Cloning and characterisation of a repetitive DNA sequence from Theileria mutans: application as a species-specific probe.

Abstract

Repetitive DNA sequences were isolated from a Theileria mutans genomic library by screening with T. mutans total DNA. DNA sequence analysis demonstrated that a section of one of the clones contained a complex series of overlapping perfect repeats ranging between 99 and 20 bp in size. The T. mutans repetitive sequence did not contain large open reading frames (ORFs), unlike T. parva Tpr repetitive DNA sequences. When used as a hybridisation probe the repetitive sequence revealed restriction-fragment-length polymorphisms (RFLPs) between the EcoRI-digested DNAs of two T. mutans stocks. The T. mutans repetitive probe gave a signal with 1 ng of purified T. mutans piroplasm DNA and detected T. mutans sequences in whole-blood DNA isolated from an experimentally infected animal when the piroplasm parasitaemia was equal to or above 0.4%. Oligonucleotide primers derived from the repetitive sequence allowed more sensitive detection of T. mutans DNA by polymerase chain reaction (PCR) amplification. Using the PCR, T. mutans DNA was amplified from an experimentally infected animal with a parasitaemia of < 0.1%.