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Antigenic analysis of Anaplasma marginale grown in bovine erythrocytes co-cultured with bovine endothelial cells
Veterinary Parasitology 94 (2000) 133–139
Short communication
Antigenic analysis of Anaplasma marginale grown in bovine erythrocytes co-cultured with bovine endothelial cells S.D. Waghela∗ , D. Melendy, D. Cruz, G.G. Wagner Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4467, USA Received 16 November 1999; received in revised form 10 August 2000; accepted 18 August 2000
Abstract Two monoclonal antibodies (mAbs) for A. marginale were used to test the antigenic integrity of A. marginale grown in vitro in bovine erythrocytes co-cultured with endothelial cells. Both the mAbs reacted in the indirect immunofluorescent antibody test with A. marginale grown in vitro and also detected the antigens in Western immunoblots of SDS-PAGE separated antigens made from A. marginale infected erythrocytes from the cultures. Furthermore, active replication was evident as [35 S]-methionine is incorporated by A. marginale present in the second passage of a culture maintained for six weeks as shown by immunoprecipitation of labeled antigens by the mAbs. This indicates that A. marginale grown in the in vitro culture system described previously [Waghela et al., Vet. Parasitol. 73 (1997) 43] maintain antigenic character, and with further development the system can be used for preparing immunogens or diagnostic antigens. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Anaplasma marginale; In vitro co-culture; Monoclonal antibody
1. Introduction Anaplasma marginale is an arthropod-transmitted rickettsia that infects erythrocytes of many ruminant species. Successful continuous in vitro growth of A. marginale would provide a more efficacious, safe and economical source of diagnostic reagents or vaccines, and especially evaluating subunit vaccines prepared from A. marginale initial body membranes (Tebele et al., 1991). Thus, it is meaningful to examine the conservation of antigens between ∗ Corresponding author. Tel.: +1-979-845-5176; fax: +1-979-862-1147. E-mail address: [email protected] (S.D. Waghela).
0304-4017/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 3 7 5 - 7
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A. marginale obtained from in vitro cultures to that obtained from blood of infected cattle. Recently, Barbet et al. (1999) described the structure conservation of surface antigens of A. marginale grown in in vitro in tick cells. Previously we described a co-culture system that supported A. marginale growth for a limited number of passages (Waghela et al., 1997). In this study, we have used monoclonal antibodies that react with 36 and 73 kDa antigens of A. marginale to show the maintenance of antigenic character of A. marginale grown in vitro in bovine erythrocytes co-cultured with endothelial cells. 2. Materials and methods 2.1. A. marginale antigen Initial bodies of A. marginale (Seymour, Texas) isolate were purified from cryopreserved infected bovine blood stabilate and disrupted with 1% (v/v) Nonidet P-40 and 0.1% (w/v) SDS in 50 mM Tris–HCl (pH 8.0) containing 5 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride and 0.1 mM N-␣-p-tosyl-l-lysine choloromethyl ketone for 30 minutes on ice, as previously described (Barbet et al., 1983). After measuring the protein content with bicinchoninic acid protein assay reagent (Pierce, Rockford, IL, USA), the lysate was stored at −79◦ C. The antigen lysate was used for the development of mAbs, SDS-PAGE, and Western blot analysis. 2.2. Monoclonal antibody BALB/c mice were immunized intrasplenically with 35 g of the A. marginale lysate. A booster with 35 g antigen lysate was given intramuscularly 14 days later, followed by an intravenous antigen dose of 35 g 4 days prior to fusion of the spleen cells with SP2/0-Ag14 mouse myeloma cells. The resulting hybridomas were grown according to the published methods (Letchworth and Appleton, 1984). Hybridoma supernatants were screened with indirect immunofluorescent antibody (IFA) test using smears of A. marginale infected blood as antigen on glass slides and rabbit anti-mouse IgG conjugated with FITC as the second antibody. Reactive hybridomas were cloned three times by limiting cell dilutions, and the supernatant from the final clone concentrated to 0.5 mg/ml and the isotype determined by radial immunodiffusion (The Binding Site, San Diego, CA, USA). 2.3. Culture of infected erythrocytes Cultures were initiated by using blood from A. marginale infected splenectomized calves as described elsewhere (Waghela et al., 1997). Periodically 5 l aliquots of sedimented erythrocytes were taken, washed two times with Dulbecco’s phosphate buffered saline (DPBS) and resuspended, in DPBS containing 0.2% ovalbumin to make smears for IFA tests. Similarly prepared erythrocytes from different passages were solubilized in 25 mM Tris, pH 6.8 containing 15% (v/v) glycerol, 2% (w/v) sodium dodecyl sulfate (SDS), 2.5% 2-β-mercaptoethanol, and a few crystals of bromophenol blue (SDS-PAGE sample buffer) for Western blot analysis.
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2.4. Metabolic labeling of cultured A. marginale and immunoprecipitation Supernatant media from a well of a second passage of a culture maintained for six weeks was removed and replaced with methionine free-MEM containing 10% FBS and 125 Ci [35 S]-methionine/ml. The percentage of infected erythrocytes at the time of addition of the radiolabel was 8%. Following an overnight incubation, the erythrocytes, at 11% of infection, were washed three times in DPBS, and the sample prepared for immunoprecipitation (Barbet et al., 1983). Uninfected erythrocytes in co-culture were similarly labeled. [35 S]-labeled proteins were reacted with either 2 g mAb or 10 l bovine serum, and the immunocomplexes precipitated with protein A/G bound to Sepharose beads. The immunoprecipitates were solubilized by boiling in SDS-PAGE sample buffer before electrophoresis. 2.5. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting A. marginale antigen lysates in SDS-PAGE sample buffer were denatured by boiling for 3 minutes and electrophoresed in a 7.5–17.5% SDS-PAGE gel with a 4% stacking gel (Barbet et al., 1983). SDS-PAGE gels were then processed for either Western blotting (Towbin and Gordon, 1984) or autoradiography (Barbet et al., 1983). Antigens separated in SDS-PAGE were electroblotted on to polyvinylidene difluoride (PVDF) membrane, which was blocked with 3% (w/v) gelatin in 10 mM Tris, pH 8.0, 150 mM NaCl containing 0.05% (v/v) Tween 20 (TBST). Following three washes in TBST, blots were reacted with 1y antibody diluted in TBST containing 1% gelatin. After washing the blots again, the 1y antibody binding was detected with either goat anti-bovine IgG or rabbit anti-mouse IgG conjugated with horse radish peroxidase, followed by the addition of 4-chloro-1-naphthol (4-CN) containing 0.015% H2 O2 as substrate.
3. Results 3.1. Reactivity of monoclonal antibody in Western blots Two different mAbs (P4H8 and P5E2) were selected by the reaction of hybridoma supernatants to A. marginale in the IFA test. In Western blot, mAb P4H8 reacted to a 36 kDa A. marginale antigen and mAb P5E2 to a 73 kDa antigen (Fig. 1). Similar reactivity of the mAbs P5E2 and P4H8 to the in vitro grown A. marginale was also observed (not shown). The control mAb reacting with Babesia bovis antigen did not react with A. marginale antigen. 3.2. Reactivity of mAb and polyclonal antibody in IFA with in vitro grown A. marginale In the IFA test, the reaction of mAbs P4H8 and P5E2 with A. marginale grown in a primary culture is shown in Fig. 2, and these two mAbs also reacted with A. marginale within endothelial cells. Fig. 2 also shows the antigen prepared from the same culture reacting to a polyclonal serum from A. marginale infected and recovered animal.
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Fig. 1. Western blot analysis showing the reactivity of mAbs to different antigens (arrows) of A. marginale derived from infected catttle. Lanes: (1) mAb P4H8; (2) mAb P6D8 (anti-Babesia bovis); (3) mAb P5E2; (4) Positive bovine serum; (5) negative bovine serum.
3.3. Immunoprecipitation The antigens of the in vitro cultured A. marginale immunoprecipitated by the mAbs P4H8 and P5E2 were of molecular mass 36 and 73 kDa respectively (Fig. 3). No immunoprecipitation was observed with similarly labeled uninfected erythrocytes co-cultured with endothelial cells (not shown).
4. Discussion Recently we have shown that A. marginale can be grown for about 16 weeks in primary cultures of erythrocytes co-cultured with endothelial cells, and that A. marginale can be passaged for limited number of sub-cultures (Waghela et al., 1997). For in vitro grown A. marginale to be useful in either diagnostic tests or better vaccines, the organism should maintain its antigenic character. Hidalgo et al. (1989) found that calves immunized with
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Fig. 2. Indirect fluorescent antibody test with mAb P4H8 (A and B); mAb P5E2 (C and D); positive bovine serum (E and F). (A, C and E)-antigen smear made from the blood of an A. marginale infected animal; (B, D and F)-antigen smear of in vitro infected erythrocytes co-cultured with endothelial cells.
A. marginale grown in tick cells produced antibodies that react with A. marginale antigens. In this study, the antigenic integrity of in vitro grown A. marginale is maintained as shown by the reactivity of two mAbs for A. marginale. These mAbs reacted in the IFA test with A. marginale grown in vitro within erythrocytes and endothelial cells, and immunoprecipitated antigens of metabolically labeled A. marginale grown in vitro. Furthermore, the same antigens were detected in Western blots of solubilized A. marginale infected erythrocytes from the cultures. One of these mAbs (P4H8) reacts with a 36 kDa antigen (MSP2) a product of a polymorphic msp2 gene (Palmer et al., 1994). This was confirmed by amplifying msp2 gene from A. marginale DNA using primers designed from the published sequence of msp2 gene (GenBank Accession number U36193.1). The gene was then cloned into an expression vector pET-32 Xa/LIC (Novagen, Madison, WI, USA). The transformed E. coli BL21 bacteria in induced cultures were used for SDS-PAGE and Western immunoblotting. The mAb P4H8 reacted with an approx. 47 kDa band which was the expected size of the fusion protein (data not shown). A conserved MSP2 pattern was recently shown to occur in various passages of A. marginale grown in embryonic cells of Ixodes scapularis (Barbet et al., 1999). However, this pattern differs in A. marginale from salivary glands of ticks, and from infected animals (Eid et al., 1996). The antigenic variation of MSP2 and other MSPs is the possible mechanism whereby A. marginale infection persists in the bovine host (Palmer et al., 2000). This study indicates that in vitro grown A. marginale can be used as an
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Fig. 3. Immunoprecipitation of [35 S]-methionine labeled initial bodies from cultured A. marginale. Lanes: (1) mAb P4H8; (2) total-labeled antigens; (3) mAb P6D8 (anti-B. bovis); (4) mAb P5E2.
antigen source in diagnostic tests or for vaccine production. The successful in vitro labeling of antigens in the second passage of a maintained co-culture confirms the viability of A. marginale in this culture system. The system would be more useful as an antigen source if several antigenic variants are expressed at each passage. However, as we reported in the previous paper (Waghela et al., 1997) that further work is necessary to obtain increasing percentage of A. marginale infected erythrocytes following each subculture for such an in vitro co-culture system to be useful.
Acknowledgements This work was supported by research grant Research Development Funding, RD-7-91; and USDA Animal Health Formula Fund, AH-8117.
References Barbet, A.F., Anderson, L.W., Palmer, G.H., McGuire, T.C., 1983. Comparison of protein synthesized by two different isolates of Anaplasma marginale. Infect. Immun. 40, 1068–1074.
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Barbet, A.F., Blentlinger, R., Lundgren, A.M., Blouin, E.F., Kocan, K.M., 1999. Comparison of surface proteins of Anaplasma marginale grown in tick cell culture, tick salivary glands and cattle. Infect. Immun. 67, 102–107. Eid, G., French, D.M., Lundgren, A.M., Barbet, A.F., McElwain, T.F., Palmer, G.H., 1996. Expression of major surface protein 2 antigenic variants during acute Anaplasma marginale rickettsemia. Infect. Immun. 66, 836–841. Hidalgo, R.J., Palmer, G.H., Jones, E.W., Brown, J.E., Ainsworth, J.A., 1989. Infectivity and antigenicity of Anaplasma marginale from tick cell culture. Am. J. Vet. Res. 50, 2033–2035. Letchworth, G.J., Appleton, A.A., 1984. Methods for production of monoclonal antibodies. United States Department of Agriculture, Handbook No. 630, 50 pp. US Government Printing Office: (1984) 421-227/10075. Palmer, G.H., Eid, G., Barbet, A.F., McGuire, T.C., McElwain, T.F., 1994. The immunoprotective Anaplasma marginale major surface protein 2 is encoded by a polymorphic multigene family. Infect. Immun. 62, 3808–3816. Palmer, G.H., Brown, W.C., Rurangirwa, F.R., 2000. Antigenic variation in the persistence and transmission of the ehrlichia Anaplasma marginale. Microb. Infect. 2, 167–176. Tebele, N., McGuire, T.C., Palmer, G.H., 1991. Induction of protective immunity by using Anaplasma marginale initial body membranes. Infect. Immunol. 59, 3199–3204. Towbin, H., Gordon, J., 1984. Immunoblotting and dot immunobinding: current status and outlook. J. Immunol. Methods 72, 313–340. Waghela, S.D., Cruz, D., Droleskey, R.E., De Loach, J.R., Wagner, G.G., 1997. In vitro cultivation of Anaplasma marginale in bovine erythrocytes co-cultured with endothelial cells. Vet. Parasitol. 73, 43–52.
Short communication
Antigenic analysis of Anaplasma marginale grown in bovine erythrocytes co-cultured with bovine endothelial cells S.D. Waghela∗ , D. Melendy, D. Cruz, G.G. Wagner Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4467, USA Received 16 November 1999; received in revised form 10 August 2000; accepted 18 August 2000
Abstract Two monoclonal antibodies (mAbs) for A. marginale were used to test the antigenic integrity of A. marginale grown in vitro in bovine erythrocytes co-cultured with endothelial cells. Both the mAbs reacted in the indirect immunofluorescent antibody test with A. marginale grown in vitro and also detected the antigens in Western immunoblots of SDS-PAGE separated antigens made from A. marginale infected erythrocytes from the cultures. Furthermore, active replication was evident as [35 S]-methionine is incorporated by A. marginale present in the second passage of a culture maintained for six weeks as shown by immunoprecipitation of labeled antigens by the mAbs. This indicates that A. marginale grown in the in vitro culture system described previously [Waghela et al., Vet. Parasitol. 73 (1997) 43] maintain antigenic character, and with further development the system can be used for preparing immunogens or diagnostic antigens. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Anaplasma marginale; In vitro co-culture; Monoclonal antibody
1. Introduction Anaplasma marginale is an arthropod-transmitted rickettsia that infects erythrocytes of many ruminant species. Successful continuous in vitro growth of A. marginale would provide a more efficacious, safe and economical source of diagnostic reagents or vaccines, and especially evaluating subunit vaccines prepared from A. marginale initial body membranes (Tebele et al., 1991). Thus, it is meaningful to examine the conservation of antigens between ∗ Corresponding author. Tel.: +1-979-845-5176; fax: +1-979-862-1147. E-mail address: [email protected] (S.D. Waghela).
0304-4017/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 3 7 5 - 7
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A. marginale obtained from in vitro cultures to that obtained from blood of infected cattle. Recently, Barbet et al. (1999) described the structure conservation of surface antigens of A. marginale grown in in vitro in tick cells. Previously we described a co-culture system that supported A. marginale growth for a limited number of passages (Waghela et al., 1997). In this study, we have used monoclonal antibodies that react with 36 and 73 kDa antigens of A. marginale to show the maintenance of antigenic character of A. marginale grown in vitro in bovine erythrocytes co-cultured with endothelial cells. 2. Materials and methods 2.1. A. marginale antigen Initial bodies of A. marginale (Seymour, Texas) isolate were purified from cryopreserved infected bovine blood stabilate and disrupted with 1% (v/v) Nonidet P-40 and 0.1% (w/v) SDS in 50 mM Tris–HCl (pH 8.0) containing 5 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride and 0.1 mM N-␣-p-tosyl-l-lysine choloromethyl ketone for 30 minutes on ice, as previously described (Barbet et al., 1983). After measuring the protein content with bicinchoninic acid protein assay reagent (Pierce, Rockford, IL, USA), the lysate was stored at −79◦ C. The antigen lysate was used for the development of mAbs, SDS-PAGE, and Western blot analysis. 2.2. Monoclonal antibody BALB/c mice were immunized intrasplenically with 35 g of the A. marginale lysate. A booster with 35 g antigen lysate was given intramuscularly 14 days later, followed by an intravenous antigen dose of 35 g 4 days prior to fusion of the spleen cells with SP2/0-Ag14 mouse myeloma cells. The resulting hybridomas were grown according to the published methods (Letchworth and Appleton, 1984). Hybridoma supernatants were screened with indirect immunofluorescent antibody (IFA) test using smears of A. marginale infected blood as antigen on glass slides and rabbit anti-mouse IgG conjugated with FITC as the second antibody. Reactive hybridomas were cloned three times by limiting cell dilutions, and the supernatant from the final clone concentrated to 0.5 mg/ml and the isotype determined by radial immunodiffusion (The Binding Site, San Diego, CA, USA). 2.3. Culture of infected erythrocytes Cultures were initiated by using blood from A. marginale infected splenectomized calves as described elsewhere (Waghela et al., 1997). Periodically 5 l aliquots of sedimented erythrocytes were taken, washed two times with Dulbecco’s phosphate buffered saline (DPBS) and resuspended, in DPBS containing 0.2% ovalbumin to make smears for IFA tests. Similarly prepared erythrocytes from different passages were solubilized in 25 mM Tris, pH 6.8 containing 15% (v/v) glycerol, 2% (w/v) sodium dodecyl sulfate (SDS), 2.5% 2-β-mercaptoethanol, and a few crystals of bromophenol blue (SDS-PAGE sample buffer) for Western blot analysis.
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2.4. Metabolic labeling of cultured A. marginale and immunoprecipitation Supernatant media from a well of a second passage of a culture maintained for six weeks was removed and replaced with methionine free-MEM containing 10% FBS and 125 Ci [35 S]-methionine/ml. The percentage of infected erythrocytes at the time of addition of the radiolabel was 8%. Following an overnight incubation, the erythrocytes, at 11% of infection, were washed three times in DPBS, and the sample prepared for immunoprecipitation (Barbet et al., 1983). Uninfected erythrocytes in co-culture were similarly labeled. [35 S]-labeled proteins were reacted with either 2 g mAb or 10 l bovine serum, and the immunocomplexes precipitated with protein A/G bound to Sepharose beads. The immunoprecipitates were solubilized by boiling in SDS-PAGE sample buffer before electrophoresis. 2.5. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting A. marginale antigen lysates in SDS-PAGE sample buffer were denatured by boiling for 3 minutes and electrophoresed in a 7.5–17.5% SDS-PAGE gel with a 4% stacking gel (Barbet et al., 1983). SDS-PAGE gels were then processed for either Western blotting (Towbin and Gordon, 1984) or autoradiography (Barbet et al., 1983). Antigens separated in SDS-PAGE were electroblotted on to polyvinylidene difluoride (PVDF) membrane, which was blocked with 3% (w/v) gelatin in 10 mM Tris, pH 8.0, 150 mM NaCl containing 0.05% (v/v) Tween 20 (TBST). Following three washes in TBST, blots were reacted with 1y antibody diluted in TBST containing 1% gelatin. After washing the blots again, the 1y antibody binding was detected with either goat anti-bovine IgG or rabbit anti-mouse IgG conjugated with horse radish peroxidase, followed by the addition of 4-chloro-1-naphthol (4-CN) containing 0.015% H2 O2 as substrate.
3. Results 3.1. Reactivity of monoclonal antibody in Western blots Two different mAbs (P4H8 and P5E2) were selected by the reaction of hybridoma supernatants to A. marginale in the IFA test. In Western blot, mAb P4H8 reacted to a 36 kDa A. marginale antigen and mAb P5E2 to a 73 kDa antigen (Fig. 1). Similar reactivity of the mAbs P5E2 and P4H8 to the in vitro grown A. marginale was also observed (not shown). The control mAb reacting with Babesia bovis antigen did not react with A. marginale antigen. 3.2. Reactivity of mAb and polyclonal antibody in IFA with in vitro grown A. marginale In the IFA test, the reaction of mAbs P4H8 and P5E2 with A. marginale grown in a primary culture is shown in Fig. 2, and these two mAbs also reacted with A. marginale within endothelial cells. Fig. 2 also shows the antigen prepared from the same culture reacting to a polyclonal serum from A. marginale infected and recovered animal.
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Fig. 1. Western blot analysis showing the reactivity of mAbs to different antigens (arrows) of A. marginale derived from infected catttle. Lanes: (1) mAb P4H8; (2) mAb P6D8 (anti-Babesia bovis); (3) mAb P5E2; (4) Positive bovine serum; (5) negative bovine serum.
3.3. Immunoprecipitation The antigens of the in vitro cultured A. marginale immunoprecipitated by the mAbs P4H8 and P5E2 were of molecular mass 36 and 73 kDa respectively (Fig. 3). No immunoprecipitation was observed with similarly labeled uninfected erythrocytes co-cultured with endothelial cells (not shown).
4. Discussion Recently we have shown that A. marginale can be grown for about 16 weeks in primary cultures of erythrocytes co-cultured with endothelial cells, and that A. marginale can be passaged for limited number of sub-cultures (Waghela et al., 1997). For in vitro grown A. marginale to be useful in either diagnostic tests or better vaccines, the organism should maintain its antigenic character. Hidalgo et al. (1989) found that calves immunized with
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Fig. 2. Indirect fluorescent antibody test with mAb P4H8 (A and B); mAb P5E2 (C and D); positive bovine serum (E and F). (A, C and E)-antigen smear made from the blood of an A. marginale infected animal; (B, D and F)-antigen smear of in vitro infected erythrocytes co-cultured with endothelial cells.
A. marginale grown in tick cells produced antibodies that react with A. marginale antigens. In this study, the antigenic integrity of in vitro grown A. marginale is maintained as shown by the reactivity of two mAbs for A. marginale. These mAbs reacted in the IFA test with A. marginale grown in vitro within erythrocytes and endothelial cells, and immunoprecipitated antigens of metabolically labeled A. marginale grown in vitro. Furthermore, the same antigens were detected in Western blots of solubilized A. marginale infected erythrocytes from the cultures. One of these mAbs (P4H8) reacts with a 36 kDa antigen (MSP2) a product of a polymorphic msp2 gene (Palmer et al., 1994). This was confirmed by amplifying msp2 gene from A. marginale DNA using primers designed from the published sequence of msp2 gene (GenBank Accession number U36193.1). The gene was then cloned into an expression vector pET-32 Xa/LIC (Novagen, Madison, WI, USA). The transformed E. coli BL21 bacteria in induced cultures were used for SDS-PAGE and Western immunoblotting. The mAb P4H8 reacted with an approx. 47 kDa band which was the expected size of the fusion protein (data not shown). A conserved MSP2 pattern was recently shown to occur in various passages of A. marginale grown in embryonic cells of Ixodes scapularis (Barbet et al., 1999). However, this pattern differs in A. marginale from salivary glands of ticks, and from infected animals (Eid et al., 1996). The antigenic variation of MSP2 and other MSPs is the possible mechanism whereby A. marginale infection persists in the bovine host (Palmer et al., 2000). This study indicates that in vitro grown A. marginale can be used as an
138
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Fig. 3. Immunoprecipitation of [35 S]-methionine labeled initial bodies from cultured A. marginale. Lanes: (1) mAb P4H8; (2) total-labeled antigens; (3) mAb P6D8 (anti-B. bovis); (4) mAb P5E2.
antigen source in diagnostic tests or for vaccine production. The successful in vitro labeling of antigens in the second passage of a maintained co-culture confirms the viability of A. marginale in this culture system. The system would be more useful as an antigen source if several antigenic variants are expressed at each passage. However, as we reported in the previous paper (Waghela et al., 1997) that further work is necessary to obtain increasing percentage of A. marginale infected erythrocytes following each subculture for such an in vitro co-culture system to be useful.
Acknowledgements This work was supported by research grant Research Development Funding, RD-7-91; and USDA Animal Health Formula Fund, AH-8117.
References Barbet, A.F., Anderson, L.W., Palmer, G.H., McGuire, T.C., 1983. Comparison of protein synthesized by two different isolates of Anaplasma marginale. Infect. Immun. 40, 1068–1074.
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Barbet, A.F., Blentlinger, R., Lundgren, A.M., Blouin, E.F., Kocan, K.M., 1999. Comparison of surface proteins of Anaplasma marginale grown in tick cell culture, tick salivary glands and cattle. Infect. Immun. 67, 102–107. Eid, G., French, D.M., Lundgren, A.M., Barbet, A.F., McElwain, T.F., Palmer, G.H., 1996. Expression of major surface protein 2 antigenic variants during acute Anaplasma marginale rickettsemia. Infect. Immun. 66, 836–841. Hidalgo, R.J., Palmer, G.H., Jones, E.W., Brown, J.E., Ainsworth, J.A., 1989. Infectivity and antigenicity of Anaplasma marginale from tick cell culture. Am. J. Vet. Res. 50, 2033–2035. Letchworth, G.J., Appleton, A.A., 1984. Methods for production of monoclonal antibodies. United States Department of Agriculture, Handbook No. 630, 50 pp. US Government Printing Office: (1984) 421-227/10075. Palmer, G.H., Eid, G., Barbet, A.F., McGuire, T.C., McElwain, T.F., 1994. The immunoprotective Anaplasma marginale major surface protein 2 is encoded by a polymorphic multigene family. Infect. Immun. 62, 3808–3816. Palmer, G.H., Brown, W.C., Rurangirwa, F.R., 2000. Antigenic variation in the persistence and transmission of the ehrlichia Anaplasma marginale. Microb. Infect. 2, 167–176. Tebele, N., McGuire, T.C., Palmer, G.H., 1991. Induction of protective immunity by using Anaplasma marginale initial body membranes. Infect. Immunol. 59, 3199–3204. Towbin, H., Gordon, J., 1984. Immunoblotting and dot immunobinding: current status and outlook. J. Immunol. Methods 72, 313–340. Waghela, S.D., Cruz, D., Droleskey, R.E., De Loach, J.R., Wagner, G.G., 1997. In vitro cultivation of Anaplasma marginale in bovine erythrocytes co-cultured with endothelial cells. Vet. Parasitol. 73, 43–52.