OF CLINICAL MICROBIOLOGY, Apr. 1991, p. 794-797 0095-1137/91/040794-04$02.00/0 Copyright © 1991, American Society for Microbiology
JOURNAL
Vol. 29, No. 4
Blocking Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Actinobacillus pleuropneumoniae Serotype 2 R.
NIELSEN,'* T. PLAMBECK,l AND N. T. FOGED2
National Veterinary Laboratory' and Animal Biotechnology Research Center,2 27 Bilowsvej, P.O. Box 373, DK 1503 Copenhagen, Denmark Received 9 November 1990/Accepted 21 January 1991
A blocking enzyme-linked immunosorbent assay (ELISA), based upon a polyclonal rabbit antiserum specific to Actinobacillus pleuropneumoniae serotype 2, was developed for the detection of antibodies to A. pleuropneumoniae serotype 2 in pigs. By testing sera from pigs experimentally infected with the 11 recognized serotypes ofA. pleuropneumoniae, the assay was proven to be specific for A. pleuropneumoniae serotype 2. With field sera from herds infected with A. pleuropneumoniae serotype 2, the assay was found to be more sensitive than the complement fixation test. Positive results were not observed with field sera from herds known to be free from Actinobacillus infection or with sera from two herds infected with either A. pleuropneumoniae serotype 6 or 8. The high diagnostic sensitivity and specificity of the blocking ELISA will make it useful in field diagnostic work.
Actinobacillus pleuropneumoniae is the cause of a contagious pleuropneumonia in swine which has become a major problem in the swine industry throughout the world. The economic losses due to A. pleuropneumoniae infection are results of deaths caused by acute illness and of lowered production in chronically infected herds (11). Until now, 12 serotypes have been recognized. The prevalence of the different types varies in different countries. Thus, in continental Europe and Japan, serotype 2 is the dominating serotype (2, 5, 8), and in Canada the incidence of this type has increased in recent years (10). Serology has proven its value in the diagnosis of A. pleuropneumoniae infection in pigs and in the control of the disease. The complement fixation (CF) test has been a valuable tool in this context in various countries, but because of its technical complexity, the test is not appropriate for use in all diagnostic laboratories. Work done in Switzerland and Canada (6, 12) has indicated a potential use for different indirect enzyme-linked immunosorbent assays (ELISAs) as alternatives to the CF test for detection of A. pleuropneumoniae infection. Thus, comparative analyses, based on sera from pigs infected with A. pleuropneumoniae, have shown good correlations between the results obtained with one of the indirect ELISAs and those obtained with the CF test (6). Several soluble antigen preparations of A. pleuropneumoniae have been used in a previously described indirect ELISA with variable results (6). In the present study, the supernatant of heat-treated A. pleuropneumoniae serotype 2 cells was used as a coating material for the wells of the microtiter plates used for the blocking ELISA. The detection of antibodies to A. pleuropneumoniae serotype 2 was based upon a competitive reaction between the serum to be tested and a polyclonal rabbit antiserum specific for A. pleuropneumoniae serotype 2.
taining 0.3% Formalin. Two milliliters of a suspension containing 109 organisms per ml was mixed with Freund incomplete adjuvant (1:1 [vol/vol]) and injected intramuscularly in 6-month-old White Landrace rabbits (mean weight, approximately 3 kg). Starting 2 weeks later, a series of immunizations were performed twice a week by intravenously injecting live A. pleuropneumoniae 4226 without adjuvant. The first injection was 0.5 ml of a bacterial suspension containing 104 organisms per ml. The following injections were 0.5 ml of a suspension containing 109 organisms per ml. In the indirect hemagglutination test and gel diffusion assay (9), this antiserum was specific for serotype 2. Blocking ELISA. Antigen for the ELISA was prepared as a supernatant of heat-treated cells (3). A 6-h culture of strain 4226 grown on PPLO agar was washed off the plates with 0.85% saline containing 0.5% Formalin. After overnight storage at 4°C, the concentration of cells was adjusted to 1010 cells per ml in Formol saline. The suspension was heated at 100°C for 1 h. The cells were removed by centrifugation, and the supernatant, filtered through 0.22-,um-pore-size membrane filters (sterile Millex 65; Millipore), was used as the
antigen. Checkerboard titrations were performed in ELISA plates to determine the optimal concentrations of antigen, serum, enzyme conjugate, and substrate. Fifty microliters of the antigen solution diluted 500 times in PBS (pH 7.2) was added to each well of flat-bottom microtiter plates (Immuno Plate II; Roskilde, Denmark). The plates were incubated overnight at 4°C and after that at room temperature for 0.5 h. The plates were blocked with 200 Rl (per well) of a blocking buffer consisting of PBS with 0.05% Tween 20 and 1% bovine serum albumin (PBS+T+BSA). Plates were incubated for 1 h at room temperature and washed three times with PBS containing 0.05% Tween 20 (PBS+T). Sera were serially diluted (start dilution, 1:4) in PBS+T+BSA, and 50 RI of diluted serum was added to the wells, which were then incubated at room temperature for 1 h. Fifty microliters of rabbit antiserum to strain 4226 (RA 4226)) was then added to each well in an optimal dilution of 1:2,000 in PBS+T+BSA. After 0.5 h at room temperature, the plates were washed three times with PBS+T. After the wash, 50 ,u of peroxidase-conjugated swine anti-rabbit immunoglobulin (Dakopatt, Copenhagen, Denmark) diluted
MATERIALS AND METHODS Immunization of rabbits. A 6-h-old culture of the serotype 2 strain 4226 of A. pleuropneumoniae grown on PPLO agar (5) was harvested in phosphate-buffered saline (PBS) con*
Corresponding author. 794
VOL. 29, 1991
ELISA FOR ANTIBODIES TO A. PLEUROPNEUMONIAE SEROTYPE 2
1:1,000 in PBS +T+BSA was added to each well. After 1 h at room temperature, the plates were washed three times with PBS+T. Fifty microliters of an o-phenylendiamine-H202 substrate solution was added to the wells. The enzyme activity was stopped after 5 min with 0.5 M H2SO4, and the A490 was measured with an automatic photometer (Kontron SLT; Labinstruments, Zurich, Switzerland). Each plate contained the following controls: (i) wells without pig test serum and RA 4226, (ii) wells in which pig test serum was replaced by PBS+T+BSA buffer, (iii) wells with a known negative serum sample, and (iv) wells with serotype 2 reference sera. The A490 for each sample was the average from duplicate wells. By interpolation the blocking titer of each test serum was calculated as the dilution resulting in a 50% reduction of the A490 obtained for the control in which serum was replaced by buffer. The relative absorbance of each test serum in a 1:4 dilution was calculated by dividing the mean A490 obtained for the serum with the mean A490 obtained for the negative control sera diluted 1:4. CF test. Antibodies to A. pleuropneumoniae serotypes 2, 6, and 8 in swine sera were measured by the modified CF test (1, 7). Test sera were heat treated at 60°C for 30 min. Normal, unheated serum from 8-week-old pigs free from A. pleuropneumoniae infection was used in a dilution of 1:150 in Veronal-buffered diluent as the reconstituting fluid for lyophilized guinea pig complement. Apart from this modification, the general procedure described in the Laboratory Branch Complement Fixation Method adapted to a microtechnique (1) was followed. Five 50% hemolytic units were used in the test, and the endpoint of serum titration was the highest dilution showing 30% hemolysis or less as compared with hemoglobin color standards (0 to 100% hemolysis). Sera. The sera tested by the blocking ELISA originated from the following sources. (i) Pigs experimentally infected with reference strains of A. pleuropneumoniae. The 11 reference strains representing serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 12 of A. pleuropneumoniae used for intranasal inoculation of pigs to provide reference immune sera were Shope 4074, S1536, S1421, M62, K17, Fem0, WF83, 405, 13261, D13039, and 8329, respectively. Eight-week-old specific pathogen-free (SPF) pigs were inoculated intranasally with 108 live organisms of an 18-h culture grown on enriched PPLO agar. Three weeks later the pigs were sacrificed and bled. Sera were stored at -20°C. (ii) Pigs experimentally infected with the Danish serotype 2 strain 4226 of A. pleuropneumonuae. The serotype 2 strain 4226 of A. pleuropneumoniae originated from a Danish feeder pig with acute pleuropneumonia. Thirty-two 8-weekold SPF pigs were inoculated intranasally with 108 live organisms from an 18-h culture of strain 4226. Sera were collected at sacrifice 3 weeks later. All pigs had lung necroses at slaughter, and A. pleuropneumoniae serotype 2 was isolated from the necroses. (iii) Pigs from a conventional herd without a history of pleuropneumonia. Forty-eight serum specimens were collected from 6-month-old pigs from a conventional breeding herd without a history of pleuropneumonia. At necropsy, pathological lesions were not observed and A. pleuropneumoniae was not isolated. The sera were negative in the CF test to all serotypes. (iv) Pigs from two SPF herds. Eighty-three serum specimens were collected from breeding animals in two SPF herds. Both herds have been monitored for several years for freedom from A. pleuropneumoniae infection by monthly
795
Relative
labsorbance 100
90 80 70 60
50 40 30 20 10
1
Reference anti-
1 2 3 4 5 6 7 8 9 10 12 type: FIG. 1. Analysis by blocking ELISA of 1:4-diluted sera from pigs intranasally infected with one of the 11 reference strains of A. pleuropneumoniae. The average A490 obtained for the negative control sera from noninfected pigs was 2.679. The A49, was significantly lowered in sera from pigs infected with A. pleuropneumoniae serotype 2, whereas none of the sera from pigs infected with the other serotypes of A. pleuropneumoniae showed any blocking effect. serum to
blood testing of breeding animals by the CF test. The 83 serum specimens used in this study were negative in the CF test for all serotypes of A. pleuropneumoniae. (v) Pigs from herds naturally infected with A. pleuropneumoniae serotype 2. Ninety-six field serum specimens were collected from breeding animals in 11 herds infected with A. pleuropneumoniae serotype 2 as diagnosed by a herd history of pleuropneumonia. The clinical diagnosis was confirmed by the finding of positive reactors to serotype 2 by the CF test. (vi) Pigs from herds infected with A. pleuropneumoniae serotype 6 or 8. Eighteen field serum specimens were collected from breeding animals in a herd naturally infected with serotype 6. This serotype was isolated from lung lesions of pigs submitted for necropsy. The sera were positive for serotype 6 in the CF test. Eighteen field serum specimens were collected from breeding animals in a herd naturally infected with serotype 8. Serotype 8 was isolated from the lungs of pigs submitted for necropsy. The sera were positive in the CF test for serotype 8. RESULTS
Serum specimens from two pigs infected with the serotype 2 reference strain S1536 of A. pleuropneumoniae had a mean relative absorbance of 10%, whereas the reference sera from pigs inoculated with serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, and 12 had a mean absorbance (+ standard deviation) of 105% + 5% (Fig. 1). Serum specimens from 32 pigs experimentally infected with A. pleuropneumoniae serotype 2 strain 4226 had a mean relative absorbance of 10% + 10%, whereas a group of 48 serum specimens collected from a conventional herd known to be free from pleuropneumonia and a group of 83 serum
796
J. CLIN. MICROBIOL.
NIELSEN ET AL. Relative absorbance
1000 f
100-
I
90-
0
10o[
80En
70-
-J
60-
.0
I
I
CL
0
4J
50-
*_4q
10
40
[
0
0
30 20 10
No A.pl. No A.pl. infection isolated SPF Cornv. Expm. Herd FIG. 2. Comparison by the serotype 2 blocking ELISA of 1:4diluted sera collected from pigs (n = 32) in an experimental (Expm.) herd infected with A. pleuropneumoniae serotype 2, from pigs (n = 48) in a conventional (Conv.) herd free from infection with A. pleuropneumoniae, and from pigs (n = 83) in two SPF herds in which A. pleuropneumoniae could not be isolated. The mean standard deviation of the A490 for each of the three groups of sera relative to the A490 for a negative control serum is shown.
Infection:
1000 10 100 Titer by CF test FIG. 3. Comparison of the titers obtained by the blocking ELISA and the CF test. All 32 serum specimens from pigs intranasally infected with A. pleuropneumoniae serotype 2 were positive in the two assays. The relation between the titers was estimated by linear regression on a double logarithmic plot. The regression line (r = 0.71) was calculated as follows: log (ELISA titer) = 0.59- log (CF test titer) + 0.83. This shows that sera with low levels of anti-serotype 2 antibodies can be detected more frequently by the ELISA than by the CF test. 1
..+1+ Serotype 2
specimens collected from SPF herds free from infection with A. pleuropneumoniae both had mean relative absorbances of 100% 10% (Fig. 2). The specific detection of antibodies to A. pleuropneumoniae serotype 2 by the blocking ELISA was further sustained with field sera collected from pigs infected with either A. pleuropneumoniae serotype 6 or 8, since the relative absorbances of these sera were 100% 10%. The 32 serum specimens collected from pigs experimentally infected with A. pleuropneumoniae serotype 2 were titrated and analyzed by both the CF test for detection of serotype 2 antibodies and the blocking ELISA. A good correlation was observed, but higher titers were observed in the ELISA for the weakly positive sera (Fig. 3). Of 96 field samples from herds infected with A. pleuropneumoniae serotype 2, 86 (90%) were positive by the blocking ELISA, with a mean relative absorbance of 10% 10%. The remaining 10 serum specimens were negative, with a relative absorbance of 96% 14%. When the same 96 serum specimens were analyzed by the CF test for detection of serotype 2 antibodies, 67 samples (70%) were seropositive (Table 1). ±
±
and control of A. pleuropneumoniae infections are available (4, 6, 12), and none of them have used a blocking ELISA. With the 50% cutoff level, the blocking ELISA with polyclonal rabbit antiserum as a detector allowed for a very specific test while maintaining a sensitivity which was higher than that of the CF test. No cross-reactions to other serotypes of A. pleuropneumoniae were seen. The specificity and sensitivity of an ELISA using bacterial antigens depend on the quality of the antigen preparation. In two reports (6, 12) EDTA-treated antigen from A. pleuropneumoniae was used for coating wells of microtiter plates in indirect ELISAs. The sensitivity and specificity of these tests was claimed to be sufficient for use in commercial herds as an aid for determining the exposure of pigs to A. pleuropneumoniae. In comparative analyses of the CF test, the indirect ELISA using different antigen preparations, and a modified tube agglutination test performed in the presence of 2-mercaptoethanol, it was found that none of the tests was
±
±
TABLE 1. Detection of antibodies to A. pleuropneumoniae serotype 2 by the CF test and the blocking ELISA No. of CF test results
ELISA result
DISCUSSION
+
The blocking ELISA described here was developed for the detection of antibodies to A. pleuropneumoniae serotype 2, which is the dominating serotype in Danish swine herds, causing approximately 70% of diagnosed outbreaks of pleu-
ropneumonia. Only a few reports
on
the ELISA
as a
tool for diagnosis
+
-
Total
67
19
86a
0
10
10
Total 67 29 96 "The 86 serum specimens resulted in a mean relative absorbance ± standard deviation of 10% ± 10% by the blocking ELISA. b The 10 serum specimens resulted in a mean relative absorbance ± standard deviation of 96% ± 14% by the blocking EUSA.
VOL. 29, 1991
ELISA FOR ANTIBODIES TO A. PLEUROPNEUMONIAE SEROTYPE 2
completely satisfactory and that a combination of at least two of these tests was necessary to interpret results (4). The favorable results obtained with the blocking ELISA described here may be due to the use of a heat-extracted antigen preparation which mainly contains important polysaccharide components in combination with a rabbit antiserum showing a high titer to these polysaccharides (9). The blocking ELISA is a realistic alternative to existing tests for monitoring herds for A. pleuropneumoniae infection as well as for eradication programs. It is therefore important that the high sensitivity and specificity found with the relatively low number of serum specimens in this study be documented with a large number of field samples. ACKNOWLEDGMENT This work was supported by grant 5.23.06 from the Danish Agricultural and Veterinary Research Council. REFERENCES 1. Casey, H. L. 1965. Standardized diagnostic complement-fixation method and adaption to micro test. Public health monograph no. 74. U.S. Department of Health, Education and Welfare, Washington, D.C. 2. Kume, K., I. Nagano, and T. Nakai. 1986. Bacteriological, serological and pathological examination of Haemophilus pleuropneumoniae infection in 200 slaughtered pigs. Jpn. J. Vet. Sci. 48:965-970. 3. Lariviere, S. Personal communication.
797
4. Martineau, G. P., R. Higgins, S. Lariviere, K. R. Mittal, J. Vaillancourt, and R. Desrosiers. 1985. Elimination and control measures for swine pleuropneumonia in Quebec. Haemophilus pleuropneumoniae compendium, p. 39-42. Sigler Printing, Ames, Iowa. 5. Nicolet, J. 1971. Sur l'hemophilose du porc. III. Differenciation serologique de Haemophilus parahaemolyticus. Zentralbl. Bakteriol. Abt. 1 Orig. 216:487-495. 6. Nicolet, J., P. Paroz, M. Krawinkler, and A. Baumgarten. 1981. An enzyme-linked immunosorbent assay, using EDTA extracted antigen for the serology of Haemophilus pleuropneumoniae. Am. J. Vet. Res. 42:2139-2142. 7. Nielsen, R. 1982. Haemophilus pleuropneumoniae infection in pigs. Thesis. National Veterinary Serum Laboratory, Copenhagen. 8. Nielsen, R. 1988. Seroepidemiology of Actinobacillus pleuropneumoniae. Can. Vet. J. 29:580-582. 9. Nielsen, R., and P. J. O'Connor. 1984. Serological characterization of 8 Haemophilus pleuropneumoniae strains and proposal of a new serotype: serotype 8. Acta Vet. Scand. 25:96-106. 10. Roussau, P., R. Assaf, G. Boulay, and M. Nsy. 1988. Immune response to an Actinobacillus pleuropneumoniae vaccine in swine. Can. Vet. J. 29:989-992. 11. Sebunya, T. N. K., and J. R. Saunders. 1983. Haemophilus pleuropneumoniae infection in swine: a review. J. Am. Vet. Med. Assoc. 182:1331-1337. 12. Wilson, P. J., C. Schipper, and E. D. Morgan. 1988. The use of an enzyme-linked immunosorbent assay for diagnosis of Actinobacillus pleuropneumoniae infection in pigs. Can. Vet. J. 29:583-584.
JOURNAL
Vol. 29, No. 4
Blocking Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Actinobacillus pleuropneumoniae Serotype 2 R.
NIELSEN,'* T. PLAMBECK,l AND N. T. FOGED2
National Veterinary Laboratory' and Animal Biotechnology Research Center,2 27 Bilowsvej, P.O. Box 373, DK 1503 Copenhagen, Denmark Received 9 November 1990/Accepted 21 January 1991
A blocking enzyme-linked immunosorbent assay (ELISA), based upon a polyclonal rabbit antiserum specific to Actinobacillus pleuropneumoniae serotype 2, was developed for the detection of antibodies to A. pleuropneumoniae serotype 2 in pigs. By testing sera from pigs experimentally infected with the 11 recognized serotypes ofA. pleuropneumoniae, the assay was proven to be specific for A. pleuropneumoniae serotype 2. With field sera from herds infected with A. pleuropneumoniae serotype 2, the assay was found to be more sensitive than the complement fixation test. Positive results were not observed with field sera from herds known to be free from Actinobacillus infection or with sera from two herds infected with either A. pleuropneumoniae serotype 6 or 8. The high diagnostic sensitivity and specificity of the blocking ELISA will make it useful in field diagnostic work.
Actinobacillus pleuropneumoniae is the cause of a contagious pleuropneumonia in swine which has become a major problem in the swine industry throughout the world. The economic losses due to A. pleuropneumoniae infection are results of deaths caused by acute illness and of lowered production in chronically infected herds (11). Until now, 12 serotypes have been recognized. The prevalence of the different types varies in different countries. Thus, in continental Europe and Japan, serotype 2 is the dominating serotype (2, 5, 8), and in Canada the incidence of this type has increased in recent years (10). Serology has proven its value in the diagnosis of A. pleuropneumoniae infection in pigs and in the control of the disease. The complement fixation (CF) test has been a valuable tool in this context in various countries, but because of its technical complexity, the test is not appropriate for use in all diagnostic laboratories. Work done in Switzerland and Canada (6, 12) has indicated a potential use for different indirect enzyme-linked immunosorbent assays (ELISAs) as alternatives to the CF test for detection of A. pleuropneumoniae infection. Thus, comparative analyses, based on sera from pigs infected with A. pleuropneumoniae, have shown good correlations between the results obtained with one of the indirect ELISAs and those obtained with the CF test (6). Several soluble antigen preparations of A. pleuropneumoniae have been used in a previously described indirect ELISA with variable results (6). In the present study, the supernatant of heat-treated A. pleuropneumoniae serotype 2 cells was used as a coating material for the wells of the microtiter plates used for the blocking ELISA. The detection of antibodies to A. pleuropneumoniae serotype 2 was based upon a competitive reaction between the serum to be tested and a polyclonal rabbit antiserum specific for A. pleuropneumoniae serotype 2.
taining 0.3% Formalin. Two milliliters of a suspension containing 109 organisms per ml was mixed with Freund incomplete adjuvant (1:1 [vol/vol]) and injected intramuscularly in 6-month-old White Landrace rabbits (mean weight, approximately 3 kg). Starting 2 weeks later, a series of immunizations were performed twice a week by intravenously injecting live A. pleuropneumoniae 4226 without adjuvant. The first injection was 0.5 ml of a bacterial suspension containing 104 organisms per ml. The following injections were 0.5 ml of a suspension containing 109 organisms per ml. In the indirect hemagglutination test and gel diffusion assay (9), this antiserum was specific for serotype 2. Blocking ELISA. Antigen for the ELISA was prepared as a supernatant of heat-treated cells (3). A 6-h culture of strain 4226 grown on PPLO agar was washed off the plates with 0.85% saline containing 0.5% Formalin. After overnight storage at 4°C, the concentration of cells was adjusted to 1010 cells per ml in Formol saline. The suspension was heated at 100°C for 1 h. The cells were removed by centrifugation, and the supernatant, filtered through 0.22-,um-pore-size membrane filters (sterile Millex 65; Millipore), was used as the
antigen. Checkerboard titrations were performed in ELISA plates to determine the optimal concentrations of antigen, serum, enzyme conjugate, and substrate. Fifty microliters of the antigen solution diluted 500 times in PBS (pH 7.2) was added to each well of flat-bottom microtiter plates (Immuno Plate II; Roskilde, Denmark). The plates were incubated overnight at 4°C and after that at room temperature for 0.5 h. The plates were blocked with 200 Rl (per well) of a blocking buffer consisting of PBS with 0.05% Tween 20 and 1% bovine serum albumin (PBS+T+BSA). Plates were incubated for 1 h at room temperature and washed three times with PBS containing 0.05% Tween 20 (PBS+T). Sera were serially diluted (start dilution, 1:4) in PBS+T+BSA, and 50 RI of diluted serum was added to the wells, which were then incubated at room temperature for 1 h. Fifty microliters of rabbit antiserum to strain 4226 (RA 4226)) was then added to each well in an optimal dilution of 1:2,000 in PBS+T+BSA. After 0.5 h at room temperature, the plates were washed three times with PBS+T. After the wash, 50 ,u of peroxidase-conjugated swine anti-rabbit immunoglobulin (Dakopatt, Copenhagen, Denmark) diluted
MATERIALS AND METHODS Immunization of rabbits. A 6-h-old culture of the serotype 2 strain 4226 of A. pleuropneumoniae grown on PPLO agar (5) was harvested in phosphate-buffered saline (PBS) con*
Corresponding author. 794
VOL. 29, 1991
ELISA FOR ANTIBODIES TO A. PLEUROPNEUMONIAE SEROTYPE 2
1:1,000 in PBS +T+BSA was added to each well. After 1 h at room temperature, the plates were washed three times with PBS+T. Fifty microliters of an o-phenylendiamine-H202 substrate solution was added to the wells. The enzyme activity was stopped after 5 min with 0.5 M H2SO4, and the A490 was measured with an automatic photometer (Kontron SLT; Labinstruments, Zurich, Switzerland). Each plate contained the following controls: (i) wells without pig test serum and RA 4226, (ii) wells in which pig test serum was replaced by PBS+T+BSA buffer, (iii) wells with a known negative serum sample, and (iv) wells with serotype 2 reference sera. The A490 for each sample was the average from duplicate wells. By interpolation the blocking titer of each test serum was calculated as the dilution resulting in a 50% reduction of the A490 obtained for the control in which serum was replaced by buffer. The relative absorbance of each test serum in a 1:4 dilution was calculated by dividing the mean A490 obtained for the serum with the mean A490 obtained for the negative control sera diluted 1:4. CF test. Antibodies to A. pleuropneumoniae serotypes 2, 6, and 8 in swine sera were measured by the modified CF test (1, 7). Test sera were heat treated at 60°C for 30 min. Normal, unheated serum from 8-week-old pigs free from A. pleuropneumoniae infection was used in a dilution of 1:150 in Veronal-buffered diluent as the reconstituting fluid for lyophilized guinea pig complement. Apart from this modification, the general procedure described in the Laboratory Branch Complement Fixation Method adapted to a microtechnique (1) was followed. Five 50% hemolytic units were used in the test, and the endpoint of serum titration was the highest dilution showing 30% hemolysis or less as compared with hemoglobin color standards (0 to 100% hemolysis). Sera. The sera tested by the blocking ELISA originated from the following sources. (i) Pigs experimentally infected with reference strains of A. pleuropneumoniae. The 11 reference strains representing serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 12 of A. pleuropneumoniae used for intranasal inoculation of pigs to provide reference immune sera were Shope 4074, S1536, S1421, M62, K17, Fem0, WF83, 405, 13261, D13039, and 8329, respectively. Eight-week-old specific pathogen-free (SPF) pigs were inoculated intranasally with 108 live organisms of an 18-h culture grown on enriched PPLO agar. Three weeks later the pigs were sacrificed and bled. Sera were stored at -20°C. (ii) Pigs experimentally infected with the Danish serotype 2 strain 4226 of A. pleuropneumonuae. The serotype 2 strain 4226 of A. pleuropneumoniae originated from a Danish feeder pig with acute pleuropneumonia. Thirty-two 8-weekold SPF pigs were inoculated intranasally with 108 live organisms from an 18-h culture of strain 4226. Sera were collected at sacrifice 3 weeks later. All pigs had lung necroses at slaughter, and A. pleuropneumoniae serotype 2 was isolated from the necroses. (iii) Pigs from a conventional herd without a history of pleuropneumonia. Forty-eight serum specimens were collected from 6-month-old pigs from a conventional breeding herd without a history of pleuropneumonia. At necropsy, pathological lesions were not observed and A. pleuropneumoniae was not isolated. The sera were negative in the CF test to all serotypes. (iv) Pigs from two SPF herds. Eighty-three serum specimens were collected from breeding animals in two SPF herds. Both herds have been monitored for several years for freedom from A. pleuropneumoniae infection by monthly
795
Relative
labsorbance 100
90 80 70 60
50 40 30 20 10
1
Reference anti-
1 2 3 4 5 6 7 8 9 10 12 type: FIG. 1. Analysis by blocking ELISA of 1:4-diluted sera from pigs intranasally infected with one of the 11 reference strains of A. pleuropneumoniae. The average A490 obtained for the negative control sera from noninfected pigs was 2.679. The A49, was significantly lowered in sera from pigs infected with A. pleuropneumoniae serotype 2, whereas none of the sera from pigs infected with the other serotypes of A. pleuropneumoniae showed any blocking effect. serum to
blood testing of breeding animals by the CF test. The 83 serum specimens used in this study were negative in the CF test for all serotypes of A. pleuropneumoniae. (v) Pigs from herds naturally infected with A. pleuropneumoniae serotype 2. Ninety-six field serum specimens were collected from breeding animals in 11 herds infected with A. pleuropneumoniae serotype 2 as diagnosed by a herd history of pleuropneumonia. The clinical diagnosis was confirmed by the finding of positive reactors to serotype 2 by the CF test. (vi) Pigs from herds infected with A. pleuropneumoniae serotype 6 or 8. Eighteen field serum specimens were collected from breeding animals in a herd naturally infected with serotype 6. This serotype was isolated from lung lesions of pigs submitted for necropsy. The sera were positive for serotype 6 in the CF test. Eighteen field serum specimens were collected from breeding animals in a herd naturally infected with serotype 8. Serotype 8 was isolated from the lungs of pigs submitted for necropsy. The sera were positive in the CF test for serotype 8. RESULTS
Serum specimens from two pigs infected with the serotype 2 reference strain S1536 of A. pleuropneumoniae had a mean relative absorbance of 10%, whereas the reference sera from pigs inoculated with serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, and 12 had a mean absorbance (+ standard deviation) of 105% + 5% (Fig. 1). Serum specimens from 32 pigs experimentally infected with A. pleuropneumoniae serotype 2 strain 4226 had a mean relative absorbance of 10% + 10%, whereas a group of 48 serum specimens collected from a conventional herd known to be free from pleuropneumonia and a group of 83 serum
796
J. CLIN. MICROBIOL.
NIELSEN ET AL. Relative absorbance
1000 f
100-
I
90-
0
10o[
80En
70-
-J
60-
.0
I
I
CL
0
4J
50-
*_4q
10
40
[
0
0
30 20 10
No A.pl. No A.pl. infection isolated SPF Cornv. Expm. Herd FIG. 2. Comparison by the serotype 2 blocking ELISA of 1:4diluted sera collected from pigs (n = 32) in an experimental (Expm.) herd infected with A. pleuropneumoniae serotype 2, from pigs (n = 48) in a conventional (Conv.) herd free from infection with A. pleuropneumoniae, and from pigs (n = 83) in two SPF herds in which A. pleuropneumoniae could not be isolated. The mean standard deviation of the A490 for each of the three groups of sera relative to the A490 for a negative control serum is shown.
Infection:
1000 10 100 Titer by CF test FIG. 3. Comparison of the titers obtained by the blocking ELISA and the CF test. All 32 serum specimens from pigs intranasally infected with A. pleuropneumoniae serotype 2 were positive in the two assays. The relation between the titers was estimated by linear regression on a double logarithmic plot. The regression line (r = 0.71) was calculated as follows: log (ELISA titer) = 0.59- log (CF test titer) + 0.83. This shows that sera with low levels of anti-serotype 2 antibodies can be detected more frequently by the ELISA than by the CF test. 1
..+1+ Serotype 2
specimens collected from SPF herds free from infection with A. pleuropneumoniae both had mean relative absorbances of 100% 10% (Fig. 2). The specific detection of antibodies to A. pleuropneumoniae serotype 2 by the blocking ELISA was further sustained with field sera collected from pigs infected with either A. pleuropneumoniae serotype 6 or 8, since the relative absorbances of these sera were 100% 10%. The 32 serum specimens collected from pigs experimentally infected with A. pleuropneumoniae serotype 2 were titrated and analyzed by both the CF test for detection of serotype 2 antibodies and the blocking ELISA. A good correlation was observed, but higher titers were observed in the ELISA for the weakly positive sera (Fig. 3). Of 96 field samples from herds infected with A. pleuropneumoniae serotype 2, 86 (90%) were positive by the blocking ELISA, with a mean relative absorbance of 10% 10%. The remaining 10 serum specimens were negative, with a relative absorbance of 96% 14%. When the same 96 serum specimens were analyzed by the CF test for detection of serotype 2 antibodies, 67 samples (70%) were seropositive (Table 1). ±
±
and control of A. pleuropneumoniae infections are available (4, 6, 12), and none of them have used a blocking ELISA. With the 50% cutoff level, the blocking ELISA with polyclonal rabbit antiserum as a detector allowed for a very specific test while maintaining a sensitivity which was higher than that of the CF test. No cross-reactions to other serotypes of A. pleuropneumoniae were seen. The specificity and sensitivity of an ELISA using bacterial antigens depend on the quality of the antigen preparation. In two reports (6, 12) EDTA-treated antigen from A. pleuropneumoniae was used for coating wells of microtiter plates in indirect ELISAs. The sensitivity and specificity of these tests was claimed to be sufficient for use in commercial herds as an aid for determining the exposure of pigs to A. pleuropneumoniae. In comparative analyses of the CF test, the indirect ELISA using different antigen preparations, and a modified tube agglutination test performed in the presence of 2-mercaptoethanol, it was found that none of the tests was
±
±
TABLE 1. Detection of antibodies to A. pleuropneumoniae serotype 2 by the CF test and the blocking ELISA No. of CF test results
ELISA result
DISCUSSION
+
The blocking ELISA described here was developed for the detection of antibodies to A. pleuropneumoniae serotype 2, which is the dominating serotype in Danish swine herds, causing approximately 70% of diagnosed outbreaks of pleu-
ropneumonia. Only a few reports
on
the ELISA
as a
tool for diagnosis
+
-
Total
67
19
86a
0
10
10
Total 67 29 96 "The 86 serum specimens resulted in a mean relative absorbance ± standard deviation of 10% ± 10% by the blocking ELISA. b The 10 serum specimens resulted in a mean relative absorbance ± standard deviation of 96% ± 14% by the blocking EUSA.
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ELISA FOR ANTIBODIES TO A. PLEUROPNEUMONIAE SEROTYPE 2
completely satisfactory and that a combination of at least two of these tests was necessary to interpret results (4). The favorable results obtained with the blocking ELISA described here may be due to the use of a heat-extracted antigen preparation which mainly contains important polysaccharide components in combination with a rabbit antiserum showing a high titer to these polysaccharides (9). The blocking ELISA is a realistic alternative to existing tests for monitoring herds for A. pleuropneumoniae infection as well as for eradication programs. It is therefore important that the high sensitivity and specificity found with the relatively low number of serum specimens in this study be documented with a large number of field samples. ACKNOWLEDGMENT This work was supported by grant 5.23.06 from the Danish Agricultural and Veterinary Research Council. REFERENCES 1. Casey, H. L. 1965. Standardized diagnostic complement-fixation method and adaption to micro test. Public health monograph no. 74. U.S. Department of Health, Education and Welfare, Washington, D.C. 2. Kume, K., I. Nagano, and T. Nakai. 1986. Bacteriological, serological and pathological examination of Haemophilus pleuropneumoniae infection in 200 slaughtered pigs. Jpn. J. Vet. Sci. 48:965-970. 3. Lariviere, S. Personal communication.
797
4. Martineau, G. P., R. Higgins, S. Lariviere, K. R. Mittal, J. Vaillancourt, and R. Desrosiers. 1985. Elimination and control measures for swine pleuropneumonia in Quebec. Haemophilus pleuropneumoniae compendium, p. 39-42. Sigler Printing, Ames, Iowa. 5. Nicolet, J. 1971. Sur l'hemophilose du porc. III. Differenciation serologique de Haemophilus parahaemolyticus. Zentralbl. Bakteriol. Abt. 1 Orig. 216:487-495. 6. Nicolet, J., P. Paroz, M. Krawinkler, and A. Baumgarten. 1981. An enzyme-linked immunosorbent assay, using EDTA extracted antigen for the serology of Haemophilus pleuropneumoniae. Am. J. Vet. Res. 42:2139-2142. 7. Nielsen, R. 1982. Haemophilus pleuropneumoniae infection in pigs. Thesis. National Veterinary Serum Laboratory, Copenhagen. 8. Nielsen, R. 1988. Seroepidemiology of Actinobacillus pleuropneumoniae. Can. Vet. J. 29:580-582. 9. Nielsen, R., and P. J. O'Connor. 1984. Serological characterization of 8 Haemophilus pleuropneumoniae strains and proposal of a new serotype: serotype 8. Acta Vet. Scand. 25:96-106. 10. Roussau, P., R. Assaf, G. Boulay, and M. Nsy. 1988. Immune response to an Actinobacillus pleuropneumoniae vaccine in swine. Can. Vet. J. 29:989-992. 11. Sebunya, T. N. K., and J. R. Saunders. 1983. Haemophilus pleuropneumoniae infection in swine: a review. J. Am. Vet. Med. Assoc. 182:1331-1337. 12. Wilson, P. J., C. Schipper, and E. D. Morgan. 1988. The use of an enzyme-linked immunosorbent assay for diagnosis of Actinobacillus pleuropneumoniae infection in pigs. Can. Vet. J. 29:583-584.
