Veterinary Microbiology, 32 (1992) 363-374 Elsevier Science Publishers B.V., Amsterdam
363
In vitro stimulation of antibody production to Mycoplasma hyopneumoniaeby porcine peripheral blood mononuclear cells Per Wallgren ~'b, G 6 r a n B61ske a and Caroline Fossum b aThe National VeterinaryInstitute, Uppsala, Sweden bDepartment of Veterinary Microbiology, Divisionfor Immunology, Swedish University of Agricultural Sciences, Biomedicum, Uppsala, Sweden (Accepted 30 January 1992)
ABSTRACT Wallgren, P., B61ske, G. and Fossum, C., 1992. In vitro stimulation of antibody production to Mycoplasma hyopneumoniae by porcine peripheral blood mononuclear cells. Vet. Microbiol., 32: 363374. A method to stimulate and detect the in vitro production of antibodies to Mycoplasma hyopneumoniae by porcine peripheral blood mononuclear cells (PBMC) was established. PBMC were cultured in microtiter plates coated with a sonicated M. hyopneumoniae whole cell antigen and the amount of antibody bound to the coating antigen was determined by an enzyme linked immunosorbent assay (ELISA). In addition, the amount of non-bound antibody was determined by testing the culture supernatants in the ELISA which detects porcine antibodies to M. hyopneumoniae. The production of antibodies, in terms of total absorbance values, was enhanced by including 2.5 ng pokeweed mitogen (PWM) per ml growth medium without altering the specificity of the assay. In a pilot experiment, the applicability of the method to follow the development of antigen-reactive cells during primary and secondary immunizations with M. hyopneumoniae was evaluated. Antigenreactive cells, identified by their ability to produce antibodies to M. hyopneumoniae in vitro, were detected seven days after the primary immunization and reached their highest antigen reactivity one week later. In comparison, antigen-reactive cells could be detected three days after the booster immunization and remained in the circulation for 2 weeks.
INTRODUCTION
The high frequency of pneumonia caused by infection with Mycoplasma hyopneumoniae during breeding of pigs is of both economic (Straw et al., 1990) and ethical interest. In Sweden, up to 22% of the piglets which have been mixed from different breeders and transported to specialized fattening Correspondence to." P. Wallgren, The National Veterinary Institute, Box 7073, S-750 05 Uppsala, Sweden.
0378-1135/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
364
P. WALLGREN ET AL.
herds already have antibodies to M. hyopneumoniae at allocation. During the first month after allocation this figure does not increase, but when reaching marketing weight 4 months later, 90% of the fattening pigs have antibodies to M. hyopneumoniae (Wallgren et al., 1990). Failure to diagnose the spread of M. hyopneumoniae infections during the first period after allocation may either be because there is no spread ofM. hyopneumoniae during this time or may be explained by a suppressed ability to mount an i m m u n e response within that time. Indication of a suppressed i m m u n e reactivity has previously been found from studies of peripheral blood mononuclear cells (PBMC) obtained from pigs at various times after allocation to fattening units. Both the ability to produce c~-interferon and the responsiveness to mitogen stimulation were reduced for PBMC obtained from newly allocated pigs (Artursson et al., 1989 ). Therefore, the release of stress hormones during transport and mixing of pigs was regarded as a possible reason for the reduced i m m u n e functions recorded in vitro. For several other species, elevated cortisol levels are proven to have a negative impact on i m m u n e functions while the pig is regarded as a relatively corticosteroid-resistant species (for review see: Frank and Griffin, 1989). Further, the sensitivity to corticosteroids differs, also in the pig, with age and between various subpopulations of lymphoid cells (Yang and Schultz, 1986 ). Consequently, the relevance of cortisol in the context of stress-mediated effects on the antibody-production can be doubtful in pigs. The aim of the present study was therefore to develop a method to measure the ability of porcine PBMC to produce antibodies to M. hyopneumoniae in vitro to enable further studies on the importance of stress on the i m m u n e reactivity in pigs. MATERIAL AND METHODS
Animals and experimental design Experiment A. an in vitro system for stimulation and quantitative determination of antibody production to M. hyopneumoniae was established with blood mononuclear cells obtained from seven conventional fattening pigs. The pigs had been housed for 20-45 days in an M. hyopneumoniae infected fattening unit.
Experiment B. the relationship between the antibody production to M. hyopneumoniae in vitro and the serological status of the animals was evaluated. For that purpose, samples were collected from 14 conventional fattening pigs, raised for 60 days in a fattening unit with known M. hyopneumoniae infection present on a herd basis. Two weeks later (day 75), samples were collected again from the same animals.
IN VITRO STIMULATION OF ANTIBODY PRODUCTION
365
Experiment C. the kinetics and magnitude of antibody production to M. hyopneumoniae after experimental immunization were studied by the in vitro method as well as in serum. Six specific pathogen free (SPF) pigs (aged six months at the beginning of the experiment) were housed at the National Veterinary Institute, Uppsala, Sweden. The animals, three boars (nos. 45, 55 and 155 ) and three gilts (nos. 43, 50 and 51 ), were immunized and sampled as described below. Immunization with M. hyopneumoniae Antigen for immunization was prepared from M. hyopneumoniae, strain V 27 (kindly provided by Dr Kobisch, Ploufragan, France) which was cultured in Friis broth with 10% SPF swine serum. The antigen preparation was further performed as described earlier (B61ske et al., 1987). The six SPF pigs were injected i.m. with 1.5 ml antigen ( 1.0 mg protein/ml) mixed with an equal volume of Freund's incomplete adjuvant on day 0 (directly after blood sampling). On day 25 the immunization was repeated with 1.5 ml M. hyopneumoniae antigen mixed with an equal volume of aluminum hydroxide adjuvant. Blood samples Blood samples were collected by jugular vein puncture using evacuated glass tubes (B-D Vacutatiner, Grenoble Cedex, France) with or without heparin ( 143 USP units) as additive. In the immunization experiment (experiment C), blood samples were collected from the six SPF animals on days 0, 7,14, 21, 28, 35, 42 and 52. Detection of antibodies to M. hyopneumoniae in porcine serum Sera were tested for the presence of antibodies to M. hyopneumoniae with an enzyme linked immunosorbent assay (ELISA) using a Tween 20 treated preparation of M. hyopneumoniae (B61ske et al., 1990) as coating antigen. Microtiter plates, M 129A (Dynatech, Chantilly, Virginia, USA), were coated with 200 IA M. hyopneumoniae antigen (0.2/tg/ml in 0.05 M carbonate buffer pH 9.6) overnight at room temperature (RT). The plates were washed three times with phosphate buffered saline (PBS) containing 0.1% Tween 20, pH 7.2 (PBS-T). Swine sera, diluted in PBS-T, were applied in volumes of 200 /zl. After 4 hours of incubation at RT, the plates were washed three times with PBS-T and 200/tl peroxidase conjugated anti-swine immunoglobulin (Dakopatts, Copenhagen, Denmark) diluted 1/6000 in PBS-T was added to each well. After 1 h of incubation at 37 °C the plates were washed again with PBST and 200/zl/well of the substrate solution (0.006% H202 and 0.10 mg/ml 3,5,3',5',-tetramethylbenzidine (Merck, Darmstadt, Germany) in 0.1 M acetate buffer pH 6.0) was added. The reaction was stopped by addition of 50 ~1 2M H 2 S O 4 and the absorbance values at 450 nm were determined. A
366
P WALLGREN ET AL.
positive swine serum standard was included on each plate and the measured absorbance values were corrected to a standard value equal to 0.9 absorbance units. Mean absorbance value for 64 SPF sera, diluted 1/ 100, plus three standard deviations plus 0.1 absorbance unit was used as the limit for positive reactions (A45o= 0.5 ). The levels of antibodies to M. hyopneumoniae are either expressed as A45o values for sera diluted 1/ 100 or as log~o ELISA titres calculated from serial tenfold dilutions of serum.
Isolation of peripheral blood mononuclear cells (PBMC) The heparinized blood, diluted 1:2 in PBS, was layered on Ficoll-Paque (Pharmacia, Uppsala, Sweden) After centrifugation at 550 g for 30 min the PBMC were collected and washed three times in PBS by centrifugation at 180 g for 10 min. After the final washing, the cells were resuspended in RPMI 1640 m e d i u m with 20 m M HEPES buffer, supplemented with glutamine (2 m M ), penicillin ( 200 I U / m l ) and 5% FCS (Myoclone; Gibco, Scotland ), i.e. growth medium. The number of cells was adjusted to 4 X 106 cells/ml growth medium.
Stimulation and quantitative determination of antibody production to M. hyopneumoniae in vitro Microtiter plates (Nunc, Roskilde, Denmark), coated with 2.0/~g/ml of a sonicated whole cell antigen of M. hyopneumoniae strain J (B61ske et al., 1990), were used to induce the production of antibodies to M. hyopneumoniae in vitro. The plates were washed with PBS-T followed by careful rinsing in PBS to remove any detergent before 4X l0 s PBMC in 0.1 ml growth medium was added to quadruplicate wells. If not otherwise indicated, 0.1 ml growth m e d i u m containing 5 ng Pokeweed mitogen (PWM: Boehringer, Mannheim, Germany) per ml was added to each well and the cells were cultured for various periods of time in a humid atmosphere with 7% CO2 at 37°C. At the end of culture, 150 ~1 of each supernatant was transferred to another microtiter plate also coated with whole cell M. hyopneumoniae antigen, and incubated for 2 h at RT. Thereafter, both the plate used for induction of antibody production and that with supernatants, were washed. Antibodies produced in vitro and bound to the two plates were then detected by the same ELISA technique as described above for detection of antibodies to M. hyopneumoniae in serum. For calculation of background values, four wells with 200/zl growth medium containing 5 ng P W M / m l , and four wells with PBS only, were included on the microtiter plate used for induction of antibody production while the positive standard serum was included on each ELISA-plate with the supernatants. After subtraction of the background values (~
363
In vitro stimulation of antibody production to Mycoplasma hyopneumoniaeby porcine peripheral blood mononuclear cells Per Wallgren ~'b, G 6 r a n B61ske a and Caroline Fossum b aThe National VeterinaryInstitute, Uppsala, Sweden bDepartment of Veterinary Microbiology, Divisionfor Immunology, Swedish University of Agricultural Sciences, Biomedicum, Uppsala, Sweden (Accepted 30 January 1992)
ABSTRACT Wallgren, P., B61ske, G. and Fossum, C., 1992. In vitro stimulation of antibody production to Mycoplasma hyopneumoniae by porcine peripheral blood mononuclear cells. Vet. Microbiol., 32: 363374. A method to stimulate and detect the in vitro production of antibodies to Mycoplasma hyopneumoniae by porcine peripheral blood mononuclear cells (PBMC) was established. PBMC were cultured in microtiter plates coated with a sonicated M. hyopneumoniae whole cell antigen and the amount of antibody bound to the coating antigen was determined by an enzyme linked immunosorbent assay (ELISA). In addition, the amount of non-bound antibody was determined by testing the culture supernatants in the ELISA which detects porcine antibodies to M. hyopneumoniae. The production of antibodies, in terms of total absorbance values, was enhanced by including 2.5 ng pokeweed mitogen (PWM) per ml growth medium without altering the specificity of the assay. In a pilot experiment, the applicability of the method to follow the development of antigen-reactive cells during primary and secondary immunizations with M. hyopneumoniae was evaluated. Antigenreactive cells, identified by their ability to produce antibodies to M. hyopneumoniae in vitro, were detected seven days after the primary immunization and reached their highest antigen reactivity one week later. In comparison, antigen-reactive cells could be detected three days after the booster immunization and remained in the circulation for 2 weeks.
INTRODUCTION
The high frequency of pneumonia caused by infection with Mycoplasma hyopneumoniae during breeding of pigs is of both economic (Straw et al., 1990) and ethical interest. In Sweden, up to 22% of the piglets which have been mixed from different breeders and transported to specialized fattening Correspondence to." P. Wallgren, The National Veterinary Institute, Box 7073, S-750 05 Uppsala, Sweden.
0378-1135/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
364
P. WALLGREN ET AL.
herds already have antibodies to M. hyopneumoniae at allocation. During the first month after allocation this figure does not increase, but when reaching marketing weight 4 months later, 90% of the fattening pigs have antibodies to M. hyopneumoniae (Wallgren et al., 1990). Failure to diagnose the spread of M. hyopneumoniae infections during the first period after allocation may either be because there is no spread ofM. hyopneumoniae during this time or may be explained by a suppressed ability to mount an i m m u n e response within that time. Indication of a suppressed i m m u n e reactivity has previously been found from studies of peripheral blood mononuclear cells (PBMC) obtained from pigs at various times after allocation to fattening units. Both the ability to produce c~-interferon and the responsiveness to mitogen stimulation were reduced for PBMC obtained from newly allocated pigs (Artursson et al., 1989 ). Therefore, the release of stress hormones during transport and mixing of pigs was regarded as a possible reason for the reduced i m m u n e functions recorded in vitro. For several other species, elevated cortisol levels are proven to have a negative impact on i m m u n e functions while the pig is regarded as a relatively corticosteroid-resistant species (for review see: Frank and Griffin, 1989). Further, the sensitivity to corticosteroids differs, also in the pig, with age and between various subpopulations of lymphoid cells (Yang and Schultz, 1986 ). Consequently, the relevance of cortisol in the context of stress-mediated effects on the antibody-production can be doubtful in pigs. The aim of the present study was therefore to develop a method to measure the ability of porcine PBMC to produce antibodies to M. hyopneumoniae in vitro to enable further studies on the importance of stress on the i m m u n e reactivity in pigs. MATERIAL AND METHODS
Animals and experimental design Experiment A. an in vitro system for stimulation and quantitative determination of antibody production to M. hyopneumoniae was established with blood mononuclear cells obtained from seven conventional fattening pigs. The pigs had been housed for 20-45 days in an M. hyopneumoniae infected fattening unit.
Experiment B. the relationship between the antibody production to M. hyopneumoniae in vitro and the serological status of the animals was evaluated. For that purpose, samples were collected from 14 conventional fattening pigs, raised for 60 days in a fattening unit with known M. hyopneumoniae infection present on a herd basis. Two weeks later (day 75), samples were collected again from the same animals.
IN VITRO STIMULATION OF ANTIBODY PRODUCTION
365
Experiment C. the kinetics and magnitude of antibody production to M. hyopneumoniae after experimental immunization were studied by the in vitro method as well as in serum. Six specific pathogen free (SPF) pigs (aged six months at the beginning of the experiment) were housed at the National Veterinary Institute, Uppsala, Sweden. The animals, three boars (nos. 45, 55 and 155 ) and three gilts (nos. 43, 50 and 51 ), were immunized and sampled as described below. Immunization with M. hyopneumoniae Antigen for immunization was prepared from M. hyopneumoniae, strain V 27 (kindly provided by Dr Kobisch, Ploufragan, France) which was cultured in Friis broth with 10% SPF swine serum. The antigen preparation was further performed as described earlier (B61ske et al., 1987). The six SPF pigs were injected i.m. with 1.5 ml antigen ( 1.0 mg protein/ml) mixed with an equal volume of Freund's incomplete adjuvant on day 0 (directly after blood sampling). On day 25 the immunization was repeated with 1.5 ml M. hyopneumoniae antigen mixed with an equal volume of aluminum hydroxide adjuvant. Blood samples Blood samples were collected by jugular vein puncture using evacuated glass tubes (B-D Vacutatiner, Grenoble Cedex, France) with or without heparin ( 143 USP units) as additive. In the immunization experiment (experiment C), blood samples were collected from the six SPF animals on days 0, 7,14, 21, 28, 35, 42 and 52. Detection of antibodies to M. hyopneumoniae in porcine serum Sera were tested for the presence of antibodies to M. hyopneumoniae with an enzyme linked immunosorbent assay (ELISA) using a Tween 20 treated preparation of M. hyopneumoniae (B61ske et al., 1990) as coating antigen. Microtiter plates, M 129A (Dynatech, Chantilly, Virginia, USA), were coated with 200 IA M. hyopneumoniae antigen (0.2/tg/ml in 0.05 M carbonate buffer pH 9.6) overnight at room temperature (RT). The plates were washed three times with phosphate buffered saline (PBS) containing 0.1% Tween 20, pH 7.2 (PBS-T). Swine sera, diluted in PBS-T, were applied in volumes of 200 /zl. After 4 hours of incubation at RT, the plates were washed three times with PBS-T and 200/tl peroxidase conjugated anti-swine immunoglobulin (Dakopatts, Copenhagen, Denmark) diluted 1/6000 in PBS-T was added to each well. After 1 h of incubation at 37 °C the plates were washed again with PBST and 200/zl/well of the substrate solution (0.006% H202 and 0.10 mg/ml 3,5,3',5',-tetramethylbenzidine (Merck, Darmstadt, Germany) in 0.1 M acetate buffer pH 6.0) was added. The reaction was stopped by addition of 50 ~1 2M H 2 S O 4 and the absorbance values at 450 nm were determined. A
366
P WALLGREN ET AL.
positive swine serum standard was included on each plate and the measured absorbance values were corrected to a standard value equal to 0.9 absorbance units. Mean absorbance value for 64 SPF sera, diluted 1/ 100, plus three standard deviations plus 0.1 absorbance unit was used as the limit for positive reactions (A45o= 0.5 ). The levels of antibodies to M. hyopneumoniae are either expressed as A45o values for sera diluted 1/ 100 or as log~o ELISA titres calculated from serial tenfold dilutions of serum.
Isolation of peripheral blood mononuclear cells (PBMC) The heparinized blood, diluted 1:2 in PBS, was layered on Ficoll-Paque (Pharmacia, Uppsala, Sweden) After centrifugation at 550 g for 30 min the PBMC were collected and washed three times in PBS by centrifugation at 180 g for 10 min. After the final washing, the cells were resuspended in RPMI 1640 m e d i u m with 20 m M HEPES buffer, supplemented with glutamine (2 m M ), penicillin ( 200 I U / m l ) and 5% FCS (Myoclone; Gibco, Scotland ), i.e. growth medium. The number of cells was adjusted to 4 X 106 cells/ml growth medium.
Stimulation and quantitative determination of antibody production to M. hyopneumoniae in vitro Microtiter plates (Nunc, Roskilde, Denmark), coated with 2.0/~g/ml of a sonicated whole cell antigen of M. hyopneumoniae strain J (B61ske et al., 1990), were used to induce the production of antibodies to M. hyopneumoniae in vitro. The plates were washed with PBS-T followed by careful rinsing in PBS to remove any detergent before 4X l0 s PBMC in 0.1 ml growth medium was added to quadruplicate wells. If not otherwise indicated, 0.1 ml growth m e d i u m containing 5 ng Pokeweed mitogen (PWM: Boehringer, Mannheim, Germany) per ml was added to each well and the cells were cultured for various periods of time in a humid atmosphere with 7% CO2 at 37°C. At the end of culture, 150 ~1 of each supernatant was transferred to another microtiter plate also coated with whole cell M. hyopneumoniae antigen, and incubated for 2 h at RT. Thereafter, both the plate used for induction of antibody production and that with supernatants, were washed. Antibodies produced in vitro and bound to the two plates were then detected by the same ELISA technique as described above for detection of antibodies to M. hyopneumoniae in serum. For calculation of background values, four wells with 200/zl growth medium containing 5 ng P W M / m l , and four wells with PBS only, were included on the microtiter plate used for induction of antibody production while the positive standard serum was included on each ELISA-plate with the supernatants. After subtraction of the background values (~
