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. Al ignment of the T. parva and T. taurotragi sequences revealed species-s pecific regions within the LSU rRNA genes. A major species-variable re gion, located between the first and second conserved secondary structu re domains of LSU rRNA, was identified. The sequence of the major spec ies-variable region was determined for T. annulata, T. buffeli, and an unclassified Theileria species isolated from buffalo. Species-specifi c oligonucleotides complementary to LSU rRNA sequences were designed f or five species of Theileria. The oligonucleotide probes discriminated between Theileria species when hybridized to amplified parasite LSU r RNA genes or directly to parasite rRNA. Three species-specific oligonu cleotides derived from nonoverlapping sequences were developed for eac h of T. parva and T. taurotragi. A simplified procedure was developed in which T. purva- and T. taurotragi-specific oligonucleotides were us ed to discriminate the two species under nonstringent hybridization an d washing conditions. Use of the nonstringent conditions also increase d the sensitivity of target detection. Comparison of Theileria species -specific oligonucleotides derived from large subunit and small subuni t rRNA genes demonstrated that, for closely related species, probes de rived from LSU rRNA sequences had improved specificity. (C) 1995 Acade mic Press, Inc.