Improved characterization of Theileria parva isolates using the polymerase chain reaction and oligonucleotide probes

Theileria parva DNA was purified from piroplasms isolated from cattle infected with 5 different East African isolates of the parasite, including the East Coast fever reference stock T. p. parva Muguga. Total cellular DNA was prepared from T. parva schizont-infected bovine lymphoblastoid cell cultures (11 isolates). Two probes, previously isolated from T. p. parva Muguga repetitive genomic DNA, were hybridized to restriction digests; closely similar restriction fragment length polymorphism (RFLP) patterns were produced, and no two isolates had the same RFLP pattern. The DNA sequences of probe PMB3, two further copies of the repeated sequence from T. p. parva Muguga, and homologous regions from T. p. parva Kiambu 4 and T. p. lawrencei 3081, were determined. Oligonucleotides were synthesized corresponding to two conserved sections flanking a region which varied between isolates. These oligonucleotides were used as primers in the polymerase chain reaction to amplify the variable region. Further oligonucleotides corresponding to sequences in this variable region were able to distinguish between isolates and no sample hybridized to both oligonucleotides. This is the first unequivocal plus/minus discrimination between groups of isolates to be achieved for T. parva.