Veterinary Immunology and Immunopathology, 29 ( ! 991 ) 31-40
31
Elsevier Science Publishers B.V., Amsterdam
Identification of immunodominant common between Anaplasma centrale and
Anaplasma marginale Varda Shkap a, Eugene Pipano a, Travis C. McGuire b and Guy H. Palmer b-~ aDepartment of Parasitology, Kimron VeterinaryInstitute, Bet Dagan, Israel bDepartment of Veterinary Microbiology and Pathology, Washington State Unirersity, Pulhnan, WA 99164-7040, USA (Accepted 7 August 1990)
ABSTRACT Shkap, V., Pipano, E., McGuire, T.C. and Palmer, G.H., 1991. Identification of immunodominant polypeptides common between Anaplasma centrale and Anaplasma marginale. Vet. hmnunol, hnmunopathol., 29:31-40. High titered antibody from rabbits immunized with Anaplasma centrale or from cattle recovered from A. centrale infection bound predominantly to several 33-36 kDa polypeptides present in both A. centrale and the Israel-NT isolate of Anaplasma marginale. High titered bovine antibody against the Israel-NT isolate ofA. marginale also reacted predominantly with A. centrale polypeptides in this size range. The immunodominance of the 33-36 kDa polypeptides and their cross-reactivity indicate that these shared epitope.~ may be primarily responsible for the cross-protective immunity between A. centrale and A. marginale. ABBREVIATIONS ELISA, enzyme-linked immunosorbent assay; SDS, sodium dodecyl sulphate.
INTRODUCTION
Bovine anaplasmosis, caused by the intraerythrocytic rickettsia Anaplasma margin.a!e~ is widespread throughout tropical and subtropical regions worldwide (Losos, 1986). Anaplasma centrale is a closely related species that causes a mild form of anaplasmosis in cattle (Theiler, 1911; Pipano et al., 1985). Significantly, prior infection with A. centrale confers protection against severe disease caused by virulent isolates of A. marginale (Theiler, 1912; Kuttier, 1967b). Consequently, deliberate infection with t~. centrale has been uti'Author to whom correspondence should be addressed.
0165-2427/91/$03.50
© 1991 m Elsevier Science Publishers B.V.
32
V. SHKAP ET AL.
lized extensively in Israel as well as Australia and countries in Africa, Asia and South America to immunize cattle against anaplasmosis (reviewed by Palmer, 1989). This cross-protective immunity indicates that protection-inducing epitopes are shared between the two species of Anaplasma and provides an immunologic basis for the development of a recombinant or synthetic peptide vaccine based upon these shared antigens. Antibody from cattle recovered from infection with A. centrale or A. marginale has been shown to cross-react with the heterologous species of Anaplasma, although at a significantly lower titer than with the homologous species (Kuttler, 1967a ). The specific antigens responsible for this cross-reactivity have not been identified. Based on the demonstration that Anaplasma organisms are susceptible to antibody mediated neutralization (Palmer et al., 1986), we hypothesize that A. centrale-induced protection against A. marginale challenge is due to induction of cross-reactive antibody. The identification of A. centrale polypeptides recognized by cross-reactive antibody would provide candidates for testing as protective immunogens against virulent A. marginale. In this paper, we report the identification of immunodominant polypeptides common to A. centrale and A. marginale. MATERIALS A N D M E T H O D S
Source of Anaplasma isolates The A. centrale isolate is the vaccine strain presently used in Israel and was originally obtained from South Africa in 1952 (Pipano et al., 1986). The Israel-NT isolate of A. marginale has been previously che:acterized morphologicaUy and partially characterized antigenically (Klopfer et aL, 10fiR; Palmer et al., 1988a). Isolates were maintained by cryopreservation in dimethylsulfoxide (Love, 1972). Seronegative splenectomized calves were inoculated with stabilate preserved Anaplasma and monitored daily for rickettsemia by using Wright stained blood smears. Infected erythrocytes were obtained during ascending rickettsemia for metabolic radiolabeling in vitro. Source of antibodies Sera were obtained from cattle recovered from experimental infection with either A. centrale or the Israel-NT isolate ofA. marginale. Sera were collected when antibody titers against intact organisms of the homologous isolate exceeded 1024 as measured by using enzyme-linked immunosorbent assay ( ELISA ) as previously described (Shkap et al., 1990 ). Preinfection sera were unreactive in the assay and were used as negative controls. Polyclonal antibodies against intact A. centrale and A. marginale ( IsraelNT isolate) were produced by repeated immunization of rabbits. Organisms were isolated from infected erythrocytes as previously described (Palmer and McGuire, 1984) and emulsified in Freund's complete adjuvant for the initial
IMMUNODOMINANT POLYPEPTIDES COMMON BETWEEN ANAPLASMA SPP.
33
immunization and in incomplete adjuvant for three subsequent immunizations. Antibody titers of sera from rabbits immunized with A. centrale were 8000 against intact A. centrale as measured by ELISA. Similarly, antibody titers from A. marginale immunized rabbits were 8000 as measured against intact A. marginale using ELISA. Sera obtained prior to immunizations were unreactive against Anaplasma as measured by ELISA and were used as negative controls. Monoclonal antibodies ANAF 19E2 and ANA050A2 are directed against A. marginale MSP-2, a 33-36 kDa surface exposed polypeptide (Palmer et al., 1985, 1988a). The generation and characterization of these antibodies has been previously described (McGuire et al., 1984). TRYP 1E l, a monoclonal antibody against the variable surface glycoprotein of Trypanosoma brucei, was used as a negative control. Supernatants from twice cloned hybridomas were concentrated to 0.1 mg/ml for use in immunoprecipitations.
Immunoblotting Immunoblotting ofA. centrale and A. marginale antigens was done as previously described in detail (Oberle et al., 1988). Briefly, organisms of each Anaplasma species were isolated from infected erythrocytes, detergent disrupted (2% sodium dodecyl sulphate (SDS), 5% 2-mercaptoethanol, pH 6.8 ), and electrophoresed on 7.5-17.5% polyacrylamide gels containing SDS. Antigens were electrophoretically transferred to nitrocellulose and reacted with either bovine sera diluted 1" 1000 or rabbit sera diluted 1" 3000. Detection of bound rabbit antibody was done by using ~25I-protein A followed by autoradiography (Oberle et al., 1988 ). Bound bovine antibody was detected by using '25i-protein G and autoradiography (Hines et ai., l ~ ) . Apparent molecular weights of antigens were determined by comparison with ~4C molecular weight standards.
Metabolic radiolabeling Anaplasma organisms were metabolically radiolabeled with 35S-methionine during short-term in vitro cultivation of infected erythrocytes. As previously described in detail, radiolabel is incorporated exclusively into Ariaplasma polypeptides (Barbet et al., 1983 ). Briefly, blood was collected from a splenectomized calf during ascending Anaplasma rickettsemia and washed with removal of the buffy coat following each 500×g centrifugation. Washed erythrocytes were diluted 1:8 in Eagle's minimal essential medium without methionine and supplemented with 10% fetal calf serum, 2 mM L-glutamine, 10 U/ml of penicillin, and l 0/~g/ml of streptomycin. Following addition of 125/~Ci 35S-methionine per milliliter of culture media, cultures were incubated at 37°C in 5% CO2 for 48 h. Erythrocytes were washed four times in Hank's balanced salt solution and then detergent lysed (50 mM Tris, 5 mM
34
V. SHKAP ET AL.
EDTA, 5 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 1% Nonidet P40).
lmmunoprecipitation Radiolabeled Anaplasma lysate was centrifuged at 135 000 Xg for 1 h, filtered through a 0.45/zm filter, and sonicated at 100 W for 10 s. Detergentsoluble polypeptides ( 10-6tdchloroacetic acid precipitable cpm per tube) were incubated with 5/tg of monoclonal antibody for 30 rain at 4 °C followed by addition of rabbit anti-mouse immunoglobulin for an additional 30 rain incubation at 4°C. Immune complexes were precipitated by using 100/zl of a 10% (v/v ) solution of Staphylococcus aureus bearing protein A. Precipitates were washed as follows: ( 1 ) four times with a 20 mM Tris buffer containing 5 mM EDTA, 100 mM NaCI, and i % Nonidet P40; (2) four times with the same buffer but containing 2 M NaCI; and (3) twice with the basic wash buffer without additional NaCI. Immune complexes were eluted by boiling in sample buffer containing SDS and 2-mercaptoethanol. Eluted antigens were separated by electrophoresis on 7.5-17.5% gradient polyacrylamide gels containing SDS. Gels were fixed and processed for fluorography as previously described. Determination of apparent molecular weights was done by comparison with ~4C molecular weight standards. RESULTS
Identification of immunodominant antigens Immunodominant antigens in A. centra!e and the Israel-NT isolate of A. marginale were identified in immunoblots based on reactivity with antibody (diluted 1:1000) induced by infection of cattle with either species. Serum antibodies from cattle recovered from A. centrale bound to multiple A. centrale polypeptides with apparent molecular sizes of 29-200 kDa (Fig. 1, Lane 1 ). Predominant high titer antibody responses were to A. centrale antigens of approximately 33-36 kDa (apparent as a doublet) and 86 kDa. When reacted against the heterologous species of Anaplasma, antibody from A. centrale-infected cattle bound primarily A. marginale polypeptides of 36 kDa (no apparent doublet) and 86 kDa, although deafly less strongly than the homologous polypeptides (Fig. 1, Lane 2). Serum from cattle infected with the Israel-NT isolate of A. marginale had hipda titers of antibody a~ainst 3336 kDa (apparent as a doublet) and 86 kDahomologous A. marginale polypeptides (Fig. 1, Lane 4). Against the heterologous A. centrale polypeptides, the dominant response was against a 33-36 kDa doublet and only slight reactivity against the larger antigens in the approximately 86 kDa range (Fig. 1, Lane 3). In contrast to the homologous antibody which bound predominantly to the larger component of the 33-36 kDa A. centrale doublet, the heterologous antibody reacted predominantly with the smaller component of the A.
IMMUNODOMINANT POLYPEPTIDES COMMON BETWEENANAPLASMA SPP.
i
2
3
•
¢:
LIP
v
35
R v
200.0 kd 92.5 kd P69.0 kd
46.0 kd I..
30.0 kd ~" ~
~
14.3 kd J.-
Fig. 1. The predominant conserved antigens recognized by cross-reactive post-infection antibody are 33-36 kDa polypeptides. Antigens from A. centrale (Lanes 1, 3, 5) or the Israel-NT isolate of A. marginale (Lanes 2, 4, 6) were electrophoretically separated on a 7.5-17.5% polyacrylamide gel and transferred to nitrocellulose. Sera from cattle recovered from acute infection were diluted 1 : 1000, reacted with the nitrocellulose, and bound antibody detected by using '251 protein G. Sera include: bovine serum following recovery from A. centrale infection (Lanes 1, 2); bovine serum following recovery from A. marginale infection (Lanes 3, 4); and bovine serum from an uninfected individual (Lanes 5, 6). Arrows in the left margin represent the position of ~4C molecular size markers.
1
3 6 =,-
2
3
4
5
6
7
aim
Fig. 2. The conserved 33-36 kDa antigens are immunodominant in immunized rabbits. Antigens from the Israel-NT isolate ofA. marginale (Lanes 1, 2, 7), A. centrale (Lanes 3, 4, 6) or uninfected bovine erythrocytes (Lane 5 ) were electrophoretically separated on a 10% polyacrylamide gel and transferred to nitrocellulose. Normal rabbit serum (Lanes 1, 3), serum from a rabbit immunized with isolated intact A. marginale Israel-NT (Lane 2), or serum from a rabbit immunized with isolated intact A. centrale (Lanes 4-7) were diluted 1 : 3000 and reacted with the nitrocellulose. Antibody binding was detected by using m2slprotein A. The arrow in the left margin indicates the apparent molecular size, in kilodaltons, of the immunodominant antigens.
36
Y. SHKAP ET AL.
12
3 456
200.0 Kd-
• 9 2 . 5 Kd- ~ N ; ~ :
i2~i: ....
46.0
30.0
Fig. 3. Identification of MSP-2 in A. centrale and the Israel-NT isolate of A. marginale. 35Smethionine radiolabeled A. centrale (Lanes 1, 3, 5) or the Israel-NT isolate of A. marginale (Lanes 2, 4, 6) were immunoprecipitated with either a negative control monoclonal antibody (TRYP 1E 1, Lanes 1, 2 ), or monoclonal antibodies against A. marginale MSP-2 (ANA050A2, Lanes 3, 4, ANAFI9E2, Lanes 5, 6). Immunoprecipitated proteins were identified by electrophoresis on 7.5-17.5% polyacrylamide gels and fluorography. Arrows in the left m a t i n represent the position of m4Cmolecular size markers.
centrale doublet (Fig. 1, Lanes 1 and 3). Preinfection sera were unreactive (Fig. 1. Lanes 5 and 6 ). To determine if these antigens were also immunodominant in rabbits immunized with intact, purified organisms, a procedure previously demonstrated to induce neutralizing antibody highly reactive with surface epitopes (Palmer and McGuire, 1984), sera were diluted 1:3000 and reacted in immunoblots. Antibody produced against A. marginale reacted predominantly with a 36 kDa A. marginale polypeptide (Fig. 2, Lane 2 ). Similarly, high titer antibody against A. centrale bound primarily to a 36 kDa A. centrale polypeptide (Fig. 2, Lanes 4 and 6). Significantly, this antibody against A. centrale reacted predominantly with a 36 kDa A. marginale polypeptide, although less strongly than with the homologous antigen (Fig. 2, Lane 7). The 36 kDa A. centrale and A. marginale antigens bound by the post-A, centrale infection sera differ slightly in their apparent molecular size (Fig. 2, Lanes 6 and 7). Preimmunization rabbit serum was unreactive with Anaplasma antigens (Fig.
IMMUNODOMINANT POLYPEPTIDES COMMON BETWEEN ANAPLASMA SPP.
37
2, Lanes 1 and 3 ). Antibody against A. centrale (Fig. 2, Lane 5 ) and antibody against A. marginale (data not shown) were unreactive with uninfected bovine erythrocytes. Identification of a 36 kDa conserved polypeptide as MSP-2 Previously a 33-36 kDa surface protein, designated MSP-2, has been identified that bears epitopes conserved among isolates of A. marginale from the U.S.A. (Palmer et al., 1988b). Based on the identification of immunodominant A. centrale and Israel-NT isolate A. marginale antigens in this molecular size range, the presence of polypeptides antigenically analogous to the A. marginale MSP-2 was examined. Radiolabeled A. centrale and A. marginale polypeptides were immunoprecipitated with monoclonal antibodies reactive with conserved MSP-2 epitopes (Fig. 3). Using either of two anti-MSP-2 monoclonal antibodies, ANA050A2 or ANAFI 9E2, a 36 kDa polypeptide was specifically immunoprecipitated from A. centrale and the Israel-NT isolate of A. marginale (Fig. 3, Lanes 3-6). The negative control monoclonal antibody, TRYP 1E 1, was not specifically reactive (Fig. 3, Lanes I and 2 ). Immunoprecipitation of the 36 kDa MSP-2 from A. marginale isolates from the U.S. has been previously published (Palmer et al., 1985). DISCUSSION
The feasibility of developing improved vaccines against anaplasmosis based on defined antigens is supported by the observation that recovery from infection induces protective immunity (reviewed in Palmer, 1989 ). Despite antigenic differences (Palmer et al., 1988a), A. centrale and A. marginale induce cross-protective immunity upon recovery from acute infection (Kuttier, 1967b, Kuttler et al., 1984). The immunologic basis for this cross-protection, shared antigens, provides a novel strategy for identification of antigens relevant to protection and very likely conserved among multiple A. marginale isolates. To identify relevant shared antigens, we used two sources of antibody: ( l ) antibody from cattle recovered from experimental infection, a procedure demonstrated to induce protection against homologous challenge (Ristic and Carson, 1977; Kuttler et al., 1984) and, following recovery from A. centrale infection, protection against A. marginale challenge including the Israel-NT isolate (Kuttler, 1967b; Pipano, 1980); and (2) antibody from rabbits immunized with intact organisms, previously demonstrated to induce neutralizing antibody highly reactive with surface epitopes (Palmer and McGuire, 1984). High titers of antibody from cattle recovered from infection with either A. marginale or A. centrale were used to identify immunodominant antigens in the size range 33-36 kDa and at approximately 86 kDa. As can be seen in Fig. l, the 86 kDa polypeptides bear common epitopes as they are recognized in both A. centrale and A. marginale by postoA, centrale infec-
38
V. SHKAP ET AL.
tion serum. In contrast, the failure of post-A, marginale infection serum to react strongly with an A. centrale 86 kDa polypeptide, in the light of strong reactivity with the homologous antigens, indicates that there are also differences in these polypeptides or their recognition by the host immune system between the two Anaplasma species. At present the basis for this unidirectional immunodominant cross-reactivity is unknown. The polypeptides in the 33-36 kDa size range are immunodominant in the po~t-infection immune response regardless of the species used to generate the infection. The appearance of a doublet in the 33-36 kDa range in both Anaplasma species indicates that the dominant response is against more than a single molecule. The larger polypeptide of the A. centrale doublet is most strongly reactive with homologous post-infection serum while the smaller polypeptide is most strongly reactive with the heterologous serum (Fig. 1 ). Similarly, two A. marginale polypeptides in this size range are strongly reactive with homologous serum while only one is reactive with the post-A, centrale infection serum (Fig. 1 ). Heterogeneity in the cross-reactive antigens is also demonstrated by the slightly different molecular sizes of the A. centrale and A. marginale polypeptides bound by antibody from rabbits immunized with purified A. centrale (Fig. 2 ). The dominance of the 33-36 kDa polypeptides in inducing cross-reactive antibody, regardless of the mode of presentation, suggests that these antigens have a significant role in cross-protective immunity. Specifically, the smaller polypeptide in the A. centrale doublet and the larger polypeptide in the A. marginale doublet are promising candidates based on their strong reactivity with heterologous bovine antibody. The multiple antigens may represent either products of a single gent with the multiple fragments res~!1~ng from processing by the organism, products of closely related genes that encoae polypeptides with shared epitopes, or different gene products. Previously, a 36 kDa polypeptide (designated MSP-2) capable of inducing protective immunity has been identified on the surface of the Florida isolate ofA. marginale (Palmer et al., 1988b). The demonstration here (Fig. 3) that an antigenicJly analogous polypeptide of 36 kDa occurs in both A. centrale and in the IsraelNT isolate of A. marginale suggests that the immunodominant polypeptides may include MSP-2. MSP-2 shares several immunochemical features with the immunodominant polypeptides identified in this paper: ( 1 ) affinity purified MSP-2 has a multiple banding pattern as identified in silver-stained polyacrylamide gels (Palmer et al., 1985); (2) the molecular size of MSP-2 varies several kilodaltons among isolates of A. marginale (Palmer et al., 1988b); and (3) smaller molecular mass fragments in the 30-36 kDa range can be identified on immunoblots reacted with monoclonal antibody against MSP-2 (Palmer et al., 1988b). Demonstration that the immunodominant cross-reactive polypeptides are composed partially or completely by MSP-2 gene products can be approached by adsorption of cross-reactive antibody with
39
IMMUNODOMINANT POLYPEPTIDES COMMON BETWEEN ,4 ~rAPLASMA SPP.
MSP-2 polypeptides. Alternatively, this approach may identify unique crossreactive polypeptides, antigenically unrelated to MSP-2, that are relevant to the induction of cross-protective immunity. ACKNOWLEDGEMENTS
We thank Hanna Bin and Alberta Brassfield for technical assistance. This work received support from The United States-Israel Binational Agricultural Research and Development Fund grant US-1561-88, The U.S. Department of Agriculture, Agricultural Research Service cooperative agreement 589AHZ-2-679, and U.S. Department of Agriculture grant 86-CRCR-l-228 I.
REFERENCES Barbet, A.F., Anderson, L.W., Palmer, G.H. and McGuire, T.C., 1983. Comparison of proteins synthesized by two different isolates of Anaplasma marginale. Infect. lmmun., 40: 10681074. Hines, S.A., McElwain, T.F., Buening, G.M. and Palmer, G.H., 1989. Molecular characterization of Babesia boris merozoite surface proteins bearing epitopes immunodominant in protected cattle. Mol. Biochem. Parasitol., 37: 1-10. Klopfer, H., Klinger, I. and Pipano, E., 1968. Observations on the morphology of Anaplasma marginale in blood films stained with new methylene blue. Refu. Vet., 25: 253-256. Kuttler, K.L., 1967a. Serological relationship ofAnaplasma marginaleand Anaplasma centrale as measured by the complement-fixation and capillary tube agglutination tests. Res. Vet. Sci., 8:207-21 I. ~ut:ier, K.L., 1967b. A :~LULUL~ J u. mmm,.u.us.,,a,.~,a,,,, ...... v . . . . . . v ....... Anaplasma centrale. Res. Vet. Sci., 8:467-471. Kuttler, K.L., Zaugg, J.L. and Johnson, L.W., 1984. Serologic and clinical responses of premunized, vaccinated, and previously infected cattle to challenge exposure by two different Anaplasma marginaleisolates. Am. J. Vet. Res., 45: 2223-2226. Losos, G.J., 1986. Anaplasmosis. In: G.J. Losos (Editor), Infectious Tropical Diseases of Domestic Animals, Longman Scientific and Technical, Essex, pp. 741-795. Love, J.N., 1972. Cryogenic preservation ofAnaplasma marginalewith dimethyl sulfoxide. Am. J. Vet. Res., 32: 2557-2560. McGuire, T.C., Palmer, G.H., Goff, W.L., Johnson, M.I. and Davis, W.C., 1984. Detection of common and isolate restricted antigens ofAnaplasma marginaleusing monocional antibodies. Infect. Immun., 45: 697-700. Oberle, S.M., Palmer, G.H., Barber, A.F. and McGuire, T.C., 1988. Molecular size variations in an immunoprotective protein complex among isolates of Anaplasma margina!e. Infect. Immun., 56: 1567-1573. Palmer, G.H., 1989. Anaplasma vaccines. In: i.G. Wright (Editor), Veterinary Protozoan and Hemoparasite Vaccines. CRC Press, Boca Raton, FL, pp. 1-29. Palmer, G.H. and McGuire, T.C., 1984. Immune serum against Anaplasma marginale initial bodies neutralizes infectivity for cattle. J. Immunol., 133:1010-1015. Palmer, G.H., Kocan, K.M., Barron, S.J., Hair, J.A., Barbet, A.F., Davis, W.C. and McGuire, T.C., 1985. Presence of common antigens, including major surface protein epitopes, between tll~
,,,~,.4,
. . . . .
..
. . . .
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V. SHKAP ET AL.
the cattle (intraerythrocytic) and tick stages of Anaplasma marginale. Infect. Immun., 50: 881-886. Palmer, G.H., Barbet, A.F., Davis, W.C. and McGuire, T.C., 1986. Immunization with an isolate-common surface protein protects cattle against anaplasmosis. Science, 231:1299-1302. Palmer, G.H., Barbet, A.F., Musoke, A.J., Rurangirwa, F., Katende, J., Pipano, E., Shkap, V., Davis, W.C. and McGuire, T.C., 1988a. Recognition of conserved surface protein epitopes on Anaplasma centraleand Anaplasma marginale isolates from Israel, Kenya and the United States. Int. J. Parasitol., 18: 33-38. Palmer, G.H., Oberle, S.M., Barbet, A.F., Davis, W.C., Goff, W.L. and McGuire, T.C., 1988b. Immunization with a 36-kilodalton surface protein induces protection against homologous and heterologous Anaplasma marginale challenge. Infect. Immun., 56:1526-1531. Pipano, E., 1980. Bovine anaplasmosis and its control. In: E. Mayer (Editor), Proc. 11th Int. Congr. Dis. of Cattle, Bergman Press, Haifa, pp. 720-726. Pipano, E., Mayer, E. and Meira, F., 1985. Comparative response of Friesian milking cows and calves to Anaplasma centrale vaccine. Brt. Vet. J., 141: 174-178. Pipano, E., Krigel, Y., Meira, F., Markovics, A. and Mayer, E., 1986. Frozen Anaplasma centrale vaccine against anaplasmosis in cattle. Brt. Vet. J., 142: 553-556. Ristic, M. and Carson, C.A., 1977. Methods of immunoprophylaxis against bovine anaplasmosis with emphasis on use of the attenuated Anaplasma marginalevaccine. In: L.H. Miller, J.J. Pino and J.J. McKeivey (Editors), Immunity to Blood Parasites of Animals and Man. Plenum, New York, NY, pp. 151-188. Shkap, V., Bin, H., Ungar-Waron, H. and Pipano, E., 1990. An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Anaplasma centrale and Anaplasma marginale. Vet. Microbiol., 25: 45-53. Theiler, A., 1911. Further investigation into anaplasmosis of South African cattle. First Report of the Director of Veterinary Research, Union of South Africa, pp. 7-46. Theiler, A., 1912. Gallsickness of imported cattle and the protective inoculation against this disease. Agric. J. Union S. Aft., 3: 1-2.
31
Elsevier Science Publishers B.V., Amsterdam
Identification of immunodominant common between Anaplasma centrale and
Anaplasma marginale Varda Shkap a, Eugene Pipano a, Travis C. McGuire b and Guy H. Palmer b-~ aDepartment of Parasitology, Kimron VeterinaryInstitute, Bet Dagan, Israel bDepartment of Veterinary Microbiology and Pathology, Washington State Unirersity, Pulhnan, WA 99164-7040, USA (Accepted 7 August 1990)
ABSTRACT Shkap, V., Pipano, E., McGuire, T.C. and Palmer, G.H., 1991. Identification of immunodominant polypeptides common between Anaplasma centrale and Anaplasma marginale. Vet. hmnunol, hnmunopathol., 29:31-40. High titered antibody from rabbits immunized with Anaplasma centrale or from cattle recovered from A. centrale infection bound predominantly to several 33-36 kDa polypeptides present in both A. centrale and the Israel-NT isolate of Anaplasma marginale. High titered bovine antibody against the Israel-NT isolate ofA. marginale also reacted predominantly with A. centrale polypeptides in this size range. The immunodominance of the 33-36 kDa polypeptides and their cross-reactivity indicate that these shared epitope.~ may be primarily responsible for the cross-protective immunity between A. centrale and A. marginale. ABBREVIATIONS ELISA, enzyme-linked immunosorbent assay; SDS, sodium dodecyl sulphate.
INTRODUCTION
Bovine anaplasmosis, caused by the intraerythrocytic rickettsia Anaplasma margin.a!e~ is widespread throughout tropical and subtropical regions worldwide (Losos, 1986). Anaplasma centrale is a closely related species that causes a mild form of anaplasmosis in cattle (Theiler, 1911; Pipano et al., 1985). Significantly, prior infection with A. centrale confers protection against severe disease caused by virulent isolates of A. marginale (Theiler, 1912; Kuttier, 1967b). Consequently, deliberate infection with t~. centrale has been uti'Author to whom correspondence should be addressed.
0165-2427/91/$03.50
© 1991 m Elsevier Science Publishers B.V.
32
V. SHKAP ET AL.
lized extensively in Israel as well as Australia and countries in Africa, Asia and South America to immunize cattle against anaplasmosis (reviewed by Palmer, 1989). This cross-protective immunity indicates that protection-inducing epitopes are shared between the two species of Anaplasma and provides an immunologic basis for the development of a recombinant or synthetic peptide vaccine based upon these shared antigens. Antibody from cattle recovered from infection with A. centrale or A. marginale has been shown to cross-react with the heterologous species of Anaplasma, although at a significantly lower titer than with the homologous species (Kuttler, 1967a ). The specific antigens responsible for this cross-reactivity have not been identified. Based on the demonstration that Anaplasma organisms are susceptible to antibody mediated neutralization (Palmer et al., 1986), we hypothesize that A. centrale-induced protection against A. marginale challenge is due to induction of cross-reactive antibody. The identification of A. centrale polypeptides recognized by cross-reactive antibody would provide candidates for testing as protective immunogens against virulent A. marginale. In this paper, we report the identification of immunodominant polypeptides common to A. centrale and A. marginale. MATERIALS A N D M E T H O D S
Source of Anaplasma isolates The A. centrale isolate is the vaccine strain presently used in Israel and was originally obtained from South Africa in 1952 (Pipano et al., 1986). The Israel-NT isolate of A. marginale has been previously che:acterized morphologicaUy and partially characterized antigenically (Klopfer et aL, 10fiR; Palmer et al., 1988a). Isolates were maintained by cryopreservation in dimethylsulfoxide (Love, 1972). Seronegative splenectomized calves were inoculated with stabilate preserved Anaplasma and monitored daily for rickettsemia by using Wright stained blood smears. Infected erythrocytes were obtained during ascending rickettsemia for metabolic radiolabeling in vitro. Source of antibodies Sera were obtained from cattle recovered from experimental infection with either A. centrale or the Israel-NT isolate ofA. marginale. Sera were collected when antibody titers against intact organisms of the homologous isolate exceeded 1024 as measured by using enzyme-linked immunosorbent assay ( ELISA ) as previously described (Shkap et al., 1990 ). Preinfection sera were unreactive in the assay and were used as negative controls. Polyclonal antibodies against intact A. centrale and A. marginale ( IsraelNT isolate) were produced by repeated immunization of rabbits. Organisms were isolated from infected erythrocytes as previously described (Palmer and McGuire, 1984) and emulsified in Freund's complete adjuvant for the initial
IMMUNODOMINANT POLYPEPTIDES COMMON BETWEEN ANAPLASMA SPP.
33
immunization and in incomplete adjuvant for three subsequent immunizations. Antibody titers of sera from rabbits immunized with A. centrale were 8000 against intact A. centrale as measured by ELISA. Similarly, antibody titers from A. marginale immunized rabbits were 8000 as measured against intact A. marginale using ELISA. Sera obtained prior to immunizations were unreactive against Anaplasma as measured by ELISA and were used as negative controls. Monoclonal antibodies ANAF 19E2 and ANA050A2 are directed against A. marginale MSP-2, a 33-36 kDa surface exposed polypeptide (Palmer et al., 1985, 1988a). The generation and characterization of these antibodies has been previously described (McGuire et al., 1984). TRYP 1E l, a monoclonal antibody against the variable surface glycoprotein of Trypanosoma brucei, was used as a negative control. Supernatants from twice cloned hybridomas were concentrated to 0.1 mg/ml for use in immunoprecipitations.
Immunoblotting Immunoblotting ofA. centrale and A. marginale antigens was done as previously described in detail (Oberle et al., 1988). Briefly, organisms of each Anaplasma species were isolated from infected erythrocytes, detergent disrupted (2% sodium dodecyl sulphate (SDS), 5% 2-mercaptoethanol, pH 6.8 ), and electrophoresed on 7.5-17.5% polyacrylamide gels containing SDS. Antigens were electrophoretically transferred to nitrocellulose and reacted with either bovine sera diluted 1" 1000 or rabbit sera diluted 1" 3000. Detection of bound rabbit antibody was done by using ~25I-protein A followed by autoradiography (Oberle et al., 1988 ). Bound bovine antibody was detected by using '25i-protein G and autoradiography (Hines et ai., l ~ ) . Apparent molecular weights of antigens were determined by comparison with ~4C molecular weight standards.
Metabolic radiolabeling Anaplasma organisms were metabolically radiolabeled with 35S-methionine during short-term in vitro cultivation of infected erythrocytes. As previously described in detail, radiolabel is incorporated exclusively into Ariaplasma polypeptides (Barbet et al., 1983 ). Briefly, blood was collected from a splenectomized calf during ascending Anaplasma rickettsemia and washed with removal of the buffy coat following each 500×g centrifugation. Washed erythrocytes were diluted 1:8 in Eagle's minimal essential medium without methionine and supplemented with 10% fetal calf serum, 2 mM L-glutamine, 10 U/ml of penicillin, and l 0/~g/ml of streptomycin. Following addition of 125/~Ci 35S-methionine per milliliter of culture media, cultures were incubated at 37°C in 5% CO2 for 48 h. Erythrocytes were washed four times in Hank's balanced salt solution and then detergent lysed (50 mM Tris, 5 mM
34
V. SHKAP ET AL.
EDTA, 5 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 1% Nonidet P40).
lmmunoprecipitation Radiolabeled Anaplasma lysate was centrifuged at 135 000 Xg for 1 h, filtered through a 0.45/zm filter, and sonicated at 100 W for 10 s. Detergentsoluble polypeptides ( 10-6tdchloroacetic acid precipitable cpm per tube) were incubated with 5/tg of monoclonal antibody for 30 rain at 4 °C followed by addition of rabbit anti-mouse immunoglobulin for an additional 30 rain incubation at 4°C. Immune complexes were precipitated by using 100/zl of a 10% (v/v ) solution of Staphylococcus aureus bearing protein A. Precipitates were washed as follows: ( 1 ) four times with a 20 mM Tris buffer containing 5 mM EDTA, 100 mM NaCI, and i % Nonidet P40; (2) four times with the same buffer but containing 2 M NaCI; and (3) twice with the basic wash buffer without additional NaCI. Immune complexes were eluted by boiling in sample buffer containing SDS and 2-mercaptoethanol. Eluted antigens were separated by electrophoresis on 7.5-17.5% gradient polyacrylamide gels containing SDS. Gels were fixed and processed for fluorography as previously described. Determination of apparent molecular weights was done by comparison with ~4C molecular weight standards. RESULTS
Identification of immunodominant antigens Immunodominant antigens in A. centra!e and the Israel-NT isolate of A. marginale were identified in immunoblots based on reactivity with antibody (diluted 1:1000) induced by infection of cattle with either species. Serum antibodies from cattle recovered from A. centrale bound to multiple A. centrale polypeptides with apparent molecular sizes of 29-200 kDa (Fig. 1, Lane 1 ). Predominant high titer antibody responses were to A. centrale antigens of approximately 33-36 kDa (apparent as a doublet) and 86 kDa. When reacted against the heterologous species of Anaplasma, antibody from A. centrale-infected cattle bound primarily A. marginale polypeptides of 36 kDa (no apparent doublet) and 86 kDa, although deafly less strongly than the homologous polypeptides (Fig. 1, Lane 2). Serum from cattle infected with the Israel-NT isolate of A. marginale had hipda titers of antibody a~ainst 3336 kDa (apparent as a doublet) and 86 kDahomologous A. marginale polypeptides (Fig. 1, Lane 4). Against the heterologous A. centrale polypeptides, the dominant response was against a 33-36 kDa doublet and only slight reactivity against the larger antigens in the approximately 86 kDa range (Fig. 1, Lane 3). In contrast to the homologous antibody which bound predominantly to the larger component of the 33-36 kDa A. centrale doublet, the heterologous antibody reacted predominantly with the smaller component of the A.
IMMUNODOMINANT POLYPEPTIDES COMMON BETWEENANAPLASMA SPP.
i
2
3
•
¢:
LIP
v
35
R v
200.0 kd 92.5 kd P69.0 kd
46.0 kd I..
30.0 kd ~" ~
~
14.3 kd J.-
Fig. 1. The predominant conserved antigens recognized by cross-reactive post-infection antibody are 33-36 kDa polypeptides. Antigens from A. centrale (Lanes 1, 3, 5) or the Israel-NT isolate of A. marginale (Lanes 2, 4, 6) were electrophoretically separated on a 7.5-17.5% polyacrylamide gel and transferred to nitrocellulose. Sera from cattle recovered from acute infection were diluted 1 : 1000, reacted with the nitrocellulose, and bound antibody detected by using '251 protein G. Sera include: bovine serum following recovery from A. centrale infection (Lanes 1, 2); bovine serum following recovery from A. marginale infection (Lanes 3, 4); and bovine serum from an uninfected individual (Lanes 5, 6). Arrows in the left margin represent the position of ~4C molecular size markers.
1
3 6 =,-
2
3
4
5
6
7
aim
Fig. 2. The conserved 33-36 kDa antigens are immunodominant in immunized rabbits. Antigens from the Israel-NT isolate ofA. marginale (Lanes 1, 2, 7), A. centrale (Lanes 3, 4, 6) or uninfected bovine erythrocytes (Lane 5 ) were electrophoretically separated on a 10% polyacrylamide gel and transferred to nitrocellulose. Normal rabbit serum (Lanes 1, 3), serum from a rabbit immunized with isolated intact A. marginale Israel-NT (Lane 2), or serum from a rabbit immunized with isolated intact A. centrale (Lanes 4-7) were diluted 1 : 3000 and reacted with the nitrocellulose. Antibody binding was detected by using m2slprotein A. The arrow in the left margin indicates the apparent molecular size, in kilodaltons, of the immunodominant antigens.
36
Y. SHKAP ET AL.
12
3 456
200.0 Kd-
• 9 2 . 5 Kd- ~ N ; ~ :
i2~i: ....
46.0
30.0
Fig. 3. Identification of MSP-2 in A. centrale and the Israel-NT isolate of A. marginale. 35Smethionine radiolabeled A. centrale (Lanes 1, 3, 5) or the Israel-NT isolate of A. marginale (Lanes 2, 4, 6) were immunoprecipitated with either a negative control monoclonal antibody (TRYP 1E 1, Lanes 1, 2 ), or monoclonal antibodies against A. marginale MSP-2 (ANA050A2, Lanes 3, 4, ANAFI9E2, Lanes 5, 6). Immunoprecipitated proteins were identified by electrophoresis on 7.5-17.5% polyacrylamide gels and fluorography. Arrows in the left m a t i n represent the position of m4Cmolecular size markers.
centrale doublet (Fig. 1, Lanes 1 and 3). Preinfection sera were unreactive (Fig. 1. Lanes 5 and 6 ). To determine if these antigens were also immunodominant in rabbits immunized with intact, purified organisms, a procedure previously demonstrated to induce neutralizing antibody highly reactive with surface epitopes (Palmer and McGuire, 1984), sera were diluted 1:3000 and reacted in immunoblots. Antibody produced against A. marginale reacted predominantly with a 36 kDa A. marginale polypeptide (Fig. 2, Lane 2 ). Similarly, high titer antibody against A. centrale bound primarily to a 36 kDa A. centrale polypeptide (Fig. 2, Lanes 4 and 6). Significantly, this antibody against A. centrale reacted predominantly with a 36 kDa A. marginale polypeptide, although less strongly than with the homologous antigen (Fig. 2, Lane 7). The 36 kDa A. centrale and A. marginale antigens bound by the post-A, centrale infection sera differ slightly in their apparent molecular size (Fig. 2, Lanes 6 and 7). Preimmunization rabbit serum was unreactive with Anaplasma antigens (Fig.
IMMUNODOMINANT POLYPEPTIDES COMMON BETWEEN ANAPLASMA SPP.
37
2, Lanes 1 and 3 ). Antibody against A. centrale (Fig. 2, Lane 5 ) and antibody against A. marginale (data not shown) were unreactive with uninfected bovine erythrocytes. Identification of a 36 kDa conserved polypeptide as MSP-2 Previously a 33-36 kDa surface protein, designated MSP-2, has been identified that bears epitopes conserved among isolates of A. marginale from the U.S.A. (Palmer et al., 1988b). Based on the identification of immunodominant A. centrale and Israel-NT isolate A. marginale antigens in this molecular size range, the presence of polypeptides antigenically analogous to the A. marginale MSP-2 was examined. Radiolabeled A. centrale and A. marginale polypeptides were immunoprecipitated with monoclonal antibodies reactive with conserved MSP-2 epitopes (Fig. 3). Using either of two anti-MSP-2 monoclonal antibodies, ANA050A2 or ANAFI 9E2, a 36 kDa polypeptide was specifically immunoprecipitated from A. centrale and the Israel-NT isolate of A. marginale (Fig. 3, Lanes 3-6). The negative control monoclonal antibody, TRYP 1E 1, was not specifically reactive (Fig. 3, Lanes I and 2 ). Immunoprecipitation of the 36 kDa MSP-2 from A. marginale isolates from the U.S. has been previously published (Palmer et al., 1985). DISCUSSION
The feasibility of developing improved vaccines against anaplasmosis based on defined antigens is supported by the observation that recovery from infection induces protective immunity (reviewed in Palmer, 1989 ). Despite antigenic differences (Palmer et al., 1988a), A. centrale and A. marginale induce cross-protective immunity upon recovery from acute infection (Kuttier, 1967b, Kuttler et al., 1984). The immunologic basis for this cross-protection, shared antigens, provides a novel strategy for identification of antigens relevant to protection and very likely conserved among multiple A. marginale isolates. To identify relevant shared antigens, we used two sources of antibody: ( l ) antibody from cattle recovered from experimental infection, a procedure demonstrated to induce protection against homologous challenge (Ristic and Carson, 1977; Kuttler et al., 1984) and, following recovery from A. centrale infection, protection against A. marginale challenge including the Israel-NT isolate (Kuttler, 1967b; Pipano, 1980); and (2) antibody from rabbits immunized with intact organisms, previously demonstrated to induce neutralizing antibody highly reactive with surface epitopes (Palmer and McGuire, 1984). High titers of antibody from cattle recovered from infection with either A. marginale or A. centrale were used to identify immunodominant antigens in the size range 33-36 kDa and at approximately 86 kDa. As can be seen in Fig. l, the 86 kDa polypeptides bear common epitopes as they are recognized in both A. centrale and A. marginale by postoA, centrale infec-
38
V. SHKAP ET AL.
tion serum. In contrast, the failure of post-A, marginale infection serum to react strongly with an A. centrale 86 kDa polypeptide, in the light of strong reactivity with the homologous antigens, indicates that there are also differences in these polypeptides or their recognition by the host immune system between the two Anaplasma species. At present the basis for this unidirectional immunodominant cross-reactivity is unknown. The polypeptides in the 33-36 kDa size range are immunodominant in the po~t-infection immune response regardless of the species used to generate the infection. The appearance of a doublet in the 33-36 kDa range in both Anaplasma species indicates that the dominant response is against more than a single molecule. The larger polypeptide of the A. centrale doublet is most strongly reactive with homologous post-infection serum while the smaller polypeptide is most strongly reactive with the heterologous serum (Fig. 1 ). Similarly, two A. marginale polypeptides in this size range are strongly reactive with homologous serum while only one is reactive with the post-A, centrale infection serum (Fig. 1 ). Heterogeneity in the cross-reactive antigens is also demonstrated by the slightly different molecular sizes of the A. centrale and A. marginale polypeptides bound by antibody from rabbits immunized with purified A. centrale (Fig. 2 ). The dominance of the 33-36 kDa polypeptides in inducing cross-reactive antibody, regardless of the mode of presentation, suggests that these antigens have a significant role in cross-protective immunity. Specifically, the smaller polypeptide in the A. centrale doublet and the larger polypeptide in the A. marginale doublet are promising candidates based on their strong reactivity with heterologous bovine antibody. The multiple antigens may represent either products of a single gent with the multiple fragments res~!1~ng from processing by the organism, products of closely related genes that encoae polypeptides with shared epitopes, or different gene products. Previously, a 36 kDa polypeptide (designated MSP-2) capable of inducing protective immunity has been identified on the surface of the Florida isolate ofA. marginale (Palmer et al., 1988b). The demonstration here (Fig. 3) that an antigenicJly analogous polypeptide of 36 kDa occurs in both A. centrale and in the IsraelNT isolate of A. marginale suggests that the immunodominant polypeptides may include MSP-2. MSP-2 shares several immunochemical features with the immunodominant polypeptides identified in this paper: ( 1 ) affinity purified MSP-2 has a multiple banding pattern as identified in silver-stained polyacrylamide gels (Palmer et al., 1985); (2) the molecular size of MSP-2 varies several kilodaltons among isolates of A. marginale (Palmer et al., 1988b); and (3) smaller molecular mass fragments in the 30-36 kDa range can be identified on immunoblots reacted with monoclonal antibody against MSP-2 (Palmer et al., 1988b). Demonstration that the immunodominant cross-reactive polypeptides are composed partially or completely by MSP-2 gene products can be approached by adsorption of cross-reactive antibody with
39
IMMUNODOMINANT POLYPEPTIDES COMMON BETWEEN ,4 ~rAPLASMA SPP.
MSP-2 polypeptides. Alternatively, this approach may identify unique crossreactive polypeptides, antigenically unrelated to MSP-2, that are relevant to the induction of cross-protective immunity. ACKNOWLEDGEMENTS
We thank Hanna Bin and Alberta Brassfield for technical assistance. This work received support from The United States-Israel Binational Agricultural Research and Development Fund grant US-1561-88, The U.S. Department of Agriculture, Agricultural Research Service cooperative agreement 589AHZ-2-679, and U.S. Department of Agriculture grant 86-CRCR-l-228 I.
REFERENCES Barbet, A.F., Anderson, L.W., Palmer, G.H. and McGuire, T.C., 1983. Comparison of proteins synthesized by two different isolates of Anaplasma marginale. Infect. lmmun., 40: 10681074. Hines, S.A., McElwain, T.F., Buening, G.M. and Palmer, G.H., 1989. Molecular characterization of Babesia boris merozoite surface proteins bearing epitopes immunodominant in protected cattle. Mol. Biochem. Parasitol., 37: 1-10. Klopfer, H., Klinger, I. and Pipano, E., 1968. Observations on the morphology of Anaplasma marginale in blood films stained with new methylene blue. Refu. Vet., 25: 253-256. Kuttler, K.L., 1967a. Serological relationship ofAnaplasma marginaleand Anaplasma centrale as measured by the complement-fixation and capillary tube agglutination tests. Res. Vet. Sci., 8:207-21 I. ~ut:ier, K.L., 1967b. A :~LULUL~ J u. mmm,.u.us.,,a,.~,a,,,, ...... v . . . . . . v ....... Anaplasma centrale. Res. Vet. Sci., 8:467-471. Kuttler, K.L., Zaugg, J.L. and Johnson, L.W., 1984. Serologic and clinical responses of premunized, vaccinated, and previously infected cattle to challenge exposure by two different Anaplasma marginaleisolates. Am. J. Vet. Res., 45: 2223-2226. Losos, G.J., 1986. Anaplasmosis. In: G.J. Losos (Editor), Infectious Tropical Diseases of Domestic Animals, Longman Scientific and Technical, Essex, pp. 741-795. Love, J.N., 1972. Cryogenic preservation ofAnaplasma marginalewith dimethyl sulfoxide. Am. J. Vet. Res., 32: 2557-2560. McGuire, T.C., Palmer, G.H., Goff, W.L., Johnson, M.I. and Davis, W.C., 1984. Detection of common and isolate restricted antigens ofAnaplasma marginaleusing monocional antibodies. Infect. Immun., 45: 697-700. Oberle, S.M., Palmer, G.H., Barber, A.F. and McGuire, T.C., 1988. Molecular size variations in an immunoprotective protein complex among isolates of Anaplasma margina!e. Infect. Immun., 56: 1567-1573. Palmer, G.H., 1989. Anaplasma vaccines. In: i.G. Wright (Editor), Veterinary Protozoan and Hemoparasite Vaccines. CRC Press, Boca Raton, FL, pp. 1-29. Palmer, G.H. and McGuire, T.C., 1984. Immune serum against Anaplasma marginale initial bodies neutralizes infectivity for cattle. J. Immunol., 133:1010-1015. Palmer, G.H., Kocan, K.M., Barron, S.J., Hair, J.A., Barbet, A.F., Davis, W.C. and McGuire, T.C., 1985. Presence of common antigens, including major surface protein epitopes, between tll~
,,,~,.4,
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..
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V. SHKAP ET AL.
the cattle (intraerythrocytic) and tick stages of Anaplasma marginale. Infect. Immun., 50: 881-886. Palmer, G.H., Barbet, A.F., Davis, W.C. and McGuire, T.C., 1986. Immunization with an isolate-common surface protein protects cattle against anaplasmosis. Science, 231:1299-1302. Palmer, G.H., Barbet, A.F., Musoke, A.J., Rurangirwa, F., Katende, J., Pipano, E., Shkap, V., Davis, W.C. and McGuire, T.C., 1988a. Recognition of conserved surface protein epitopes on Anaplasma centraleand Anaplasma marginale isolates from Israel, Kenya and the United States. Int. J. Parasitol., 18: 33-38. Palmer, G.H., Oberle, S.M., Barbet, A.F., Davis, W.C., Goff, W.L. and McGuire, T.C., 1988b. Immunization with a 36-kilodalton surface protein induces protection against homologous and heterologous Anaplasma marginale challenge. Infect. Immun., 56:1526-1531. Pipano, E., 1980. Bovine anaplasmosis and its control. In: E. Mayer (Editor), Proc. 11th Int. Congr. Dis. of Cattle, Bergman Press, Haifa, pp. 720-726. Pipano, E., Mayer, E. and Meira, F., 1985. Comparative response of Friesian milking cows and calves to Anaplasma centrale vaccine. Brt. Vet. J., 141: 174-178. Pipano, E., Krigel, Y., Meira, F., Markovics, A. and Mayer, E., 1986. Frozen Anaplasma centrale vaccine against anaplasmosis in cattle. Brt. Vet. J., 142: 553-556. Ristic, M. and Carson, C.A., 1977. Methods of immunoprophylaxis against bovine anaplasmosis with emphasis on use of the attenuated Anaplasma marginalevaccine. In: L.H. Miller, J.J. Pino and J.J. McKeivey (Editors), Immunity to Blood Parasites of Animals and Man. Plenum, New York, NY, pp. 151-188. Shkap, V., Bin, H., Ungar-Waron, H. and Pipano, E., 1990. An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Anaplasma centrale and Anaplasma marginale. Vet. Microbiol., 25: 45-53. Theiler, A., 1911. Further investigation into anaplasmosis of South African cattle. First Report of the Director of Veterinary Research, Union of South Africa, pp. 7-46. Theiler, A., 1912. Gallsickness of imported cattle and the protective inoculation against this disease. Agric. J. Union S. Aft., 3: 1-2.
