BOVINE BABESIOSIS: VACCINES BABESIOSES DOS BOVINOS: VACINAS IGNACIO E. ECHAIDE 1

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1 BOVINE BABESIOSIS: VACCINES BABESIOSES DOS BOVINOS: VACINAS IGNACIO E. ECHAIDE 1 ABSTRACT Live vaccines based on attenuated Babesia bovis and B. bigemina strains are currently used to protect cattle against babesiosis. Nevertheless they have several drawbacks represented by the risk of virulence reversion, contamination with pathogens, and the difficulties to industrialize the production. The generation of alternative vaccines, requires increasing the knowledge about the mechanisms of Babesia infection and the pathways to induce bovine protective immune response against Babesia. Different approaches have been followed to select candidate antigens from the initial empirical selection to the current search of new candidates using proteomic and genomic tools. It has been suggested that new vaccines will require the inclusion of multiple conserved antigens, and the appropriate adjuvant to modulate the cellular immune response. Importantly, the immune protection induced by molecular vaccines must be challenged with heterologous Babesia strains. There still no subunit or DNA vaccine available to protect cattle against babesiosis. For other protozoan diseases, attenuated live organisms modified by gamma irradiation or genetically modified are under consideration. Babesia live vaccines will continue providing artificial endemic stability to cattle herds until new vaccines can be available, however they should be improved. KEYWORDS: Babesia bovis, Babesia bigemina, vaccines. Bovine babesiosis is caused by different species of Babesia, apicomplexan hemoparasites naturally transmitted by at least four genera of ticks (UILENBERG, 2006). These vector ticks are distributed in tropical and subtropical areas of the world where near 400 million cattle are raised (GUGLIELMONE, 1995). In South America, Riphicephalus (Boophilus ) microplus, the definitive vector of Babesia bovis and B. 1 Instituto Nacional de Tecnología Agropecuaria CC Rafaela. Santa Fe. Argentina. iechaide@rafaela.inta.gov.ar

2 bigemina, is endemic in a variable zone north S (WRIGHT; RIDDLES, 1989). B. bovis may produce high mortality rates among susceptible cattle. The disease is characterized by fever, anemia, hypotensive shock syndrome, encephalitis and frequently death. Nervous signs and respiratory distress occurs when parasitized erythrocytes are sequestered in the capillary beds of the brain and lungs, respectively, resulting in low peripheral parasitemia (BROWN; PALMER, 1999). Fever, anemia and hemoglobinurea are clinical signs that characterize babesiosis caused by B. bigemina (CALLOW, 1979). Costs due to babesiosis have been estimated considering mortality, abortions, loss of milk/meat production and control measures. The estimated economic losses provoked by babesiosis in the Argentinean cattle industry was nearly US$ 30 million per year. Cattle raised in endemic areas are considered under risk of suffering babesiosis outbreaks; however, when the vector is profuse and the Babesia inoculation rate is high, they naturally acquire protective immunity to Babesia infection before 10 months of age (endemic stability). Young calves are protected from early infections by antibodies passively acquired from colostrums. Afterwards, infections produced between four and nine months of age are usually mild and generate long lasting immune protection (CALLOW, 1979). The relative resistance is associated to age and cattle breeds. The susceptibility of bovine to babesiosis is age-inverse. All breeds are susceptible to babesiosis, however, Bos indicus cattle carry lower tick burden and experience milder clinical symptoms than Bos taurus (BOCK et al., 2004). Variations in R. microplus population caused by climate, geographic modifications or uncontrolled use of acaricides decreases the inoculation rates of Babesia spp. to calves and consequently increase the risk of babesiosis outbreaks if they are exposed later in life (endemic instability) (BOCK et al., 1999). Outbreaks caused by B. bigemina are not as frequently observed and could be attributed to a lower pathogenicity of this species (GUGLIELMONE, 1994). Some B. bigemina outbreaks have been observed in herds with low prevalence of B. bovis. The introduction of cattle to babesiosis endemic areas, due to agricultural expansion and the desire to improve the genetic fitness of local cattle, increases the demand for preventive methods to protect the naive cattle against babesiosis. Although vaccines are not the only tool to prevent babesiosis, they constitute the most rational and economical way to provide protection to susceptible bovines. Before 1964, the protection of cattle from babesiosis was based mainly on the inoculation of susceptible cattle with blood from Babesia spp. carriers or from

3 splenectomized calves. This method frequently required chemotherapy to avoid death and involved the potential risk to transmit other pathogenic microorganisms (ROGERS et al., 1988). These early vaccines were significantly improved when attenuated Babesia strains and new diagnosis techniques to identify blood contaminants were introduced. Babesia live vaccines generate immune protection that persists for at least 3 years for B. bovis and 16 month for B. bigemina after infections were eliminated by chemosterilization (DE WAAL, 2006 ). In 1964, Australian researchers observed that B. bovis strains became less virulent than the original isolate after the syringe made successive passages of parasitized blood through several splenectomized calves (CALLOW et al., 1979). This method of attenuation may result from selective enrichment of a less pathogenic subpopulation of merozoites that are typically eliminated by the spleen in eusplenic bovines. Less pathogenic merozoites would be unable to modify the surface of parasitized erythrocytes (knobs), thus preventing the localization in the endothelial lining of capillaries (sequestration). This is most likely a useful mechanism to avoid the spleen (SCHETTERS et al., 1999). Alternatively, the attenuation may be a consequence of the down-regulation of virulence genes. The inoculation of these strains to naive cattle - particularly calves - produces a mild clinical reaction and induces immune protection against natural and experimental challenges with heterologous virulent strains (ECHAIDE et al., 1993a, b). Attenuation of B. bigemina was obtained after two to three syringe passages of parasitized blood placed four to six months apart among calves splenectomized after the chronic infection was established (DALGLIESH et al., 1981). The mechanism of attenuation, however, is still unknown. In Argentina, the inoculation of cattle with blood from Babesia carriers was the only method to immunize cattle until Attenuated strains were available in 1985 and the number of vaccinated cattle has continuously increased since then. B. bovis R1A and B. bigemina S1A attenuated strains were multiplied in splenectomized calves until 1992, when merozoites were multiplied in vitro (MASP) at the National Institute for Agricultural Technology (INTA) near Rafaela city (31 16 south). These strains have maintained the attenuated phenotype and immunogenicity after several passages in vitro, and have generated immune protection against the challenge with virulent heterologous strains at least six months after vaccination (ECHAIDE et al., 1993a, b). Between 1993 and April of 2008, approximately 1.8 million doses of live chilled vaccine based on B. bovis and B. bigemina were multiplied on in vitro cultures and were

4 inoculated. The demand of this vaccine has been continuously increasing since 1993 in coincidence with the cattle movement into endemic areas as well as environmental changes provoked by the humans or nature. The B. bovis R1A strain lost the capability of being transmitted by ticks (MANGOLD et al., 1993). This may have occurred to the B. bigemina S1A strain as well (still under evaluation). Reversion from attenuated to virulent phenotype or significant lack of protection after natural challenges has not been observed. Nevertheless, a brief study of babesiosis in vaccinated bovines completed in 1996 showed that an apparent failure of protection occurred in 0.09% of 105,000 vaccinated cattle (GUGLIELMONE; VANZINI, 1999). Long lasting protection induced by live vaccines against heterologous Babesia challenges has been the most effective feature maintaining its market value. Among the recognized drawbacks, Babesia live vaccines have the risk of dissemination of pathogenic microorganisms particularly viruses (ROGERS et al., 1988), the risk of reversion from an attenuated to virulent state or the lost of immunogenicity, as well as the need of continuous cold due to its short shelf-life. Field Babesia virulent strains were able to cause outbreaks of babesiosis (i.e., breakthrough isolates) in vaccinated cattle in Australia (BOCK et al., 1995; DE VOS, 1978). Several hypotheses has been given to explain the events that occurred in 1966, 1976, and (BOCK et al., 1995). It was suggested that immune pressure may play a role in selection in these parasites (LEROITH et al., 2005). Similar lack of immunogenicity was reported in South Africa (DE VOS, 1978). To minimize the probability that splenectomized or normal bovine blood donors were carriers of any infectious microorganisms, new diagnostic methods and techniques with high sensitivity and specificity have become available (e.g. PCR to identify DNA from bovine leukemia virus). This technique should be combined with serology and the inoculation of susceptible experimental animals, and the herds source of donor cattle must be free of transmissible diseases. To maintain the attenuated state and immunogenicity of live vaccines, each vaccine batch should be prepared using strains with a similar number of in vitro or in vivo passages followed by the periodic inoculation of naïve cattle. Shelf-life may be increased by freezing Babesia live vaccines in liquid nitrogen (CALLOW et al., 1997). Frozen vaccines require a higher concentration of parasites, and their efficiency depend on the cryoprotectant selected. Evaluations have been carried out using dimethyl sulfoxide (DMSO), glycerol or polyvinylp yrrolidone (PVP) (CALLOW et al., 1997; DALGLIESH et al., 1990).

5 Dimethyl sulfoxide is an excellent cryoprotectant for Babesia; however, it requires a short time between thawing and inoculation to avoid a toxic effect. Glycerol requires a longer period than DMSO of stabilization before freezing, however it provides hours of stability after being thawed (MANGOLD et al., 1990). Polyvinylpyrrolidone has no toxicity and it is useful when freezing Babesia from culture or any evaluated strain preserved as master stabilates. Frozen live vaccines provide the possibility to assess the infectivity, stability of strains attenuation, protective capability and lack of contaminants. To solve the problem of breakthrough isolates, new attenuated B. bovis strains have been introduced. It has been observed that live vaccines are protective when including at least two subpopulations of Babesia (BOCK et al., 2001). Developments of subunit vaccines or DNA based vaccines have been explored as the alternative to live vaccines. The understanding of both the innate mechanisms of resistance to acute B. bovis infection in young animals and the acquired immune mechanisms that continuously control the parasitaemia in persistently infected cattle will help devise strategies to induce a protective immune response by vaccination. Immunity to Babesia infection requires both innate and acquired responses. Acquired immunity resulting from immunization or persistent infection is dependent on both antibody and Th-cells (TAYLOR-ROBINSON, 1995). Th-cells provide the cytokines needed for high-affinity immunoglobulin maturation and the activation of macrophages. The resolution of an acute infection in naive calves infected with B. bovis parasites depends on a strong innate immune response that is based on the activation of macrophages via IFN -?, primarily in the spleen. This results in the killing of the parasites by phagocytosis and in the production of toxic macrophage metabolites, including nitric oxide (NO) (EAST et al., 1997). Using in vitro culture, it was observed that the secretory products from activated macrophages are toxic for B. bovis (MONTEALEGRE et al., 1985). Although passively administered antibodies were protective against in vivo challenge, exposure of the parasitized erythrocytes to antibodies in vitro had no effect on parasite viability. This suggested that in vivo, the antibodies act as an opsonin for activated macrophages. The higher parasitemias achieved in splenectomized calves inoculated with Babesia and the reactivation of babesiosis in carriers after the splenectomy provides evidence that reticuloendothelial cells from the spleen are needed to clear parasitized erythrocytes (WRIGHT; GOODGER, 1988). Six month old calves from non-endemic areas or those born from naive cows are relatively resistant to developing the severe form of B. bovis that is

6 typically observed in susceptible adults upon initial infection (TRUEMAN; BLIGHT, 1978; GOFF et al., 2001). Experimental results have suggested that the age-related resistance to B. bovis is partially mediated by an early IL-12 and NK cell-mediated IFN-? response. This activa tes macrophages to produce sufficient NO to kill the parasites early enough to prevent the pathological consequences of infection in order to allow adaptive immune responses to follow. Different molecules from pathogenic microorganisms stimulate innate immune responses by binding to mammalian Toll-like receptors (TLRs) (TAKEDA; AKIRA, 2005). The innate immune response begins when TLRs expressed on a variety of leukocyte subsets identify conserved molecules associated to different pathogen-associated molecular patterns (PAMPs), resulting in cytokine and co-stimulatory molecule expression by antigen-presenting cells required for the initiation of adaptive immune responses. Examples of PAMPs and their respective TLRs from different organisms include bacterial lipopolysaccharide (TLR4), poly G motifs of DNA (TLR8), non-methylated CpG motifs of DNA (TLR9), and profilin (TLR11). B. bovis merozoite extracts non-specifically activated proliferation of bovine peripheral blood mononuclear cells (PBMC), in part due to the presence of nonmethylated CpG motifs (BROWN et al., 1998). It has been suggested that merozoite extracts, B. bovis lipid, and purified DNA from B. bovis, activate macrophages to produce IL-1 ß, IL-12, TNF-a and NO (SHODA et al., 2001). Since Babesia sporozoites and merozoites infect only erythrocytes, cells that do not express MHC antigens, the adaptive immune response depend on the presence of parasite antigens to CD4 + T-lymphocytes provided by professional antigen-presenting cells. The control of infections would be based on two processes: 1) the destruction of infected erythrocytes by activated splenic macrophages, and 2) in the neutralizing antibodies directed against surface antigens of B. bovis free merozoites (MSA) and parasitized erythrocyte (VESA) (ALLRED; AL-KHEDERY, 2004) or p200 in B. bigemina (SHOMPOLE et al., 1995). Both of these immune mechanisms depend on CD4+ T cells. Characterizing the antigen-specific T cell response in cattle protected from Babesia infection has revealed that CD4+ T lymphocytes respond in vitro to B. bovis and B. bigemina antigens (BROWN; LOGAN, 1992; RODRÍGUEZ et al., 1996). IFN-? expressed by CD4+ helper T cells specific for B. bovis and B. bigemina were found to enhance IgG 2 secretion by B-lymphocytes. The strong opsonizing activity of IgG2 is also required for parasite elimination to control babesiosis (MCGUIRE et al., 1979). Definitive in vivo experiments in bovines, demonstrating effector functions of

7 either activated macrophages or CD4+ T cells in protective immunity to B. bovis and B. bigemina infections, have yet to be performed. The observation that killed parasites or parasite extracts are able to partially protect bovines against challenge with pathogenic strains has encouraged the investigation to develop subunit vaccines. Antigens designed or copied to make vaccines against Babesia should be expressed by merozoites or sporozoites during natural infections and should be reachable by the effectors of immune system. This is why the main candidates are surface proteins or molecules that are secreted by these stages during the infection. Conversely, the sequences or overall structure and the function of these proteins should be conserved between isolates from different geographic zones (LEROITH et al., 2005). Finally, the selected sequences should include B and T cell epitopes, since the protective immune response has a humoral and cellular base. It is also possible to find conserved sequences or structures in polymorphic proteins. The first evaluations to select the candidates were based on in vitro tests to assess the capability of mono- or polyclonal antibodies that would neutralize the erythrocytes invasion process (seroneutralization). The capability of the molecules to induce lymphocyte activation measured by the type of cytokines expressed was also evaluated (LEROITH et al., 2006). The best evaluation, however, is the challenge of vaccinated bovines with heterologous virulent strains of Babesia. Different strategies have been used to identify molecules potentially capable of generating protective responses against Babesia. The first selection approach was based on immunodominance defined by hyperimmune sera obtained from infected bovines. Many of these immunodominant antigens have been shown be incapable of generating protection after the heterologous challenge, despite its high antibody response (ECHAIDE et al., 1993a, b). It has been proposed that immunodominant antigens would only distract the immune system, and would therefore facilitate the persistence of infections. This prompted the search and evaluation of antigens represented by small sequences or epitopes unrecognized by the immune system of the host based on the rationale that such sequences would be relevant for the parasite survival. It was also suggested that the combination of these subdominant antigens (antigenic diversity) would be the base of a potential protective subunit vaccine. Moreover, the current knowledge of the immune mechanisms required to protect against Babesia challenges suggested that selected antigens should include B and T cell epitopes (BROWN et al., 2006). Subunit vaccines will also require the appropriate adjuvants.

8 B. bovis merozoite antigens were fractioned, and each fraction was tested for induction of protective immunity in cattle. Five B. bovis merozoite antigens were identified that were neither serologically immunodominant nor particularly abundant (WRIGHT et al., 1992). Four proteins were defined as immunogenic even though no serologically immunodominant SBP1 (HINES et al., 1995) were localized in the spherical body of merozoites, and T21B4 as well as two less defined proteins described as 12D3 (which includes T cell epitopes) and 11C5 localized in the rhoptries. All were assumed to be secreted by merozoites. The recombinant form of these proteins was evaluated alone or combined with either Quil-A saponin or in Freund s adjuvant. Bo th were able to generate partial protection, but the better degree of immune protection was achieved when more than one protein was given (WRIGHT et al., 1992). These results encouraged researchers to continue with careful evaluations of new molecules and combinations. Vaccine candidates such as merozoite surface antigens (MSA) and rhoptry-associated proteins (RAP) were identified by bovine immune serum or monoclonal antibodies. The rationale for this approach was to target molecules considered vital for erythrocyte invasion and was verified in vitro using previously generated neutralizing antibodies. The relevant B. bovis antigenic proteins identified using sera from carrier cattle recovered from babesiosis were RAP-1 (60-kDa), MSA-1 (42-kDa), and MSA-2 (44- kda). Antibodies against MSA-1 were able to neutralize erythrocyte invasion by merozoites in vitro (HINES et al., 1992; HINES et al., 1995), however it failed to elicit protective immunity in cattle against the homologous strain challenge (MCELWAIN et al., 1991). The relevance of antigenic variation of MSA-1 in immune evasion was shown in a recent study comparing msa-1 genes in Australian K and T vaccine strains with 14 vaccine breakthrough isolates, wherein a significant genetic variation was found (LEROITH et al., 2005). Predicted amino acid sequence differences as low as 18.7% resulted in a complete lack of antibody cross-reactivity between MSA-1 from vaccine strains and their respective breakthrough isolates. The MSA-2 proteins are encoded by a family of four tandem genes (FLORIN- CHRISTENSEN et al., 2002; BERENS et al., 2005) partially conserved among geographically distant strains. Bovine polyclonal antibodies specific for MSA- 2a1/MSA-2a2, MSA-2b, and MSA-2c also hampered the invasion of merozoites to erythrocytes in vitro (WILKOWSKY et al., 2003). For all MSA-1 and MSA-2 molecules, the predicted overall structure, and presumably the function, appears to be

9 conserved despite sequence polymorphism (LEROITH et al., 2005). All MSA family members appear to be glycosylphosphatidyl-inositol (GPI)-anchored. In other protozoa, including trypanosomes and malarial parasites, GPI anchors are known to activate innate production of pro-inflammatory cytokines, which can have protective or pathological consequences. Studies have suggested that the structure of the protein, rather than sequence per se, was important for inducing protective immunity. The antibody-based proteomic approach also identified several B. bovis secreted spherical body proteins, designated SBP -1, SBP-2, and SBP-3 (HINES et al., 1995, RUEF et al., 2000). Nevertheless, their functions and protective capacity as vaccine antigens have yet to be determined. In B. bigemina, the antigens identified by antibodies directed against the merozoite surface are the RAP-1 family proteins (BROWN; PALMER, 1999; SUAREZ et al., 1998), gp45, and gp55 proteins (MCELWAIN et al., 1991). Evaluated in vivo, native RAP-1a and gp45 proteins conferred partial protection following the challenge with the homologous B. bigemina strain (MCELWAIN et al., 1991; BROWN et al., 1998). B. bovis RAP-1 was evaluated in three different vaccination and challenge trials at INTA (Argentina) by performing 1) recombinant fusion protein (RAP-1-MBP) emulsed in Freund s adjuvant, 2) DNA recombinant plasmid, and 3) rap-1 gene cloned into Mycobacterium bovis (rbcg). In each experiment, immunity was challenged with the heterologous S2P virulent strain of B. bovis. No protection was observed for any of the formulations assessed (unpublished data). Another strategy to identify potentially important vaccine candidates was to isolate CD4+ T memory cells from cattle that had recovered from B. bovis infection (BROWN; PALMER, 1999). This allowed the identification of heat shock proteins (Hsp) 70, Hsp90 (RUEF et al., 2000), and Hsp20 (BROWN et al., 2001). From recovered cattle, Hsp20 was able to elicit detectable T-lymphoproliferative responses. B. bovis RAP-1 was also shown to highly stimulate T memory cells (BROWN et al., 1996), as was B. bigemina RAP-1a (MCGUIRE et al., 1979; BROWN et al., 1998). B. bovis RAP-1 contains numerous T cell epitopes recognized by CD4+ T cells from infected and recovered cattle. Immunization and challenge studies were performed with recombinant B. bovis RAP-1 (NORIMINE et al., 2003) and Hsp20. Protein was administered repeatedly with IL-12 and RIBI adjuvant until reproducibly strong CD4+ T-lymphocyte proliferation and IFN-? secretion were observed. All vaccinates yielded strong cellmediated and IgG1 and IgG2 responses, but none of the cattle were protected against

10 the development of disease or parasitaemia following challenge with the virulent T2Bo strain (NORIMINE et al., 2003). Recently, the availability of genomic sequences of B. bovis has allowed the identification of vaccine candidate antigens by genetic identity with homologous proteins in other protozoa, notably Plasmodium falciparum. BLAST analysis was used to identify two genes coding for the proteins of interest (GAFFAR et al., 2004a; b). One encodes the AMA-1 a micronemal protein expressed on the surface of merozoites (HOWELL et al., 2001; HOWELL et al., 2003). B. bovis AMA-1 was identified as a low-abundance protein, and a 69-kDa form of the protein was found to be secreted upon invasion of erythrocytes in vitro (GAFFAR et al., 2004a). Rabbit antisera raised against B. bovis AMA-1 peptides significantly blocked invasion by B. bovis in vitro. The analysis of an EST library allowed the identification of a 75-kDa antigen of the apicomplexan thrombospondin- related anonymous protein (TRAP) family (GAFFAR et al., 2004b). These families of proteins are believed to function as host cell binding proteins (SULTAN et al., 1997). Antisera raised against TRAP peptides significantly inhibited erythrocyte invasion. Control of babesiosis should be based in the effort to integrate the management of the tick vector, the host and the hemoparasites. The use of acaricides, the election of a tick resistant breed of cattle, the evaluation of endemic stability status and the strategic use of vaccines are possible methods to help control babesiosis. The different strategies to develop subunit or recombinant Babesia vaccines have increased the knowledge necessary to generate the protective immunogens that could replace the currently used live vaccine. These strategies, however, have not provided enough results to overcome the immunization and heterologous challenge trials, and the immune protection achieved remains insufficient. The challenge is to develop a vaccine innocuous to young and adult cattle and protective enough to resist virulent heterologous infection transmitted by ticks under field conditions. New DNA constructs that include multiple antigens or immunogenic epitopes of different proteins should be evaluated. Prime-boost immunization with DNA followed by a recombinant virus expressing selected proteins is an example of an alternative method that combines two expression systems (FUKUMOTO et al., 2007). Nevertheless, it is also important to minimize the need for repeated vaccinations. Immunization protocols should include adjuvants to specifically enhance the required type-1 immune response (BROWN; PALMER, 1999), such as DNA based adjuvants (CpG oligonucleotides) for DNA

11 vaccines. CpG oligonucleotides have shown to activate bovine B cells to proliferate and secrete significant levels of IgG2. DNA/subunit vaccines continue to be a primary research objective. Live vaccines currently used in different countries should be improved to minimize their well known drawbacks. The multiplication of attenuated Babesia strains on in vitro cultures that produce live vaccines provides the advantages of utilizing sera and erythrocytes from continuously analyzed bovine donors, achievement of high parasitemias by the established due date, and uses the minimal inclusion of cells other than erythrocytes. Immunogenicity and attenuation status of strains should be evaluated periodically and a previously established number of subcultures should not be exceeded. References ALLRED, D.R. ; AL-KHEDERY, B. Antigenic variation and cytoadhesion in Babesia bovis and Plasmodium falciparum: different logics achieve the same goal. MBP., 134:27-35, BERENS, S.J.; BRAYTON, K.A.; MOLLOY, J.B.; BOCK, R.E. ; LEW, A.E. ; MCELWAIN, T.F. Merozoite surface antigen 2 proteins of Babesia bovis vaccine breakthrough isolates contain a unique hypervariable region composed of degenerate repeats. Infect. Immun., 73: , BOCK, R.E. ; DE VOS, A.J.; LEW, A.E.; KINGSTONE, T.G.; FRASER, I.R. Studies on failure of T strain live Babesia vaccine. Aust. Vet. J. 72: , BOCK, R.; KINGSTON, T.G.; DE VOS, A.J. Effect of breed of cattle on transmission rate and innate resistance to infection with Babesia bovis and B. bigemina transmitted by Boophilus microplus. Aust. Vet. J. 77: ; BOCK, R.E. ; DE VOS, A.J. Immunity following use of Australian tick fever vaccine: a review of the evidence. Aust. Vet. J., 79: , BOCK, R.; JACKSON, L.; DE VOS, A.J.; JORGENSEN, W. Babesiosis of cattle. Parasitol., 129:S247- S269, BROWN, W.C.; MCELWAIN, T.F.; RUEF, B.J.; SUAREZ, C.E.; SHKAP, V.; CHITKO-MCKOWN, C.G.; TUO, W.; RICE-FICHT, A.C.; PALMER, G.H. Babesia bovis rhoptry associated protein- 1 is immunodominant for T helper cells of immune cattle and contains T cell epitopes conserved among geographically distant B. bovis strains. Infect. Immun.; 64: , BROWN, W.C.; ESTES, D.M.; CHANTLER, S.E.; KEGERREIS, K.A.; SUAREZ, C.E. DNA and a CpG oligonucleotide derived from Babesia bovis are mitogenic for B cells. Infect. Immun., 66: , BROWN, W.C., LOGAN, K.S. Babesia bovis: Bovine helper T cell lines reactive with soluble and membrane antigens of merozoites. Exp. Parasitol., 74: , 1998.

12 BROWN, W.C.; MCELWAIN,T.F.; HÖTZEL, I.; SUAREZ, C.E.; PALMER, G.H. Helper T cell epitopes encoded by the Babesia bigemina rap-1 gene family in the constant and variant domains are conserved among parasite strains. Infect. Immun., 66: , BROWN, W.C.; PALMER, G.H. Designing blood stage vaccines against Babesia bovis and Babesia bigemina. Parasitol. Today, 15: , BROWN, W.C.; RUEF, B.J.; NORIMINE, J.; KEGERREIS, K.A.; SUAREZ, C.E.; CONLEY, P.G. ; STICH, R.W.; CARSON, K.H.; RICE-FICHT, A.C. A novel 20-kilodalton protein conserved in Babesia bovis and B. bigemina stimulates memory CD4+ T lymphocyte responses in B. bovisimmune cattle. MBP; 118:97-109, BROWN, W.C.; NORIMINE, J.; GOFF, W.L.; SUAREZ, C.E.; MCELWAIN, T:F: Prospects for recombinant vaccines against Babesia bovis and related parasites. Parasite Immunol. ; 28: , CALLOW, L.L. ; MELLORS, L.T.; MC GREGOR, W. Reduction in virulence of Babesia bovis due to rapid passage in splenectomized cattle. Int. J. Parasitol., 9: , CALLOW, L.L. Some aspects of epidemiology and control of bovine babesiosis in Australia. J. S. Afr. Vet. Assoc. 50: , CALLOW, L.L. ; DALGLIESH, R.J.; DE VOS, A.J. Development of effective living vaccines against bovine babesiosis -the longest field trial? Int. J. Parasitol., 27:747-67, DALGLIESH, R.J.: CALLOW, L.L.; MELLORS, L.T. ; MCGREGOR, W. Development of a highly infective Babesia bigemina vaccine of reduced virulence. Aust.Vet.J., 57:8-11, DALGLIESH, R.J.; JORGENSEN, W.K.; DE VOS, A.J. Australian frozen vaccines for the control of babesiosis and anaplasmosis in cattle -a review. Trop. Anim. Health Prod.,22:44-52, DALGLIESH, R.J. Babesiosis. In: Immunology and molecular biology of parasitic infections, ed. Warren KS. Oxford: Blackwell, pp , DE VOS, A.J. Immunogenicity and pathogenicity of three South African strain of Babesia bovis in Bos indicus cattle. Onderstepoort J. Vet. Res., 45: , DE WAAL, D.T.; COMBRINK, M.P. Live vaccines against bovine babesiosis. Vet. Parasitol., 138: EAST, I.J.; ZAKRZEWSKI, H.; GALE, K.R.; LEATCH, G.; DIMMOCK, C.M.; THOMAS, M.B.; WALTISBUHL, D.J. Vaccination against Babesia bovis: T cells from protected and unprotected animals show different cytokine profiles. Int. J. Parasitol., 27: , ECHAIDE, I.E.; DE ECHAIDE S.T.; GUGLIELMONE A.A. Live and soluble antigens for cattle protection to Babesia bigemina. Vet. Parasitol., 51:35-40, 1993a. ECHAIDE, I.E.; ECHAIDE, S.T. DE; MANGOLD, A.J.; GUGLIELMONE, A.A. Live and soluble antigens from in vitro culture to vaccinate cattle against Babesia bovis. Proc. IX International Veterinary Hemoparasite Disease Conference. Mérida, México. October, 1993, p.13, 1993b.

13 FLORIN-CHRISTENSEN, M.; SUAREZ, C.E.; HINES, S.A.; PALMER, G.H.; BROWN, W.C.; MCELWAIN, T.F. The Babesia bovis merozoite surface antigen 2 locus contains four tandemly arranged and expressed genes encoding immunologically distinct proteins. Infect. Immun. 70: , FUKUMOTO, S.; TAMAKI, Y.; OKAMURA, M.; BANNAI, H.; YOKOYAMA, N.; SUZUKI, T. ; IGARASHI, I.; SUZUKI, H.; XUAN, X. Prime-boost immunization with DNA followed by a recombinant vaccinia virus expressing P50 induced protective immunity against Babesia gibsoni infection in dogs. Vaccine; 25: GAFFAR, F.R.; YATSUDA, A.P.; FRANSSEN, F.F.; DE VRIES, E. Erythrocyte invasion by Babesia bovis merozoites is inhibited by polyclonal antisera directed against peptides derived from a homologue of Plasmodium falciparum apical membrane antigen 1. Infect. Immun. 72: , GAFFAR, F.R.; YATSUDA, A.P.; FRANNSEN, F.F; DE VRIES, E. A Babesia bovis merozoite protein with a domain architecture highly similar to the thrombospondin-related anonymous protein (TRAP) present in Plasmodium sporozoites. MBP; 136:25-34, GOFF, W.L. ; JOHNSON, W.C.; PARISH, S.M.; BARRINGTON, G.M.; TUO, W.; VALDEZ, R.A. The age-related immunity in cattle to Babesia bovis infection involves the rapid induction of interleukin-12, interferon-?, and inducible nitric oxide synthase mrna expression in the spleen. Parasite Immunol. 23: , GUGLIELMONE, A.A. Epidemiología y control de los hemoparásitos (Babesia y Anaplasma) en la Argentina. In. A. Nari and C. Fiel (Ed.) Enfermedades parasitarias de importancia económica en bovinos. Bases epidemiológicas para su prevención y control en Argentina y Uruguay. Hemisferio Sur, Montevideo, Uruguay, pp , GUGLIELMONE, A.A. Epidemiology of babesiosis and anaplasmosis in South and Central America. Vet. Parasitol., 57: , GUGLIELMONE, A.A.; VANZINI, V.R. Análisis de fracasos en la prevención de la anaplasmosis y la babesiosis en bovinos inoculados con vacunas vivas. Rev. Med. Vet., 80:66-68, HINES, S.A.; PALMER, G.H.; JASMER, D.P.; MCGUIRE, T.C.; MCELWAIN, T.F. Neutralizationsensitive merozoite surface antigens of Babesia bovis encoded by members of a polymorphic gene family. MBP; 55: HINES, S.A.; PALMER, G.H.; BROWN, W.C.; MCELWAIN, T.F. ; SUAREZ, C.E.; VIDOTTO, O.; RICE-FICHT, A.CGenetic and antigenic characterization of Babesia bovis merozoite spherical body protein Bb-1. MBP. 69: , HINES, S.A.; PALMER, G.H.; JASMER, D.P.; GOFF, W.L.; MCELWAIN, T.F. Immunization of cattle with recombinant Babesia bovis merozoite surface antigen-1. Infect. Immun., 63: , 1995.

14 HOWELL, S.; WITHERS-MARTINEZ, C.; KOCKEN, C.H. ; THOMAS, A.W.; BLACKMAN, M.J. Proteolytic procession and primary structure of Plasmodium falciparum apical membrane antigen-1. J. Biol. Chem. 276: , HOWELL, S.A.; WELL, I.; FLECK, S.L.; KETTLEBOROUGH, C.; COLLINS, C.R.; BLACKMAN, M.J. A single malaria merozoite serine protease mediates shedding of multiple surface proteins by juxtamembrane cleavage. J. Biol. Chem. 278: , LEROITH, T. ; BRAYTON, K.A.; MOLLOY, J.B.; BOCK, R.E. ; HINES, S.A.; LEW, A.E. ; MCELWAIN, T.F. Sequence variation and immunologic cross-reactivity among Babesia bovis merozoite surface antigen 1 proteins from vaccine strains and vaccine breakthrough isolates. Infect. Immun., 73: , LEROITH, T., BERENS, S., BRAYTON, K.A., HINES, S.A., BROWN, W.C., NORIMINE, J., MCELWAIN, T.F. The Babesia bovis merozoite surface antigen 1 hypervariable region induces surface-reactive antibodies that block invasion. Infect. Immun., 74:3663-7, MCGUIRE, T.C.; MUSOKE, A.J. ; KURTII, T. Functional properties of bovine IgG1 and IgG2: interaction with complement, macrophages, neutrophils, and skin. Immunology, 9; 38: , MANGOLD, A.J.; AGUIRRE, D.H.; GUGLIELMONE, A.A. Post-thawing viability of vaccines for bovine babesiosis and anaplasmosis cryopreserved with glycerol. Vet. Parasitol. 37:301-6, MANGOLD, A.J.; AGUIRRE, D.H.; CAFRUNE, M.M.; DE ECHAIDE, S.T.; GUGLIELMONE, A.A. Evaluation of the infectivity of a vaccinal and a pathogenic Babesia bovis strain from Argentina to Boophilus microplus. Vet. Parasitol., 51:143-8, MCELWAIN, T.F.; PERRYMAN, L.E.; MUSOKE, A.J.; MCGUIRE, T.C. Molecular characterization and immunogenic ity of neutralization sensitive Babesia bigemina merozoite surface proteins. MBP, 47: , MONTEALEGRE, F.; LEVY, M.G.; RISTIC, M.; JAMES, M.A. Growth inhibition of Babesia bovis in culture by secretions from bovine mononuclear phagocytes. Infect. Immun. 50, , NORIMINE, J.; MOSQUEDA, J.; SUAREZ, C.E.; PALMER, G.H.; MCELWAIN, T.F.; MBASSA, G. ; BROWN, W.C. Stimulation of T helper cell IFN-gamma and IgG responses specific for Babesia bovis rhoptry associated protein 1 (RAP-1) or a RAP-1 protein lacking the carboxy terminal repeat region is insufficient to provide protective immunity against virulent B. bovis challenge. Infect. Immun. 71: , RODRÍGUEZ, S.D.; PALMER, G.H.; MCELWAIN, T.F.; MCGUIRE, T.C.; RUEF, B.J.; CHITKO- MCKOWN, M.G.; BROWN, W.C. CD4+ T helper lymphocyte responses against Babesia bigemina rhoptry associated protein-1. Infect- Immun., 64: , ROGERS, R.J.; DIMMOCK, C.K.; DE VOS, A.J.; RODWELL, B.J. Bovine leucosis virus contamination of a vaccine produced in vivo against bovine babesiosis and anaplasmosis. Aust. Vet. J. 65:285-7, 1988.

15 RUEF, B.J.; DOWLING, S.C.; CONLEY, P.G.; PERRYMAN, L.E.; BROWN, W.C.; JASMER, D.P. ; RICE-FICHT, A.C. A unique Babesia bovis spherical body protein is conserved among geographic isolates and localizes to the infected erythrocyte membrane. MBP; 105:1-12, RUEF, B.J.; WARD,T.J.; OXNER, C.R.; CONLEY, P.G. ; BROWN, W.C.; RICE-FICHT, A.C. Phylogenetic analysis with newly characterized Babesia bovis hsp70 and hsp90 provides strong support for paraphyly with the piroplasms. MBP, 109:67-72, SCHETTERS, T.P.M.; ELING, W.M.C. Can Babesia infections be used as a model for cerebral malaria? Parasitol. Today, 15: , SHODA, L.K.M. ; KEGERREIS, K.A.; SUAREZ, C.E. ; RODITI, I.; CORRAL, R.S.; BERTOT, G.M.; NORIMINE, J.; BROWN, W.C. DNA from protozoan parasites Babesia bovis, Trypanosoma cruzi and T. brucei is mitogenic for B lymphocytes and stimulates macrophage expression of interleukin-12, tumor necrosis factor-alpha, and nitric oxide. Infect. Immun., 69: , SHOMPOLE, S.; PERRYMAN, L.E.; RURANGIRWA, F.R.; MCELWAIN, T.F.; JASMER, D.P. ; MUSOKE, A.J.; WELLS, C.W.; MCGUIRE, T.C. Monoclonal antibody to a conserved epitope on proteins encoded by Babesia bigemina and present on the surface of intact infected erythrocytes. Infect Immun., 63: , SUAREZ, C.E.; PALMER, G.H.; HÖTZEL, I.; MCELWAIN, T.F. Structure, sequence, and transcriptional analysis of the Babesia bovis rap-1 multigene locus. MBP, 93: , SULTAN, A.A.; THATHY, V.; FREVERT, U.; ROBSON, K.J. ; CRISANTI, A.; NUSSENZWEIG, V.; NUSSENZWEIG, R.S. ; MÉNARD, R. TRAP is necessary for gliding motility and infectivity of Plasmodium sporozoites. Cell, 90: , TAKEDA, K.; AKIRA, S. TOLL-LIKE RECEPTORS IN INNATE IMMUNITY. INT. IMMUNOL., 17:1-14, TAYLOR-ROBINSON, A.W. Regulation of immunity to malaria: valuable lessons learned from murine models. Parasitol. Today, 11: , TRUEMAN, K.F.; BLIGHT, G.W. The effect of age on resistance of cattle to Babesia bovis. Aust. Vet. J. 54: , UILENBERG, G. Babesia-A historical overview. Vet. Parasitol., 138:3-10, WILKOWSKY, S.E.; FARBER, M.; ECHAIDE, I.; TORIONI DE ECHAIDE, S.; ZAMORANO, P.I. ; DOMINGUEZ, M.; SUAREZ, C.E.; FLORIN-CHRISTENSEN, M. Babesia bovis merozoite surface protein-2c (MSA-2c) contains highly immunogenic, conserved B-cell epitopes that elicit neutralization-sensitive antibodies in cattle. MBP, 127: , WRIGHT, I.G. ; GOODGER, B.V. In: Babesiosis of domestic animals and man (Ristic, M., Ed.), CRC Press, pp , 1988

16 WRIGHT, I.G.; RIDDLES, P.W. Biotechnology in ticks-borne diseases. Present Status, future perspectives. FAO -UN Biotechnology for live stock production. Plenum, N.Y., pp , (1989). WRIGHT, I.G. ; CASU, R.; COMMINS, M.A.; DALRYMPLE, B.P.; GALE, K.R.; GOODGER, B.V. ; RIDDLES, P.W.; WALTISBUHL, D.J.; ABETZ, I.; BERRIE, D.A.; et al. The development of a recombinant Babesia vaccine. Vet. Parasitol.; 44: 3-13, 1992.