Molecular Identification and Genotyping of Theileria Orientalis Type 3 (Buffeli) Isolated from Cattle Using Nested-PCR Assay in Guilan Province, Iran

Protozoan parasites of the genus Theileria are tick-borne parasites that have been found in many species of mammals. More than a dozen species of Theileria have been found in cattle, water buffalo, sheep, and goats. Theileria orientalis is a non-pathogenic blood protozoan parasite that was detected and identified during a regular investigation of piroplasmida infection in indigenous cattle in the spring of 2019 in Northern Provinces of Iran. In total, 92 blood samples were collected from different areas of Guilan and Mazandaran Provinces, Iran during the spring. The Giemsa stained blood smears did not show any parasitic infection; however, T. orientalis was identified by 18S rRNA gene polymerase chain reaction (PCR) and DNA sequencing. The specific sequenced DNA for T. orientalis was registered in GenBank under the accession number MN453385. The partial 18S rRNA gene sequence of the obtained DNA showed 100% nucleotide identity with reference sequences for the T. orientalis that have been registered from Europe, Africa, and Asia. Additionally, molecular phylogenetic studies have shown that T. orientalis Iran GC98-01 isolate belongs to nonpathogenic T. orientalis type 3 (buffeli). In this study, the indigenous Bos indicus cattle were detected as asymptomatic carriers of Theileria spp. infection. Here, we identified and genotyped T. orientalis for the first time as T. orientalis type 3 (buffeli) in Iran using molecular phylogenetic analysis and registered the 18S rRNA gene sequence of the T. orientalis GC98-01 isolate in GenBank.  Moreover, rare T. annulata infection was detected in cattle using semi-nested PCR in Mazandaran (Miankaleh peninsula). The T. orientalis can be differentiated from other Theileria and Babesia haemoprotozoan parasites by specific molecular assays.