Vaccines against Theileria parva

Vaccines against Theileria parva SUBHASH MORZARIA,a VISH NENE, RICHARD BISHOP, AND ANTHONY MUSOKE International Livestock Research Institute, P.O. Box 30709, Nairobi, Kenya

ABSTRACT: Bovine theileriosis caused by Theileria parva continues to be a major economic problem in many parts of Eastern, Southern, and Central Africa. Due to the unsustainable nature of the present control method—using toxic acaricides to kill ticks—alternative control methods are being sought. Live vaccines are being used in many countries in the region. These vaccines are based on the infective sporozoite stage of the parasite. Sporozoites are inoculated in cattle with simultaneous administration of a long-acting formulation of oxytetracycline. These vaccines are poorly adopted in the region, mainly because of problems associated with the use of live parasites. An experimental recombinant vaccine based on a sporozoite surface antigen (p67) has been developed. Immunization with this antigen induces neutralizing antibodies and, under laboratory conditions, this technique protects approximately 70% of the immunized cattle to a defined needle challenge. The efficacy of the vaccine is currently being evaluated under field challenge in Kenya. Since a vaccine based on a single antigen may not be sustainable under field conditions, a search for schizont antigens that induce protective cell-mediated immune responses continues. It is expected that the ultimate vaccine against theileriosis will incorporate a mixture of several antigens derived from both sporozoite and schizont stages, contributing to robust immunity.

INTRODUCTION Theileria parva, a tick transmitted protozoan parasite, causes a severe lymphoproliferative disease in cattle variously known as East Coast fever, January disease or corridor disease. The main vector of the parasite is a three-host tick Rhipicephalus appendiculatus. The disease is present in 10 countries in Eastern and Central Africa, however the vector is more widely distributed than the parasite and there is a potential danger of the disease spreading to other areas. Theileria parva is highly pathogenic to cattle and mortality in Bos taurus cattle and their crosses can approach 100%. The parasite is considered to be the major factor in inhibiting the introduction of highly productive taurine breeds of cattle in subSaharan Africa. Where such cattle are maintained successfully in Africa, it is through heavy cost to the farmer, involving the use of toxic and expensive chemicals to kill the vector. Estimated annual losses are around US$190 million.1 The most common form of the disease in cattle due to T. parva is East Coast fever (ECF). It is characterized by high mortality in exotic cattle and large numbers of schizonts and piroplasms. A mild form of the disease, known as January disease, occurs aAddress for correspondence. Voice: +254-2-630743; fax: +254-2-631499. [email protected]

464

MORZARIA et al.: VACCINES AGAINST THEILERIA PARVA

465

predominantly in Zimbabwe. This disease occurs seasonally, and the outbreaks, coinciding with the emergence from diapause of adult ticks in January, are usually characterized by high morbidity and low mortality with low numbers of parasites. The main vector of January disease is Rhipicephalus zambesiensis. The African buffalo plays an important role in the epidemiology of the disease since it is a natural host that does not suffer from the disease but remains a constant source of infection for ticks. The disease that occurs in cattle after the introduction of T. parva from buffalo is referred to as corridor disease, and is characterized by high mortality and low parasitosis.

IMMUNITY TO ECF Cattle that recover from ECF develop solid, strain-specific immunity that lasts for several years.2 Experiments have shown that passive transfer of antibodies to the schizont and piroplasm stages from immune to naïve cattle is not protective. There is now strong evidence that protective immunity is mediated by the generation of MHC class I restricted cytotoxic T cells (CTLs) specific for schizont-infected lymphocytes.3 This has been supported by the demonstration that passive transfer of CD8+ enriched cells from the responding lymph to its naïve twin can protect against challenge4 and there is also good correlation between appearance of CTLs in blood and clearance of schizont parasitemia. By analyzing cellular responses to different parasite isolates it appears that lack of cross protection is due to the specificity of the CTL response to infection and the occurrence of parasite antigenic diversity.5 There is also a hierarchy in the restricting MHC class I allele and, thus, bias in the crossprotective capacity.6 Apart from the CD8+ CTL responses, CD4+ T cell proliferative responses to schizont-infected cells and cell lysates can be detected in immunized cattle. There is evidence that CD4+ T cell help is required for induction and recall of parasitespecific CTL and some T helper cells have been shown to exhibit cytolytic activity comparable to CTLs but their protective role is unknown7 Other cell types such as γδ and NK cells have been shown to respond to T. parva infection and challenge of immune cattle, but their role in immunity remains to be determined.8 Recent studies have shown that antibodies against the sporozoite stage of the parasite are generated in animals that are experimentally as well as naturally infected.9 These antibodies, which are of the IgG2 subclass, neutralize sporozoite infectivity to lymphocytes in vitro and thus may contribute to immunity.10 Vaccines against ECF have been considered the most sustainable way of controlling the disease. Two vaccines have been developed over the years; a live vaccine referred to as the infection and treatment method (infection with sporozoites and treatment with an antitheilerial drug), and a subunit experimental vaccine based on a sporozoite specific antigen. Both are described in this paper.

466

ANNALS NEW YORK ACADEMY OF SCIENCES

IMMUNIZATION BY THE INFECTION AND TREATMENT METHOD The observation that cattle can be immunized by prolonged (30 days) Aureomycin (Cyanamid) treatment during tick induced T. parva infection,11 formed the basis for the development of the infection and treatment method of immunization. Several researchers have improved the method of producing mild infections in cattle by using laboratory-reared, experimentally infected ticks to challenge cattle while under drug cover. However, one of the difficulties in immunizing by this method was that the infectivity of ticks could not be critically evaluated and thus the method was not totally reliable. An important breakthrough in T. parva vaccine research has been the development of a method for harvesting and cryopreserving sporozoites.12 This has enabled cattle to be infected with a uniform and reproducible dose of sporozoite stabilates. Brown et al.13 were the first to exploit this method to successfully immunize cattle with a defined, potentially lethal sporozoite challenge and four daily treatments of N-pyrrolidinomethyl tetracycline. This method of immunization was further refined with the introduction of a long acting oxytetracycline (TLA) (Terramycin, Pfizer, LA), enabling the use of a single dose of the drug to be given concurrently with a sporozoite challenge to induce solid immunity.14 The method, as it is currently used for immunizing cattle against T. parva, involves the use of selected stock(s) of parasite stabilates, inoculated subcutaneously over a lymph node (usually parotid or prescapular) and a simultaneous intramuscular injection of TLA at 20 mg/ kg body weight. The TLA provides therapeutic cover for up to five days. Several formulations of long acting oxytetracyclines are available through various commercial companies. However, only a few laboratories produce vaccine stabilates that are well characterized, quality controlled, and in sufficient quantities for large scale immunization. The protective immunity engendered through the infection and treatment method of immunization is cell mediated, principally effected by CD8+ cytotoxic T lymphocytes. The immunity is effective against high doses of homologous challenge and, in absence of further challenge, the immunity lasts for over 36 months.2 A number of immunizing stocks have been developed over the years and are being used in various parts of ECF endemic areas. The Katete stock has been used successfully in the Eastern Province of Zambia. The Marikebuni stock that was isolated from the Kilifi District of Kenya protects against a large number of stocks from Eastern Africa and is being sold as a vaccine stock by a commercial company in Kenya. The Boleni stock from Zimbabwe generally produces mild infection and provides wide protection against a number of strains from Eastern and Central Africa. The Boleni stock is unique among the immunizing parasites because it does not require the use of oxytetracyclines. Currently the stock is being routinely used for immunization of cattle against January disease in Zimbabwe. The Serengetitransformed, Kiambu 5, and Muguga stocks have been combined to produce a combination referred to as “Muguga cocktail” that provides wider protection than any of the three stocks used individually. The cocktail has been used in cattle in Malawi, Zambia, Uganda, and Tanzania.

MORZARIA et al.: VACCINES AGAINST THEILERIA PARVA

467

Important Factors Affecting the Efficacy of the Infection and Treatment Method Immunizing Stocks Theileria parva stocks comprise a mixture of parasite genotypes; the quality and spectrum of protection provided by a stock depend on the immunogenic composition of the various populations in a stock. In cross immunity studies that have been described, it is interesting to note that only a small proportion of immunized animals (between 25–30%) undergo severe disease (breakthrough) when challenged with a different immunogenic stock.15 Since the combination of parasites that is obtained at any given time is random and no one parasite stock is identical to another, the capacity of immunizing parasites to protect against various field populations may vary greatly. For example, the Marikebuni stock protects against Muguga and Mariakani challenges, whereas the Boleni stock protects against Muguga and Marikebuni challenges, but not against the Mariakani stock.16 It is clear that selection of an immunizing stock becomes difficult since a number of expensive cross-immunity tests have to be performed to identify a stock that will protect against a large number of T. parva stocks. Even if a stock is selected carefully in this manner, it may not provide the expected protection when used in the field. Thus, due to dynamic nature of parasite populations in the field the selection of immunizing stocks based on limited cross-immunity and field trials is not totally reliable. Response to Tetracycline Some parasite stocks cannot be controlled by one dose of long-acting oxytetracycline to produce a mild ECF reaction. This is may be due to a high immunization dose of sporozoites and/or to the virulence of parasite stock. Determining an optimum immunization dose requires titration of the candidate stabilate against a fixed dose of the drug in cattle. However, there is evidence that dilution of a stabilate may result in loss or reduction in some important immunizing parasite components from the original stock. This has been known to happen in both the Muguga and Marikebuni stocks. Generally it will be difficult to consistently achieve an ideal immunization dose of a stabilate that will not cause severe reactions and yet stimulate 100% protection. A compromise, to err on the side of either safety or efficacy, may have to be accepted in such circumstances. Cattle Breed Effects Susceptibility to T. parva infection varies among cattle breeds. It is known that Bos indicus cattle are more resistant to infection than the Bos taurus breeds. This is often reflected in the way different breeds respond to the infection and treatment immunization. The optimum immunizing stabilate dose determined for Boran cattle may produce severe ECF reaction in exotic cattle. This factor needs to be kept in mind when developing and applying the vaccine in the field. Conversely, an immunization dose selected for Bos taurus cattle may be inadequate for the Bos indicus type of cattle in that the response to some of the components of the stabilate may be reduced or excluded in these breeds.

468

ANNALS NEW YORK ACADEMY OF SCIENCES

Age, Weight and Dose Related Effects It has been observed that ECF reactions observed in cattle may vary according to the age of animals. For example, an approximate LD70 determined for T. parva Muguga stock (ILRI stabilate 3087) in young Boran cattle 3–6 months of age, was altered to an approximate LD 60 when used in cattle over one year of age (S. Morzaria, A. Musoke, and T. Dolan, unpublished observation). It is not clear if this difference in infectivity was due to different age susceptibility, or due to the different dose of stabilate given in relation to the size of the animals. This needs further investigation since it could have an impact on the development and application of the infection and treatment method of immunization. Carrier State and Sexual Recombination Animals that recover following infection with T. parva and after infection and treatment immunization become carriers.17 Thus, there is a possibility that the live immunization method may introduce new strains that might break through the animals immune to local parasite stocks. Recently, it has been shown that sexual cycle is obligatory in the development of T. parva and that genetic polymorphism occurs due to recombination.18 This raises the possibility that the immunizing parasites may recombine with the local parasite with the emergence of several new genotypes. Studies need to be performed in order to determine the biological impact of introducing new parasite stocks in the epidemiology of ECF and long term efficacy of live vaccines.

VARIATIONS OF INFECTION AND TREATMENT IMMUNIZATION METHOD Infection and Treatment using Antitheilerial Naphthoquinones When parasites cannot be controlled with oxytetracyclines, other antitheilerial drugs provide options for immunization. The two drugs that have been extensively investigated are parvaquone (2-cyclohexyl-3-hydroxy-1,4-naphthoquinone) and buparvaquone (2-hydroxy-3-(trans-4-t-butylcyclohexylmethyl)1,4-naphthoquinone). The former drug is used in animals that are infected with an immunizing parasite stock and then treated early in the clinical disease. This treatment at 20 mg/kg bw is usually instituted when infected animals show two days of pyrexia and schizont parasitosis.19 Similarly, buparvaquone at 2.5 mg/kg can also be used to treat cattle early in the infection. One of the disadvantages of this method is that immunized animals have to be monitored closely for pyrexia and schizont parasitosis to ensure that the treatment is not delayed. Buparvaquone has also been used in the infection and treatment method in the same way as TLA. Buparvaquone is given at 2.5 mg/kg BW simultaneously with the immunizing stabilate. This method needs further investigation because the drug can prevent the establishment of infection in immunized animals and such animals remain susceptible to further T. parva challenge.

MORZARIA et al.: VACCINES AGAINST THEILERIA PARVA

469

Immunization of Cattle by Exposure to Natural Challenge While under Long Acting Oxytetracycline Cover This approach to immunization can be used in areas where supply of stabilate is difficult due lack of adequate infrastructure. The method involves natural exposure of cattle to tick challenge during a period of 4–5 weeks while they are under TLA treatment, given once a week at the dose of 20mg/kg.20 One of the advantages of this method is that animals are immunized by the local stocks that exist in the area and thus there is no danger of introducing new immunological stocks. However, the method is empirical in that, for animals to develop immunity, it is imperative that they get exposed to T. parva challenge during drug cover. This cannot be guaranteed, especially in areas where tick/parasite challenge is seasonal and not consistent.

IMMUNIZATION WITH THE RECOMBINANT ANTIGEN P67 In addition to the cellular immunity acquired due to infection there is also an antibody response to sporozoite antigens. Multiple infections lead to development of antibodies that neutralize the establishment of schizont infected cell line in vitro, an activity that is present in sera collected from cattle in ECF endemic areas. By development of monoclonal antibodies (mAb) it has been shown that sporozoite neutralizing antibodies detect a 67-kD protein.21 This molecule is the major component of the sporozoite surface and is not expressed in either schizonts or piroplasms.21 Immunization with recombinant forms of this molecule (r-p67), derived from either bacterial or insect cells, induces high levels of sporozoite neutralizing antibodies in cattle. In vivo immunization studies have shown that approximately 30% of immunized cattle are nonreactors to a needle LD70 sporozoite challenge, 40% experience a mild disease reaction from which they recover, and the remaining 30% suffer severe disease and are clinically indistinguishable from controls.9 Transient parasitosis and mild clinical reactions are also observed in challenge of cattle immunized by infection and treatment and such responses constitute immunity to ECF. Hence, p67 is able to routinely induce immunity at a level of about 70%. An important aspect in developing an antisporozoite vaccine that has probably contributed to the success of this approach, although the protective capacity of naturally acquired p67 immune responses under field conditions is unknown, is that the severity of ECF is dependent on sporozoite dose. Thus, a neutralizing antibody response has to only reduce the exposure to sporozoites, but not eliminate it totally. In fact the mild disease reaction is desirable because the recovery phase is associated with development of a CTL response. These animals will, therefore, contain protective responses directed against both sporozoites and schizonts. The antibody response to r-p67 has been studied in some detail and data have been accumulated from more than 300 cattle. All cattle develop high levels of ELISA antibody titers to p67 (titers range between 1: 60,000 and 1: 640,000) and have the capacity to neutralize sporozoite infectivity in vitro (titers range between 1: 500 and 1 : 1000) but there is no correlation between the level of antibody, the isotype response, and immunity to ECF.21 CD4+ T cell lines and clones that respond to r-p67 are being established, but it is not yet known if these relate to immunity.

470

ANNALS NEW YORK ACADEMY OF SCIENCES

The peptide specificity of six of seven sporozoite neutralizing mAbs has been determined by Pepscan analysis using synthetic overlapping peptides of p67. Together, the mAbs identified five epitopes, three located between residues 105 and 229, and two between 617 and 639 of p67, suggesting that these sequences are exposed on the sporozoite surface.21 Bovine antibodies to one of these peptides neutralized sporozoite infectivity, confirming the validity of this approach for defining the potential relevance of the response to immunity. A pepscan analysis was also carried out with sera from animals inoculated with r-p67. Bovine anti-p67 sera from 16 cattle that were immune to sporozoite challenge, was tested individually and compared with samples from 15 cattle that were susceptible to challenge. Antibody reactivity was higher in sera from cattle that were immune to ECF, compared to those that were susceptible, but the differences were not statistically significant. The central region of p67, residues 313–583, appeared to be nonantigenic as far as the presence of linear peptide epitopes is concerned. The pepscan data has indicated that the bovine antibody response to linear epitopes identified by sporozoite inhibitory mAbs was generally poor. Thus, it may be possible to improve the efficacy of the experimental vaccine by focusing the antibody response to these epitopes. An interesting aspect of the p67 based vaccine is that, in contrast to the antigens that are the target of CTLs, the sporozoite antigen appears to be invariant among parasites isolates maintained in cattle. Indeed, cross-protection experiments have shown that cattle immunized with r-p67 derived from the Muguga stock are protected against the immunologically different Marikebuni stock as well as the homologous stock. Polymorphism in p67 is, however, evident in parasites isolated from buffalo,22 the wildlife reservoir, although the relevance of this to immunity is unknown. Ongoing field trials being carried out in different epidemiological situations will reveal the level of protection against natural tick challenge and suitability of the p67based vaccine against ECF. However, it is clear that for a robust vaccine a combination of antigens that invoke both humoral as well as cell mediated immune responses is desirable.

VACCINE STRATEGIES UNDER DEVELOPMENT Identification of Genes Encoding Schizont Antigens Recognized by CTLs For the induction of effective and robust immunity against Theileria parva it would be desirable to induce immune responses against both the sporozoite and schizont stages. Schizont-specific bovine CTLs recognize parasite antigens expressed as peptides complexed with bovine MHC class I molecules on the surface of the infected cell. Thus, assays need to be developed that detect these antigens and have to be based on engaging the T cell receptor on the CTL in either cytolytic or cytokine release assays. Two approaches can be adopted for identifying schizont antigens carrying CTL epitopes. The first is to start with a defined protein and to ask whether it contains CTL epitopes and the other is to use parasite specific CTLs and to use these to determine the epitopes bound by the CTLs. The first requires the capacity to induce an antigen CTL response followed by an assay to detect the response on parasitized host cells. One of the ways of achieving this is to use a live delivery system such as

MORZARIA et al.: VACCINES AGAINST THEILERIA PARVA

471

vaccinia virus for candidate antigens. The second approach is complicated since several thousand parasite molecules need to be screened in order to identify the CTL antigen. Currently, DNA based and protein based approaches are being undertaken in an attempt to identify schizont antigens using parasite specific CTLs. The first approach involves the transfection of parasite DNA into an appropriate antigen presenting cell (APC). Following parasite gene expression in the APC, the target cells are subjected to CTL assay. Once a cloned parasite DNA molecule is found to sensitize the APC then it can be used as above in recombinant vaccinia virus to test for CTL induction and subsequent protection. The second approach requires sophisticated protein biochemistry involving the physical isolation of peptides from schizont-infected cells, fractionation of these peptides by HPLC, testing of the fractions for ability to sensitize APC to lysis by CTLs and then performing peptide sequence analysis to determine the sequence of the relevant peptide. Once identified, synthetic oligonucleotides can be made in order to clone the full length gene encoding the peptide, alternatively minigene constructs can be made in vaccinia virus. Both approaches have been successfully used in the identification of tumor antigens expressed in the context of MHC class I molecules.23

CONCLUSIONS Two methods of immunization against T. parva infection are described. The infection and treatment method is now being used in many parts of Eastern, Central and Southern Africa. The most important component of this method is the availability of well characterized stabilates. For the production of stabilates on a commercial scale a number of issues need to be considered such as safety, standards, and quality assurance. The infection and treatment method works if used properly under supervision. However, a number of logistic problems exist in the delivery of stabilates. Of these, probably the most important is the requirement for a cold chain, which is not practical in the context of poorly developed infrastructure in the area where the vaccine is most needed. The second method of immunization described is based on a recombinant antigen. This is still an experimental vaccine and is under development. The fact that animals can be protected using a subunit vaccine provides cause for optimism. In the long term, it is possible that other parasite antigens that induce protective immunity may need to be included in the vaccine. In this regard the schizont antigens that are the targets of CTL responses are prime vaccine candidates.

ACKNOWLEDGMENT This is ILRI publication number 990145. REFERENCES 1. M UKHEBI, A.W., B.D. P ERRY & R. K RUSKA. 1992. Estimating economic losses caused by theileriosis and the economics of its control in Africa. Prev. Vet. Med. 12: 73–85.

472

ANNALS NEW YORK ACADEMY OF SCIENCES

2. B URRIDGE, M.J., S.P. M ORZARIA, M.P. C UNNINGHAM & C.G.D. B ROWN. 1972. Duration of immunity to East Coast fever (Theileria parva infection of cattle). Parasitol. 64: 511–515. 3. M ORRISON, W.I., B.M. G ODDEERIS, A.J. T EALE, C.M. G ROOCOCK, J.S. K EMP & D.A. S TAGG. 1987. Cytotoxic T-cell elicited in cattle challenged with Theileria parva (Muguga): evidence for restriction by class I MHC determinant and parasite-strain specificity. Parasite Immunol. 9: 563–578. 4. M CK EEVER, D.J., E.L.N. T ARACHA, E.L. I NNES, N.D. M ACH UGH, E. A WINO, B.M. G ODDEERIS & W.I. M ORRISON. 1994. Adoptive transfer of immunity to Theileria parva in the CD8+ fraction of responding efferent lymph. Proc. Nat. Acad. Sci. USA 91: 1959–1963. 5. T ARACHA, E.L., B.M. G ODDEERIS, S.P. M ORZARIA & W.I. M ORRISON. 1995. Parasite strain specificity of bovine cytotoxic T cell responses to Theileria parva is determined primarily by immunodominance. J. Immunol. 155: 4854–4860. 6. G ODDEERIS, B.M., P.G. T OYE & W.I. M ORRISON, 1990. Strain specificity of bovine Theileria parva-specific cytotoxic T cells is determined by the phenotype of the restricting class I MHC. Immunology 69: 38–44. 7. B ALDWIN, C.L., K.P. I AMS, W.C. B ROWN & D.J. G RAB. 1992. Theileria parva: CD4+ helper and cytotoxic T-cell clones react with a schizont-derived antigen associated with the surface of Theileria parva-infected lymphocytes. Exp. Parasitol. 75: 19–30. 8. B ALDWIN, C.L., S.J. B LACK, W.C. B ROWN, P.A. C ONRAD, B.M. G ODDEERIS, S.W. K INUTHIA, P.A. L ALOR, N.D. M ACH UGH, W.I. M ORRISON, S.P. M ORZARIA, J. N AESSENS & J. N EWSON. 1988. Bovine T. cells, B cells, and null cells are transformed by the protozoan parasite Theileria parva. Infect. Immun. 56: 462–467. 9. M USOKE, A.J., S.P. M ORZARIA, C. N KONGE, E., J ONES & V. N ENE. 1992. A recombinant sporozoite surface antigen of Theileria parva induces protection in cattle. Proc. Nat. Acad. Sci. USA 89: 514–518. 10. M USOKE, A.J., V.M. N ANTLYA, G. B USCHER, R.A. M ASAKE & B. O TIM. 1982. Bovine immune responses to Theileria parva: neutralising antibodies to sporozoites. Immunology 45: 663–668. 11. N EITZ, W.O. 1953. Aureomycin in Theileria pava infection. Nature 171: 34–35. 12. C UNNINGHAM, M.P., C.G.D. B ROWN, M.J. B URRIDGE & R.E. P URNELL. 1973. Cryopreservation of infective particles of Theileria parva. Int. J. Parasitol. 3: 583–587. 13. B ROWN, C.G.D., D.E. R ADLEY, M.P. C UNNINGHAM, I.M. K IRIMI, S.P. M ORZARIA & A.J. M USOKE. 1977. Immunization against East Coast fever (Theileria parva infection of cattle) by infection and treatment: Chemoprophylaxis with N-pyrrolidinomethyl tetracycline. Tropenmed. Parasitol. 28: 342–348. 14. R ADLEY, D.E. 1981. Infection and treatment method of immunization. In Advances in the Control of Theileriosis. A.D. Irvin, M.P. Cunningham & A.S. Young, Eds.: 227– 237. Martinus Nijhoff, The Hague. 15. R ADLEY, D.E., C.G.D. B ROWN & M.J. B URRIDGE. 1975. East Coast Fever. 1. Chemoprophylactic immunisation of cattle against Theileria parva (Muguga) and five Theileria strains. Vet. Parasitol. 1: 35–41. 16. I RVIN, A.D., S.P. M ORZARIA, F.C. M UNATSWA & R.A.I. N ORVAL. 1989. Immunization of cattle with a Theileria parva bovis stock from Zimbabwe protects against challenge with virulent T. p. parva and T. p. lawrencei stocks from Kenya. Vet. Parasitol. 32: 271–278. 17. Y OUNG, A.S., B.L. L EITCH & R.M. N EWSON. 1981. The occurrence of Theileria parva carrier state in cattle from an East Coast fever endemic area of Kenya. In Advances in the Control of Theileriosis. A.D. Irvin, M.P. Cunningham & A.S. Young, Eds.: 60–62. Martinus Nijhoff, The Hague. 18. M ORZARIA, S.P., J.R. Y OUNG, P.R. S POONER, T.T. D OLAN, A.S. Y OUNG & R.P. B ISHOP. 1992. Evidence of a sexual cycle in Theileria parva and characterisation of recombinants. In First International Conference on Tick-borne Pathogens at the HostVector Interface—An agenda for Research: Proceedings and Abstracts. U.G. Munderloh & T.J. Kurtti, Eds.: 71–74. University of Minnesota College of Agriculture, St. Paul, Minnesota.

MORZARIA et al.: VACCINES AGAINST THEILERIA PARVA

473

19. D OLAN, T.T., A. L INYONYI, S.K. M BOGO & A.S. Y OUNG. 1984. Comparison of longacting oxytetracycline and parvaquone in immunization against East Coast fever by infection and treatment. Res. Vet. Sci. 37: 175–178. 20. C HUMO, R.S., A.D. I RVIN, S.P. M ORZARIA, J. K ATENDE & R.E. P URNELL. 1989. Longacting oxytetracycline prophylaxis to protect susceptible cattle introduced into an area of Kenya with endemic East Coast fever. Vet. Rec. 124: 219–222. 21. N ENE, V., E. G OBRIGHT, R. B ISHOP, S. M ORZARIA & A. M USOKE. 1999. Linear peptide specificity of bovine antibody responses to p67 of Theileria parva and sequence diversity of sporozoite-neutralizing epitopes: implications for a vaccine. Infect. Immun. 67(3): 1261–1266. 22. N ENE, V., A. M USOKE, E. G OBRIGHT & S. M ORZARIA. 1996. Conservation of the sporozoite p67 vaccine antigen in cattle-derived Theileria parva stocks with different crossimmunity profiles. Infect. Immun. 64: 2056–2061. 23. C OX, A.L., J. S KIPPER, Y. C HEN, R.A. H ENDERSON, T.L. D ARROW, J. S HABANOWITZ, V.H. E NGELHARD, D.F. H UNT & C.L. S LINGLLUFF, J R. 1994. Identification of a peptide recognised by five melanoma-specific cytotoxic T cell lines. Science 264: 716– 719.