Veterinary Microbiology, 22 (1990) 161-170 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
161
In Vitro P r o d u c t i o n of A n t i b o d i e s to B o v i n e Virus D i a r r h o e a V i r u s by P e r i p h e r a l B l o o d M o n o n u c l e a r Cells from Cattle I m m u n i z e d by Infection B. LARSSON 1, C. FOSSUM 2 and N. JUNTTI 3
'Department of Cattle and Sheep Diseases and 2Department of Veterinary Microbiology, Section of Virology, Swedish University of Agricultural Sciences, Box 7019, S-750 07 Uppsala (Sweden) 3Department of Virology, The National Veterinary Institute, Biomedicum, Uppsala (Sweden) (Accepted 17 October 1989)
ABSTRACT Larsson, B., Fossum, C. and Juntti, N., 1990. In vitro production of antibodies to bovine virus diarrhoea virus by peripheral blood mononuclear cells from cattle immunized by infection. Vet. Microbiol., 22: 161-170. A method for in vitro production of antibodies to bovine virus diarrhoea virus (BVDV) by peripheral blood mononuclear cells (PBMC) was developed. The PBMC were cultured in microtitre plates coated with detergent-solubilized BVDV and the supernatants were tested in an enzyme-linked immunosorbent assay which detects IgG antibodies to BVDV. Following incubation of PBMC with an optimal concentration of pokeweed mitogen for 5 days, antibodies to BVDV were detected in culture supernatants of PBMC from immune cattle, but not in supernatants of PBMC from seronegative cattle, from persistently BVDV-infected cattle or from a 5-day-old calf that received BVDV antibodies via colostrum. This antibody-production assay may therefore be used as a tool to discriminate between passively acquired antibodies and those actively induced.
INTRODUCTION
Cows infected with bovine virus diarrhoea virus (BVDV) during early pregnancy seroconvert but may give birth to persistently viraemic calves. As a rule, these calves are seronegative to BVDV and are therefore thought to be immunotolerant to the agent. Persistently infected calves appear to be more susceptible to opportunistic infections and constitute the population at risk of developing a fatal condition called mucosal disease (for a review, see Baker, 1987). The BVDV infects the cells of the immune system, and results of several studies indicate a hampered function of lymphocytes derived from infected cattle (Johnson and Muscoplat, 1973; Reggiardo and Kaeberle, 1981; Roth et al., 1986; Larsson, 1988; Larsson et al., 1988). However, there is no information
0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
162
B. LARSSON ET AL.
available about the ability of lymphocytes from immune and non-immune cattle to produce antibodies to BVDV in vitro. The aims of the present study were to develop a method for production of antibodies to BVDV by bovine peripheral blood mononuclear cells (PBMC), and to examine whether this production in vitro reflects the humoral immune status in vivo. MATERIALSAND METHODS Cattle Blood was collected from 15 cattle of Swedish Red and White breed. Three of the animals (nos. 1 to 3) belonged to a farm where all animals were seronegative to BVDV. Three animals (nos. 4 to 6) were considered to be persistently infected with non-cytopathic BVDV since the agent was isolated from their sera on two occasions 2 months apart, and they had no or only a low level of serum antibodies to BVDV. Eight of the animals (nos. 7 to 14) were seropositive to BVDV and were from the same herds as the persistently infected cattle. Animal no. 15 was a 5-day-old colostrum-fed offspring to a seropositive mother (no. 14). Detection of serum antibodies to B VD V Serum was tested for the presence of antibodies to BVDV by an indirect enzyme-linked immunosorbent assay (ELISA) as described before (Juntti et al., 1987 ). Briefly, microtitre plates were coated with detergent-solubilized cytopathic BVDV strain Singer (BVDV-ELISA plates), and sera were assayed in 4-fold dilutions (1:10 to 1:2560). Serum antibodies which bound to the BVDV were detected by a horseradish peroxidase (HRP)-conjugated monoclonal antibody (MAb) to bovine IgG. Isolation of cells Peripheral blood mononuclear cells (PBMC) were isolated from cattle as described before (Johnson and Morein, 1977). The PBMC were washed three times in phosphate-buffered saline and resuspended in RPMI-1640 growth medium containing glutamine (2mM), 2-mercaptoethanol (5 X 10- ~ M) and 7.5% heat-inactivated foetal calf serum (FCS) free from detectable IgG antibodies, which was tested as previously described (Juntti et al., 1987).
IN VITRO PRODUCTION OF ANTIBODIES TO BOVINE VIRUS DIARRHOEA VIRUS
163
Cell cultures for production of antibodies to B VD V To determine the optimal conditions for the generation of antibodies in vitro, PBMC were cultured at various concentrations in volumes of 200 #l per well in BVDV-ELISA plates and in the presence or absence of pokeweed mitogen (PWM; Boehringer Mannheim, Mannheim, F.R.G. ). The final concentration of PBMC ranged from 2.5 X 105 to 10 X 106 cells per ml and that of PWM from 0.12 pg to 8ttg per ml (4-step titration). Each combination was set up in hexaplicate, and cultures found to be of appropriate combination were incubated for 2, 5, 8 and 11 days, respectively, at 37°C.
Assays for detection of antibodies to B VD V generated in vitro After incubation of the cells, the amount of antibodies which had bound to the solid-phase antigens during incubation of cells and the amount of antibodies in supernatant were assayed.
Antibodies bound to the solid-phase antigens. The amount of antibodies was measured by adding the HRP-conjugated MAb to bovine IgG after thorough washing of the plates. The final steps of the ELISA were then performed as described above for detection of antibodies to BVDV in serum. The magnitude of antibody production was indicated by the mean absorbance value for each culture. Antibodies in supernatant. Supernatants from each combination were pooled and tested for antibodies to BVDV in an indirect ELISA. Some supernatants were also examined in a virus neutralization test (VNT) and in a competitive ELISA. The indirect ELISA was performed as described above for serum, and supernatants were assayed undiluted and in 2-fold dilutions up to 1 : 16. The antibody titre of a supernatant was determined as the highest dilution giving an absorbance value above the mean absorbance value + 3 S.D. (n = 6) of control medium (supplemented medium incubated without cells). The VNT was performed as previously described for serum (Larsson et al., 1988) and supernatants were tested undiluted and in dilutions of 1:2 and 1:4. The competitive ELISA was performed as previously described for serum (Juntti et al., 1987). However, the MAb to BVDV (VD-1) was used unconjugated. In brief, equal volumes (50~1) per well of a supernatant (undiluted and 2-fold dilutions up to 1 : 16) and the MAb VD-1, diluted 1 : 1000, were incubated in BVDV-ELISA plates. The amount of MAb VD-1 which bound to the solidphase antigens was detected by a HRP anti-mouse conjugate (Dakopatts, Denmark) diluted 1:1000.
164
B. LARSSON ET AL.
RESULTS
Factors influencing the in vitro production of antibodies to B VD V Concentration of PWM. Results from initial experiments showed that antibody production to BVDV was infrequently observed when PBMC from seropositive cows were cultured in BVDV-coated microtitre plates. In an attempt to enhance the antibody production, PWM was added to final concentrations that ranged from 0.12 pg to 8 #g per ml. As shown in Fig. 1, antibody production became pronounced at PWM concentrations ranging from about 0.5 ng to 0.1 ~g per ml with an optimum of 2 ng per ml. None of the various PWM concentrations elicted detectable antibody production when PBMC were cultured without BVDV, i.e., in an uncoated microtitre plate. Period of incubation. The time course of antibody production was studied for PBMC isolated from two animals, one seropositive and one seronegative to BVDV. The PBMC were cultured in growth medium alone or with the addition of PWM (2.5, 0.25 and 0.025 #g per ml). The incubation was interrupted after 2, 5, 8, and 11 days and the antibody titre to BVDV in pooled supernatants for each set of culture was determined in the indirect ELISA. Antibody production was not detected in 2-day cultures of PBMC (titre < 1 ) but was observed after prolonged incubation periods of the PBMC isolated from the seropositive an0.70.64J 0.5-
0
"0
0.4-
@ 0 .,4 4J
0.30.20.i
----[--/ I 0
I
I
0.12 0.48 1.9
I
7.6
I
31
I
I
I
0.12 0.49 2.0
pg
I
7.8
ng PWM
I
31
L
I
I
I
0.12 0.50 2.0 8.0
/ug
(per m l )
Fig. 1. T h e influence of various concentrations of P W M on in vitro production of antibodies to B V D V as indicated by the optical density of undiluted culture supernatants as measured in indirect E L I S A . T h e P B M C were derived from an a n i m a l seropositive to B V D V and were cultured (2 X 10 6 cells per ml) for 5 days in a microtitre plate coated w i t h B V D V ( Q ) or in an uncoated microtitre plate ( O ). T h e dotted line indicates the optical density ( m e a n + 3 S.D., n = 6 ) of control m e d i u m (derived from cultures w i t h o u t P B M C ) .
IN VITRO PRODUCTIONOF ANTIBODIESTO BOVINEVIRUSDIARRHOEAVIRUS
165
TABLE1
Antibody titre to BVDV in pooled supernatants (n = 6) of peripheral blood mononuclear cells ( P B M C ) cultured for various days in microtitre plates coated with BVDV antigen and stimulated with different concentrations of pokeweed mitogen (PWM) PBMC from animal no2 13
PWM
Antibody titre in culture supernatantb
(/tg/ml)
0 0.025 0.25 2.5 0 0.025 0.25 2.5
Day 2
Day 5
Day 8
Day 11
161
In Vitro P r o d u c t i o n of A n t i b o d i e s to B o v i n e Virus D i a r r h o e a V i r u s by P e r i p h e r a l B l o o d M o n o n u c l e a r Cells from Cattle I m m u n i z e d by Infection B. LARSSON 1, C. FOSSUM 2 and N. JUNTTI 3
'Department of Cattle and Sheep Diseases and 2Department of Veterinary Microbiology, Section of Virology, Swedish University of Agricultural Sciences, Box 7019, S-750 07 Uppsala (Sweden) 3Department of Virology, The National Veterinary Institute, Biomedicum, Uppsala (Sweden) (Accepted 17 October 1989)
ABSTRACT Larsson, B., Fossum, C. and Juntti, N., 1990. In vitro production of antibodies to bovine virus diarrhoea virus by peripheral blood mononuclear cells from cattle immunized by infection. Vet. Microbiol., 22: 161-170. A method for in vitro production of antibodies to bovine virus diarrhoea virus (BVDV) by peripheral blood mononuclear cells (PBMC) was developed. The PBMC were cultured in microtitre plates coated with detergent-solubilized BVDV and the supernatants were tested in an enzyme-linked immunosorbent assay which detects IgG antibodies to BVDV. Following incubation of PBMC with an optimal concentration of pokeweed mitogen for 5 days, antibodies to BVDV were detected in culture supernatants of PBMC from immune cattle, but not in supernatants of PBMC from seronegative cattle, from persistently BVDV-infected cattle or from a 5-day-old calf that received BVDV antibodies via colostrum. This antibody-production assay may therefore be used as a tool to discriminate between passively acquired antibodies and those actively induced.
INTRODUCTION
Cows infected with bovine virus diarrhoea virus (BVDV) during early pregnancy seroconvert but may give birth to persistently viraemic calves. As a rule, these calves are seronegative to BVDV and are therefore thought to be immunotolerant to the agent. Persistently infected calves appear to be more susceptible to opportunistic infections and constitute the population at risk of developing a fatal condition called mucosal disease (for a review, see Baker, 1987). The BVDV infects the cells of the immune system, and results of several studies indicate a hampered function of lymphocytes derived from infected cattle (Johnson and Muscoplat, 1973; Reggiardo and Kaeberle, 1981; Roth et al., 1986; Larsson, 1988; Larsson et al., 1988). However, there is no information
0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
162
B. LARSSON ET AL.
available about the ability of lymphocytes from immune and non-immune cattle to produce antibodies to BVDV in vitro. The aims of the present study were to develop a method for production of antibodies to BVDV by bovine peripheral blood mononuclear cells (PBMC), and to examine whether this production in vitro reflects the humoral immune status in vivo. MATERIALSAND METHODS Cattle Blood was collected from 15 cattle of Swedish Red and White breed. Three of the animals (nos. 1 to 3) belonged to a farm where all animals were seronegative to BVDV. Three animals (nos. 4 to 6) were considered to be persistently infected with non-cytopathic BVDV since the agent was isolated from their sera on two occasions 2 months apart, and they had no or only a low level of serum antibodies to BVDV. Eight of the animals (nos. 7 to 14) were seropositive to BVDV and were from the same herds as the persistently infected cattle. Animal no. 15 was a 5-day-old colostrum-fed offspring to a seropositive mother (no. 14). Detection of serum antibodies to B VD V Serum was tested for the presence of antibodies to BVDV by an indirect enzyme-linked immunosorbent assay (ELISA) as described before (Juntti et al., 1987 ). Briefly, microtitre plates were coated with detergent-solubilized cytopathic BVDV strain Singer (BVDV-ELISA plates), and sera were assayed in 4-fold dilutions (1:10 to 1:2560). Serum antibodies which bound to the BVDV were detected by a horseradish peroxidase (HRP)-conjugated monoclonal antibody (MAb) to bovine IgG. Isolation of cells Peripheral blood mononuclear cells (PBMC) were isolated from cattle as described before (Johnson and Morein, 1977). The PBMC were washed three times in phosphate-buffered saline and resuspended in RPMI-1640 growth medium containing glutamine (2mM), 2-mercaptoethanol (5 X 10- ~ M) and 7.5% heat-inactivated foetal calf serum (FCS) free from detectable IgG antibodies, which was tested as previously described (Juntti et al., 1987).
IN VITRO PRODUCTION OF ANTIBODIES TO BOVINE VIRUS DIARRHOEA VIRUS
163
Cell cultures for production of antibodies to B VD V To determine the optimal conditions for the generation of antibodies in vitro, PBMC were cultured at various concentrations in volumes of 200 #l per well in BVDV-ELISA plates and in the presence or absence of pokeweed mitogen (PWM; Boehringer Mannheim, Mannheim, F.R.G. ). The final concentration of PBMC ranged from 2.5 X 105 to 10 X 106 cells per ml and that of PWM from 0.12 pg to 8ttg per ml (4-step titration). Each combination was set up in hexaplicate, and cultures found to be of appropriate combination were incubated for 2, 5, 8 and 11 days, respectively, at 37°C.
Assays for detection of antibodies to B VD V generated in vitro After incubation of the cells, the amount of antibodies which had bound to the solid-phase antigens during incubation of cells and the amount of antibodies in supernatant were assayed.
Antibodies bound to the solid-phase antigens. The amount of antibodies was measured by adding the HRP-conjugated MAb to bovine IgG after thorough washing of the plates. The final steps of the ELISA were then performed as described above for detection of antibodies to BVDV in serum. The magnitude of antibody production was indicated by the mean absorbance value for each culture. Antibodies in supernatant. Supernatants from each combination were pooled and tested for antibodies to BVDV in an indirect ELISA. Some supernatants were also examined in a virus neutralization test (VNT) and in a competitive ELISA. The indirect ELISA was performed as described above for serum, and supernatants were assayed undiluted and in 2-fold dilutions up to 1 : 16. The antibody titre of a supernatant was determined as the highest dilution giving an absorbance value above the mean absorbance value + 3 S.D. (n = 6) of control medium (supplemented medium incubated without cells). The VNT was performed as previously described for serum (Larsson et al., 1988) and supernatants were tested undiluted and in dilutions of 1:2 and 1:4. The competitive ELISA was performed as previously described for serum (Juntti et al., 1987). However, the MAb to BVDV (VD-1) was used unconjugated. In brief, equal volumes (50~1) per well of a supernatant (undiluted and 2-fold dilutions up to 1 : 16) and the MAb VD-1, diluted 1 : 1000, were incubated in BVDV-ELISA plates. The amount of MAb VD-1 which bound to the solidphase antigens was detected by a HRP anti-mouse conjugate (Dakopatts, Denmark) diluted 1:1000.
164
B. LARSSON ET AL.
RESULTS
Factors influencing the in vitro production of antibodies to B VD V Concentration of PWM. Results from initial experiments showed that antibody production to BVDV was infrequently observed when PBMC from seropositive cows were cultured in BVDV-coated microtitre plates. In an attempt to enhance the antibody production, PWM was added to final concentrations that ranged from 0.12 pg to 8 #g per ml. As shown in Fig. 1, antibody production became pronounced at PWM concentrations ranging from about 0.5 ng to 0.1 ~g per ml with an optimum of 2 ng per ml. None of the various PWM concentrations elicted detectable antibody production when PBMC were cultured without BVDV, i.e., in an uncoated microtitre plate. Period of incubation. The time course of antibody production was studied for PBMC isolated from two animals, one seropositive and one seronegative to BVDV. The PBMC were cultured in growth medium alone or with the addition of PWM (2.5, 0.25 and 0.025 #g per ml). The incubation was interrupted after 2, 5, 8, and 11 days and the antibody titre to BVDV in pooled supernatants for each set of culture was determined in the indirect ELISA. Antibody production was not detected in 2-day cultures of PBMC (titre < 1 ) but was observed after prolonged incubation periods of the PBMC isolated from the seropositive an0.70.64J 0.5-
0
"0
0.4-
@ 0 .,4 4J
0.30.20.i
----[--/ I 0
I
I
0.12 0.48 1.9
I
7.6
I
31
I
I
I
0.12 0.49 2.0
pg
I
7.8
ng PWM
I
31
L
I
I
I
0.12 0.50 2.0 8.0
/ug
(per m l )
Fig. 1. T h e influence of various concentrations of P W M on in vitro production of antibodies to B V D V as indicated by the optical density of undiluted culture supernatants as measured in indirect E L I S A . T h e P B M C were derived from an a n i m a l seropositive to B V D V and were cultured (2 X 10 6 cells per ml) for 5 days in a microtitre plate coated w i t h B V D V ( Q ) or in an uncoated microtitre plate ( O ). T h e dotted line indicates the optical density ( m e a n + 3 S.D., n = 6 ) of control m e d i u m (derived from cultures w i t h o u t P B M C ) .
IN VITRO PRODUCTIONOF ANTIBODIESTO BOVINEVIRUSDIARRHOEAVIRUS
165
TABLE1
Antibody titre to BVDV in pooled supernatants (n = 6) of peripheral blood mononuclear cells ( P B M C ) cultured for various days in microtitre plates coated with BVDV antigen and stimulated with different concentrations of pokeweed mitogen (PWM) PBMC from animal no2 13
PWM
Antibody titre in culture supernatantb
(/tg/ml)
0 0.025 0.25 2.5 0 0.025 0.25 2.5
Day 2
Day 5
Day 8
Day 11
