Simultaneous Detection and Differentiation of Ovine Piroplasms in Hyaloma anatolicum using PCR-RFLP

Hyaloma spp. has been described as the most abundant species that transmitted ovine piroplasms in Fars province. The traditional methods of diagnosis infected ticks vectors are based on morphological characteristics that have some problems. The best methods for detection and characterization of the protozoan parasites in ticks are the molecular biological methods. The aim of this study was to simultaneous detection of Theileria lestoquardi, Theileria ovis, Theileria annulata and Babesia ovis in H.a.anatolicum using PCR-RFLP technique. The primers were designed from conserved regions of 18S rRNA gene. The amplified DNA fragments of T.ovis, T.lestoquardi, T.annulata and B.ovis in salivary gland of H.a.anatolicum was ranged from 384 to 425 bp. PCR-RFLP bands pattern with restriction enzyme HpaII, MspI of T.ovis revealed two fragments of 270 and 155 bp size, the T.lestoquardi yielded 222 and 199 bp size and T.annulata yielded 143 and 277 bp sized fragments. The restriction enzyme HpaII, MspI can not digest PCR product of B.ovis, for discriminate this species from the other Theileria species the HhaI, cfoI was used. The enzyme digestion of B.ovis specific product using HhaI, cfoI yielded 161 and 223 bp but the other Theileria species remained undigested with this