JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1992,
p.
346-350
Vol. 30, No. 2
0095-1137/92/020346-05$02.00/0 Copyright © 1992, American Society for Microbiology
Characterization of Pseudorabies Virus Antibody Responses in Young Swine after Infection and Vaccination by Using an Immunoglobulin M Antibody Capture Enzyme-Linked Immunosorbent Assay MONTE B. MCCAW,l* TOM W. MOLITOR,2 AND H. S. JOO2 Department of Food Animal and Equine Medicine, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, North Carolina 27606,1 and Large Animal Clinical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 551082 Received 13 September 1991/Accepted 7 November 1991 An immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MACELISA) was developed for the detection of pseudorabies virus (PRV)-specific IgM antibody in swine sera because false-positive reactions frequently occurred when sera from older swine were tested with an indirect IgM enzyme-linked immunosorbent assay. Monoclonal mouse anti-swine IgM was used as the capturing antibody, and rabbit anti-PRV hyperimmune gamma globulin was used as the indicating antibody. Sera from non-PRV-infected, experimentally infected, vaccinated and challenged, passively immune and challenged, and naturally infected swine were evaluated. The PRV MACELISA had a specificity of 95% and was as sensitive and reproducible as previously reported in direct assays. An antibody response was still detectable with the MACELISA 21 days after inoculation. The PRV MACELISA did not detect a consistent antibody response in sera from swine vaccinated with either killed-PRV or modified live-virus vaccines but did detect an antibody response in sera from passively immune pigs after challenge with virulent PRV. These results indicated that the PRV MACELISA may be useful for the rapid serodiagnosis of recent PRV infection in swine. 10 to 12, 20 to 24, and >52 weeks) from each of five nonvaccinated PRV-free herds. Group 2 consisted of sera from animals experimentally infected intranasally with PRV strain 4892 (6). Sixteen seronegative 8-week-old pigs were intranasally inoculated with either 104.0 or 1060 50% tissue culture infective dose (TCID50), and sera were collected biweekly for 3 weeks. Group 3 consisted of six seronegative 8-week-old pigs vaccinated with either modified live-virus or killed-PRV vaccine. All pigs were challenged intranasally 28 days later with 106-5 TCID50 of Shope strain PRV. Sera were collected biweekly throughout the experiment. Group 4 consisted of 10 passively immune 5-week-old pigs (serum neutralization antibody titers, 1:4 to 1:8) that nursed PRVimmune sows. Four pigs each were infected with 104-5 or 106-5 TCID50 of Shope strain PRV and bled biweekly. The remaining two passively immune pigs were housed in the same room and served as contact-infected controls. Group 5 consisted of 16 sows in a swine herd that was experiencing a PRV epizootic. Acute- and convalescent-phase sera were collected, respectively, 10 and 52 days after the onset of clinical signs from these sows. Control sera used in each plate tested were obtained as follows. Negative control serum was a pool of sera from five of the 4-week-old pigs in group 1. Positive control serum was collected from a pig 10 days after experimental infection with Shope strain PRV. Antisera and conjugates. Mouse monoclonal anti-swine IgM and IgG ascites were generous gifts from Prem Paul, Iowa State University (9, 10). Rabbit anti-PRV hyperimmune gamma globulin was prepared by immunizing rabbits three times intramuscularly with PRV antigen mixed with a mineral oil adjuvant. Seven days after the last immunization, the rabbits were euthanatized and the sera were collected and precipitated with saturated ammonium sulfate. The precipitate was resuspended in distilled water and dialyzed
Immunoglobulin M (IgM) antibody is the first class of immunoglobulin produced in response to an infection and can be detected for a short period of time after infection (12). An assay that detects pseudorabies virus (PRV)-specific IgM antibody in serum samples may be used to identify pigs recently infected with PRV. The purpose of this report was to describe an IgM antibody capture enzyme-linked immunosorbent assay (MAC ELISA) for the detection of PRV-specific IgM antibody in swine sera and to characterize the PRV-specific IgM antibody responses in naturally or experimentally infected, passively immune, or vaccinated swine. MATERIALS AND METHODS Virus and vaccine. Virulent PRV strain 4892 was a gift from John Kluge and Howard Hill, Iowa State University. Shope strain PRV was obtained from the National Animal Disease Center, Ames, Iowa. Stock PRV was prepared in
pig kidney (PK-15) cells by routine methods. Modified live-virus and killed-PRV vaccines were purchased from Norden Laboratories, Lincoln, Nebr., and were administered in accordance with the manufacturer's recommendations. Preparation of PRV antigen. PRV antigen was prepared from cultured PRV-infected PK-15 cells (5). Sera. The following groups of sera were used. Group 1 consisted of 200 serum samples previously determined to be seronegative for PRV by serum neutralization (3) and was used to establish the cutoff between positive and negative values in the MACELISA. The 200 serum samples originated from 10 pigs each of four different age groups (4 to 6, *
Corresponding author. 346
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exhaustively against phosphate-buffered saline (PBS, pH 7.2). This solution was absorbed by gentle agitation with pelleted, uninfected PK-15 cells for 2 h at 37°C. Following centrifugation at 2,000 x g for 15 min, the supernatant was collected and stored at -20°C in aliquots. Goat anti-mouse IgG-IgM-IgA-peroxidase (Cappel Inc., Durham, N.C.) was used as the conjugate in the indirect PRV IgG enzyme-linked immunosorbent assay (ELISA). Goat anti-rabbit IgG-peroxidase (Kirkegaard and Perry Laboratories, Gaithersburg, Md.) was used as the conjugate in the PRV MACELISA. Indirect PRV IgG ELISA. The indirect PRV IgG ELISA was performed similarly to the method of Martin et al. (8). Polystyrene ELISA plates (Immulon I; Dynatech Laboratories, Alexandria, Va.) were coated with 100 ,ul of a 1:400 dilution of PRV antigen in coating buffer (50 mM carbonatebicarbonate buffer [pH 9.6]) and incubated overnight at 4°C. All plates were washed three times with PBS containing 0.05% Tween 20 between each step. Sera diluted (1:50) in PBS were added in triplicate. Negative and positive control sera were included on each plate. The plates were incubated for 45 min at 22°C and washed, and 100 ,ul of mouse monoclonal anti-swine IgG ascites (1:2,500) in PBS was added per well. The plates were incubated for 3 h at 37°C and washed. Goat anti-mouse IgG-IgA-IgM conjugate (0.04 mg/ ml; diluted 1:960 in PBS) was added to each plate, and the plates were incubated for 45 min at 22°C. The plates were washed, substrate (o-phenylenediamine dihydrochloride in citrate-phosphate buffer with H202) was added, and the plates were incubated for 5 to 10 min. The reaction was stopped by the addition of 5 N H2SO4. The plates were read on a Dynatech ELISA reader (MR 580; incidence wavelength, 540 nm; reference wavelength, 490 nm). The results were expressed as the sample optical density (OD) ratio (mean sample OD/mean negative control OD). MACELISA. MACELISA plates were coated with mouse monoclonal anti-swine IgM ascites (1:500; 100 ,ul per well) in coating buffer. The plates were incubated overnight at 4°C and washed three times. Diluted sera (1:40) were added in triplicate, and the plates were incubated for 3 h at 37°C. Negative and positive control sera were included on each plate. The plates were washed five times, and PRV antigen (1:500 in PBS; 100 ,ul per well) was added. The plates were incubated at 4°C overnight and washed four times. Rabbit anti-PRV (1:20,000 in PBS; 100 p.l per well) with 0.1% normal mouse ascites was added to each plate. After 2 h of incubation at 22°C, the plates were washed six times and goat anti-rabbit IgG-peroxidase conjugate (0.04 mg/ml diluted 1:480 in PBS; 100 p.l per well) was added. The plates were incubated for 2 h at room temperature and washed five times, and o-phenylenediamine dihydrochloride substrate was added. The reaction was stopped by the addition of 5 N H2SO4. The plates were read as described for the indirect ELISA. The results were expressed as the sample OD ratio. RESULTS Specificity of the PRV MACELISA. To evaluate the specificity of the PRV MACELISA, we tested group 1 sera. The mean age group OD ratios ranged from 1.016 to 1.080, with standard deviations of less than or equal to +0.141. The PRV MACELISA results for the 200 uninfected-pig serum samples were used to set a positive/negative cutoff OD ratio. The PRV MACELISA was 95% specific when the positive/ negative OD ratio was fixed at 1.28. Only 10 of the 200 serum samples had OD ratios higher than 1.28 in the MACELISA.
347
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0
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CA
o
1.00
0
20 40
80 160 320 640 1280 2560 5120
Serum Dilution FIG. 1. Dilution effect on MACELISA OD ratios of a serum sample collected from a 6-week-old pig 10 days after experimental infection with strain 4892 PRV (0) and a serum sample collected from an uninfected 6-week-old pig (0).
Acute- and convalescent-phase sera from naturally infected sows (group 5 sera) were also tested by the MACELISA. The mean OD ratios for the acute and convalescent phases were 1.92 and 1.07 with 87.5 and 0.0% of the sera testing positive, respectively. Eleven of the 16 acute-phase sow serum samples had OD ratios of .1.50, and 14 of the 16 convalescent-phase sow sera had OD ratios of .1.10. The remaining two convalescent-phase sow sera had OD ratios of 1.28) in a 1:5,120 dilution of serum collected 10 days after experimental infection of a seronegative 6-week-old pig (Fig. 1). Reproducibility. The reproducibility of the PRV MAC ELISA was evaluated with one negative (group 1) and three positive (group 2) serum samples. Nine replicates of each serum sample were tested in the same plate on each of four different days. The within-plate coefficient of variation ranged from 6.13 to 15.38%, and the between-day coefficient of variation ranged from 19.06 to 29.35% (Table 1). Antibody response after experimental PRV infection. Sera collected from 16 seronegative pigs experimentally infected with strain 4892 PRV were tested by the MACELISA and the indirect IgG ELISA. An antibody response was detected in sera from four of these pigs 3 days postinfection (p.i.) by the MACELISA. These sera had OD ratios of 1.31 to 1.35. The group mean antibody response was first detected 7 days p.i. by the MACELISA and 10 days p.i. by the indirect IgG ELISA (Fig. 2). The mean MACELISA response peaked at 10 days p.i. and was still detectable (OD ratio, >1.28) at 21
348
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J. CLIN. MICROBIOL.
TABLE 1. Reproducibility of the PRV MACELISA Test'
Serum
Coefficient of
Within-plate
Negative Weakly positive Intermediately positive
Strongly positive
15.38 10.26 7.54 6.13
Negative Weakly positive Intermediately positive Strongly positive
19.06 20.70 20.17 29.35
Between-day
variation (%)
2.5-
;
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days p.i. The group mean indirect IgG ELISA response began to rise 10 days p.i. and was highest at 21 days p.i. Antibody response after vaccination and subsequent challenge. PRV-specific IgM and IgG antibody responses were evaluated in serial serum samples collected from six pigs vaccinated with commercially available products and challenged with virulent PRV at 28 days after vaccination. No IgM antibody response was detected in any of the vaccinated pigs up to 17 days postvaccination (Fig. 3). On days 18 and 21 postvaccination, the sera from one pig had a strong response in the PRV MACELISA. A weak IgM antibody response was detected in sera collected 10 and 14 days postchallenge. An IgG antibody response was first detected 24 days postvaccination in both groups of pigs (Fig. 3). A high IgG antibody response was observed from 7 days postchallenge on in both groups of vaccinated pigs. Antibody response after experimental infection in passively immune pigs. An IgM antibody response was detected at 10 days p.i. in all passively immune PRV-challenged pigs and both passively immune, contact-infected, unchallenged pigs (Fig. 4). There appeared to be no difference in IgM antibody response between pigs challenged with 106-5 or 104-5 TCID50 of Shope strain PRV. The IgM antibody response seen in the two passively immune, contact-infected pigs tended to be weaker than that seen in the challenged pigs. The IgM antibody response was still detectable up to 20 days p.i. in
A.
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both challenged and unchallenged groups. The IgG antibody levels continued to decrease until 10 to 14 days postchallenge in sera from pigs in both challenged and unchallenged groups (Fig. 4). The subsequent rapid rise in IgG antibody levels was approximately the same for all three treatment groups; however, the antibody response for the contactinfected pigs tended to be weaker.
5
DISCUSSION 0
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DAYS POSTINFECTION FIG. 2. Group mean antibody responses in sera collected serially from 16 seronegative 6-week-old pigs infected intranasally with either 1040 or 106. TCID50 of strain 4892 PRV, as tested by the PRV MACELISA (0) or the indirect PRV IgG ELISA (0).
The MACELISA developed in this study appeared to be a specific and sensitive assay for the detection of PRV-specific IgM antibodies. The selected cutoff OD ratio of 1.28 was 95% specific when sera from PRV-free pigs (group 1 sera) were tested by the MACELISA. At this cutoff value, 14 of 16 of the acute-phase serum samples collected from naturally infected sows were positive in the PRV MACELISA and all of the convalescent-phase serum samples were negative. The MACELISA was at least as sensitive as previously reported PRV IgM antibody tests, detecting antibody responses in 25% of pigs at 3 days p.i. (4, 8, 11, 13). An antibody response was still detectable in 75% of pigs 21 days
p.i. The reproducibility of the PRV MACELISA was similar to those of ELISAs used to evaluate sera from patients possibly infected with Toxoplasma gondii or rubella virus (1,
MACELISA FOR MEASURING SWINE ANTIBODY RESPONSES TO PRV
VOL. 30, 1992
a
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349
have been recently infected by PRV, since unchallenged, passively immune, contact-infected pigs showed an antibody response similar to that of challenged, passively immune penmates. The MACELISA appears to be a useful tool for the detection of pigs recently infected with PRV. At this time, the detection of PRV-specific IgM antibody indicates that initial infection occurred approximately 7 to 21 days previously. Additional work is needed to determine whether weak PRV-specific IgM antibody responses with low PRVspecific IgG antibody levels can be used to differentiate between early PRV infection in passively immune piglets versus late PRV infection in nonimmune pigs approximately 21 days p.i. However, this type of antibody response probably indicates early infection if the pigs are displaying clinical signs near the time of blood collection or if they are greater than 14 weeks of age. More experiments are needed to conclude whether the MACELISA is reliable for the detection of recent PRV infection of previously vaccinated swine. However, the MACELISA appears to be suitable for monitoring the PRV infection status of passively immune
pigs. ACKNOWLEDGMENTS
a
2.0 °0 1.0, of. 09
This work was supported by USDA grant 85-CSRS-2-2585. Special thanks go to Robert L. Vessella.
0.0,
0 7 14 21 28 DAYS POST-CHALLENGE FIG. 4. Group mean antibody responses in sera collected serially from 10 passively immune 5-week-old pigs. Four pigs each were infected intranasally with 1065 (A) or 104-5 (0) TCID50 of Shope strain PRV. Two pigs were left as contact-infected controls (*). All sera were assayed by the MACELISA (A) and the indirect PRV IgG ELISA (B).
2, 7, 15). The within-plate reproducibility of the MACELISA (expressed as the coefficient of variation) ranged from 6.13 to 15.38%. The between-day reproducibility of the MAC ELISA varied from 19.06 to 29.35%. Coating new plates daily, rather than coating many plates at one time and storing them for later use, may have contributed to the high coefficients of variation for sera tested on different days with the MACELISA (1). The MACELISA did not consistently detect an antibody response in sera from vaccinated pigs. The PRV vaccines used may have not stimulated an antibody response strong enough to be detected, but stronger IgM antibody responses after vaccination have been detected in other studies (11, 13, 14). A MACELISA antibody response was detected following challenge in passively immune pigs; this response occurred later, was of a lower magnitude, and was of a shorter duration than that in infected pigs. These results agree with those of Westenbrink and Kimman (16), who investigated the IgM antibody response in passively immune calves subsequently infected with bovine respiratory syncytial virus. The dose of PRV used for challenge did not have a significant effect on the IgM antibody response in this study (P > 0.05). Also, the MACELISA may be effective for determining whether passively immune pigs
1.
2.
3.
4.
5.
6.
7.
8. 9.
REFERENCES Blomberg, J., I. Nilsson, and M. Andersson. 1983. Viral antibody screening system that uses a standardized single dilution immunoglobulin G enzyme immunoassay with multiple antigens. J. Clin. Microbiol. 17:1081-1091. Enders, G., and F. Knotek. 1985. Comparison of the performance and reproducibility of various serological methods and diagnostic kits for the detection of rubella antibodies. J. Virol. Methods 11:1-14. Hill, H. T., R. A. Crandeli, C. L. Kanitz, J. P. McAdaragh, G. L. Seawright, R. F. Solorzano, and W. C. Stewart. 1977. Recommended minimum standards for diagnostic tests employed in the diagnosis of pseudorabies (Aujeszky's disease), p. 375-390. In 20th Annual Proceedings of the American Association of Veterinary Laboratory Diagnosticians. American Association of Veterinary Laboratory Diagnosticians, Inc., Madison, Wis. Iglesias, J. G., and J. W. Harkness. 1988. Studies of transplacental and perinatal infection with two clones of a single Aujeszky's disease (pseudorabies) virus isolate. Vet. Microbiol. 16:243-254. Joo, H. S., T. W. Molitor, and A. D. Leman. 1984. Radial immunodiffusion enzyme assay for detection of antibodies to pseudorabies virus in swine serum. Am. J. Vet. Res. 45:20962098. Kluge, J. P., Y. Niyo, L. D. Miller, H. T. Hill, and E. C. Pirtle. 1986. Comparison of lesions induced by three Aujeszky's disease viral isolates, p. 338. In Proceedings of the International Pig Veterinary Society Meeting. American Association of Swine Practitioners, Des Moines, lowa. Konishi, E., and J. Takahashi. 1983. Reproducible enzymelinked immunosorbent assay with a magnetic processing system for diagnosis of toxoplasmosis. J. Clin. Microbiol. 17:225231. Martin, S., R. C. Wardley, and A. I. Donaldson. 1983. Serological response of pigs infected with Aujeszky's disease virus. Res. Vet. Sci. 35:227-233. Paul, P. S., W. Mengeling, C. E. Maistrom, and R. A. Van Deusen. 1989. Production and characterization of monoclonal antibodies to porcine immunoglobulin gamma, alpha, and light
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chains. Am. J. Vet. Res. 50:471-479. 10. Paul, P. S., R. A. Van Deusen, and W. L. Mengeling. 1985. Monoclonal precipitating antibodies to porcine immunoglobulin M. Vet. Immunol. Immunopathol. 8:311-328. 11. Rodak, L., B. Smid, L. Valicek, and E. Jurak. 1987. Four-layer enzyme immunoassay (EIA) detection of differences in IgG, IgM and IgA antibody response to Aujeszky's disease virus in infected and vaccinated pigs. Vet. Microbiol. 13:121-133. 12. Schluederberg, A. 1965. Immune globulins in human viral infections. Nature (London) 205:1232-1233. 13. Snyder, M. L. 1986. Serological differentiation of IgM and IgG antibodies to PRV, p. 134-143. In Proceedings of the Livestock Conservation Institute. Livestock Conservation Institute, Madison, Wis.
J. CLIN. MICROBIOL. 14.
van Zaane, D., R. A. M. Brouwers, J. T. van Oirschot, J. M. A. Pol, and T. G. Kimman. 1989. Mucosal immunity of Aujeszky's disease, p. 139-149. In J. T. van Oirschot (ed.), Vaccination and control of Aujeszky's disease. Kluwer Academic Publishers, Boston. 15. Vernon, S. D., and P. A. Webb. 1985. Recent vesicular stomatitis virus infection detected by immunoglobulin M antibody capture enzyme-linked immunosorbent assay. J. Clin. Microbiol. 22:582-586. 16. Westenbrink, F., and T. G. Kimman. 1987. Immunoglobulin M-specific enzyme-linked immunosorbent assay for serodiagnosis of bovine respiratory syncytial virus infections. Am. J. Vet. Res. 48:1132-1137.
p.
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0095-1137/92/020346-05$02.00/0 Copyright © 1992, American Society for Microbiology
Characterization of Pseudorabies Virus Antibody Responses in Young Swine after Infection and Vaccination by Using an Immunoglobulin M Antibody Capture Enzyme-Linked Immunosorbent Assay MONTE B. MCCAW,l* TOM W. MOLITOR,2 AND H. S. JOO2 Department of Food Animal and Equine Medicine, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, North Carolina 27606,1 and Large Animal Clinical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 551082 Received 13 September 1991/Accepted 7 November 1991 An immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MACELISA) was developed for the detection of pseudorabies virus (PRV)-specific IgM antibody in swine sera because false-positive reactions frequently occurred when sera from older swine were tested with an indirect IgM enzyme-linked immunosorbent assay. Monoclonal mouse anti-swine IgM was used as the capturing antibody, and rabbit anti-PRV hyperimmune gamma globulin was used as the indicating antibody. Sera from non-PRV-infected, experimentally infected, vaccinated and challenged, passively immune and challenged, and naturally infected swine were evaluated. The PRV MACELISA had a specificity of 95% and was as sensitive and reproducible as previously reported in direct assays. An antibody response was still detectable with the MACELISA 21 days after inoculation. The PRV MACELISA did not detect a consistent antibody response in sera from swine vaccinated with either killed-PRV or modified live-virus vaccines but did detect an antibody response in sera from passively immune pigs after challenge with virulent PRV. These results indicated that the PRV MACELISA may be useful for the rapid serodiagnosis of recent PRV infection in swine. 10 to 12, 20 to 24, and >52 weeks) from each of five nonvaccinated PRV-free herds. Group 2 consisted of sera from animals experimentally infected intranasally with PRV strain 4892 (6). Sixteen seronegative 8-week-old pigs were intranasally inoculated with either 104.0 or 1060 50% tissue culture infective dose (TCID50), and sera were collected biweekly for 3 weeks. Group 3 consisted of six seronegative 8-week-old pigs vaccinated with either modified live-virus or killed-PRV vaccine. All pigs were challenged intranasally 28 days later with 106-5 TCID50 of Shope strain PRV. Sera were collected biweekly throughout the experiment. Group 4 consisted of 10 passively immune 5-week-old pigs (serum neutralization antibody titers, 1:4 to 1:8) that nursed PRVimmune sows. Four pigs each were infected with 104-5 or 106-5 TCID50 of Shope strain PRV and bled biweekly. The remaining two passively immune pigs were housed in the same room and served as contact-infected controls. Group 5 consisted of 16 sows in a swine herd that was experiencing a PRV epizootic. Acute- and convalescent-phase sera were collected, respectively, 10 and 52 days after the onset of clinical signs from these sows. Control sera used in each plate tested were obtained as follows. Negative control serum was a pool of sera from five of the 4-week-old pigs in group 1. Positive control serum was collected from a pig 10 days after experimental infection with Shope strain PRV. Antisera and conjugates. Mouse monoclonal anti-swine IgM and IgG ascites were generous gifts from Prem Paul, Iowa State University (9, 10). Rabbit anti-PRV hyperimmune gamma globulin was prepared by immunizing rabbits three times intramuscularly with PRV antigen mixed with a mineral oil adjuvant. Seven days after the last immunization, the rabbits were euthanatized and the sera were collected and precipitated with saturated ammonium sulfate. The precipitate was resuspended in distilled water and dialyzed
Immunoglobulin M (IgM) antibody is the first class of immunoglobulin produced in response to an infection and can be detected for a short period of time after infection (12). An assay that detects pseudorabies virus (PRV)-specific IgM antibody in serum samples may be used to identify pigs recently infected with PRV. The purpose of this report was to describe an IgM antibody capture enzyme-linked immunosorbent assay (MAC ELISA) for the detection of PRV-specific IgM antibody in swine sera and to characterize the PRV-specific IgM antibody responses in naturally or experimentally infected, passively immune, or vaccinated swine. MATERIALS AND METHODS Virus and vaccine. Virulent PRV strain 4892 was a gift from John Kluge and Howard Hill, Iowa State University. Shope strain PRV was obtained from the National Animal Disease Center, Ames, Iowa. Stock PRV was prepared in
pig kidney (PK-15) cells by routine methods. Modified live-virus and killed-PRV vaccines were purchased from Norden Laboratories, Lincoln, Nebr., and were administered in accordance with the manufacturer's recommendations. Preparation of PRV antigen. PRV antigen was prepared from cultured PRV-infected PK-15 cells (5). Sera. The following groups of sera were used. Group 1 consisted of 200 serum samples previously determined to be seronegative for PRV by serum neutralization (3) and was used to establish the cutoff between positive and negative values in the MACELISA. The 200 serum samples originated from 10 pigs each of four different age groups (4 to 6, *
Corresponding author. 346
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exhaustively against phosphate-buffered saline (PBS, pH 7.2). This solution was absorbed by gentle agitation with pelleted, uninfected PK-15 cells for 2 h at 37°C. Following centrifugation at 2,000 x g for 15 min, the supernatant was collected and stored at -20°C in aliquots. Goat anti-mouse IgG-IgM-IgA-peroxidase (Cappel Inc., Durham, N.C.) was used as the conjugate in the indirect PRV IgG enzyme-linked immunosorbent assay (ELISA). Goat anti-rabbit IgG-peroxidase (Kirkegaard and Perry Laboratories, Gaithersburg, Md.) was used as the conjugate in the PRV MACELISA. Indirect PRV IgG ELISA. The indirect PRV IgG ELISA was performed similarly to the method of Martin et al. (8). Polystyrene ELISA plates (Immulon I; Dynatech Laboratories, Alexandria, Va.) were coated with 100 ,ul of a 1:400 dilution of PRV antigen in coating buffer (50 mM carbonatebicarbonate buffer [pH 9.6]) and incubated overnight at 4°C. All plates were washed three times with PBS containing 0.05% Tween 20 between each step. Sera diluted (1:50) in PBS were added in triplicate. Negative and positive control sera were included on each plate. The plates were incubated for 45 min at 22°C and washed, and 100 ,ul of mouse monoclonal anti-swine IgG ascites (1:2,500) in PBS was added per well. The plates were incubated for 3 h at 37°C and washed. Goat anti-mouse IgG-IgA-IgM conjugate (0.04 mg/ ml; diluted 1:960 in PBS) was added to each plate, and the plates were incubated for 45 min at 22°C. The plates were washed, substrate (o-phenylenediamine dihydrochloride in citrate-phosphate buffer with H202) was added, and the plates were incubated for 5 to 10 min. The reaction was stopped by the addition of 5 N H2SO4. The plates were read on a Dynatech ELISA reader (MR 580; incidence wavelength, 540 nm; reference wavelength, 490 nm). The results were expressed as the sample optical density (OD) ratio (mean sample OD/mean negative control OD). MACELISA. MACELISA plates were coated with mouse monoclonal anti-swine IgM ascites (1:500; 100 ,ul per well) in coating buffer. The plates were incubated overnight at 4°C and washed three times. Diluted sera (1:40) were added in triplicate, and the plates were incubated for 3 h at 37°C. Negative and positive control sera were included on each plate. The plates were washed five times, and PRV antigen (1:500 in PBS; 100 ,ul per well) was added. The plates were incubated at 4°C overnight and washed four times. Rabbit anti-PRV (1:20,000 in PBS; 100 p.l per well) with 0.1% normal mouse ascites was added to each plate. After 2 h of incubation at 22°C, the plates were washed six times and goat anti-rabbit IgG-peroxidase conjugate (0.04 mg/ml diluted 1:480 in PBS; 100 p.l per well) was added. The plates were incubated for 2 h at room temperature and washed five times, and o-phenylenediamine dihydrochloride substrate was added. The reaction was stopped by the addition of 5 N H2SO4. The plates were read as described for the indirect ELISA. The results were expressed as the sample OD ratio. RESULTS Specificity of the PRV MACELISA. To evaluate the specificity of the PRV MACELISA, we tested group 1 sera. The mean age group OD ratios ranged from 1.016 to 1.080, with standard deviations of less than or equal to +0.141. The PRV MACELISA results for the 200 uninfected-pig serum samples were used to set a positive/negative cutoff OD ratio. The PRV MACELISA was 95% specific when the positive/ negative OD ratio was fixed at 1.28. Only 10 of the 200 serum samples had OD ratios higher than 1.28 in the MACELISA.
347
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o
1.00
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20 40
80 160 320 640 1280 2560 5120
Serum Dilution FIG. 1. Dilution effect on MACELISA OD ratios of a serum sample collected from a 6-week-old pig 10 days after experimental infection with strain 4892 PRV (0) and a serum sample collected from an uninfected 6-week-old pig (0).
Acute- and convalescent-phase sera from naturally infected sows (group 5 sera) were also tested by the MACELISA. The mean OD ratios for the acute and convalescent phases were 1.92 and 1.07 with 87.5 and 0.0% of the sera testing positive, respectively. Eleven of the 16 acute-phase sow serum samples had OD ratios of .1.50, and 14 of the 16 convalescent-phase sow sera had OD ratios of .1.10. The remaining two convalescent-phase sow sera had OD ratios of 1.28) in a 1:5,120 dilution of serum collected 10 days after experimental infection of a seronegative 6-week-old pig (Fig. 1). Reproducibility. The reproducibility of the PRV MAC ELISA was evaluated with one negative (group 1) and three positive (group 2) serum samples. Nine replicates of each serum sample were tested in the same plate on each of four different days. The within-plate coefficient of variation ranged from 6.13 to 15.38%, and the between-day coefficient of variation ranged from 19.06 to 29.35% (Table 1). Antibody response after experimental PRV infection. Sera collected from 16 seronegative pigs experimentally infected with strain 4892 PRV were tested by the MACELISA and the indirect IgG ELISA. An antibody response was detected in sera from four of these pigs 3 days postinfection (p.i.) by the MACELISA. These sera had OD ratios of 1.31 to 1.35. The group mean antibody response was first detected 7 days p.i. by the MACELISA and 10 days p.i. by the indirect IgG ELISA (Fig. 2). The mean MACELISA response peaked at 10 days p.i. and was still detectable (OD ratio, >1.28) at 21
348
McCAW ET AL.
J. CLIN. MICROBIOL.
TABLE 1. Reproducibility of the PRV MACELISA Test'
Serum
Coefficient of
Within-plate
Negative Weakly positive Intermediately positive
Strongly positive
15.38 10.26 7.54 6.13
Negative Weakly positive Intermediately positive Strongly positive
19.06 20.70 20.17 29.35
Between-day
variation (%)
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days p.i. The group mean indirect IgG ELISA response began to rise 10 days p.i. and was highest at 21 days p.i. Antibody response after vaccination and subsequent challenge. PRV-specific IgM and IgG antibody responses were evaluated in serial serum samples collected from six pigs vaccinated with commercially available products and challenged with virulent PRV at 28 days after vaccination. No IgM antibody response was detected in any of the vaccinated pigs up to 17 days postvaccination (Fig. 3). On days 18 and 21 postvaccination, the sera from one pig had a strong response in the PRV MACELISA. A weak IgM antibody response was detected in sera collected 10 and 14 days postchallenge. An IgG antibody response was first detected 24 days postvaccination in both groups of pigs (Fig. 3). A high IgG antibody response was observed from 7 days postchallenge on in both groups of vaccinated pigs. Antibody response after experimental infection in passively immune pigs. An IgM antibody response was detected at 10 days p.i. in all passively immune PRV-challenged pigs and both passively immune, contact-infected, unchallenged pigs (Fig. 4). There appeared to be no difference in IgM antibody response between pigs challenged with 106-5 or 104-5 TCID50 of Shope strain PRV. The IgM antibody response seen in the two passively immune, contact-infected pigs tended to be weaker than that seen in the challenged pigs. The IgM antibody response was still detectable up to 20 days p.i. in
A.
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both challenged and unchallenged groups. The IgG antibody levels continued to decrease until 10 to 14 days postchallenge in sera from pigs in both challenged and unchallenged groups (Fig. 4). The subsequent rapid rise in IgG antibody levels was approximately the same for all three treatment groups; however, the antibody response for the contactinfected pigs tended to be weaker.
5
DISCUSSION 0
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DAYS POSTINFECTION FIG. 2. Group mean antibody responses in sera collected serially from 16 seronegative 6-week-old pigs infected intranasally with either 1040 or 106. TCID50 of strain 4892 PRV, as tested by the PRV MACELISA (0) or the indirect PRV IgG ELISA (0).
The MACELISA developed in this study appeared to be a specific and sensitive assay for the detection of PRV-specific IgM antibodies. The selected cutoff OD ratio of 1.28 was 95% specific when sera from PRV-free pigs (group 1 sera) were tested by the MACELISA. At this cutoff value, 14 of 16 of the acute-phase serum samples collected from naturally infected sows were positive in the PRV MACELISA and all of the convalescent-phase serum samples were negative. The MACELISA was at least as sensitive as previously reported PRV IgM antibody tests, detecting antibody responses in 25% of pigs at 3 days p.i. (4, 8, 11, 13). An antibody response was still detectable in 75% of pigs 21 days
p.i. The reproducibility of the PRV MACELISA was similar to those of ELISAs used to evaluate sera from patients possibly infected with Toxoplasma gondii or rubella virus (1,
MACELISA FOR MEASURING SWINE ANTIBODY RESPONSES TO PRV
VOL. 30, 1992
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349
have been recently infected by PRV, since unchallenged, passively immune, contact-infected pigs showed an antibody response similar to that of challenged, passively immune penmates. The MACELISA appears to be a useful tool for the detection of pigs recently infected with PRV. At this time, the detection of PRV-specific IgM antibody indicates that initial infection occurred approximately 7 to 21 days previously. Additional work is needed to determine whether weak PRV-specific IgM antibody responses with low PRVspecific IgG antibody levels can be used to differentiate between early PRV infection in passively immune piglets versus late PRV infection in nonimmune pigs approximately 21 days p.i. However, this type of antibody response probably indicates early infection if the pigs are displaying clinical signs near the time of blood collection or if they are greater than 14 weeks of age. More experiments are needed to conclude whether the MACELISA is reliable for the detection of recent PRV infection of previously vaccinated swine. However, the MACELISA appears to be suitable for monitoring the PRV infection status of passively immune
pigs. ACKNOWLEDGMENTS
a
2.0 °0 1.0, of. 09
This work was supported by USDA grant 85-CSRS-2-2585. Special thanks go to Robert L. Vessella.
0.0,
0 7 14 21 28 DAYS POST-CHALLENGE FIG. 4. Group mean antibody responses in sera collected serially from 10 passively immune 5-week-old pigs. Four pigs each were infected intranasally with 1065 (A) or 104-5 (0) TCID50 of Shope strain PRV. Two pigs were left as contact-infected controls (*). All sera were assayed by the MACELISA (A) and the indirect PRV IgG ELISA (B).
2, 7, 15). The within-plate reproducibility of the MACELISA (expressed as the coefficient of variation) ranged from 6.13 to 15.38%. The between-day reproducibility of the MAC ELISA varied from 19.06 to 29.35%. Coating new plates daily, rather than coating many plates at one time and storing them for later use, may have contributed to the high coefficients of variation for sera tested on different days with the MACELISA (1). The MACELISA did not consistently detect an antibody response in sera from vaccinated pigs. The PRV vaccines used may have not stimulated an antibody response strong enough to be detected, but stronger IgM antibody responses after vaccination have been detected in other studies (11, 13, 14). A MACELISA antibody response was detected following challenge in passively immune pigs; this response occurred later, was of a lower magnitude, and was of a shorter duration than that in infected pigs. These results agree with those of Westenbrink and Kimman (16), who investigated the IgM antibody response in passively immune calves subsequently infected with bovine respiratory syncytial virus. The dose of PRV used for challenge did not have a significant effect on the IgM antibody response in this study (P > 0.05). Also, the MACELISA may be effective for determining whether passively immune pigs
1.
2.
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4.
5.
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7.
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REFERENCES Blomberg, J., I. Nilsson, and M. Andersson. 1983. Viral antibody screening system that uses a standardized single dilution immunoglobulin G enzyme immunoassay with multiple antigens. J. Clin. Microbiol. 17:1081-1091. Enders, G., and F. Knotek. 1985. Comparison of the performance and reproducibility of various serological methods and diagnostic kits for the detection of rubella antibodies. J. Virol. Methods 11:1-14. Hill, H. T., R. A. Crandeli, C. L. Kanitz, J. P. McAdaragh, G. L. Seawright, R. F. Solorzano, and W. C. Stewart. 1977. Recommended minimum standards for diagnostic tests employed in the diagnosis of pseudorabies (Aujeszky's disease), p. 375-390. In 20th Annual Proceedings of the American Association of Veterinary Laboratory Diagnosticians. American Association of Veterinary Laboratory Diagnosticians, Inc., Madison, Wis. Iglesias, J. G., and J. W. Harkness. 1988. Studies of transplacental and perinatal infection with two clones of a single Aujeszky's disease (pseudorabies) virus isolate. Vet. Microbiol. 16:243-254. Joo, H. S., T. W. Molitor, and A. D. Leman. 1984. Radial immunodiffusion enzyme assay for detection of antibodies to pseudorabies virus in swine serum. Am. J. Vet. Res. 45:20962098. Kluge, J. P., Y. Niyo, L. D. Miller, H. T. Hill, and E. C. Pirtle. 1986. Comparison of lesions induced by three Aujeszky's disease viral isolates, p. 338. In Proceedings of the International Pig Veterinary Society Meeting. American Association of Swine Practitioners, Des Moines, lowa. Konishi, E., and J. Takahashi. 1983. Reproducible enzymelinked immunosorbent assay with a magnetic processing system for diagnosis of toxoplasmosis. J. Clin. Microbiol. 17:225231. Martin, S., R. C. Wardley, and A. I. Donaldson. 1983. Serological response of pigs infected with Aujeszky's disease virus. Res. Vet. Sci. 35:227-233. Paul, P. S., W. Mengeling, C. E. Maistrom, and R. A. Van Deusen. 1989. Production and characterization of monoclonal antibodies to porcine immunoglobulin gamma, alpha, and light
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chains. Am. J. Vet. Res. 50:471-479. 10. Paul, P. S., R. A. Van Deusen, and W. L. Mengeling. 1985. Monoclonal precipitating antibodies to porcine immunoglobulin M. Vet. Immunol. Immunopathol. 8:311-328. 11. Rodak, L., B. Smid, L. Valicek, and E. Jurak. 1987. Four-layer enzyme immunoassay (EIA) detection of differences in IgG, IgM and IgA antibody response to Aujeszky's disease virus in infected and vaccinated pigs. Vet. Microbiol. 13:121-133. 12. Schluederberg, A. 1965. Immune globulins in human viral infections. Nature (London) 205:1232-1233. 13. Snyder, M. L. 1986. Serological differentiation of IgM and IgG antibodies to PRV, p. 134-143. In Proceedings of the Livestock Conservation Institute. Livestock Conservation Institute, Madison, Wis.
J. CLIN. MICROBIOL. 14.
van Zaane, D., R. A. M. Brouwers, J. T. van Oirschot, J. M. A. Pol, and T. G. Kimman. 1989. Mucosal immunity of Aujeszky's disease, p. 139-149. In J. T. van Oirschot (ed.), Vaccination and control of Aujeszky's disease. Kluwer Academic Publishers, Boston. 15. Vernon, S. D., and P. A. Webb. 1985. Recent vesicular stomatitis virus infection detected by immunoglobulin M antibody capture enzyme-linked immunosorbent assay. J. Clin. Microbiol. 22:582-586. 16. Westenbrink, F., and T. G. Kimman. 1987. Immunoglobulin M-specific enzyme-linked immunosorbent assay for serodiagnosis of bovine respiratory syncytial virus infections. Am. J. Vet. Res. 48:1132-1137.
