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Development and validation of an indirect enzyme immunoassay for detection of antibody to Anaplasma marginale in bovine sera
veterinary parasitology ELSEVIER
Veterinary Parasitology67 (1996) 133-142
Development and validation of an indirect enzyme immunoassay for detection of antibody to Anaplasma marginale in bovine sera K. Nielsen
a,.
, P. Smith a , D. Gall a S.T. de Eshaide b9 G. Wagner c, A. Dajer d
aAgriculture and Agri-Food Canada, Animal Diseases Research Institute, 3851 FallowfieMRoad, Nepean, Ont., Canada K2H 8P9 b INTA, CC 22, CP 2300, Rafaela, Santa Fe, Argentina c Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A & M University, College Station, TX 77843, USA a College of Veterinary Medicine, University of Yucatan, Merida, Mexico
Received 12 February 1996; accepted 16 May 1996
Abstract An indirect enzyme immunoassay (IELISA) for detection of bovine antibody activity to Anaplasma marginale was developed. This assay used a crude antigen prepared from erythrocytes of infected calves, immobilized in a polystyrene matrix and a mouse monoclonal antibody to bovine IgG 1, conjugated with horseradish peroxidase. Negative sera (n = 1842) were tested and the diagnostic specificity was 98.4 + 0.6% before retesting 29 positive samples. After retesting, eight samples remained positive and the specificity was calculated to be 99.6 + 0.3%. The diagnostic sensitivity, using 831 serum samples collected from naturally or experimentally infected cattle in Argentina, 370 from Mexico and 525 sera from experimentally vaccinated or infected cattle from Texas, was 87.3 +__1.6%. Keywords: Anaplasma marginale; Anaplasmosis;ELISA; Serology; Validation
1. Introduction Anaplasmosis is an acute to chronic infection of ruminants caused by rickettsia of the genus Anaplasma, with Anaplasma marginale being the most common. It is a disease
* Correspondingauthor. 0304-4017/96/$15.00 Copyright © 1996 Elsevier Science B.V. All fights reserved. Pll S0304-4017(96)01042-4
134
K. Nielsen et al. / Veterinary Parasitology 67 (1996) 133-142
most common to tropical and subtropical areas and is exotic to Canada. However, it is necessary for Agriculture and Agri-Food Canada to maintain a surveillance program for this disease and to be in a state of readiness for its detection should an outbreak occur. The complement fixation test is currently used for diagnosis. However, because of problems with antigen preparations and its lack of sensitivity, a suitable alternative serological test was sought. Anaplasmosis in the acute state can be diagnosed by microscopic examination of blood smears and visual detection of the parasite. Parasitemia is frequently low in subclinical and chronic infections. It is possible to detect the presence of the organism by nucleic acid probes as well (Eriks et al., 1989; Goff et al., 1988; Shompole et al., 1989; Goff et al., 1990; Aboytes-Torres and Buening, 1990). Presumptive diagnostic tests have been developed to measure antibody activity in serum. The tests most frequently used are agglutination, complement fixation, immunofluorescence tests and enzyme immunoassay. Initially, a non-modified card test was used, however, because of its relative insensitivity (Gonzalez et al., 1978), a modified card agglutination test was developed (Todorovic et al., 1977). The modified card test was found to be more sensitive than the complement fixation test, but was less specific (Todorovic et al., 1977; Maas et al., 1986). The lack of sensitivity of the complement fixation test was thought to be in part due to its inability to detect isotypes of antibody other than IgGl (McGuire et al., 1978) which is probably the reason it fails to detect animals in the carrier state (Goff et al., 1990; Luther et al., 1980). While the indirect fluorescent antibody test showed good correlation with a nucleic acid probe (Goff et al., 1990), it is generally considered to be of relatively low specificity (Gonzalez et al., 1978). A number of enzyme immunoassays have been described (Thoen et al., 1980; Barry et al., 1986; Winkler et al., 1987; Nakamura et al., 1988; Dugzun et al., 1988; Montenegro-James et al., 1990; Shkap et al., 1990), using a variety of antigen preparations and several different detection systems. In general, the enzyme immunoassays are considered to be more sensitive and specific than the conventional assays for antibody, especially when detecting antibody in the carrier state of the disease. Therefore, it was decided that an enzyme immunoassay would be the most reasonable alternative to the complement fixation test as an assay for detection of antibody activity. The purpose of the present investigation was to develop and validate an indirect enzyme immunoassay which uses the complement fixation antigen and a monoclonal anti-bovine IgG 1 antibody, conjugated with horseradish peroxidase.
2. Materials and methods
Sera: Sera collected from across Canada (n = 1842) were used as negative samples. Except for portions in the west of the country (the last case was diagnosed in 1979), anaplasmosis has not been diagnosed in Canada. These sera were therefore presumed to be negative for antibody to A. marginale. Positive samples were obtained from animals either naturally or experimentally infected with A. marginale (demonstrated by microscopic observation) from Argentina (n = 831) and Mexico (n = 370). Sera from Texas (n = 525) originated from vaccina-
K. Nielsen et al. / Veterinary Parasitology 67 (1996) 133-142
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tion experiments from animals either vaccinated or infected with A. marginale. All sera were stored at - 2 0 ° C until tested. Sera from Argentina were pretreated with one half volume of 0.2 M glycine-HCl buffer, pH 2.5 for 18 to 24 h and then neutralized with the same volume of 0.3 M tfis-HCl buffer, pH 7.2, giving a dilution of 1:3 of the serum. This predilution was considered when test dilutions were prepared. Antigen: (Antigen was prepared and supplied by NVSL, USDA, Ames, IO). An A. marginale stabilate, stored in liquid nitrogen, was used to infect an 80-100 kg splenectomized Holstein-Fresian calf. Parasitemia was monitored and when 30% parasitemia was reached (about 30 days), the animals was exsanguinated and the washed and packed erythrocytes injected into a second splenectomized calf. When parasitemia reached 50%, the second calf was exsanguinated and the washed and packed erythrocytes injected into several splenectomized calves. Parasitemia was monitored and when 75% was reached (in about 5 days), erythrocytes were harvested. Washed cells (50% in saline) were processed in a French pressure cell at 1200 PSI. The cell lysate was centrifuged at 16000 g and the sediment resuspended in saline and homogenized in a tissue grinder. A Giemsa stain was performed to ensure that the erythrocytes were lysed. The homogenate was again centrifuged at 16 000 g and the pellet homogenized in a tissue grinder. The antigen was again centrifuged at 16 000 g and the pellet resuspended in 0.2 M acetate buffer, pH 5.0 containing 0.15 M NaC1 and 0.1% methyi-p-hydroxybenzoate. The antigen concentration was adjusted to 3.5 to 5% v / v . The antigen preparation was stored at 4°C. Modified card agglutination test: Kits containing antigen, bovine serum factor and all necessary equipment were generously supplied by the USDA. Complement fixation test: Antigen prepared as above was used to test doubling dilutions of serum in the presence of three hemolytic units of guinea pig serum. The mixture was incubated at 37°C for 1 h in a 96 well plate. An indicator system of sheep erythrocytes sensitized with rabbit antibody was added and hemolysis assessed visually. Enzyme immunoassay procedure: Polystyrene 96 well plates (NUNC 2-69620, GIBCO-BRL, Burlington, Ont.) were coated with 100/.~l of antigen diluted 1:8000 in 0.1 M phosphate buffer, pH 6.3 and containing 0.15 M NaCl and 7.5 mM of each of ethylenodiamino N,N,N',N',tetraacetic acid disodium salt (EDTA) and ethylene glycol bis(beta-aminoethyl ether)N,N,N',N', tetraacetic acid (EGTA) ( P B S / E D T A / E G T A ) . Plates were sealed and incubated at 4°C overnight and then frozen at - 2 0 ° C . For use, plates were thawed at 37°C for 1 h and allowed to equilibrate to room temperature. Several batches of antigen coated plates were stored at - 20°C for up to 12 months and tested at intervals to ascertain stability of the antigen. After four wash cycles using 0.1 M phosphate buffer, pH 7.2 and containing 0.15 M NaC1 and 0.05% Tween 20 (PBST), 100ul of serum, diluted l:100 in P B S / E D T A / E G T A was added to duplicate wells. Control sera, also diluted 1:100 in P B S / E D T A / E G T A were added to quadruplicate wells. A strongly positive serum that gave an absorbance of approximately 1.0 after 10 min of incubation with the substrate/chromogen was selected (AMC + + ). A serum found to give a low level non-specific interaction in the ELISA was included as it was recognized that if this serum gave an absorbance value above a certain level, the assay was not functioning properly (AMC + ). This serum later replaced the AMC - and a positive serum with a
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low antibody level was used as the AMC + . A negative serum ( A M C - ) was used initially and a buffer control containing no serum (AMCc) was also included. Incubation proceeded for 1 h at 25°C. After four further wash cycles with PBST, 100 pA of a mouse monoclonal antibody, specific for an epitope of bovine IgGl conjugated with horseradish peroxidase, and appropriately diluted in PBST, was added to each well for 1 h at 25°C. After an additional four wash cycles with PBST, 75ul of TMB substrate/chromogen solution were added to each well with constant shaking. After l0 min of development, 75 /xl of 1 M H 2 S O 4 w e r e added to each well to stop the reaction and optical densities were assessed at 450 nm in a spectrophotometer. Data: Software for the analysis of the data was developed. This software converted optical density measurements to percent positivity of the positive control serum (AMC + + ) by the following formula: mean absorbance of test sample × 100 %P=
mean absorbance o f A M C + +
In addition, the software kept track of all the average percent positivity values of the four controls. In the complement fixation test, the reciprocal of the final dilution to result in 50% lysis of the sensitized erythrocytes was considered the titer of the serum. A titer of 5 was considered significant. In the card agglutination test, a serum was considered positive if visible agglutination of the antigen occurred in 4 min. Test specificity was determined using: true negative observations × 100 % specificity =
true negative 4- false positive observations "
Test sensitivity was calculated using: true positive observations × 100 % sensitivity =
true positive 4- false negative observations "
Receiver Operator Characteristics (ROC analysis) were calculated using Systat (Stenson, 1988).
3. Results Low molarity, alkaline carbonate buffer, classically used for passive attachment of antigens to polystyrene could not be used with the A. marginale antigen as the stability of antigen coated plates deteriorated in less than 2 weeks. This was detected by a
Fig. 1. Frequencydistribution of 1842 Canadian sera (considered to be negative for antibody to A. marginale, open bars) and samples from animals exposed to A. marginale either by vaccinationor infection (closed bars). The X-axis represents the IELISA results in 10% increments of positivity and the Y-axis represents the number of sera in each increment. The positive sera were modified card test positive. (A) Positive samples from Argentina. (B) Positive samples from Mexico. (C) Positive samples from Texas.
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K. Nielsen et al./Veterinary Parasitology 67 (1996) 133-142
decrease in the AMC + + percent positivity and an increase in both the AMC + and the AMC - , but mostly in the former. This would, therefore, give rise to false positive reactions. When the antigen was suspended in buffer at a neutral pH, the stability
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K. Nielsen et al. / Veterinary Parasitology 67 (1996) 133-142
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Table 1 Results of testing a total of 1726 serum samples originating from cattle vaccinated against or infected with Anaplasma marginale in the modified card test (MCT), the complement fixation test (CFT) and the indirect enzyme immunoassay (1ELISA) MCT
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6 24 190 220
99.3 93.5 63.8 87.3
improved and no deviations were noted over a 9 month test period. Addition of divalent cation chelators (EDTA and EGTA) to the antigen-coating buffer, reduced non-specific (or specific interaction through erythrocyte isoantigen-antibody complex formation) serum protein interaction. The negative cut-off was established using the 99th percentile of the sorted data of the 1842 negative serum samples. The cut-off was set at 29% P. From these sera, only eight false positive reactions remained, resulting in an assay specificity of 99.6 + 0.3%.
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K. Nielsen et al. / Veterinary Parasitology 67 (1996) 133-142
Table 2 Result of testing a total of 1842 Canadian sera (assumed to be negative for antibody to A. marginale) in the indirect ELISA, the MCT and the CFT n
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a 160 randomly selected sera from the negative group were tested by the modified card agglutination and the complement fixation tests. Anticomplementary sera were considered positive b or negative c. The specificity o f the m o d i f i e d card agglutination and the c o m p l e m e n t fixation tests was also d e t e r m i n e d , by using a r a n d o m l y selected subset o f the n e g a t i v e sera. T h e s e data are presented in T a b l e 2. Sensitivity values w e r e d e t e r m i n e d using the m o d i f i e d card agglutination test, the c o m p l e m e n t fixation test and the I E L I S A using k n o w n e x p o s u r e (natural, e x p e r i m e n t a l or vaccinal) or o b s e r v a t i o n o f the causative o r g a n i s m in b l o o d smears as the reference for positivity. T h e s e data are tabulated in T a b l e 1. F r e q u e n c y distributions using the 1842 n e g a t i v e sera and the positive sera f r o m A r g e n t i n a , M e x i c o and T e x a s individually, are presented in Fig. 1A, B and C. Plots o f
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Fig. 3. Quality control data for the IELISA for antibody to A. marginale. AMC + + represents data for the strong positive serum sample used to calculate the percent positivity for all data. AMC + is a weak positive serum selected to give results just above the cut-off value selected. A M C - is a negative serum control and AMCc is the buffer control. Each point represents the average of four determinations on a single 96 well plate. The solid lines represent + or - two standard deviations from the mean.
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K, Nielsen et a L / Veterinary Parasiwlogy 67 (1996) 133-142
the ROC analysis are presented in Fig. 2A, B and C for the same data. Fig. 3 represents the quality control data for the strong positive, the weak positive and the negative sera as well as the buffer control for each of 60 plates tested (except for the weak positive serum for which data from 20 plates were available due to a change in the serum used).
4. Discussion
Based on a cut-off value of 29% positivity of a strong positive control serum, AMC + +, the indirect ELISA procedure described was both sensitive and specific (87.3% and 99.6%, respectively, Tables 1 and 2). The assay was developed and validated on a large number of negative sera as well as a substantial number of sera from A. marginale exposed cattle. Frequency distributions and ROC analysis of the data are presented in Fig. 1 and Fig. 2. Fig. 1A showed some overlap between negative and positive (from Argentina) results and this is confirmed with the data derived from sera obtained from Mexico and Texas (Fig. 1B and Fig. 1C). This makes it difficult to establish an exact cut-off value. From the ROC analysis, the diagonal intercept for the negative and sera from Argentina was 23% positivity (Fig. 2A). Based on the sera from Canada and those from Mexico (Fig. 2B) or Texas (Fig. 2C) sera, the diagonal intercept was at 19 and 9.5%, respectively. If these values were selected, the assay specificity would be too low for Canadian requirements. Therefore, a cut-off of 29% positivity was selected to maximize specificity, but with some loss in sensitivity, especially with the sera from Texas. The overall sensitivity and specificity values are in agreement with those published by Montenegro-James et al., 1990; Dugzun et al., 1988 and Barry et al., 1986. The results obtained with the complement fixation test were unexpectedly low, however, the modified card test, as supplied by the USDA, gave excellent results for sensitivity 89.5%) and specificity (97.5%). In the previous studies, the complement fixation test was found to give a specificity of 96.2 to 99.9% and a sensitivity of 66.7%. In the current study, the sensitivity ranged from 64.5 to 26.7% and the specificity was 88.8% or 100% (if anticomplementary sera were considered positive or negative, respectively). These data are presented in Table 1. The low sensitivity may be a result of different A. marginale isolates giving rise to antibodies that react differently with the isolate used for preparation of the antigen (Kuttler and Winward, 1984). This may also explain the variation in sensitivity and specificity of the enzyme immunoassay with the samples from Texas, compared with the samples from Argentina and Mexico. The enzyme immunoassay described utilized an antigen which was fairly simple to prepare from infected erythrocytes and did not require a great deal of manipulation. This resulted in assay characteristics very similar to those using recombinant or highly purified antigens. The antigen preparation was initially intended for the complement fixation test, however, when stored for any length of time at 4 or - 25°C, the antigen became anticomplementary and activated complement in the absence of antibody. This antigen preparation cannot be frozen in its liquid format for use in the complement fixation test or the indirect enzyme immunoassay. It was, however, noted that 96 well plates, coated with the antigen could be frozen without immediate loss of
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141
antigen activity but deteriorated over a 2-4 week period. This deterioration resulted in an increase in non-specific activity of some sera and therefore false positive reactions. This observation resulted in the addition of a non-specifically binding serum, the AMC + , as a control that ensured non-specific reactivity remained at a minimum, below 20%P. Also, the use of a phosphate buffer containing divalent cation chelating agents as a coating buffer for the antigen resulted in antigen stability in frozen plates of at least 12 months and also reduced non-specific interaction with serum proteins. The remainder of the indirect enzyme immunoassay followed a standardized format except that divalent cation chelators were also incorporated into the serum diluent. This reduced non-specific binding of proteins to the antigen and resulted in fewer false positive reactions.
Acknowledgements The authors wish to acknowledge the generosity of David Olsen NSVL for the procedure for antigen preparation. We also wish to acknowledge the USDA for providing the kits for performing the modified card agglutination test. Some of the antigen used in this study was prepared by K. Malkin, ADRI. The authors wish to thank J. Bosse and W. Kelly for their valuable contributions to this work.
References Aboytes-Torres, R. and Buening, G.M., 1990. Development of a recombinant Anaplasma marginale DNA probe. In: P.S. Paul (Editor), Nucleic Acid Probes, 24: 391-408. Barry, D.N., Parker, R3., De Vos, A.J., Dunster, P. and RodweU, BJ., 1986. A microplate enzyme linked immunosorbent assay for measuring antibody to Anaplasma marginale in cattle serum. Austral. Vet. J., 63: 76-79. Dugzun, A., Schuntner, C.A., Wright, I.G., Lentch, G. and Waltisbuhl, DJ., 1988. A sensitive ELISA method for the diagnosis of Anaplasma marginale infections. Vet. Parasitol., 29: 1-7. Eriks, I.S., Palmer, G.H., McGuire, T.C., Ailred, D.R. and Barbet, A.F., 1989. Detection and quantitation of Anaplasma marginale in carrier cattle by using a nucleic acid probe. J. Clin. Microbiol., 27: 279-284. Goff, W.L., Barbet, A.F., Stiller, D., Palmer, G.H., Knowles, D.P., Kocan, K.M., Gorham, J.R. and McGuire, T.C., 1988. Detection of Anaplasma marginale-infected tick vectors by using a cloned DNA probe. Proc. Nat. Acad. Sci. USA, 85: 919-923. Goff, W.L., Stiller, D., Roeder, R.A., Johnson, L.W., Falk, D., Gotham, J.R. and McGuire, T.C., 1990. Comparison of a DNA probe, complement fixation and indirect immonofluorescence tests for diagnosing Anaplasma marginale in suspected carrier cattle. Vet. Microbiol., 24: 381-390. Gonzalez, E., Long, R. and Todorovic, R., 1978. Comparisons of the complement fixation, indirect fluorescent antibody and card agglutination tests for the diagnosis of bovine anaplasmosis. Am. J. Vet. Res., 39: 1538-1541. Kuttler, K. and Winward, L., 1984. Serologic comparison of four Anaplasma isolates as measured by the complement fixation test. Vet. Microbiol., 9:181-186. Luther, D.G., Cox, H.U. and Nelson, W.O., 1980. Comparisons of serotests with calf inoculations for detection of carriers in anaplasmosis-vaceinated cattle. Am. J. Vet. Res., ,11: 2085-2086. Maas J., Lincoln, S.D., Coan, M.E., Kuttler, K.L., Zaug, J.D. and Stiller, D., 1986. Epidemioiogic aspects of bovine anaplasmosis in semiarid range conditions of south central Idaho. Am. J. Vet. Res., 47: 528-533. MeGuire, T.C., Musoke, A.J. and Kurtti, T., 1978. Functional properties of bovine lgG I and IgG2: Interaction with complement, macrophages, neutrophils and skin. Immunol., 38: 249-256.
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Montenegro-James, S., Guillan, A.T., Ma, S.J., Tapang, P., Abdel-Gawad, A., Toro, M. and Ristic, M., 1990. Use of the DOT enzyme linked immunosorbent assay with isolated Anaplasma marginale initial bodies for serodiagnosis of anaplasmosis in cattle. Am. J. Vet. Res., 51: 1518-1521. Nakamura, Y., Shimizu, S., Minami, T. and Ito, S., 1988. Enzyme-linked immunosorbent assay for detection of antibodies to Anaplasrna centrale. Japan. J. Vet. Sci., 50: 933-935. Shkap, V., Bin, H., Ungar-Waron, H. and Pipano, E., 1990. An enzyme linked immunosorbent assay (ELISA) for detection of antibodies to Anaplasma centrale and Anaplasma marginale. Vet. Microbiol., 25: 45-53. Shompole, S., Waghela, S.D., Rurangirwa, F.R. and McGuire, T.C., 1989. Cloned DNA probes identify Anaplasma ovis in goats and reveal high prevalence of infection. J. Clin. Microbiol., 27: 2730-2735. Stenson, H., 1988. SIGNAL: a supplementary module for SYSTAT and SYSGRAPH. SYSTAT Inc., Evanston, II. Thoen, C.O, Blackburn, B., Mills, K., Lomme, J. and Hopkins, M.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. Todorovic R., Long, R. and McCallon, B., 1977. Comparison of the rapid card agglutination test with the complement fixation test for the diagnosis of Anaplasma marginale infection in cattle in Colombia. Vet. Microbiol., 2: 167-177. 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. Micrnbiol., 25: 633-636.
Veterinary Parasitology67 (1996) 133-142
Development and validation of an indirect enzyme immunoassay for detection of antibody to Anaplasma marginale in bovine sera K. Nielsen
a,.
, P. Smith a , D. Gall a S.T. de Eshaide b9 G. Wagner c, A. Dajer d
aAgriculture and Agri-Food Canada, Animal Diseases Research Institute, 3851 FallowfieMRoad, Nepean, Ont., Canada K2H 8P9 b INTA, CC 22, CP 2300, Rafaela, Santa Fe, Argentina c Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A & M University, College Station, TX 77843, USA a College of Veterinary Medicine, University of Yucatan, Merida, Mexico
Received 12 February 1996; accepted 16 May 1996
Abstract An indirect enzyme immunoassay (IELISA) for detection of bovine antibody activity to Anaplasma marginale was developed. This assay used a crude antigen prepared from erythrocytes of infected calves, immobilized in a polystyrene matrix and a mouse monoclonal antibody to bovine IgG 1, conjugated with horseradish peroxidase. Negative sera (n = 1842) were tested and the diagnostic specificity was 98.4 + 0.6% before retesting 29 positive samples. After retesting, eight samples remained positive and the specificity was calculated to be 99.6 + 0.3%. The diagnostic sensitivity, using 831 serum samples collected from naturally or experimentally infected cattle in Argentina, 370 from Mexico and 525 sera from experimentally vaccinated or infected cattle from Texas, was 87.3 +__1.6%. Keywords: Anaplasma marginale; Anaplasmosis;ELISA; Serology; Validation
1. Introduction Anaplasmosis is an acute to chronic infection of ruminants caused by rickettsia of the genus Anaplasma, with Anaplasma marginale being the most common. It is a disease
* Correspondingauthor. 0304-4017/96/$15.00 Copyright © 1996 Elsevier Science B.V. All fights reserved. Pll S0304-4017(96)01042-4
134
K. Nielsen et al. / Veterinary Parasitology 67 (1996) 133-142
most common to tropical and subtropical areas and is exotic to Canada. However, it is necessary for Agriculture and Agri-Food Canada to maintain a surveillance program for this disease and to be in a state of readiness for its detection should an outbreak occur. The complement fixation test is currently used for diagnosis. However, because of problems with antigen preparations and its lack of sensitivity, a suitable alternative serological test was sought. Anaplasmosis in the acute state can be diagnosed by microscopic examination of blood smears and visual detection of the parasite. Parasitemia is frequently low in subclinical and chronic infections. It is possible to detect the presence of the organism by nucleic acid probes as well (Eriks et al., 1989; Goff et al., 1988; Shompole et al., 1989; Goff et al., 1990; Aboytes-Torres and Buening, 1990). Presumptive diagnostic tests have been developed to measure antibody activity in serum. The tests most frequently used are agglutination, complement fixation, immunofluorescence tests and enzyme immunoassay. Initially, a non-modified card test was used, however, because of its relative insensitivity (Gonzalez et al., 1978), a modified card agglutination test was developed (Todorovic et al., 1977). The modified card test was found to be more sensitive than the complement fixation test, but was less specific (Todorovic et al., 1977; Maas et al., 1986). The lack of sensitivity of the complement fixation test was thought to be in part due to its inability to detect isotypes of antibody other than IgGl (McGuire et al., 1978) which is probably the reason it fails to detect animals in the carrier state (Goff et al., 1990; Luther et al., 1980). While the indirect fluorescent antibody test showed good correlation with a nucleic acid probe (Goff et al., 1990), it is generally considered to be of relatively low specificity (Gonzalez et al., 1978). A number of enzyme immunoassays have been described (Thoen et al., 1980; Barry et al., 1986; Winkler et al., 1987; Nakamura et al., 1988; Dugzun et al., 1988; Montenegro-James et al., 1990; Shkap et al., 1990), using a variety of antigen preparations and several different detection systems. In general, the enzyme immunoassays are considered to be more sensitive and specific than the conventional assays for antibody, especially when detecting antibody in the carrier state of the disease. Therefore, it was decided that an enzyme immunoassay would be the most reasonable alternative to the complement fixation test as an assay for detection of antibody activity. The purpose of the present investigation was to develop and validate an indirect enzyme immunoassay which uses the complement fixation antigen and a monoclonal anti-bovine IgG 1 antibody, conjugated with horseradish peroxidase.
2. Materials and methods
Sera: Sera collected from across Canada (n = 1842) were used as negative samples. Except for portions in the west of the country (the last case was diagnosed in 1979), anaplasmosis has not been diagnosed in Canada. These sera were therefore presumed to be negative for antibody to A. marginale. Positive samples were obtained from animals either naturally or experimentally infected with A. marginale (demonstrated by microscopic observation) from Argentina (n = 831) and Mexico (n = 370). Sera from Texas (n = 525) originated from vaccina-
K. Nielsen et al. / Veterinary Parasitology 67 (1996) 133-142
135
tion experiments from animals either vaccinated or infected with A. marginale. All sera were stored at - 2 0 ° C until tested. Sera from Argentina were pretreated with one half volume of 0.2 M glycine-HCl buffer, pH 2.5 for 18 to 24 h and then neutralized with the same volume of 0.3 M tfis-HCl buffer, pH 7.2, giving a dilution of 1:3 of the serum. This predilution was considered when test dilutions were prepared. Antigen: (Antigen was prepared and supplied by NVSL, USDA, Ames, IO). An A. marginale stabilate, stored in liquid nitrogen, was used to infect an 80-100 kg splenectomized Holstein-Fresian calf. Parasitemia was monitored and when 30% parasitemia was reached (about 30 days), the animals was exsanguinated and the washed and packed erythrocytes injected into a second splenectomized calf. When parasitemia reached 50%, the second calf was exsanguinated and the washed and packed erythrocytes injected into several splenectomized calves. Parasitemia was monitored and when 75% was reached (in about 5 days), erythrocytes were harvested. Washed cells (50% in saline) were processed in a French pressure cell at 1200 PSI. The cell lysate was centrifuged at 16000 g and the sediment resuspended in saline and homogenized in a tissue grinder. A Giemsa stain was performed to ensure that the erythrocytes were lysed. The homogenate was again centrifuged at 16 000 g and the pellet homogenized in a tissue grinder. The antigen was again centrifuged at 16 000 g and the pellet resuspended in 0.2 M acetate buffer, pH 5.0 containing 0.15 M NaC1 and 0.1% methyi-p-hydroxybenzoate. The antigen concentration was adjusted to 3.5 to 5% v / v . The antigen preparation was stored at 4°C. Modified card agglutination test: Kits containing antigen, bovine serum factor and all necessary equipment were generously supplied by the USDA. Complement fixation test: Antigen prepared as above was used to test doubling dilutions of serum in the presence of three hemolytic units of guinea pig serum. The mixture was incubated at 37°C for 1 h in a 96 well plate. An indicator system of sheep erythrocytes sensitized with rabbit antibody was added and hemolysis assessed visually. Enzyme immunoassay procedure: Polystyrene 96 well plates (NUNC 2-69620, GIBCO-BRL, Burlington, Ont.) were coated with 100/.~l of antigen diluted 1:8000 in 0.1 M phosphate buffer, pH 6.3 and containing 0.15 M NaCl and 7.5 mM of each of ethylenodiamino N,N,N',N',tetraacetic acid disodium salt (EDTA) and ethylene glycol bis(beta-aminoethyl ether)N,N,N',N', tetraacetic acid (EGTA) ( P B S / E D T A / E G T A ) . Plates were sealed and incubated at 4°C overnight and then frozen at - 2 0 ° C . For use, plates were thawed at 37°C for 1 h and allowed to equilibrate to room temperature. Several batches of antigen coated plates were stored at - 20°C for up to 12 months and tested at intervals to ascertain stability of the antigen. After four wash cycles using 0.1 M phosphate buffer, pH 7.2 and containing 0.15 M NaC1 and 0.05% Tween 20 (PBST), 100ul of serum, diluted l:100 in P B S / E D T A / E G T A was added to duplicate wells. Control sera, also diluted 1:100 in P B S / E D T A / E G T A were added to quadruplicate wells. A strongly positive serum that gave an absorbance of approximately 1.0 after 10 min of incubation with the substrate/chromogen was selected (AMC + + ). A serum found to give a low level non-specific interaction in the ELISA was included as it was recognized that if this serum gave an absorbance value above a certain level, the assay was not functioning properly (AMC + ). This serum later replaced the AMC - and a positive serum with a
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low antibody level was used as the AMC + . A negative serum ( A M C - ) was used initially and a buffer control containing no serum (AMCc) was also included. Incubation proceeded for 1 h at 25°C. After four further wash cycles with PBST, 100 pA of a mouse monoclonal antibody, specific for an epitope of bovine IgGl conjugated with horseradish peroxidase, and appropriately diluted in PBST, was added to each well for 1 h at 25°C. After an additional four wash cycles with PBST, 75ul of TMB substrate/chromogen solution were added to each well with constant shaking. After l0 min of development, 75 /xl of 1 M H 2 S O 4 w e r e added to each well to stop the reaction and optical densities were assessed at 450 nm in a spectrophotometer. Data: Software for the analysis of the data was developed. This software converted optical density measurements to percent positivity of the positive control serum (AMC + + ) by the following formula: mean absorbance of test sample × 100 %P=
mean absorbance o f A M C + +
In addition, the software kept track of all the average percent positivity values of the four controls. In the complement fixation test, the reciprocal of the final dilution to result in 50% lysis of the sensitized erythrocytes was considered the titer of the serum. A titer of 5 was considered significant. In the card agglutination test, a serum was considered positive if visible agglutination of the antigen occurred in 4 min. Test specificity was determined using: true negative observations × 100 % specificity =
true negative 4- false positive observations "
Test sensitivity was calculated using: true positive observations × 100 % sensitivity =
true positive 4- false negative observations "
Receiver Operator Characteristics (ROC analysis) were calculated using Systat (Stenson, 1988).
3. Results Low molarity, alkaline carbonate buffer, classically used for passive attachment of antigens to polystyrene could not be used with the A. marginale antigen as the stability of antigen coated plates deteriorated in less than 2 weeks. This was detected by a
Fig. 1. Frequencydistribution of 1842 Canadian sera (considered to be negative for antibody to A. marginale, open bars) and samples from animals exposed to A. marginale either by vaccinationor infection (closed bars). The X-axis represents the IELISA results in 10% increments of positivity and the Y-axis represents the number of sera in each increment. The positive sera were modified card test positive. (A) Positive samples from Argentina. (B) Positive samples from Mexico. (C) Positive samples from Texas.
137
K. Nielsen et al./Veterinary Parasitology 67 (1996) 133-142
decrease in the AMC + + percent positivity and an increase in both the AMC + and the AMC - , but mostly in the former. This would, therefore, give rise to false positive reactions. When the antigen was suspended in buffer at a neutral pH, the stability
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K. Nielsen et al. / Veterinary Parasitology 67 (1996) 133-142
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Table 1 Results of testing a total of 1726 serum samples originating from cattle vaccinated against or infected with Anaplasma marginale in the modified card test (MCT), the complement fixation test (CFT) and the indirect enzyme immunoassay (1ELISA) MCT
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139
K. Nielsen et al. / Veterinary Parasitology 67 (1996) 133-142
Table 2 Result of testing a total of 1842 Canadian sera (assumed to be negative for antibody to A. marginale) in the indirect ELISA, the MCT and the CFT n
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K, Nielsen et a L / Veterinary Parasiwlogy 67 (1996) 133-142
the ROC analysis are presented in Fig. 2A, B and C for the same data. Fig. 3 represents the quality control data for the strong positive, the weak positive and the negative sera as well as the buffer control for each of 60 plates tested (except for the weak positive serum for which data from 20 plates were available due to a change in the serum used).
4. Discussion
Based on a cut-off value of 29% positivity of a strong positive control serum, AMC + +, the indirect ELISA procedure described was both sensitive and specific (87.3% and 99.6%, respectively, Tables 1 and 2). The assay was developed and validated on a large number of negative sera as well as a substantial number of sera from A. marginale exposed cattle. Frequency distributions and ROC analysis of the data are presented in Fig. 1 and Fig. 2. Fig. 1A showed some overlap between negative and positive (from Argentina) results and this is confirmed with the data derived from sera obtained from Mexico and Texas (Fig. 1B and Fig. 1C). This makes it difficult to establish an exact cut-off value. From the ROC analysis, the diagonal intercept for the negative and sera from Argentina was 23% positivity (Fig. 2A). Based on the sera from Canada and those from Mexico (Fig. 2B) or Texas (Fig. 2C) sera, the diagonal intercept was at 19 and 9.5%, respectively. If these values were selected, the assay specificity would be too low for Canadian requirements. Therefore, a cut-off of 29% positivity was selected to maximize specificity, but with some loss in sensitivity, especially with the sera from Texas. The overall sensitivity and specificity values are in agreement with those published by Montenegro-James et al., 1990; Dugzun et al., 1988 and Barry et al., 1986. The results obtained with the complement fixation test were unexpectedly low, however, the modified card test, as supplied by the USDA, gave excellent results for sensitivity 89.5%) and specificity (97.5%). In the previous studies, the complement fixation test was found to give a specificity of 96.2 to 99.9% and a sensitivity of 66.7%. In the current study, the sensitivity ranged from 64.5 to 26.7% and the specificity was 88.8% or 100% (if anticomplementary sera were considered positive or negative, respectively). These data are presented in Table 1. The low sensitivity may be a result of different A. marginale isolates giving rise to antibodies that react differently with the isolate used for preparation of the antigen (Kuttler and Winward, 1984). This may also explain the variation in sensitivity and specificity of the enzyme immunoassay with the samples from Texas, compared with the samples from Argentina and Mexico. The enzyme immunoassay described utilized an antigen which was fairly simple to prepare from infected erythrocytes and did not require a great deal of manipulation. This resulted in assay characteristics very similar to those using recombinant or highly purified antigens. The antigen preparation was initially intended for the complement fixation test, however, when stored for any length of time at 4 or - 25°C, the antigen became anticomplementary and activated complement in the absence of antibody. This antigen preparation cannot be frozen in its liquid format for use in the complement fixation test or the indirect enzyme immunoassay. It was, however, noted that 96 well plates, coated with the antigen could be frozen without immediate loss of
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antigen activity but deteriorated over a 2-4 week period. This deterioration resulted in an increase in non-specific activity of some sera and therefore false positive reactions. This observation resulted in the addition of a non-specifically binding serum, the AMC + , as a control that ensured non-specific reactivity remained at a minimum, below 20%P. Also, the use of a phosphate buffer containing divalent cation chelating agents as a coating buffer for the antigen resulted in antigen stability in frozen plates of at least 12 months and also reduced non-specific interaction with serum proteins. The remainder of the indirect enzyme immunoassay followed a standardized format except that divalent cation chelators were also incorporated into the serum diluent. This reduced non-specific binding of proteins to the antigen and resulted in fewer false positive reactions.
Acknowledgements The authors wish to acknowledge the generosity of David Olsen NSVL for the procedure for antigen preparation. We also wish to acknowledge the USDA for providing the kits for performing the modified card agglutination test. Some of the antigen used in this study was prepared by K. Malkin, ADRI. The authors wish to thank J. Bosse and W. Kelly for their valuable contributions to this work.
References Aboytes-Torres, R. and Buening, G.M., 1990. Development of a recombinant Anaplasma marginale DNA probe. In: P.S. Paul (Editor), Nucleic Acid Probes, 24: 391-408. Barry, D.N., Parker, R3., De Vos, A.J., Dunster, P. and RodweU, BJ., 1986. A microplate enzyme linked immunosorbent assay for measuring antibody to Anaplasma marginale in cattle serum. Austral. Vet. J., 63: 76-79. Dugzun, A., Schuntner, C.A., Wright, I.G., Lentch, G. and Waltisbuhl, DJ., 1988. A sensitive ELISA method for the diagnosis of Anaplasma marginale infections. Vet. Parasitol., 29: 1-7. Eriks, I.S., Palmer, G.H., McGuire, T.C., Ailred, D.R. and Barbet, A.F., 1989. Detection and quantitation of Anaplasma marginale in carrier cattle by using a nucleic acid probe. J. Clin. Microbiol., 27: 279-284. Goff, W.L., Barbet, A.F., Stiller, D., Palmer, G.H., Knowles, D.P., Kocan, K.M., Gorham, J.R. and McGuire, T.C., 1988. Detection of Anaplasma marginale-infected tick vectors by using a cloned DNA probe. Proc. Nat. Acad. Sci. USA, 85: 919-923. Goff, W.L., Stiller, D., Roeder, R.A., Johnson, L.W., Falk, D., Gotham, J.R. and McGuire, T.C., 1990. Comparison of a DNA probe, complement fixation and indirect immonofluorescence tests for diagnosing Anaplasma marginale in suspected carrier cattle. Vet. Microbiol., 24: 381-390. Gonzalez, E., Long, R. and Todorovic, R., 1978. Comparisons of the complement fixation, indirect fluorescent antibody and card agglutination tests for the diagnosis of bovine anaplasmosis. Am. J. Vet. Res., 39: 1538-1541. Kuttler, K. and Winward, L., 1984. Serologic comparison of four Anaplasma isolates as measured by the complement fixation test. Vet. Microbiol., 9:181-186. Luther, D.G., Cox, H.U. and Nelson, W.O., 1980. Comparisons of serotests with calf inoculations for detection of carriers in anaplasmosis-vaceinated cattle. Am. J. Vet. Res., ,11: 2085-2086. Maas J., Lincoln, S.D., Coan, M.E., Kuttler, K.L., Zaug, J.D. and Stiller, D., 1986. Epidemioiogic aspects of bovine anaplasmosis in semiarid range conditions of south central Idaho. Am. J. Vet. Res., 47: 528-533. MeGuire, T.C., Musoke, A.J. and Kurtti, T., 1978. Functional properties of bovine lgG I and IgG2: Interaction with complement, macrophages, neutrophils and skin. Immunol., 38: 249-256.
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Montenegro-James, S., Guillan, A.T., Ma, S.J., Tapang, P., Abdel-Gawad, A., Toro, M. and Ristic, M., 1990. Use of the DOT enzyme linked immunosorbent assay with isolated Anaplasma marginale initial bodies for serodiagnosis of anaplasmosis in cattle. Am. J. Vet. Res., 51: 1518-1521. Nakamura, Y., Shimizu, S., Minami, T. and Ito, S., 1988. Enzyme-linked immunosorbent assay for detection of antibodies to Anaplasrna centrale. Japan. J. Vet. Sci., 50: 933-935. Shkap, V., Bin, H., Ungar-Waron, H. and Pipano, E., 1990. An enzyme linked immunosorbent assay (ELISA) for detection of antibodies to Anaplasma centrale and Anaplasma marginale. Vet. Microbiol., 25: 45-53. Shompole, S., Waghela, S.D., Rurangirwa, F.R. and McGuire, T.C., 1989. Cloned DNA probes identify Anaplasma ovis in goats and reveal high prevalence of infection. J. Clin. Microbiol., 27: 2730-2735. Stenson, H., 1988. SIGNAL: a supplementary module for SYSTAT and SYSGRAPH. SYSTAT Inc., Evanston, II. Thoen, C.O, Blackburn, B., Mills, K., Lomme, J. and Hopkins, M.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. Todorovic R., Long, R. and McCallon, B., 1977. Comparison of the rapid card agglutination test with the complement fixation test for the diagnosis of Anaplasma marginale infection in cattle in Colombia. Vet. Microbiol., 2: 167-177. 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. Micrnbiol., 25: 633-636.