Veterinary Microbiology, 25 (1990) 45-53 Elsevier Science Publishers B.V., Amsterdam
45
An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Anaplasma centrale and Anaplasma marginale Varda Shkap*, Hanna Bin, Hanna Ungar-Waron and E. Pipano Department of Parasitology, Kimron Veterinary Institute, P.O.B. 12, Bet-Dagan, Israel (Accepted 19 February 1990)
ABSTRACT Shkap, V., Bin, H., Ungar-Waron, H. and Pipano, E., 1990. An enzyme-linked immunoassay (ELISA) for the detection of antibodies to Anaplasma centrale and A. marginale. Vet. Microbiol., 25:45-53. An enzyme-linked immunoassay (ELISA) was applied to detect antibodies to A. centrale and A. marginale using homologous and heterologous antigens. The assay was compared with the indirect fluorescent antibody (IFA) test, and although a similar degree of sensitivitywas obtained, the ELISA test had several advantages. Partially purified Anaplasma initial bodies used for antigen preparations contained negligible amounts of residual erythrocytic material, and did not interfere with the specificity of the ELISA. The antigenic similarity between A. marginale and A. centrale was further substantiated by cross-reactivity obtained with heterologous antigens in both ELISA and IFA tests, and antibodies produced during natural infection with A. marginale were indistinguishablein both tests from those produced following vaccination with A. centrale.
INTRODUCTION
Anaplasmosis is a hemoparasitic disease of cattle caused by the intraerythrocytic rickettsia Anaplasma marginale. In Australia, South Africa and Israel anaplasmosis is controlled by immunization with a live vaccine containing the less virulent species A. centrale (Callow, 1976; Bigalke, 1980; Pipano, 1980). Diagnosis of acute anaplasmosis is usually based on direct microscopic detection of the organisms in Giemsa-stained blood smears. The animal response to infection has been measured by a variety of serological methods such as card agglutination (CA) and capillary agglutination tests, complement fixation, and the indirect fluorescent antibody (IFA) technique (Ristic, 1968; Pipano et al., 1972; Gonzalez et al., 1978; Kuttler and Winward, 1984; Goff et al., 1985; Montenegro-James et al., 1985 ). The enzymelinked immunosorbent assay (ELISA) has been introduced for detecting an*To whom correspondence should be addressed.
0378-1135/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
46
V. S H K A P ET AL.
tibodies to A. marginale (Thoen et al., 1980; Barry et al., 1986; Winkler et al., 1987; Nakamura et al., 1988a; Duzgun et al., 1988 ), using various antigenic preparations. Barry et al. (1986) and Nakamura et al. (1988b) measured antibodies in serum samples obtained from a small number of calves infected with A. centrale using heterologous A. marginale antigen. In this study we present the results of an ELISA procedure for the detection and titration of antibodies to both A. centrale and A. marginale, employing homologous and heterologous antigens. MATERIALS AND METHODS
Antigens Antigens for ELISA were prepared by partial purification of initial bodies ofA. marginale and A. centrale in infected erythrocytes, as described by Palmer and McGuire (1984). Briefly, blood from infected cattle at 30-40% parasitemia was collected into an equal volume of cold PBS, pH 7.2, and washed three times with cold PBS by centrifugation at 2600 Xg for 20 min. The buffy coat was removed after each centrifugation. The packed erythrocytes were resuspended in an equal volume of PBS containing 30% DMSO and stored at - 70 ° C. About 40 ml of erythrocytes were thawed at 37 ° C and further washed seven or eight times with cold PBS by centrifugation at 10 0 0 0 × g for 15 min to remove hemoglobin. A pink pellet of erythrocytic debris was removed before the last washing, and the final sediment was resuspended in 2 ml PBS and sonicated at 100 W for 2 min. The sonicate was washed twice, centrifuged at 2600g for 15 min, the supernatant discarded and the pellet, containing purified initial bodies, was resuspended in 0.4 ml PBS. Aliquots of antigen in 50/tl volumes were stored at - 7 0 ° C. Uninfected erythrocytes were processed similarly. The antigen for the IFA test was prepared from erythrocytes parasitized with either A. marginale orA. centrale. One part of blood from infected calves was collected into ten parts of cold PBS containing 0.4% sodium citrate, washed five times with cold PBS and separated by centrifugation at 1600g for 20 min after each wash. The final pellet was resuspended in PBS containing 1.5% bovine serum albumin (BSA) and thin blood smears were prepared. The slides were air-dried, fixed in acetone for 15 min, wrapped in Whatman filter paper and stored at - 70 oC. SDS-PAGE Partially purified initial bodies were solubilized by boiling for 5 min in sample buffer containing 0.025 M Tris-glycine, pH 6.8, 2% w/v SDS, 15% v~ v glycerol, 2.5% fl-mercaptoethanol and a few crystals of bromphenol blue. SDS-PAGE was performed in 7.5-17.5% polyacrylamide gradient slab gels
AN I-LISA FOR A. CENTRALE AND A. MARGINALE
47
and stained with 0.075% Coomassie blue. A preparation of uninfected erythrocytes was included.
Serum samples Sera were collected from 142 cattle irrespective of age, located on a farm where routine vaccination of 6- to 8-month-old calves with live A. centrale organisms was performed. Twenty four imported Anaplasma-free animals were bled while in the quarantine station and their sera used as negative controis. Eleven splenectomized calves were experimentally infected with live A. centrale and seven others with A. marginale organisms. Sera having an IFA titer of 1 : 1024 were collected and used as positive reference sera. Eight additional sera from splenectomized calves having antibody titers of 1 : 1024 against Babesia bigemina, B. bovis, Theileria annulata, and four with titers up to 1:4000 to Besnoitia besnoiti and Toxoplasma gondii were also tested for nonspecific activity against Anaplasma.
Protein determination The protein content of antigens used in the ELISA was determined with the Bio-Rad protein assay reagents (Bio-Rad, Richmond, CA) using BSA as standard.
Serological tests For the ELISA test, microtiter plates (Dynatech) were coated with antigens diluted in 0.05 M carbonate/bicarbonate buffer, pH 9.6, and incubated for 18 h at 4°C. After three washes with PBS-0.05% Tween-20 (PT), the plates were dried and stored at 4 °C until use. Blocking of nonspecific binding sites was performed with 5% horse serum diluted in PT for 2 h at 37°C. Duplicate serum samples at four-fold dilutions in PT were added to the wells and incubated for 18 h at 4 ° C. After three additional washes, peroxidase-labeled rabbit anti-bovine IgG conjugate (Bio-Makor, Israel), diluted 1 : 2000 in P T 0.2% BSA, was added and the plate incubated for 2 h at 37 ° C. An additional wash was carried out, followed by addition of the substrate, consisting of 1 m g / m l of azino di-[3-ethyl-benzthiazoline] sulfonate in a solution containing 0.1 M citric acid, 0.2 M phosphate buffer, pH 4.5, and 0.05% hydrogen peroxide. All reagents were in 100-~1 aliquots per well. The reaction was allowed to proceed for 1 h at room temperature, and the absorbance values were read at 405 n m on an ELISA reader (microplate autoreader EL309, BioTek Instruments).
IFA procedure Frozen antigen-coated slides were allowed to thaw for 30 min at 37 °C in a container supplied with silica gel. Circles were marked on the slides with nail polish. Four-fold dilutions of serum samples in PBS were applied to the slides
48
V. SHKAP ET AL.
in duplicate, and incubated for 30 min at 37°C in a humid chamber. The slides were washed twice in PBS, dried between filter paper and allowed to react for 30 min at 37°C with rabbit anti-bovine IgG fluorescein conjugate (Bio-Makor) diluted 1:75. Slides were washed and dried as before, covered with PBS-glycerol ( 1 : 1 ) and examined under a fluorescence microscope. In both ELISA and IFA assays, the optimal dilutions obtained and subsequently used were those exhibiting the best resolution between reference sera resulting from checkerboard titrations (Voller et al., 1976). Results of the ELISA were expressed as the highest dilution of serum producing an optical density (O.D.) at 405 nm higher by 2 s.d. than that obtained with negative control serum at the same dilution. The endpoint in IFA was the highest dilution of serum still exhibiting fluorescence with the Anaplasma organisms.
Statistics Data were analysed by Student's paired t-test (Weisbrot, 1985 ). RESULTS Coomassie-stained protein bands obtained from Anaplasma-infected or uninfected erythrocytes and separated by SDS-PAGE are shown in Fig. 1. Multiple bands were observed in the preparations of isolated initial bodies, while only a few bands, albeit weakly stained, could be detected in the material obtained from uninfected erythrocytes. The optimal concentration of the coating antigen was determined from the dosage-dependent absorbance curves (Fig. 2). The highest resolution between positive and negative sera was obtained at an antigen concentration of 5 pg/ml. Similar O.D. readings were recorded under the same experimental conditions with increasing concentrations of antigen. The optimal working dilution of the conjugate chosen was 1 : 2000, as determined at a constant concentration of antigen (5 # g / m l ) and by means of positive and negative reference sera (data not shown). ELISA titration curves (Fig. 3) were established from the mean absorbance reading obtained by the reactivity of both A. centrale and A. marginale antigens with 24 negative serum samples collected from imported cattle not previously exposed to Anaplasma, 11 anti-A, centrale and seven anti-A, marginale reference positive sera. The mean O.D. value of negative sera at the lowest serum dilution ( 1 : 64 ) was less than 0.310 with either antigen. In the homologous A. centrale system mean O.D. readings were five times higher than the mean negative control at a serum dilution of 1 : 64, four times higher at 1:256, and three times higher at 1 : 1024. The mean O.D. readings of the positive sera in the A. marginale homologous system were twice the negative serum values at 1:64 and 1:256, but only 1.5 times higher at 1 : 1024. The same sera tested by IFA showed 8.3% false positive reactions at 1 : 64.
AN ELISA FOR A. CENTRALE AND A. MARGINALE
MAc
116
49
Am Ni
-
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-
~4~~
~ ,
29- i
Fig. 1. SDS-PAGE ofA. centrale (Ac), A. rnarginale (Am) and uninfected (Ni) erythrocytes stained with Coomassie Brilliant Blue G-250. M, Molecular weight standard markers ( X 10-3).
Similar results were obtained by ELISA and IFA run in parallel using A. centrale or A. marginale antigens (Figs. 4A and B). Results of 142 serum samples obtained from cattle vaccinated with A. centrale and collected at times ranging from 1 week to 1 year after vaccination showed no statistically significant difference between the two tests when tested with the A. centrale antigen (t--0.277, which is much lower than the critical value of 1.980). Twenty serum samples (13.8%) with titers up to 1:256 by IFA were negative by ELISA, while only three sera (2.06%) negative by IFA were positive by ELISA (Fig. 4A ). Similar results were recorded when a total of 48 samples were tested with A. marginale antigen (Fig. 4b). Fig. 5 shows cross-reactions obtained by both ELISA and IFA employing A. centrale and A. rnarginale antigens on 96 previously calibrated positive
50
V. S H K A P ET AL.
1.2 C:E 1.0
Lo 0.8 ,e 0.6
# ~
~~ 0 . 4
d
- ° ~ °
~ °
o
0.2
0
1 2.5 ~i 7.5 10 ANTIGEN CONCENTRATION(.gml)
Fig. 2. Standardization of antigen concentration for coating ELISA microplates. Positive serum at 1:64 ( - 0 - ) and 1:256 ( - O - ) dilutions. Negative serum diluted 1:64 ( - O - ) and 1:256
(-@-). 1.8
1.4
E ¢-. LO O 1.0
0 0.6
0.2
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L
il
1:64 Serum
1 : 256 dilution
1:1024
Fig. 3. Mean absorbance readings (O.D. at 405 nm) for A. centrale (Ac) and A. marginale (Am) antigens reacting with reference Ac-positive ([]), Am-positive ( [] ) and negative ( [] ) sera in ELISA.
sera collected from cattle vaccinated against A. centrale. Parallel results were obtained at 1 : 64 and 1:256 by both methods, while at 1 : 1024 dilution 0.8% o f the sera were positive by IFA with the heterologous A. marginale antigen. Negative reactions against both A. centrale and A. marginale antigens were obtained with sera having positive titers to B. besnoiti and T. gondii (up to 1:4000 and 1:256, respectively). In contrast, sera from cattle with titers up to 1 : 1024 against T. annulata, B. ovis and B. bigemina, gave slightly higher O.D. readings than the m e a n negative control value at 1 : 64 (0.350 vs. 0.315 ), but these were still low when c o m p a r e d with the ELISA values obtained with the positive reference sera shown in Fig. 3.
AN ELISA FORA. CENTRALE A N D A. MARGINALE
(n = 142)
A
51
B
(n=48)
m J
uJ 1024 -
••
>03
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-.
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rr
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::::: 256
!!!!!!~ 10~4
RECIPROCAL
... I
n~.at TITER
256
I
1024
BY I F A
Fig. 4. Comparison between IFA and ELISA titers using A. centrale (A) and A. marginale (B) antigens. Serum samples were collected, irrespective of age, from cattle on a farm where routine vaccination with A. centrale organisms was performed annually in 6- to 8-month-old animals. A
80 6O
40 I-,,~ 2 0 U,,I n0 n" U.I
I
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~ 80
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256
1024
RECIPROCAL ANTIBODY TITER
Fig. 5. Percentage of cross-reactivity obtained between A. centrale-positive post-vaccination sera (n = 96 ), homologous A. centrale ( [] ) and heterologous A. marginale ( [] ) antigens by ELISA (A) and IFA (B). DISCUSSION I n this s t u d y we d e m o n s t r a t e t h e v a l i d i t y o f t h e E L I S A c o m p a r e d w i t h I F A f o r t h e s e r o d i a g n o s i s o f a n a p l a s m o s i s . T h e E L I S A offers a n u m b e r o f a d v a n -
52
V. SHKAP ET AL.
tages. It requires minute amounts of antigen, and the results are objective and recorded automatically, thus facilitating large-scale field screening and vaccination follow-up. The specificity of the assay was assessed by lack of reactivity exhibited by sera from cattle infected with Besnoitia or Toxoplasma, although some absorbance background was recorded with positive sera to such blood protozoans as Theileria and Babesia. This was due to the presence of residual erythrocytic impurities accompanying the antigenic material, as seen with preparations of uninfected erythrocytes run on SDS-PAGE (Fig. 1 ). However, the O.D. values obtained were slightly higher than the mean of negative controls and are therefore negligible. Similar nonspecific reactivity with erythrocytic material in various serological tests for detection of anti-Anaplasma antibodies were reported by Montenegro et al. ( 1981 ), MontenegroJames et al. (1985) and Barry et al. (1986). Coating of ELISA plates with known amounts of antigen, based on protein estimation, enables better standardization of the test than can be obtained with IFA, in which preparation of blood smears with unevenly spread infected erythrocytes might impede reliable and objective interpretation. Indeed, we observed a total of 8.3% false positive reactions by IFA at 1 : 64, which is similar to results reported by Gonzales et al. (1978). We consider the relatively high number of positive reactions obtained by IFA on field sera to reflect a subjective bias in the evaluation of fluorescence; this is the main disadvantage of the test. Although there are conflicting reports regarding the degree of sensitivity obtained by the complement fixation, IFA, card agglutination and plate agglutination tests (Wilson et al., 1978; Gonzales et al,. 1978; Goff et al,. 1985; MontenegroJames et al., 1985), our results indicate a similar degree of sensitivity for ELISA and IFA. The antigenic relationship between A. margina[e and A. centrale was further substantiated in this study by cross-reaction experiments carried out by ELISA and IFA. Specific antisera exhibited higher homologous reactivity at higher dilutions (Fig. 5 ). Thus, when a serum tested against both Anaplasma antigens exhibits a higher end-point to A. marginale than to A. centrale, one can assume that the animal has contracted an infection with A. marginale. A similar degree of reactivity towards both antigens at high serum dilutions may also be indicative of natural infection with A. marginale before or after vaccination with A. centrale. Specific antibodies towards A. centrale or A. marginale as infecting parasites could not be detected, despite using homologous antigens. In a similar study, Amerault and Roby ( 1971 ) reported that postvaccination antibodies could not be distinguished from those produced by natural infection using CF or CA tests, and Barry et al. (1986) did not observe differential reactivity betwee~ antisera to A. centrale or A. marginale in the microplate ELISA employing an A. marginale antigen. It therefore appears that further investigation is n~eded to obtain clear-cut identification of antibodies produced by either natural infection or by vaccination.
AN ELISA FOR A. CENTRALE AND A. MARGINALE
53
REFERENCES Amerault, T.E. and Roby, T.O., 1971. Card agglutination and complement-fixation reactions after vaccination of cattle against anaplasmosis. J. Am. Vet. Med. Assoc., 159:1749-1751. Barry, D.N., Parker, R.J., De Vos, A.J., Dunster, P. and Rodwell, B.J., 1986. A microplate enzyme-linked immunosorbent assay for measuring antibody to Anaplasma marginale in cattle serum. Aust. Vet. J., 63: 76-79. Bigalke, R., 1980. The control of the ticks and tick-borne diseases of cattle in South Africa. Zimbabwe Vet. J., 11: 20-22. Callow, L., 1976. Tick-borne livestock diseases and their vectors. III. Australian methods of vaccination against anaplasmosis and babesiosis. World Anim. Rev., 18:9-15. Duzgun, A., Schuntner, C., Wright, I., Leatch, G. and Waltisbuhl, D., 1988. A sensitive ELISA technique for the diagnosis ofAnaplasma marginale infections. Vet. Parasitol., 29: 1-7. Goff, W.L., Johnson, B.S. and Kuttler, K.L,. 1985. Development of an indirect fluorescent antibody test, using microfluometry as a diagnostic test for bovine anaplasmosis. Am. J. Vet. Res., 46: 1080-1084. Gonzales, E.F., Long, R.F. and Todorovic, R.A., 1978. Comparisons of the complement fixation, indirect fluorescent antibody, and card agglutination test for the diagnosis of bovine anaplasmosis. Am. J. Vet. Res., 39: 1538-1541. Kuttler, K.L. and Winward, L.D., 1984. Serologic comparison of 4 Anaplasma isolates as measured by the complement-fixation test. Vet. Microbiol., 9:181-186. Montenegro-James, S., James, M.A. and Ristic, M., 1985. Modified indirect fluorescent antibody test for the serodiagnosis ofAnaplasma marginale infections in cattle. Am. J. Vet. Res., 46: 2401-2403. Montenegro, S., James, M.A., Levy, M.G., Preston, H.D., Esparza, H. and Ristic, M., 1981. Utilization of culture-derived soluble antigen in the latex agglutination test for bovine babesiosis and anaplasmosis. Vet. Parasitol., 8:291-297. Nakamura, Y., Shimizu Shinya, Minami, T. and Ito S. 1988a. Enzyme-linked immunosorbent assay using solubilized antigen for detection of antibodies to Anaplasma marginale. Trop. Anita. Health Prod., 20: 259-266. Nakamura, Y., Shimizu Shinya, Minami, T. and Ito S., 1988b. Enzyme-linked immunosorbent assay for detection of antibodies to Anaplasma centrale. Jpn. J. Vet. Sci., 50: 933-935. Palmer, G.H. and McGuire, T.C., 1984. Immune serum against Anaplasma marginale initial bodies neutralizes infectivity for cattle. J. Immunol., 133:1010-1015. Pipano, E., Klinger, I. and Weisman, Y., 1972. Application of the capillary agglutination test for anaplasmosis in cattle vaccinated with Anaplasma centrale. Refu. Vet., 29:166-169. Pipano, E., 1980. Bovine anaplasmosis and its control. In: Mayer, E.. (Editor), Proceedings of an International Congress on Diseases of Cattle. Bregman Press, Israel, pp. 720-726. Ristic, M., 1968. Anaplasmosis. In: Weinman, D. and Ristic, M. (Editors), Infectious Blood Diseases of Man and Animals. Academic Press Inc., New York, pp. 478-542. Thoen, C.O., Blackburn, B., Mills, K., Lomme, J. and Marry, P., 1980. Enzyme-linked immunosorbent assay for detecting antibodies in cattle in a herd in which anaplasmosis was diagnosed. J. Clin. Microbiol., 11: 499-502. Voller, A., Bidwell, D.F., Bartlett, A., Fleck, D.G., Perkins, M. and Oladehin, B., 1976. A microplate enzyme-immunoassay for Toxoplasma antibody. J. Clin. Pathol., 29:150-153. Weisbrot, I.M. (Editor), 1985. Statistics for the Clinical Laboratory. Lippincott, J.B., Company, East Washington Square, Philadelphia, PA, 198 pp. Wilson, A.J., Trueman, K.F., Spinks, G. and McSorbey, F., 1978. A comparison of 4 serological tests in the detection ofhumoral antibodies to anaplasmosis in cattle. Aust. Vet. J., 54: 383386. Winkler, G.C., Brown, G.M. and Lutz, H., 1987. Detection of antibodies to Anaplasma marginale by an improved enzyme-linked immunosorbent assay with sodium dodecyl sulfate-disrupted antigen. J. Clin. Microbiol., 25: 633-636.
45
An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Anaplasma centrale and Anaplasma marginale Varda Shkap*, Hanna Bin, Hanna Ungar-Waron and E. Pipano Department of Parasitology, Kimron Veterinary Institute, P.O.B. 12, Bet-Dagan, Israel (Accepted 19 February 1990)
ABSTRACT Shkap, V., Bin, H., Ungar-Waron, H. and Pipano, E., 1990. An enzyme-linked immunoassay (ELISA) for the detection of antibodies to Anaplasma centrale and A. marginale. Vet. Microbiol., 25:45-53. An enzyme-linked immunoassay (ELISA) was applied to detect antibodies to A. centrale and A. marginale using homologous and heterologous antigens. The assay was compared with the indirect fluorescent antibody (IFA) test, and although a similar degree of sensitivitywas obtained, the ELISA test had several advantages. Partially purified Anaplasma initial bodies used for antigen preparations contained negligible amounts of residual erythrocytic material, and did not interfere with the specificity of the ELISA. The antigenic similarity between A. marginale and A. centrale was further substantiated by cross-reactivity obtained with heterologous antigens in both ELISA and IFA tests, and antibodies produced during natural infection with A. marginale were indistinguishablein both tests from those produced following vaccination with A. centrale.
INTRODUCTION
Anaplasmosis is a hemoparasitic disease of cattle caused by the intraerythrocytic rickettsia Anaplasma marginale. In Australia, South Africa and Israel anaplasmosis is controlled by immunization with a live vaccine containing the less virulent species A. centrale (Callow, 1976; Bigalke, 1980; Pipano, 1980). Diagnosis of acute anaplasmosis is usually based on direct microscopic detection of the organisms in Giemsa-stained blood smears. The animal response to infection has been measured by a variety of serological methods such as card agglutination (CA) and capillary agglutination tests, complement fixation, and the indirect fluorescent antibody (IFA) technique (Ristic, 1968; Pipano et al., 1972; Gonzalez et al., 1978; Kuttler and Winward, 1984; Goff et al., 1985; Montenegro-James et al., 1985 ). The enzymelinked immunosorbent assay (ELISA) has been introduced for detecting an*To whom correspondence should be addressed.
0378-1135/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
46
V. S H K A P ET AL.
tibodies to A. marginale (Thoen et al., 1980; Barry et al., 1986; Winkler et al., 1987; Nakamura et al., 1988a; Duzgun et al., 1988 ), using various antigenic preparations. Barry et al. (1986) and Nakamura et al. (1988b) measured antibodies in serum samples obtained from a small number of calves infected with A. centrale using heterologous A. marginale antigen. In this study we present the results of an ELISA procedure for the detection and titration of antibodies to both A. centrale and A. marginale, employing homologous and heterologous antigens. MATERIALS AND METHODS
Antigens Antigens for ELISA were prepared by partial purification of initial bodies ofA. marginale and A. centrale in infected erythrocytes, as described by Palmer and McGuire (1984). Briefly, blood from infected cattle at 30-40% parasitemia was collected into an equal volume of cold PBS, pH 7.2, and washed three times with cold PBS by centrifugation at 2600 Xg for 20 min. The buffy coat was removed after each centrifugation. The packed erythrocytes were resuspended in an equal volume of PBS containing 30% DMSO and stored at - 70 ° C. About 40 ml of erythrocytes were thawed at 37 ° C and further washed seven or eight times with cold PBS by centrifugation at 10 0 0 0 × g for 15 min to remove hemoglobin. A pink pellet of erythrocytic debris was removed before the last washing, and the final sediment was resuspended in 2 ml PBS and sonicated at 100 W for 2 min. The sonicate was washed twice, centrifuged at 2600g for 15 min, the supernatant discarded and the pellet, containing purified initial bodies, was resuspended in 0.4 ml PBS. Aliquots of antigen in 50/tl volumes were stored at - 7 0 ° C. Uninfected erythrocytes were processed similarly. The antigen for the IFA test was prepared from erythrocytes parasitized with either A. marginale orA. centrale. One part of blood from infected calves was collected into ten parts of cold PBS containing 0.4% sodium citrate, washed five times with cold PBS and separated by centrifugation at 1600g for 20 min after each wash. The final pellet was resuspended in PBS containing 1.5% bovine serum albumin (BSA) and thin blood smears were prepared. The slides were air-dried, fixed in acetone for 15 min, wrapped in Whatman filter paper and stored at - 70 oC. SDS-PAGE Partially purified initial bodies were solubilized by boiling for 5 min in sample buffer containing 0.025 M Tris-glycine, pH 6.8, 2% w/v SDS, 15% v~ v glycerol, 2.5% fl-mercaptoethanol and a few crystals of bromphenol blue. SDS-PAGE was performed in 7.5-17.5% polyacrylamide gradient slab gels
AN I-LISA FOR A. CENTRALE AND A. MARGINALE
47
and stained with 0.075% Coomassie blue. A preparation of uninfected erythrocytes was included.
Serum samples Sera were collected from 142 cattle irrespective of age, located on a farm where routine vaccination of 6- to 8-month-old calves with live A. centrale organisms was performed. Twenty four imported Anaplasma-free animals were bled while in the quarantine station and their sera used as negative controis. Eleven splenectomized calves were experimentally infected with live A. centrale and seven others with A. marginale organisms. Sera having an IFA titer of 1 : 1024 were collected and used as positive reference sera. Eight additional sera from splenectomized calves having antibody titers of 1 : 1024 against Babesia bigemina, B. bovis, Theileria annulata, and four with titers up to 1:4000 to Besnoitia besnoiti and Toxoplasma gondii were also tested for nonspecific activity against Anaplasma.
Protein determination The protein content of antigens used in the ELISA was determined with the Bio-Rad protein assay reagents (Bio-Rad, Richmond, CA) using BSA as standard.
Serological tests For the ELISA test, microtiter plates (Dynatech) were coated with antigens diluted in 0.05 M carbonate/bicarbonate buffer, pH 9.6, and incubated for 18 h at 4°C. After three washes with PBS-0.05% Tween-20 (PT), the plates were dried and stored at 4 °C until use. Blocking of nonspecific binding sites was performed with 5% horse serum diluted in PT for 2 h at 37°C. Duplicate serum samples at four-fold dilutions in PT were added to the wells and incubated for 18 h at 4 ° C. After three additional washes, peroxidase-labeled rabbit anti-bovine IgG conjugate (Bio-Makor, Israel), diluted 1 : 2000 in P T 0.2% BSA, was added and the plate incubated for 2 h at 37 ° C. An additional wash was carried out, followed by addition of the substrate, consisting of 1 m g / m l of azino di-[3-ethyl-benzthiazoline] sulfonate in a solution containing 0.1 M citric acid, 0.2 M phosphate buffer, pH 4.5, and 0.05% hydrogen peroxide. All reagents were in 100-~1 aliquots per well. The reaction was allowed to proceed for 1 h at room temperature, and the absorbance values were read at 405 n m on an ELISA reader (microplate autoreader EL309, BioTek Instruments).
IFA procedure Frozen antigen-coated slides were allowed to thaw for 30 min at 37 °C in a container supplied with silica gel. Circles were marked on the slides with nail polish. Four-fold dilutions of serum samples in PBS were applied to the slides
48
V. SHKAP ET AL.
in duplicate, and incubated for 30 min at 37°C in a humid chamber. The slides were washed twice in PBS, dried between filter paper and allowed to react for 30 min at 37°C with rabbit anti-bovine IgG fluorescein conjugate (Bio-Makor) diluted 1:75. Slides were washed and dried as before, covered with PBS-glycerol ( 1 : 1 ) and examined under a fluorescence microscope. In both ELISA and IFA assays, the optimal dilutions obtained and subsequently used were those exhibiting the best resolution between reference sera resulting from checkerboard titrations (Voller et al., 1976). Results of the ELISA were expressed as the highest dilution of serum producing an optical density (O.D.) at 405 nm higher by 2 s.d. than that obtained with negative control serum at the same dilution. The endpoint in IFA was the highest dilution of serum still exhibiting fluorescence with the Anaplasma organisms.
Statistics Data were analysed by Student's paired t-test (Weisbrot, 1985 ). RESULTS Coomassie-stained protein bands obtained from Anaplasma-infected or uninfected erythrocytes and separated by SDS-PAGE are shown in Fig. 1. Multiple bands were observed in the preparations of isolated initial bodies, while only a few bands, albeit weakly stained, could be detected in the material obtained from uninfected erythrocytes. The optimal concentration of the coating antigen was determined from the dosage-dependent absorbance curves (Fig. 2). The highest resolution between positive and negative sera was obtained at an antigen concentration of 5 pg/ml. Similar O.D. readings were recorded under the same experimental conditions with increasing concentrations of antigen. The optimal working dilution of the conjugate chosen was 1 : 2000, as determined at a constant concentration of antigen (5 # g / m l ) and by means of positive and negative reference sera (data not shown). ELISA titration curves (Fig. 3) were established from the mean absorbance reading obtained by the reactivity of both A. centrale and A. marginale antigens with 24 negative serum samples collected from imported cattle not previously exposed to Anaplasma, 11 anti-A, centrale and seven anti-A, marginale reference positive sera. The mean O.D. value of negative sera at the lowest serum dilution ( 1 : 64 ) was less than 0.310 with either antigen. In the homologous A. centrale system mean O.D. readings were five times higher than the mean negative control at a serum dilution of 1 : 64, four times higher at 1:256, and three times higher at 1 : 1024. The mean O.D. readings of the positive sera in the A. marginale homologous system were twice the negative serum values at 1:64 and 1:256, but only 1.5 times higher at 1 : 1024. The same sera tested by IFA showed 8.3% false positive reactions at 1 : 64.
AN ELISA FOR A. CENTRALE AND A. MARGINALE
MAc
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49
Am Ni
-
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-
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Fig. 1. SDS-PAGE ofA. centrale (Ac), A. rnarginale (Am) and uninfected (Ni) erythrocytes stained with Coomassie Brilliant Blue G-250. M, Molecular weight standard markers ( X 10-3).
Similar results were obtained by ELISA and IFA run in parallel using A. centrale or A. marginale antigens (Figs. 4A and B). Results of 142 serum samples obtained from cattle vaccinated with A. centrale and collected at times ranging from 1 week to 1 year after vaccination showed no statistically significant difference between the two tests when tested with the A. centrale antigen (t--0.277, which is much lower than the critical value of 1.980). Twenty serum samples (13.8%) with titers up to 1:256 by IFA were negative by ELISA, while only three sera (2.06%) negative by IFA were positive by ELISA (Fig. 4A ). Similar results were recorded when a total of 48 samples were tested with A. marginale antigen (Fig. 4b). Fig. 5 shows cross-reactions obtained by both ELISA and IFA employing A. centrale and A. rnarginale antigens on 96 previously calibrated positive
50
V. S H K A P ET AL.
1.2 C:E 1.0
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~~ 0 . 4
d
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~ °
o
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Fig. 2. Standardization of antigen concentration for coating ELISA microplates. Positive serum at 1:64 ( - 0 - ) and 1:256 ( - O - ) dilutions. Negative serum diluted 1:64 ( - O - ) and 1:256
(-@-). 1.8
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il
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Fig. 3. Mean absorbance readings (O.D. at 405 nm) for A. centrale (Ac) and A. marginale (Am) antigens reacting with reference Ac-positive ([]), Am-positive ( [] ) and negative ( [] ) sera in ELISA.
sera collected from cattle vaccinated against A. centrale. Parallel results were obtained at 1 : 64 and 1:256 by both methods, while at 1 : 1024 dilution 0.8% o f the sera were positive by IFA with the heterologous A. marginale antigen. Negative reactions against both A. centrale and A. marginale antigens were obtained with sera having positive titers to B. besnoiti and T. gondii (up to 1:4000 and 1:256, respectively). In contrast, sera from cattle with titers up to 1 : 1024 against T. annulata, B. ovis and B. bigemina, gave slightly higher O.D. readings than the m e a n negative control value at 1 : 64 (0.350 vs. 0.315 ), but these were still low when c o m p a r e d with the ELISA values obtained with the positive reference sera shown in Fig. 3.
AN ELISA FORA. CENTRALE A N D A. MARGINALE
(n = 142)
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51
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Fig. 4. Comparison between IFA and ELISA titers using A. centrale (A) and A. marginale (B) antigens. Serum samples were collected, irrespective of age, from cattle on a farm where routine vaccination with A. centrale organisms was performed annually in 6- to 8-month-old animals. A
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RECIPROCAL ANTIBODY TITER
Fig. 5. Percentage of cross-reactivity obtained between A. centrale-positive post-vaccination sera (n = 96 ), homologous A. centrale ( [] ) and heterologous A. marginale ( [] ) antigens by ELISA (A) and IFA (B). DISCUSSION I n this s t u d y we d e m o n s t r a t e t h e v a l i d i t y o f t h e E L I S A c o m p a r e d w i t h I F A f o r t h e s e r o d i a g n o s i s o f a n a p l a s m o s i s . T h e E L I S A offers a n u m b e r o f a d v a n -
52
V. SHKAP ET AL.
tages. It requires minute amounts of antigen, and the results are objective and recorded automatically, thus facilitating large-scale field screening and vaccination follow-up. The specificity of the assay was assessed by lack of reactivity exhibited by sera from cattle infected with Besnoitia or Toxoplasma, although some absorbance background was recorded with positive sera to such blood protozoans as Theileria and Babesia. This was due to the presence of residual erythrocytic impurities accompanying the antigenic material, as seen with preparations of uninfected erythrocytes run on SDS-PAGE (Fig. 1 ). However, the O.D. values obtained were slightly higher than the mean of negative controls and are therefore negligible. Similar nonspecific reactivity with erythrocytic material in various serological tests for detection of anti-Anaplasma antibodies were reported by Montenegro et al. ( 1981 ), MontenegroJames et al. (1985) and Barry et al. (1986). Coating of ELISA plates with known amounts of antigen, based on protein estimation, enables better standardization of the test than can be obtained with IFA, in which preparation of blood smears with unevenly spread infected erythrocytes might impede reliable and objective interpretation. Indeed, we observed a total of 8.3% false positive reactions by IFA at 1 : 64, which is similar to results reported by Gonzales et al. (1978). We consider the relatively high number of positive reactions obtained by IFA on field sera to reflect a subjective bias in the evaluation of fluorescence; this is the main disadvantage of the test. Although there are conflicting reports regarding the degree of sensitivity obtained by the complement fixation, IFA, card agglutination and plate agglutination tests (Wilson et al., 1978; Gonzales et al,. 1978; Goff et al,. 1985; MontenegroJames et al., 1985), our results indicate a similar degree of sensitivity for ELISA and IFA. The antigenic relationship between A. margina[e and A. centrale was further substantiated in this study by cross-reaction experiments carried out by ELISA and IFA. Specific antisera exhibited higher homologous reactivity at higher dilutions (Fig. 5 ). Thus, when a serum tested against both Anaplasma antigens exhibits a higher end-point to A. marginale than to A. centrale, one can assume that the animal has contracted an infection with A. marginale. A similar degree of reactivity towards both antigens at high serum dilutions may also be indicative of natural infection with A. marginale before or after vaccination with A. centrale. Specific antibodies towards A. centrale or A. marginale as infecting parasites could not be detected, despite using homologous antigens. In a similar study, Amerault and Roby ( 1971 ) reported that postvaccination antibodies could not be distinguished from those produced by natural infection using CF or CA tests, and Barry et al. (1986) did not observe differential reactivity betwee~ antisera to A. centrale or A. marginale in the microplate ELISA employing an A. marginale antigen. It therefore appears that further investigation is n~eded to obtain clear-cut identification of antibodies produced by either natural infection or by vaccination.
AN ELISA FOR A. CENTRALE AND A. MARGINALE
53
REFERENCES Amerault, T.E. and Roby, T.O., 1971. Card agglutination and complement-fixation reactions after vaccination of cattle against anaplasmosis. J. Am. Vet. Med. Assoc., 159:1749-1751. Barry, D.N., Parker, R.J., De Vos, A.J., Dunster, P. and Rodwell, B.J., 1986. A microplate enzyme-linked immunosorbent assay for measuring antibody to Anaplasma marginale in cattle serum. Aust. Vet. J., 63: 76-79. Bigalke, R., 1980. The control of the ticks and tick-borne diseases of cattle in South Africa. Zimbabwe Vet. J., 11: 20-22. Callow, L., 1976. Tick-borne livestock diseases and their vectors. III. Australian methods of vaccination against anaplasmosis and babesiosis. World Anim. Rev., 18:9-15. Duzgun, A., Schuntner, C., Wright, I., Leatch, G. and Waltisbuhl, D., 1988. A sensitive ELISA technique for the diagnosis ofAnaplasma marginale infections. Vet. Parasitol., 29: 1-7. Goff, W.L., Johnson, B.S. and Kuttler, K.L,. 1985. Development of an indirect fluorescent antibody test, using microfluometry as a diagnostic test for bovine anaplasmosis. Am. J. Vet. Res., 46: 1080-1084. Gonzales, E.F., Long, R.F. and Todorovic, R.A., 1978. Comparisons of the complement fixation, indirect fluorescent antibody, and card agglutination test for the diagnosis of bovine anaplasmosis. Am. J. Vet. Res., 39: 1538-1541. Kuttler, K.L. and Winward, L.D., 1984. Serologic comparison of 4 Anaplasma isolates as measured by the complement-fixation test. Vet. Microbiol., 9:181-186. Montenegro-James, S., James, M.A. and Ristic, M., 1985. Modified indirect fluorescent antibody test for the serodiagnosis ofAnaplasma marginale infections in cattle. Am. J. Vet. Res., 46: 2401-2403. Montenegro, S., James, M.A., Levy, M.G., Preston, H.D., Esparza, H. and Ristic, M., 1981. Utilization of culture-derived soluble antigen in the latex agglutination test for bovine babesiosis and anaplasmosis. Vet. Parasitol., 8:291-297. Nakamura, Y., Shimizu Shinya, Minami, T. and Ito S. 1988a. Enzyme-linked immunosorbent assay using solubilized antigen for detection of antibodies to Anaplasma marginale. Trop. Anita. Health Prod., 20: 259-266. Nakamura, Y., Shimizu Shinya, Minami, T. and Ito S., 1988b. Enzyme-linked immunosorbent assay for detection of antibodies to Anaplasma centrale. Jpn. J. Vet. Sci., 50: 933-935. Palmer, G.H. and McGuire, T.C., 1984. Immune serum against Anaplasma marginale initial bodies neutralizes infectivity for cattle. J. Immunol., 133:1010-1015. Pipano, E., Klinger, I. and Weisman, Y., 1972. Application of the capillary agglutination test for anaplasmosis in cattle vaccinated with Anaplasma centrale. Refu. Vet., 29:166-169. Pipano, E., 1980. Bovine anaplasmosis and its control. In: Mayer, E.. (Editor), Proceedings of an International Congress on Diseases of Cattle. Bregman Press, Israel, pp. 720-726. Ristic, M., 1968. Anaplasmosis. In: Weinman, D. and Ristic, M. (Editors), Infectious Blood Diseases of Man and Animals. Academic Press Inc., New York, pp. 478-542. Thoen, C.O., Blackburn, B., Mills, K., Lomme, J. and Marry, P., 1980. Enzyme-linked immunosorbent assay for detecting antibodies in cattle in a herd in which anaplasmosis was diagnosed. J. Clin. Microbiol., 11: 499-502. Voller, A., Bidwell, D.F., Bartlett, A., Fleck, D.G., Perkins, M. and Oladehin, B., 1976. A microplate enzyme-immunoassay for Toxoplasma antibody. J. Clin. Pathol., 29:150-153. Weisbrot, I.M. (Editor), 1985. Statistics for the Clinical Laboratory. Lippincott, J.B., Company, East Washington Square, Philadelphia, PA, 198 pp. Wilson, A.J., Trueman, K.F., Spinks, G. and McSorbey, F., 1978. A comparison of 4 serological tests in the detection ofhumoral antibodies to anaplasmosis in cattle. Aust. Vet. J., 54: 383386. Winkler, G.C., Brown, G.M. and Lutz, H., 1987. Detection of antibodies to Anaplasma marginale by an improved enzyme-linked immunosorbent assay with sodium dodecyl sulfate-disrupted antigen. J. Clin. Microbiol., 25: 633-636.
