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Conservation of merozoite membrane and apical complex B cell epitopes among Babesia bigemina and Babesia bovis strains isolated in Brazil
veterinary parasitology ELSEVIER
Veterinary Parasitology61 (1996) 21-30
Conservation of merozoite membrane and apical complex B cell epitopes among Babesia bigemina and Babesia boris strains isolated in Brazil Claudio R. Madruga a, Carlos E. Suarez b, Terry F. McElwain b, Guy H. Palmer b,, a Centro Nacional de Pesquisa de Gado de Corte, EMBRAPA, Caixa Postal 154, CEP 79080-970, Campo Grande, MS, Brazil b Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, WA 99164-7040 USA
Received 15 November1994; accepted 9 February1995
Abstract Babesia merozoite polypeptides bear surface exposed and neutralization-sensitive B cell epitopes and have been shown to induce partial protection against experimental challenge. Variation in these epitopes has been examined in a limited number of strains. In this study, utilizing strains of Babesia bovis and Babesia bigemina from Matto Grosso do Sul in Brazil, we examined the conservation of epitopes bound by monoclonal antibodies developed against Mexico strains of B. bovis and B. bigemina. Apical complex B-cell epitopes, previously shown to be species-specific but common among otherwise antigenically distinct strains, were also conserved between clones of the Mexico strains and the Matto Grosso do Sul strains of each Babesia species. Mexico strain polypeptides bearing these epitopes were recognized by sera from cattle infected with the Matto Grosso do Sul strains. Two distinct epitopes on the B. boris neutralization-sensitive merozoite surface antigen-1 (MSA-1) were also conserved between the Mexico Mo7 clone and the Matto Grosso do Sul strain, in contrast to previous studies which demonstrated variability among strains. Sera from cattle with B. bovis infections naturally acquired in Matto Grosso do Sul bound Mexico Mo7 MSA-1, demonstrating that conserved MSA-1 epitopes were recognized by the bovine immune system. Similarly, merozoite surface epitopes on the B. bigemina 45 kDa and 55 kDa glycoproteins were conserved on the Matto Grosso do Sul strain of B. bigemina. Keywords: Babesia bigemina; Babesia boris; Merozoite; Apical complex; B cell epitopes
* Correspondingauthor. 0304-4017/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0304-4017(95)00809-8
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I. Introduction Babesiosis is a tick-borne disease widespread in cattle herds within tropical and subtropical regions worldwide (McCosker, 1981). In Central and South America, 70% of the cattle live within areas infested with the vector Boophilus microplus (Lopez, 1977). Brazil contains approximately 100 x 10 ~ cattle, one-sixth of the world's cattle population, with most of the highly productive cattle located in the southern states (Montenegro-James, 1992). Prevalence of both Babesia boris and B. bigemina in this region is high with mean values of > 75% reported in each of the states of Matto Grosso do Sul, Minas Gerais, and Rio Grande do Sul (Madruga et al., 1983; Patarroyo et al., 1987; Leite, 1988). Annual direct economic losses due to babesiosis in Rio Grande do Sul were estimated at US $99 million in 1984 (Madruga et al., 1984). Consequently, in Brazil and throughout South America, development of improved control for babesiosis is a high priority. Immunoprophylaxis provides a method for control both within enzootically unstable areas and for introduction of susceptible cattle into enzootically stable areas. The efficacy of current live vaccines, composed of B. boris or B. bigemina strains of low virulence, or antigenically defined subunit vaccines requires that the challenge strains contain the epitopes targeted by the immune response. For both B. boris and B. bigemina these targets may include surface B cell epitopes distributed diffusely in the merozoite outer membrane, or focally, as is characteristic of epitopes derived from apical complex organelles (Palmer and McElwain, 1995). In this manuscript, a panel of monoclonal antibodies (mAb) defined against biological clones of the Mexico strains of B. boris or B. bigemina were used to identify conserved and variable B cell epitopes on recently isolated strains from Matto Grosso do Sul, Brazil. In addition, the ability of cattle experimentally infected with Matto Grosso strains of B. bigemina or B. boris and cattle with field acquired B. boris or B. bigemina infections to respond to conserved B cell epitopes on the Mexico clones was determined.
2. Materials and methods
2.1. Babesia bovis and Babesia bigemina strains The Mo 7 biological clone of a Mexico strain of B. boris and the JG-29 clone of a Mexico strain of B. bigemina, used as reference strains in this study, were obtained by limiting dilution cloning using in vitro continuous erythrocyte culture as previously described (Rodriguez et al., 1986; Hines et al., 1989; McElwain et al., 1991). The Matto Grosso do Sul strain of B. boris was isolated from Boophilus microplus obtained in an enzootic area by feeding larvae on a seronegative splenectomized calf for five days (Kessler et al., 1987). The Matto Grosso do Sul strain of B. bigemina was isolated similarly with the exception that B. microplus nymphs were fed on a seronegative splenectomized calf (Kessler et al., 1987). The Matto Grosso do Sul strain of B. boris or B. bigemina were then obtained from the infected calf during acute parasitemia and the species identify confirmed using morphologic characteristics (Purnell, 1981). All strains
C.R. Madrugaet al. / VeterinaryParasitology61 (1996)21-30
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were maintained as cryopreserved stabilates as described for B. boris (Palmer et al., 1982) and B. bigemina (Vega et al., 1985). 2.2. Monocional antibodies (mAbs)
MAbs against B. boris were produced by immunization of mice with Mexico strain merozoites as described (Goff et al., 1988; Reduker et al., 1989). Each mAb reacts with the surface of live merozoites of the Mexico strain (Goff et al., 1988; Reduker et al., 1989). The mAbs used and their reactivity with the Mexico strain were: 23/28.57, reactive with a 16 kDa apical polypeptide (Reduker et al., 1989); BABB 35A4, 23/3.16, and 23/10.36, reactive with three distinct epitopes on the 42 kDa merozoite surface antigen-1 (MSA-1) (Palmer et al., 1991); 23/70.174, reactive with the 44 kDa MSA-2 (Jasmer et al., 1992); and 23/53.156 and BABB75A4, reactive with two distinct epitopes on the 60 kDa rhoptry associated protein-1 (RAP-l) (Suarez et al., 1993). MAbs against B. bigemina were produced by immunization with Mexico strain merozoites and each mAb reacts with the surface of live merozoites of the Mexico strain (McElwain et al., 1987, 1991). The mAbs used and their reactivity with the Mexico strain were: 14/16.1.7, reactive with the 58 kDa RAP-1 (Machado et al., 1993); 14/1.3.2, reactive with the 45 kDa merozoite surface glycoprotein (gp45) (McElwain et al., 1987); and 14/20.3.9, reactive with the 55 kDa merozoite surface glycoprotein (gp55) (McElwain et al., 1987). TryplE1, reactive with a variable surface glycoprotein of Trypanosoraa brucei, was used as a negative control mAb. All mAbs were super° natants from twice-cloned hybridomas. 2.3. Indirect immunofluorescence assay (IFA)
MAb binding was assessed using IFA with B. boris or B. bigemina infected erythrocytes. Blood was collected from splenectomized calves experimentally infected with the Matto Grosso do Sul strain of B. boris (11.5% parasitemia)' or the Matto Grosso do Sul strain of B. bigeraina (7.0% parasitemia) prior to the sixth day post-inoculation. Smears were fixed in phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and 3.7% formaldehyde and incubated for 30 min at 4°C with each mAb hybridoma supernatant. Following three washes in PBS containing 0.5% BSA, the smears were incubated for 30 rain with fluorescein isothiocyanate conjugated goat antibody against mouse immunoglobulin. Finally, smears were washed and examined by epifluorescent microscopy. 2.4. Bovine sera
Serum against the Mexico strain of B. boris was obtained from a non-splenectomized calf recovered from acute infection with the Mo7 clone of the Mexico strain. The reactivity of this serum with defined Mexico strain polypeptides and its ability to neutralize in vitro growth of the Mexico strain merozoites has been described previously (Hines et al., 1989, 1992). Pre-infection serum was used as a negative control. Seronegative calves, raised in isolation, were splenectomized and then inoculated with
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109 parasitized erythrocytes of the Matto Grosso do Sul strain of B. boris. Sera were collected prior to intravenous inoculation and then following recovery from acute babesiosis. Cattle within enzootic regions of Matto Grosso do Sul with naturally acquired B. boris infections were identified by seropositivity in the IFA test (Johnston et al., 1973), and used as a source of carrier sera. Serum against the Mexico strain of B. bigemina was obtained from a non-splenectomized calf following recovery from acute infection with the Mexico strain. The reactivity of this serum with defined Mexico strain polypeptides has been described previously (McElwain et al., 1991; Ushe et al., 1994). Pre-infection serum was used as a negative control. Seronegative splenectomized calves were experimentally infected with 10 9 B. bigemina parasitized erythrocytes of the Matto Grosso do Sul strain. Sera were collected prior to intravenous inoculation and then following recovery from acute babesiosis. Cattle with B. bigemina infections naturally acquired within Matto Grosso do Sul were identified by seropositivity in the IFA test (Ross and Lohr, 1968) and used as a source of carrier sera.
2.5. Radiolabeling and immunoprecipitation Mexico strain Mo-7 B. bovis merozoite proteins were metabolically labeled with 10 /xCi per well of [35S]-methionine during in vitro continuous culture using methioninedeficient medium (Goff et al., 1988). Mexico strain B. bigemina were grown in short-term culture and labeled by addition of 100 /xCi [3SS]-methionine per 109 cells (McElwain et al., 1987). The radiolabeled proteins (10 6 cpm [35S] per reaction) were incubated with 5 / z g of each mAb or 5/xl of undiluted bovine sera and precipitated using protein G-Sepharose as previously described (Suarez et al., 1991). The immunoprecipitated proteins were eluted by boiling for 3 min in SDS-PAGE sample buffer and then electrophoresed in 7.5% to 17.5% polyacrylamide gels. Gels were fixed and dried and immunoprecipitated proteins identified by fluorography. Molecular size standards included a4C labeled myosin (200 kDa), phosphorylase b (92.5 kDa), bovine serum albumin (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa) and lysozyme (14.3 kDa).
2.6. B. bovis MSA-1 ELISA Recombinant MSA-1, expressed and purified from E. coli as described previously (Hines et al., 1992), was used to coat microtiter plates at 150 ng per well in a pH 9.6 Na2CO 3 \ N a H C O 3 buffer. Plates were blocked by incubation with phosphate buffered saline (PBS) containing 0.1% gelatin at 37°C for 2 h. Plates were washed with PBS containing 0.2% tween-20. Bovine sera were diluted 1:10 in pH 7.2 PBS and 50#1 added per well. Following incubation at 37°C for 1 h, the plates were rinsed with PBS-0.2% tween-20 and horseradish peroxidase linked protein G was added and incubated for 1 h. The plates were again rinsed and 5-aminosalicylic acid added as a substrate and allowed to react for 45 min. Sera with an optical density at 440 nm that was > 3x the optical density of negative control sera were scored as positive.
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3. Results and discussion
Conservation of surface exposed B cell epitopes between the Mexico strain and the Matto Grosso do Sul B. bovis strain was determined using surface reactive mAbs in immunofluorescence. The reactivity and binding pattern of each mAb with the Matto Grosso do Sul strain is indicated in Table 1. The conservation of the B cell epitopes on the 16 kDa apical polypeptide and the 60 kDa RAP-1 is consistent with previous studies indicating that these apical complex and surface exposed epitopes are also present on clones and strains from Argentina, Australia, and Israel (Palmer et al., 1991; Suarez et al., 1994). The Matto Grosso do Sul strain also expressed two of three distinct MSA-1 epitopes and one MSA-2 epitope present on the Mexico strain of B. bovis (Table 1). Mexico strain MSA-1 and MSA-2 epitopes have been previously shown to be expressed on a genetically distinct Texas strain, but were not conserved among more geographically distant strains including two strains from Argentina (Palmer et al., 1991; Suarez et al., 1994). The expression of the MSA-1 and MSA-2 epitopes by the Matto Grosso do Sul strain suggests that antigenic variability in these merozoite membrane surface polypeptides may be less extreme than previously thought. The loss of expression of a single MSA-1 epitope but retention of two distinct MSA-1 epitopes has not been previously described and indicates that antigenic variation, attributed to a frame-shift mutation between the Mexico and Australia L strains (Hines et al., 1992), does not necessarily involve all surface exposed epitopes. To determine if cattle infected with the Matto Grosso do Sul strain developed antibody that recognized conserved epitopes on specific B. boris polypeptides, sera from infected animals were used to immunoprecipitate [3SS]-methionine labeled Mexico Mo7 polypeptides (Fig. 1). Reactivity with specific polypeptides was determined by comparison with co-migrating polypeptides precipitated with the defined mAbs (data not shown). Serum from an animal infected with the Mo7 Mexico clone reacted with multiple homologous Mexico strain polypeptides (Fig. 1, lane 2), as previously described (Hines et al., 1989). Sera from cattle experimentally infected with the Matto
Table 1 Reactivity of surface binding monoclonal antibodies developed against the Mexico strain with the Matto Grosso do Sul strain of Babesia boris MAb
Polypeptide reactivity
Binding to Matto Grosso do Sul strain a
TryplE1 23/28.57 BABB35A4 23/3.16 23/10.36 23/70.174 23/53.156 BABB75A4
T. brucei b 16 kDa MSA-1; 42 kDa MSA- 1; 42 kDa MSA-1; 42 kDa MSA-2; 44 kDa RAP-l; 60 kDa RAP-I; 60 kDa
Negative Apical merozoite Homogenous merozoite membrane Homogenous merozoite membrane Negative Homogenous merozoite membrane Apical merozoite Apical merozoite
intraerythrocytic merozoites. b TryplE1 binds a Trypanosoma brucei VSG and was used as a negative control. a B i n d i n g to
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C.R. Madruga et al. / Veterinary Parasitology 61 (1996) 21-30 1 2 3 4 5 6 7 2oolb 921~ 691~ 4,-- RAP1 481~
"'MSA 1 3o1~ 141b
Fig. l. B. bot:is merozoite polypeptides recognized by sera from infected cattle. Mexico clone Mo-7 B. bovis merozoites were metabolically labeled with [3Ss] methionine, immunoprecipitated with each bovine serum, and bound polypeptides identified using SDS-PAGE and flurography. Lane assignments are as follows: negative bovine serum (lane 1); serum from a Mexico strain infected animal (lane 2); sera from splenectomized cattle following acute infection with the Matto Grosso do Sul strain (lanes 3,6,7); and sera from cattle with B. boris infection naturally acquired within Matto Grosso do Sul (lanes 4,5). Molecular size markers (kDa) are in the left margin and the position of the 60 kDa RAP-1 and the 42 kDa MSA-1 are indicated in the right margin.
Grosso do Sul strain also recognized multiple polypeptides in the heterologous Mexico strain (Fig. 1, lanes 3,6,7) as did sera from carrier cattle with naturally acquired infections (Fig. 1, lanes 4,5). Interestingly, the 60 kDa RAP-1 polypeptide was immunoprecipitated by sera from the acutely infected splenectomized cattle but not by the naturally infected carrier cattle (Fig. 1). The reactivity of the sera from splenectomized cattle indicates that the spleen is not required to generate an antibody response to RAP-1. The absence of antibodies against RAP-1 in the sera of carrier cattle may be associated with low merozoite number during persistent infection as compared to the high parasitemia during the acute phase of infection. Alternatively, the carrier cattle, which were naturally infected with B. boL, is within an enzootic area of Matto Grosso do Sul, may have been infected with a strain antigenically distinct from the Matto Grosso do Sul strain used in the experimental infections. Specific reactivity of the heterologous sera to Mexico strain MSA-1 was confirmed using a recombinant MSA-1 ELISA. Serum from the positive control Mexico strain infected animal had an OD of 0.337 (4.4x negative control OD). Heterologous sera from two carrier cattle also bound MSA-1; sera from the animals represented in Fig. 1, lane 5, and Fig. 1, lane 4, had ODs of 0.327 (4.3x negative control OD) and 0.247 (3.2x negative control OD), respectively. Sera from the acutely infected splenectomized cattle did not react with MSA-1, despite the presence of MSA-1 epitopes on the Matto Grosso do Sul strain used to initiate infection. The lack of binding of the sera from the splenectomized cattle suggests that, unlike R A P - l , the spleen may have a critical
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Table 2 Reactivity of surface binding monoclonal antibodies developed against the Mexico strain with the Matto Grosso do Sul strain of Babesia bigemina MAb Polypeptide Binding to Matto Grosso reactivity do Sul strain a TryplE1 T. brucei b Negative 14/1.3.2 gp45; 45 kDa Homogenousmerozoitemembrane 14/20.3.9 gp55; 55 kDa Punctate merozoitemembrane 14/16.1.7 RAP-I; 58 kDa Apical merozoite Binding to intraerythrocyticmerozoites. b TryplE1 binds a Trypanosoma brucei VSG and was used as a negativecontrol. a
function in generating antibody to MSA-1 early after infection. This is consistent with previous results showing that non-splenectomized calves develop antibody to MSA-1 as early as 7 - 1 0 days postinfection (T.F. McElwain et al., unpublished data, 1991). The reactivity of the sera from the carrier cattle with naturally acquired infections confirms that MSA-1 B cell epitopes conserved between the Mexico strain and strains within Matto Grosso do Sul are recognized by infected cattle. Immunization of cattle with Mexico strain MSA-1 induces antibody that inhibits growth of both the Mexico and Texas strains (Hines et al., 1992). Whether the inhibition-sensitive epitopes are conserved on strains from Matto Grosso do Sul or elsewhere in South America is unknown. The reactivity and binding pattern of each anti-B, bigemina mAb with the Matto Grosso do Sul strain of B. bigemina is indicated in Table 2. The reactivity of mAb 14/16.1.7, which binds a neutralization-sensitive epitope on the 58 kDa RAP-1 (Figueroa et al., 1991; Ushe et al., 1994), with the Matto Grosso do Sul strain was expected as this epitope has previously been shown to be conserved among strains from Argentina, Kenya, Mexico, Puerto Rico, and St. Croix (McElwain et al., 1991; Suarez et al., 1994). The epitopes on the Mexico strain 45 kDa and 55 kDa surface glycoproteins, shown here to be conserved with the Matto Grosso do Sul strain, frequently vary among strains as shown by their lack of conservation in all other examined strains with the exception of the 55 kDa polypeptide epitope in Texcoco, an additional strain isolated in Mexico (McElwain et al., 1991). Sera from cattle infected with the Matto Grosso do Sul strain of B. bigemina immunoprecipitated multiple [35S] labeled Mexico strain polypeptides (Fig. 2). Predominant merozoite surface polypeptides (apparent molecular sizes of 72, 58, 55, 45 and 36 kDa) were immunoprecipitated by sera from cattle infected with the homologous Mexico strain (Fig. 2, lane 7), splenectomized cattle acutely infected with the Matto Grosso do Sul strain (Fig. 2, lanes 2,3,6), and cattle with B. bigemina infections naturally acquired within Matto Grosso do Sul (Fig. 2, lanes 4,5). There were no consistent differences in the major polypeptides recognized by sera from acutely infected splenectomized cattle versus chronically infected cattle. The recognition of these Mexico strain polypeptides by the heterologous sera indicates that the conserved B cell epitopes, including those on RAP-1 and gp45, are recognized by the immune system following acute or chronic infection.
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C.R. Madruga et al. / Veterinary Parasitology 61 (1996) 21-30 1 2 3 4 5 6 7 8 200 I~ 92 ~ , 69 ID-
~RAP1 --gp45
46~
30 ~ ~
/~
~4~O
Fig. 2. B. bigemina merozoite polypeptides recognized by sera from infected cattle. Mexico clone JG-29 B. bigemina merozoites were metabolically labeled with [35S] methionine, immunoprecipitated with each bovine serum, and bound polypeptides identified using SDS-PAGE and fluorography. Lane assignments are as follows: negative bovine serum (lane 8); serum from a Mexico strain infected animal (lane 7); sera from splenectomized cattle following acute infection with the Matto Grosso do Sul strain (lanes 2,3,6); and sera from cattle with B. bigemina infection naturally acquired within Matto Grosso do Sul (lanes 4,5). Molecular size markers are in lane 1 and are designated in the left margin (kDa). The position of the 58 kDa RAP-1 and gp45 are indicated in the right margin.
In summary, the conservation of merozoite surface exposed B cell epitopes, including epitopes not previously shown to be conserved among geographically distinct strains, and the immunogenicity of these epitopes suggests that Babesia merozoite antigenic variation may be less widespread than previously believed. The widespread conservation of apical complex surface exposed epitopes, and specifically the neutralization sensitive epitope on RAP-I, support the development and testing of immunogens based on these molecules.
Acknowledgments This work was supported by the USDA NRICGP No. 92-37204-8180 and the Centro Nacional de Pesquisa de Gado de Corte, EMBRAPA. We acknowledge the assistance of S.G.T. Pepper.
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Suarez, C.E., McEIwain, T.F., Echaide, I., Toriani de Echaide, S. and Palmer, G.H., 1994. Interstrain conservation of Babesial RAP-I surface exposed B-cell epitopes despite rap-1 genomic polymorphism. Infect. lmmun., 62: 3576-3579. Suarez, C.E., Palmer, G.H., Hines, S.A. and McElwain, T.F., 1993. Immunogenic B-cell epitopes of Babesia bot,is rhoptry-associated protein 1 are distinct from sequences conserved between species. Infect. Immun., 61: 3511-3517. Suarez, C.E., Palmer, G.H., Jasmer, D.P., Hines, S.A., Perryman, L.E. and McElwain T.F., 1991. Characterization of the gene encoding a 60-kilodalton Babesia bol,is merozoite protein with conserved and surface exposed epitopes. Mol. Biochem. Parasitol.. 46: 45-52. Ushe, T.C., Palmer, G.H., Sotomayor, L., Figueroa, J.V., Buening, G.M., Perryman, L.E. and McElwain, T.F., 1994. Antibody response to a Babesia bigemina RAP-1 surface exposed and neutralization sensitive epitopes in immune cattle. Infect. Immun., 62: 5698-5701. Vega, C.A., Buening, G.M., Rodriguez, S.D., Carson, C.A. and McLaughlin, K., 1985. Cryopreservation of Babesia bigemina for in vitro cultivation. Am. J. Vet. Rcs., 46: 421-423.
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Conservation of merozoite membrane and apical complex B cell epitopes among Babesia bigemina and Babesia boris strains isolated in Brazil Claudio R. Madruga a, Carlos E. Suarez b, Terry F. McElwain b, Guy H. Palmer b,, a Centro Nacional de Pesquisa de Gado de Corte, EMBRAPA, Caixa Postal 154, CEP 79080-970, Campo Grande, MS, Brazil b Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, WA 99164-7040 USA
Received 15 November1994; accepted 9 February1995
Abstract Babesia merozoite polypeptides bear surface exposed and neutralization-sensitive B cell epitopes and have been shown to induce partial protection against experimental challenge. Variation in these epitopes has been examined in a limited number of strains. In this study, utilizing strains of Babesia bovis and Babesia bigemina from Matto Grosso do Sul in Brazil, we examined the conservation of epitopes bound by monoclonal antibodies developed against Mexico strains of B. bovis and B. bigemina. Apical complex B-cell epitopes, previously shown to be species-specific but common among otherwise antigenically distinct strains, were also conserved between clones of the Mexico strains and the Matto Grosso do Sul strains of each Babesia species. Mexico strain polypeptides bearing these epitopes were recognized by sera from cattle infected with the Matto Grosso do Sul strains. Two distinct epitopes on the B. boris neutralization-sensitive merozoite surface antigen-1 (MSA-1) were also conserved between the Mexico Mo7 clone and the Matto Grosso do Sul strain, in contrast to previous studies which demonstrated variability among strains. Sera from cattle with B. bovis infections naturally acquired in Matto Grosso do Sul bound Mexico Mo7 MSA-1, demonstrating that conserved MSA-1 epitopes were recognized by the bovine immune system. Similarly, merozoite surface epitopes on the B. bigemina 45 kDa and 55 kDa glycoproteins were conserved on the Matto Grosso do Sul strain of B. bigemina. Keywords: Babesia bigemina; Babesia boris; Merozoite; Apical complex; B cell epitopes
* Correspondingauthor. 0304-4017/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0304-4017(95)00809-8
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I. Introduction Babesiosis is a tick-borne disease widespread in cattle herds within tropical and subtropical regions worldwide (McCosker, 1981). In Central and South America, 70% of the cattle live within areas infested with the vector Boophilus microplus (Lopez, 1977). Brazil contains approximately 100 x 10 ~ cattle, one-sixth of the world's cattle population, with most of the highly productive cattle located in the southern states (Montenegro-James, 1992). Prevalence of both Babesia boris and B. bigemina in this region is high with mean values of > 75% reported in each of the states of Matto Grosso do Sul, Minas Gerais, and Rio Grande do Sul (Madruga et al., 1983; Patarroyo et al., 1987; Leite, 1988). Annual direct economic losses due to babesiosis in Rio Grande do Sul were estimated at US $99 million in 1984 (Madruga et al., 1984). Consequently, in Brazil and throughout South America, development of improved control for babesiosis is a high priority. Immunoprophylaxis provides a method for control both within enzootically unstable areas and for introduction of susceptible cattle into enzootically stable areas. The efficacy of current live vaccines, composed of B. boris or B. bigemina strains of low virulence, or antigenically defined subunit vaccines requires that the challenge strains contain the epitopes targeted by the immune response. For both B. boris and B. bigemina these targets may include surface B cell epitopes distributed diffusely in the merozoite outer membrane, or focally, as is characteristic of epitopes derived from apical complex organelles (Palmer and McElwain, 1995). In this manuscript, a panel of monoclonal antibodies (mAb) defined against biological clones of the Mexico strains of B. boris or B. bigemina were used to identify conserved and variable B cell epitopes on recently isolated strains from Matto Grosso do Sul, Brazil. In addition, the ability of cattle experimentally infected with Matto Grosso strains of B. bigemina or B. boris and cattle with field acquired B. boris or B. bigemina infections to respond to conserved B cell epitopes on the Mexico clones was determined.
2. Materials and methods
2.1. Babesia bovis and Babesia bigemina strains The Mo 7 biological clone of a Mexico strain of B. boris and the JG-29 clone of a Mexico strain of B. bigemina, used as reference strains in this study, were obtained by limiting dilution cloning using in vitro continuous erythrocyte culture as previously described (Rodriguez et al., 1986; Hines et al., 1989; McElwain et al., 1991). The Matto Grosso do Sul strain of B. boris was isolated from Boophilus microplus obtained in an enzootic area by feeding larvae on a seronegative splenectomized calf for five days (Kessler et al., 1987). The Matto Grosso do Sul strain of B. bigemina was isolated similarly with the exception that B. microplus nymphs were fed on a seronegative splenectomized calf (Kessler et al., 1987). The Matto Grosso do Sul strain of B. boris or B. bigemina were then obtained from the infected calf during acute parasitemia and the species identify confirmed using morphologic characteristics (Purnell, 1981). All strains
C.R. Madrugaet al. / VeterinaryParasitology61 (1996)21-30
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were maintained as cryopreserved stabilates as described for B. boris (Palmer et al., 1982) and B. bigemina (Vega et al., 1985). 2.2. Monocional antibodies (mAbs)
MAbs against B. boris were produced by immunization of mice with Mexico strain merozoites as described (Goff et al., 1988; Reduker et al., 1989). Each mAb reacts with the surface of live merozoites of the Mexico strain (Goff et al., 1988; Reduker et al., 1989). The mAbs used and their reactivity with the Mexico strain were: 23/28.57, reactive with a 16 kDa apical polypeptide (Reduker et al., 1989); BABB 35A4, 23/3.16, and 23/10.36, reactive with three distinct epitopes on the 42 kDa merozoite surface antigen-1 (MSA-1) (Palmer et al., 1991); 23/70.174, reactive with the 44 kDa MSA-2 (Jasmer et al., 1992); and 23/53.156 and BABB75A4, reactive with two distinct epitopes on the 60 kDa rhoptry associated protein-1 (RAP-l) (Suarez et al., 1993). MAbs against B. bigemina were produced by immunization with Mexico strain merozoites and each mAb reacts with the surface of live merozoites of the Mexico strain (McElwain et al., 1987, 1991). The mAbs used and their reactivity with the Mexico strain were: 14/16.1.7, reactive with the 58 kDa RAP-1 (Machado et al., 1993); 14/1.3.2, reactive with the 45 kDa merozoite surface glycoprotein (gp45) (McElwain et al., 1987); and 14/20.3.9, reactive with the 55 kDa merozoite surface glycoprotein (gp55) (McElwain et al., 1987). TryplE1, reactive with a variable surface glycoprotein of Trypanosoraa brucei, was used as a negative control mAb. All mAbs were super° natants from twice-cloned hybridomas. 2.3. Indirect immunofluorescence assay (IFA)
MAb binding was assessed using IFA with B. boris or B. bigemina infected erythrocytes. Blood was collected from splenectomized calves experimentally infected with the Matto Grosso do Sul strain of B. boris (11.5% parasitemia)' or the Matto Grosso do Sul strain of B. bigeraina (7.0% parasitemia) prior to the sixth day post-inoculation. Smears were fixed in phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and 3.7% formaldehyde and incubated for 30 min at 4°C with each mAb hybridoma supernatant. Following three washes in PBS containing 0.5% BSA, the smears were incubated for 30 rain with fluorescein isothiocyanate conjugated goat antibody against mouse immunoglobulin. Finally, smears were washed and examined by epifluorescent microscopy. 2.4. Bovine sera
Serum against the Mexico strain of B. boris was obtained from a non-splenectomized calf recovered from acute infection with the Mo7 clone of the Mexico strain. The reactivity of this serum with defined Mexico strain polypeptides and its ability to neutralize in vitro growth of the Mexico strain merozoites has been described previously (Hines et al., 1989, 1992). Pre-infection serum was used as a negative control. Seronegative calves, raised in isolation, were splenectomized and then inoculated with
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109 parasitized erythrocytes of the Matto Grosso do Sul strain of B. boris. Sera were collected prior to intravenous inoculation and then following recovery from acute babesiosis. Cattle within enzootic regions of Matto Grosso do Sul with naturally acquired B. boris infections were identified by seropositivity in the IFA test (Johnston et al., 1973), and used as a source of carrier sera. Serum against the Mexico strain of B. bigemina was obtained from a non-splenectomized calf following recovery from acute infection with the Mexico strain. The reactivity of this serum with defined Mexico strain polypeptides has been described previously (McElwain et al., 1991; Ushe et al., 1994). Pre-infection serum was used as a negative control. Seronegative splenectomized calves were experimentally infected with 10 9 B. bigemina parasitized erythrocytes of the Matto Grosso do Sul strain. Sera were collected prior to intravenous inoculation and then following recovery from acute babesiosis. Cattle with B. bigemina infections naturally acquired within Matto Grosso do Sul were identified by seropositivity in the IFA test (Ross and Lohr, 1968) and used as a source of carrier sera.
2.5. Radiolabeling and immunoprecipitation Mexico strain Mo-7 B. bovis merozoite proteins were metabolically labeled with 10 /xCi per well of [35S]-methionine during in vitro continuous culture using methioninedeficient medium (Goff et al., 1988). Mexico strain B. bigemina were grown in short-term culture and labeled by addition of 100 /xCi [3SS]-methionine per 109 cells (McElwain et al., 1987). The radiolabeled proteins (10 6 cpm [35S] per reaction) were incubated with 5 / z g of each mAb or 5/xl of undiluted bovine sera and precipitated using protein G-Sepharose as previously described (Suarez et al., 1991). The immunoprecipitated proteins were eluted by boiling for 3 min in SDS-PAGE sample buffer and then electrophoresed in 7.5% to 17.5% polyacrylamide gels. Gels were fixed and dried and immunoprecipitated proteins identified by fluorography. Molecular size standards included a4C labeled myosin (200 kDa), phosphorylase b (92.5 kDa), bovine serum albumin (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa) and lysozyme (14.3 kDa).
2.6. B. bovis MSA-1 ELISA Recombinant MSA-1, expressed and purified from E. coli as described previously (Hines et al., 1992), was used to coat microtiter plates at 150 ng per well in a pH 9.6 Na2CO 3 \ N a H C O 3 buffer. Plates were blocked by incubation with phosphate buffered saline (PBS) containing 0.1% gelatin at 37°C for 2 h. Plates were washed with PBS containing 0.2% tween-20. Bovine sera were diluted 1:10 in pH 7.2 PBS and 50#1 added per well. Following incubation at 37°C for 1 h, the plates were rinsed with PBS-0.2% tween-20 and horseradish peroxidase linked protein G was added and incubated for 1 h. The plates were again rinsed and 5-aminosalicylic acid added as a substrate and allowed to react for 45 min. Sera with an optical density at 440 nm that was > 3x the optical density of negative control sera were scored as positive.
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3. Results and discussion
Conservation of surface exposed B cell epitopes between the Mexico strain and the Matto Grosso do Sul B. bovis strain was determined using surface reactive mAbs in immunofluorescence. The reactivity and binding pattern of each mAb with the Matto Grosso do Sul strain is indicated in Table 1. The conservation of the B cell epitopes on the 16 kDa apical polypeptide and the 60 kDa RAP-1 is consistent with previous studies indicating that these apical complex and surface exposed epitopes are also present on clones and strains from Argentina, Australia, and Israel (Palmer et al., 1991; Suarez et al., 1994). The Matto Grosso do Sul strain also expressed two of three distinct MSA-1 epitopes and one MSA-2 epitope present on the Mexico strain of B. bovis (Table 1). Mexico strain MSA-1 and MSA-2 epitopes have been previously shown to be expressed on a genetically distinct Texas strain, but were not conserved among more geographically distant strains including two strains from Argentina (Palmer et al., 1991; Suarez et al., 1994). The expression of the MSA-1 and MSA-2 epitopes by the Matto Grosso do Sul strain suggests that antigenic variability in these merozoite membrane surface polypeptides may be less extreme than previously thought. The loss of expression of a single MSA-1 epitope but retention of two distinct MSA-1 epitopes has not been previously described and indicates that antigenic variation, attributed to a frame-shift mutation between the Mexico and Australia L strains (Hines et al., 1992), does not necessarily involve all surface exposed epitopes. To determine if cattle infected with the Matto Grosso do Sul strain developed antibody that recognized conserved epitopes on specific B. boris polypeptides, sera from infected animals were used to immunoprecipitate [3SS]-methionine labeled Mexico Mo7 polypeptides (Fig. 1). Reactivity with specific polypeptides was determined by comparison with co-migrating polypeptides precipitated with the defined mAbs (data not shown). Serum from an animal infected with the Mo7 Mexico clone reacted with multiple homologous Mexico strain polypeptides (Fig. 1, lane 2), as previously described (Hines et al., 1989). Sera from cattle experimentally infected with the Matto
Table 1 Reactivity of surface binding monoclonal antibodies developed against the Mexico strain with the Matto Grosso do Sul strain of Babesia boris MAb
Polypeptide reactivity
Binding to Matto Grosso do Sul strain a
TryplE1 23/28.57 BABB35A4 23/3.16 23/10.36 23/70.174 23/53.156 BABB75A4
T. brucei b 16 kDa MSA-1; 42 kDa MSA- 1; 42 kDa MSA-1; 42 kDa MSA-2; 44 kDa RAP-l; 60 kDa RAP-I; 60 kDa
Negative Apical merozoite Homogenous merozoite membrane Homogenous merozoite membrane Negative Homogenous merozoite membrane Apical merozoite Apical merozoite
intraerythrocytic merozoites. b TryplE1 binds a Trypanosoma brucei VSG and was used as a negative control. a B i n d i n g to
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C.R. Madruga et al. / Veterinary Parasitology 61 (1996) 21-30 1 2 3 4 5 6 7 2oolb 921~ 691~ 4,-- RAP1 481~
"'MSA 1 3o1~ 141b
Fig. l. B. bot:is merozoite polypeptides recognized by sera from infected cattle. Mexico clone Mo-7 B. bovis merozoites were metabolically labeled with [3Ss] methionine, immunoprecipitated with each bovine serum, and bound polypeptides identified using SDS-PAGE and flurography. Lane assignments are as follows: negative bovine serum (lane 1); serum from a Mexico strain infected animal (lane 2); sera from splenectomized cattle following acute infection with the Matto Grosso do Sul strain (lanes 3,6,7); and sera from cattle with B. boris infection naturally acquired within Matto Grosso do Sul (lanes 4,5). Molecular size markers (kDa) are in the left margin and the position of the 60 kDa RAP-1 and the 42 kDa MSA-1 are indicated in the right margin.
Grosso do Sul strain also recognized multiple polypeptides in the heterologous Mexico strain (Fig. 1, lanes 3,6,7) as did sera from carrier cattle with naturally acquired infections (Fig. 1, lanes 4,5). Interestingly, the 60 kDa RAP-1 polypeptide was immunoprecipitated by sera from the acutely infected splenectomized cattle but not by the naturally infected carrier cattle (Fig. 1). The reactivity of the sera from splenectomized cattle indicates that the spleen is not required to generate an antibody response to RAP-1. The absence of antibodies against RAP-1 in the sera of carrier cattle may be associated with low merozoite number during persistent infection as compared to the high parasitemia during the acute phase of infection. Alternatively, the carrier cattle, which were naturally infected with B. boL, is within an enzootic area of Matto Grosso do Sul, may have been infected with a strain antigenically distinct from the Matto Grosso do Sul strain used in the experimental infections. Specific reactivity of the heterologous sera to Mexico strain MSA-1 was confirmed using a recombinant MSA-1 ELISA. Serum from the positive control Mexico strain infected animal had an OD of 0.337 (4.4x negative control OD). Heterologous sera from two carrier cattle also bound MSA-1; sera from the animals represented in Fig. 1, lane 5, and Fig. 1, lane 4, had ODs of 0.327 (4.3x negative control OD) and 0.247 (3.2x negative control OD), respectively. Sera from the acutely infected splenectomized cattle did not react with MSA-1, despite the presence of MSA-1 epitopes on the Matto Grosso do Sul strain used to initiate infection. The lack of binding of the sera from the splenectomized cattle suggests that, unlike R A P - l , the spleen may have a critical
C.R. Madruga et al. / Veterinary Parasitology 61 (1996) 21-30
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Table 2 Reactivity of surface binding monoclonal antibodies developed against the Mexico strain with the Matto Grosso do Sul strain of Babesia bigemina MAb Polypeptide Binding to Matto Grosso reactivity do Sul strain a TryplE1 T. brucei b Negative 14/1.3.2 gp45; 45 kDa Homogenousmerozoitemembrane 14/20.3.9 gp55; 55 kDa Punctate merozoitemembrane 14/16.1.7 RAP-I; 58 kDa Apical merozoite Binding to intraerythrocyticmerozoites. b TryplE1 binds a Trypanosoma brucei VSG and was used as a negativecontrol. a
function in generating antibody to MSA-1 early after infection. This is consistent with previous results showing that non-splenectomized calves develop antibody to MSA-1 as early as 7 - 1 0 days postinfection (T.F. McElwain et al., unpublished data, 1991). The reactivity of the sera from the carrier cattle with naturally acquired infections confirms that MSA-1 B cell epitopes conserved between the Mexico strain and strains within Matto Grosso do Sul are recognized by infected cattle. Immunization of cattle with Mexico strain MSA-1 induces antibody that inhibits growth of both the Mexico and Texas strains (Hines et al., 1992). Whether the inhibition-sensitive epitopes are conserved on strains from Matto Grosso do Sul or elsewhere in South America is unknown. The reactivity and binding pattern of each anti-B, bigemina mAb with the Matto Grosso do Sul strain of B. bigemina is indicated in Table 2. The reactivity of mAb 14/16.1.7, which binds a neutralization-sensitive epitope on the 58 kDa RAP-1 (Figueroa et al., 1991; Ushe et al., 1994), with the Matto Grosso do Sul strain was expected as this epitope has previously been shown to be conserved among strains from Argentina, Kenya, Mexico, Puerto Rico, and St. Croix (McElwain et al., 1991; Suarez et al., 1994). The epitopes on the Mexico strain 45 kDa and 55 kDa surface glycoproteins, shown here to be conserved with the Matto Grosso do Sul strain, frequently vary among strains as shown by their lack of conservation in all other examined strains with the exception of the 55 kDa polypeptide epitope in Texcoco, an additional strain isolated in Mexico (McElwain et al., 1991). Sera from cattle infected with the Matto Grosso do Sul strain of B. bigemina immunoprecipitated multiple [35S] labeled Mexico strain polypeptides (Fig. 2). Predominant merozoite surface polypeptides (apparent molecular sizes of 72, 58, 55, 45 and 36 kDa) were immunoprecipitated by sera from cattle infected with the homologous Mexico strain (Fig. 2, lane 7), splenectomized cattle acutely infected with the Matto Grosso do Sul strain (Fig. 2, lanes 2,3,6), and cattle with B. bigemina infections naturally acquired within Matto Grosso do Sul (Fig. 2, lanes 4,5). There were no consistent differences in the major polypeptides recognized by sera from acutely infected splenectomized cattle versus chronically infected cattle. The recognition of these Mexico strain polypeptides by the heterologous sera indicates that the conserved B cell epitopes, including those on RAP-1 and gp45, are recognized by the immune system following acute or chronic infection.
28
C.R. Madruga et al. / Veterinary Parasitology 61 (1996) 21-30 1 2 3 4 5 6 7 8 200 I~ 92 ~ , 69 ID-
~RAP1 --gp45
46~
30 ~ ~
/~
~4~O
Fig. 2. B. bigemina merozoite polypeptides recognized by sera from infected cattle. Mexico clone JG-29 B. bigemina merozoites were metabolically labeled with [35S] methionine, immunoprecipitated with each bovine serum, and bound polypeptides identified using SDS-PAGE and fluorography. Lane assignments are as follows: negative bovine serum (lane 8); serum from a Mexico strain infected animal (lane 7); sera from splenectomized cattle following acute infection with the Matto Grosso do Sul strain (lanes 2,3,6); and sera from cattle with B. bigemina infection naturally acquired within Matto Grosso do Sul (lanes 4,5). Molecular size markers are in lane 1 and are designated in the left margin (kDa). The position of the 58 kDa RAP-1 and gp45 are indicated in the right margin.
In summary, the conservation of merozoite surface exposed B cell epitopes, including epitopes not previously shown to be conserved among geographically distinct strains, and the immunogenicity of these epitopes suggests that Babesia merozoite antigenic variation may be less widespread than previously believed. The widespread conservation of apical complex surface exposed epitopes, and specifically the neutralization sensitive epitope on RAP-I, support the development and testing of immunogens based on these molecules.
Acknowledgments This work was supported by the USDA NRICGP No. 92-37204-8180 and the Centro Nacional de Pesquisa de Gado de Corte, EMBRAPA. We acknowledge the assistance of S.G.T. Pepper.
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Suarez, C.E., McEIwain, T.F., Echaide, I., Toriani de Echaide, S. and Palmer, G.H., 1994. Interstrain conservation of Babesial RAP-I surface exposed B-cell epitopes despite rap-1 genomic polymorphism. Infect. lmmun., 62: 3576-3579. Suarez, C.E., Palmer, G.H., Hines, S.A. and McElwain, T.F., 1993. Immunogenic B-cell epitopes of Babesia bot,is rhoptry-associated protein 1 are distinct from sequences conserved between species. Infect. Immun., 61: 3511-3517. Suarez, C.E., Palmer, G.H., Jasmer, D.P., Hines, S.A., Perryman, L.E. and McElwain T.F., 1991. Characterization of the gene encoding a 60-kilodalton Babesia bol,is merozoite protein with conserved and surface exposed epitopes. Mol. Biochem. Parasitol.. 46: 45-52. Ushe, T.C., Palmer, G.H., Sotomayor, L., Figueroa, J.V., Buening, G.M., Perryman, L.E. and McElwain, T.F., 1994. Antibody response to a Babesia bigemina RAP-1 surface exposed and neutralization sensitive epitopes in immune cattle. Infect. Immun., 62: 5698-5701. Vega, C.A., Buening, G.M., Rodriguez, S.D., Carson, C.A. and McLaughlin, K., 1985. Cryopreservation of Babesia bigemina for in vitro cultivation. Am. J. Vet. Rcs., 46: 421-423.