Skip to main content Accessibility help

Generation and characterization of cloned Theileria parva parasites

  • S. P. Morzaria (a1), T. T. Dolan (a1), R. A. I. Norval (a1), R. P. Bishop (a1) and P. R. Spooner (a1)...


A 3-step procedure for cloning Theileria parva parasites was developed. The first step involved the in vitro infection of a fixed number of bovine lymphocytes with titrated sporozoites. The cell lines obtained from infections initiated using sporozoite/lymphocyte ratios below 1:100 were then selected for cloning as these contained schizont-infected cells, each of which was derived from infection with a single sporozoite. In the second step, these cell lines were cloned by limiting dilution. As sporozoites infect lymphocytes and transform to induce clonal multiplication, this step produced infected cell lines containing both cloned parasites and cloned lymphocytes. In the third step, the cloned cell lines were used to infect cattle and isolation of the parasite in ticks was made during piroplasm parasitaemia. Finally, sporozoites were harvested from infected ticks and used for further characterization. Sporozoites derived from cloned cell lines of T. parva Muguga, Marikebuni, Boleni, Uganda and buffalo-derived 7014 were characterized using monoclonal antibody profiles, DNA restriction fragment length polymorphism detected using repetitive and telomeric probes, in vivo infectivity and, in one case, cross-immunity studies. Additionally, several distinct schizont-infected lymphocyte clones were isolated from the Muguga, Mariakani and buffalo-derived 7014 stocks. The combined results of the characterization revealed that the cloning procedure selected clones of T. parva from the parental stocks which were known to contain a mixture of genetically different parasite populations. The cloning method and the clones generated will be of value in studies of the biology of the parasite and in elucidating the strain specificity of immune responses in cattle.



Hide All
Allsopp, B. A.Allsopp, M. T. E. P. (1988). Theileria parva: genomic DNA studies reveal intra-specific sequence diversity. Molecular and Biochemical Parasitology 28, 7784.
Anonymous (1989 a). Classification of Theileria parva reactions in cattle. In Theileriosis in Eastern, Central and Southern Africa (ed. Dolan, T. T.), pp. 187–8. Nairobi: ILRAD.
Anonymous (1989 b). Nomenclature in Theileria. In Theileriosis in Eastern, Central and Southern Africa (ed. Dolan, T. T.), pp. 182–3. Nairobi: ILRAD.
Bailey, K. P. (1960). Notes on the rearing of Rhipicephalus appendiculatus and their infection with Theileria parva for experimental transmission. Bulletin of Epizootic Diseases of Africa 8, 3343.
Bishop, R. P., Sohanpal, B. K., Allsopp, B. A., Spooner, P. R., Dolan, T. T. & Morzaria, S. P. (1993). Detection of polymorphisms among Theileria parva stocks using repetitive, telomeric and ribosomal DNA probes and anti-schizont monoclonal antibodies. Parasitology 107, 1931.
Bishop, R., Sohanpal, B., Kariuki, D. P., Young, A. S., Nene, V., Baylis, H., Allsopp, B. A., Spooner, P. R., Dolan, T. T. & Morzaria, S. P. (1992). Detection of a carrier state in Theileria Parva-infected cattle by the polymerase chain reaction. Parasitology 104, 215–32.
Bishop, R. P., Spooner, P. R., Kanhai, G. K, Kiarie, J., Latif, A. A., Hove, T., Masaka, S. & Dolan, T. T. (1994). Molecular characterization of Theileria parasites: application to the epidemiology of theileriosis in Zimbabwe. Parasitology 109, 573–81.
Brown, C. G. D. (1979). Propagation of Theileria. In Practical Tissue Culture Applications (ed. Maramorosch, K. & Hirumi, H.), pp. 223–54. New York: Academic Press.
Brown, C. G. D. (1981). Application of in vitro techniques to vaccination against theileriosis. In Advances in the Control of Theileriosis (ed. Irvin, A. D., Cunningham, M. P. & Young, A. S.), pp. 104–19. The Hague: Martinus Nijhoff.
Brown, C. G. D., Radley, D. E., Cunningham, M. P., Kirimi, I. M., Morzaria, S. P. & Musoke, A. J. (1977). Immunization against East Coast fever (Theileria parva infection of cattle) by infection and treatment: chemoprophylaxis with N-pyrrolidinomethyl tetracycline. Tropenmedizin und Parasitologie 28, 342–8.
Brown, C. G. D., Cunningham, M. P., Joyner, L. P., Purnell, R. E., Branagan, D., Corry, G. L. & Bailey, K. P. (1978). Theileria parva: significance of leukocytes for infecting cattle. Experimental Parasitology 45, 5564.
Buscher, G., Morrison, W. I. & Nelson, R. T. (1984). Titration in cattle of infectivity and immunogenicity of autologous cell lines infected with Theileria parva. Veterinary Parasitology 15, 2938.
Buscher, G. & Otim, B. (1986). Quantitative studies on Theileria parva in the salivary gland of Rhipicephalus appendiculatus adults: quantitation and prediction of infections. International Journal for Parasitology 16, 93100.
Clevers, H., MacHugh, N. D., Bensaid, A., Dunlap, S., Baldwin, C. L., Kaushal, A., Iams, K., Howard, C. J. & Morrison, W. I. (1990). Identification of a bovine surface antigen uniquely expressed on CD4-CD8-T cell receptor g/d+ T lymphocytes. European Journal of Immunology 20, 809–17.
Conrad, P. A., Iams, K., Brown, W. C., Sohanpal, B. & Moiyoi, O. Ole (1987). DNA probes detect genomic diversity in Theileria parva stocks. Molecular and Biochemical Parasitology 25, 213–26.
Conrad, P. A., Moiyoi, O. K. Ole, Baldwin, C. L., Dolan, T. T., O'callaghan, C. J., Njamunggeh, R. E. G., Grootenhuis, J. G., Stagg, D. A., Leitch, B. L. & Young, A. S. (1989). Characterization of buffalo-derived theilerial parasites with monoclonal antibodies and DNA probes. Parasitology 98, 179–88.
Cunningham, M. P., Brown, C. G. D., Burridge, M. J. & Purnell, R. E. (1973). Cyropreservation of infective particles of Theileria parva. International Journal for Parasitology 3, 583–7.
Cunningham, M. P., Brown, C. G. D., Burridge, M. J., Musoke, A. J., Purnell, R. E., Radley, D. E. & Sempebwa, C. (1974). East Coast fever: titration in cattle of suspensions of Theileria parva derived from ticks. British Veterinary Journal 130, 336–45.
Dolan, T. T., Teale, A. J., Stagg, D. A., Kemp, S. J., Cowan, K. M., Young, A. S., Groocock, C. M., Leitch, B. L., Brown, C. G. D. & Spooner, R. L. (1984). A histocompatibility barrier to immunization against East Coast fever using Theileria Parva-infected lymphoblastoid cell lines. Parasite Immunology 6, 243–50.
Ellis, J. A., Baldwin, C. L., MacHugh, N. D., Bensaid, A., Teale, A. J., Goddeeris, B. M. & Morrison, W. I. (1986). Characterisation by a monoclonal antibody and functional analysis of a subset of bovine T lymphocytes that express BoT8, a molecule analogous to human CD8. Immunology 58, 351–8.
Fawcett, D. W., Buscher, G. & Doxsey, S. (1982). Salivary gland of the tick vector of East Coast fever. III. The ultrastructure of sporogony in Theileria parva. Tissue and Cell 14, 183206.
Fawcett, D. W., Doxsey, S., Stagg, D. A. & Young, A. S. (1982). The entry of sporozoites of Theileria parva into bovine lymphocytes in vitro. Electron microscopic observations. European Journal of Cell Biology 27, 1021.
Feinberg, A. P. & vogelstein, B. (1983). A technique for radiolabelling DNA restriction fragments to high specific activity. Analytical Biochemistry 132, 613.
Fujisaki, K., Irvin, A. D., Voigt, W. P., Leitch, B. L. & Morzaria, S. P. (1988). The establishment of infection in the salivary glands of Rhipicephalus appendiculatus ticks by transplantation of kinetes of Theileria parva and the potential use of the method for parasite cloning. International Journal for Parasitology 18, 75–8.
Goddeeris, B. M., Katende, J. M., Irvin, A. D. & Chumo, R. S. C. (1982). Indirect fluorescent antibody test for experimental and epizootiological studies on East Coast fever (Theileria parva infection of cattle). Evaluation of cell culture schizont antigen fixed and stored in suspension. Research in Veterinary Science 33, 360–5.
Irvin, A. D. (1987). Characterisation of species and strains of Theileria. Advances in Parasitology 26, 145–79.
Irvin, A. D., Dobbelaere, D. A. E., Mwamachi, D. M., Minami, M., Spooner, P. R. & Ocama, J. G. R. (1983). Immunisation against East Coast fever: correlation between monoclonal antibody profiles of Theileria parva stocks and cross-immunity in vivo. Research in Veterinary Science 35, 341–6.
Kinuthia, S. W. (1987). Expression of parasite specific receptors on bovine lymphocyte target cells for Theileria parva. Ph.D. thesis, Université Libre de Bruxelles, Belgium.
Lalor, P. A., Morrison, W. I., Goddeeris, B. M., Jack, R. J. & Black, S. J. (1986). Monoclonal antibodies identify phenotypically and functionally distinct cell types in the bovine lymphoid system. Veterinary Immunology and Immunopathology 13, 121–40.
Lawrence, J. A. & Mackenzie, P. K. I. (1980). Isolation of a non-pathogenic Theileria of cattle transmitted by Rhipicephalus appendiculatus. Zimbabwe Veterinary Journal 11, 2735.
Maniatis, T., Fritsch, E. G. & Sambrook, J. (1982). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory.
Minami, T., Spooner, P. R., Irvin, A. D., Ocama, J. G. R., Dobbelaere, D. A. E. & Fujinaga, T. (1983). Characterisation of stocks of Theileria parva by monoclonal antibody profiles. Research in Veterinary Science 35, 334–40.
Morrison, W. I. (1988). A comparison of the responses of cattle inoculated with purified T or B lymphocytes infected with Theileria parva. In Annual Scientific Report, pp. 20. Nairobi: ILRAD.
Morrison, W. I., Buscher, G., Emery, D. L., Nelson, R. T. & Murray, M. (1981). The kinetics of infection with Theileria parva in cattle and the relevance to the development of immunity. In Advances in the Control of Theileriosis (ed. Irvin, A. D., Cunningham, M. P. & Young, A. S.), pp. 311–26. The Hague: Martinus Nijhoff.
Morzaria, S. P., Spooner, P. R., Bishop, R. P., Musoke, A. J. & Young, J. R. (1990). SfiI and NotI polymorphisms in Theileria stocks detected by pulsed field gel electrophoresis. Molecular and Biochemical Parasitology 40, 203–12.
Morzaria, S. P. & Young, J. R. (1992). Restriction mapping of the genome of the protozoan parasite Theileria parva. Proceedings of the National Academy of Sciences, USA 89, 5241–5.
Morzaria, S. P., Young, J. R., Spooner, P. R., Dolan, T. T., Young, A. S. & Bishop, R. P. (1992). Evidence of a sexual cycle in Theileria parva and characterisation of recombinants. In First International Conference on Tick-Borne Pathogens at the Host–Vector Interface –An Agenda for Research: Proceedings and Abstracts (ed. Munderloh, U. G. & Kurtti, T. J.), pp. 71–4. St Paul, Minnesota: University of Minnesota College of Agriculture.
Mukhebi, A. W., Perry, B. D. & Kruska, R. (1992). Estimated economics of theileriosis control in Africa. Preventive Veterinary Medicine 12, 7385.
Nelson, R. T. & Hirumi, H. (1982). In vitro cloning of Theileria-infected bovine lymphoblastoid cells: standardisation and characterisation. In Advances in the Control of Theileriosis (ed. Irvin, A. D., Cunningham, M. P. & Young, A. S.), pp. 120–1. The Hague: Martinus Nijhoff.
Pinder, M. & Hewett, R. (1980). Monoclonal antibodies detect antigenic diversity in Theileria parva parasites. Journal of Immunology 124, 1000–1.
Pipano, E. (1981). Schizonts and tick stages in immunisation against Theileria annulata infection. In Advances in the Control of Theileriosis (ed. Irvin, A. D., Cunningham, M. P. & Young, A. S.), pp. 242–52. The Hague: Martinus Nijhoff.
Pirie, H. M., Jarrett, W. F. H. & Crighton, G. W. (1970). Studies on vaccination against East Coast fever using macroschizonts. Experimental Parasitology 27, 343–9.
Radley, D. E. (1981). Infection and treatment method of immunisation against theileriosis. In Advances in the Control of Theileriosis (ed. Irvin, A. D., Cunningham, M. P. & Young, A. S.), pp. 227–37. The Hague: Martinus Nijhoff.
Radley, D. E., Brown, C. G. D., Burridge, M. J., Cunningham, M. P., Kirimi, I. M., Purnell, R. E. & Young, A. S. (1975). East Coast fever. 1. Chemoprophylactic immunisation of cattle against Theileria parva (Muguga) and five theilerial strains. Veterinary Parasitology 1, 3541.
Rodriguez, S. D., Buening, G. M., Green, T. J. & Carson, C. A. (1983). Cloning of Babesia bovis by in vitro cultivation. Infection and Immunity 42, 1518.
Rosario, V. E. (1981). Cloning of naturally occurring mixed infections of malaria parasites. Science 212, 1037–8.
Shapiro, S. Z., Fujisaki, K., Morzaria, S. P., Webster, P., Fujinaga, T., Spooner, P. R. & Irvin, A. D. (1987). A life-cycle stage-specific antigen of Theileria parva recognized by anti-macroschizont monoclonal antibodies. Parasitology 94, 2937.
Spooner, P. R. (1990). The effects of oxytetracyline on Theileria parva in vitro. Parasitology 100, 1117.
Tracer, W., Tershakovec, M., Lyandvert, L., Stanley, H., Lanners, N. & Gubert, E. (1981). Clones of the malaria parasites Plasmodium falciparum obtained by microscopic selection: their characterisation with regard to knobs, chloroquine sensitivity, and formation of gametocytes. Proceedings of the National Academy of Sciences, USA 78, 6527–30.
Toye, P. G., Goddeeris, B. M., Iams, K., Musoke, A. J. & Morrison, W. I. (1991). Characterisation of a polymorphic immunodominant molecule in sporozoites and schizonts of Theileria parva. Parasite Immunology 13, 4962.
Young, A. S., Leitch, B. L., Stagg, D. A. & Dolan, T. T. (1983). Identification of Theileria infections in living salivary glands of ticks. Parasitology 86, 519–28.


Related content

Powered by UNSILO

Generation and characterization of cloned Theileria parva parasites

  • S. P. Morzaria (a1), T. T. Dolan (a1), R. A. I. Norval (a1), R. P. Bishop (a1) and P. R. Spooner (a1)...


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed.