Mar 5, 1988 - Idaho) were collected from cattle in Honduras that had recovered from tick-induced field infection with a native B. bovis isolate. [35S]methionine ...
Vol. 56, No. 6
INFECTION AND IMMUNITY, June 1988, p. 1658-1660
0019-9567/88/061658-03$02.00/0 Copyright © 1988, American Society for Microbiology
Identification of Babesia bigemina and Babesia bovis Merozoite Proteins with Isolate- and Species-Common Epitopes Recognized by Antibodies in Bovine Immune Sera TERRY F. McELWAIN,lt* GUY H. PALMER,lt WILL L. GOFF,2 AND TRAVIS C. McGUIRE' Department of Veterinary Microbiology and Pathology' and Animal Disease Research Unit, U.S. Department of Agriculture,2 Washington State University, Pullman, Washington 99164 Received 16 November 1987/Accepted
5
March 1988
Immunoprecipitation of radiolabeled antigens with bovine antisera indicated that many Babesia bigemina and Babesia bovis merozoite proteins contain isolate-common epitopes, while at least 16 B. bigemina and 8 B. bovis proteins contain species-cross-reactive epitopes. One immunogenic, isolate-common, and species-specific candidate diagnostic protein from each species was identified.
Bovine babesiosis caused by Babesia bigemina and Babesia bovis continues to be a significant deterrent to livestock production in countries with tropical or subtropical climates (17). Observations made in studies on immunoprophylaxis against and diagnosis of these parasites have indicated that there are antigenic similarities and differences between the two species (1, 6, 7, 10, 13, 14, 20, 22, 23), as well as among various isolates of the same species (1-4, 8, 9, 15, 16, 18, 19, 21), that may have important functional roles in the induction of protective immunity and antibody-based diagnosis. In this report, the identification, relative immunogenicity, and isolate or species cross-reactivity of biosynthetically radiolabeled, Mexico isolate B. bigemina and B. bovis merozoite proteins immunoprecipitated by bovine immune sera are
weights of 14,500 to >200,000. Serial dilution of these antisera resulted in a decrease in the number of proteins recognized. The reactivity of most B. bigemina proteins either was lost rapidly when B85 antiserum was diluted or decreased in proportion to the dilution. However, B. bigemina proteins with relative molecular weights of -240,000, 135,000, and 72,000 were immunoprecipitated by antiserum diluted 1:32, and the 72,000-molecular-weight protein was precipitated by antiserum diluted 1:64. Similar results were obtained in immunoprecipitations of radiolabeled B. bovis proteins with dilutions of C151 antiserum. Proteins reactive with serum diluted 1:40 had relative molecular weights of 145,000, 120,000 (doublet), and 42,000, while the 42,000molecular-weight protein was still recognized by serum diluted 1:80. Heterologous immunoprecipitations. In a previous study (18), bovine serum obtained from an animal experimentally infected with a Kenya isolate of B. bigemina immunoprecipitated Mexico B. bigemina isolate proteins that comigrated with all but one protein precipitated by B85 antiserum. One of the three proteins immunoprecipitated by the highest dilutions of B85 antiserum in homologous immunoprecipitations (72,000 molecular weight) was also precipitated by a 1:10 dilution of antibodies against the Kenya isolate. Similarly, five different antisera obtained from cattle after recovery from acute infection with B. bovis in Honduras were able to immunoprecipitate most of the Mexico isolate B. bovis proteins precipitated by C151 antiserum (Fig. 1). The 120,000- and 42,000-molecular-weight proteins recognized by 1:40 dilutions of C151 antiserum were also recognized by 1:25 dilutions of the Honduran antisera (Fig. 1). Interspecies immunoprecipitations. The results of interspecies immunoprecipitations are shown in Fig. 2. Antiserum B85, which had an indirect fluorescent-antibody (18) titer of 1:1,600 against the Mexico B. bigemina isolate, reacted with the Mexico B. bovis isolate at a titer of 1:64. Antiserum C151 (anti-B. bovis Mexico isolate) had indirect fluorescent-antibody titers of 1:5,120 and 1:640 against B. bovis and B. bigemina, respectively. Each antiserum also immunoprecipitated a number of [35S]methionine-radiolabeled proteins of the other species. The 240,000- and 135,000-molecularweight proteins recognized by a 1:32 dilution of B85 antiserum were among the 16 B. bigemina proteins precipitated by C151 antiserum. Four of the eight B. bovis proteins immunoprecipitated by B85 antiserum (120,000, 59,000, 53,000,
described. Antisera B85 and C151, which were used for immunoprecipitations, were collected from cattle with intact spleens 25 (B85) and 60 (C151) days after experimental infection with cryopreserved (12, 18) Mexico isolate blood stabilates of B. bigemina and B. bovis, respectively. Heterologous B. bovis isolate antisera (provided by J. L. Zaugg, Animal Disease Research Unit, U.S. Department of Agriculture, Caldwell, Idaho) were collected from cattle in Honduras that had recovered from tick-induced field infection with a native B. bovis isolate. [35S]methionine radiolabeling of B. bigemina in short-term culture has been previously described (18). B. bovis was similarly labeled in microaerophilus stationary-phase culture by incubation in methionine-deficient medium for 18 to 24 h with 10 ,uCi of [35S]methionine per ml (5, 11). The parasite specificity of proteins labeled under these conditions and the inability of uninfected erythrocytes to incorporate radiolabel have been previously established (9, 18). Antigen preparation, radioimmunoprecipitation, sodium dodecyl sulfatepolyacrylamide gel electrophoresis, and fluorography have been previously described in detail (18). Homologous immunoprecipitations. Antisera B85 and C151 immunoprecipitated homologous Mexico isolate proteins biosynthetically labeled with [35S]methionine with molecular * Corresponding author. t Present address: Department of Infectious Diseases, University of Florida, Gainesville, FL 32610. t Present address: Department of Comparative and Experimental Pathology, University of Florida, Gainesville, FL 32610.
1658
NOTES
VOL. 56, 1988 1
4
3
2
5
6
7
9
a
10
1659
12
11
200.000 > .l &an&
damomms
&Wm
awww-o
49k
V_
-
120 kOa
92.500 b
69,000
00
46,000 >
t
-42 kDa _-
3 0
..
30,000 b
14,300 IN'
FIG. 1. Immunoprecipitation of Mexico B. bovis isolate proteins by antisera against a Honduras B. bovis isolate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-radiolabeled proteins immunoprecipitated by preinoculation C151 serum (lane 1), undiluted C151 immune serum (lane 2), and five different antisera against a Honduras isolate either undiluted (lanes 3, 5, 7, 9, and 11) or diluted 1:25 (lanes 4, 6, 8, 10, and 12) is shown. Proteins of 120 and 42 kilodaltons (kDa) with dominant isolate-common epitopes are indicated on the right. 14C-labeled molecular weight standards (Amersham Corp., Arlington Heights, Ill.) used for molecular weight comparisons are indicated on the left and consisted of myosin, 200,000 molecular weight; phosphorylase b, 92,500; bovine serum albumin, 69,000; ovalbumin, 46,000; carbonic anhydrase, 30,000; and lysozyme, 14,300.
and 19,000 molecular weight) also had isolate-common epitopes. In addition, the 120,000-molecular-weight protein was one of the proteins recognized by C151 serum antibodies diluted 1:40. The ability of antibodies against heterologous geographic isolates to immunoprecipitate proteins from the Mexico B. bigemina and B. bovis isolates indicates the conservation of at least one and probably more epitopes between proteins from the heterologous isolates. The conservation of these epitopes is extensive, as many Mexico isolate proteins of each species were precipitated by antisera against a different
2
1
3
4
5
6
7
8
9
200.000 -
9 2,500 1
-
::
_
69,000 _-
46,000
*
1
-
30.000
14.300 *-_
FIG. 2. Immunoprecipitation of Mexico B. bigemina and B. bovis isolate proteins by antisera against the heterologous species. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-radiolabeled proteins of B. bigemina (lane 4) immunoprecipitated by preinoculation B85 serum (lane 3), B85 immune serum against B. bigemina (lane 2), and C151 immune serum against B. bovis (lane 1) is shown, as are similar profiles of radiolabeled B. bovis proteins (lane 6) immunoprecipitated by preinoculation C151 serum (lane 7), a 1:10 dilution of C151 immune serum against B. bovis (lane 8), and B85 immune serum against B. bigemina (lane 9). Lane 5 is blank. Arrows adjacent to lanes 1 and 9 indicate the major B. bigemina and B. bovis proteins containing species-cross-reactive epitopes. Molecular weight markers (see legend to Fig. 1) are indicated on the left.
geographic isolate of the same species. Two of the three highly immunogenic Mexico B. bigemina isolate proteins recognized by B85 antiserum (240,000 and 72,000 molecular weight) were also precipitated by 1:10 dilutions of antibodies against the Kenya isolate (18). The 42,000-molecular-weight Mexico B. bovis protein, which was highly immunogenic in immunoprecipitations with C151 antiserum, was precipitated by all five of the undiluted and three of the diluted Honduras antisera. The presence and possible functional importance of species-cross-reactive epitopes between B. bigemina and B. bovis and the relative molecular weights of several proteins bearing these epitopes have been previously reported (1, 20, 23). By using radiolabeled antigen and immunoprecipitation, additional B. bigemina and B. bovis proteins with speciescross-reactive epitopes have been identified in this study. Up to 16 B. bigemina and 8 B. bovis Mexico isolate radiolabeled proteins could be immunoprecipitated by antisera against the other species. Among the highly immunogenic B. bigemina proteins recognized by B85 antiserum, only one (the 72,000-molecular-weight protein) is both isolate common and species specific. A single B. bovis protein of 42,000 molecular weight recognized by C151 antiserum fulfills the same criteria. Based on these results, the two proteins are candidate antigens for species-specific, antibody-based diagnosis. Immunoassays are in progress to more extensively test the immunogenicity, epitope conservation, and recognition of these proteins (after immunoaffinity purification) with statistically significant numbers of acute-phase, convalescentphase, and carrier antisera. This work was supported by USDA-ARS-Hemoparasitic Diseases Research Unit Cooperative Agreement 58-9AH2-2-663, USDACSRS-Caribbean Basin Advisory Group 86-CRSR-2-2842, Public Health Service pathobiology training grant GM-07853 and immunology training grant AI-07025 from the National Institutes of Health, and Washington Technology Center Agreement 13N-2540-0345. LITERATURE CITED 1. Callow, L. L. 1977. Vaccination against bovine babesiosis, p. 121-149. In L. H. Miller, J. A. Pino, and J. J. McKelvey (ed.),
1660
2. 3.
4.
5.
6. 7. 8.
9.
10.
11. 12.
NOTES
Immunity to blood parasites of animals and man. Plenum Publishing Corp., New York. Cowman, A. F., P. Timms, and D. J. Kemp. 1984. DNA polymorphisms and subpopulations in Babesia bovis. Mol. Biochem. Parasitol. 11:91-103. Dalgliesh, R. J., L. L. Callow, K. T. Mellors, and W. McGregor. 1981. Development of a highly infective Babesia bigemina vaccine of reduced virulence. Aust. Vet. J. 57:8-11. De Vos, A. J., M. P. Combrink, and R. Bessenger. 1982. Babesia bigemina vaccine: comparison of the efficacy and safety of Australian and South African strains under experimental conditions in South Africa. Onderstepoort J. Vet. Res. 49:155-158. Goff, W. L., and C. E. Yunker. 1986. Babesia bovis: increased percentage parasitized erythrocytes in cultures and assessment of growth by incorporation of [3H]hypoxanthine. Exp. Parasitol. 62:202-210. Goodger, B. V. 1971. Preparation and preliminary assessment of purified antigens in the passive haemagglutination test for bovine babesiosis. Aust. Vet. J. 47:251-256. Goodger, B. V. 1973. Further studies of haemagglutinating antigens of Babesia bigemina. Aust. Vet. J. 49:81-84. Goodger, B. V., M. A. Commins, I. G. Wright, and G. B. Mirre. 1984. Babesia bovis: vaccination of cattle against heterologous challenge with fractions of lysate from infected erythrocytes. Z. Parasitenkd. 70:321-329. Kahl, L. P., R. F. Anders, B. J. Rodwell, P. Timms, and G. F. Mitchell. 1982. Variable and common antigens of Babesia bovis parasites differing in strain and virulence. J. Immunol. 129: 1700-1705. Leeflang, P., and N. M. Perie. 1972. Comparative immunofluorescent studies on 4 Babesia species of cattle. Res. Vet. Sci. 13:342-346. Levy, M. G., and M. Ristic. 1980. Babesia bovis: continuous cultivation in a microaerophilous stationary phase culture. Science 207:1218-1220. Love, J. N. 1972. Cryogenic preservation of Anaplasma marginale with dimethyl sulfoxide. Am. J. Vet. Res. 33:2557-2560.
INFECT. IMMUN.
13. Mahoney, D. F. 1962. Bovine babesiosis: diagnosis of infection by a complement fixation test. Aust. Vet. J. 38:48-52. 14. Mahoney, D. F. 1967. Bovine babesiosis: preparation and assessment of complement fixing antigens. Exp. Parasitol. 20:232-241. 15. Mahoney, D. F., and I. G. Wright. 1976. Babesia argentina: immunization of cattle with a killed antigen against infection with a heterologous strain. Vet. Parasitol. 2:273-282. 16. Mahoney, D. F., I. G. Wright, and B. V. Goodger. 1981. Bovine babesiosis: the immunization of cattle with fractions of erythrocytes infected with Babesia bovis (syn B. argentina). Vet. Immunol. Immunopathol. 2:145-156. 17. McCosker, P. J. 1981. The global importance of babesiosis, p. 1-24. In M. Ristic and J. P. Kreier (ed.), Babesiosis. Academic Press, Inc., New York. 18. McElwain, T. F., L. E. Perryman, W. C. Davis, and T. C. McGuire. 1987. Antibodies define multiple proteins with epitopes exposed on the surface of live Babesia bigemina merozoites. J. Immunol. 138:2298-2304. 19. Montenegro-James, S., M. Toro Benitez, E. Leon, R. Lopez, and M. Ristic. 1985. Heterologous strain immunity in bovine babesiosis using a culture-derived soluble Babesia bovis immunogen. Vet. Parasitol. 18:321-337. 20. Smith, R. D., E. Molinar, F. Larios, J. Monroy, F. Trigo, and M. Ristic. 1980. Bovine babesiosis: pathogenicity and heterologous species immunity of tick-borne Babesia bovis and B bigemina infections. Am. J. Vet. Res. 41:1957-1965. 21. Timms, P., R. J. Dalgliesh, D. N. Barry, C. K. Dimmock, and B. J. Rodwell. 1983. Babesia bovis: comparison of culturederived parasites, non-living antigen and conventional vaccine in the protection of cattle against heterologous challenge. Aust. Vet. J. 60:75-77. 22. Todorovic, R. A. 1975. Serologic diagnosis of babesiosis: a review. Trop. Anim. Health Prod. 7:1-14. 23. Wright, I. G., B. V. Goodger, G. Leatch, J. H. Aylward, K. Rode-Bramanis, and D. J. Waltisbuhl. 1987. Protection of Babesia bigemina-immune animals against subsequent challenge with virulent Babesia bovis. Infect. Immun. 55:363-368.
