Baljinder Sohanpal

DNA probes detect genomic diversity in Theileria parva stocks

Different stocks of Theileria parva were analysed for restriction fragment length polymorphisms by agarose gel electrophoresis, orthogonal-field-alternation gel electrophoresis (OFAGE) and Southern hybridization with DNA probes. Polymorphisms seen with DNA from purified piroplasms of different T. parva stocks, after digestion with restriction enzymes, were more clearly apparent with OFAGE than with standard agarose gel electrophoresis. Genomic differences between these theilerial parasites were investigated further using three DNA probes, which were selected from a genomic library of T. parva (Muguga) piroplasm DNA cloned in lambda gt11. All three clones hybridized to T. parva DNA in preparations from schizont-infected bovine lymphoblastoid cells and to DNA from intraerythrocytic piroplasms. These probes did not, however, hybridize under high stringency conditions to DNA prepared from uninfected bovine lymphoblasts, T. mutans piroplasms, or bovine lymphoblasts infected with T. annulata or T. taurotragi. The five Kenyan stocks of T. parva that were tested showed characteristic hybridization patterns with these DNA probes. Our results show that DNA probes can be used to distinguish selected stocks of T. parva by hybridization to DNA either from intraerythrocytic piroplasms taken from infected cattle, or from isolates of schizont-infected bovine lymphoblastoid cells that are maintained continuously in vitro.

Discrimination betweenTheileria parva andT. taurotragi in the salivary glands ofRhipicephalus appendiculatus ticks using oligonucleotides homologous to ribosomal RNA sequences

Theileria parva, an apicomplexan protozoan parasite, causes a disease of cattle called East Coast fever (ECF), which is economically important in eastern, central and southern Africa (Norval et al. 1992). The main field vector of T. parva is the brown ear tick Rhipicephalus appendiculatus. T. taurotragi, a parasite of eland (Taurotragus oryx) that infects cattle (Young et al. 1977; Grootenhuis et al. 1979) and a wide range of wild ungulates (Stagg et al. 1983), is highly infective to R. appendiculatus (Young et al. 1980). Ticks from the field are often infected with T. taurotragi and T. parva (Morzaria 1989). Morphological differences between sporozoites of the two Theileria species are not reliably discernable using light microscopy. A method of distinguishing between T. parva and T. taurotragi infection in ticks is therefore essential for quantitative epidemiology studies and the development of mathematical models for theileriosis in Africa. Recently the development of oligonucleotides derived from ribosomal RNA (rRNA) sequences that can distinguish species of Plasmodium (Waters and McCutchan 1989) and Theileria (Allsopp et al. 1993) has been reported. Herein we describe the use of speciesspecific ribosomal oligonucleotides for discrimination between T. parva and T. taurotragi sporozoites by hybridisation to parasite rRNA in infected R. appendiculatus salivary glands. Uninfected nymphal R. appendicuIatus ticks were fed either on cattle experimentally infected with a sporozoite stabilate (3087) of T. parva Muguga (Brocklesby et al. 1961) or on an eland that had been bred in captivity and experimentally infected with T. taurotragi by ticks fed on the naturally infected eland 7695 (T. Dolan et al., unpublished data). Ticks were maintained according to Bailey (1960) at 24 ~ C until needed. Sporogony was induced by feeding ticks on rabbits for 4 days, and infected

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

Repetitive DNA sequences were isolated from aTheileria mutans genomic library by screening withT. 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. TheT. mutans repetitive sequence did not contain large open reading frames (ORFs), unlikeT. parva Tpr repetitive DNA sequences. When used as a hybridisation probe the repetitive sequence revealed restriction-fragment-length polymorphisms (RFLPs) between theEcoRI-digested DNAs of twoT. mutans stocks. TheT. mutans repetitive probe gave a signal with 1 ng of purifiedT. mutans piroplasm DNA and detectedT. 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 ofT. 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%.

Genetic fingerprinting of Theileria parva using a telomeric DNA probe

Abstract A cloned Theileria parva telomeric DNA sequence, designated pTpUtel, was used to characterize T. parva stocks and clones by hybridization to EcoRI-digested DNA. Eight of the nine T. parva stocks tested were discriminated by the telomeric restriction-fragment-length polymorphisms (RFLPs). Two isolates derived from buffalo 7014 by tick feeding on different occasions were also differentiated using the probe. The probe gave comparable results on purified piroplasm or schizont-infected lymphocyte DNA and did not cross-hybridize with uninfected bovine DNA. The telomeric restriction pattern of a cloned T. parva parasite remained identical after four passages through ticks and cattle. The telomeric sequence therefore represents a useful additional tool for analysis of theileriosis epidemiology.