Inrernukvwl JournalforParosirology Vol. 20, No. 3, pp. 341-345, Printed in Great Errfain
1990
0
002@-7519/90 P3.M) + 0.00 Pergamon Press p/c Sociefyfor Porosifology
1990 Awlralian
A SLIDE ENZYME-LINKED IMMUNOSORBENT ASSAY (SELISA) FOR THE DIAGNOSIS OF BABESIA BO VIS INFECTIONS AND FOR THE SCREENING OF ~~~~~~~-SPECIFIC MONOCLONAL ANTIBODIES and BRIAN V. GOODGER~
MERCY W. KUNGU*$
* Department of Parasitology, University of Queensland, St. Lucia, Queensland 4067, Australia t CSIRO, Division of Tropical Animal Production, Long Pocket Laboratories, Private Bag No. 3, P.O., Indooroopilly, Queensland 4068, Australia (received 7 Augusf 1989; accepted 1 January 1990)
Abstract-KuNc%u M. W. and GWDGER B. V. 1990. A slide enzyme-linked immunosorbent assay (SELISA) for the diagnosis of&besia bovbinfections and for the screening of &b&a-specific monoclonal antibodies. InfernafiondJ~urnalforParusifology20: 341-345. A slide enzyme-linked immunosorbent assay (SELISA), a mo~fication of the standard ELISA technique, was developed for detection of Bubesiabovis antibodies in bovine sera. Smears of B. b&s-infected blood were used as the source of antigen in the test which was read using a light microscope. Monoclonal antibodies to defined B. bovis antigens were used to demonstrate the cellular specificity of the test. The SELISA was shown to be as sensitive as existing non-enzyme based serological tests for B. bovis. Comparative to the conventional ELISA technique, it was more economical and technically simpler, thus making it an ideal test for field application. INDEX KEY WORDS: Enzyme-linked immunosorbent assay; slide ELISA: indirect fluorescent antibody test; indirect haemagglutination test; Eabesiu bovis; diagnosis; monoclonal antibodies.
SELISA is sensitive, specific, economical and very convenient to perform. A comparison of the sensitivity and specificity of this test with that of the IHA and IFA tests is also reported here.
INTRODUCTION IN recent years a number of serological tests for the
diagnosis of babesiosis have been developed. These tests include the complement fixation test; various agglutination tests including indirect haemagglutination (IHA) and parasitized erythrocyte agglutination tests; gel precipitation; indirect fluorescent antibody technique (IFAT); enzyme immunoassay and radioimmunoassay techniques (reviewed by Callow, 1984; Wright, Schuntner & Goodger, 1986). For any serological test to be reliable and useful it must meet certain criteria. It should be highly specific, highly sensitive, reproducible, inexpensive, quick and safe to perform (Wright et al., 1986). The IFAT and the enzyme-linked immunosorbent assay (ELISA) are the most frequently used in the diagnosis of babesial infections. The IFAT, though sensitive, has been shown to lack specificity as some negative sera can have high titres (154, see Johnston, Pearson & Leatch, 1973) and is also limited by the subjective nature of result interpretation and by operator fatigue problems. The ELBA, on the other hand, has been shown to be very sensitive and specific and is simple to perform (Waltisbuhl, Goodger, Wright, Commins & Mahoney, 1987). A modification of the standard ELISA technique, the slide ELISA (SELISA), is reported here. The $ To whom all correspondence should be addressed.
MATERIALS AND METHODS Preparation of antigen slides for the IFAT and the slide EUSA. Whole blood, containing l&15% infected erythrocytes, was collected in EDTA from a Babe& bovis-
infected calf. After centrifugation at 3000 g for 10 min the cells were washed three times in 0.02 M-PBS pH 7.2. The washed cells were then reconstituted to a 50% suspension in 0.5% bovine serum albumin and PBS. Thin smears were prepared with a cytocentrifuge (Cytosmear; Perkin &Elmer). The smears were air-dried, fixed in anhydrous acetone for 5 min, wrapped in aluminium foil and stored at - 70°C for at least 1 year. Test sera. Cattle were obtained from tick-free areas and their sera shown to be negative for 3. bovis antibody by IFAT (Johnston et al., 1973) and ELISA (Waltisbuhl et al., 1987). Twenty-two such cattle were given a single infective dose of B. bovis by either syringe-passage or by tick transmission (Mahoney, Wright & Goodger, 1979). The cattle, along with 10 negative controls, were maintained in a tick-free environment for 4 years and serum samples obtained on a monthly basis. Twenty-seven of the sera, eight of which were negative, were used to evaluate the sensitivity and specificity of SELISA and to compare the IHA test (Goodger & Mahoney, 1974). IFAT (Johnston et al., 1973) and the SELISA. Monoclonal antibodies (Mabs). The Mabs were raised to partly purified B. bovis fractions known to be protective from
341
342
M. W. KUNGYJ and B. V. GOODGER
various vaccination experiments at the CSIRO laboratories (Wright, White, Tracey-Patte, Donaldson, Goodger, Waltisbuhl & Mahoney, 1983). These Mabs were tested by both the SELISA and the IFAT to assess and compare cellular specificity. Tissue culture fluid (RPM1 1640 + 10% foetal calf serum) was used as a negative control. Secondary sera. Conjugated antisera to bovine and mouse IgG were obtained commercially (Kirkegaard & Perry, U.S.A.). Horse-radish peroxidase conjugates were used in the ELISA and the SELISA while fluorescein isothiocyanate (FITC) conjugates were used in the IFAT. Substrate. The substrate of choice for the SELISA was 4chloro-1-naphthol. The working solution contained l-3 mg substrate dissolved in 2 ml methanol, to which was added 10 ml 0.02 M-Tris buffered saline pH 7.4 and 40 ~1 H,O,. The slide enzyme-linked immunosorbent a.woy (SELISA) procedure. A series of defined experiments to determine the optimum operating conditions for the test was undertaken. The series included tests to determine incubation times, temperatures and dilutions of primary and secondary antisera. The following procedure was finally used. The antigen slides were thawed at 37°C for 10 min before use. Each smear was divided into eight to 10 rectangular compartments using an oil-based marker pen. Circular areas of approximately 5 mm diameter were marked in each compartment with a diamond pencil. Test sera were doubly diluted in PBS containing 2% horse serum and 15 ~1 aliquots of each dilution placed in each circle in a known sequence. The slides were then placed in a moist chamber and incubated
overnight at 4 or 22°C for 90 min. The samples were washed with PBS, the slides given two 5-min rinses in fresh PBS and air-dried. Fifteen microlitres of optimally diluted (normally l/50 to I/100) horse-radish peroxidase-conjugated secondary antibody was then added to each test circle and incubated in a moist chamber at 37’C for 30 min. The slides were washed twice for 5 min with Tris buffered saline and air-dried. One drop (15~1) of the substrate was added and allowed to react in a moist chamber for 20 min at room temperature. The substrate was washed off with distilled water, the slides airdried and mounted in glycerol-PBS (9 vol. glycerol:1 vol. PBS) and examined using an oil-immersion x 90 objective fitted to a light microscope. Endogenous peroxidase in tissue samples can cause false positive reactions and attempts were made to inhibit such reactions in the SELISA by chemical means using hydrogen peroxide or sodium azide, or by pH adjustment (Malorny, Bildau & Sorg, 1988). Attempts to inhibit endogenous peroxidase in the SELISA were unsuccessful even when the chemicals were tried at different concentrations for varying periods of time; there was a loss of antigenicity, as was indicated by reduction in intensity of staining. Likewise attempts to counter-stain the non-infected erythrocytes with dyes such as Eosin-Azure-II solution resulted in loss of the substrate staining, and accordingly the slides were examined without counter-staining. Statisrics. Statistical comparisons were made using correlation coefficients of the IHA test, the IFAT and the SELISA results on the 27 animals tested at both 12 and 36 months post-infection (p.i.).
TABLE ~-RESULTSFORTHEBOVINESERAREACTIONSBYTHEIHA,TI~EIFA~ES~SANDTHESELISA Test
IFAT
IHA Months
Serum sample No.* I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
SELISA
post-infection
12
36
12
36
12
36
I+ 2+ I+ 2+ 3+ 3+ 3+ 2+ 3+ I+ I+ 1+ I+ 1+ I+ 1+ 2+ 2+ I+
2+ 1+ 2+ 2+ 3+ 2+ 3+ 2+ 3+ 1-t 2+
I+ 2+ 1+ 1+ 3+ I+ 1+ I+ 2+ I+ 2+ _
1+ 1+ 1+ I+ 2+ I+ 1+ 2+ I+ I+ I+ -
I+ 1+ 3+ z!z 3+ I+ 1+ 2+ 2+ f I+ _
1+ 1+ 1-t 1+ 3+ It I+ 2+ 2+ I+ I+ _
1+
I+ 2+
2+ I+ 1-t
3+ 3+ If I+
f 1+ 1+ 3+ 3+ 2+ 2+
t+ 2+ + 3+ 3+ 1+ I+
2+ 1+ 2+ 2f I+ I+
f _ i _ _
- Negative; f doubtful; * Serum samples obtained
_
-
I + weak positive; 2 + positive; 3 + strong positive. from infected (l-19)
and non-infected
(2&27) cattle
_ * _
343
Slide ELISA RESULTS The IFAT The negative sera showed faint staining of both the infected cells and the parasites when used at low dilutions of up to 1:50. Above this dilution, any fluorescence observed was considered specific. An overall score for each reaction was given after examination of the staining at the different dilutions (Table 1). The staining patterns given by different Mabs with B. &v&infected erythrocytes are shown in Table 2.
negative except on two occasions when faint reactions were obtained at 150. The individual reactions are shown in Table 1. Comparison of IHA test, IFAT and SELISA The results obtained by these three tests are compared in Table 1. The pair of tests giving the closest results were IFAT and SELISA. In addition, the SELISA method gave the closest agreement between 12 and 36 months (see Table 1). A statistical analysis of these comparisons is detailed in Table 3. DISCUSSION
TABLE
SELISA
Z-THE
INFECTED
ERYTHROCYTES
STAINING WITH
PATTERN
OF
Babesia-
MONOCLONAL
DIFFERENT
ANTIBODIES*
Mabs A B C D E
Staining pattern (specificity) Infected erythrocyte especially the cell membrane Infected erythrocyte and parasite Punctate staining of the parasite Whole parasite Parasite membrane
* A similar staining pattern was obtained with the IFA technique.
The SELISA The positiveenegative dilution cut-off for bovine sera was determined to be 1:SO using incubation times of 1.5 h at room temperature (22°C). Incubation overnight at 4°C gave similar results. A positive reaction was indicated by bluish-purple staining of the B. bovis-infected erythrocytes or of the parasite, or of both, depending on the specificity of the staining antibody (Fig. 1). In a negative reaction, no staining of either the infected cells or the parasite occurred. The intensity of the staining was used to classify the reactions into negative, weak positive, positive or strong and positive. Negative and positive reactions were easily discriminated at 1:50 dilution. The negative control for the Mabs (tissue culture fluid) gave no staining. Of the sera from infected cattle maintained under tick-free conditions lSjl9 were positive and 2/19 were & (doubtful) 12 months after the infection, while at 3 years after infection 17/19 were positive (results of two animals were inconsistent). Sera from the uninfected cattle maintained under tick-free conditions were all
TABLE
J-CORRELATION
OF SCORES BETWEEN DIFFERENT
12 AND 36 MONTHS Tests
IHA and IFA IHA and SELISA IFA and SELISA * P < 0.05.
TESTS AT
POST-INFECTION
Correlation 36 months p.i. 12 months pi. 0.64 0.56 0.85*
0.42 0.56 0.79;
The SELISA was found to be extremely sensitive as it detected antibodies to B. bovis for at least 36 months (see Table 1) after a single infection (no tests were done on sera beyond this period). Moreover comparison with the IFA and the IHA tests results showed that the SELISA was also sensitive and relatively consistent in detecting antibodies not only at 12 months but also at 36 months post-infection (Tables 1 and 3). The diversity in the staining patterns with different Mabs (Table 2) demonstrated the unique and precise cellular specificity of the test and confirmed the data previously obtained when these Mabs were screened by IFAT. Such information cannot be gleaned with the conventional ELISA or the IHA test. This also makes the SELISA an ideal test in screening hybridomas as Mabs can be selected not only on their avidity but also on their specificity. The SELISA has other advantages over most existing serological tests for diagnosis of babesiosis. The IFAT requires an expensive fluorescent microscope and it is also known that stray ultra-violet light can present a health hazard. This test lacks specificity depending on the species, is tedious to interpret and may cause operator fatigue (Todorovic & Carson, 1981; Callow, 1984; Schuntner & Wright, 1989). The IHA was a good test for B. bovis but was a difficult test to perform (Wright et al., 1986). The SELISA should be ideally suited for field application if blood smears can be stored, because only a conventional light microscope is required and this could be illuminated using batteries. In comparison with the conventional ELISA, the SELISA is cheap, very convenient to perform, and more importantly, the completed test slides can be kept for at least 1 week at room temperature (22°C) for later confirmation of results. During the development of the SELISA technique, a similar test to the SELISA for the diagnosis of malaria in man, the malaria immunoperoxidase assay, was reported (Lim, 1988). This test was shown to be as sensitive and specific as the IFAT in detecting antimalaria antibodies and was more convenient to perform. While similar to the Babesia SELISA in performance, no detailed information is available regarding its long-term sensitivity. The SELISA should be applicable in the diagnosis of other protozoan diseases of man and animals,
344
M. W. KUNG’U and B. V. GOODGER
FIG. 1.Scale bars, 13 pm. The SELISA staining of Babesia bovis-infected erythrocytes after reaction with: (a) B. bovis positive serum. Both the parasites and the infected erythrocytes stain strongly. (b) Monoclonal antibody A. The parasites do not stain but the infected erythrocytes, especially the erythrocytic membrane stain strongly. (c) Monoclonal antibody C. The parasites stain strongly while the erythrocytes do not.
Slide ELISA provided that particulate antigen, preferably whole parasite, can be easily obtained and without loss of antigenicity.
of the stored
Acknowledgements-We are grateful to the Australian International Development issistance Bureau and the CSIRO-University of Queensland Research Program for financial support, and to Drs I. G. Wright and H. M. D. Hoyte for reading the manuscript. REFERENCES CALLOW L. L. 1984. Piroplasms. In: Animal Health in Australia, Protozoa1 and Rickettsial Diseases, Vol. 51, pp. 121-169. Australian Government Publishing Service, Canberra. GRUDGER B. V. & MAHONEY D. F. 1974. Evaluation of the passive haemagglutination test for the diagnosis of Babesia argentina infection in cattle. Australian Veterinary Journal 50: 246-249. JOHNSTON L. A. Y., PEARSON, R. V. & LEATCH G. 1973. Evaluation of an indirect fluorescent antibody test for detecting Babesia argentina infection in cattle. Australian Veterinary Journal 49: 373-377. LIM T. S. 1988. A sensitive malaria immunoperoxidase assay for the detection of Plasmodium falciparum antibody. American Journal of Tropical Medicine and Hygiene 38: 255-257. MAHONEY D. F., WRIGHT I. G. & GOODGER B. V. 1979. Immunity in cattle to Babesia bovis after single infections with parasites of various origin. Australian Veterinary
345
Journal55: 10-12. MALORNYU., BILDAUH. & SORG C. 1988. Efficient inhibition of endogenous peroxidase without antigen denaturation in immunohistochemistry. Journal of Immunological Methods 111: 101-107. SCHUNTNERC. A. &WRIGHT I. G. 1989. Detection of Babesia bigemina in cattle by a radioimmunoassay incorporating specifically depleted antigen. Veterinary Parasitology 31: 229-24 1. TODOROVIC R. A & CARSON C. A. 1981. Methods for measuring the immunological response to Babesia. In: Babesiosis (Edited bv RISTIC M. & KREIER J. P.).I, __ DD. 381410. Academic Press; New York. WALTISBUHLD. J., GRUDGER B. V., WRIGHT I. G., COMMINS M. A. & MAHONEY D. F. 1987. An enzyme-linked immunosorbent assay to diagnose Babesia bovis infection in cattle. Parasitology Research 73: 126131. WRIGHT I. G., WHITE M., TRACEY-PALE P. D., DONALDSONR. A.. GOODGER B. V.. WALTISBUHLD. J. & MAHONEY D. F. 1983. Babesia bovii: isolation of a protective antigen by using monoclonal antibodies. Infection and Immunity 41: 244-250. WRIGHT I. G., SCHUNTNERC. A. & GRUDGER B. V. 1986. Application of nuclear and related techniques to the diagnosis and control of tick-borne parasitic diseases of livestock. In: Nuclear and Related Techniques in Animal Production and Health, Proceedings of an International Symposium on the use of Nuclear Techniques in Studies of Animal Production and Health in Different Environments, Vienna, 1986, pp. 341-350. International Atomic Energy Agency, Austria.
1990
0
002@-7519/90 P3.M) + 0.00 Pergamon Press p/c Sociefyfor Porosifology
1990 Awlralian
A SLIDE ENZYME-LINKED IMMUNOSORBENT ASSAY (SELISA) FOR THE DIAGNOSIS OF BABESIA BO VIS INFECTIONS AND FOR THE SCREENING OF ~~~~~~~-SPECIFIC MONOCLONAL ANTIBODIES and BRIAN V. GOODGER~
MERCY W. KUNGU*$
* Department of Parasitology, University of Queensland, St. Lucia, Queensland 4067, Australia t CSIRO, Division of Tropical Animal Production, Long Pocket Laboratories, Private Bag No. 3, P.O., Indooroopilly, Queensland 4068, Australia (received 7 Augusf 1989; accepted 1 January 1990)
Abstract-KuNc%u M. W. and GWDGER B. V. 1990. A slide enzyme-linked immunosorbent assay (SELISA) for the diagnosis of&besia bovbinfections and for the screening of &b&a-specific monoclonal antibodies. InfernafiondJ~urnalforParusifology20: 341-345. A slide enzyme-linked immunosorbent assay (SELISA), a mo~fication of the standard ELISA technique, was developed for detection of Bubesiabovis antibodies in bovine sera. Smears of B. b&s-infected blood were used as the source of antigen in the test which was read using a light microscope. Monoclonal antibodies to defined B. bovis antigens were used to demonstrate the cellular specificity of the test. The SELISA was shown to be as sensitive as existing non-enzyme based serological tests for B. bovis. Comparative to the conventional ELISA technique, it was more economical and technically simpler, thus making it an ideal test for field application. INDEX KEY WORDS: Enzyme-linked immunosorbent assay; slide ELISA: indirect fluorescent antibody test; indirect haemagglutination test; Eabesiu bovis; diagnosis; monoclonal antibodies.
SELISA is sensitive, specific, economical and very convenient to perform. A comparison of the sensitivity and specificity of this test with that of the IHA and IFA tests is also reported here.
INTRODUCTION IN recent years a number of serological tests for the
diagnosis of babesiosis have been developed. These tests include the complement fixation test; various agglutination tests including indirect haemagglutination (IHA) and parasitized erythrocyte agglutination tests; gel precipitation; indirect fluorescent antibody technique (IFAT); enzyme immunoassay and radioimmunoassay techniques (reviewed by Callow, 1984; Wright, Schuntner & Goodger, 1986). For any serological test to be reliable and useful it must meet certain criteria. It should be highly specific, highly sensitive, reproducible, inexpensive, quick and safe to perform (Wright et al., 1986). The IFAT and the enzyme-linked immunosorbent assay (ELISA) are the most frequently used in the diagnosis of babesial infections. The IFAT, though sensitive, has been shown to lack specificity as some negative sera can have high titres (154, see Johnston, Pearson & Leatch, 1973) and is also limited by the subjective nature of result interpretation and by operator fatigue problems. The ELBA, on the other hand, has been shown to be very sensitive and specific and is simple to perform (Waltisbuhl, Goodger, Wright, Commins & Mahoney, 1987). A modification of the standard ELISA technique, the slide ELISA (SELISA), is reported here. The $ To whom all correspondence should be addressed.
MATERIALS AND METHODS Preparation of antigen slides for the IFAT and the slide EUSA. Whole blood, containing l&15% infected erythrocytes, was collected in EDTA from a Babe& bovis-
infected calf. After centrifugation at 3000 g for 10 min the cells were washed three times in 0.02 M-PBS pH 7.2. The washed cells were then reconstituted to a 50% suspension in 0.5% bovine serum albumin and PBS. Thin smears were prepared with a cytocentrifuge (Cytosmear; Perkin &Elmer). The smears were air-dried, fixed in anhydrous acetone for 5 min, wrapped in aluminium foil and stored at - 70°C for at least 1 year. Test sera. Cattle were obtained from tick-free areas and their sera shown to be negative for 3. bovis antibody by IFAT (Johnston et al., 1973) and ELISA (Waltisbuhl et al., 1987). Twenty-two such cattle were given a single infective dose of B. bovis by either syringe-passage or by tick transmission (Mahoney, Wright & Goodger, 1979). The cattle, along with 10 negative controls, were maintained in a tick-free environment for 4 years and serum samples obtained on a monthly basis. Twenty-seven of the sera, eight of which were negative, were used to evaluate the sensitivity and specificity of SELISA and to compare the IHA test (Goodger & Mahoney, 1974). IFAT (Johnston et al., 1973) and the SELISA. Monoclonal antibodies (Mabs). The Mabs were raised to partly purified B. bovis fractions known to be protective from
341
342
M. W. KUNGYJ and B. V. GOODGER
various vaccination experiments at the CSIRO laboratories (Wright, White, Tracey-Patte, Donaldson, Goodger, Waltisbuhl & Mahoney, 1983). These Mabs were tested by both the SELISA and the IFAT to assess and compare cellular specificity. Tissue culture fluid (RPM1 1640 + 10% foetal calf serum) was used as a negative control. Secondary sera. Conjugated antisera to bovine and mouse IgG were obtained commercially (Kirkegaard & Perry, U.S.A.). Horse-radish peroxidase conjugates were used in the ELISA and the SELISA while fluorescein isothiocyanate (FITC) conjugates were used in the IFAT. Substrate. The substrate of choice for the SELISA was 4chloro-1-naphthol. The working solution contained l-3 mg substrate dissolved in 2 ml methanol, to which was added 10 ml 0.02 M-Tris buffered saline pH 7.4 and 40 ~1 H,O,. The slide enzyme-linked immunosorbent a.woy (SELISA) procedure. A series of defined experiments to determine the optimum operating conditions for the test was undertaken. The series included tests to determine incubation times, temperatures and dilutions of primary and secondary antisera. The following procedure was finally used. The antigen slides were thawed at 37°C for 10 min before use. Each smear was divided into eight to 10 rectangular compartments using an oil-based marker pen. Circular areas of approximately 5 mm diameter were marked in each compartment with a diamond pencil. Test sera were doubly diluted in PBS containing 2% horse serum and 15 ~1 aliquots of each dilution placed in each circle in a known sequence. The slides were then placed in a moist chamber and incubated
overnight at 4 or 22°C for 90 min. The samples were washed with PBS, the slides given two 5-min rinses in fresh PBS and air-dried. Fifteen microlitres of optimally diluted (normally l/50 to I/100) horse-radish peroxidase-conjugated secondary antibody was then added to each test circle and incubated in a moist chamber at 37’C for 30 min. The slides were washed twice for 5 min with Tris buffered saline and air-dried. One drop (15~1) of the substrate was added and allowed to react in a moist chamber for 20 min at room temperature. The substrate was washed off with distilled water, the slides airdried and mounted in glycerol-PBS (9 vol. glycerol:1 vol. PBS) and examined using an oil-immersion x 90 objective fitted to a light microscope. Endogenous peroxidase in tissue samples can cause false positive reactions and attempts were made to inhibit such reactions in the SELISA by chemical means using hydrogen peroxide or sodium azide, or by pH adjustment (Malorny, Bildau & Sorg, 1988). Attempts to inhibit endogenous peroxidase in the SELISA were unsuccessful even when the chemicals were tried at different concentrations for varying periods of time; there was a loss of antigenicity, as was indicated by reduction in intensity of staining. Likewise attempts to counter-stain the non-infected erythrocytes with dyes such as Eosin-Azure-II solution resulted in loss of the substrate staining, and accordingly the slides were examined without counter-staining. Statisrics. Statistical comparisons were made using correlation coefficients of the IHA test, the IFAT and the SELISA results on the 27 animals tested at both 12 and 36 months post-infection (p.i.).
TABLE ~-RESULTSFORTHEBOVINESERAREACTIONSBYTHEIHA,TI~EIFA~ES~SANDTHESELISA Test
IFAT
IHA Months
Serum sample No.* I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
SELISA
post-infection
12
36
12
36
12
36
I+ 2+ I+ 2+ 3+ 3+ 3+ 2+ 3+ I+ I+ 1+ I+ 1+ I+ 1+ 2+ 2+ I+
2+ 1+ 2+ 2+ 3+ 2+ 3+ 2+ 3+ 1-t 2+
I+ 2+ 1+ 1+ 3+ I+ 1+ I+ 2+ I+ 2+ _
1+ 1+ 1+ I+ 2+ I+ 1+ 2+ I+ I+ I+ -
I+ 1+ 3+ z!z 3+ I+ 1+ 2+ 2+ f I+ _
1+ 1+ 1-t 1+ 3+ It I+ 2+ 2+ I+ I+ _
1+
I+ 2+
2+ I+ 1-t
3+ 3+ If I+
f 1+ 1+ 3+ 3+ 2+ 2+
t+ 2+ + 3+ 3+ 1+ I+
2+ 1+ 2+ 2f I+ I+
f _ i _ _
- Negative; f doubtful; * Serum samples obtained
_
-
I + weak positive; 2 + positive; 3 + strong positive. from infected (l-19)
and non-infected
(2&27) cattle
_ * _
343
Slide ELISA RESULTS The IFAT The negative sera showed faint staining of both the infected cells and the parasites when used at low dilutions of up to 1:50. Above this dilution, any fluorescence observed was considered specific. An overall score for each reaction was given after examination of the staining at the different dilutions (Table 1). The staining patterns given by different Mabs with B. &v&infected erythrocytes are shown in Table 2.
negative except on two occasions when faint reactions were obtained at 150. The individual reactions are shown in Table 1. Comparison of IHA test, IFAT and SELISA The results obtained by these three tests are compared in Table 1. The pair of tests giving the closest results were IFAT and SELISA. In addition, the SELISA method gave the closest agreement between 12 and 36 months (see Table 1). A statistical analysis of these comparisons is detailed in Table 3. DISCUSSION
TABLE
SELISA
Z-THE
INFECTED
ERYTHROCYTES
STAINING WITH
PATTERN
OF
Babesia-
MONOCLONAL
DIFFERENT
ANTIBODIES*
Mabs A B C D E
Staining pattern (specificity) Infected erythrocyte especially the cell membrane Infected erythrocyte and parasite Punctate staining of the parasite Whole parasite Parasite membrane
* A similar staining pattern was obtained with the IFA technique.
The SELISA The positiveenegative dilution cut-off for bovine sera was determined to be 1:SO using incubation times of 1.5 h at room temperature (22°C). Incubation overnight at 4°C gave similar results. A positive reaction was indicated by bluish-purple staining of the B. bovis-infected erythrocytes or of the parasite, or of both, depending on the specificity of the staining antibody (Fig. 1). In a negative reaction, no staining of either the infected cells or the parasite occurred. The intensity of the staining was used to classify the reactions into negative, weak positive, positive or strong and positive. Negative and positive reactions were easily discriminated at 1:50 dilution. The negative control for the Mabs (tissue culture fluid) gave no staining. Of the sera from infected cattle maintained under tick-free conditions lSjl9 were positive and 2/19 were & (doubtful) 12 months after the infection, while at 3 years after infection 17/19 were positive (results of two animals were inconsistent). Sera from the uninfected cattle maintained under tick-free conditions were all
TABLE
J-CORRELATION
OF SCORES BETWEEN DIFFERENT
12 AND 36 MONTHS Tests
IHA and IFA IHA and SELISA IFA and SELISA * P < 0.05.
TESTS AT
POST-INFECTION
Correlation 36 months p.i. 12 months pi. 0.64 0.56 0.85*
0.42 0.56 0.79;
The SELISA was found to be extremely sensitive as it detected antibodies to B. bovis for at least 36 months (see Table 1) after a single infection (no tests were done on sera beyond this period). Moreover comparison with the IFA and the IHA tests results showed that the SELISA was also sensitive and relatively consistent in detecting antibodies not only at 12 months but also at 36 months post-infection (Tables 1 and 3). The diversity in the staining patterns with different Mabs (Table 2) demonstrated the unique and precise cellular specificity of the test and confirmed the data previously obtained when these Mabs were screened by IFAT. Such information cannot be gleaned with the conventional ELISA or the IHA test. This also makes the SELISA an ideal test in screening hybridomas as Mabs can be selected not only on their avidity but also on their specificity. The SELISA has other advantages over most existing serological tests for diagnosis of babesiosis. The IFAT requires an expensive fluorescent microscope and it is also known that stray ultra-violet light can present a health hazard. This test lacks specificity depending on the species, is tedious to interpret and may cause operator fatigue (Todorovic & Carson, 1981; Callow, 1984; Schuntner & Wright, 1989). The IHA was a good test for B. bovis but was a difficult test to perform (Wright et al., 1986). The SELISA should be ideally suited for field application if blood smears can be stored, because only a conventional light microscope is required and this could be illuminated using batteries. In comparison with the conventional ELISA, the SELISA is cheap, very convenient to perform, and more importantly, the completed test slides can be kept for at least 1 week at room temperature (22°C) for later confirmation of results. During the development of the SELISA technique, a similar test to the SELISA for the diagnosis of malaria in man, the malaria immunoperoxidase assay, was reported (Lim, 1988). This test was shown to be as sensitive and specific as the IFAT in detecting antimalaria antibodies and was more convenient to perform. While similar to the Babesia SELISA in performance, no detailed information is available regarding its long-term sensitivity. The SELISA should be applicable in the diagnosis of other protozoan diseases of man and animals,
344
M. W. KUNG’U and B. V. GOODGER
FIG. 1.Scale bars, 13 pm. The SELISA staining of Babesia bovis-infected erythrocytes after reaction with: (a) B. bovis positive serum. Both the parasites and the infected erythrocytes stain strongly. (b) Monoclonal antibody A. The parasites do not stain but the infected erythrocytes, especially the erythrocytic membrane stain strongly. (c) Monoclonal antibody C. The parasites stain strongly while the erythrocytes do not.
Slide ELISA provided that particulate antigen, preferably whole parasite, can be easily obtained and without loss of antigenicity.
of the stored
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