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Comparative evaluation of enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Dourine
r~,terinaryParasitology, 39 ( 1 9 9 1 ) 2 3 3 - 2 3 9 Elsevier Science Publishers B.V., A m s t e r d a m
233
Comparative evaluation of enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Dourine D.A. Wassail, R.J.F. Gregory and L.P. Phipps Central Veterinat:vLaboratoo', New Haw, Weybridge, KTI5 3NB, UK (Accepted 31 January 1991 )
ABSTRACT Wassail, D.A., Gregory, R.J.F. and Phipps, L.P., 1991. Comparative evaluation of enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Dourine. 12,t. Parasitol.. 39: 233-239. The detection of antibodies against Trypanosoma equiperdum in 689 equid sera was compared by enzyme-linked immunosorbent assay (ELISA), the complement fixation test (CFT) and an indirect immunofluorescent test (IIF). CFT was the least sensitive technique, susceptible to anti-complementary factors and the most technically demanding. IIF was more sensitive, but was only suitable lbr testing limited numbers of samples. In this study, ELISA was the most sensitive test, the least labour intensive and lends itself to a considerable degree of automation. It is suggested that ELISA would be relatively easy to standardise between laboratories and an ELISA protocol could be adopted as the internationally approved test for equine health certification.
INTRODUCTION
Dourine is a chronic or acute disease of breeding solipeds, transmitted directly from animal to animal during coitus. The causal organism is Trypanosoma equiperdum. Definitive diagnosis requires the correct recognition of clinical symptoms and identification of the parasite. This is rarely possible because not only can the clinical signs be confused with other conditions such as coital exanthema, but symptomless infections can occur. The trypanosomes are difficult to find in affected tissues and are only fleetingly present in the bloodstream. Humoral antibodies are present in infected animals whether they show clinical signs or not. Therefore, diagnosis is based on clinical evidence supported by serology and the international horse trade increasingly relies on immunodiagnosis for health certification for export. Other workers have evaluated a range of techniques for the serological diagnosis of Dourine and concluded that the complement fixation test (CFT), 0 3 0 4 - 4 0 1 7 / 9 0 / $ 0 3 . 5 0 © 1990 Elsevier Science Publishers B.V. All rights reserved.
234
D.A. WASSALLEI AL.
the indirect immunofluorescent test (IIF) and the enzyme-linked immunosorbent assay (ELISA) are the most reliable and appropriate for routine diagnostic procedures (Caporale et al., 1981; Williamson et al., 1988). This report describes a comparison of these tests using a large panel of equid sera. MATERIALS AND METHODS
Antigen Antigens for CFT and IIF were prepared from trypanosomes recovered from rats previously inoculated with live T. equiperdum from cryopreserved stock. When the parasitaemia was at its highest, blood was collected (using heparin as an anticoagulant), filtered and the trypanosomes were separated from the red blood cells by differential centrifugation. A detailed method has been published by the Ministry of Agriculture, Fisheries and Food (M.A.F.F.) ( 1986 ). A soluble ELISA antigen was prepared by initially diluting the CFT antigen 1/20 in the plate coating buffer (0.1 M carbonate buffer, pH 9.6), stirring at 4°C overnight and sonicating in a MSE Model 2-7B sonicator at Amplitude 3 ( 11-12 llm peak to peak) for 4 × 20 s. The sonicate was then centrifuged at 10 0 0 0 × g for 30 min at 4°C. The protein concentration of the supernatant was estimated by spectrophotometry at 280 nm against albumin standards and adjusted to 5/tg ml- 1 in the coating buffer.
Sent m Four hundred and forty-nine horse and 240 mule sera were obtained from Italy where they were collected as part of a survey of diseases of racehorses and wild equids in 1982. A standard high-titre positive control serum was obtained from a horse experimentally infected with the same strain of trypanosome used to produce the antigen. Serum was recovered in a large volume 9 weeks post-infection, lyophilised and stored in 2 ml aliquots at 4 ° C. Standard negative control serum was a pooled sample obtained from native British horses with no experience of the disease and stored similarly to the positive control sera.
Assays CFT was carried out in 96U microwell plates (Nunc, Roskilde, Denmark). Test and control sera were diluted 1/5 with veronal buffer and incubated at 58°C for 30 min to inactivate their complement. Working dilutions of antigen, complement and rabbit haemolytic serum (RHS) were determined by
COMPARATIVE EVALUATION OF ELISA FOR DOURINE SERODIAGNOSIS
235
titration and dilutions prepared so that each serum sample was incubated with 2 units of antigen and complement for 1 h at 37°C, with the addition of a 3% suspension of sheep red blood cells (SRBC) sensitised with 1 unit of RHS. Fixation was assessed by estimating the proportion of cells not lysed. IIF was carried out on glass slides coated with a suspension of T. equiperdum in phosphate-buffered saline at a dilution that gave about 25 parasites per field at a magnification of X 400. The slides were air-dried and stored at - 2 0 ° C. After incubation with serum dilutions, the slides are incubated with a fluorescein-labelled sheep anti-horse immunoglobulin and examined under UV illumination. The detailed methods employed for the CFT and IIF are described in M.A.F.F. (1986). In addition, a 'reduced system' CFT (CFTRS) was performed, which differed from the standard CFT by using a single m i n i m u m haemolytic dose unit of complement and a 2.3% suspension of sensitised SRBC. This system is routinely used in this laboratory, together with the conventional CFT, to detect weak positive reactions. ELISA was carried out in EIA microtitration plates (Flow Laboratories, Rickmansworth, UK) using the method of Sinclair and Wassall (1983), modified according to the following procedure. Antigen (0.1 ml) was added to each well and left overnight at 4°C; 0.1 ml of blocking solution (0.1 M phosphate-buffered saline containing 0.05% Tween 20 (PBS-T)) and 1% bovine serum albumin (BSA) were then added and the plate gently rotated at room temperature (RT) for 30 min. The plate was then washed five times with PBS-T in a Microplate washer (Flow Laboratories). Serum (0.1 ml) diluted 1/80 in PBS-T/1% BSA was added, the plate gently rotated at RT for 30 min and subsequently washed. Then 0.1 ml of rabbit anti-horse immunoglobulin (Nordic Immunology, Maidenhead, UK) diluted 1/1000 in PBS-T/ 5% BSA was added and the plate rotated and washed as before. Goat antirabbit immunoglobulin conjugated to horseradish peroxidase (0.1 ml) (Nordic) diluted 1/2000 in PBS-T/5% BSA was added and the plate rotated and washed. A 0.1 ml volume of substrate (5-amino salicyclic acid prepared as described by Ruitenberg et al. (1976) ) was then added and the plate rotated. Finally, 0.025 ml of 1 M sodium hydroxide was added to stop the reaction and the optical density (OD) read at 450 n m in a Titertek Multiskan (Flow Laboratories ).
Interpretation of results CFT The sera were screened at a dilution of 1/5 and those showing more than 50% fixation at this dilution were deemed to be positive. IIF Sera showing strong fluorescence of the parasites at a dilution of 1/80 and above were considered positive.
236
D.A. WASSALL ET M_ 1 Positive Negative
0,8
I
0,6 g cJ 0 0.4 i I
0.2
I
20
80
320 1280 RECIPROCAL SERUM DILUTIONS
5120
20480
Fig, 1. O D o f c o n t r o l s e r u m d i l u t i o n s .
TABLE 1 R e s u l t s o f a s s a y s w i t h f o u r s e r o l o g i c a l tests p e r f o r m e d on 689 e q u i d s e r a S e r u m No.
1-
CFT
CFT-RS
I1F
ELISA
-
+/-
+
+
+ + +
9
-
1()- 15
-
1 6 - 18 1 9 - 21 ~
+/_
-
+/+
23 262931 32 37.~ 53
+/+/+/+/+/+ AC AC
+/+/-+/+/-+ + AC A("
+/+/+ + +
AC
AC
+/-
+
+/-
+/-
+
--
--
25 28 30 36 51 72
73 74 75-689
--
-
+ + + + -
+ / - i n c o n c l u s i v e result. :kC = a n t i - c o m p l e m e n t a D sera.
ELISA
Working dilutions of antigen and i m m u n o g l o b u l i n s were determined by chequerboard titration. Figure 1 shows the titration of the control sera against
COMPARATIVE EVALUATION OF ELISA FOR DOURINE SERODIAGNOSIS
237
the optimum reagent concentrations. A dilution of 1/80 was utilised in the test and an OD greater than 0.2 (30% of the positive control sera) was considered positive. A trace of fixation or fluorescence at the appropriate dilution was considered an inconclusive result and designated + / - in Table 1. RESULTS
Six hundred and fifteen out of the 689 sera tested proved to be negative by all methods. The results of the remaining 74 sera are given in Table 1. Samples numbered 53-74 were among the sera collected from mules. Fifteen sera (37-51 ) showed positive antibody titres with every tests. IIF gave an inconclusive result with 13 sera ( 1-9, 16-18 and 74) previously considered negative by CFT, of which ten (1-9 and 74) gave positive ELISA results. Three sera ( 19-21 ) were designated positive by EL1SA alone. Seven sera (22 and 31-36 ) which gave inconclusive results with CFT were found to be positive by IIF and ELISA, and a further two samples (29-30) also inconclusive by IIF were identified as positive with ELISA. A group of 22 sera with anti-complementary activity (52-73) revealed one sample (73) which gave an inconclusive IIF, but a positive ELISA result. DISCUSSION
This investigation indicates that IIF and ELISA are more sensitive than CFT for the detection of serum antibodies against T. equiperdum. It is possible that this is due to complement-fixing antibody levels declining relatively early during the infection, as has been observed in cases of eqaine piroplasmosis (Mahoney, 1964 ). An excellent correlation between the IIF and ELISA was demonstrated by the fact that all the 27 sera found to be positive by IIF ( 10-15, 22 and 31-51 ) also gave a positive result with ELISA. Other workers have noted that although ELISA has a satisfactory concordance ratio with CFT (Caporale et al., 1981 ) and can be used to supplement CFT (Williamson et al., 1988 ), a few samples gave positive results with CFT, but were negative with ELISA. In contrast, the ELISA used in this study confirmed the results of all the CFT-positive sera (37-51 ). This improvement may be due to differences in the antigen preparation or the use of an intermediate antibody in this method. Our observations, therefore, suggest that ELISA is more reliable than CFT with normal sera and can detect antibodies in anti-complementary sera. Further work is continuing to determine whether the samples that gave a positive result with the ELISA technique alone do demonstrate a specific antibody response to T. equiperdum. CFT is the basis for health certification in many countries. Although there is no internationally accepted protocol, it has been used almost exclusively as
238
D.A. WASSALL ET AL.
the serological test for health certification purposes since its development by Watson in 1915. Inconclusive results are not uncommon, however, particularly with mule and donkey sera (Williamson and Herr, 1986 ). The test itself is technically demanding and labour intensive, particularly in terms of the preparation and titration of reagents. Anti-complementary factors in equid sera commonly invalidate the results. The concentrations of the reagents in the detection system have to be routinely and accurately determined. Red blood cells must be collected on a regular basis from sheep that have proved to be suitable donors. The test is standardised on the principle of excess reagent quantities so that the system can tolerate some anti-complementary effects whilst maintaining sufficient sensitivity to detect low antibody titres. In practice, we have found it necessary to devise a 'reduced system' assay in which low titres are more evident because a larger proportion of the available complement and cells are used up than in an excess reagent system. In this study, the reduced system proved to be of value in confirming the positive diagnosis of Samples 32-36, deemed inconclusive by CFT, and in the case of Serum 74 where the CFT result was negative. An IIF test is frequently used as a confirmatory test for Dourine. The test employed at this laboratory is a simple modification of the IIF for equine piroplasmosis (Donnelly et al., 1980). Immunofluorescence provides a reliable and sensitive technique, but it is also labour intensive and therefore only suitable for testing small numbers of samples. Estimating the intensity of fluorescence demands experience on the part of the observer and is difficult to standardise between observers. It is, therefore, extremely difficult to standardise the test between laboratories. ELISA is a very sensitive technique and appears to perform well in this system. It lends itself to a considerable degree of automation which makes it suitable for the routine testing of large numbers of samples. The technique also has the advantage of being relatively economical with reagents, particularly with regard to the antigen; an important consideration since the antigen is expensive and laborious to produce. These results suggest that ELISA should now be considered as an internationally approved test for Dourine. Further work is in progress to develop an ELISA for equine piroplasmosis. A triple test which detects antibodies against T. equiperdum, Babesia equi and Babesia caballi would greatly facilitate routine testing for equine health certification. ACKNOWLEDGEMENTS
We wish to thank Dr. E. Lillini of the lstituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Rome, Italy, for supplying us with serum from his survey of equid diseases in 1982, and J.D.E. Pow and K.T.A. Thaw for their expert technical assistance.
COMPARATIVE EVALUATION OF ELISA FOR DOURINE SERODIAGNOSIS
239
REFERENCES Caporale, V.P., Biancifiori, F., Frescura, F., DiMatteo, A., Nannini, D. and Urbani, G., 1981. Comparison of various tests for the serological diagnosis of Trypanosoma equiperdum infection in the horse. Comp. Immun. Microbiol. Infect. Dis., 4: 243-246. Donnelly, J., Joyner, L.P., Graham-Jones, O. and Ellis, C.P., 1980. A comparison of complement fixation and immunofluorescent antibody tests in a survey of the prevalence of Babesia equi and Babesia caballi in horses in the Sultanate of Oman. Trop. Anita. Health Prod., 12: 50-60. Mahoney, D.F., 1964. Bovine babesiosis: An assessment of the significance of complement fixing antibodies based upon experimental infection. Aust. Vet. J., 40: 369-375. Ministry of Agriculture, Fisheries and Food, 1986. Manual of Veterinary Parasitological Laboratory Techniques. 3rd Edn. HMSO, London. Ruitenberg, E.J., Steerenberg, P.A., Brosi, B.J.M. and Buys, J., 1976. Reliability of the enzymelinked immunosorbent assay (ELISA) for the serodiagnosis of Trichinella spiralis infections in conventionally raised pigs. J. Immunol. Methods, 10: 67-83. Sinclair, I.J. and Wassail, D.A., 1983. Enzyme-linked immunosorbent assay for the detection of antibodies to ttypoderrna boris in cattle. Res. Vet. Sci., 34:251-252. Watson, E.A., 1915. Dourine and the complement fixation test. Parasitology, 8:156-183. Williamson, C.C. and Herr, S., 1986. Dourine in southern Africa, 1981-1984. Serological findings from the Veterinary Research Institute, Onderstepoort. J. S. Afr. Vet. Assoc., 57: 163165. Williamson, C.C., Stoltsz, W.H., Mattheus, A. and Schiele, G.J., 1988. An investigation into alternative methods for the serodiagnosis ofDourine. Onderstepoort. J. Vet. Res,, 55:117119.
233
Comparative evaluation of enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Dourine D.A. Wassail, R.J.F. Gregory and L.P. Phipps Central Veterinat:vLaboratoo', New Haw, Weybridge, KTI5 3NB, UK (Accepted 31 January 1991 )
ABSTRACT Wassail, D.A., Gregory, R.J.F. and Phipps, L.P., 1991. Comparative evaluation of enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Dourine. 12,t. Parasitol.. 39: 233-239. The detection of antibodies against Trypanosoma equiperdum in 689 equid sera was compared by enzyme-linked immunosorbent assay (ELISA), the complement fixation test (CFT) and an indirect immunofluorescent test (IIF). CFT was the least sensitive technique, susceptible to anti-complementary factors and the most technically demanding. IIF was more sensitive, but was only suitable lbr testing limited numbers of samples. In this study, ELISA was the most sensitive test, the least labour intensive and lends itself to a considerable degree of automation. It is suggested that ELISA would be relatively easy to standardise between laboratories and an ELISA protocol could be adopted as the internationally approved test for equine health certification.
INTRODUCTION
Dourine is a chronic or acute disease of breeding solipeds, transmitted directly from animal to animal during coitus. The causal organism is Trypanosoma equiperdum. Definitive diagnosis requires the correct recognition of clinical symptoms and identification of the parasite. This is rarely possible because not only can the clinical signs be confused with other conditions such as coital exanthema, but symptomless infections can occur. The trypanosomes are difficult to find in affected tissues and are only fleetingly present in the bloodstream. Humoral antibodies are present in infected animals whether they show clinical signs or not. Therefore, diagnosis is based on clinical evidence supported by serology and the international horse trade increasingly relies on immunodiagnosis for health certification for export. Other workers have evaluated a range of techniques for the serological diagnosis of Dourine and concluded that the complement fixation test (CFT), 0 3 0 4 - 4 0 1 7 / 9 0 / $ 0 3 . 5 0 © 1990 Elsevier Science Publishers B.V. All rights reserved.
234
D.A. WASSALLEI AL.
the indirect immunofluorescent test (IIF) and the enzyme-linked immunosorbent assay (ELISA) are the most reliable and appropriate for routine diagnostic procedures (Caporale et al., 1981; Williamson et al., 1988). This report describes a comparison of these tests using a large panel of equid sera. MATERIALS AND METHODS
Antigen Antigens for CFT and IIF were prepared from trypanosomes recovered from rats previously inoculated with live T. equiperdum from cryopreserved stock. When the parasitaemia was at its highest, blood was collected (using heparin as an anticoagulant), filtered and the trypanosomes were separated from the red blood cells by differential centrifugation. A detailed method has been published by the Ministry of Agriculture, Fisheries and Food (M.A.F.F.) ( 1986 ). A soluble ELISA antigen was prepared by initially diluting the CFT antigen 1/20 in the plate coating buffer (0.1 M carbonate buffer, pH 9.6), stirring at 4°C overnight and sonicating in a MSE Model 2-7B sonicator at Amplitude 3 ( 11-12 llm peak to peak) for 4 × 20 s. The sonicate was then centrifuged at 10 0 0 0 × g for 30 min at 4°C. The protein concentration of the supernatant was estimated by spectrophotometry at 280 nm against albumin standards and adjusted to 5/tg ml- 1 in the coating buffer.
Sent m Four hundred and forty-nine horse and 240 mule sera were obtained from Italy where they were collected as part of a survey of diseases of racehorses and wild equids in 1982. A standard high-titre positive control serum was obtained from a horse experimentally infected with the same strain of trypanosome used to produce the antigen. Serum was recovered in a large volume 9 weeks post-infection, lyophilised and stored in 2 ml aliquots at 4 ° C. Standard negative control serum was a pooled sample obtained from native British horses with no experience of the disease and stored similarly to the positive control sera.
Assays CFT was carried out in 96U microwell plates (Nunc, Roskilde, Denmark). Test and control sera were diluted 1/5 with veronal buffer and incubated at 58°C for 30 min to inactivate their complement. Working dilutions of antigen, complement and rabbit haemolytic serum (RHS) were determined by
COMPARATIVE EVALUATION OF ELISA FOR DOURINE SERODIAGNOSIS
235
titration and dilutions prepared so that each serum sample was incubated with 2 units of antigen and complement for 1 h at 37°C, with the addition of a 3% suspension of sheep red blood cells (SRBC) sensitised with 1 unit of RHS. Fixation was assessed by estimating the proportion of cells not lysed. IIF was carried out on glass slides coated with a suspension of T. equiperdum in phosphate-buffered saline at a dilution that gave about 25 parasites per field at a magnification of X 400. The slides were air-dried and stored at - 2 0 ° C. After incubation with serum dilutions, the slides are incubated with a fluorescein-labelled sheep anti-horse immunoglobulin and examined under UV illumination. The detailed methods employed for the CFT and IIF are described in M.A.F.F. (1986). In addition, a 'reduced system' CFT (CFTRS) was performed, which differed from the standard CFT by using a single m i n i m u m haemolytic dose unit of complement and a 2.3% suspension of sensitised SRBC. This system is routinely used in this laboratory, together with the conventional CFT, to detect weak positive reactions. ELISA was carried out in EIA microtitration plates (Flow Laboratories, Rickmansworth, UK) using the method of Sinclair and Wassall (1983), modified according to the following procedure. Antigen (0.1 ml) was added to each well and left overnight at 4°C; 0.1 ml of blocking solution (0.1 M phosphate-buffered saline containing 0.05% Tween 20 (PBS-T)) and 1% bovine serum albumin (BSA) were then added and the plate gently rotated at room temperature (RT) for 30 min. The plate was then washed five times with PBS-T in a Microplate washer (Flow Laboratories). Serum (0.1 ml) diluted 1/80 in PBS-T/1% BSA was added, the plate gently rotated at RT for 30 min and subsequently washed. Then 0.1 ml of rabbit anti-horse immunoglobulin (Nordic Immunology, Maidenhead, UK) diluted 1/1000 in PBS-T/ 5% BSA was added and the plate rotated and washed as before. Goat antirabbit immunoglobulin conjugated to horseradish peroxidase (0.1 ml) (Nordic) diluted 1/2000 in PBS-T/5% BSA was added and the plate rotated and washed. A 0.1 ml volume of substrate (5-amino salicyclic acid prepared as described by Ruitenberg et al. (1976) ) was then added and the plate rotated. Finally, 0.025 ml of 1 M sodium hydroxide was added to stop the reaction and the optical density (OD) read at 450 n m in a Titertek Multiskan (Flow Laboratories ).
Interpretation of results CFT The sera were screened at a dilution of 1/5 and those showing more than 50% fixation at this dilution were deemed to be positive. IIF Sera showing strong fluorescence of the parasites at a dilution of 1/80 and above were considered positive.
236
D.A. WASSALL ET M_ 1 Positive Negative
0,8
I
0,6 g cJ 0 0.4 i I
0.2
I
20
80
320 1280 RECIPROCAL SERUM DILUTIONS
5120
20480
Fig, 1. O D o f c o n t r o l s e r u m d i l u t i o n s .
TABLE 1 R e s u l t s o f a s s a y s w i t h f o u r s e r o l o g i c a l tests p e r f o r m e d on 689 e q u i d s e r a S e r u m No.
1-
CFT
CFT-RS
I1F
ELISA
-
+/-
+
+
+ + +
9
-
1()- 15
-
1 6 - 18 1 9 - 21 ~
+/_
-
+/+
23 262931 32 37.~ 53
+/+/+/+/+/+ AC AC
+/+/-+/+/-+ + AC A("
+/+/+ + +
AC
AC
+/-
+
+/-
+/-
+
--
--
25 28 30 36 51 72
73 74 75-689
--
-
+ + + + -
+ / - i n c o n c l u s i v e result. :kC = a n t i - c o m p l e m e n t a D sera.
ELISA
Working dilutions of antigen and i m m u n o g l o b u l i n s were determined by chequerboard titration. Figure 1 shows the titration of the control sera against
COMPARATIVE EVALUATION OF ELISA FOR DOURINE SERODIAGNOSIS
237
the optimum reagent concentrations. A dilution of 1/80 was utilised in the test and an OD greater than 0.2 (30% of the positive control sera) was considered positive. A trace of fixation or fluorescence at the appropriate dilution was considered an inconclusive result and designated + / - in Table 1. RESULTS
Six hundred and fifteen out of the 689 sera tested proved to be negative by all methods. The results of the remaining 74 sera are given in Table 1. Samples numbered 53-74 were among the sera collected from mules. Fifteen sera (37-51 ) showed positive antibody titres with every tests. IIF gave an inconclusive result with 13 sera ( 1-9, 16-18 and 74) previously considered negative by CFT, of which ten (1-9 and 74) gave positive ELISA results. Three sera ( 19-21 ) were designated positive by EL1SA alone. Seven sera (22 and 31-36 ) which gave inconclusive results with CFT were found to be positive by IIF and ELISA, and a further two samples (29-30) also inconclusive by IIF were identified as positive with ELISA. A group of 22 sera with anti-complementary activity (52-73) revealed one sample (73) which gave an inconclusive IIF, but a positive ELISA result. DISCUSSION
This investigation indicates that IIF and ELISA are more sensitive than CFT for the detection of serum antibodies against T. equiperdum. It is possible that this is due to complement-fixing antibody levels declining relatively early during the infection, as has been observed in cases of eqaine piroplasmosis (Mahoney, 1964 ). An excellent correlation between the IIF and ELISA was demonstrated by the fact that all the 27 sera found to be positive by IIF ( 10-15, 22 and 31-51 ) also gave a positive result with ELISA. Other workers have noted that although ELISA has a satisfactory concordance ratio with CFT (Caporale et al., 1981 ) and can be used to supplement CFT (Williamson et al., 1988 ), a few samples gave positive results with CFT, but were negative with ELISA. In contrast, the ELISA used in this study confirmed the results of all the CFT-positive sera (37-51 ). This improvement may be due to differences in the antigen preparation or the use of an intermediate antibody in this method. Our observations, therefore, suggest that ELISA is more reliable than CFT with normal sera and can detect antibodies in anti-complementary sera. Further work is continuing to determine whether the samples that gave a positive result with the ELISA technique alone do demonstrate a specific antibody response to T. equiperdum. CFT is the basis for health certification in many countries. Although there is no internationally accepted protocol, it has been used almost exclusively as
238
D.A. WASSALL ET AL.
the serological test for health certification purposes since its development by Watson in 1915. Inconclusive results are not uncommon, however, particularly with mule and donkey sera (Williamson and Herr, 1986 ). The test itself is technically demanding and labour intensive, particularly in terms of the preparation and titration of reagents. Anti-complementary factors in equid sera commonly invalidate the results. The concentrations of the reagents in the detection system have to be routinely and accurately determined. Red blood cells must be collected on a regular basis from sheep that have proved to be suitable donors. The test is standardised on the principle of excess reagent quantities so that the system can tolerate some anti-complementary effects whilst maintaining sufficient sensitivity to detect low antibody titres. In practice, we have found it necessary to devise a 'reduced system' assay in which low titres are more evident because a larger proportion of the available complement and cells are used up than in an excess reagent system. In this study, the reduced system proved to be of value in confirming the positive diagnosis of Samples 32-36, deemed inconclusive by CFT, and in the case of Serum 74 where the CFT result was negative. An IIF test is frequently used as a confirmatory test for Dourine. The test employed at this laboratory is a simple modification of the IIF for equine piroplasmosis (Donnelly et al., 1980). Immunofluorescence provides a reliable and sensitive technique, but it is also labour intensive and therefore only suitable for testing small numbers of samples. Estimating the intensity of fluorescence demands experience on the part of the observer and is difficult to standardise between observers. It is, therefore, extremely difficult to standardise the test between laboratories. ELISA is a very sensitive technique and appears to perform well in this system. It lends itself to a considerable degree of automation which makes it suitable for the routine testing of large numbers of samples. The technique also has the advantage of being relatively economical with reagents, particularly with regard to the antigen; an important consideration since the antigen is expensive and laborious to produce. These results suggest that ELISA should now be considered as an internationally approved test for Dourine. Further work is in progress to develop an ELISA for equine piroplasmosis. A triple test which detects antibodies against T. equiperdum, Babesia equi and Babesia caballi would greatly facilitate routine testing for equine health certification. ACKNOWLEDGEMENTS
We wish to thank Dr. E. Lillini of the lstituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Rome, Italy, for supplying us with serum from his survey of equid diseases in 1982, and J.D.E. Pow and K.T.A. Thaw for their expert technical assistance.
COMPARATIVE EVALUATION OF ELISA FOR DOURINE SERODIAGNOSIS
239
REFERENCES Caporale, V.P., Biancifiori, F., Frescura, F., DiMatteo, A., Nannini, D. and Urbani, G., 1981. Comparison of various tests for the serological diagnosis of Trypanosoma equiperdum infection in the horse. Comp. Immun. Microbiol. Infect. Dis., 4: 243-246. Donnelly, J., Joyner, L.P., Graham-Jones, O. and Ellis, C.P., 1980. A comparison of complement fixation and immunofluorescent antibody tests in a survey of the prevalence of Babesia equi and Babesia caballi in horses in the Sultanate of Oman. Trop. Anita. Health Prod., 12: 50-60. Mahoney, D.F., 1964. Bovine babesiosis: An assessment of the significance of complement fixing antibodies based upon experimental infection. Aust. Vet. J., 40: 369-375. Ministry of Agriculture, Fisheries and Food, 1986. Manual of Veterinary Parasitological Laboratory Techniques. 3rd Edn. HMSO, London. Ruitenberg, E.J., Steerenberg, P.A., Brosi, B.J.M. and Buys, J., 1976. Reliability of the enzymelinked immunosorbent assay (ELISA) for the serodiagnosis of Trichinella spiralis infections in conventionally raised pigs. J. Immunol. Methods, 10: 67-83. Sinclair, I.J. and Wassail, D.A., 1983. Enzyme-linked immunosorbent assay for the detection of antibodies to ttypoderrna boris in cattle. Res. Vet. Sci., 34:251-252. Watson, E.A., 1915. Dourine and the complement fixation test. Parasitology, 8:156-183. Williamson, C.C. and Herr, S., 1986. Dourine in southern Africa, 1981-1984. Serological findings from the Veterinary Research Institute, Onderstepoort. J. S. Afr. Vet. Assoc., 57: 163165. Williamson, C.C., Stoltsz, W.H., Mattheus, A. and Schiele, G.J., 1988. An investigation into alternative methods for the serodiagnosis ofDourine. Onderstepoort. J. Vet. Res,, 55:117119.