- Email: [email protected]
Conserved recombinant antigens of Anaplasma marginale and Babesia equi for serologic diagnosis
veterinary parasitology ELSEVIER
Veterinary Parasitology 57 ( 1995 ) 93-96
Conserved recombinant antigens of Anaplasma marginale and Babesia equi for serologic diagnosis D.P. Knowles a'*, L.E. Perryman b, T.F. McElwain c, L.S. Kappmeyer a, D. Stiller~, G.H. Palmerb, E.S. Visser d, S.G. Hennageff, W.C. Davis b, T.C. M c G u i r e b aAnimal Disease Research Unit, AgriculturalResearch Service, US Department of Agriculture, Pullman, WA 99164-7030, USA bDepartment of Veterinary Microbiology and Pathology, WashingtonState University, Pullman, WA 99164-7040, USA cWashington Animal Disease Diagnostic Laboratory, WashingtonState University, Pullman, WA 99164-7040, USA dOnderstepoort VeterinaryInstitute, Onderstepoort O110, South Africa eNational VeterinaryServices Laboratories, Animal and Plant Health Inspection Service, US Department of Agriculture, Ames, IA 50010, USA
Abstract
The competitive inhibition ELISA (CI-ELISA) format overcomes problems associated with antigen purity since the specificity of the CI-ELISA depends solely on the monoclonal antibody (mAb) used. Therefore, the CI-ELISA format is well suited for use with recombinant antigens. Molecular clones expressing a conserved 19 kDa protein of Anaplasma marginale and a 34 kDa protein of Babesia equi were derived and characterized. The 19 kDa A. marginale protein, conserved in all recognized Anaplasma species, and present in the infected tick salivary gland, was reactive with all bovine immune sera tested. The 34 kDa B. equi Protein contains a protein epitope bound by antibody in equine immune sera from 19 countries. Monoclonal antibodies reactive with these proteins were derived and applied with recombinant copies of the 19 kDa A. marginale and 34 kDa B. equi proteins in a CI-ELISA format.
Keywords:Anaplasma marginale; Babesia equi; Diagnosis-Protozoa
* Corresponding author. 0304-4017/95/$09.50 © 1995 Elsevier Science B,V. All rights reserved SSD10304-4017 (94) 03113-4
94
D.P. Knowles et al. / Veterinary Parasitology 5 7 (1995) 93-96
Anaplasmosis, a vector-borne rickettsial disease of cattle, is caused by Anaplasma marginale (Theiler, 1910) and Anaplasma centrale (Theiler, 1911). Clinical disease is characterized by anemia, weight loss, abortion and death (A1derink and Dietrich, 1981 ). Survivors are lifelong carriers of the rickettsia (Swift and Thomas, 1983 ). The tick-borne hemoprotozoan Babesia equi causes disease that affects horses worldwide (Laveran, 1901; Friedhoff, 1982; Schein, 1988 ). Infection followed by fever, anemia and icterus can occur when uninfected horses are moved into endemic areas or infected horses are moved into nonendemic areas in which an appropriate tick vector is present (Knowles, 1988 ). Horses that survive initial infection are lifelong carriers ofB. equi (Holbrook, 1969 ). Eventual control ofA. marginale infection will require both an effective vaccine and identification of carrier cattle (McGuire et al., 1991 ). Two possible methods for routine carrier identification are a nucleic acid probe for hybridization of infected cattle blood or the detection ofA. marginale-specific antibody in serum. Hybridization of DNA extracted from blood with an A. marginale-specific nucleic acid probe does not always detect known carriers (Eriks et al., 1989 ), because of cyclic changes in rickettsemia levels (Kieser et al., 1990). Carrier identification by antibody requires that infected cattle never clear the rickettsia. Indefinite persistence of A. marginale in infected cattle has been documented (Ristic, 1969; Kieser et al., 1990). Current serologic tests for anaplasmosis are not widely used, primarily because the error rate is high (McGuire et al., 1991 ). One problem with current tests is false positive results caused by erythrocyte contamination of the A. marginale antigen used in the tests (Amerault and Roby, 1968; Duzgun et al., 1988 ), and the presence of anti-erythrocyte antibody in the sera of some cattle. Infection with B. equi is a problem for the importation and exportation of horses. Many countries, including the United States, do not allow importation ofB. equiinfected horses (Friedhoff, 1982 ). Since 1969, the US Department of Agriculture has used the complement fixation test (CFT) to identify B. equi infected horses (Hirato et al., 1945; Frerichs et al., 1969a,b). The limitations associated with the CFT, including the inability to evaluate sera with anticomplement activity, have been described (McGuire et al., 1971; Knowles et al., 1991 ). Also, since it has not been possible to continuously cultivate B. equi in vitro, antigen for CFT must be produced by the infection of splenectomized horses. First described for use in detecting anti-bluetongue virus antibody (Anderson, 1984), the CI-ELISA format has also been used to detect antibodies to Cowdria ruminantium, A. marginale and B. equi (Jongejan et al., 1991; Knowles et al., 1991; Visser et al., 1992). Components of the CI-ELISA include a monoclonal antibody (mAb) and corresponding epitope. The mAb must possess an appropriate binding affinity such that polyclonal antibody can replace it in the competitive reaction. Desirable epitope characteristics include a linear peptide composition, immunodominance, and conservation among isolates. The former is an important characteristic in cases where production of the epitope by molecular cloning and expression in bacteria is necessary. Since the specificity of the CIELISA depends entirely on the mAb used, the test is well suited for use with re-
D.P. Knowles et al. / Veterinary Parasitology 57 (1995) 93-96
95
combinant antigens. If the recombinant antigen is expressed in sufficient quantity to provide an appropriate signal:noise ratio after application to the ELISA plate as a bacterial lysate, additional purification of the recombinant antigen is not necessary. In an effort to improve the serological detection ofA. marginale and B. equi, recombinant antigens with the above described characteristics and corresponding mAbs were derived (Knowles et al., 1992; Visser et al., 1992). A 19 kDa A. marginale surface protein, conserved in all recognized Anaplasma species (Visser et al., 1992), and present in the infected tick salivary gland, was shown to be reactive with all bovine immune sera tested. A 34 kDa B. equi protein that contains a surface protein epitope recognized by antibody in equine immune sera from 19 countries (Knowles et al., 1992), has demonstrated utility in the CIELISA format (Anderson, 1984; Knowles et al., 1991, 1992). In parasitic infections that are persistent, the CI-ELISA format can be used to serologicaily detect carders. An important attribute of the CI-ELISA format is the ease with which it can be used with recombinant antigens. This is especially important in those parasitic diseases for which diagnostic antigen cannot be produced in cell culture. Furthermore, the production of recombinant antigen in Escherichia coli precludes the necessity of infecting animals for antigen production.
References Alderink, F.J. and Dietrich, R., 1981. Anaplasmosis in Texas: epidemiologic and economic data from the questionnaire survey. In: Proc. 7th National Anaplasmosis Conference, Mississippi State University Press, pp. 27-44. Amerault, T.E. and Roby, T.O., 1968. A rapid card agglutination test for bovine anaplasmosis. J. Am. Vet. Med. Assoc., 153: 1828-1834. Anderson, J., 1984. Use of monoclonal antibody in a blocking ELISA to detect group specific antibodies to bluetongue virus. J. Immunol. Methods, 74: 139-149. Duzgun, A , Schunter, C.A., Wright, I.G., Leatch, G. and Waltisbuhl, D.J., 1988. A sensitive ELISA technique for the diagnosis ofAnaplasma marginale infections. Vet. Parasitol., 29: 1-7. Eriks, I.S., Palmer, G.H., McGuire, T.C., Allred, D.R. and Barber, A.F., 1989. Detection and quantitation ofAnaplasma marginale in carder cattle by using a nucleic acid probe. J. Clin. Microbiol., 27: 279-284. Frerichs, W.M., Holbrook, A.A, and Johnson, A.J., 1969a. Equine piroplasmosis: production of antigens for the complement fixation test. Am. J. Vet. Res., 30:1337-1341. Frerichs, W.M., Holbrook, A.A. and Johnson, A.J., 1969b. Equine piroplasmosis: complement fixation titers of horses infected with Babesia caballi. Am. J. Vet. Res., 30: 697-702. Friedhoff, K.T., 1982. The piroplasms of Equidae-significance for international commerce. Bed. Muench. Tieraerztl. Wochenschr., 95: 368-374. Hirato, K., Nonomiya, N., Uwano, T. and Kuth, T., 1945. Studies on the complement fixation reaction for equine piroplasmosis. Jpn. J. Vet. Sci., 7:197-205. Holbrook, A.A., 1969. Biology of equine piroplasmosis. J. Am. Vet. Med. Assoc., 155: 453-454. Jongejan, F., Thielemann, M.J.C., de Groot, M., van Kooten, P.J.S. and van der Zeijst, B.A.M., 1991. Competitive enzyme-linked immunosorbent assay for heartwater using monoclonal antibodies to a Cowdria ruminantium-specific 32-kilodalton protein. Vet. Microbiol., 28:199-211.
96
D.P. Knowles et al. / VeterinaryParasitology 5 7 (1995) 93-96
Kieser, S.T., Eriks, I.S. and Palmer, G.H., 1990. Cyclic rickettsemia during persistent Anaplasma marginale infection in cattle. Infect. Immun., 58:1117-1119. Knowles, D.P., Perryman, L.E., Kappmeyer, L.S. and Hennager, S.G., 1991. Detection of equine antibody to Babesia equi merozoite proteins by a monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay. J. Clin. Microbiol., 29: 2056-2058. Knowles, D.P., Kappmeyer, L.S., Stiller, D., Hennager, S.G. and Perryman, L.E., 1992. Antibody to a recombinant merozoite protein epitope identifies horses infected with Babesia equi. J. Clin. Microbiol., 30:3122-3126. Knowles, R.C., i 988. Equine babesiosis: epidemiology, control and chemotherapy. Equine Vet. Sci., 8: 61-64. Laveran, A., 1901. Contribution ~ll'~tude de Piroplasma equi. R. Soc. Biol., 53: 385-388. McGuire, T.C., VanHoosier, G.L. and Henson, J.B., 1971. The complement-fixation reaction in equine infectious anemia: demonstration of inhibition by IgG (T). J. Immunol., 107:1738-1744. McGuire, T.C., Davis, W.C., Brassfield, A.L., McElwain, T.F. and Palmer, G.H., 1991. Identification of Anaplasma marginale long-term carrier by detection of serum antibody to isolated MSP-3. J. Clin. Microbiol., 29: 788-793. Ristic, M., 1960. Anaplasmosis, Adv. Vet. Sci., 6:111-192. Schein, E., 1988. Equine babesiosis. In: M. Ristic (Editor), Babesiosis of Domestic Animals and Man. CRC Press, Boca Raton, FL, pp. 197-208. Swift, B.L. and Thomas, G.M., 1983. Bovine anaplasmosis: elimination of the carrier state with injectable long-acting oxytetracycline. J. Am. Vet. Med. Assoc., 183: 63-65. Theiler, A., 1910. Anaplasma marginale (genus nov. et species nov. ). Un nouveau protozaire du Betail. Bull. Soc. Pathoi. Exot., 3:135. Theiler, A., 1911. Further investigations into anaplasmosis of South African cattle. In: First Report of the Director of Veterinary Research, Union of South Africa, pp. 7-76. Visser, E.S., McGuire, T.C., Palmer, G.H., Davis, W.C., Shkap, V., Pipano, E. and Knowles, D.P., 1992. The Anaplasma marginale msp5 gene encodes a 19-kilodalton protein conserved in all recognized Anaplasma species. Infect. Immun., 60:5139-5144.
Veterinary Parasitology 57 ( 1995 ) 93-96
Conserved recombinant antigens of Anaplasma marginale and Babesia equi for serologic diagnosis D.P. Knowles a'*, L.E. Perryman b, T.F. McElwain c, L.S. Kappmeyer a, D. Stiller~, G.H. Palmerb, E.S. Visser d, S.G. Hennageff, W.C. Davis b, T.C. M c G u i r e b aAnimal Disease Research Unit, AgriculturalResearch Service, US Department of Agriculture, Pullman, WA 99164-7030, USA bDepartment of Veterinary Microbiology and Pathology, WashingtonState University, Pullman, WA 99164-7040, USA cWashington Animal Disease Diagnostic Laboratory, WashingtonState University, Pullman, WA 99164-7040, USA dOnderstepoort VeterinaryInstitute, Onderstepoort O110, South Africa eNational VeterinaryServices Laboratories, Animal and Plant Health Inspection Service, US Department of Agriculture, Ames, IA 50010, USA
Abstract
The competitive inhibition ELISA (CI-ELISA) format overcomes problems associated with antigen purity since the specificity of the CI-ELISA depends solely on the monoclonal antibody (mAb) used. Therefore, the CI-ELISA format is well suited for use with recombinant antigens. Molecular clones expressing a conserved 19 kDa protein of Anaplasma marginale and a 34 kDa protein of Babesia equi were derived and characterized. The 19 kDa A. marginale protein, conserved in all recognized Anaplasma species, and present in the infected tick salivary gland, was reactive with all bovine immune sera tested. The 34 kDa B. equi Protein contains a protein epitope bound by antibody in equine immune sera from 19 countries. Monoclonal antibodies reactive with these proteins were derived and applied with recombinant copies of the 19 kDa A. marginale and 34 kDa B. equi proteins in a CI-ELISA format.
Keywords:Anaplasma marginale; Babesia equi; Diagnosis-Protozoa
* Corresponding author. 0304-4017/95/$09.50 © 1995 Elsevier Science B,V. All rights reserved SSD10304-4017 (94) 03113-4
94
D.P. Knowles et al. / Veterinary Parasitology 5 7 (1995) 93-96
Anaplasmosis, a vector-borne rickettsial disease of cattle, is caused by Anaplasma marginale (Theiler, 1910) and Anaplasma centrale (Theiler, 1911). Clinical disease is characterized by anemia, weight loss, abortion and death (A1derink and Dietrich, 1981 ). Survivors are lifelong carriers of the rickettsia (Swift and Thomas, 1983 ). The tick-borne hemoprotozoan Babesia equi causes disease that affects horses worldwide (Laveran, 1901; Friedhoff, 1982; Schein, 1988 ). Infection followed by fever, anemia and icterus can occur when uninfected horses are moved into endemic areas or infected horses are moved into nonendemic areas in which an appropriate tick vector is present (Knowles, 1988 ). Horses that survive initial infection are lifelong carriers ofB. equi (Holbrook, 1969 ). Eventual control ofA. marginale infection will require both an effective vaccine and identification of carrier cattle (McGuire et al., 1991 ). Two possible methods for routine carrier identification are a nucleic acid probe for hybridization of infected cattle blood or the detection ofA. marginale-specific antibody in serum. Hybridization of DNA extracted from blood with an A. marginale-specific nucleic acid probe does not always detect known carriers (Eriks et al., 1989 ), because of cyclic changes in rickettsemia levels (Kieser et al., 1990). Carrier identification by antibody requires that infected cattle never clear the rickettsia. Indefinite persistence of A. marginale in infected cattle has been documented (Ristic, 1969; Kieser et al., 1990). Current serologic tests for anaplasmosis are not widely used, primarily because the error rate is high (McGuire et al., 1991 ). One problem with current tests is false positive results caused by erythrocyte contamination of the A. marginale antigen used in the tests (Amerault and Roby, 1968; Duzgun et al., 1988 ), and the presence of anti-erythrocyte antibody in the sera of some cattle. Infection with B. equi is a problem for the importation and exportation of horses. Many countries, including the United States, do not allow importation ofB. equiinfected horses (Friedhoff, 1982 ). Since 1969, the US Department of Agriculture has used the complement fixation test (CFT) to identify B. equi infected horses (Hirato et al., 1945; Frerichs et al., 1969a,b). The limitations associated with the CFT, including the inability to evaluate sera with anticomplement activity, have been described (McGuire et al., 1971; Knowles et al., 1991 ). Also, since it has not been possible to continuously cultivate B. equi in vitro, antigen for CFT must be produced by the infection of splenectomized horses. First described for use in detecting anti-bluetongue virus antibody (Anderson, 1984), the CI-ELISA format has also been used to detect antibodies to Cowdria ruminantium, A. marginale and B. equi (Jongejan et al., 1991; Knowles et al., 1991; Visser et al., 1992). Components of the CI-ELISA include a monoclonal antibody (mAb) and corresponding epitope. The mAb must possess an appropriate binding affinity such that polyclonal antibody can replace it in the competitive reaction. Desirable epitope characteristics include a linear peptide composition, immunodominance, and conservation among isolates. The former is an important characteristic in cases where production of the epitope by molecular cloning and expression in bacteria is necessary. Since the specificity of the CIELISA depends entirely on the mAb used, the test is well suited for use with re-
D.P. Knowles et al. / Veterinary Parasitology 57 (1995) 93-96
95
combinant antigens. If the recombinant antigen is expressed in sufficient quantity to provide an appropriate signal:noise ratio after application to the ELISA plate as a bacterial lysate, additional purification of the recombinant antigen is not necessary. In an effort to improve the serological detection ofA. marginale and B. equi, recombinant antigens with the above described characteristics and corresponding mAbs were derived (Knowles et al., 1992; Visser et al., 1992). A 19 kDa A. marginale surface protein, conserved in all recognized Anaplasma species (Visser et al., 1992), and present in the infected tick salivary gland, was shown to be reactive with all bovine immune sera tested. A 34 kDa B. equi protein that contains a surface protein epitope recognized by antibody in equine immune sera from 19 countries (Knowles et al., 1992), has demonstrated utility in the CIELISA format (Anderson, 1984; Knowles et al., 1991, 1992). In parasitic infections that are persistent, the CI-ELISA format can be used to serologicaily detect carders. An important attribute of the CI-ELISA format is the ease with which it can be used with recombinant antigens. This is especially important in those parasitic diseases for which diagnostic antigen cannot be produced in cell culture. Furthermore, the production of recombinant antigen in Escherichia coli precludes the necessity of infecting animals for antigen production.
References Alderink, F.J. and Dietrich, R., 1981. Anaplasmosis in Texas: epidemiologic and economic data from the questionnaire survey. In: Proc. 7th National Anaplasmosis Conference, Mississippi State University Press, pp. 27-44. Amerault, T.E. and Roby, T.O., 1968. A rapid card agglutination test for bovine anaplasmosis. J. Am. Vet. Med. Assoc., 153: 1828-1834. Anderson, J., 1984. Use of monoclonal antibody in a blocking ELISA to detect group specific antibodies to bluetongue virus. J. Immunol. Methods, 74: 139-149. Duzgun, A , Schunter, C.A., Wright, I.G., Leatch, G. and Waltisbuhl, D.J., 1988. A sensitive ELISA technique for the diagnosis ofAnaplasma marginale infections. Vet. Parasitol., 29: 1-7. Eriks, I.S., Palmer, G.H., McGuire, T.C., Allred, D.R. and Barber, A.F., 1989. Detection and quantitation ofAnaplasma marginale in carder cattle by using a nucleic acid probe. J. Clin. Microbiol., 27: 279-284. Frerichs, W.M., Holbrook, A.A, and Johnson, A.J., 1969a. Equine piroplasmosis: production of antigens for the complement fixation test. Am. J. Vet. Res., 30:1337-1341. Frerichs, W.M., Holbrook, A.A. and Johnson, A.J., 1969b. Equine piroplasmosis: complement fixation titers of horses infected with Babesia caballi. Am. J. Vet. Res., 30: 697-702. Friedhoff, K.T., 1982. The piroplasms of Equidae-significance for international commerce. Bed. Muench. Tieraerztl. Wochenschr., 95: 368-374. Hirato, K., Nonomiya, N., Uwano, T. and Kuth, T., 1945. Studies on the complement fixation reaction for equine piroplasmosis. Jpn. J. Vet. Sci., 7:197-205. Holbrook, A.A., 1969. Biology of equine piroplasmosis. J. Am. Vet. Med. Assoc., 155: 453-454. Jongejan, F., Thielemann, M.J.C., de Groot, M., van Kooten, P.J.S. and van der Zeijst, B.A.M., 1991. Competitive enzyme-linked immunosorbent assay for heartwater using monoclonal antibodies to a Cowdria ruminantium-specific 32-kilodalton protein. Vet. Microbiol., 28:199-211.
96
D.P. Knowles et al. / VeterinaryParasitology 5 7 (1995) 93-96
Kieser, S.T., Eriks, I.S. and Palmer, G.H., 1990. Cyclic rickettsemia during persistent Anaplasma marginale infection in cattle. Infect. Immun., 58:1117-1119. Knowles, D.P., Perryman, L.E., Kappmeyer, L.S. and Hennager, S.G., 1991. Detection of equine antibody to Babesia equi merozoite proteins by a monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay. J. Clin. Microbiol., 29: 2056-2058. Knowles, D.P., Kappmeyer, L.S., Stiller, D., Hennager, S.G. and Perryman, L.E., 1992. Antibody to a recombinant merozoite protein epitope identifies horses infected with Babesia equi. J. Clin. Microbiol., 30:3122-3126. Knowles, R.C., i 988. Equine babesiosis: epidemiology, control and chemotherapy. Equine Vet. Sci., 8: 61-64. Laveran, A., 1901. Contribution ~ll'~tude de Piroplasma equi. R. Soc. Biol., 53: 385-388. McGuire, T.C., VanHoosier, G.L. and Henson, J.B., 1971. The complement-fixation reaction in equine infectious anemia: demonstration of inhibition by IgG (T). J. Immunol., 107:1738-1744. McGuire, T.C., Davis, W.C., Brassfield, A.L., McElwain, T.F. and Palmer, G.H., 1991. Identification of Anaplasma marginale long-term carrier by detection of serum antibody to isolated MSP-3. J. Clin. Microbiol., 29: 788-793. Ristic, M., 1960. Anaplasmosis, Adv. Vet. Sci., 6:111-192. Schein, E., 1988. Equine babesiosis. In: M. Ristic (Editor), Babesiosis of Domestic Animals and Man. CRC Press, Boca Raton, FL, pp. 197-208. Swift, B.L. and Thomas, G.M., 1983. Bovine anaplasmosis: elimination of the carrier state with injectable long-acting oxytetracycline. J. Am. Vet. Med. Assoc., 183: 63-65. Theiler, A., 1910. Anaplasma marginale (genus nov. et species nov. ). Un nouveau protozaire du Betail. Bull. Soc. Pathoi. Exot., 3:135. Theiler, A., 1911. Further investigations into anaplasmosis of South African cattle. In: First Report of the Director of Veterinary Research, Union of South Africa, pp. 7-76. Visser, E.S., McGuire, T.C., Palmer, G.H., Davis, W.C., Shkap, V., Pipano, E. and Knowles, D.P., 1992. The Anaplasma marginale msp5 gene encodes a 19-kilodalton protein conserved in all recognized Anaplasma species. Infect. Immun., 60:5139-5144.