The beta-tubulin gene of Babesia and Theileria parasites is an informativemarker for species discrimination

A fragment of the beta -tubulin gene was polymerase chain reaction (PCR) am plified from genomic DNAs of Babesia bovis, Babesia bigemina, Babesia diver gens, Babesia major, Babesia caballi, Babesia equi, Babesia microti, Theile ria annulata and Theileria sergenti, Single amplification products were obt ained for each of these species, but the size of the amplicons varied from 310 to 460 bp, Sequence analysis revealed that this variation is due to the presence of a single intron, which ranged from 20 to 170 bp. The extensive genetic variability at the beta -tubulin locus has been exploited to devel op two types of species identification assays. The first assay can be used on samples containing mostly parasite DNA, like those prepared from infecte d erythrocytes. Following PCR amplification, the species identification is obtained directly from the size of the products (for Babesia species infect ing human or horse) or using a simple PCR-restriction fragment length polym orphism (RFLP) protocol (for Babesia species infecting cattle). The second assay can be used on samples prepared from whole blood, that contain both p arasite and host DNAs. In this case, due to the strong conservation of the beta -tubulin gene, co-amplification of a gene fragment from the host DNA w as observed. A nested PCR assay was developed for the specific amplificatio n of parasite DNA, using a primer designed to span the exon-intron boundary . Direct identification of Babesia species infecting human and horse is aga in obtained after the electrophoretic separation of the amplification produ cts, while for Babesia and Theileria species infecting cattle, differentiat ion is based on a nested PCR-RFLP protocol. These methods may be used for t he simultaneous identification of horses and cattle carrying multiple paras ites by means of a single PCR or using the PCR-RFLP protocol. (C) 2000 Publ ished by Elsevier Science Ltd. on behalf of the Australian Society for Para sitology.