An insight into misidentification of the small-subunit ribosomal RNA (18S rRNA) gene sequences of Theileria spp. as Theileria annulata

Background: There had been isolated reports of the presence of novel Theileria annulata genotypes based on the 18S rRNA gene sequence data from India, Pakistan and Saudi Arabia; but, these studies were restricted to limited field samples. Additionally, no comparative study has been conducted on all the isolates of this parasite from different countries whose sequences are available in the nucleotide databases. Therefore, we aimed to study the genetic diversity of T. annulata based on all available nearly complete 18S rRNA gene sequences in the GenBank (TM). Out of a total of 312 gene sequences of T. annulata available in the NCBI database, only 70 nearly complete sequences (> 1527 bp) were used for multiple sequence alignment. Results: The maximum likelihood tree obtained using TN93 + G + I model manifested two major clades. All the valid host-cell transforming Theileria species clustered in one clade. The T. annulata designated sequences occupying this clade clustered together, excluding two isolates (DQ287944 and EU083799), and represented the true T. annulata sequences (n = 54). DQ287944 and EU083799 exhibited close association with Theileria lestoquardi. In addition, 14 Indian sequences formed a large monophyletic group with published Theileria orientalis sequences. The broad range of sequence identity (95.8-100%) of T. annulata designated sequences indicated the presence of different Theileria spp. A closer analysis revealed the presence of three Theileria spp., namely, T. annulata, T. orientalis, and two isolates (DQ287944 and EU083799) closely related to T. lestoquardi. The true T. annulata sequences manifested 98.8-100% nucleotide identity within them. EU083799 and 14 misidentified Indian T. annulata sequences exhibited the highest similarity with T. lestoquardi (98.6-98.8%) and T. orientalis (98.0-99.9%) in comparison with the other Theileria spp. of domestic and wild ruminants. Conclusion: In the course of analyzing the genetic diversity of T. annulata, we identified the nearly complete 18S rRNA gene sequences of other Theileria spp. that have not only been misidentified as T. annulata in the GenBank (TM), but are also published as T. annulata. Moreover, a high level of sequence conservation was noticed in the 18S rRNA gene of true T. annulata and T. orientalis sequences.