Bovine theileriosis is a destructive disease affect the livestock production in Egypt. In the current study 210 cattle and 37 buffaloes from different localities in Upper Egypt were collected and tested by Tams1 target based polymerase chain reaction (Tams1 PCR) and Reverse Line Blot (RLB) assay that target 18S rRNA gene of Theileria spp. flowed by sequencing and phylogenetic analysis for identification of theileria species in both cattle and buffaloes. Tams-1 PCR confirmed the infection in 46.19% and 8.11% while RLB assay confirmed the infection in 65.24% and 51.35% in cattle and buffaloes, respectively. RLB was used as reference test to evaluate Tams-1 PCR assay as a diagnostic tool used for diagnosis of bovine theileriosis, the sensitivity of this test was (70.80% and 15.79%) in cattle and buffaloes, respectively while its specificity was 100% in both animal’s species. The PCR products were hybridized on Biodyne C membrane which containing the spacer-oligonucleotide sequences of Theileria spp. Phylogenetic analysis was carried out by using neighbor joining (NJ) method. The obtained results concluded that Assiut and AL-Fayoum governorates have different species of the theileria protozoan parasite include Theileria annulata, Theileria lestoquardi, Theileria uilenbergi and Theileria ovis. On the other hand in AL-Wadi AL-Jadid governorate Theileria annulata is the only species founded. All partial sequences were deposited in the GenBankTM databases and available under accession numbers as following T. annulata (GenBankTM: KF781307, GenBankTM: KF922216&GenBank TM:KF922219), T. lestoquardi (GenBankTM:KF771185), T. ovis (GenBankTM:KF922218) and T. uilenbergi (GenBankTM:KF781308, GenBankTM:KF771184, GenBank TM: KF922215 & GenBank TM: KF922217).
Tams-1 PCR, RLB, 18S rRNA, Neighbor joining.