~2eterinaryMicrobiology, 28 ( 1991 ) 199-211 Elsevier Science Publishers B.V., A m s t e r d a m


Competitive enzyme-linked immunosorbent assay for heartwater using monoclonal antibodies to a Cowdria ruminantium-specific 32-kilodalton protein

F. Jongejan a, M.J.C. Thielemans a, M. De Groot a, P.J.S. Van Kooten b and B.A.M. V a n D e r Z e i j s t c aDepartment of Tropical Veterinary Medicine and Protozoology, bDepartment of Veterinary Immunology, CDepartment of Bacteriology, Institute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, P.O. Box 80.165, 3508 TD Utrecht, Netherlands. (Accepted 10 January 1991 )

ABSTRACT Jongejan, F., Thielemans, 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 rurninantiurn-specific 32-kilodalton protein. Vet. Microbiol. 28:199-211. Hybridomas producing monoclonal antibodies (mAb) to Cowdria ruminantium were raised. Four mAbs of the IgG isotype reacted in western blots with a 32-kilodalton Cowdria protein (Cr32), which had previously been shown to be conserved and immunodominant. A fifth mAb of the lgM isotype recognized a 40-kDa Cowdria protein. The latter mAb was negative in an indirect fluorescent antibody test (IFA), whereas the other four were positive, mAb No. 4F10B4 showed the strongest signal in western blots using three different stocks of Cowdria. lmmuno-gold labeling of Cowdria organisms in vitro using 4F10B4 showed that Cr32 has surface-exposed antigenic determinants. Using mAb 4F10B4, a competitive ELISA was developed which detected specific Cowdria antibodies in goat, sheep and cattle sera. Antibodies in animal sera competed with binding of mAb 4F 10B4 ~o a crude sonicated Cowdria antigen obtained from infected endothelial cell cultures. The competition ELISA (CELISA) detected antibodies in 55 out of 70 (79%) goats experimentally infected with one of eight different Cowdria stocks. Fourteen out of the 15 sera which were shown negative in the CELISA were also negative in the IFA. Nevertheless, all 15 sera recognized some epitopes of the immunodominant Cowdria-specific 32 kDa protein as judged from their reaction with this protein in western blots. Overall, there was 89% agreement between CELISA and IFA considering all 70 goat sera. Moreover, antibodies were detected in nine out of nine sheep infected with one of three different stocks of Cowdria and in sera from calves experimentally infected by two different strains of heartwater. There were no cross-reactions with Ehrlichia phagocytophila antibodies in goat sera, nor with Anaplasma marginale antibodies in bovine sera. Lack of cross-reactivity and detection of antibodies to eight geographically widely distributed slocks of Cowdria, makes the competition ELISA a promising test for use in heartwater endemic areas.


© 1991 - - Elsevier Science Publishers B.V.




Heartwater, caused by the tick-borne rickettsia Cowdria ruminantium, is an infectious disease of domestic and wild ruminants in sub-Saharan Africa and in the Caribbean (Uilenberg, 1983). To study the epidemiology of the disease, a reliable serodiagnostic test is of prime importance. Several serological assays for C. ruminantium have been reported, such as a capillary flocculation test (Ilemobade and Blotkamp, 1976) and a complement fixation test (Du Plessis, 1982; Musisi and Hussein, 1985), but neither of these types has shown the required sensitivity and specificity. Furthermore, an indirect fluorescent antibody assay (IFA) using infected peritoneal mouse macrophages (Du Plessis, 1981 ) has shown its usefulness in serological monitoring of experimental animals (Du Plessis et al., 1989), but difficulties with the interpretation of the results obtained in epidemiological surveys have been attributed to cross-reactions with Ehrlichia spp. (Camus, 1987; Du Plessis et al., 1987). The specificity of the IFA developed by Logan (1987) based on infected neutrophil cultures has also been limited due to cross-reactions between Cowdria and Ehrlichia (Logan et al., 1986; Jongejan et al., 1989). Enzyme-linked immunosorbent assays (ELISA) with Cowdria antigens isolated from sheep brain or Ambylomma hebraeum ticks by wheat germ lectine affinity chromatography (Viljoen et al., 1985 ) or Percoll density gradient centrifugation (Neitz et al., 1986) have also been reported. The main obstacles for these assays have been the availability of purified Cowdria antigen. Recently, endothelial cell cultures infected with Cowdria have become available (Bezuidenhout, 1987), which provide a convenient source of antigen for use in serological assays such as IFA (Martinez et al., 1990). For use in ELISA, unpurified Cowdria antigens harvested from such infected endothelial cultures did not give satisfactory results due to high background levels obtained with pre-immune sera (M.J.C. Thielemans and F. Jongejan, unpublished observations). These aspecific reactions associated with impure antigen can be avoided by making use of the specificity of monoclonal antibodies (mAbs) (Hewitt et al., 1982). Competitive ELISA, mediated by mAb, has recently been reported by Dahl et al. (1987) and was also used for the serodiagnosis of E. risticii, the causal agent of equine monocytic ehrlichiosis (Shankarappa et al., 1989 ). We recently demonstrated that Cowdria contains and i m m u n o d o m i n a n t and antigenically conserved protein of 32 kDa and suggested basing serodiagnosis of heartwater on this protein (Jongejan and Thielemans, 1989 ). In this paper we report the production and characterization of monoclonal antibodies directed against the 32 kDa protein of Cowdria (Cr32). These mAbs were used to determine the localisation of the Cr32 protein and to develop a competitive ELISA for the detection of circulating antibodies in ruminants infected with heartwater.




Rickettsiae Eight stocks of C. ruminantium were used; one from Senegal (Jongejan et al., 1988), one from Zambia (Lutale; Jongejan et al., 1988), one from Sudan ( U m Banein; Jongejan et al., 1984), one from the Caribbean Island of Guadeloupe (Gardel; Uilenberg et al., 1985 ) and four stocks from South Africa: Ball 3 (Haig, 1952 ), K/imm (Du Plessis and KiJmm, 1971 ), Kwanyanga (Mackenzie and Van Rooyen, 1981 ) and the Welgevonden stock described by Du Plessis (1985). All isolates were stored as infected blood stabilates under liquid nitrogen and their infectivity was tested by intravenous inoculation into experimental goats or sheep. Smears were made of brain cortex of each animal which had died, and stained with Diff-Quik (Merz & Dada AG, Switzerland). The presence of rickettsial inclusion bodies in endothelial cells of the capillaries confirmed death was due to heartwater. Two isolates ofAnaplasma marginale were used, one from Texas, USA and one from Nigeria. Both isolates were stored as infected blood stabilates under liquid nitrogen and used to initiate infection of splenectomized Bos taurus calves (De Kxoon et al., in press). An isolate of E. phagocytophila (Ameland) (Uilenberg et al., 1979 ) originated from sheep and has been previously used in immunofluorescence studies with Cowdria (Jongejan et al., 1989).

Antisera Goats (Saanen breed) and sheep (Tesselaar breed) were infected intravenously with 2-ml aliquots of thawed blood stabilate containing one of the eight different Cowdria stocks. The clinical reaction was treated with Terramycin LA R (Pfizer) as described (Uilenberg, 1983). Calves (B. taurus) were either infected with fresh blood or by Amblyomma ticks. Homologous challenge inocula were administered 4-6 weeks post infection. Sera were prepared on a weekly basis and stored at - 2 0 ° C until further use. Pre-infection sera from goats, sheep and cattle were used as controls. Antibodies to A. marginale were raised in 4 splenectomized calves, which were infection with blood stabilate and treated with Terramycin LA r~ (Pfizer). Sera were collected 4 to 6 weeks post inoculation (p.i.) and stored at - 20 ° C. Those sera with antibody titres of 5210 against A. marginale in the IFA test were used in this study (De Kroon et al., 1990). Nine Saanen goats were infected with E. phagocytophila using blood stabilate and sera were collected 3-4 weeks after the goats had spontaneously recovered from the infection. High antibody titres to E. phagocytophila were demonstrated with IFA as described previously (Jongejan et al, 1989).



In vitro cultivation Bovine endothelial cells were isolated from umbilical cord arteries (Van de Wiel et al., 1989), with minor modifications. The bovine umbilical endothelial (BUE) cells were grown in RPMI 1640, supplemented with penicillin (100 I U / m l ) , streptomycin (100/~g/ml), amphotericin B (1.25 /~g/ml), HEPES buffer (20 m M ) , L-glutamine (2 m M ) and 10% newborn calf serum. Infection of BUE cell cultures with Cowdria ( Senegal, Welgevonden, U m Banein and Lutale stocks) was initiated by inoculation of 0.5 ml aliquots of infected BUE culture supernatant which had been stored in sucrose-phosphate-glutamate (SPG) (Bovarnick et al., 1950) at-80 ° C. The cultures were incubated at 37°C on a rocking platform (10 cycles/min) maintained in Cowdria growth m e d i u m which consisted of Glasgow Minimal essential medium (GMEM), supplemented with antibiotics, HEPES buffer (20 m M ) (pH 7.0-7.2 ), L-glutamine (2 mM ), 10% newborn calf serum and 2.6 g/1 tryptose phosphate broth. Monoclonal antibodies Six- to eight-week old Balb/C mice were injected intravenously with 0.2 ml of BUE culture supernatant containing elementary bodies of the mouse-pathogenic Welgevonden isolate of Cowdria. The mice showed signs of sickness on day 9 p.i. and moribund mice were sacrificed on day 11 p.i. Spleen cells from two mice were fused with SP2/0 myeloma cells ( 1:1 ) by using 40% polyethylene glycol 1500 (Sigma Chemical Co., St. Louis, USA) as the fusing agent. After incubation for 18 h at 37°C and 5% CO2, 0.1 ml of selective m e d i u m was added, which consisted of RPMI 1640 m e d i u m with 10% fetal bovine serum, hypoxanthine, aminopterin and thymidine. Hybridoma supernates were screened with immunofluorescence and western blotting using Cowdria antigens from BUE cells. Cells from antibody-positive wells were expanded, cryopreserved, cloned and recloned by limiting dilution. Isotyping was carried out in an Ouchterlony test using rabbit antiserum specific for each of the respective mouse IgG sub-classes. Ascites was produced in Balb/C mice by intraperitoneal injection of 2 × 106 hybrid cells per mouse, which had been treated with 500 mg Pristane (0.5 ml; Aldrich Chemical Co., Inc., Milwaukee, Wisc. USA) 2 weeks previously. Ten ml ascitic fluid was collected from three moribund mice, centrifuged 10 min at 1000 g and stored in 200/A aliquots at - 80°C. The titer was 8 × l 0 4 determined by competitive ELISA. Fluorescent antigen BUE cultures infected with Cowdria were centrifuged for 10 min at 10 000 g. The pellet was resuspended in PBS, spotted onto microscope slides and fixed in acetone. The slides were incubated with two-fold titrations of antisera from goats immunised against Cowdria or with undiluted hybridoma su-



pernatant. FITC-labelled rabbit-anti-goat or FITC-rabbit-anti-mouse immunoglobulins (Nordic Diagnostic Laboratory, Tilburg, The Netherlands) were used as second antibody and fluorescence was observed with an Olympus BH2RFL microscope.

Western blotting analysis BUE cells heavily infected by Cowdria (Senegal, Welgevonden, Lutale or U M Banein stock) were collected and together with elementary bodies in the supernatant centrifuged at 10 000 g for 10 min at 4 ° C, resuspended in SPG buffer and stored at - 8 0 ° C. The material was washed in SPG, ultrasonically disrupted in SPG on ice with a Branson Sonifier in 4 cycles of 15 sec and the protein content determined according to Bradford (1976). Sonicates of infected endothelial cell cultures were subjected to SDS-PAGE on 7.5-20% polyacrylamide gradient gels under reducing conditions. Western blotting was carried out as described previously (Jongejan and Thielemans, 1989). Briefly, electrophoretic transfer was accomplished overnight. Thereafter, the blots were stained in 0.2% Ponceau S in 3% trichloracetic acid, cut into strips and destained. Blots were quenched, washed and incubated with goat antisera diluted 1:100 or undiluted hybridoma supernatant. Binding of antibody was localized by rabbit-anti-goat or rabbit-anti-mouse immunoglobulins conjugated with horseradish peroxidase (Dakopatts, Glostrup, Denmark) diluted 1:750 for 90 min. Binding of conjugate was visualized by adding sodium-nitroprusside, o-dianisidine and hydrogen peroxide as substrate.

Electron microscopy The immunogold method for electron microscopy was adapted after Faulk and Taylor (1971 ). A suspension of Cowdria elementary bodies and dislodged infected BUE cells was placed in a microcentrifuge tube and pelleted at 10 000 g for 10 min in an Eppendorf microcentrifuge. The pellet was washed once with phosphate-buffered saline (PBS), reacted with mAb 4F 10B4 (hybridoma supernatant) 1:1 and incubated at 37 °C for 45 min, pelleted, washed twice with PBS and incubated with colloidal gold (particle size 10 n m in diameter) that was conjugated to anti-mouse IgG (Janssen Pharmaceutica, Beersen, Belgium). Cowdria elementary bodies and infected BUE cells were pelleted and fixed for 60 min at 4°C in 1.25% formaldehyde, 2.5% glutaraldehyde, 0.03% CaCI2 and 0.03% picric acid in 0.05% cacodylate buffer o f p H 7.4 (Ito and Rikihisa, 1981 ). The specimens were washed in 0.2 M cacodylate buffer embedded in 3% agar, post-fixed for 60 min with 2% osmium-tetroxide in 0.1% cacodylate buffer (pH 7.4) at room temperature, washed again, dehydrated in graded concentrations of ethanol and embedded in Epon. Sections of 60-90 nm were cut with a diamond knife using an ultramicrotome, then stained with lead citrate and examined using a Zeiss EM-10A transmission electron microscope on 60 kV.


F. J O N G E J A N


CELISA Sonicates of endothelial cells heavily infected by Cowdria (Senegal) were prepared as described for western blotting. Checkerboard titrations were conducted to determine optimal concentrations of mAb and antisera (data not shown). Aliquots of 10/tg/ml of protein were applied to microtitre plates in carbonate-bicarbonate buffer at pH 9.6 at 4°C overnight. Antigen-coated plates were washed in distilled water containing 0.05% Tween 20 and blocked with PBS/gelatine 0.25%. After washing of the plates mAb 4F10B4 was applied simultaneously with test serum both at 1:100 final dilution and incubated for 60 min at 37°C. Next, peroxidase-labeled rabbit-anti-mouse immunoglobulins (Dakopatts) diluted 1:500 was added and incubated for 60 min at 37 ° C. After the plates were washed, 100 ~tl ofABTS substrate solution (Sigma Chemicals, USA) was dispensed into each well and the optical density was measured at 405 nm after 30 min incubation. RESULTS

Five mAbs were raised against Cowdria. Four mAbs, of which two (4F10B4 and 1E3H10) were of the IgG3 isotype and the two others (3D8H1 and kDa ~





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Competitive enzyme-linked immunosorbent assay for heartwater using monoclonal antibodies to a Cowdria ruminantium-specific 32-kilodalton protein.

Hybridomas producing monoclonal antibodies (mAb) to Cowdria ruminantium were raised. Four mAbs of the IgG isotype reacted in western blots with a 32-k...
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