Ahedor, B.Otgonsuren, D.Zhyldyz, A.et al.2024-02-192024-02-192023https://doi.org/10.1186/s13071-023-06045-zhttp://ugspace.ug.edu.gh:8080/handle/123456789/41318Research ArticleBackground Theileria equi causes equine piroplasmosis, an economically signifcant disease that afects horses and other equids worldwide. Based on 18S ribosomal RNA (18S rRNA sequences), T. equi can be classifed into fve genotypes: A, B, C, D, and E. These genotypes have implications for disease management and control. However, no conventional polymerase chain reaction (PCR) assays are available to diferentiate the genotypes of T. equi. To over come this limitation, we developed and evaluated PCR assays specifc for the detection of each T. equi genotype. Methods A pair of forward and reverse primers, specifcally targeting the 18S rRNA sequence of each genotype, was designed. The genotype-specifc PCR assays were evaluated for their specifcity using plasmids containing inserts of the 18S rRNA sequence of each genotype. Subsequently, the assays were tested on 270 T. equi-positive equine blood DNA samples (92 from donkeys in Sri Lanka and 178 from horses in Paraguay). 18S rRNA sequences derived from the PCR amplicons were analyzed phylogenetically. Results Each genotype-specifc PCR assay accurately targeted the intended genotype, and did not produce any amplicons when 18S rRNA from other T. equi genotypes or genomic DNA of Babesia caballi or uninfected horse blood was used as the template. Previous studies employing PCR sequencing methods identifed T. equi genotypes C and D in the Sri Lankan samples, and genotypes A and C in the Paraguayan samples. In contrast, our PCR assay demonstrated exceptional sensitivity by detecting four genotypes (A, C, D, and E) in the Sri Lankan samples and all fve genotypes in the Paraguayan samples. All the Sri Lankan samples and 93.3% of the Paraguayan samples tested positive for at least one genotype, further emphasizing the sensitivity of our assays. The PCR assays also had the abil ity to detect co-infections, where multiple genotypes in various combinations were detected in 90.2% and 22.5% of the Sri Lankan and Paraguayan samples, respectively. Furthermore, the sequences obtained from PCR amplicons clustered in the respective phylogenetic clades for each genotype, validating the specifcity of our genotype-specifc PCR assays. Conclusions The genotype-specifc PCR assays developed in the present study are reliable tools for the diferential detection of T. equi genotypes.enEquine piroplasmosisGenotypePolymerase chain reactionTheileria equiArticle