Theileria: improved species discrimination using oligonucleotides derived from large subunit ribosomal RNA sequences

Partial nucleotide sequences of large subunit ribosomal RNA (LSU rRNA) coding genes of Theileria parva and T. taurotragi were determined. Alignment of the T. parva and T. taurotragi sequences revealed species-specific regions within the LSU rRNA genes. A major species-variable region, located between the first and second conserved secondary structure domains of LSU rRNA, was identified. The sequence of the major species-variable region was determined for T. annulata, T. buffeli, and an unclassified Theileria species isolated from buffalo. Species-specific oligonucleotides complementary to LSU rRNA sequences were designed for five species of Theileria. The oligonucleotide probes discriminated between Theileria species when hybridized to amplified parasite LSU rRNA genes or directly to parasite rRNA. Three species-specific oligonucleotides derived from nonoverlapping sequences were developed for each of T. parva and T. taurotragi. A simplified procedure was developed in which T. parva- and T. taurotragi-specific oligonucleotides were used to discriminate the two species under nonstringent hybridization and washing conditions. Use of the nonstringent conditions also increased the sensitivity of target detection. Comparison of Theileria species-specific oligonucleotides derived from large subunit and small subunit rRNA genes demonstrated that, for closely related species, probes derived from LSU rRNA sequences had improved specificity.