Molecular detection, prevalence, and phylogenetic characterization of Theileria lestoquardi in goats using conventional PCR and merozoite surface protein gene sequencing in Iraq

Abstract

Background: Infection with Theileria lestoquardi represents one of the most pathogenic forms of ovine and caprine theileriosis in the Middle East. The disease is transmitted by ixodid ticks, such as Hyalomma anatolicum, and is associated with fever, lymphadenopathy, anemia, and considerable economic losses. Molecular tools (like polymerase chain reaction (PCR)) provide higher sensitivity and specificity than microscopic diagnosis and allow accurate epidemiological and phylogenetic characterization. Aim: The present study was conducted to determine the molecular prevalence of T. lestoquardi in goats from Al-Qadisiyah Governorate, Iraq, evaluate its distribution according to sex, age, and month of sampling, and characterize local isolates through sequencing and phylogenetic analysis of the merozoite surface protein (MSP) gene. Methods: Out total of 100 blood samples were collected from goats (multiple herds) between September and December 2025. Genomic DNA was extracted using a commercial kit. Conventional PCR was performed targeting the merozoite surface protein gene using specific primers that amplified a 785 bp fragment. Amplicons were visualized on 1.5% agarose gel. Ten positive samples were selected for sequencing. Sequence alignment and phylogenetic analysis were performed using MEGA11 software based on the Maximum Likelihood method and Tamura–Nei model. According to the PCR findings, the prevalence rates were analyzed according to sex, age, and month using Chi-square test, with statistical significance set at P ≤ 0.05. Results: Out of 100 examined goats, 35 (35%; 95% CI: 25.7–44.3%) were positive for T. lestoquardi. Infection was slightly higher in females (36.36%) than males (33.33%), with no statistically significant association between sex and infection (P > 0.05). According to age, goats <1 year showed the highest prevalence (50%), followed by 1–3 years (28.57%) and >3 year (20%), with a significant association between age and infection rate (P ≤ 0.05). Monthly distribution revealed the highest prevalence in September (48%), followed by October (40%), November (32%), and December (20%) (September–December 2025), showing a significant decline toward winter months (P ≤ 0.05). Sequencing analysis of partial MSP gene fragments confirmed all isolates as T. lestoquardi, showing 97.68–99.54% identity with global strains from Egypt, Iran, Pakistan, and India. Phylogenetic analysis demonstrated close clustering of Iraqi isolates with Middle Eastern strains, indicating limited genetic divergence. Conclusion: The study confirmed endemicity of T. lestoquardi among goats with significant age- and season-related variation. Molecular and phylogenetic analyses demonstrated high genetic similarity between Iraqi isolates and regional strains, highlighting the epidemiological continuity of the parasite within neighboring countries. This helps targeting tick control measures during high-risk seasons and paying attention to younger animals.