. equi and Babesia caballi were used, followed by species-specific primers generating 435 bp amplicons. Sequenced PCR-positive samples revealed high homology (99.52-100% in India, 95.89-100% globally), with all Indian isolates classified as genotype A. The EMA-1 gene phylogenetic analysis confirmed the insights from the 18S rRNA gene analysis. A median-joining network of 18S rRNA identified 46 global haplotypes, with Hap_1 as the most common, showing geographic clustering. Brazil had the most haplotypes (n = 13), followed by Sri Lanka (n = 11), Israel (n = 9), South Africa and China (n = 6 each), Switzerland (n = 4), and India (n = 3). The star-like network indicates rapid population expansion and the emergence of new haplotypes. Low overall nucleotide diversity (π = 0.172 ± 0.007) but high haplotype diversity (Hd = 1.0 ± 0.005) across T. equi genotypes suggests minor haplotype distinctions and recent population expansion. AMOVA highlighted greater genetic variation among genotypes (78.61%) than within (21.39%), with significant differentiation (Fst >0.33). The logistic regression model identified summer season (OR = 3.129; p = 0.012), tick infestation (OR = 2.988; p = 0.031), presence of ruminants on the farm (OR = 5.865; p = 0.004), anaemia (OR = 4.231; p < 0.001), and red urine (OR = 4.892; p = 0.017) as significant predictors of T. equi infection in equids. The rainy season, age, sex, management, and body condition were not significant predictors (p > 0.05).