Veterinary Immunology and Immunopathology, 28 ( 1991 ) 81-87
81
Elsevier Science Publishers B.V., Amsterdam
Effect of carrageenan on humoral and protective immune responses of chicks vaccinated with Newcastle disease virus I K. Nachimuthu, P. Richard Masillamony and V.D. Padmanaban Department of Microbiology, Madras Veterinary College, Madras 600 007, India (Accepted 16 May ! 990)
ABSTRACT Nachimuthu, K., Masillamony, P.R. and Padmanaban, V.D., 1991. Effect ofcarrageenan on humoral and protective immune responses of chicks vaccinated with Newcastle disease virus. Vet.lmmunol. lmmunopathol., 28:81-87. The effect of carrageenan (CGN) treatment on the generation of humorai and protective immune response to Newcastle disease virus vaccine was investigated in chicks. Carrageenan treatment significantly impaired the primary humoral immune response in vaccinated chi~:ks. Further, the protective immune response in CGN-treated chicks was only 50% of that in the control vaccinated chicks. However, the post-challenge response was not altered by drug treatment, lndomethacin, a potent inhibitor of prostaglandin synthesis, was able to reverse the immunosuppressive effect of CGN. This finding suggests that prostaglandins might be involved in CGN-mediated immunosuppression in chicks.
INTRODUCTION
Carrageenan (CGN), has been found to suppress the immune response to several thymus-dependent (T-dependent) antigens (Aschheim and Raffel, 1972; Ishizaka et al., 1977; Rumjanek et al., 1977; Murthy and Rap,land, 1984a). In contrast, the in vitro humoral response to thymus-independent (T-independent) antigens, such as lipopolysaccharides, polyvinyl pyrrolidone, trinitrophenylated DEAE dextron and Brucella abortus, was not affected by CGN (lshizaka et al., 1977; Elfaki et al., 1987). The immunosuppressive effect of CGN has been attributed to its cytopathic effect on the mononuclear phagocytic system. It does not appear to be cytotoxic to T or B lymphocytes (Thomson and Fowler, 1981; Elfaki et al., 1987 ). Although the mechanism by which CGN mediates suppression is not completely understood, it has been observed that indomethacin (IDN) a po~Part of the Ph.D. Thesis submitted by the Senior Author to the Tamil Nadu Agricultural University, Coimbatore-3.
0165-2427/9 !/$03.50
© 1991 - - Elsevier Science Publishers B.V.
82
K. NACHIMUTHU ET AL.
tent inhibitor of prostaglandin release (Goodwin and Webb, 1980) could le" verse the effect of CGN on the immune response (Bash anrl Cochran, 1980; Palanivelu, 1987). Thus it vias proposed that CON caJi mediate suppression by altering the prostaglandin level in macrophages (Bash and Cochran, 1980}. In the present study, we have investigated the effect of CGN treatment in chicks vaccinated with Newcastle disease virus (NDV) 011 the post-vaccination and post-challenge immune response. To further elucidate the mecha.nism of action of CGN we studied the effect of IDN on this system. MATERIALS AND METHons
Experimental birds White Leghorn chicks were obtained from the Poultry Research Station, Madras, and were free of adventitious infections. They were maintained under isolation in chick brooders and given water and feed ad libitum. Lighting in the chick brooder was in accordance with standard recommendations for subtropical zones.
Carrageenan GN type II (iota carrageenan; Sigma, U.S.A.) was dissolved by boiling in 0.1 M phosphate-buffer~d saline (PBS; pH 7.3) at a concentration of 10 mg/ ml, and was stored at -20°C until use.
Indomethacin Ten mg ofindoroethacin (Sigma) was dissolved in 1 ml ofethanol and then diluted in 19 ml of PBS shortly before use (Quan et at, 1980). The chicks received three intraperitoneal (lP) injections of 50 /lg IDN in 0.1 ml solution at 24-h intervals. The control chicks received three IP injections of 0.1 rolof 5% ethanol in PBS.
Strain a/vaccine and experimental procedure The La Sota strain ofNDV, with a titre of 106.74 EIDso/ml, was obtained from the Institute of Veterinary Biological Products, Pune, India. Chicks of groups I, II and III were vaccinated IP with 0.1 rol of a 1 in 10 dilution of the La Sota strain of NDV after injecting the final dose of CGN. The control chicks of group IV were associated IP. Group V consisted of unvaccinated control chicks (Table I). The brooders were assigned randomly to treatments, and the experiment consisted of two replicates. All chicks shown in Table I we"re of the same hatch. These chicks were allotted randomly to studies of the post-vaccination response and post-challenge response, as shown in Tables 2-4. The chicks were anaesthetised with ether and killed by dislocation of the neck. Serum and spleen samples were collected and tested 2 weeks after vaccination.
83
EFFECf OF CARRAGEENAN ON CHICKS WITH NOV
TABLE 1 Vaccination schedule Group
Treatment
Age of chicks at vaccination
Rout of vaccination
Number of chicks
I
CGN CGN+IDN
24 days 24 days 14 days 24 days
IP IP IP IP
30 30
2 3 4 5
ION NO
Control
30 30 30
Carrageenan treatment The procedure described by Murthy and Ragland (1984b) was followed. CGN was administered to 60 chicks at the rate of0.5 rngjchick per day IP for 3 consecutive days before g~ving the antigen. On the day of the experiment, the 4th and final dose of 0.5 illg of CGN was administered about 1 h before injecting the vaccine. Thus a total of 2 mg of CGN was administered in four equal parts. The CGl\T-treatcd chicks, along with other chicks, were grouped as shown in Table 1.
Microhaemagglutination inhibition test The microhaernagglutination inhibition (MHI) test was conducted following the procedure of Timms and Alexander (1977). Laxbro V-bottom, microloops and droppers were used for the test. The La Sota strain ofNDV was used as the indicator virus. Normal saline was used as diluent, and a 1% suspension of chicken erythrocytes was used as indicator in the test system (Nachimuthu et aI., 1989b).
Passive haemolytic plaque-forming cell assay Plaque-forming cells (PFC) were detected following established methods (Cunningham and Szenberg, 1968) with some modifications (Nachimuthu et aI., 1989a). the number ofPFCj 106 vinble spleen cells was calculated.
Challenge with virulent virus The control group (V) and the vaccinated chicks (groups I-IV) were challenged intramuscularly with 100 50% chick mortality dose (CMD so ) of virulent NDV obtained from the Institute of Veterinary Preventive Medicine, Ranipet, India, on the 14th day after vaccination.
84
K. NACHIMUTHU ET AL.
RESULTS
Suppressive effect ofcarrageenan and indomethacin treatment on the primary antibody response to ND V vaccination The MHI test and PFC assay were carrieo out i 4 cl~ys after vaccination and again 14 days after challenge with virulent NDV. As shown in Table 2, CON treatment significantly reduced the primary antibody response in NDVvaccinated chicks. Both the MHI antibody 11tre and the PFe response were found to be significantly lower in the CON-treated glOUpS than in other groups (P 0.(5). ·*This group differs significantly from the rest (P 0.05 ). *"'This group differs significantly from the rest (P< 0.01).
85
EFFECT OJ' ;:;..RRAl:;cENAN ON CHICKS WITH NDV
TABI.E4 Carrageenan-mediated suppression of the protective immune response induced by Newcastle disease virus vaccine and its reversal by indomethacin treatment Treatment before vaccination
PBS CGN CGN+ION ION No vaccination
Number ofchicks Used 9
12 15 15 15
Percent protection Affected 0 7 13 14 15
Died 0 6 3 ? IS
100.0** 50.0** 80.0...... 86.7** 0.0
**Highly significant compared with non-vaccinated group (P< 0.D1 ).
Effect ofcarrageenan and indomethacin on the protective immune response in NDV-vaccinated chicks The effect ofCGN on the generation of the protective immune response to NOV vaccination was studied. All normal vaccinated chicks withstood challenge (Table 4). In the CGN-treated group, 50% of animals were protected compared with 86.7% in the ION-treated group and 80% in the group treated with CON and ION. The percentage of chicks surviving ill all the vaccinated groups was highly significantly different from that in the unvaccinated group (P
81
Elsevier Science Publishers B.V., Amsterdam
Effect of carrageenan on humoral and protective immune responses of chicks vaccinated with Newcastle disease virus I K. Nachimuthu, P. Richard Masillamony and V.D. Padmanaban Department of Microbiology, Madras Veterinary College, Madras 600 007, India (Accepted 16 May ! 990)
ABSTRACT Nachimuthu, K., Masillamony, P.R. and Padmanaban, V.D., 1991. Effect ofcarrageenan on humoral and protective immune responses of chicks vaccinated with Newcastle disease virus. Vet.lmmunol. lmmunopathol., 28:81-87. The effect of carrageenan (CGN) treatment on the generation of humorai and protective immune response to Newcastle disease virus vaccine was investigated in chicks. Carrageenan treatment significantly impaired the primary humoral immune response in vaccinated chi~:ks. Further, the protective immune response in CGN-treated chicks was only 50% of that in the control vaccinated chicks. However, the post-challenge response was not altered by drug treatment, lndomethacin, a potent inhibitor of prostaglandin synthesis, was able to reverse the immunosuppressive effect of CGN. This finding suggests that prostaglandins might be involved in CGN-mediated immunosuppression in chicks.
INTRODUCTION
Carrageenan (CGN), has been found to suppress the immune response to several thymus-dependent (T-dependent) antigens (Aschheim and Raffel, 1972; Ishizaka et al., 1977; Rumjanek et al., 1977; Murthy and Rap,land, 1984a). In contrast, the in vitro humoral response to thymus-independent (T-independent) antigens, such as lipopolysaccharides, polyvinyl pyrrolidone, trinitrophenylated DEAE dextron and Brucella abortus, was not affected by CGN (lshizaka et al., 1977; Elfaki et al., 1987). The immunosuppressive effect of CGN has been attributed to its cytopathic effect on the mononuclear phagocytic system. It does not appear to be cytotoxic to T or B lymphocytes (Thomson and Fowler, 1981; Elfaki et al., 1987 ). Although the mechanism by which CGN mediates suppression is not completely understood, it has been observed that indomethacin (IDN) a po~Part of the Ph.D. Thesis submitted by the Senior Author to the Tamil Nadu Agricultural University, Coimbatore-3.
0165-2427/9 !/$03.50
© 1991 - - Elsevier Science Publishers B.V.
82
K. NACHIMUTHU ET AL.
tent inhibitor of prostaglandin release (Goodwin and Webb, 1980) could le" verse the effect of CGN on the immune response (Bash anrl Cochran, 1980; Palanivelu, 1987). Thus it vias proposed that CON caJi mediate suppression by altering the prostaglandin level in macrophages (Bash and Cochran, 1980}. In the present study, we have investigated the effect of CGN treatment in chicks vaccinated with Newcastle disease virus (NDV) 011 the post-vaccination and post-challenge immune response. To further elucidate the mecha.nism of action of CGN we studied the effect of IDN on this system. MATERIALS AND METHons
Experimental birds White Leghorn chicks were obtained from the Poultry Research Station, Madras, and were free of adventitious infections. They were maintained under isolation in chick brooders and given water and feed ad libitum. Lighting in the chick brooder was in accordance with standard recommendations for subtropical zones.
Carrageenan GN type II (iota carrageenan; Sigma, U.S.A.) was dissolved by boiling in 0.1 M phosphate-buffer~d saline (PBS; pH 7.3) at a concentration of 10 mg/ ml, and was stored at -20°C until use.
Indomethacin Ten mg ofindoroethacin (Sigma) was dissolved in 1 ml ofethanol and then diluted in 19 ml of PBS shortly before use (Quan et at, 1980). The chicks received three intraperitoneal (lP) injections of 50 /lg IDN in 0.1 ml solution at 24-h intervals. The control chicks received three IP injections of 0.1 rolof 5% ethanol in PBS.
Strain a/vaccine and experimental procedure The La Sota strain ofNDV, with a titre of 106.74 EIDso/ml, was obtained from the Institute of Veterinary Biological Products, Pune, India. Chicks of groups I, II and III were vaccinated IP with 0.1 rol of a 1 in 10 dilution of the La Sota strain of NDV after injecting the final dose of CGN. The control chicks of group IV were associated IP. Group V consisted of unvaccinated control chicks (Table I). The brooders were assigned randomly to treatments, and the experiment consisted of two replicates. All chicks shown in Table I we"re of the same hatch. These chicks were allotted randomly to studies of the post-vaccination response and post-challenge response, as shown in Tables 2-4. The chicks were anaesthetised with ether and killed by dislocation of the neck. Serum and spleen samples were collected and tested 2 weeks after vaccination.
83
EFFECf OF CARRAGEENAN ON CHICKS WITH NOV
TABLE 1 Vaccination schedule Group
Treatment
Age of chicks at vaccination
Rout of vaccination
Number of chicks
I
CGN CGN+IDN
24 days 24 days 14 days 24 days
IP IP IP IP
30 30
2 3 4 5
ION NO
Control
30 30 30
Carrageenan treatment The procedure described by Murthy and Ragland (1984b) was followed. CGN was administered to 60 chicks at the rate of0.5 rngjchick per day IP for 3 consecutive days before g~ving the antigen. On the day of the experiment, the 4th and final dose of 0.5 illg of CGN was administered about 1 h before injecting the vaccine. Thus a total of 2 mg of CGN was administered in four equal parts. The CGl\T-treatcd chicks, along with other chicks, were grouped as shown in Table 1.
Microhaemagglutination inhibition test The microhaernagglutination inhibition (MHI) test was conducted following the procedure of Timms and Alexander (1977). Laxbro V-bottom, microloops and droppers were used for the test. The La Sota strain ofNDV was used as the indicator virus. Normal saline was used as diluent, and a 1% suspension of chicken erythrocytes was used as indicator in the test system (Nachimuthu et aI., 1989b).
Passive haemolytic plaque-forming cell assay Plaque-forming cells (PFC) were detected following established methods (Cunningham and Szenberg, 1968) with some modifications (Nachimuthu et aI., 1989a). the number ofPFCj 106 vinble spleen cells was calculated.
Challenge with virulent virus The control group (V) and the vaccinated chicks (groups I-IV) were challenged intramuscularly with 100 50% chick mortality dose (CMD so ) of virulent NDV obtained from the Institute of Veterinary Preventive Medicine, Ranipet, India, on the 14th day after vaccination.
84
K. NACHIMUTHU ET AL.
RESULTS
Suppressive effect ofcarrageenan and indomethacin treatment on the primary antibody response to ND V vaccination The MHI test and PFC assay were carrieo out i 4 cl~ys after vaccination and again 14 days after challenge with virulent NDV. As shown in Table 2, CON treatment significantly reduced the primary antibody response in NDVvaccinated chicks. Both the MHI antibody 11tre and the PFe response were found to be significantly lower in the CON-treated glOUpS than in other groups (P 0.(5). ·*This group differs significantly from the rest (P 0.05 ). *"'This group differs significantly from the rest (P< 0.01).
85
EFFECT OJ' ;:;..RRAl:;cENAN ON CHICKS WITH NDV
TABI.E4 Carrageenan-mediated suppression of the protective immune response induced by Newcastle disease virus vaccine and its reversal by indomethacin treatment Treatment before vaccination
PBS CGN CGN+ION ION No vaccination
Number ofchicks Used 9
12 15 15 15
Percent protection Affected 0 7 13 14 15
Died 0 6 3 ? IS
100.0** 50.0** 80.0...... 86.7** 0.0
**Highly significant compared with non-vaccinated group (P< 0.D1 ).
Effect ofcarrageenan and indomethacin on the protective immune response in NDV-vaccinated chicks The effect ofCGN on the generation of the protective immune response to NOV vaccination was studied. All normal vaccinated chicks withstood challenge (Table 4). In the CGN-treated group, 50% of animals were protected compared with 86.7% in the ION-treated group and 80% in the group treated with CON and ION. The percentage of chicks surviving ill all the vaccinated groups was highly significantly different from that in the unvaccinated group (P
