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Effects of Different Doses of Sheep Erythrocytes on the Humoral Immune Response of Chicken Lines Selected for High or Low Antibody Production
Effects of Different Doses of Sheep Erythrocytes on the Humoral Immune Response of Chicken Lines Selected for High or Low Antibody Production M. B. KREUKNIET and A. J. van der ZIJPP Department of Animal Husbandry, Agricultural University P.O. Box 338, 6700 AH Wageningen, The Netherlands (Received for publication March 8, 1989)
1990 Poultry Science 69:608-614 INTRODUCTION
In mice, bidirectional selective breeding studies have been carried out to gain a better understanding about the genetic variation in disease resistance (Biozzi et al, 1979, 1984). Differences in antibody production within lines selected for high (H) or low (L) antibody production to sheep red blood cells (SRBC) could be attributed to a difference between these lines in terms of antigen handling by macrophages (Biozzi et al, 1979, 1984) and in the multiplication rate of B-lymphocytes (Bcells, Biozzi et al, 1984). A higher catabolic rate in the macrophages from L line mice caused reduced effectiveness in triggering Bcells to antibody response against SRBC. A larger threshold dose of SRBC was required in order to establish a detectable antibody response in the L line mice. The differences in antibody response between the lines were more pronounced at low doses (Biozzi et al, 1984). In chicken lines selected for antibody production after intravenous (i.v.) immunization with SRBC, the largest differences in antibody production between the H and L lines
were found when the lines were immunized with the dosage used for selection (Ubosi et al, 1985; Gross, 1986). Thus, the selection dose was also the optimal dose for these selection lines. However, when immunized with lower doses, the differences in antibody response between the lines were larger than after immunization with doses above those used for selection (Gross, 1986). Differences between the lines in the kinetics of the immune response were not determined (Ubosi et al., 1985; Gross, 1986). In chickens, selection for anti-SRBC production after intramuscular (i.m.) injections of cairageenan (van der Zijpp et al., 1988) or for colloidal carbon (van der Zijpp et al, 1989) did not produce any difference between the lines in terms of antigen-handling by macrophages. However, more immunocompetent cells were detected in the H line spleen than in the L line spleen (Donker, 1989). The present study was conducted in order to evaluate the influences from the dose of SRBC on the humoral responses of chicken lines selected for anti-SRBC production after i.m. administration of SRBC. In a previous study,
608
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ABSTRACT A study was conducted to determine the influence caused by the dose of sheep red blood cells (SRBC) on the humoral response of chicken lines selected for high (H) or low (L) antibody production to SRBC. The chicks were of the 5th selection generation. Both sexes were used. The primary doses of SRBC used were: 5 x l O - 4 ^ x 10 -3 ,5 x 10~2,25 x 10 -2 , and 5 x 10"1 mL packed cells, resuspended with phosphatebuffered saline (PBS) to 1 mL, and injected intramuscularly (im). All chicks were reimmunized im with 5 x 10 _1 mL packed cells in 1 mL of PBS. Throughout the experiment, H line chicks had higher titers than L line chicks. The level of primary total and 2-mercaptoethanol (2ME)-resistant titers followed the dose level. However, in total titers, the interactions between line and dose were seen at days 3 and 5 postimmunization. This was caused by a deviation in the ranking of the doses in the L line. Moreover, the kinetics of the primary response differed between the lines. Generally, in H line the peak number for the titers was reached earlier in the response period. The optimum response in terms of the total number of titers in the secondary response generally was inverse to the primary dose level. However, from Day 7 of the secondary response onward, the dose effects were influenced by line. For Line L, no effects for dose on the responding antibody level was seen. The 2ME-resistant titers followed the dose level in the secondary response. Line differences in the reaction to the primary dose level will influence the effectiveness of vaccinations, which should be kept in mind when chickens are bred for disease resistance. (Key words: selection lines, doses, sheep red blood cells, antibody)
609
DOSE EFFECTS ON HUMORAL RESPONSE
using White Leghorn laying hens, a dose of 5 x 10-4 mL of SRBC did not invoke an immune response (van der Zijpp, 1978). Dosages of SRBC ranging from 5 x 10 -4 mL to 5 x 10 -1 mL of SRBC were used in the current study in order to demonstrate line differences from the threshold dose to doses inducing a normal immune response. MATERIALS AND METHODS
One-hundred chicks of the 5th generation chicks selected for high (H) and low (L) antibody production to SRBC (van der Zijpp and Nieuwland, 1986) were used. Selection was performed on a base population of ISA-Warren hybrids, (medium-heavy, brown, egg layers). The selection criterion was total agglutination titer 5 days after i.m. immunization with 25 x 10~2 mL of packed-cell SRBC, resuspended to 1 mL in a phosphate-buffered saline solution (PBS), at 37 days of age. Lines and sexes were equally divided over the 5 dosage groups, without any replicates (n = 5 per subgroup). After hatching, all chicks were vaccinated against Mareks disease, infectious bronchitis, infectious bursal disease, and Newcastle disease at 0, 1, 15, and 22 days of age, respectively. The chicks were housed in twodeck battery cages, with a maximum bird density of 10 chicks per cage. The sexes were kept separately. Feed and water were available ad libitum.
Immunization At 31 days of age, the chicks were bilaterally injected with two portions (.5 mL each) of the SRBC dose, into the Musculus pectoralis (breast muscles). Ten chicks, five cockerels, and five pullets were injected with each SRBC dose. At 59 days of age, all 100 chicks were reimmunized i.m. with 5 x 10 -1 mL of packed cells, resuspended to 1 mL with PBS. Preparation of Plasma Samples All chicks were repeatedly bled from the wing vein at 0, 3, 5, 7, 10, and 14 days postimmunization (PI) and at 0, 3, 5, 7, 10, and 13 days postreimmunization (PR). After centrifugation (1,200 x g), plasma was collected and stored and -20 C until assayed. Hemagglutination Assay Antibody titers (both total and 2-mercaptoethanol (2ME)-resistant antibody titers) were determined, using a microliter procedure (van der Zijpp and Leenstra, 1980). The number of titers was expressed as the log2 of the highest plasma dilution giving total agglutination. Statistical Analysis
Sheep Erythrocyte Doses Five doses of SRBC were used: 5 x 10 -1 ,25 x 10-2, 5 x 10-2, 5 x 10"3, and 5 x 1(H mL of
The titers were analyzed using the general linear model procedure (SAS Institute, 1985), with the following model:
TABLE 1. Analysis of variance (sum of squares) for the primary total titers of chicken lines selected for high or low antibody production, immunized with different doses sheep red blood cells (SRBC) Days after immunization Source of variation
0
3
5
7
10
14
Line, 1 df Dose SRBC, 4 df Line by dose, 4 df Remainder, 90 df
1.0 2.1 2.9 55^
28.1*** 81.1*** 16.1** 72.1
98.0*** 401.1*** 22.1* 190.1
136.9*** 310.1*** 8.9 249.1
70.6*** 81.6*** 1.8 235.8
17.6** 20.4* 11.0 182.0
*P<.05. **P<01. ***P<.001.
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Experimental Chicks
packed cells, all resuspended to 1 mL with PBS. The SRBC were obtained in a heparin solution from five, unrelated, Texel sheep and were washed three times in physiological saline (.9% NaCl).
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ijkl = (i. + Li + Dj + St + LxDy + LxSjk
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where: value of the 1th chick: primary or secondary titer; population mean; effect of the i"1 line (i = H, L); Li Dj = effect of the j * dose; (j = 5 x 10-1, 25 x 10-2, 5 x lfr 2 , 5 x 10- 3 , 5 x 1(H mL of SRBC); m S k = effect of the k sex (k = male, female); LxDii = two-way interaction effect of the i* line and j * dose (LxSjk, DxSjk) as LxDjj, but between other main effects); LxDxSyk = as above, but three-way interaction; and e ijkl = residual error. Y
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FIGURE 1. The kinetics of the primary total antibody response in chicken lines selected for high (H) or low (L) of the responses were apparent (Figure 1). The response to sheepredblood cells (SRBC) after intramuscu- responses were higher for the H line; and lar immunization (PI) with five different doses of SRBC.
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generally, the peak number of titers was reached sooner after immunization than for the L line. The height of the response was also influenced by the SRBC dose administered: a lower dose of SRBC was followed by a smaller anti-SRBC response. However, within days, the differences between H and L lines tended to be smaller at the low doses. On Days 3 and 5 PI, significant (Table 1) interactions between line and the SRBC dose were found, caused by a deviation in the ranking of the L line. In the H line, a descending sequence of the SRBC doses administered was followed by a similar sequence in the antibody level. But in the L line chicks, the dose of 25 x 10-2 mL of SRBC caused higher total titers than the 5 x lO -1 mL SRBC dose (Figures 1 and 2).
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Primary 2-Mercaptoethanol Titers
Secondary Total Titers A complex model became necessary in order to fit the results of the total number of titers after reimmunization. The titers were affected by line, dose, and sex at most days, as well as by the interactions of line by sex, and line by dose (Table 3). In general, the H line chicks had higher total titers, but the H line pullets had a higher (.2 to 2 titer points) response than the cockerels of this line. The sexes in the L line did not differ. In addition to differences in the height of the response, differences in the kinetics between the lines were also apparent. In general, the peak titers for the H line were reached on Day 5 PR, followed by a decrease in titer of .5 (Table 4). The L line titers on Days 5 and 7 PR were hardly different. Furthermore, the height of the response for the H line was affected by the primary SRBC dose administered: the response being more or less inverse to the primary dose level; for the L line, no relationship between titers and dose could be detected.
Effects of line and dose were found on secondary IgG titers. Again, Line H had higher titers. Dose effects were not consistent. In general, the highest primary dose of SRBC also gave the highest secondary IgG response (Table 2). No differences between the lines in terms of responses to the doses were detected. DISCUSSION
The objectives of the present study were to evaluate line differences in antibody responses to SRBC doses, ranging from threshold doses to ones inducing a normal immune response. Although White Leghorns did not respond to 5 x 10"4 mL of SRBC, this dose level did induce an antibody response in the selection lines used in the present study. The dose range of 5 x lO"4 to 5 x 10-1 mL of SRBC in the present study did not include the threshold dose for either line. When the height of the response (which was influenced by the dose level) was not considered, the kinetics of the antibody response to all doses of SRBC were consistent with the normal observations for these selection lines (van der Zijpp et al, 1988, 1989; Donker, 1989). The IgG-type antibodies appeared relatively late, compared with other results (Martin et al., 1988). However, when the selection lines in the present study were immunized i.v., IgG-type antibodies could also be detected earlier in the response pattern (Donker, 1989), indicating an effect for the immunization route. The higher titers for the H line, within dose, agree with the usual observations in these
TABLE 3. Analysis of variance (sum of squares) for the secondary total titers of chicken lines selected for high or low antibody production, immunized with different doses of sheep red blood cells (SRBC) Days after reimmunization Source of variation
3
5
7
10
13
Line, 1 df Dose of SRBC, 4 df Sex, 1 df Line by dose of SRBC, 4 df Line by sex, 1 df Remainder, 88 df
37.2*** 4.2 0.1 1.8 2.3 72.6
110.3*** 30.4* 18.5** 12.4 18.5** 194.7
75.7*** 397*** 3.6 18.7* 10.9** 136.2
59.3*** 12.7 7.3* 18.3* 7.3* 132.1
13.0*** 4.5 10.2** 10.5* 17.6*** 89.1
*P<.05. **P<01. ***P<.001.
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No IgG-type antibodies were detected at Day 0 and 3 PI only. And at Day 5 PI and only when 5 x 10-' or 25 x 10-2 mL of SRBC was injected, very small amounts of IgG could be detected in only a few chicks (H < 1.8; L < .2). At Days 7, 10, and 14 PI, the line and the dose affected the number of 2ME-resistant titers. Immunization with low doses of SRBC was followed by a low IgG-type response (Table 2). The response sequence followed the order of the SRBC doses. Except for consistendy lower titers in the L line chicks, no differences between lines were found in the responses to the various doses of SRBC.
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selection lines (van der Zijpp and Nieuwland, 1986; van der Zijpp et al., 1988, 1989). In mice (Biozzi et al., 1984) and chickens (Ubosi et al., 1985; Gross, 1986), differences between selection lines have been more pronounced when these lines were immunized with low doses of SRBC. Biozzi et al. (1984) attributed this outcome to the higher catabolic rate of L line macrophages, leaving less SRBC to trigger B-cell responses. This hypothesis is not supported by data from the present study; the differences between primary antibody titers of the present selection lines even tended to be smaller at low doses (Figure 2). Moreover, when the macrophages of both lines were killed with carrageenan (van der Zijpp et al., 1988), the line differences were maintained. Also, no line differences in the activity of phagocytes could be determined (M. B. Kreukniet, Agricultural University, P.O. Box 338, Wageningen, The Netherlands, unpublished data), giving strong evidence that mechanisms other than antigen-handling by macrophages are responsible for the differences in the present chicken lines. Moreover, a higher number of direct plaque-forming cells in H line chicks was found (Donker, 1989). In general, the primary immune response followed the level of SRBC dose, while the secondary response in Line H showed an inverse response sequence. In Line L, no order in the total antibody level could be detected. As is obvious, these differences between lines in relation to the primary dose level could have negative consequences for the effectiveness of the vaccination, which should be kept in mind, when chickens are bred for disease resistance.
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ACKNOWLEDGMENTS
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The authors thank Miss K. van Belzen and Mr. M.G.B. Nieuwland for their technical assistance and Mrs. M.S.J, van der Wal and Mrs. A.E.M.M. van Hapert for typing the manuscript.
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REFERENCES
*
Biozzi, G., D. Mouton, O. A. Sant'Anna, H. C. Passos, M. Gennari, M. H. Reis, V.C.A. Ferreira, A. M. Heuman, Y. Bouthillier, O. M. Ibanez, C. Stiffel, and M. Siquera, 1979. Genetics of immunoresponsiveness to natural antigens in the mouse. Curr. Top. Microbiol. Immunol. 85:31-98. Biozzi, G., D. Mouton, C. Stiffel, and Y. Bouthillier, 1984. A major role of macrophage in quantitative genetic regu-
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on May 29, 2015
i n — CN oo r- co wo oc a\ co ~ ~ CN -H -H -H -H +1 -H -H +1 -H
613
614
KREUKNIET AND van der ZIJPP van derZijpp, A. J., 1978. The humoral immune response of the chicken. Pages 92-96 in: Proc. Zodiac-Symp. Adaptation. Pudoc Wageningen, The Netherlands. van der Zijpp, A. J„ and F. R. Leenstra, 1980. Genetic analysis of the humoral immune response of White Leghorn chicks. Poultry Sci. 59:1363-1369. vanderZijpp, A. J., and M.G.B. Nieuwland, 1986. Immunological characterisation of lines selected for high and low antibody production. Proc. 7th Eur. Poult. Conf., Paris, France. 1:211-215. van der Zijpp, A. J„ T. R. Scott, B. Glick, and M. B. Kreukniet, 1988. Interference with the humoral immune response in diverse genetic lines of chickens: I. the effect of carrageenan. Vet. Immunol. Immunopathol. 20:53-60 van der Zijpp, A. J., T. R. Scott, B. Glick, and M. B. Kreukniet, 1989. Interference with the humoral immune response in diverse genetic lines of chicken: II. the effect of colloidal carbon. Vet. Immunol. Immunopathol. 23:187-194.
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on May 29, 2015
lation of immunoresponsiveness and antiinfectious immunity. Adv. Immunol. 36:189-234. Donker, R. A., 1989. Page 203 in: Thermal influences on antibody production and metabolism in chicken lines divergently selected for immune responsiveness. Ph.D. diss. Univ. of Wageningen, The Netherlands. Gross, W. B., 1986. Effects of dose of antigen and social environment on antibody response of high and low antibody response chickens. Poultry Sci. 65:687-692. Martin, A., F.M.A. McNabb, and P. B. Siegel, 1988. Thiouracil and antibody titers of chickens from lines divergently selected for antibody response to sheep erythrocytes. Dev. Comp. Immunol. 12:611-619. SAS Institute, 1985. SAS User's Guide: Statistics Version 5 Edition. SAS Inst. Inc., Cary, NC. Ubosi, C. O., E. A. Dunnington, W. B. Gross, and P. B. Siegel, 1985. Divergent selection of chickens for antibody response to sheep erythrocytes: Kinetics of primary and secondary immunizations. Avian Dis. 29: 347-355.
1990 Poultry Science 69:608-614 INTRODUCTION
In mice, bidirectional selective breeding studies have been carried out to gain a better understanding about the genetic variation in disease resistance (Biozzi et al, 1979, 1984). Differences in antibody production within lines selected for high (H) or low (L) antibody production to sheep red blood cells (SRBC) could be attributed to a difference between these lines in terms of antigen handling by macrophages (Biozzi et al, 1979, 1984) and in the multiplication rate of B-lymphocytes (Bcells, Biozzi et al, 1984). A higher catabolic rate in the macrophages from L line mice caused reduced effectiveness in triggering Bcells to antibody response against SRBC. A larger threshold dose of SRBC was required in order to establish a detectable antibody response in the L line mice. The differences in antibody response between the lines were more pronounced at low doses (Biozzi et al, 1984). In chicken lines selected for antibody production after intravenous (i.v.) immunization with SRBC, the largest differences in antibody production between the H and L lines
were found when the lines were immunized with the dosage used for selection (Ubosi et al, 1985; Gross, 1986). Thus, the selection dose was also the optimal dose for these selection lines. However, when immunized with lower doses, the differences in antibody response between the lines were larger than after immunization with doses above those used for selection (Gross, 1986). Differences between the lines in the kinetics of the immune response were not determined (Ubosi et al., 1985; Gross, 1986). In chickens, selection for anti-SRBC production after intramuscular (i.m.) injections of cairageenan (van der Zijpp et al., 1988) or for colloidal carbon (van der Zijpp et al, 1989) did not produce any difference between the lines in terms of antigen-handling by macrophages. However, more immunocompetent cells were detected in the H line spleen than in the L line spleen (Donker, 1989). The present study was conducted in order to evaluate the influences from the dose of SRBC on the humoral responses of chicken lines selected for anti-SRBC production after i.m. administration of SRBC. In a previous study,
608
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on May 29, 2015
ABSTRACT A study was conducted to determine the influence caused by the dose of sheep red blood cells (SRBC) on the humoral response of chicken lines selected for high (H) or low (L) antibody production to SRBC. The chicks were of the 5th selection generation. Both sexes were used. The primary doses of SRBC used were: 5 x l O - 4 ^ x 10 -3 ,5 x 10~2,25 x 10 -2 , and 5 x 10"1 mL packed cells, resuspended with phosphatebuffered saline (PBS) to 1 mL, and injected intramuscularly (im). All chicks were reimmunized im with 5 x 10 _1 mL packed cells in 1 mL of PBS. Throughout the experiment, H line chicks had higher titers than L line chicks. The level of primary total and 2-mercaptoethanol (2ME)-resistant titers followed the dose level. However, in total titers, the interactions between line and dose were seen at days 3 and 5 postimmunization. This was caused by a deviation in the ranking of the doses in the L line. Moreover, the kinetics of the primary response differed between the lines. Generally, in H line the peak number for the titers was reached earlier in the response period. The optimum response in terms of the total number of titers in the secondary response generally was inverse to the primary dose level. However, from Day 7 of the secondary response onward, the dose effects were influenced by line. For Line L, no effects for dose on the responding antibody level was seen. The 2ME-resistant titers followed the dose level in the secondary response. Line differences in the reaction to the primary dose level will influence the effectiveness of vaccinations, which should be kept in mind when chickens are bred for disease resistance. (Key words: selection lines, doses, sheep red blood cells, antibody)
609
DOSE EFFECTS ON HUMORAL RESPONSE
using White Leghorn laying hens, a dose of 5 x 10-4 mL of SRBC did not invoke an immune response (van der Zijpp, 1978). Dosages of SRBC ranging from 5 x 10 -4 mL to 5 x 10 -1 mL of SRBC were used in the current study in order to demonstrate line differences from the threshold dose to doses inducing a normal immune response. MATERIALS AND METHODS
One-hundred chicks of the 5th generation chicks selected for high (H) and low (L) antibody production to SRBC (van der Zijpp and Nieuwland, 1986) were used. Selection was performed on a base population of ISA-Warren hybrids, (medium-heavy, brown, egg layers). The selection criterion was total agglutination titer 5 days after i.m. immunization with 25 x 10~2 mL of packed-cell SRBC, resuspended to 1 mL in a phosphate-buffered saline solution (PBS), at 37 days of age. Lines and sexes were equally divided over the 5 dosage groups, without any replicates (n = 5 per subgroup). After hatching, all chicks were vaccinated against Mareks disease, infectious bronchitis, infectious bursal disease, and Newcastle disease at 0, 1, 15, and 22 days of age, respectively. The chicks were housed in twodeck battery cages, with a maximum bird density of 10 chicks per cage. The sexes were kept separately. Feed and water were available ad libitum.
Immunization At 31 days of age, the chicks were bilaterally injected with two portions (.5 mL each) of the SRBC dose, into the Musculus pectoralis (breast muscles). Ten chicks, five cockerels, and five pullets were injected with each SRBC dose. At 59 days of age, all 100 chicks were reimmunized i.m. with 5 x 10 -1 mL of packed cells, resuspended to 1 mL with PBS. Preparation of Plasma Samples All chicks were repeatedly bled from the wing vein at 0, 3, 5, 7, 10, and 14 days postimmunization (PI) and at 0, 3, 5, 7, 10, and 13 days postreimmunization (PR). After centrifugation (1,200 x g), plasma was collected and stored and -20 C until assayed. Hemagglutination Assay Antibody titers (both total and 2-mercaptoethanol (2ME)-resistant antibody titers) were determined, using a microliter procedure (van der Zijpp and Leenstra, 1980). The number of titers was expressed as the log2 of the highest plasma dilution giving total agglutination. Statistical Analysis
Sheep Erythrocyte Doses Five doses of SRBC were used: 5 x 10 -1 ,25 x 10-2, 5 x 10-2, 5 x 10"3, and 5 x 1(H mL of
The titers were analyzed using the general linear model procedure (SAS Institute, 1985), with the following model:
TABLE 1. Analysis of variance (sum of squares) for the primary total titers of chicken lines selected for high or low antibody production, immunized with different doses sheep red blood cells (SRBC) Days after immunization Source of variation
0
3
5
7
10
14
Line, 1 df Dose SRBC, 4 df Line by dose, 4 df Remainder, 90 df
1.0 2.1 2.9 55^
28.1*** 81.1*** 16.1** 72.1
98.0*** 401.1*** 22.1* 190.1
136.9*** 310.1*** 8.9 249.1
70.6*** 81.6*** 1.8 235.8
17.6** 20.4* 11.0 182.0
*P<.05. **P<01. ***P<.001.
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on May 29, 2015
Experimental Chicks
packed cells, all resuspended to 1 mL with PBS. The SRBC were obtained in a heparin solution from five, unrelated, Texel sheep and were washed three times in physiological saline (.9% NaCl).
610
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where: value of the 1th chick: primary or secondary titer; population mean; effect of the i"1 line (i = H, L); Li Dj = effect of the j * dose; (j = 5 x 10-1, 25 x 10-2, 5 x lfr 2 , 5 x 10- 3 , 5 x 1(H mL of SRBC); m S k = effect of the k sex (k = male, female); LxDii = two-way interaction effect of the i* line and j * dose (LxSjk, DxSjk) as LxDjj, but between other main effects); LxDxSyk = as above, but three-way interaction; and e ijkl = residual error. Y
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Primary Total Titers Differences between the lines in the kinetics
FIGURE 1. The kinetics of the primary total antibody response in chicken lines selected for high (H) or low (L) of the responses were apparent (Figure 1). The response to sheepredblood cells (SRBC) after intramuscu- responses were higher for the H line; and lar immunization (PI) with five different doses of SRBC.
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Primary 2-Mercaptoethanol Titers
Secondary Total Titers A complex model became necessary in order to fit the results of the total number of titers after reimmunization. The titers were affected by line, dose, and sex at most days, as well as by the interactions of line by sex, and line by dose (Table 3). In general, the H line chicks had higher total titers, but the H line pullets had a higher (.2 to 2 titer points) response than the cockerels of this line. The sexes in the L line did not differ. In addition to differences in the height of the response, differences in the kinetics between the lines were also apparent. In general, the peak titers for the H line were reached on Day 5 PR, followed by a decrease in titer of .5 (Table 4). The L line titers on Days 5 and 7 PR were hardly different. Furthermore, the height of the response for the H line was affected by the primary SRBC dose administered: the response being more or less inverse to the primary dose level; for the L line, no relationship between titers and dose could be detected.
Effects of line and dose were found on secondary IgG titers. Again, Line H had higher titers. Dose effects were not consistent. In general, the highest primary dose of SRBC also gave the highest secondary IgG response (Table 2). No differences between the lines in terms of responses to the doses were detected. DISCUSSION
The objectives of the present study were to evaluate line differences in antibody responses to SRBC doses, ranging from threshold doses to ones inducing a normal immune response. Although White Leghorns did not respond to 5 x 10"4 mL of SRBC, this dose level did induce an antibody response in the selection lines used in the present study. The dose range of 5 x lO"4 to 5 x 10-1 mL of SRBC in the present study did not include the threshold dose for either line. When the height of the response (which was influenced by the dose level) was not considered, the kinetics of the antibody response to all doses of SRBC were consistent with the normal observations for these selection lines (van der Zijpp et al, 1988, 1989; Donker, 1989). The IgG-type antibodies appeared relatively late, compared with other results (Martin et al., 1988). However, when the selection lines in the present study were immunized i.v., IgG-type antibodies could also be detected earlier in the response pattern (Donker, 1989), indicating an effect for the immunization route. The higher titers for the H line, within dose, agree with the usual observations in these
TABLE 3. Analysis of variance (sum of squares) for the secondary total titers of chicken lines selected for high or low antibody production, immunized with different doses of sheep red blood cells (SRBC) Days after reimmunization Source of variation
3
5
7
10
13
Line, 1 df Dose of SRBC, 4 df Sex, 1 df Line by dose of SRBC, 4 df Line by sex, 1 df Remainder, 88 df
37.2*** 4.2 0.1 1.8 2.3 72.6
110.3*** 30.4* 18.5** 12.4 18.5** 194.7
75.7*** 397*** 3.6 18.7* 10.9** 136.2
59.3*** 12.7 7.3* 18.3* 7.3* 132.1
13.0*** 4.5 10.2** 10.5* 17.6*** 89.1
*P<.05. **P<01. ***P<.001.
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on May 29, 2015
No IgG-type antibodies were detected at Day 0 and 3 PI only. And at Day 5 PI and only when 5 x 10-' or 25 x 10-2 mL of SRBC was injected, very small amounts of IgG could be detected in only a few chicks (H < 1.8; L < .2). At Days 7, 10, and 14 PI, the line and the dose affected the number of 2ME-resistant titers. Immunization with low doses of SRBC was followed by a low IgG-type response (Table 2). The response sequence followed the order of the SRBC doses. Except for consistendy lower titers in the L line chicks, no differences between lines were found in the responses to the various doses of SRBC.
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selection lines (van der Zijpp and Nieuwland, 1986; van der Zijpp et al., 1988, 1989). In mice (Biozzi et al., 1984) and chickens (Ubosi et al., 1985; Gross, 1986), differences between selection lines have been more pronounced when these lines were immunized with low doses of SRBC. Biozzi et al. (1984) attributed this outcome to the higher catabolic rate of L line macrophages, leaving less SRBC to trigger B-cell responses. This hypothesis is not supported by data from the present study; the differences between primary antibody titers of the present selection lines even tended to be smaller at low doses (Figure 2). Moreover, when the macrophages of both lines were killed with carrageenan (van der Zijpp et al., 1988), the line differences were maintained. Also, no line differences in the activity of phagocytes could be determined (M. B. Kreukniet, Agricultural University, P.O. Box 338, Wageningen, The Netherlands, unpublished data), giving strong evidence that mechanisms other than antigen-handling by macrophages are responsible for the differences in the present chicken lines. Moreover, a higher number of direct plaque-forming cells in H line chicks was found (Donker, 1989). In general, the primary immune response followed the level of SRBC dose, while the secondary response in Line H showed an inverse response sequence. In Line L, no order in the total antibody level could be detected. As is obvious, these differences between lines in relation to the primary dose level could have negative consequences for the effectiveness of the vaccination, which should be kept in mind, when chickens are bred for disease resistance.
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ACKNOWLEDGMENTS
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The authors thank Miss K. van Belzen and Mr. M.G.B. Nieuwland for their technical assistance and Mrs. M.S.J, van der Wal and Mrs. A.E.M.M. van Hapert for typing the manuscript.
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REFERENCES
*
Biozzi, G., D. Mouton, O. A. Sant'Anna, H. C. Passos, M. Gennari, M. H. Reis, V.C.A. Ferreira, A. M. Heuman, Y. Bouthillier, O. M. Ibanez, C. Stiffel, and M. Siquera, 1979. Genetics of immunoresponsiveness to natural antigens in the mouse. Curr. Top. Microbiol. Immunol. 85:31-98. Biozzi, G., D. Mouton, C. Stiffel, and Y. Bouthillier, 1984. A major role of macrophage in quantitative genetic regu-
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on May 29, 2015
i n — CN oo r- co wo oc a\ co ~ ~ CN -H -H -H -H +1 -H -H +1 -H
613
614
KREUKNIET AND van der ZIJPP van derZijpp, A. J., 1978. The humoral immune response of the chicken. Pages 92-96 in: Proc. Zodiac-Symp. Adaptation. Pudoc Wageningen, The Netherlands. van der Zijpp, A. J„ and F. R. Leenstra, 1980. Genetic analysis of the humoral immune response of White Leghorn chicks. Poultry Sci. 59:1363-1369. vanderZijpp, A. J., and M.G.B. Nieuwland, 1986. Immunological characterisation of lines selected for high and low antibody production. Proc. 7th Eur. Poult. Conf., Paris, France. 1:211-215. van der Zijpp, A. J„ T. R. Scott, B. Glick, and M. B. Kreukniet, 1988. Interference with the humoral immune response in diverse genetic lines of chickens: I. the effect of carrageenan. Vet. Immunol. Immunopathol. 20:53-60 van der Zijpp, A. J., T. R. Scott, B. Glick, and M. B. Kreukniet, 1989. Interference with the humoral immune response in diverse genetic lines of chicken: II. the effect of colloidal carbon. Vet. Immunol. Immunopathol. 23:187-194.
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on May 29, 2015
lation of immunoresponsiveness and antiinfectious immunity. Adv. Immunol. 36:189-234. Donker, R. A., 1989. Page 203 in: Thermal influences on antibody production and metabolism in chicken lines divergently selected for immune responsiveness. Ph.D. diss. Univ. of Wageningen, The Netherlands. Gross, W. B., 1986. Effects of dose of antigen and social environment on antibody response of high and low antibody response chickens. Poultry Sci. 65:687-692. Martin, A., F.M.A. McNabb, and P. B. Siegel, 1988. Thiouracil and antibody titers of chickens from lines divergently selected for antibody response to sheep erythrocytes. Dev. Comp. Immunol. 12:611-619. SAS Institute, 1985. SAS User's Guide: Statistics Version 5 Edition. SAS Inst. Inc., Cary, NC. Ubosi, C. O., E. A. Dunnington, W. B. Gross, and P. B. Siegel, 1985. Divergent selection of chickens for antibody response to sheep erythrocytes: Kinetics of primary and secondary immunizations. Avian Dis. 29: 347-355.