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Evaluation of the currently used diagnostic procedures for the detection of Brucella melitensis in sheep
Small Ruminant Research 31 (1998) 1±6
Evaluation of the currently used diagnostic procedures for the detection of Brucella melitensis in sheep Z. Bercovicha,*, L. GuÈlerb, T. Baysalb, B.E.C. Schreudera, F.G. van Zijdervelda a
Department of Bacteriology, Institute for Animal Science and Health (ID-DLO ) Edelhertweg 15, P. O. Box 65, 8200 AB Lelystad, The Netherlands b Veteriner Kontrol ve Arastirma EnstituÈsuÈ MuÈduÈrluÈguÈ, Konya, Turkey Accepted 13 March 1998
Abstract A study was conducted to determine whether the use of the enzyme-linked immunosorbent assay (ELISA) improves detection of brucellosis in individual sheep. Sera from 132 sheep that aborted due to B. melitensis were used to assess the ef®cacy of the ELISA to detect brucellosis in sheep. ELISA results were compared with those of the serum agglutination test (SAT), complement ®xation test (CFT), Rose Bengal Plate test (RBPT) and the skin delayed-type hypersensitivity (SDTH). Sera from 11132 sheep free from brucellosis were tested with the ELISA to determine its speci®city. The results show little difference in diagnostic ef®cacy between SAT, CFT, RBPT and the SDTH test, although the skin test indicated infection in some seronegative sheep. A combination of CFT and SAT, or CFT and RBPT, or CFT and SDTH tests indicated brucellosis in 75± 88% of the infected animals, whereas the ELISA indicated infection in 97±100% of those animals. Seventeen (0.2%) from the 11132 negative sheep sera tested positive with the ELISA, indicating a 99.8% speci®city. We concluded that screening sheep for brucellosis with ELISA improves Brucella diagnosis, and in areas of low prevalence it will help to maintain ¯ocks of sheep free from brucellosis. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Brucella melitensis; ELISA; Serological diagnosis; Delayed-type hypersensitivity test
1. Introduction Brucella melitensis, the causal organism of Malta fever in man, has not yet been isolated from animals in the Netherlands where the milk ring test, serum agglutination test (SAT) and complement ®xation test (CFT) are used for the surveillance for brucellosis. Although the Netherlands have been free from brucellosis for many years, trade in animals with various *Corresponding author. Tel.: +0320 238150; fax: +0320 238153.
countries greatly increases the possibility of introducing brucellosis in healthy ¯ocks. Therefore, a sensitive test that indicates possibly infected sheep is of great value. The milk ring test that is widely used to detect brucellosis in dairy cattle is not sensitive enough to detect brucellosis in sheep (Shimi and Tabatabayi, 1981). The SAT, although adequate to detect acute infection, may fail to agglutinate with antibodies present in sheep sera, or give a marked prozone phenomenon (Renoux et al., 1956; Goguer, 1965;
0921-4488/98/$ ± see front matter # 1998 Elsevier Science B.V. All rights reserved. PII S0921-4488(98)00111-4
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Z. Bercovich et al. / Small Ruminant Research 31 (1998) 1±6
Le Pennec, 1967). The use of the Rose Bengal Plate test (RBPT) which is considered a valuable screening test (Farina, 1985) is less effective than the CFT detecting brucellosis in individual sheep (Joint FAO/WHO Expert Committee on Brucellosis, Technical report 740, 1986). Further, its ef®cacy is in¯uenced by cell concentration and the standardization procedure of the antigen (Blasco et al., 1994a; Hosie et al., 1985). The CFT which is considered the most effective test for diagnosing brucellosis in sheep (Joint FAO/WHO Expert Committee on Brucellosis, Technical report 740, 1986) has no particular advantage over the SAT performed in a hypertonic environment of 5±20% NaCl. Moreover, some infected animals may elude detection with the CFT (Karmann and Schloz, 1956; Levchenco and Drozhzhin, 1958; Blasco et al., 1994b). However, other studies demonstrate the value of the CFT, and the skin delayed-type hypersensitivity (SDTH) test for the detection of brucellosis in sheep (Ebadi and Zowghi, 1983; Ebadi, 1984; Loquerie and Durand, 1984). Although the currently in use serological tests effectively detect brucellosis on a ¯ock basis, they are not always reliable for detecting infection in an individual animal. This is a major problem in areas of low prevalence of brucellosis, and where trade in sheep may introduce brucellae in areas free from brucellosis. Since neither a single serological test nor a combination of several serological tests can detect all infected sheep in a ¯ock, it is opportune to assess whether the use of the enzyme-linked immunosorbent assay (ELISA) improves detection of infected sheep. While the ef®cacy of the ELISA to detect Brucella ovis in rams has been extensively evaluated in recent years, relatively little information is available on the ef®cacy of the ELISA to detect sheep infected with B. melitensis. The objectives of the present study were to compare ELISA results with those of the SAT, CFT, RBPT and the SDTH tests, and to establish whether the use of the ELISA is a meaningful addition to the diagnosis of sheep brucellosis. 2. Material and methods 2.1. Animals Two ¯ocks of fat-tailed sheep, in the Konya province of Turkey, from which B. melitensis was isolated
from aborted fetuses were chosen for this study. Group A, with a history of B. melitensis infection and vaccination against brucellosis with Rev 1, consisted of 55 sheep that aborted. Group B, with a history of B. melitensis infection and no vaccination against brucellosis, consisted of 77 sheep that aborted. Group C consisted of 11132 Brucella-free Dutch sheep. 2.2. Serum samples and serological tests Although the aborting sheep were marked at the time of abortion they were ear-tagged a few weeks later, when blood samples were collected for serological examination. Sera from those sheep were tested with the SAT, CFT, RBPT and ELISA. The 11132 sera from brucellosis-free sheep were tested with the ELISA only and the results were used to calculated the speci®city of the assay. The SAT was done with an antigen prepared from B. abortus S-99. The antigen, diluted in hypertonic phenol-saline (5% NaCl), was standardized to give 50% agglutination at a ®nal dilution of 1:500 with the second international standard anti-Brucella abortus serum. The SAT was carried out according to the procedure used at the brucellosis research laboratory of ID-DLO, Lelystad, the Netherlands. A 75% agglutination or more in a serum diluted 1:15 was considered positive. The CFT was done according to Hill (1963). The sera were inactivated at 608C for 55 min to prevent anti-complementary activity. Sera giving 100% inhibition of hemolysis at a serum dilution 1:10 or higher were considered positive. The RBPT was performed with an antigen produced by RhoÃne MeÂrieux, Lyon, France, kindly provided by Dr. B. Garin-Bastuij, CNEVA Maisons-Alfort, France. The test, using 50 ml serum and 50 ml antigen, containing 4±5% cells, was done on a glass plate. After the test sera and antigen were mixed, the plate was shaken for 20 s every minute for 4 min. Sera that tested negative or doubtfully positive after 4 min were read again 4 min later. Test results were recorded as positive or negative. Animals were regarded infected if they tested positive in CFT and either SAT, RBPT or SDTH. The indirect ELISA was performed as described by Bercovich and Taaijke (1990) with some modi®cations. Brucella abortus strain 99 grown on trypticase-
Z. Bercovich et al. / Small Ruminant Research 31 (1998) 1±6
soy agar at 378C was used to prepare the antigen. The bacterial growth was harvested with sterile saline, autoclaved for 25 min at 1218C, cooled to room temperature and ®ltered through a sterile gauze to remove agar particles. The bacteria were then disrupted by soni®cation for 20 min at 20 Kc/s on a 500 W disintegrator. The resulting suspension, supplemented with 0.1% formaldehyde (®nal concentration), was stored in stock solution at 4±68C. 2.2.1. Buffers A 0.01 M phosphate-buffered saline (PBS) pH 6.3 was used to dilute the antigen. Dilution of the test serum was made in 0.01 M PBS pH 7.2 containing 0.05% Tween 20. The washing solution consisted of demineralized water with 0.05% (v/v) Tween 20. Horseradish peroxidase conjugated mouse monoclonal anti-bovine IgG1 (CVI-BIgG1, 15.8.1) that detects bovine as well as ovine IgG1 was diluted in 0.5 M PBS pH 7.2 containing 0.05% Tween 20. The substrate that was prepared by dissolving 1 g 5-amino-2-hydroxybenzoic acid (Merck, art. 819019) in 1 l 0.01 M PBS containing 0.04 g Na2EDTA had a pH of 5.9±6.0. The substrate solution was divided into 9 ml amounts and kept at ÿ208C. Prior to use 0.005% of fresh H2O2 was added. The working dilutions of antigen and conjugate were determined by checker-board analysis using a sheep serum containing 1000 CFT units as positive control. A pool of 20 sera from Brucella-free sheep was used as negative control. After the optimal antigen and conjugate concentration was determined, a cut-off point for the ELISA that is suitable for use in the ®eld was established with the aid of 500 sheep sera that tested negative with the CFT and RBPT. As sensitivity and speci®city may vary according to the reagents used in the assay, we chose as a cut-off point an extinction value equal to 4 units/mlÿ1. The ELISA plates were then ®lled with 0.1 ml diluted antigen, sealed and incubated overnight at 48C. The coated plates were then stored at ÿ208C until used. 2.2.2. The ELISA was performed as follows After defrosting the antigen in a 378C waterbath the plates were washed ®ve times with a washing solution (total amount approximately 500 ml). Test sera were diluted 1:50 with PBS buffer pH. 7.2. This buffer was also used to ®ll the wells of the coated plates with
3
0.1 ml buffer. Diluted serum (0.1 ml) was then dispensed in Row A and serial two-fold dilutions of test and control sera were made. After 1 h incubation at 378C the plates were washed ®ve times and 0.1 ml of diluted mouse monoclonal anti-bovine IgG1 conjugate was added to each well. After 1 h incubation at 378C, the plates were washed ®ve times and 0.1 ml substrate and H2O2 solution was added to each well. After overnight incubation at room temperature, the plates were shaken on a micro-shaker, and the results were recorded on an Easy reader EAR 400 at 450 nm (SLTLabinstruments, Austria). 2.3. Brucellin and SDTH test The brucellin used in this study was the same as that used in cattle in studies described earlier (Bercovich and Ter Laak, 1990; Bercovich et al., 1996). The Brucella abortus strain 45/20 was used to prepare the brucellin according to Bercovich et al. (1995). The protein concentration of the brucellin was determined by the Kjeldahl method and standardized in phosphate buffer pH 7.2 to contain 1.0 mg protein/mlÿ1. The 132 sheep that aborted in ¯ocks A and B and 30 Brucella-free Dutch sheep were tested with the SDTH test. All sheep were injected intradermally with 0.1 ml brucellin (100 mg protein) in the lower eyelid; some infected sheep were also injected in the tail. A clearly manifest asymmetry of the head caused by a visible swelling of the lower eyelid or a palpable swelling in the tail at the injection site were considered a reaction elicited by the injection with brucellin. 3. Results Thirty-seven of the 55 (67.2%) sheep in ¯ock A, and 48 of the 77 (62.3%) sheep in ¯ock B tested positive with all the tests. The percentage of infected animals that tested positive with each of the tests is summarized in Table 1. The serological response of the sheep that did not test positive in all the tests is summarized in Table 2. The combination of CFT and SAT failed to indicate infection in six sheep in each of the infected ¯ocks. The CFT and RBPT failed to indicate infection in seven sheep in ¯ock A and four in ¯ock B, while the CFT and SDTH test failed to indicate infection in
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Z. Bercovich et al. / Small Ruminant Research 31 (1998) 1±6
Table 1 Percentage of infected animals that tested positive with the SAT, CFT, ELISA RBPT and SDTH Serological test Flock A Flock B
SAT
CFT
ELISA
RBPT
SDTH test
78.2 88.3
85.4 88.3
98.2 97.5
78.1 83.1
85.4 76.6
respectively ®ve and 13 sheep in ¯ocks A and B. The ELISA tested positive in all but one (39, ¯ock B) of the aborting animals. Seventeen of the 11132 sera from Brucella-free sheep tested positive with the ELISA at 4±32 units. Two of those sheep, each in a different ¯ock, tested with the CFT respectively at 20 and 100 units. Although differences in skin swellings at the injection site of the brucellin were observed among the animals that aborted, all swelling were clearly visible and palpable. All the Brucella-free sheep tested negative with the SDTH test. 4. Discussion The objectives of the present study were to compare the ELISA results with those of the SAT, CFT, RBPT and the SDTH test, and to establish whether the use of the ELISA is a meaningful addition to the diagnosis of sheep brucellosis. Detection of brucellosis with serological tests in areas with low prevalence is problematic because serum titres of some infected animals sharply decline after infection, or because infected sheep do not react in the commonly used serological tests (Morgan, 1974). With this in mind, the SDTH test which is independent of humoral antibody and more sensitive than SAT and CFT was also included in this study (Ebadi and Zowghi, 1983). Since diagnostic errors occur, the relative sensitivity and speci®city of a test are used to assess its ef®cacy to detect infection. These criteria can only be implemented if a gold standard is used to establish the true disease state of the animal. Isolation of Brucella could be a gold standard as it unequivocally establishes the cause of infection. However, bacteriological examination cannot always be relied on to prove the presence or absence of infection in individual animals (Unel et al., 1969; Alton et al., 1975;
Table 2 Serological and SDTH test results of sheep that did not test positive in all tests Sheep number Flock A 4 13 20 22 28 30 31 32 35 39 40 41 46 47 48 51 54 55 Flock B 7 8 10 13 17 18 24 32 33 37 38 40 39 42 44 51 52 56 61 63 64 66 67 70 71 72 73 74 76
SAT units
CFT units
ELISA units
Rose Bengal
SDTH test
120 <30 30 960 120 <30 <30 <30 <30 <30 <30 30 60 <30 <30 <30 <30 <30
200 <20 <20 200 200 <20 50 100 <20 <20 <20 200 200 20 <20 50 <20 100
512 8 4 512 512 16 128 256 32 16 8 512 512 64 8 64 64 64
ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ
ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ
240 <30 <30 30 <30 <30 30 30 30 960 <30 <30 30 60 960 30 30 <30 <30 120 240 <30 30 30 120 30 73 240 120
200 <20 <20 <20 <20 50 20 200 <20 100 50 200 <20 200 200 100 50 <20 <20 100 200 <20 50 50 200 50 50 100 200
512 16 4 16 16 256 64 512 16 64 128 512 <4 512 512 128 64 4 32 256 256 8 32 32 256 64 128 128 256
ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ
ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ
Z. Bercovich et al. / Small Ruminant Research 31 (1998) 1±6
Corbel et al., 1980). Therefore, the presumptive diagnosis provided by the serological tests is usually accepted as indication for brucellosis. Although the sheep in this study were marked after abortion, bacteriologic test results could not be related to the individual animal because they were not ear-tagged at the time of abortion. Consequently, the presence of brucellosis was indirectly established by the isolation of B. melitensis from the aborted fetuses, but the infection status of individual animals was determined by the serological tests or the SDTH test. We also considered serologic test results of the vaccinated sheep indicative of infection because the immune response to Rev 1 vaccination usually disappears 3± 5 months after vaccination (Falade, 1983; Madsen, 1993). Our study shows little difference in diagnostic ef®ciency between the SAT, CFT, RBPT and the SDTH test on a ¯ock basis (Table 1). With the exception of the ELISA, the tests detected brucellosis in 76± 88% of the animals con®rming the observation that no single test effectively detects brucellosis (Mahajan and Kulshreshtha, 1991). This clearly suggests the need to use several serological tests. However, a combined used of CFT and SAT indicated infection in, respectively, 89 and 92.2% of aborting sheep in ¯ocks A and B, whereas a combination of the CFT and the RBPT indicated infection in 87.3% of the sheep in ¯ock A and 93.6% in ¯ock B (Table 2). These results clearly differ from the results of JimeÂneÂz de BagueÂs et al. (1992) showing complete agreement between CFT and RBPT. While the SDTH test indicated infection in sheep that tested negative with SAT, CFT and the RBPT (Table 2), the combination of CFT and the SDTH indicated infection in respectively only 91 and 83.2% of the sheep in ¯ock A and B. Further, our study did not con®rm the ef®cacy of the RBPT or the SDTH test reported in other studies (Farina, 1985; Blasco et al., 1994b), although it con®rmed the relative ef®cacy of the CFT to detect brucellosis (Blasco et al., 1994b). In practice, it is expected that a highly sensitive test will identify most diseased animals. Likewise, a speci®c test is expected to correctly identify most truenegative animals. But, since speci®city is inversely related to sensitivity the norm to indicate infection has to be a compromise between those two parameters. Since an undetected infected animal constitutes a
5
hazard, and over-condemning of animals is undesirable, we set the cutoff point of the ELISA to detect 4 ELISA units/ml. Using this norm the ELISA indicated infection in 131/132 of the animals that aborted. Further, the value of the ELISA is demonstrated by the indication of infection in seronegative sheep 40, 48, 10, 56 and 66 (Table 2). This study also shows that only a few sera tested false-positive with the ELISA. A sample of 11132 sheep sera revealed 17 (0.2%) sheep of which 8, 6 and 3 sheep tested in the ELISA 4, 8 and 32 units respectively. Although two of those sheep also tested positive with the CFT, ELISA results were considered falsepositive because, retrospectively, those and all other ¯ocks proved to be free from brucellosis. This, and the size of the examined sample clearly indicate a 99.8% speci®city. In this respect the diagnostic speci®city of the ELISA proved very satisfactory in that the cutoff point value of <4 units/mlÿ1 may truly represent negative ewes. As not all the animals of the infected ¯ocks were tested with the ELISA, calculation of the sensitivity could not be done. Nevertheless, the sensitivity of the ELISA found in other studies (JimeÂneÂz de BagueÂs et al., 1992; Delgado et al., 1995), and the speci®city of the assay found in this study conclusively demonstrate the reliability of the assay. These ®ndings clearly endorse the use of the assay for the detection of infected sheep in areas of low prevalence. It can be inferred, therefore, that the ELISA is an excellent choice on which to base the surveillance for brucellosis in sheep. Any tendency of `over condemning' of ELISA-positive animals is compensated by the knowledge that a very high percentage of the potentially infected animals have been identi®ed and can be removed. Acknowledgements We thank Toos Dekker and Bert Tetenburg for their assistance with the serological examination. References Alton, G.G., Maw, J., Rogerson, B.A., McPherson, G.G., 1975. The serological diagnosis of bovine brucellosis: An evaluation of the complement fixation, serum agglutination and rose bengal tests. Aust. Vet. J. 51, 57±63.
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Bercovich, Z., Taaijke, R., 1990. Enzyme immunoassay using mouse monoclonal anti-bovine antibodies for the detection of Brucella abortus antibodies in cow milk. J. Vet. Med. B 37, 753±759. Bercovich, A., Ter Laak, E.A., 1990. An evaluation of the Delayedtype hypersensitivity test for the diagnosis of brucellosis in individual cattle: A field study. Vet. Microbiol. 22, 241±248. Bercovich, Z., Eger, A., Dekker, T., Haagsma, J., 1995. Production of Brucella allergens and evaluation of their biological activity in a guinea pigs bio-assay. J. Vet. Med. B 42, 19±27. Bercovich, Z., Dekker, T., Eger, A., Haagsma, J., 1996. A comparison of the potency of several Brucella allergens to detect brucellosis in cattle. Vet. Res. Comm. 20, 141±151. Blasco, J.M., Garin-Bastuij, B., Martin, C.M., Gerbier, G., Fanlo, J., JimeÂneÂz de BagueÂs, M.P., Cau, C., 1994a. Efficacy of the Rose Bengal and complement fixation antigens for the diagnosis of Brucella melitensis infection in sheep and goats. Vet. Rec. 134, 415±420. Blasco, J.M., Marin, C., JimeÂnez de BagueÂs, M., BarbereÂn, M., Hernandez, A., Molina, L., Velasco, J., Diaz, R., Moriyon, I., 1994b. Evaluation of allergic and serological tests for diagnosing Brucella melitensis infection in sheep. J. Clin. Microbiol. 32, 1835±1840. Corbel, M.J., Scott, A.C., Ross, H.M., 1980. Properties of a cellwall-defective variant of Brucella abortus of bovine origin. J. Hyg. 85, 103±113. Delgado, S., Fernandez, M., Carmenes, P., 1995. Evaluation of an enzyme-linked immunosorbent assay for the detection of sheep infected and vaccinated with Brucella melitensis. J. Vet. Diag. Investig. 7, 206±209. Ebadi, A., Zowghi, E., 1983. The use of allergic test in the diagnosis of Brucella melitensis infection in sheep. Br. Vet. J. 139, 456±462. Ebadi, A., 1984. Evaluation of the allergic test in the diagnosis of brucellosis in sheep. Develop. Biol. Standard. 56, 387±391. Falade, S., 1983. Serological response of sheep to Brucella melitensis Rev 1 vaccine. 30, 546±551. Farina, R., 1985. Current serological methods in Brucella melitensis diagnosis. In: Verger, J.M., Plommet, M. (Eds.), Brucella melitensis. Martinus Nijhoff, Dordrecht, pp. 139±146. Goguer, M., 1965. Comparison of the plate and tube agglutination tests for brucellosis in sheep. Vet. Bull. 35, Abstr., 3710. Hill, W.K.W., 1963. Standardization of the complement fixation test for brucellosis. Bull. Off. Int. Epiz. 60, 401±417.
Hosie, B.D., Al-Bakri, O.M., Futter, R.J., 1985. Survey of Brucellosis in goats and sheep in the Yemen Arab Republic: Comparison of tests for Brucella melitensis infection in sheep. Trop. Anim. Health Prod. 17, 93±99. JimeÂneÂz de BagueÂs, M.P., Marin, C.M., Blasco, J.M., Moriyon, I., Gamazo, C., 1992. An ELISA with Brucella LPS antigen for the diagnosis of Brucella melitensis infection in sheep and for the evaluation of serologic responses following subcutaneous or conjunctival Brucella melitensis strain Rev 1 vaccination. Vet. Microbiol. 30, 233±241. Karmann, P., Schloz, H.D., 1956. Beitrag zur BekaÈmpfung des Maltafiebers der Schafe in Lande Nordrhein-Westfalen. Mh. Tierheilk. 8, 127±134. Le Pennec, J., 1967. Serology of ovine brucellosis. Vaccination and infection antibodies negative to Wright's agglutination test but positive to the complement fixation test. Vet Bull., 37. Abstr. 3571. Levchenco, I.D., Drozhzhin, V.N., 1958. Use in outbreaks of abortion in sheep of the agglutination test with 10% NaCl. 28, Abstr. 1006. Loquerie, R., Durand, M.P., 1984. Clinical application of a skin test for delayed hypersensitivity, using a commercial allergen, for detecting brucellosis in sheep and goats. Develop. Biol. Stand. 56, 407±410. Madsen, M., 1993. The serological response to vaccination with Brucella abortus strain 19 and Brucella melitensis Rev 1, as measured by Rose Bengal, serum agglutination and complement fixation tests. Zimbabwe Vet. J. 24, 13±20. Mahajan, N.K., Kulshreshtha, R.C., 1991. Comparison of serological tests for Brucella melitensis infection in sheep. Trop. Anim. Health Pro. 23, 11±16. Morgan, W.J.B., 1974. Brucellosis of small ruminants. Diagnosis, control and eradication. Bull. Off. Int. Epiz. 82, 93±96. Renoux, G., Alton, G., Mahaffey, L.W., 1956. Etude sur la brucellose ovine et caprine: v. ReÂactions seÂrologiques dans le sang de breÂbis reÂcemment par Brucella melitensis. Arch Inst. Pasteur, Tunis. 33, 33±41. Shimi, A., Tabatabayi, A.H., 1981. Pathological, bacteriological and serological responses of ewes experimentally infected with Brucella melitensis. Bull. Off. Int. Epiz. 39(11/12), 1411±422. Unel, S., Williams, C.F., Stableforth, A.W., 1969. Relative value of the agglutination test, complement fixation test and Coombs test in the detection of Brucella melitensis infection in sheep. J. Comp. Pathol. 79, 155±159.
Evaluation of the currently used diagnostic procedures for the detection of Brucella melitensis in sheep Z. Bercovicha,*, L. GuÈlerb, T. Baysalb, B.E.C. Schreudera, F.G. van Zijdervelda a
Department of Bacteriology, Institute for Animal Science and Health (ID-DLO ) Edelhertweg 15, P. O. Box 65, 8200 AB Lelystad, The Netherlands b Veteriner Kontrol ve Arastirma EnstituÈsuÈ MuÈduÈrluÈguÈ, Konya, Turkey Accepted 13 March 1998
Abstract A study was conducted to determine whether the use of the enzyme-linked immunosorbent assay (ELISA) improves detection of brucellosis in individual sheep. Sera from 132 sheep that aborted due to B. melitensis were used to assess the ef®cacy of the ELISA to detect brucellosis in sheep. ELISA results were compared with those of the serum agglutination test (SAT), complement ®xation test (CFT), Rose Bengal Plate test (RBPT) and the skin delayed-type hypersensitivity (SDTH). Sera from 11132 sheep free from brucellosis were tested with the ELISA to determine its speci®city. The results show little difference in diagnostic ef®cacy between SAT, CFT, RBPT and the SDTH test, although the skin test indicated infection in some seronegative sheep. A combination of CFT and SAT, or CFT and RBPT, or CFT and SDTH tests indicated brucellosis in 75± 88% of the infected animals, whereas the ELISA indicated infection in 97±100% of those animals. Seventeen (0.2%) from the 11132 negative sheep sera tested positive with the ELISA, indicating a 99.8% speci®city. We concluded that screening sheep for brucellosis with ELISA improves Brucella diagnosis, and in areas of low prevalence it will help to maintain ¯ocks of sheep free from brucellosis. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Brucella melitensis; ELISA; Serological diagnosis; Delayed-type hypersensitivity test
1. Introduction Brucella melitensis, the causal organism of Malta fever in man, has not yet been isolated from animals in the Netherlands where the milk ring test, serum agglutination test (SAT) and complement ®xation test (CFT) are used for the surveillance for brucellosis. Although the Netherlands have been free from brucellosis for many years, trade in animals with various *Corresponding author. Tel.: +0320 238150; fax: +0320 238153.
countries greatly increases the possibility of introducing brucellosis in healthy ¯ocks. Therefore, a sensitive test that indicates possibly infected sheep is of great value. The milk ring test that is widely used to detect brucellosis in dairy cattle is not sensitive enough to detect brucellosis in sheep (Shimi and Tabatabayi, 1981). The SAT, although adequate to detect acute infection, may fail to agglutinate with antibodies present in sheep sera, or give a marked prozone phenomenon (Renoux et al., 1956; Goguer, 1965;
0921-4488/98/$ ± see front matter # 1998 Elsevier Science B.V. All rights reserved. PII S0921-4488(98)00111-4
2
Z. Bercovich et al. / Small Ruminant Research 31 (1998) 1±6
Le Pennec, 1967). The use of the Rose Bengal Plate test (RBPT) which is considered a valuable screening test (Farina, 1985) is less effective than the CFT detecting brucellosis in individual sheep (Joint FAO/WHO Expert Committee on Brucellosis, Technical report 740, 1986). Further, its ef®cacy is in¯uenced by cell concentration and the standardization procedure of the antigen (Blasco et al., 1994a; Hosie et al., 1985). The CFT which is considered the most effective test for diagnosing brucellosis in sheep (Joint FAO/WHO Expert Committee on Brucellosis, Technical report 740, 1986) has no particular advantage over the SAT performed in a hypertonic environment of 5±20% NaCl. Moreover, some infected animals may elude detection with the CFT (Karmann and Schloz, 1956; Levchenco and Drozhzhin, 1958; Blasco et al., 1994b). However, other studies demonstrate the value of the CFT, and the skin delayed-type hypersensitivity (SDTH) test for the detection of brucellosis in sheep (Ebadi and Zowghi, 1983; Ebadi, 1984; Loquerie and Durand, 1984). Although the currently in use serological tests effectively detect brucellosis on a ¯ock basis, they are not always reliable for detecting infection in an individual animal. This is a major problem in areas of low prevalence of brucellosis, and where trade in sheep may introduce brucellae in areas free from brucellosis. Since neither a single serological test nor a combination of several serological tests can detect all infected sheep in a ¯ock, it is opportune to assess whether the use of the enzyme-linked immunosorbent assay (ELISA) improves detection of infected sheep. While the ef®cacy of the ELISA to detect Brucella ovis in rams has been extensively evaluated in recent years, relatively little information is available on the ef®cacy of the ELISA to detect sheep infected with B. melitensis. The objectives of the present study were to compare ELISA results with those of the SAT, CFT, RBPT and the SDTH tests, and to establish whether the use of the ELISA is a meaningful addition to the diagnosis of sheep brucellosis. 2. Material and methods 2.1. Animals Two ¯ocks of fat-tailed sheep, in the Konya province of Turkey, from which B. melitensis was isolated
from aborted fetuses were chosen for this study. Group A, with a history of B. melitensis infection and vaccination against brucellosis with Rev 1, consisted of 55 sheep that aborted. Group B, with a history of B. melitensis infection and no vaccination against brucellosis, consisted of 77 sheep that aborted. Group C consisted of 11132 Brucella-free Dutch sheep. 2.2. Serum samples and serological tests Although the aborting sheep were marked at the time of abortion they were ear-tagged a few weeks later, when blood samples were collected for serological examination. Sera from those sheep were tested with the SAT, CFT, RBPT and ELISA. The 11132 sera from brucellosis-free sheep were tested with the ELISA only and the results were used to calculated the speci®city of the assay. The SAT was done with an antigen prepared from B. abortus S-99. The antigen, diluted in hypertonic phenol-saline (5% NaCl), was standardized to give 50% agglutination at a ®nal dilution of 1:500 with the second international standard anti-Brucella abortus serum. The SAT was carried out according to the procedure used at the brucellosis research laboratory of ID-DLO, Lelystad, the Netherlands. A 75% agglutination or more in a serum diluted 1:15 was considered positive. The CFT was done according to Hill (1963). The sera were inactivated at 608C for 55 min to prevent anti-complementary activity. Sera giving 100% inhibition of hemolysis at a serum dilution 1:10 or higher were considered positive. The RBPT was performed with an antigen produced by RhoÃne MeÂrieux, Lyon, France, kindly provided by Dr. B. Garin-Bastuij, CNEVA Maisons-Alfort, France. The test, using 50 ml serum and 50 ml antigen, containing 4±5% cells, was done on a glass plate. After the test sera and antigen were mixed, the plate was shaken for 20 s every minute for 4 min. Sera that tested negative or doubtfully positive after 4 min were read again 4 min later. Test results were recorded as positive or negative. Animals were regarded infected if they tested positive in CFT and either SAT, RBPT or SDTH. The indirect ELISA was performed as described by Bercovich and Taaijke (1990) with some modi®cations. Brucella abortus strain 99 grown on trypticase-
Z. Bercovich et al. / Small Ruminant Research 31 (1998) 1±6
soy agar at 378C was used to prepare the antigen. The bacterial growth was harvested with sterile saline, autoclaved for 25 min at 1218C, cooled to room temperature and ®ltered through a sterile gauze to remove agar particles. The bacteria were then disrupted by soni®cation for 20 min at 20 Kc/s on a 500 W disintegrator. The resulting suspension, supplemented with 0.1% formaldehyde (®nal concentration), was stored in stock solution at 4±68C. 2.2.1. Buffers A 0.01 M phosphate-buffered saline (PBS) pH 6.3 was used to dilute the antigen. Dilution of the test serum was made in 0.01 M PBS pH 7.2 containing 0.05% Tween 20. The washing solution consisted of demineralized water with 0.05% (v/v) Tween 20. Horseradish peroxidase conjugated mouse monoclonal anti-bovine IgG1 (CVI-BIgG1, 15.8.1) that detects bovine as well as ovine IgG1 was diluted in 0.5 M PBS pH 7.2 containing 0.05% Tween 20. The substrate that was prepared by dissolving 1 g 5-amino-2-hydroxybenzoic acid (Merck, art. 819019) in 1 l 0.01 M PBS containing 0.04 g Na2EDTA had a pH of 5.9±6.0. The substrate solution was divided into 9 ml amounts and kept at ÿ208C. Prior to use 0.005% of fresh H2O2 was added. The working dilutions of antigen and conjugate were determined by checker-board analysis using a sheep serum containing 1000 CFT units as positive control. A pool of 20 sera from Brucella-free sheep was used as negative control. After the optimal antigen and conjugate concentration was determined, a cut-off point for the ELISA that is suitable for use in the ®eld was established with the aid of 500 sheep sera that tested negative with the CFT and RBPT. As sensitivity and speci®city may vary according to the reagents used in the assay, we chose as a cut-off point an extinction value equal to 4 units/mlÿ1. The ELISA plates were then ®lled with 0.1 ml diluted antigen, sealed and incubated overnight at 48C. The coated plates were then stored at ÿ208C until used. 2.2.2. The ELISA was performed as follows After defrosting the antigen in a 378C waterbath the plates were washed ®ve times with a washing solution (total amount approximately 500 ml). Test sera were diluted 1:50 with PBS buffer pH. 7.2. This buffer was also used to ®ll the wells of the coated plates with
3
0.1 ml buffer. Diluted serum (0.1 ml) was then dispensed in Row A and serial two-fold dilutions of test and control sera were made. After 1 h incubation at 378C the plates were washed ®ve times and 0.1 ml of diluted mouse monoclonal anti-bovine IgG1 conjugate was added to each well. After 1 h incubation at 378C, the plates were washed ®ve times and 0.1 ml substrate and H2O2 solution was added to each well. After overnight incubation at room temperature, the plates were shaken on a micro-shaker, and the results were recorded on an Easy reader EAR 400 at 450 nm (SLTLabinstruments, Austria). 2.3. Brucellin and SDTH test The brucellin used in this study was the same as that used in cattle in studies described earlier (Bercovich and Ter Laak, 1990; Bercovich et al., 1996). The Brucella abortus strain 45/20 was used to prepare the brucellin according to Bercovich et al. (1995). The protein concentration of the brucellin was determined by the Kjeldahl method and standardized in phosphate buffer pH 7.2 to contain 1.0 mg protein/mlÿ1. The 132 sheep that aborted in ¯ocks A and B and 30 Brucella-free Dutch sheep were tested with the SDTH test. All sheep were injected intradermally with 0.1 ml brucellin (100 mg protein) in the lower eyelid; some infected sheep were also injected in the tail. A clearly manifest asymmetry of the head caused by a visible swelling of the lower eyelid or a palpable swelling in the tail at the injection site were considered a reaction elicited by the injection with brucellin. 3. Results Thirty-seven of the 55 (67.2%) sheep in ¯ock A, and 48 of the 77 (62.3%) sheep in ¯ock B tested positive with all the tests. The percentage of infected animals that tested positive with each of the tests is summarized in Table 1. The serological response of the sheep that did not test positive in all the tests is summarized in Table 2. The combination of CFT and SAT failed to indicate infection in six sheep in each of the infected ¯ocks. The CFT and RBPT failed to indicate infection in seven sheep in ¯ock A and four in ¯ock B, while the CFT and SDTH test failed to indicate infection in
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Z. Bercovich et al. / Small Ruminant Research 31 (1998) 1±6
Table 1 Percentage of infected animals that tested positive with the SAT, CFT, ELISA RBPT and SDTH Serological test Flock A Flock B
SAT
CFT
ELISA
RBPT
SDTH test
78.2 88.3
85.4 88.3
98.2 97.5
78.1 83.1
85.4 76.6
respectively ®ve and 13 sheep in ¯ocks A and B. The ELISA tested positive in all but one (39, ¯ock B) of the aborting animals. Seventeen of the 11132 sera from Brucella-free sheep tested positive with the ELISA at 4±32 units. Two of those sheep, each in a different ¯ock, tested with the CFT respectively at 20 and 100 units. Although differences in skin swellings at the injection site of the brucellin were observed among the animals that aborted, all swelling were clearly visible and palpable. All the Brucella-free sheep tested negative with the SDTH test. 4. Discussion The objectives of the present study were to compare the ELISA results with those of the SAT, CFT, RBPT and the SDTH test, and to establish whether the use of the ELISA is a meaningful addition to the diagnosis of sheep brucellosis. Detection of brucellosis with serological tests in areas with low prevalence is problematic because serum titres of some infected animals sharply decline after infection, or because infected sheep do not react in the commonly used serological tests (Morgan, 1974). With this in mind, the SDTH test which is independent of humoral antibody and more sensitive than SAT and CFT was also included in this study (Ebadi and Zowghi, 1983). Since diagnostic errors occur, the relative sensitivity and speci®city of a test are used to assess its ef®cacy to detect infection. These criteria can only be implemented if a gold standard is used to establish the true disease state of the animal. Isolation of Brucella could be a gold standard as it unequivocally establishes the cause of infection. However, bacteriological examination cannot always be relied on to prove the presence or absence of infection in individual animals (Unel et al., 1969; Alton et al., 1975;
Table 2 Serological and SDTH test results of sheep that did not test positive in all tests Sheep number Flock A 4 13 20 22 28 30 31 32 35 39 40 41 46 47 48 51 54 55 Flock B 7 8 10 13 17 18 24 32 33 37 38 40 39 42 44 51 52 56 61 63 64 66 67 70 71 72 73 74 76
SAT units
CFT units
ELISA units
Rose Bengal
SDTH test
120 <30 30 960 120 <30 <30 <30 <30 <30 <30 30 60 <30 <30 <30 <30 <30
200 <20 <20 200 200 <20 50 100 <20 <20 <20 200 200 20 <20 50 <20 100
512 8 4 512 512 16 128 256 32 16 8 512 512 64 8 64 64 64
ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ
ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ
240 <30 <30 30 <30 <30 30 30 30 960 <30 <30 30 60 960 30 30 <30 <30 120 240 <30 30 30 120 30 73 240 120
200 <20 <20 <20 <20 50 20 200 <20 100 50 200 <20 200 200 100 50 <20 <20 100 200 <20 50 50 200 50 50 100 200
512 16 4 16 16 256 64 512 16 64 128 512 <4 512 512 128 64 4 32 256 256 8 32 32 256 64 128 128 256
ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ
ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ
Z. Bercovich et al. / Small Ruminant Research 31 (1998) 1±6
Corbel et al., 1980). Therefore, the presumptive diagnosis provided by the serological tests is usually accepted as indication for brucellosis. Although the sheep in this study were marked after abortion, bacteriologic test results could not be related to the individual animal because they were not ear-tagged at the time of abortion. Consequently, the presence of brucellosis was indirectly established by the isolation of B. melitensis from the aborted fetuses, but the infection status of individual animals was determined by the serological tests or the SDTH test. We also considered serologic test results of the vaccinated sheep indicative of infection because the immune response to Rev 1 vaccination usually disappears 3± 5 months after vaccination (Falade, 1983; Madsen, 1993). Our study shows little difference in diagnostic ef®ciency between the SAT, CFT, RBPT and the SDTH test on a ¯ock basis (Table 1). With the exception of the ELISA, the tests detected brucellosis in 76± 88% of the animals con®rming the observation that no single test effectively detects brucellosis (Mahajan and Kulshreshtha, 1991). This clearly suggests the need to use several serological tests. However, a combined used of CFT and SAT indicated infection in, respectively, 89 and 92.2% of aborting sheep in ¯ocks A and B, whereas a combination of the CFT and the RBPT indicated infection in 87.3% of the sheep in ¯ock A and 93.6% in ¯ock B (Table 2). These results clearly differ from the results of JimeÂneÂz de BagueÂs et al. (1992) showing complete agreement between CFT and RBPT. While the SDTH test indicated infection in sheep that tested negative with SAT, CFT and the RBPT (Table 2), the combination of CFT and the SDTH indicated infection in respectively only 91 and 83.2% of the sheep in ¯ock A and B. Further, our study did not con®rm the ef®cacy of the RBPT or the SDTH test reported in other studies (Farina, 1985; Blasco et al., 1994b), although it con®rmed the relative ef®cacy of the CFT to detect brucellosis (Blasco et al., 1994b). In practice, it is expected that a highly sensitive test will identify most diseased animals. Likewise, a speci®c test is expected to correctly identify most truenegative animals. But, since speci®city is inversely related to sensitivity the norm to indicate infection has to be a compromise between those two parameters. Since an undetected infected animal constitutes a
5
hazard, and over-condemning of animals is undesirable, we set the cutoff point of the ELISA to detect 4 ELISA units/ml. Using this norm the ELISA indicated infection in 131/132 of the animals that aborted. Further, the value of the ELISA is demonstrated by the indication of infection in seronegative sheep 40, 48, 10, 56 and 66 (Table 2). This study also shows that only a few sera tested false-positive with the ELISA. A sample of 11132 sheep sera revealed 17 (0.2%) sheep of which 8, 6 and 3 sheep tested in the ELISA 4, 8 and 32 units respectively. Although two of those sheep also tested positive with the CFT, ELISA results were considered falsepositive because, retrospectively, those and all other ¯ocks proved to be free from brucellosis. This, and the size of the examined sample clearly indicate a 99.8% speci®city. In this respect the diagnostic speci®city of the ELISA proved very satisfactory in that the cutoff point value of <4 units/mlÿ1 may truly represent negative ewes. As not all the animals of the infected ¯ocks were tested with the ELISA, calculation of the sensitivity could not be done. Nevertheless, the sensitivity of the ELISA found in other studies (JimeÂneÂz de BagueÂs et al., 1992; Delgado et al., 1995), and the speci®city of the assay found in this study conclusively demonstrate the reliability of the assay. These ®ndings clearly endorse the use of the assay for the detection of infected sheep in areas of low prevalence. It can be inferred, therefore, that the ELISA is an excellent choice on which to base the surveillance for brucellosis in sheep. Any tendency of `over condemning' of ELISA-positive animals is compensated by the knowledge that a very high percentage of the potentially infected animals have been identi®ed and can be removed. Acknowledgements We thank Toos Dekker and Bert Tetenburg for their assistance with the serological examination. References Alton, G.G., Maw, J., Rogerson, B.A., McPherson, G.G., 1975. The serological diagnosis of bovine brucellosis: An evaluation of the complement fixation, serum agglutination and rose bengal tests. Aust. Vet. J. 51, 57±63.
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Bercovich, Z., Taaijke, R., 1990. Enzyme immunoassay using mouse monoclonal anti-bovine antibodies for the detection of Brucella abortus antibodies in cow milk. J. Vet. Med. B 37, 753±759. Bercovich, A., Ter Laak, E.A., 1990. An evaluation of the Delayedtype hypersensitivity test for the diagnosis of brucellosis in individual cattle: A field study. Vet. Microbiol. 22, 241±248. Bercovich, Z., Eger, A., Dekker, T., Haagsma, J., 1995. Production of Brucella allergens and evaluation of their biological activity in a guinea pigs bio-assay. J. Vet. Med. B 42, 19±27. Bercovich, Z., Dekker, T., Eger, A., Haagsma, J., 1996. A comparison of the potency of several Brucella allergens to detect brucellosis in cattle. Vet. Res. Comm. 20, 141±151. Blasco, J.M., Garin-Bastuij, B., Martin, C.M., Gerbier, G., Fanlo, J., JimeÂneÂz de BagueÂs, M.P., Cau, C., 1994a. Efficacy of the Rose Bengal and complement fixation antigens for the diagnosis of Brucella melitensis infection in sheep and goats. Vet. Rec. 134, 415±420. Blasco, J.M., Marin, C., JimeÂnez de BagueÂs, M., BarbereÂn, M., Hernandez, A., Molina, L., Velasco, J., Diaz, R., Moriyon, I., 1994b. Evaluation of allergic and serological tests for diagnosing Brucella melitensis infection in sheep. J. Clin. Microbiol. 32, 1835±1840. Corbel, M.J., Scott, A.C., Ross, H.M., 1980. Properties of a cellwall-defective variant of Brucella abortus of bovine origin. J. Hyg. 85, 103±113. Delgado, S., Fernandez, M., Carmenes, P., 1995. Evaluation of an enzyme-linked immunosorbent assay for the detection of sheep infected and vaccinated with Brucella melitensis. J. Vet. Diag. Investig. 7, 206±209. Ebadi, A., Zowghi, E., 1983. The use of allergic test in the diagnosis of Brucella melitensis infection in sheep. Br. Vet. J. 139, 456±462. Ebadi, A., 1984. Evaluation of the allergic test in the diagnosis of brucellosis in sheep. Develop. Biol. Standard. 56, 387±391. Falade, S., 1983. Serological response of sheep to Brucella melitensis Rev 1 vaccine. 30, 546±551. Farina, R., 1985. Current serological methods in Brucella melitensis diagnosis. In: Verger, J.M., Plommet, M. (Eds.), Brucella melitensis. Martinus Nijhoff, Dordrecht, pp. 139±146. Goguer, M., 1965. Comparison of the plate and tube agglutination tests for brucellosis in sheep. Vet. Bull. 35, Abstr., 3710. Hill, W.K.W., 1963. Standardization of the complement fixation test for brucellosis. Bull. Off. Int. Epiz. 60, 401±417.
Hosie, B.D., Al-Bakri, O.M., Futter, R.J., 1985. Survey of Brucellosis in goats and sheep in the Yemen Arab Republic: Comparison of tests for Brucella melitensis infection in sheep. Trop. Anim. Health Prod. 17, 93±99. JimeÂneÂz de BagueÂs, M.P., Marin, C.M., Blasco, J.M., Moriyon, I., Gamazo, C., 1992. An ELISA with Brucella LPS antigen for the diagnosis of Brucella melitensis infection in sheep and for the evaluation of serologic responses following subcutaneous or conjunctival Brucella melitensis strain Rev 1 vaccination. Vet. Microbiol. 30, 233±241. Karmann, P., Schloz, H.D., 1956. Beitrag zur BekaÈmpfung des Maltafiebers der Schafe in Lande Nordrhein-Westfalen. Mh. Tierheilk. 8, 127±134. Le Pennec, J., 1967. Serology of ovine brucellosis. Vaccination and infection antibodies negative to Wright's agglutination test but positive to the complement fixation test. Vet Bull., 37. Abstr. 3571. Levchenco, I.D., Drozhzhin, V.N., 1958. Use in outbreaks of abortion in sheep of the agglutination test with 10% NaCl. 28, Abstr. 1006. Loquerie, R., Durand, M.P., 1984. Clinical application of a skin test for delayed hypersensitivity, using a commercial allergen, for detecting brucellosis in sheep and goats. Develop. Biol. Stand. 56, 407±410. Madsen, M., 1993. The serological response to vaccination with Brucella abortus strain 19 and Brucella melitensis Rev 1, as measured by Rose Bengal, serum agglutination and complement fixation tests. Zimbabwe Vet. J. 24, 13±20. Mahajan, N.K., Kulshreshtha, R.C., 1991. Comparison of serological tests for Brucella melitensis infection in sheep. Trop. Anim. Health Pro. 23, 11±16. Morgan, W.J.B., 1974. Brucellosis of small ruminants. Diagnosis, control and eradication. Bull. Off. Int. Epiz. 82, 93±96. Renoux, G., Alton, G., Mahaffey, L.W., 1956. Etude sur la brucellose ovine et caprine: v. ReÂactions seÂrologiques dans le sang de breÂbis reÂcemment par Brucella melitensis. Arch Inst. Pasteur, Tunis. 33, 33±41. Shimi, A., Tabatabayi, A.H., 1981. Pathological, bacteriological and serological responses of ewes experimentally infected with Brucella melitensis. Bull. Off. Int. Epiz. 39(11/12), 1411±422. Unel, S., Williams, C.F., Stableforth, A.W., 1969. Relative value of the agglutination test, complement fixation test and Coombs test in the detection of Brucella melitensis infection in sheep. J. Comp. Pathol. 79, 155±159.