Prospects for subunit vaccines against tick borne diseases

BI: vet.J. (1996). 152, 621

REVIEW P R O S P E C T S F O R S U B U N I T VACCINES AGAINST TICK-BORNE DISEASES

A.J. MUSOKE, G. H. PALMER*, T. F. McELWAIN*, V. NENE and D. McKEEVER International l.ivestod¢ Research Institute, P.O. Box 30709, Nairobi, Kenya; and * Department o/Microbiolokq, and Pathologl,, Washington State Lhtiw,rsit~,, Pullman, USA

SUMMARY

Tick-borne parasites are a serious i m p e d i m e n t to the improvement of livestock production in the developing world. The major parasites affecting cattle inclnde Theileria parva, T. annulata, Babesia bigemina, B. bovis, Anaplasma marginale and Cowdda ruminantium. Tile control of these infections is d e p e n d e n t on the use of acaricides to decrease transmission by the tick vectors, and immunization of susceptible animals with live vaccines. The use of acaricide is h a m p e r e d by the development of resistance, and live vaccines require cold chain facilities, which are generally unreliable in developing countries. There is therefore a n e e d for improved vaccines that can circumvent these problems. There is a subunit vaccine being developed for 7: pa~va based on the major surface antigen of the sporozoite (p67). A similar antigen, SPAG 1, has been identified as a candidate for 7: annulata. Although several candidate antigens have been identified for Babesia spp., progress towards development of a subunit vaccine based on these antigens has been h a m p e r e d by polymorphism a m o n g isolates and between species, and lack of knowledge of the i m m u n e effector mechanisms responsible for protection. The search for protective antigens of A. mmginale has focused on outer m e m b r a n e proteins; immunization with a variety of these antigens alone or in combination, has yielded promising results. As with Babesia, fllrther definition of i m m u n e effector mechanisms is n e e d e d to optimize immunization strategies. The work on identifying the protective antigens of C. ruminantium is in its embryonic stages; however, two antigens have been identified and are currently being evaluated. There is high expectancy for subunit vaccines for all these diseases; however there is n e e d for further work to elucidate the i m m u n e mechanisms in order to select appropriate antigen delivery systems. Kz~a,opms: Subunit; vaccine; Theileria; Babesia; rickettsia.

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© 1996 Bailli6reTindall

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INTRODUCTION

Development of the livestock industry in many third world countries has been severely hindered by tick-borne diseases. The major protozoan pathogens responsible for these diseases in cattle are Theileria pmva and Theilevia annulata, which cause East Coast fever (ECF) and tropical theileriosis, and Babesia b~gemina and Babesia boris, which cause redwater fever. The rickettsial organisms Cozodria ruminantium and Anaplasma mm~nale are responsible for heartwater and anaplasmosis. The life cycles and biology of these parasites have been described in detail in various publications (see reviews by Ristic, 1977; Uilenberg, 1983; Young & Morzaria, 1986; hwin & Morrison, 1987) and will not be discussed here. East Coast fever is of major economic importance throughout eastern, central and southern Africa, with annual losses in 1989 estimated at U.S.$168 million (Mukhebi et al., 1992). 71 annulata is of even greater importance and occurs frolTl north Africa to China (see review by Ii-vin & Morrison, 1987). There are no recent figures for losses due to 71 annulata, but approximately 500 million cattle are at risk of contracting the disease. Anaplasmosis and babesiosis are the most prevalent of the haemoparasitic infections, with enzootic regions on all six continents (Losos, 1986a, b). Losses due to A. mmg4nale, (Goodger et al., 1979), B. bigemina and B. bovis arise from high morbidity and mortality, the cost of control and treatment, and constraints to the livestock importation trade. C. ntminantium is widespread in sub-Saharan Africa and has been introduced to the Caribbean islands (see review by Uilenberg et al., 1993). The close proximity of these islands to the United States of America and South and Central Alnerica creates a threat of the spread of the disease to these countries. The disease is currently ranked third in importance behind theileriosis and trypanosomiasis in Africa but its economic impact has not been evaluated. All of the above tick-borne pathogens constitute a major impediment to the introduction of more productive Bos taurus cattle into endemic areas because of their high susceptibility to infection. This review will focus on recent research towards the development of subunit vaccines against these diseases and comment the complications of the introduction of such vaccines in the field.

CURRENT METHODS OF C O N T R O L OF TICK-BORNE DISEASES

The prevailing strategy for the prevention of tick-borne diseases in susceptible stock is based on the control of the tick vectors using acaricides. This strategy is hampered by the development of acaricide resistance in tick populations, the financial strain of acaricide purchase and the need for maintenance of control facilities. Additional concerns relate to environmental contamination. Although vaccines are available against these tick-borne diseases, they are all based on the use of live organisms and depend on cold chain support, which in many endemic areas is unreliable. Currently, the only practical method of immunization against ECF is infection with cryopreserved sporozoites and simultaneous treatment with long acting formulations of oxytetracycline (Radley, 1981). Use of this regime is limited by the fact that protection is only ensured against the immunizing stocks (Radley, 1981). All effective vaccine based on inoculation of schi-

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zont-infected cells is available for T. annulata (Pipano, 1981) and is in widespread use. This vaccine affords solid protection against a large number of isolates, although breakthrough infections have occasionally been observed when immunized animals are exposed to heterologous parasites (Hashemi-Fesharki & Shal-Del, 1973). Attempts to develop a similar vaccine for T. pa)va were frustrated by the requirement for large numbers of cells to effect immunization and observations of severe infections in a proportion of cattle following inoculation (Brown et aL, 1978; Dolan et al., 1982). The most common and effective current vaccines against both anaplasmosis and babesiosis utilize live organisms to establish post-infection immunity associated with a carrier state (premunition) (Brock et al., 1965; Kuttler, 1967; Schmidt, 1973; Vizcaino et al., 1980). Despite their effectiveness, these vaccines have a number of biological and technical shortcomings that have provided the impetus to develop improved inactivated vaccines. These shortcomings include: (i) potential contamination of blood-based vaccines with known pathogens such as bovine leukosis virus, bluetongue virus, Theile)ia, or heterologous Anaplasma or Babesia (Rogers el al., 1988); (ii) potential contamination and widespread direct transmission of unidentified or newly emergent pathogens; (iii) variation in virulence resulting in morbidity in inoculated adult cattle (Lohr, 1969; Henry et aL, 1983; Potgeiter & van Rensburg, 1983; and (iv) loss of infectivity resulting in failure to induce premunition. The risks have prevented licensing of live blood-based vaccines in the United States of America although whole A. marginale vaccines are available (Brock et al., 1965) and, in general, provide partial protection against acute anaplasmosis. However, the usefulness of these inactivated vaccines outside the United States of America is limited by their high cost and incomplete protection relative to premunition. The current method of immunization against heartwater is inoculation with blood from infected sheep and subsequent treatment with tetracyclines (Bezuidenhout & Oberem, 1985). Vaccination against B. bigemina and B. bovis is achieved using organisms of reduced virulence (Mahoney et aL, 1979; Dalgliesh et al., 1981). Both of these vaccines suffer from the same disadvantages associated with those used against Anaplasma.

PROGRESS TOWARDS DEVELOPMENT OF SUBUNIT VACCINES

T. parva and T. a n n u l a t a The approaches used to identify antigens for subunit vaccine development are, in general, guided by the type of immune responses that are likely to mediate protection. Two such immune mechanisms have been identified for T. parva. The immunity that protects the animal against the pathogenic schizont stage of this parasite is not d e p e n d e n t on antibody (Muhammed et al., 1975). The development of techniques for in vitro transformation of bovine lymphocytes with T. parva and the maintenance of infected cell lines in culture (Brown et al., 1973) were the most significant advances in allowing the elucidation of cellular immune mechanisms against the parasite. It was quickly demonstrated that immune peripheral blood lymphocytes proliferated in the presence of autologous infected cells, and

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that these cultures contained parasite-specific killing activity (Pearson el aL, 1979). The availability of a comprehensive range of bovine tissue ~ping sera allowed the demonstration that this activity was restricted by class I elements of the bovine MHC (Morrison el aL, 1987). It was also established that the effector cells were CD8 + T-lymphocytes, which confirmed that cattle deploy classical cytolytic T-lymphocytes against 7". p m v a (Goddeeris el aL, 1986). Prominent parasite-specific C D 4 + T-cell responses have also been detected in imnmne cattle (Baldwin el aL, 1987; Brown el aL, 1989), although the effector thnction of these cells has not been established. Kinetic analyses of parasite-specific CTL activity' revealed that it is temporally associated with the clearance of parasitosis in immune cattle (Morrison el aL, 1987), which was consistent with it being involved in the resolution of infection. More direct evidence for the role of CTL in parasite clearance was provided by the ol)sel-vation that protection could be transferred hetween immnne and nai've monozygotic twin cah'es undergoing lethal challenge in the CD8 + fi-action of responding efferent lymph (McKeever el al., 1994). Altlmugh humoral responses are not implicated in immunity of recovered cattle, animals do develop sporozoite nentralizing antibodies after multiple exposure to the parasite (Musoke et aL, 1984). This observation gave rise to the belief that sporozoite surt:ace antigens might constitute neutralizing vaccine candidates. The techniques for the identification of antigens recognized by antibodies are well established and for this reason considerable progress has heen made in determining the immnnizing potential of sporozoite antigens of 7". parva. Antisera from cattle in ECF-endemic areas are polyspecific and on immunoblots bind to several sporozoite molecules, inchlding proteins of relative molecular mass (Mr) 105, 85, 67, 55 and 31 kilo-Daltons (kDa). Candidate vaccine antigens were identified fl'om this repertoire of sporozoite antigens by raising monoclonal antibodies (mAl3s) that neutralized sporozoite infectivity (Dobbelaere el aL, 1984; Musoke et al., 1984). The majori D' of these mAbs bound to a sporozoite stagespecifc 67 kDa molecule (p67) that is expressed on the surface membrane of the parasite. DNA coding for p67 was identified bv immunoscreening a lambda gtl 1 cDNA expression libra D, derived fi'om sporoblasts of the Muguga stock of 7". pmva. It was established that the gene encoding p67 is present in a single copy and is split into two exons by a 29 1313intron (Nene el at, 1992). An Eschedchia coil recombinant filsion protein, NSl-p67, containing all 709 amino acid residues of p67 fused to the first 85 residues of NSI, a non-structural protein of influenza virus A, has been used in cattle imnmnization experiments. A semi-purified preparation of NSl-p67 formulated in saponin was used to immnnize nine cattle. All cattle developed high titres of IgG antibodies that bound to native p67 on imnaunoblots and neutralized sporozoite infectivity (Musoke el al., 1992). The immunized aniInals together with nine unimnmlaized controls were challenged with a 70% lethal dose of a 71 p m v a (Muguga) sporozoite stabilate. Of the nine immtinized animals, lout did not react to challenge and had no detectable parasitosis. Two cattle undenvent a mild febrile reaction with transient schizont parasitosis. The remaining three immunized cattle and the nine control animals all developed severe ECF (Musoke el at, 1992). Hence, six of nine cattle were immune to ECF. Current efforts are directed towards increasing the degree of protection by improving the immunization scheme. Additional studies are focused

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on the immune mechanisms responsible for protection in these cattle, since immunity does not appeal- to correlate with antibody titres (Musoke et al., 1992). Imnaunity engendered by recombinant p67 is likely to be directed against the sporozoite stage of the parasite, since the antigen is not expressed by the subsequent stages. Antigenic diversity between different populations of T. parva is well recognized and is believed to be responsible for breakthrough infections in cattle immunized by infection and treatment (Radley, 1981). An important feature of p67 that relates to its use as a vaccine is that its sequence is highly conserved among different cattle isolates of T. paTva and we have recently demonstrated that cattle immunized with recombinant p67 are protected against challenge with a heterologous cattle-derived parasite (A. J. Musoke, S. P. Morzaria & V. Nene, unpublished observations). However, the p67 gene sequence of buffalo-derived parasites contains two small regions of polymorphism (V. Nene & E. Gobright, unpublished observations) and the impact of this finding, if any, on vaccine effectiveness is under investigation. While attempts are being made to increase the level of protection achieved with recombinant p67, a vaccine that incorporates antigens of the schizont, the target of parasite-specific CTL, may provide more long lasting immunity in the field. Two methods are currently available for identifying antigens that are expressed in the context of MHC class I molecules. The most direct approach is the isolation, fractionation and sequence analysis of peptides associated with MHC class I molecules. Once a peptide of defined sequence is shown to sensitize target cells to lysis by specific CTLs, then the gene encoding the peptide can be isolated using synthetic oligonucleotides as probes. The second approach depends on the expression of parasite genes in transfected target cells. This strategy uses cDNAs expressed ira transiently transfected cells expressing appropriate MHC molecules, in coinbination with lytic or cytokine release assays. Both of these strategies have been successfully applied to the identification of tumour and x4ral antigens, (R6tzschke et al., 1990; Van Bleek & Nathenson, 1990; Traversari et aL, 1992; Brichard el al., 1993; Slingluff et al., 1993; Coulie et al., 1994) and are applicable to the search for antigens of 7". pa,va that provoke parasite-specific CTL responses. In tel'ms of vaccine potential, the schizont antigen should ideally be conserved among different parasite stocks. Should this not be the case, a cocktail of antigens would be required to give as wide a cover as possible. In addition, because of the processing requirements of class I MHC-restricted antigen presentation, incorporation of a schizont antigen component ira recombinant vaccine against T. pa)va will require the use of appropriate antigen delivery systems such as those based on recombinant viruses or bacteria (Brochier el al., 1991; Chat_field et aL, 1992). Similar strategies to those described above can be applied to the development of subunit vaccines for the control of 7". annulata infections. In fact, a recombinant form of a sporozoite molecule, SPAG1, has been shown to induce sporozoite neutralizing antibodies (Williamson et al., 1989), although the protective capacity of this molecule has not been fully evaluated. One of the clinical manifestations of 7: annulata infection is severe and prolonged anaemia, which is thought to be due to the merozoite stage of the parasite. This has led to the belief that antigens of merozoites could be used to induce immune responses that might prevent erythrocyte invasion and therefore anaemia (Hall et al., 1990). Two merozoite

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antigens, namely the 30 kDa (Glascodine et al., 1990) and Mag 1 (Hall et al., 1990) have been identified, and availability of the genes encoding these antigens will make it possible to evaluate their potential role in the induction of protective responses. Babesia b i g e m i n a a n d Babesia bovis The ability of inactivated merozoites to induce protection against challenge with homologous and heterologous isolates of B. boris (Mahoney, 1967a, b; Mahoney & Wright, 1976) and B. bigemina (Wright et al., 1987) established the basis for development of a killed vaccine. However, meeting the goal of a standardized vaccine that can be economically produced in recombinant proka13,otic or eukalyotic hosts requires definition of the critical epitopes responsible for protection. Although the specific regulatory and effector mechanisms required for protective immunity in babesiosis are unknown, it is clear that both antibody and T-lymphocyte-naediated mechanisnls are involved (Mahoney, 1967b; Mahoney el aL, 1979; Meeusen et aL, 1984; Zivkovic et aL, 1984). Consequently, research in the past decade has focused on identification and testing of individual and defined sets of babesial antigens and determination of B- and T-cell epitope conservation among different isolates of each Babesia spp. Three general approaches have been followed. The first approach uses sequential fractionation of merozoite antigens with testing of each fraction by immunization and challenge (Wright et al., 1992). The second approach identifies babesial molecules as potential tax-gets of the protective immune response based on their exposure to antibodies (Howard el al., 1980; McElwain et al., 1987; Hines et al., 1989). The third strategy is based on their proposed function in erythrocyte invasion and parasitism, followed by characterization of their epitopes and ability to induce protective inamtmity (Palmer & McElwain, 1995). Sequential fi-actionation of complex merozoite antigen preparations followed by immunization has resulted in the definition of three B. boris antigens, designated 12D3, 11C5 and 21B4, that are individually capable of inducing protection against virulent challenge (Wright et aL, 1992). hnmunization of cattle with a combination of recombinant 11C5 and 12D3 resulted in a marked reduction in the mean maximum parasitaemia upon challenge (Wright et aL, 1992). The severity of anaemia did not appear to be significantly different among vaccinated and unvaccinated cattle, but fewer of the vaccinated cattle required treatment. In an attempt to enhance vaccine efficacy, the third recombinant antigen, 21B4, was added to create a trivalent immunogen. In a field trial, complicated by the occurrence of concomitant B. bigemina challenge, the trivalent vaccine induced protection against B. bovis parasitaemia and anaemia (Gale et al., 1992b). Importantly, there were no significant side effects reported for either the recombinant bivalent or trivalent vaccines in contrast to the anaemia induced by use of a live vaccine in the same trial (Wright et al., 1992). The critical epitopes on these three molecules have not been identified. Identification of homologue molecules from B. bigemina has been noted, but detailed studies have not been published (Wright et al., 1992). Potential targets of the protective immune response to B. bovis and B. bigemina include the surface of sporozoites and extracellular merozoites and the surface of the parasitized erythrocyte. Epitopes on these surfaces are susceptible to antibody

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mediated clearance, which may involve activated effector cells. Antibody independent non-MHC restricted lymphocyte lysis of infected erythrocytes or merozoites, as described for Toxoplasma gondii (Khan et al., 1991), may also be directed against three epitopes. In contrast to the sporozoite surface, which has not been characterized, a number of antigens on both the free merozoite surface and the surface of erythrocytes parasitized with B. bigemina or B. bovis merozoites have been identified (reviewed in Pahner & McElwain, 1995). A primal-y drawback to vaccine development using antigens expressed on the merozoite surface is the extensive epitope variation among isolates. This problem has been clearly shown for the neutralization-sensitive Merozoite Surface Antigen1 (MSA-1) on B. boris (Pahner et al., 1991; Hines et al., 1992) and the analogous 45 kDA glycoprotein on B. bigemina (McElwain et al., 1991). No conserved MSA-1 B- or T-cell epitopes have been identified among isolates from Australia, Israel and Mexico (Palmer et al., 1991; Brown et al., 1993a, 1993b; Shkap et al., 1994), while only limited B-cell epitope conservation has been detected between isolates in Mexico and Brazil (Madruga et al., in press). Marked B-cell epitope variation of the 45 kDa B. bigemina glycoprotein, which induces reduction in peak parasitaemia upon challenge of immunized cattle, also occurs among isolates (McElwain et al., 1991). This high degree of antigenic polymorphism also applied to parasite molecules expressed on the external surface of the infected erythrocyte (Allred et al., 1993b). Two parasite derived molecules, described as 113 kDa and 128 kDa polypeptides, were shown to vary antigenically and structurally within a single animal following infection with a well-characterized clone. At least two partially characterized antigens on the surface of B. bigemina parasitized erythrocytes appear to be more broadly conserved both within and among isolates. These include an erythrocyte surface antigen complex, which is conserved among isolates from Kenya, Mexico, Puerto Rico and St Croix (Shompole et al., 1994), and a 17.5/76 kDa complex shown to be conserved among five isolates from Brazil, the Caribbean and Mexico (Vidotto et al., submitted). The erythrocyte surface antigen complex has been shown to induce antibody that promotes macrophage phagocytosis of parasitized elythrocytes (Shompole et al., 1995). However neither antigen has yet been tested for ability to induce protection. That erythrocyte surface antigens are capable of inducing protection is indicated by the B. bovis 11C5 antigen. This antigen, initially identified by fractionation and incorporated into the Australian trivalent immunogen, is expressed on the infected erythrocyte and can induce partial protection in immunized cattle (Goodger et al., 1992; Wright et al., 1992). The third approach that has been used to identify potential immunogens of Babesia spp. is based on their required role in erythrocyte invasion and parasitism. The apical complex organelles, micronemes, rhoptries, and dense granules or spherical bodies, are the defining feature of all apicomplexan parasites and appear to mediate key events in erythrocyte invasion and parasitism (reviewed in Perkins, 1992). In Babesia and Plasmodium spp., rhoptry proteins have been shown to be the target of neutralizing antibodies and to be capable of inducing protection against parasitemia in vivo (reviewed in Perkins et al., 1992; Palmer & McElwain, 1995). The Rhoptry Associated Protein-1 (RAP-l) is encoded by a family of genes that are significantly conserved among Babesia spp. (Suarez et al.,

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1991; Dalrymple et al., 1993). RAP-l, B-cell epitopes are expressed within the rhoptry and at the apical surface of the free merozoite (McElwain et al., 1987; Goff et al., 1988). Importantly, these surface epitopes, in contrast to MSA-1 epitopes, are highly conserved among isolates. B. bigemina RAP-1 epitopes are conserved among isolates from Argentina, Brazil, Kenya, Mexico, Puerto Rico and St Croix (McElwain et al., 1991; Suarez et al., 1994; Madruga el. al., in press) while B. bovis RAP-1 epitopes are conserved among isolates from Argentina, Australia, Brazil, Israel and Mexico (Palmer et al., 1991; Shkap et al., 1994; Suarez et al., 1994; Madruga et al., in press). Monoclonal antibodies against a conserved B. btqemina RAP-1 epitope inhibit merozoite growth in vitro (Figueroa & Buening, 1991) and RAP-1 immunized cattle develop significantly reduced parasitemia upon challenge (McElwain el al., 1991). In addition, B. boris RAP-1 is also a c o m p o n e n t (designated as 21B4) of the trivalent recombinant vaccine shown to induce partial protection against challenge in Australia (Gale et al., 1992b). Although the RAP-1 genes of B. bigemina and B. boris have regions of highly conserved sequence, compatible with mediating a common step in invasion of bovine erythrocytes, there is no significant conservation of RAP-1 epitopes between the species (Suarez el al., 1993). Therefore a different RAP-1 immunogen is needed for protection against each Babesia spp. Development of recombinant RAP-1 immunogens may require inclusion of multiple loci encoding RAP-1 in each species (Mishra et al., 1992; DahTmple et al., 1993; Suarez et al., 1994), as recent unpublished data indicates that surface exposed B-cell epitopes are locus specific. The gene encoding a second B. bovis apical complex protein, designated the Bb-1 spherical body protein or By80, has also been cloned (Brown et al., 1993b; DahTmple et al., 1993; Hines et al., 1995) and shown to be one of the components of a protectioninducing fraction (Dalrymple et al., 1993). In contrast to RAP-l, however, Bb-1 polymorphism among isolates occurs and involves at least one T-cell epitope that is not conserved between Australia and Mexico isolates (Brown et al., 1993b; Hines et al., 1995). Clearly the approaches used to identify candidate vaccine components for B. bigemina and B. bovis overlap, and at least one of the identified immunogens, RAP1, meets criteria for all three approaches: presence in a protection-inducing fraction, exposure to the immune system on the merozoite surface, and isolate conservation, as expected for a molecule with a required function in erythrocyte invasion. In addition, the 11C5, 12D3, ESA-1 and the 17.5/76kDa complex appear to be additional candidates for vaccine development. To date Babesia spp. immunogens have been evaluated by their ability to induce protection against challenge (Palmer & McElwain, 1995). However it should be emphasized that the lack of knowledge regarding the specific regulatory and effector mechanisms required for protective immunity is a primary constraint to development of recombinant vaccines that induce protection comparable to that induced by premunition. Improved understanding of immune mechanisms should allow investigators to enhance the level of immunity induced by univalent or multivalent immunogens by cytokine modulation of the immune response (Brown & RiceFicht, 1994), enabling antigenically defined vaccines to replace premunition as the predominant method of immunoprophylaxis for babesiosis.

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Anaplasma marginale The development of vaccines by expression of critical A. marginale epitopes in recombinant cultivable bacteria would substantially reduce vaccine production expenses and improve vaccine standardization. In contrast to the multi-stage protozoan lifecycle, the rickettsia A. ma~ginale has a single stage within the mammalian host. A. ma~ginale invades and replicates in mature erythrocytes but, unlike Babesia spp., does not appear to deposit or insert pathogen-encoded molecules on the eD, throcyte surface (Palmer, 1989a). In addition, the erythrocyte does not bear the required MHC molecules needed for classical lymphocyte-mediated cytotoxicity of the infected host cells as do lymphocytes infected with Theileria spp. (Goddeeris et aL, 1986). Consequently, the infected erythrocyte itself does not appear to be a target for the protective immune response and vaccine development studies have primarily targeted the surface of the extracellular A. marginale (Palmer & McElwain, 1995). Rickettsial outer membrane polypeptides are surface exposed and may be involved in critical functions such as nutrient transport and attachment and invasion of host cells. Importantly, A. marginale outer membrane epitopes are exposed to antibodies which may block invasion (McGarey & Allred, 1994) or mediate antibody-dependent effector mechanisms such as opsonization followed by rickettsial killing (Cantor et aL, 1993). Experimental support for the importance of outer membrane polypeptides is demonstrated by the ability of cattle immunized with purified outer membranes to control acute rickettsaemia upon virulent challenge (Tebele et aL, 1991; Palmer et al., 1994a). The A. marginale outer membrane contains multiple polypeptides of which at least six are both surface exposed and contain immunogenic B-cell epitopes (Palmer & McGuire, 1984; Tebele et al., 1991). These six outer membrane polypeptides have been designated major surface proteins (MSP) and include MSP-la, MSP-lb, MSP-2, MSP-3, MSP-4 and MSP-5. Identification of which MSP epitopes are the critical targets of the protective immune response has been limited by the inability to cultivate erythrocyte stages of A. marginale in vitro and the lack of sufficient syngeneic calves to conduct passive transfer of antigen-specific T-lymphocytes. However, in vitro el-ythrocyte binding studies have identified MSP-la, MSPlb, MSP-2 and MSP-4 as putative adhesins that may be required for erythrocyte invasion (McGarey & Allred, 1994; McGarey et al., 1994). Specifically, monoclonal and polyclonal antibodies against these MSPs partially or completely block A. marginale binding to bovine erythrocytes, but not erythrocytes of other species. In addition to identifying these MSPs as vaccine candidates, the use of monoclonal antibodies has allowed the identification of specific determinants that may be targeted to prevent erythrocyte invasion. Consistent with the findings that monoclonal antibody ANA22B1, specific for MSP-la, blocks erythrocyte binding, in vitro incubation of this antibody with purified A. ma~ginale organisms reduces infectivity for calves (Pahner et al., 1986). Unfortunately, no studies have been reported that identify MSP epitopes that stimulate T-lymphocyte responses. Whether for enhancement of the antibody response to critical B-cell epitopes or for aug-. menting cellular effector mechanisms, identification of these epitopes will be critically important to progress in vaccine development. Whether or not MSP epitopes are conserved among different A. marginale isolates is an important consideration in developing vaccines for deployment in

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regions where multiple antigenically variant strains occur. Isolates of A. m.a)ginah, differ in antigenicity, morpholo~,, virulence, tick transmissibility and abilitT to induce cross-protection against heterologons isolates (reviewed in Palmer, 1989a). As expected from these phenotypic differences, genomic and protein structural differences have been identified among isolates (Allred et al., 1993a; Barbet & Allred, 1991; Barbet et aL, 1983; Visser et al., 1992; Eriks el aL, 1994; Pahner el al., 1994b). In studies involving over 20 isolates, B-cell epitopes conserved among all isolates have been identified on MSP-la, MSP-4 and MSP-5 (McGuire et aL, 1984; Palmer et aL, 1988b; Tebele et aL, 1991; Oberle et al.., 1993; Eriks el al., 1994). However, MSPla is distinguished by marked polymorphism in domains that include a neutralization-sensitive epitope (Allred et al., 1993b). This variation in the neutralizationsensitive region may alter its affiniff for eD,throcyte binding or its susceptibility to neutralization. In contrast to MSP-la, MSP-4 and MSP-5, which are encoded by single-copy genes, MSP-2 is encoded by an extensively polymorphic gene family that results in expression of variant surface polypeptides with marked amino acid and B-cell epitope polymorphism (Pahner et al., 1994b). Consequently, the expression of individual MSP-2 B-cell epitopes is highly variable, both within and among isolates of A. ma)ginaLe. This variation provides a basis for antigenic variation that may affect the efficacy of recombinant vaccines based on a single or limited set of MSP-2 molecules or, alternatively, may be involved in the prolonged persistence ofA. ma)~naLe in cattle or infection of the tick vectors. Immunization of cattle with native MSP-2 or native or recombinant MSP-1 induces protection, defined as statistically significant reduction in rickettsaemia and anaemia as compared to adjuvant-immunized control cattle, against A. marginale challenge (Pahner et al., 1986, 1988a, 1989b). hnmunit), induced by immunization with either native MSP-1, a complex of MSP-la and MSP-lb, or native MSP-2 protected against both homologous and beterologous isolate challenges. Although these immunization trials indicate that outer membrane polypeptides are targets of the protective immune response against acute anaplasmosis, the level of protection is not uniform among individual vaccinates and complete protection against rickettsaemia and anaemia is achieved in only a minor subset of vaccinated cattle. The remaining vaccinates are either partially protected (developing a reduced rickettsaemia as compared to adjuvant-immunized controls) or unprotected. It is still unclear whether immunization with MSP-1 or MSP-2 can be optimized by rigorous evaluation of variables such as adjuvant, dose and schedule of vaccination, or whether additional or different immunogens are required. The dichotomy in the level and uniformity of protection induced by individual MSP immunogens (Palmer et al., 1989b; Barbet et al., 1991; Tebele et al., 1991) as compared to whole organism killed vaccines and outer membrane immunogens may reflect the need for a multi-component vaccine to induce a broad B- and T-cell polyclonal response. Alternatively, antigen processing and presentation of individual and membrane-complexed MSPs may be quite different. Within the outer membrane MSP-2 occurs as a tetramer and MSP-5 as a dimer (Vidotto et al., 1994). In addition, MSP-la and MSP-lb are disulphide-bonded and non-covalently linked to MSP-5. These intramolecular and intermolecular relationships are not maintained

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in purified recombinant polypeptides and may be critical in inducing the appropriate immune responses. Consequently, presentation of recombinant MSPs in a membrane context, either in a recombinant bacterial vector or in an artificial membrane such as an ISCOM or liposome, may he essential to enhance efficacy of individual MSPs. A primm T constraint to development of MSP vaccines as an alternative to killed vaccines or prenmnition is the lack of knowledge regarding the specific immune effectors that control rickettsaemia during acute infection. It is clear that T-lymphocytes are involved in protective immunity (Buening, 1976; Carson el aL, 1976; Corrier el al., 1981; Gale el aL, 1992a) but the identity of the relevant T-lymphocyte subsets, the critical functions they mediate, and the determinants that best induce the required responses are unknown. The efficacy of premunition in inducing and maintaining protective immunity indicates that antigen composition, antigen presentation and processing, and cytokine regulation of the immnne response are critically important in vaccine developlnent. An understanding of each of these factors should not only provide a basis for development of an effective recombinant vaccine against anaplasmosis but enhance our ability to control other persistent bacterial pathogens affecting animals and hulnans. Cowdria r u m i n a n t i u m As with 7: pa)va, animals that recover fl'om (7,. ruminantium infection are solidly protected against homologous challenge (Nei~ & Alexander, 1941; Du Plessis et al., 1984). However, the nature of the protective immunity against C. ruminanlium is poorly understood (Uilenberg, 1983; Stewart, 1987). The organism sun,fives within vascular endothelial cells of infected animals and the clinical signs of the disease are associated with the resultant increase in vascular permeability (Clark, 1962; Du Plessis, 1975; Prozesky, 1987). The agent is thus hidden from the hnmoral immune response when it is most pathogenic. After inoculation with C. ruminanlium, antibody responses are detected in cattle at the height of the febrile reaction (Semu el aL, 1992). These responses are probably induced by the rickettsaemia that follows rupture of infected cells but antibody titres are not correlated with the immune status of the animal (Du Plessis et al., 1984). In addition, experiments involving the transfer of immune gamma-globulin have revealed no evidence of protection (Alexander, 1931; Du Plessis, 1970). Together with the intracellular location of the organism, these observations have led to the belief that immunity is likely to be mediated by cellular responses directed at the infected cell. Further research into the nature of protective immunity against C. ~tm.inantium in ruminants will therefore inevitably draw heavily on information gathered from work on T. pa~va. The cellular studies outlined for T. pa)va necessitated the development and adaptation of a considerable nmnber of techniques and reagents. These are applicable to the study of the bmdne cellular immune mechanisms involved in protection against any pathogen. With the availability of techniques for in vitro culture of C. ruminantium, the application of these systems should provide information on the role of CTL in protection within a short time. Should this population of Tcells be implicated in protection, the methods under development for the identifi-

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cation of CTL antigens of T. p m v a will be applicable. Recent evidence that growth of the agent is heaxily influenced by certain cytokines (Mahan et aL, 1994; Totte et al., 1994) suggests that other T-cell populations might also be involved in protection. In addition, studies in goats and sheep have shown that immunization with killed Cowdria organisms confers protection against homologous challenge (Martinez el al., 1994; Mahan el aL, 1995). Because these responses are induced without active infection, it is unlikely that they are effected bv class I MHC-restricted cells.

CONCLUSION

A successful recombinant vaccine against a specific tick-borne disease should ideally induce immunity at least comparable to that engendered by available live vaccines in the degree of protection and longevity of immuni~. It is clear that in each of the parasites under discussion, none of the recombinant antigens evaluated todate fnlfils this requirenaent. The failure of" recombinant antigens to induce solid protection, in spite of promising results with native proteins, may reflect the inabili~' of the expression systems currently employed to make secondary modifications to the expressed products that are important for imnaune recognition. In addition, these antigens may only be partly responsible for protective immunity; other antigens may be essential for the induction of appropriate responses. There is therefore a need for a continuing effort in the characterization of imnlune mechanisms responsible for protection in these diseases. This contention is supported by recent observations on malaria that suggest that cellular immune mechanisms 1nay play a greater role in protection than previously believed (Tsuji el al., 1990; Nardin & Nussensweig, 1993). A major consideration in designing vaccines for haemoparasites is that many of these organisms have at least two lifecvcle stages in the host animal. A successful vaccine may need to include antigens of different lifecvcle stages. For example, an effective vaccine lor ECF might require sporozoite and schizont antigen components (Musoke et aL, 1992; McKeever & Morrison, 1994). Similarly, for tickborne parasites where polymorphic antigens have been identified, it may be useflfl to expand the search for conserved antigens expressed bv other lifecycle stages, in particular the tick derived stages. As candidate vaccine antigens are identified for these pathogens, the identification of appropriate antigen delive W systems will become increasingly important. With the expansion of knowledge of the inductive requirements of the immune responses, delive D, systems are available that engender the appropriate humoral or cellular immune responses. The choice of formulation is heavily influenced by the nature of the desired response. Additional criteria that must be applied relate to the infrastructure and financial capacity in developing countries. Most of the diseases under discussion are prevalent in countries where resources are limited. The live antigen delive D, systems based on recombinant viruses and bacteria hold many advantages for these countries. Expression constructs can be manipulated to optimize the desired immune mechanisms, and, in theo D, at least, multiple antigen vaccines can be constructed with relative ease. More importantly, live

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antigen delive D, vectors a b r o g a t e the n e e d for p r o d u c t i o n a n d purification o f large quantities o f antigen, a n d so are i n h e r e n t l y less expensive. C u r r e n t fears o f e m d r o n m e n t a l c o n t a m i n a t i o n with genetically e n g i n e e r e d m i c r o - o r g a n i s m s are likely to diminish with the d e v e l o p m e n t o f highly a t t e n u a t e d vector isolates c o u p l e d with successful field trials o f existing live r e c o m b i n a n t vaccines; rabies vaccines based on r e c o m b i n a n t p o x viruses have now b e e n d e p l o y e d successfully in the field in b o t h E m ' o p e a n d the USA (Brochier et aL, 1991; Pastoret & Brochier, 1992).

ACKNOWLEDGEMENTS We are grateful to o u r colleagues at ILRI for discussions a n d criticisms offered d u r i n g the p r e p a r a t i o n o f this m a n u s c r i p t . This is ILRI p u b l i c a t i o n n u m b e r 1442.

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GAI.F, K. R., WRl(,lrr, I. G. & RJDDX.F.S,P. W. (1992b). Vaccination against Babesia bovis using antigens produced by recombinant DNA technology. In Recent Developments in the Control 0fAnaplasmosis, Babesiosis, and Cowdriosis, pp. 113-9, ed..T.T. Dolan, Proceedings of a Workshop held at ILRAD, Nairobi, Kenya. Gt..~,S¢:ODINE,J.,TETI.EY,L., TAIT, A., BROWN,D. & SHIELS,B. (1990). Developmental expression of a Theile~a annulata merozoite surface antigen. Molecular and Biochemical Parasitology40, 105-12. GOI)I)I-ERIS,B. M., MORRISON,W. I., TF~\I.I-:,A.J., BENSAID,A. & BAI.DWIN,C. L. (1986). Bovine cytoxic T-cell clones specific for cells infected with the protozoan parasite Theileria parva: parasite strain specificity and class I major histocompatibility complex restriction. Proceedinff~ Natural Academy of Sciences USA 83, 5238--42. Gow:, W. L., D,.wls, W. C. & P,-~LMF.R,G. H. (1988). Identification of Babesia bovis merozoite surface antigens using immtme bovine sera and monoclonal antibodies. Infection and hnmunitv 56, 2363-8. GOOD¢;Fm13. V., WALTISBUIII,D.J., Wm(;HT, I. G. & WHITE,M. (1992). Babesia bovis: analysis of and preliminary vaccination studies with a defined infected erythrocyte membrane binding antigen. International Journal of Parasitology 22, 533-5. GOOD¢;I-;R,W.j., CARPI-NTER,J. & RJE,\I,~'4N,H. (1979). Estimation of economic loss associated with anaplasmosis in California beef cattle. Journal of American Veterina~. Medicine Association 174, 1333-6. HALt., F. R., WHJJ..XMSON,S., BROWN,C. G. D., HUSSAID,K., TAx'r, A. & SHIEI.S,B. R. (1990). In Recent Developments in Research and Conlrol of Theileria annulata, pp. 89-93, ed. T. T. Dolan. Proceedings of a Workshop held at ILRAD, Nairobi, Kenya. HASHEMx-FEsHARKI,R. & SHAt)-D~:t., F. (1973). Vaccination of calves and milking cows with different strains of Theileria annulata. American Journal of Veterinmy Research 34, 1465-9. Ht-NRV, E. T., NORMAN,B. B., FLY,D. E., WIC:H,~L,U'~N,R. W. & YORK,S. M. (1983). Effects and use of a modified live Anaplasma mmginalevaccine in beef heifers in California. Journal of American Vete~qna~yMedical Association 183, 66-9. HINI-:s, S. A., PAI.MER,G. H. & BROWN,W. C. (1995). Genetic and antigenic characterization of Babesia bovis merozoite spherical body protein Bb-1. Molecular and Biochemical Parasitolo©' 69, 149-59. Hlyl.:s, S. A., M(:EI.w..\IN,T. F., BUENIN(;,G. M. & P?d.MER,G. H. (1989). Molecular characterization of Babesia bovis merozoite surface proteins bearing epitopes immunodominant in protected cattle. Molecular and Biochemical Parasitology 7, 1-10. HINI.s, S. A., P.¢t..~II-R, G. H., J~.SMER, D. P., McGuIRE, T. C. & McELw.~dN, T. F. (1992). Neutralization-sensitive merozoite surface antigens of Babesia bovis encoded by members of a polymorphic gene family. Molecular and Biochemical Parasitology 55, 85-94. HowAm), R.J., RODWELL,B.J., SMrrH, P. M., CALt.OW,L. L. & MITCHELL,G. F. (1980). Comparison of the surface proteins and glycoproteins on erythroproteins of calves before and during infection with Babesia bovis.Journal of Protozoology 27, 241-7. IRvIY, A. D. 8,: MORRISON,W. I. (1987). Immunopathology, immunology and immunoprophlaLxis of Theileria infectio~as. In Immune Responses in Parasitic Infections: Immunology. bnmunopatholo~, and hnmunoprophlaxis Vol. III, pp. 223-274, ed. E. J. L. Soulsby, CRC Press. KJ~..\x, I., ELy, K. H. & K-XSPFR,L. H. (1991). A purified parasite antigen (p30) mediated CD8 + T cell immunity against fatal 7bxoplasma gondii infection in mice. Journal oflmmunolo©, 7, 3501-9. KuIq'I.I'R, K. L. (1967). A study of the immunological relationship of Anaplasma marginale and Anaplasma centrale. Research in VetelqnaO, Science 8, 467-71. LOHR, K. F. (1969). Immunisierung gegen babesiose und anaplasmose von 40 nach Kenya importierten Charollais-Rindern und Bericht uber Erscheinungen der Photosensibilitat bei disen Tieren. Z. VeterinarnzedB16, 40-6. Losos, G. J. (1986a). Anaplasmosis. In h~fectious Tropical Diseases of Domestic Animals, pp. 742-795, ed. G.J. Losos. Essex, UK: Longman Press. Losos, G.J. (1986b). Babesiosis. In: Infectious Tropical Diseases of Domestic Animals, pp. 4-97. ed. G.J. Losos. Essex, UK: Longman Press.

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Mt'W~JHCBI,A. W., Pl.:m~v, B. D. & Km,s~;, R. (1992). Estimated economics o f Theileriosis control in Africa. Preventive Velerilm~3' Medicine 12, 73-85. Mt'sc)kl.:, A..]., N..~xrl'xs:\, V. M., Rtlt.\x(;,mv.L F. R. & Bts(:lH.l~, (;. (1984). Evidence t o r a common protective anligen determinant on sporozoites o f several 7"heileHa pmva strains. lmmu,olo~' 52, 231-8. Mt'soKl-, A..]., Mc)I~Z\RI.LS. P., NKO:,,:(;I.:,(;. G., Joyl.:s, E. & NI.:NI.:,V. (1992). A reco,nbinant sporozoite slnlhce antigen of Theih,ria pm~,a induces protection in cattle. Proceeding~of the Nalural Academ~' ~)/.S'cie~tce(l iS'A) 89, 514-8. NAIl.[}IX, E. H. & NtsSl.ZXZWH(;, R. S. (1993). T cell responses to pre-el3'throcytic stages of malaria: Role in protection and vaccine developed against pre-el3,throcyte stages. Annual Review of hnmu ~tolo/,9' 11,687-727. NHrz, ~'. O. 8¢ AI.I':X.\Xl)ER,R. A. (1941). The inmnmization of cattle against heartwater. ./our~tal (q[.%'oulhAfrican Veterinmv MMicine Association 12, 103-11. NI.:xI.;, V., IAxls, K. P., (;()l',Rl(;llr, E. g: MrS()KE, A. J. (1992). Characterisation of the gene encoding a candidate vaccine antigen of Theileria pm~,a sporozoites. Molecular and Biochemical Pamsiloh)g9' 51, 17-28. OBl.:ml.:, S. M., P.\].xn-m G. H. & B.-~Rm.:],A. F. (1993). Expression and immune recognition of tile conserved attaplasma mmg~inale surface protein MSP-4. h~/ectio, at~d Immuttilv 61, 5245-51. P.\LmR, G. H. (1989a). Anaplasma Vaccines. In l~terinar~, Protozoan a*~d Hemoparasite Vaccim<~,pp. 1-29, ed. I. G. Wright. Boca Raton, FL: CRC Press. PAI.MI-R,(;. H. & M(:(;t'IRI':, T. C. (1984). l m n m n e serttm against Anaplasma mmginale initial bodies neutralizes infectivity in cattle.Jou~val ofhnmunolo©, 133, 1010-5. Pai.m'.R, G. H. & M(:ELw.-\Jx, T. F. (1995). Molecnlar basis for vaccine development against anaplasmosis and babesiosis, l,),terinmyPamsitolo©,57, 233-53. P..XI..~JE:R,G. H., B.-\RI~r[,A. F., D:wls, h r. C. & M(:Gt'wRE:,T. C. (1986). hmnunization with an isolate-co,ninon surface protein protects cattle against anaplasmosis. Sdeme 23, 1299-302. PAL.XII'~R,G. H., Om.:ml.:, S. M., Baam.:r, A. F., D..wls, W. C., G()vv, W. L. & M(:Gt'IRL T. C. (1988a). Immunization with a 36-kilodalton surface protein induces protection against hontoh)gous and heterologous Ana/)lasma malginab" challenge. InJ?ction and lmmuni O, 56, 1526-31. P:\l.m:n, G. H., B..xm~.:r, A. F., C.~×IOm G. H. & M(:Gt~ni-, T. C. (1989b). hnmunization of cattle with tile MSP-1 surlace protein complex induces protection against a structuralh' variant Anaplasma mmiffinale isolate. Infi'clion and Immuni(~, 57, 3666-9. PAIJMER, G. H., MtN()I)Z.\NA, D., TIiI',].:I.I -, N., UsI[I (, T. & M(:EI.wAIN,T. F. (1994a). Heterologous strain challenge of cattle immunized with Anaplasma mmginale outer membranes. VeterinaO, Immunolo©' a++dlmmunopatholo©' 42, 265-73. P.-\t.x~v,, G. H., E[t), G., B..~Rm.:-r,A. F., McGt'~RL T. C. & McEl.w..\tx, T. F. (1994b). The immunoprotective Anaplasma mmx~inaleMajor Sul+face Protein 2 (MSP-2) is encoded by a polymorphic mnhigene family. Infi,ction and hnmu++it+,62, 3808-16. P..\l.m.R, G. H., B..\Rm-T,A. F., Mt's~+K~.:,A.J. et al. (ltJ88b). Recognition of conserved surface protein epitopes on A,aplasma centrale and Anaplasma mmginale isolates fi-om Israel, Kenya and the United States. International.Journal of Pamsitolo©' 18, 33-8. P.+u.m:R, G. H., McE~.w..ux, T. F., PEam+,ax, L. E. et al. (1991). Strain variation of Babesia boris merozoite surface exposed epitopes, h+feclion and Immunity, 59, 3340-2. PasTo~Et, P. P. & BRO(:*HFR,B. (1992). Development of a recombinant vaccines-rabies vaccine for oral vaccination of foxes against rabies. Developments in Biolo~cal Standardization 79, 105-11. PI-ARSOX,T. W., LL'NI}IN,L. B., Dolts, T. T. & ST.\CO, D. A. (1979). Cell-mediated immnniB' to Theileria transformed cell lines. Nature 281,678-80. PrRK[NS, M. E. (1992). Rhopu T organelles of apicomplexan parasites. Parasitolo~, Today 8; 28-32. Pw..\x(), E. (1981). Schizonts and tick stages in immunization against 7"heile~a annulata infection. In Advances in the ('ontml of Theilerosis, eds A. D. Irvin, M. P. Cunningham & A. S. Young. pp. 242-252, The Hague: Martinus Nijhoff.

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POTt;ErrEe,, F. T. & VaN RENSBUR(:, L. (1983). Infectivily, virulence and imnmnogellici~, of A naplasma ten trale live vaccine. O~lders/epoortJ0u rnal of l:eterina ~y Research 50, 29-31. PROZESKV, L. (1987). Tile pathology of heartwater. III. A review. Ondersteporl Journal of Veterinmy Medicine 54, 319-25. R..xDu'v, D. E. (1981). Infection and treatment method of immunisation against theileriosis. In Advances in the Control ofTheih, riosis, pp. 227-37, eds A. D. Irvin, M. P. Cunningham & A. S. Young. The Hague: Martinus N!jhoff. RasTm, M. (1977). Bovine anaplasmosis. In Parasite Protozoa, pp. 235-50, ed. J. P. I(a'eier. New York: Academic Press Inc. RO(;ERS, R.J., DIMMoc~, C. K.. DEVos, A.J. & ROllWI.:[.I.,B.J. (1988). Bovine leucosis virus contamination of a vaccine p , o d u c e d in vivo against bovine babesiosis and anaplasmosis. Australian Veterinao, Jom~ta165, 285-7. RC)TZSCHKI-,O., E u.~;, K., DH~I-s, K., ScHn.t), H. & Nom).x, M. (1990). Isolation and analysis of naturally processed viral peptides as recognized by cytotoxic T-cells. 348-350 Schmidt, H. (1973). Anaplasmosis in callle. Journal (?f Amerirrm l:r,lerina~y Medical Assodation 90, 723-36. ScH,xmrr, H. (1973). Anaplasmosis in cattle..]oul~al o/Amerira~ l.:ete~4~aO, Medical Assodalion 90, 723-36. SI.:Mt', S. M., M..xH..xx,S. M., Yt×KI-R, C. E. & Bt'Rmlml-, M.J. (1992). Development mad persistence of Cowdria r~tmina~lium specific antibodies following experimental infection of cattle, as detected by the indirect fluorescent antibody test. Veterinary' hnmunolo~' and Immunopatholo©, 33, 339-52. SIIKAP, V., PII'AN(), E., McELw.-xlx, T. F. et al. (1994). C,'oss-protective immnni~, induced by Babesia bovis clones with antigenically urn'elated variable merozoite surface antigens (VMSA). l:etemm~)' hnmunolo©, and hnmunopathol:(©' 41,367-74. SHOMPOI.E, S., McEl.walx, T. F., JAS.~,.:R, D. P. el al. (1994). Identification of Bab~ia bigemina infected el'ythrocyte surface antigens containing epitopes consen,ed a m o n g strains. Parasite Immunolo~, 16, 119-27. Siio.~wol.E, S., PERR~.~IA×,L. E., McEt.wAIx, T. F. et al. (1995). Monoclonal antibody to a consen,ed epitope on protein encoded by Babesia bigemina and present on the surface of intact infected el-ythrocytes, hTfi,ction and Immu~it~, 63, 3507-13. SI.IN(;I.t'F'I:, C. L. Jr., Cox, L. A., HIqNI)ERSI.)N,A. R., I~t'NT, F. D. & EN(;EI.II.-\RI),H. V. (1993). Recognition of humml melano cells by HLA-A2.l-restricled cvtotoxic T lymphocytes is mediated by at least six shared peptide epitopes. TheJournal :?/"lmmunolog9, 150, 2955-63. STEWART, C. G. (1987). Specific immnni~, in farm animals to heartwater. O~ulerstepoort Journal of Veterinm), Research 54, 341-2. Sc..\~rz, C. E., M(:Et.w..ux, T. F., SrEpm-xs, E. B., M~s~,~\, V. S. & PaL~Ee, G. H. (1991). Sequence conservation a m o n g merozoite apical complex proteins of Babesia boris, Babesia bigemina, and other apicomplexa. MolerTdar and Biochemical Parasitolo~, 49, 329-32. SUAItEZ, C. E., P..\I,MI.:R,G. H., HINES, S. A. 8c M(:EI w:xlx, T. F. (1993). h n m u n o g e n i c B-cell epitopes of Babesia boris rhopt~3,-associated protein 1 are distinct from sequences conserved between species. Infection and hnmunitv 61,3511-7. St'.¢m.:z, C. E., M(:El.w..uy, T. F., E(:HawE, I., Tom..xx~ t)l.: Ecll..~un.: & P.-u..m-~, G. H. (1994). Interstrain consela:ation of babesial RAP-1 surface exposed B-cell epitopes despite rapl genomic polymorphism, h~fection and Immunity 62, 3576-9. TEmpt.r, N., McGclm;., T. C. & P..xLx~, G. H. (1991). Induction of protective immunity using Anaplasma mm~nale initial body membranes. Infi, ctim~ and bnmuni(~, 59, 3199-204. TonE, P., Jo.~(;l-I.ax, F., ~)t-Gm.;~-,A. L. & W~m.:t,e~;,J. (1994). Production of alpha interferon in Cowd~Ja ruminantium--infected cattle and its effect on infected endothelial cell cultures. Infeaion and hnmunity 62, 2600-4. TRAVF.RSAI~.I,C., BRU(;t;I';N, VAN DI".R P., E'~I'~NI)E,V:xNDEN B., HAINAUT,P. & LEMOINE, C. (1992). Transfection and expression of a gene coding for a h u m a n melanoma antigen recognized by autologous cytolytic T-lymphocytes. Immuno-genetirs 35, 145-52. TsKj~, M., RoMEo, P., NUESENZWEIt;,R. S. &: ZAVAIA,F. (1990). CD4 + cytolytic T-cell clone confers protection .against routine malaria.Journal of Experimental Medicine 172, 1353-7.

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639

UU.I:.XBER~:,G. (1983). Hea,-twate, (Cowdria ruminantium infection): current status. Advances in Velerinmy Sciem'e and Comparilive Medicine 97, 427-80. Uu.Exm-rc., G., Domu.:l.t.:rl.:, D. A. E., m.: Gl.:~:, A. L. & KocJl, H.J. ( 1 9 9 3 ) . Progress in Research on Tickborne diseases: Theileriosis and Hearnvater. Veterinmy Quarterly 15, 48-54. VAx BI.H-K, M. G. & N.-vnIvxsox, G. S. (1990). Isolation of an endogenously processed immunodominant viral peptide from the class I H-2K molecule. Nature 348, 213-6. Vmorro, M. C., M~:Gt'lrE, T. C., McEl.wAlx, T. F., PAI..~n.:R,G. H. & Kxowta.:s, D. P. (1994). Intermolecula, ,-elationships of major surface proteins of Anaplasma ma~ginale, h{feclion and Immu~dll, 62, 2940-6. VID()I-I(), O., ~'[[:EI.w.\IN,T. F., MA(:II.-\Df),R. Z., PEI~RYMAN,L. E., St'AREZ,C. E. & P.-\I.MER,G. H. (1995). Babesia bigemina: parasitized eD, throcytes. (Submitted). VIssI-R, E. S., M¢:Gtlm-, T. C. & PAI.XU.:R,G. H. (1992). The A,~ap&sma mm~nal*, rasp 5 gene encodes a 19-kilodalton protein conserved in all recognized Anaplasma species, h{fi, ction a ~ d l m m u nil1,

60, 5 1 3 9 - 4 4 .

\,qzc:,ux¢), O., CORmt~R, D. E., T~:Rr~V,M. K. el al. (1980). Comparison of three methods of immunization against bovine anaplasmosis: evaluation of protection afforded against challenge exposure. A merican./ournal of 1.'eterinaU Research 41, 1066-8. ~;ll.iJ.-\Msox, S., T.\IT, A., BROWN, D. el al. (1989). Theileria a,mulata sporozoite surface antigen expressed in Es¢'herichia coil elicits neutralizing antibody. Proceedings of Natural Acadetl O' qf Science USA 86, 4639-43. Wmcarr, I. G., CAst, R., Coxmlxs, M. A. el al. (1992). The development of a recombinant Babesia vaccine. Velerinm3' Parasilologq' 44, 3-13. Wmca rr, I. G., Go~m~a-R, B. V., LF.xn:ll, G., A~a.w.xm~,J. H., RcmE-1L.~xl..~xls,K. & W.~t.rlsmlu., D. J. (1987). Ba&~ia bigemina: protection of immune animals against subsequent challenge with virulent Babesia boris. Infection and Immunilr 155, 364-8. Yotx~,, A. S. & MoRz..\m.x,S. P. (1986). Biology of I~abesia. Parasitolo©' Todm' 2, 211-9. Zn'~ovu:, D., SHxI.:x, W., Kt'u., H., Al.u~Rs-v.\x Bl.:Xfxu.:l.,C. M. G. & SI'~:~sxUm-R,J. E. (1984). Immunin' to Babesia in mice. I. Adoptive transfer of immunity to Babesia mdhaini with immtme spleen cells and the effect of irradiation on the protection of immune mice. Veledna U hnmunolo~' and hnmunopatholo~' 5, 343-57.

(,4cceptedJor publitwlion 6 ,Vovember1995)