Veterinary Parasitology, 44 (1992) 321-327 Elsevier Science Publishers B.V., Amsterdam
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Short Communication Development and characterization of monoclonal antibodies to first-generation merozoites of Eimeria bovis* Paula J. Haeber, David S. Lindsay and Byron L. Blagburn Department of Pathobiology, College of Feterinary Medicine, Auburn University, Auburn, AL 36849-5519, USA (Accepted 18 December 1991 )
ABSTRACT Haeber, P.J., Lindsay, D.S. and Blagburn, B.L., 1992. Development and characterization of monoclonal antibodies to first-generation merozoites of Eimeria boris. Vet. Parasitol., 44: 321-327. Merozoites of Eimeria bovis were harvested from bovine monocyte cell cultures and used to immunize BALB/C mice. Spleens from immunized mice were removed and the cells fused with mouse myeloma cells. Supernates from resulting hybridoma cell lines were examined for antibodies to firstgeneration E. boris merozoites using an indirect immunofluorescent antibody (IFA) assay. Three positive cell lines were identified and cloned by limiting dilution. All three cell lines produced immunoglobulins of the IgG1 isotype that recognized antigens in the anterior half to two-thirds of the merozoites. Specificity of the monoclonal antibodies was examined with the IFA assay against sporozoites of E. bovis, sporozoites and merozoites of Eimeria papillata from mice and Eimeria tenella from chickens, sporozoites ofIsospora suis from pigs, and tachyzoites of Toxoplasma gondii and Neospora caninum from cell cultures. Monoclonal antibodies from the three clones reacted with the anterior end of E. boris sporozoites, but did not react with the other parasites examined. None of the monoclonal antibodies reacted with merozoite antigens in immunoblots.
INTRODUCTION
Bovine coccidiosis is an economically important disease of cattle throughout the world (Fitzgerald, 1972, 1975; Ernst and Benz, 1986). Although 13 species are known to infect cattle, Eimeria zuernii and Eimeria bovis are the most important, and are the species that are usually associated with clinical Correspondence to: D.S. Lindsay, Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849-5519, USA. *This study was supported in part by College of Veterinary Medicine Grant FAH-DR-AL-V227. Published as College of Veterinary Medicine Publication No. 2271.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
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bovine coccidiosis (Ernst and Benz, 1986). The hallmark of the disease is severe diarrhea; feces are liquid and contain blood, mucus, and strands of mucosal tissue. Surviving cattle develop immunity following primary infections (Hughes et al., 1989). The life cycle ofE. bovis differs only slightly from other species of coccidia. Following the ingestion of sporulated oocysts, very large first-generation schizonts ( 1.2 × 105 merozoites per schizont ) develop within endothelial cells of central lacteals in the jejunum and ileum. Merozoites released from these schizonts initiate a second generation of schizogony that occurs in the cecum and colon. Sexual stages also develop in the cecum and colon and are associated with the onset of clinical signs. Researchers have generated monoclonal antibodies (MAB) to the sporozoite stage ofE. bovis (Whitmire et al., 1988; Lindsay et al., 1989; Speer et al., 1989). These MAB have been used to characterize antigens, examine interactions of sporozoites and host cells, examine tissues for extraintestinal stages, and examine specificity of MAB generated against other parasites (Whitmire et al., 1988; Speer and Whitmire, 1989; Lindsay et al., 1989, 1990a, 1991). Because first-generation merozoites are produced in large numbers and precede the pathogenic sexual stages, it would appear advantageous to characterize antigens associated with this stage of the parasite. An immune response which destroys significant numbers of first-generation merozoites would prevent the development of clinical coccidiosis by decreasing the numbers of sexual stages produced. The purpose of this study was to produce MAB against first-generation merozoites ofE. bovis, identify their isotypes and specific antigens with which they react, and examine the specificity of the MAB for E. bovis and other coccidian parasites. MATERIALS AND METHODS
Oocysts of E. bovis were collected from the feces of experimentally inoculated Holstein bull calves, sporulated in 2.5% ( w / v ) potassium dichromate solution, cleaned and concentrated by flotation in Sheather sugar solution and stored at 4°C in potassium dichromate solution until used for inoculation of cell cultures. Bovine monocyte cells (Speer et al., 1985 ) were used to produce first-generation merozoites of E. bovis. Bovine monocyte cells were grown to monolayers in 25 cm 2 tissue culture flasks in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% ( v / v ) fetal bovine serum, 1 mM sodium pyruvate, 5 × 10 -2 mM 2-mercaptoethanol, 100 U ml-~ penicillin G, 100/tg m1-1 streptomycin sulfate, and 0.25/tg ml -~ amphotericin B. Cells were maintained in identical medium in which the fetal bovine serum was
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reduced to 2%. Cell cultures were grown and maintained at 37°C in a 95% air-5% CO2 atmosphere. Sporozoites ofE. bovis were excysted from oocysts using standard excystation techniques (Doran and Vetterling, 1967). Sporozoites were separated from oocysts and sporocyst walls by passage over a nylon fiber column (Bontemps and Yvore, 1974 ). Bovine monocyte cell cultures were inoculated with 1.25 × 105 sporozoites and the resulting merozoites collected from the supernatant on days 12-16 post inoculation. The merozoites were washed in phosphate-buffered saline (pH 7.2 ) and pooled for murine inoculations or for use as antigen in an indirect immunofluorescent antibody (IFA) assay. Two, 4-week-old, female, BALB/C mice received primary immunizations with freshly prepared merozoites and boosting immunizations with frozen merozoites. For the primary immunizations, one mouse received 2 × 106 merozoites intraperitoneally and one mouse received 2 × 106 merozoites, that had been emulsified in Freund's complete adjuvant, subcutaneously. Boosting inoculations were given 2, 4, and 11 weeks after the primary inoculation in an identical manner, except that merozoites were emulsified in Freund's incomplete adjuvant for the inoculation given at 2 weeks after primary immunization. No other immunizations were given using adjuvant. Spleens were removed from the two mice 3 days after the fourth immunization, the cells pooled, and a fusion performed. Spleen cells were fused with mouse P3-X63-AG8 myeloma cells using techniques previously described (Danforth, 1982, 1983; Danforth and Augustine, 1983 ). The resulting hybridoma cell lines were screened against E. bovis merozoites in the IFA assay (see below). Positive hybridoma cell lines were expanded and cloned by limiting dilution. Supernatants containing the MAB were collected and stored at - 20 ° C until used. The isotypes of the MAB were determined using a commercially produced Ouchterlony immunodiffusion kit (ICN Biomedicals, Inc., Costa Mesa, CA). Indirect immunofluorescent antibody assays were performed on air-dried parasites. Merozoites of E. boris (3 × 104 per spot) were air-dried onto multispot microscope slides and stored at - 2 0 ° C until used in the IFA assay. Undiluted hybridoma supernatants were incubated with the slides for 30 min and the slides washed three times for 5 min in phosphate-buffered saline. Slides were then incubated for 30 min with a 1 : 250 dilution of fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG, IgA, and IgM (heavy and light chains) (Kirkegaard and Perry Laboratories, Gaithersburg, MD) in phosphate-buffered saline. Slides were washed three times for 5 min, mounted in 90% glycerol in phosphate-buffered saline and viewed with an epifluorescent microscope. The specificity of the MAB were tested against E. bovis sporozoites, Eimeria tenella sporozoites and merozoites, Eimeria papillata sporozoites and merozoites, and Isospora suis sporozoites in the IFA assay. Additionally, ta-
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chyzoites of the RH isolate of Toxoplasma gondii and tachyzoites of the NC1 isolate ofNeospora caninum were harvested from bovine monocyte cell cultures and examined in the IFA assay. Although, T. gondii and N. caninum are not typical enteric coccidia they were included because they are important parasites of domestic animals (Dubey, 1990). Polyacrylamide gel electrophoresis was performed using 7.5% and 12.0% continuous gels and the separated proteins transferred to nitrocellulose sheets using methods similar to those described by others (Laemmli, 1970; Towbin et al., 1979 ). Approximately 4 × 106 merozoites were heated in sample buffer (2% sodium dodecyl sulfate, 10% glycerol, 6.26× 10 -2 M tris (hydroxymethyl) aminomethane, and 5 × 10-2 mM 2-mercaptoethanol ) for 5 min prior to addition to each sample well. These merozoites were collected from the jejunum and ileum of an experimentally inoculated calf as described (Reduker and Speer, 1986). An IgG1 monoclonal antibody, MAB 4GE9A, to the refractile body ofE. bovis was used as a negative control (Lindsay et al., 1989, 1990a). Positive controls consisted of a cross-reactive monoclonal IgG 1 MAB, MAB 2AET, generated against E. bovis sporozoites (Lindsay et al., 1989 ) or a cross-reactive IgG 1 MAB, MAB TG2AB2, generated against tachyzoites of the RH isolate of T. gondii (Lindsay et al., 1990b); these react to several bands of E. bovis first-generation merozoite proteins in immunoblots. Biotinylated molecular weight markers (Bio-Rad Laboratories, Richmond, CA) were used to estimate molecular weights, and the avidin-biotin peroxidase system was used to identify bound antibodies (Vector Laboratories, Burlingame, CA).
RESULTS
Three hybridoma cell lines producing antibodies to E. bovis merozoites were identified and cloned. Ouchterlony immunodiffusion indicated that all three MAB were of the IgG1 isotype. All three MAB recognized epitopes present on the anterior half to two-thirds of E. bovis merozoites in the IFA assay. Occasionally, some merozoites were observed that did not react in the IFA assay. A similar pattern of binding was observed when E. boris sporozoites were examined in the IFA assay, but the binding was usually localized to the anterior quarter of the sporozoite. None of the other coccidian parasites examined reacted with any of the MAB in the IFA assay. None of the E. bovis merozoite MAB recognized proteins transferred to nitrocellulose sheets. The negative control MAB to E. bovis sporozoite refractile body was also non-reactive. The positive control cross-reactive MAB both reacted in the immunoblots.
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DISCUSSION
The MAB produced against E. bovis merozoites reacted with homologous sporozoites, but not with sporozoites, merozoites or tachyzoites of other coccidia or coccidia-like organisms. The MAB generated in this study can therefore be characterized as species specific and stage cross-reactive. Other studies with Eimeria species have demonstrated MAB that cross-reacted similarly with sporozoites and merozoites, (Danforth and McAndrew, 1987; Whitmire et al., 1988; Lindsay et al., 1989), and sporozoites and oocyst walls and sporocysts (Speer et al., 1983, 1989) of the eliciting species, when sporozoites were used to immunize mice. Analysis by immunofluorescence indicated that the MAB produced in the present study reacted with the anterior end of both sporozoites and merozoites ofE. bovis. Because cellular organelles responsible for host cell invasion by coccidia are located in the anterior end of the parasites (Chobotar and Scholtyseck, 1982 ), it is possible that the MAB are reacting to structures involved in host cell penetration. Researchers have recently identified a MAB that binds to the anterior portion of the invasive stage of several different coccidial parasites and to motile stages of seven species of Plasmodium (Taylor et al., 1990 ). They postulated that the MAB recognized a highly conserved antigen present on many stages of parasites within the phylum Apicomplexa. The MAB produced in the present study were specific for E. bovis, but were not specific for a single developmental stage of the parasite. Combined results of these studies indicate that parasite-specific antigens, as well as cross-reactive antigens, are present on the anterior end of motile stages of coccidial parasites. Because the E. bovis MAB did not react with merozoite antigens following immunoblot analysis in the present study, we could not determine whether they were binding to the same or different antigens on or within merozoites. However, because the positive control MAB reacted in the immunoblots, lack of reactivity was not due to technical problems such as inefficient transfer or inadequate protein concentration. It is not unusual for MAB that are generated using native proteins to fail to recognize denatured antigens (Goding, 1986).
ACKNOWLEDGMENTS
We thank Dr. C.A. Sundermann, Department of Zoology and Wildlife Sciences, Auburn University for providing oocysts ofE. tenella and E. papillata.
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REFERENCES Bontemps, M. and Yvore, P., 1974. Technique de purification de suspensions de sporozoites d'Eimeria sur colonee de fibers synthetiques. Ann. Rech. Vet., 5:109-113. Chobotar, B. and Scholtyseck, E., 1982. Ultrastructure. In: P.L. Long (Editor), The biology of the Coccidia. University Park Press, Baltimore, MD, pp. 101-165. Danforth, H.D., 1982. Development of hybridoma-produced antibodies directed against Eimeria tenella and E. mitis. J. Parasitol., 68: 392-397. Danforth, H.D., 1983. Use of monoclonal antibodies directed against Eimeria tenella sporozoites to determine stage specificity and in vitro effect on parasite penetration and development. Am. J. Vet. Res., 44: 1722-1727. Danforth, H.D. and Augustine, P.C., 1983. Specificity and cross reactivity of immune serum and hybridoma antibodies to various species of avian coccidia. Poult. Sci., 62:2145-2151. Danforth, H.D. and McAndrew, S.J., 1987. Hybridoma antibody characterization of stage-specific and stage cross-reactive antigens of Eimeria tenella. J. Parasitol., 73:985-992. Doran, D.J. and Vetterling, J.M., 1967. Cultivation of the turkey coccidium Eimeria rneleagrimitis Tyzzer, 1929, in mammalian cell cultures. Proc. Helminthol. Soc. Wash., 34: 59-65. Dubey, J.P., 1990. Neospora caninum: a look at a new Toxoplasma-like parasite of dogs and other animals. Comp. Cont. Ed. Pract. Vet., 12: 653-663. Ernst, J.V. and Benz, G.W., 1986. Intestinal coccidiosis in cattle. Vet. Clin. N. Am. Food Anim. Pract., 2: 283-291. Fitzgerald, P.R., 1972. The economics of bovine coccidiosis. Feedstuffs, 44: 28-29. Fitzgerald, P.R., 1975. The significance of bovine coccidiosis as a disease in the United States. Bovine Pract., 11: 28-33. Goding, J.W., 1986. Monoclonal Antibodies: Principles and Practice. Second Edition. Academic Press, New York, 199 pp. Hughes, H.P.A., Whitmire, W.M. and Speer, C.A., 1989. Immunity patterns during acute infection by Eimeria bovis. J. Parasitol., 75: 86-91. Laemmli, V.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. Lindsay, D.S., Dubey, J.P., Augustine, P.C., Danforth, H.D. and Fayer, R., 1989. Specificity and cross-reactivity of hybridoma antibodies generated against Eimeria boris sporozoites. Vet. Parasitol., 32:145-151. Lindsay, D.S., Dubey, J.P. and Fayer, R., 1990a. Extraintestinal stages ofEimeria bovis in calves and attempts to induce relapse of clinical disease. Vet. Parasitol., 36: 1-9. Lindsay, D.S., Dubey, J.P. and Blagburn, B.L., 1990b. Toxoplasma gondii: characterization of monoclonal antibodies generated against tachyzoites. J. Ala. Acad. Sci., 61: 144. Lindsay, D.S., Dubey, J.P. and Fayer, R., 1991. Immunohistologic examination of monoclonal antibodies generated against Eimeria bovis sporozoites for reactivity to meronts and sexual stages ofE. bovis and other eimerian parasites. Am. J. Vet. Res., 52: 239-242. Reduker, D.W. and Speer, C.A., 1986. Isolation and purification ofEimeria bovis (Apicomlexa: Eimeriidae) first generation merozoites. Can. J. Zool., 66: 2478-2480. Speer, C.A. and Whitmire, W.M., 1989. Shedding of the immunodominant P20 surface antigen ofEimeria bovis sporozoites. Infect. Immunol., 57: 999-1001. Speer, C.A., Wong, R.B. and Schenkel, R.H., 1983. Ultrastructural localization of monoclonal IgG antibodies for antigenic sites ofEimeria tenella oocysts, sporocysts and sporozoites. J. Protozool., 30: 548-554. Speer, C.A., Thammnana, P. and Schenkel, R.H., 1989. Ultrastructural localization of antigenic sites on sporozoites, sporocysts, and oocysts of Eimeria acervulina and Eimeria tenella by monoclonal IgG and IgM antibodies. J. Parasitol., 75: 92-97. Speer, C.A., Reduker, D.W., Burgess, D.E., Whitmire, W.M. and Splitter, G.A., 1985. Lym-
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phokine-induced inhibition of growth of Eimeria bovis and Eimeria papillata (Apicomplexa) in cultured bovine monocytes. Infect. Immunol., 50: 566-571. Taylor, D.W., Evans, C.B., Aley, S.B., Barta, J.R. and Danforth, H.D., 1990. Identification of an apically-located antigen that is conserved in sporozoan parasites. J. Protozool., 37: 540545. Towbin, H., Staehelin, T. and Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76: 4350-4354. Whitmire, W.M., Kyle, J.E., Speer, C.A. and Burgess, D.E., 1988. Inhibition of penetration of cultured cells by Eimeria boris sporozoites by monoclonal immunoglobulin G antibodies against the parasite surface protein p20. Infect. Immunol., 56: 2538-2543.
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Short Communication Development and characterization of monoclonal antibodies to first-generation merozoites of Eimeria bovis* Paula J. Haeber, David S. Lindsay and Byron L. Blagburn Department of Pathobiology, College of Feterinary Medicine, Auburn University, Auburn, AL 36849-5519, USA (Accepted 18 December 1991 )
ABSTRACT Haeber, P.J., Lindsay, D.S. and Blagburn, B.L., 1992. Development and characterization of monoclonal antibodies to first-generation merozoites of Eimeria boris. Vet. Parasitol., 44: 321-327. Merozoites of Eimeria bovis were harvested from bovine monocyte cell cultures and used to immunize BALB/C mice. Spleens from immunized mice were removed and the cells fused with mouse myeloma cells. Supernates from resulting hybridoma cell lines were examined for antibodies to firstgeneration E. boris merozoites using an indirect immunofluorescent antibody (IFA) assay. Three positive cell lines were identified and cloned by limiting dilution. All three cell lines produced immunoglobulins of the IgG1 isotype that recognized antigens in the anterior half to two-thirds of the merozoites. Specificity of the monoclonal antibodies was examined with the IFA assay against sporozoites of E. bovis, sporozoites and merozoites of Eimeria papillata from mice and Eimeria tenella from chickens, sporozoites ofIsospora suis from pigs, and tachyzoites of Toxoplasma gondii and Neospora caninum from cell cultures. Monoclonal antibodies from the three clones reacted with the anterior end of E. boris sporozoites, but did not react with the other parasites examined. None of the monoclonal antibodies reacted with merozoite antigens in immunoblots.
INTRODUCTION
Bovine coccidiosis is an economically important disease of cattle throughout the world (Fitzgerald, 1972, 1975; Ernst and Benz, 1986). Although 13 species are known to infect cattle, Eimeria zuernii and Eimeria bovis are the most important, and are the species that are usually associated with clinical Correspondence to: D.S. Lindsay, Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849-5519, USA. *This study was supported in part by College of Veterinary Medicine Grant FAH-DR-AL-V227. Published as College of Veterinary Medicine Publication No. 2271.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
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bovine coccidiosis (Ernst and Benz, 1986). The hallmark of the disease is severe diarrhea; feces are liquid and contain blood, mucus, and strands of mucosal tissue. Surviving cattle develop immunity following primary infections (Hughes et al., 1989). The life cycle ofE. bovis differs only slightly from other species of coccidia. Following the ingestion of sporulated oocysts, very large first-generation schizonts ( 1.2 × 105 merozoites per schizont ) develop within endothelial cells of central lacteals in the jejunum and ileum. Merozoites released from these schizonts initiate a second generation of schizogony that occurs in the cecum and colon. Sexual stages also develop in the cecum and colon and are associated with the onset of clinical signs. Researchers have generated monoclonal antibodies (MAB) to the sporozoite stage ofE. bovis (Whitmire et al., 1988; Lindsay et al., 1989; Speer et al., 1989). These MAB have been used to characterize antigens, examine interactions of sporozoites and host cells, examine tissues for extraintestinal stages, and examine specificity of MAB generated against other parasites (Whitmire et al., 1988; Speer and Whitmire, 1989; Lindsay et al., 1989, 1990a, 1991). Because first-generation merozoites are produced in large numbers and precede the pathogenic sexual stages, it would appear advantageous to characterize antigens associated with this stage of the parasite. An immune response which destroys significant numbers of first-generation merozoites would prevent the development of clinical coccidiosis by decreasing the numbers of sexual stages produced. The purpose of this study was to produce MAB against first-generation merozoites ofE. bovis, identify their isotypes and specific antigens with which they react, and examine the specificity of the MAB for E. bovis and other coccidian parasites. MATERIALS AND METHODS
Oocysts of E. bovis were collected from the feces of experimentally inoculated Holstein bull calves, sporulated in 2.5% ( w / v ) potassium dichromate solution, cleaned and concentrated by flotation in Sheather sugar solution and stored at 4°C in potassium dichromate solution until used for inoculation of cell cultures. Bovine monocyte cells (Speer et al., 1985 ) were used to produce first-generation merozoites of E. bovis. Bovine monocyte cells were grown to monolayers in 25 cm 2 tissue culture flasks in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% ( v / v ) fetal bovine serum, 1 mM sodium pyruvate, 5 × 10 -2 mM 2-mercaptoethanol, 100 U ml-~ penicillin G, 100/tg m1-1 streptomycin sulfate, and 0.25/tg ml -~ amphotericin B. Cells were maintained in identical medium in which the fetal bovine serum was
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reduced to 2%. Cell cultures were grown and maintained at 37°C in a 95% air-5% CO2 atmosphere. Sporozoites ofE. bovis were excysted from oocysts using standard excystation techniques (Doran and Vetterling, 1967). Sporozoites were separated from oocysts and sporocyst walls by passage over a nylon fiber column (Bontemps and Yvore, 1974 ). Bovine monocyte cell cultures were inoculated with 1.25 × 105 sporozoites and the resulting merozoites collected from the supernatant on days 12-16 post inoculation. The merozoites were washed in phosphate-buffered saline (pH 7.2 ) and pooled for murine inoculations or for use as antigen in an indirect immunofluorescent antibody (IFA) assay. Two, 4-week-old, female, BALB/C mice received primary immunizations with freshly prepared merozoites and boosting immunizations with frozen merozoites. For the primary immunizations, one mouse received 2 × 106 merozoites intraperitoneally and one mouse received 2 × 106 merozoites, that had been emulsified in Freund's complete adjuvant, subcutaneously. Boosting inoculations were given 2, 4, and 11 weeks after the primary inoculation in an identical manner, except that merozoites were emulsified in Freund's incomplete adjuvant for the inoculation given at 2 weeks after primary immunization. No other immunizations were given using adjuvant. Spleens were removed from the two mice 3 days after the fourth immunization, the cells pooled, and a fusion performed. Spleen cells were fused with mouse P3-X63-AG8 myeloma cells using techniques previously described (Danforth, 1982, 1983; Danforth and Augustine, 1983 ). The resulting hybridoma cell lines were screened against E. bovis merozoites in the IFA assay (see below). Positive hybridoma cell lines were expanded and cloned by limiting dilution. Supernatants containing the MAB were collected and stored at - 20 ° C until used. The isotypes of the MAB were determined using a commercially produced Ouchterlony immunodiffusion kit (ICN Biomedicals, Inc., Costa Mesa, CA). Indirect immunofluorescent antibody assays were performed on air-dried parasites. Merozoites of E. boris (3 × 104 per spot) were air-dried onto multispot microscope slides and stored at - 2 0 ° C until used in the IFA assay. Undiluted hybridoma supernatants were incubated with the slides for 30 min and the slides washed three times for 5 min in phosphate-buffered saline. Slides were then incubated for 30 min with a 1 : 250 dilution of fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG, IgA, and IgM (heavy and light chains) (Kirkegaard and Perry Laboratories, Gaithersburg, MD) in phosphate-buffered saline. Slides were washed three times for 5 min, mounted in 90% glycerol in phosphate-buffered saline and viewed with an epifluorescent microscope. The specificity of the MAB were tested against E. bovis sporozoites, Eimeria tenella sporozoites and merozoites, Eimeria papillata sporozoites and merozoites, and Isospora suis sporozoites in the IFA assay. Additionally, ta-
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chyzoites of the RH isolate of Toxoplasma gondii and tachyzoites of the NC1 isolate ofNeospora caninum were harvested from bovine monocyte cell cultures and examined in the IFA assay. Although, T. gondii and N. caninum are not typical enteric coccidia they were included because they are important parasites of domestic animals (Dubey, 1990). Polyacrylamide gel electrophoresis was performed using 7.5% and 12.0% continuous gels and the separated proteins transferred to nitrocellulose sheets using methods similar to those described by others (Laemmli, 1970; Towbin et al., 1979 ). Approximately 4 × 106 merozoites were heated in sample buffer (2% sodium dodecyl sulfate, 10% glycerol, 6.26× 10 -2 M tris (hydroxymethyl) aminomethane, and 5 × 10-2 mM 2-mercaptoethanol ) for 5 min prior to addition to each sample well. These merozoites were collected from the jejunum and ileum of an experimentally inoculated calf as described (Reduker and Speer, 1986). An IgG1 monoclonal antibody, MAB 4GE9A, to the refractile body ofE. bovis was used as a negative control (Lindsay et al., 1989, 1990a). Positive controls consisted of a cross-reactive monoclonal IgG 1 MAB, MAB 2AET, generated against E. bovis sporozoites (Lindsay et al., 1989 ) or a cross-reactive IgG 1 MAB, MAB TG2AB2, generated against tachyzoites of the RH isolate of T. gondii (Lindsay et al., 1990b); these react to several bands of E. bovis first-generation merozoite proteins in immunoblots. Biotinylated molecular weight markers (Bio-Rad Laboratories, Richmond, CA) were used to estimate molecular weights, and the avidin-biotin peroxidase system was used to identify bound antibodies (Vector Laboratories, Burlingame, CA).
RESULTS
Three hybridoma cell lines producing antibodies to E. bovis merozoites were identified and cloned. Ouchterlony immunodiffusion indicated that all three MAB were of the IgG1 isotype. All three MAB recognized epitopes present on the anterior half to two-thirds of E. bovis merozoites in the IFA assay. Occasionally, some merozoites were observed that did not react in the IFA assay. A similar pattern of binding was observed when E. boris sporozoites were examined in the IFA assay, but the binding was usually localized to the anterior quarter of the sporozoite. None of the other coccidian parasites examined reacted with any of the MAB in the IFA assay. None of the E. bovis merozoite MAB recognized proteins transferred to nitrocellulose sheets. The negative control MAB to E. bovis sporozoite refractile body was also non-reactive. The positive control cross-reactive MAB both reacted in the immunoblots.
MAB AGAINST FIRST-GENERATION MEROZOITES OF E. BOVIS
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DISCUSSION
The MAB produced against E. bovis merozoites reacted with homologous sporozoites, but not with sporozoites, merozoites or tachyzoites of other coccidia or coccidia-like organisms. The MAB generated in this study can therefore be characterized as species specific and stage cross-reactive. Other studies with Eimeria species have demonstrated MAB that cross-reacted similarly with sporozoites and merozoites, (Danforth and McAndrew, 1987; Whitmire et al., 1988; Lindsay et al., 1989), and sporozoites and oocyst walls and sporocysts (Speer et al., 1983, 1989) of the eliciting species, when sporozoites were used to immunize mice. Analysis by immunofluorescence indicated that the MAB produced in the present study reacted with the anterior end of both sporozoites and merozoites ofE. bovis. Because cellular organelles responsible for host cell invasion by coccidia are located in the anterior end of the parasites (Chobotar and Scholtyseck, 1982 ), it is possible that the MAB are reacting to structures involved in host cell penetration. Researchers have recently identified a MAB that binds to the anterior portion of the invasive stage of several different coccidial parasites and to motile stages of seven species of Plasmodium (Taylor et al., 1990 ). They postulated that the MAB recognized a highly conserved antigen present on many stages of parasites within the phylum Apicomplexa. The MAB produced in the present study were specific for E. bovis, but were not specific for a single developmental stage of the parasite. Combined results of these studies indicate that parasite-specific antigens, as well as cross-reactive antigens, are present on the anterior end of motile stages of coccidial parasites. Because the E. bovis MAB did not react with merozoite antigens following immunoblot analysis in the present study, we could not determine whether they were binding to the same or different antigens on or within merozoites. However, because the positive control MAB reacted in the immunoblots, lack of reactivity was not due to technical problems such as inefficient transfer or inadequate protein concentration. It is not unusual for MAB that are generated using native proteins to fail to recognize denatured antigens (Goding, 1986).
ACKNOWLEDGMENTS
We thank Dr. C.A. Sundermann, Department of Zoology and Wildlife Sciences, Auburn University for providing oocysts ofE. tenella and E. papillata.
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