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

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

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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.

0378-1135/91/$03.50

© 1991 - - Elsevier Science Publishers B.V.

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INTRODUCTION

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.

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MATERIALS AND METHODS

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).

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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-

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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.

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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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Fig. 1. Western blot of extracts of umbilical endothelial cell cultures (BU E) infected with three stocks of C. ruminantium probed with mAbs. Lanes 1 to 5 contain antigen of Cowdria (Senegal). Lane 1; positive anti-Cowdria (Senegal) goat serum, Lane 2; mAb IE3H10, Lane 3; 1E5H8, Lane 4; mAb 4F10B4, Lane 5; mAb 3D8H1. Lanes 6 to 10 contain antigen of the Cowdria strain Welgevonden. Lane 6; positive anti-Cowdria (Welgevonden) goat serum, Lane 7; mAb I E3HI0, Lane 8; mAb IE5H8, Lane 9; mAb 4F10B4, Lane 10; mAb 3D8H1. Lanes 11 to 15 contain Cowdria (Um Banein stock) antigen. Lane 11; positive anti-Cowdria (Urn Banein) goat serum, Lane 12; mAb IE3HI0, Lane 13; mAb lE5H8, Lane 14; mAb 4F10B4, Lane 15; mAb 3D8H1. Molecular mass markers in kDa are indicated on the left.

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Fig. 2. Electron micrograph showing Cr32-specific epitope detection on the surface of elementary bodies of Cowdria by immunogold labeling with mAb 4F10B4. (39 000 × ).

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Fig. 3. The reactivity ofsera from goats, immunised with 8 different heartwater stocks, in competition ELISA using mAb 4F10B4. Lane 1; negative control sera from 27 non-infected goats, Lane 2-9; Sera of Cowdria-infected animals (no. of goats between brackets). Lane 2; Senegal stock ( 16 ), Lane 3; Welgevonden stock (6), Lane 4; Um Banein stock ( 1 ), Lane 5; Lutale stock (2), Lane 6; Kiimm (8), Lane 7; Kwanyanga (16), Lane 8; Ball 3 (12), and Lane 9; Gardel (9). Lane 10 shows the reactivity of nine anti-E, phagocytophila goat sera. The dashed base line is based upon the mean OD value of 27 negative control sera of 0.742 with a standard deviation of 0.073. After providing for 2 standard deviation units, the base-line was fixed on 0.598.

1E5H8) of the IgG2b isotype, were directed against epitopes on a 32-kDa Cowdria protein (Cr32) as demonstrated by western blotting (Fig. 1 ). A fifth mAb (4B2F3H 12 ) of the IgM isotype recognized a 40-kDa Cowdria protein (not shown). All four anti-Cr32 mAbs were reactive in an indirect fluorescent antibody assay (IFA), but the anti-40 kDa mAb was negative, mAb 4F10B4 showed the strongest signal in western blots using three different

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1 2 3 4 5 6 7 8 9 101112131415161718 Fig. 4. Western blot analysis with sera from goats immunised against different stocks of Cowdria ruminantium. Fifteen sera are tested which were shown to be negative in CELISA (Fig. 3 ). Lane 1; Senegal antiserum (negative in CELISA, but positive in IFA), Lanes 2-15 show Cowdria antisera which were negative in CELISA and IFA. Lane 2; Senegal antiserum, Lane 3-5; Welgevonden, Lane 6; U m Banein, Lane 7-10; Kwanyanga, Lane 11-13; Ball 3, Lane 14-15; Gardel. Lane 16; Positive antiserum (Senegal stock), Lane 17; Positive antiserum (Welgevonden stock), Lane 18; negative control serum. Molecular mass markers in kDa are indicated on the left. TABLE 1 Comparison of results ~ of CELISA 2 and IFA 3 for sera from 70 goats experimentally infected with Cowdria ruminanlium CELISA result

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~Nos. of samples 2Overall seroprevalence CELISA 55/70 (79%) 3percent agreement with IFA 62/70 (89%)

stocks of Cowdria (Fig. 1 ) and also recognized Cr32 in a fourth stock of Cowdria (Lutale) (not shown). The 1E3H 10 and 3D8H1 mAbs recognized Cr32 of the S stock (lane 2 and lane 5, respectively) and the W stock (lane 7 and land 10, resp.), but not in the UB isolate of Cowdria (lane 12 and lane 15,

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C O M P E T I T I V E ELISA F O R H E A R T W A T E R

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Fig. 6. The reactivity of sera from cattle in CELISA. Lane 1; 25 non-infected control calves, Lane 2; sera from calves experimentally infected with 2 different Cowdria stocks (Lutale and Senegal ), Lane 3; Anaplasma marginale sera with titres of 5210 against isolates from Texas and Nigeria. Mean OD value of 0.549 based on 25 non-infected control calves minus two times the standard deviation provides a base line of 0.461.

resp. ). mAb 1E5H8 recognized all three Cowdria antigens, but signals on S and UB were very weak. Immune-gold staining of viable Cowdria organisms in BUE cultures showed gold particles attached to the outer surface of the rickettsiae (Fig. 2 ). The CELISA detected antibodies in 55 out of 70 (79%) goats infected with

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one of eight different Cowdria stocks (Fig. 3). Although 14 out of 15 sera, which were shown negative in the CELISA, were also negative in the IFA, all 15 samples recognized the immunodominant Cowdria-specific 32 kDa protein in western blots (Fig. 4) (Table 1 ). Overall, there was 89% agreement between CELISA and IFA with all 70 goat sera tested (Table 1 ). Antibodies against Cowdria were also detected in nine out of nine sheep immunised with either the Senegal, Welgevonden or Ball 3 stock using CELISA (Fig. 5). Moreover, anti-CR32 antibodies were also detected in six out of six calves experimentally infected with one of two different isolates of Cowdria (Fig. 6, lane 2 ). Furthermore, there were no cross-reactions in the CELISA with antisera from nine goats experimentally infected with E. phagocytophila (Fig. 3, lane 10). Finally, no cross-reactions were detected in sera obtained from four calves with antibody titres of 5120 against Anaplasma marginale (Fig. 6, lane 3 ). DISCUSSION

This is the first report describing mAbs against C. ruminantium. These mAbs may be valuable tools for antigenic and molecular analysis of this rickettsia. Four of the five specific hybridomas that were obtained could be stabilized, whereas mAb 4B2F3H 12 of isotype IgM which recognized a 40-kDa protein was either lost during further cloning or ceased antibody production. The reason that four out of five mAbs recognized epitopes on the immunodominant 32-kDa protein from C. ruminantium (Jongejan and Thielemans, 1989 ), may have been due to our method of immunization of mice. We used virulent rickettsiae from endothelial cell culture supernatant without adjuvant to infect mice. These mice were moribund at the time their spleen cells were fused. This may have been the reason for the efficient recognition of the Cr32 molecule, because it is known that this protein is also strongly recognized in mice immunized against the mouse-pathogenic KiJmm and Kwanyanga stocks (Jongejan and Thielemans, 1989 ). We do not know yet, whether thefour anti-Cr32 mAbs possibly recognize the same or overlapping epitopes on the Cr32 molecule. Localisation of proteins with immuno-gold labeling using mAbs has been reported for Chlamydia trachomatis (Kuo and Chi, 1987). Incubation of mAbs 4F10B4 with naive Cowdria organisms in vitro facilitated the subsequent membrane-bound localisation of the Cr32 epitope with gold-conjugated second antibody. This shows that the Cr32 protein has surface-exposed antigenic determinants, which was already suspected on the basis of its immunodominant recognition in Cowdria infections. The demonstration of serotypes in Cowdria with the IFA on infected neutrophils stressed the need for a species specific test (Jongejan et al., 1989). For use in the IFA, Cowdria antigens obtained from endothelial cell cultures were found to be superior over those obtained from infected mouse macro-

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phages or cultures neutrophils. Moreover, serotype-specificity was less pronounced using extracellular elementary bodies obtained from endothelial cell cultures (Martinez et al., 1990). The results with the competitive ELISA showed that serotype-specificity does not play a role, which may be expected since Cr32 is a conserved protein from C. ruminantium present in all isolates of Cowdria tested thus far (Jongejan, 1990). Comparison of the CELISA with IFA results for goat sera showed 89% agreement between both tests. The CELISA detected antibodies in seven goat sera, which were negative in the IFA (Table 1 ), indicating that the CELISA is somewhat more sensitive than the IFA. Both tests failed to detect antibodies in 14 sera which were obtained from goats immunised against different Cowdria stocks (Table 1; Fig. 3). However, when these sera were tested in western blots they all appeared to recognize the Cr32 protein (Fig. 4). A possible explanation is that polyclonal goat antisera recognize a different epitope on the Cr32 molecule than the monoclonal antibody. The western blot appears to be a more sensitive assay for the detection of antibodies against Cowdria-specific protein. However, western blotting is not very practical for large scale serological testing. Moreover, the advantage of the described CELISA is that sera from many animal species can be tested, except mouse. For instance, nine sheep immunised against heartwater were clearly distinguished from the non-infected control sheep (Fig. 5 ). Moreover, in six out of six calves infected with one of two different isolates of Cowdria were anti-Cr32 antibodies detected (Fig. 6). Our preliminary results with sera from wild animals indicate that the competition ELISA could also be used to determine the involvement of wildlife in the epidemiology of heartwater. Finally, there were no cross-reactions in the CELISA with antisera from goats experimentally infected with E. phagocytophila (Fig. 3 ) and no crossreactions were detected in sera with high titres against A. marginale (Fig. 6 ). E. phagocytophila sera which have previously been shown to cross-react in the IFA with Cowdria-infected neutrophils, apparently did not recognize any epitope on the Cr32 molecule. Although it seems unlikely that any other ehrlichial agents will recognize epitopes on this Cowdria-specific protein, further studies on this aspect of the CELISA are required, especially with E. bovis and E. ondiri which occur sympatrically with C. ruminantium. The results presented here suggest that the competitive ELISA can be used for examining for the presence of antibodies to C. ruminantium in domestic ruminants and that the test is versatile enough to be used in epidemiological field studies in heartwater endemic areas. ACKNOWLEDGEM

ENTS

The research presented here was supported by the European Community (Directorate General XII) under contract TS2-0115-C entitled; "Integrated

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Control of Cowdriosis and Dermatophilosis of Ruminants". Prof. Dr. F. Uilenberg, IEMVT, is thanked for his critical comments on the manuscript.

REFERENCES Bezuidenhout, J.D., 1987. The present state of Cowdria ruminantium cultivation in cell lines. Onderstepoort J. Vet. Res., 54: 205-210. Bovarnick, M.R., Miller, J.C. and Snyder, J.C., 1950. The influence of certain salts, amino acids, sugars and proteins on the stability of rickettsiae. J. Bacteriol., 59: 509-522. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254. Camus, E., 1987. Contribution a l'etude 6pidemiologique de al cowdriose (Cowdria ruminantium) en Guadeloupe. These Doct. -es- Sci., Universit6 de Paris-Sud, Centre d'Orsay, 202 pp. Dahl, R.J., Woods, W.H. and Johnson, A.M., 1987. Recognition by the human immune system of candidate vaccine epitopes of Toxoplasma gondii measured by a competitive ELISA. Vaccine, 5: 187-191. Du Plessis, J.L., 1981. The application of the indirect fluorescent antibody test to the serology of heartwater. Proc. Int. Cong. Tick Biology and Control, Rhodes University, 27-29 January 1981, Grahamstown, pp. 47-52. Du Plessis, J.L., 1982. Mice infected with a Cowdria ruminantium-like agent as a model in the study ofheartwater. D.V.Sc.Thesis, University of Pretoria, 157 pp. Du Plessis, J.L., 1985. A method for determining the Cowdria ruminantium infection rate of Amblyomma hebraeum: effects in mice infected with tick homogenates. Onderstepoort J. Vet. Res., 52: 55-61. Du Plessis, J.L. and Kiimm, N.A.L., 1971. The passage of Cowdria ruminantiutn in mice. J. S. Aft. Med. Assoc., 42: 217-221. Du Plessis, J.L., Camus, E., Oberem, P.T. and Malan, L., 1987. Heartwater serology: some problems with the interpretation of results. Onderstepoort J. Vet. Res., 54: 327-329. Du Plessis, J.L., Van Gas, L., Olivier, J.A. and Bezuidenhout, J.D., 1989. The heterogenicity of Cowdria ruminantium stocks: Cross-immunity and serology in sheep and pathogenicity to mice. Onderstepoort J. Vet. Res., 56:195-201. Faulk, W.P. and Taylor, G.M., 1971. An immunocolloid method for the electron microscope. Immunochem., 8:1081-1083. Haig, D.A., 1952. Note on the use of the white mouse for the transport of strains of heartwater. J. S. Aft. Vet. Med. Assoc., 23: 167-170. Hewitt, J., Coates, A.R.M., Mitchison, D.A. and Ivanyi, J., 1982. The use of routine monoclonal antibodies without purification of antigen in the serodiagnosis of tuberculosis. J. Immunol. Methods, 55:205-211. Ilemobade, A.A. and Blotkamp, J., 1976. Preliminary observations on the use of the capillary flocculation test for the diagnosis of heartwater (Cowdria ruminantium infection). Res. Vet. Sci., 21: 370-372. Ito, S. and Rikihisa, Y., 1981. Techniques for electron microscopy of rickettsiae. In: W. Burgdorfer and R.L. Anacker (Editors), Rickettsiae and Rickettsial Diseases, Academic Press, pp. 213-227. Jongejan, F. 1990. Tick/host interactions and disease transmission with special reference to Cowdria ruminantium (Rickettsiales). Ph.D. Thesis, University of Utrecht, The Netherlands, 199 pp.

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