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Brucella abortus Antibody Response of White Leghorn Chickens Selected for High and Low Antibody Responsiveness to Sheep Erythrocytes
Brucella abortus Antibody Response of White Leghorn Chickens Selected for High and Low Antibody Responsiveness to Sheep Erythrocytes T. R. SCOTT,1 E. A. DUNNINGTON,2 and P. B. SIEGEL2 Department of Poultry Science, P.O. Box 340379, Clemson University, Clemson, South Carolina 29634-0379 and Poultry Science Department, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 ABSTRACT Three experiments were conducted with lines of chickens selected for high (HA) and low (LA) antibody response to sheep erythrocytes to determine their antibody responses following primary immunization with Brucella abortus. In Experiment 1, HA chicks responded to immunization with a higher total titer than LA chicks at 7 d postimmunization. At both 5 and 7 d following immunization 2-mercaptoethanol-resistant (IgG) titers were higher in HA than LA chicks. There also was a significant sex effect at 7 d in the first experiment, with males having higher total titers than females. In the second experiment, HA chicks had higher total titers at 5 d postimmunization than LA chicks, but IgG titers were similar for both lines. Total and IgG titers of HA chicks used in Experiment 3 were significantly higher than those of LA chicks at 5 and 7 d postimmunization. Both HA and LA chicks exhibited divergent antibody responses to B. abortus although they had originally been selected for antibody responsiveness to sheep erythrocytes. (Key words: chicken, selected lines, sex, antibody responses, Brucella abortus) 1994 Poultry Science 73:346-349
INTRODUCTION To achieve divergent separation of lines of chickens selected for antibody responsiveness, it has been common practice to utilize the polyvalent, T-dependent antigen SRBC to elicit a humoral immune response (Siegel and Gross, 1980; van der Zijpp et al, 1987). Biozzi et al. (1979), with their murine high and low antibody lines, showed that selection for erythrocyte stimulation resulted in a nonspecific effect for many types of antigens. The murine
Received for publication June 21, 1993. Accepted for publication October 14, 1993. 1 Clemson University. To whom correspondence should be addressed. 2 Virginia Polytechnic Institute and State University.
lines were high and low responders to various heterologous erythrocytes, proteins, haptens, bacterial polysaccharides, histocompatibility and tumor antigens, viruses, bacteria, and parasites. A recent paper by Heller et al. (1992) reports similar nonspecific effects for chicken lines selected for early (HC) or late (LC) antibody responses to Escherichia coli. The present experimentation was undertaken to examine antibody production in high (HA) and low (LA) antibody response lines of chickens (Siegel and Gross, 1980) following antigen stimulation with Brucella abortus, a Type-1 T-independent antigen (Mosier and Subbarao, 1982). MATERIALS AND METHODS Three experiments were conducted with chicks from parents selected for 17
346
RESEARCH NOTE
generations for high (HA) and low (LA) antibody response (Siegel and Gross, 1980). The chicks were housed in battery cages and provided ad libitum access to feed and water for the duration of each experiment. At 42 d of age in the first experiment, five males and five females from each line were immunized intravenously in the wing vein with .1 mL of B. abortus stock solution.3 Blood samples were drawn from the wing vein at 3, 5, and 7 d postimmunization. In Experiments 2 and 3, 15 males from each line were immunized and bled as described for Experiment 1. Serum samples were recovered from clotted blood following centrifugation, heat-inactivated at 56 C, and used in an assay to determined hemagglutination titers (McCorkle and Glick, 1980). Both total and 2-mercaptoethanol-resistant (IgG) titers were determined. Titers were expressed as the log2 of the reciprocal of the highest serum dilution showing visible agglutination. Data were analyzed by analysis of variance within days postimmunization using the General Linear Models (GLM) procedure of SAS® (SAS Institute, 1985). The model for the first experiment included the effects due to line, sex, and the line by sex interaction; but the model for the second and third experiments included only the line effect. Treatment means were further separated by least significant difference.
347
titers to B. abortus, but there was no line by sex interaction at any time postimmunization in Experiment 1. In Experiment 2, HA chicks had significantly higher (P £ .011) total titers against B. abortus than LA chicks at 5 d postimmunization (Table 1). The persistence observed in the first experiment was not evident in this experiment. The IgG titers were highest at 5 d postimmunization, with the difference between lines approaching significance (P <, .076). In Experiment 3, both total and IgG titers of HA chicks were higher than those of LA chicks at 5 and 7 d postimmunization (P < .001 for total and IgG titers at both sampling times). Total titers remained at the same level in both lines from 5 to 7 d postimmunization, whereas IgG titers of both lines continued to rise through 7 d postimmunization. Summarizing the three experiments, there were no differences between lines for titers at 3 d postimmunization and in no case were titers higher for LA than HA chicks at 5 and 7 d postimmunization. In 8 of 12 comparisons at 5 and 7 d postimmunization, titers were significantly greater for Line HA than LA chicks. These results are consistent with the previous observation in these lines that HA chicks had higher total titers to B. abortus at 27 d postimmunization than LA chicks (Dunnington et ah, 1992). Both that report and the present findings show that Line HA and LA chicks can exhibit respective high and low antibody responses against B. RESULTS AND DISCUSSION abortus even though they were selected for Antibody titers 3 d postimmunization antibody responsiveness to SRBC. Brucella abortus is classified as a Type-1 were similar between both lines within all experiments (Table 1). In Experiment 1 the T-independent antigen, which stimulates B mean total titer was lower (P < .008) for cells with little assistance from T helper LA than HA chicks at 7 but not 5 d cells, but does require macrophage-hke postimmunization. This pattern resulted adherent accessory cells to induce antifrom persistence of the antibody response body formation (Mosier and Subbarao, from 5 to 7 d postimmunization in HA but 1982). In lines of mice selected for antinot LA chicks. The IgG titers of HA chicks body responsiveness to erythrocyte antiwere higher at both 5 (P < .011) and 7 (P < gens, their divergent antibody responses .014) d postimmunization. A sex effect (P to the various T-dependent and T< .038) was present only at 7 d postimmu- independent antigens were due, in part, to nization with the males having the higher differences in activities of macrophages of total titers (9.9 ± .2) than females (9.3 ± .2). the respective lines (Biozzi et ah, 1979). The HA males had the highest antibody Van der Zijpp et ah (1988, 1989), on the other hand, did not believe that their lines of chickens selected for high and low antibody response differed in macrophage 3 ability to respond to antigenic challenge. Difco Laboratories, Detroit, MI 48232.
348
SCOTT ET AL.
TABLE 1. Mean ± SEM total and 2-mercaptoethanol (IgG)-resistant antibody titers (log2) of High (HA) and Low (LA) antibody response lines of chickens to Brucella abortus antigen at 5 and 7 d postimmunization
Experiment
Days . postimmunization HA
Total
IgG LA
HA
LA
1
3 5 7
5.7 ± .2 10.4 ± .2 10.0 ± .2"
5.9 ± .2 10.0 ± .2 9.2 ± .2b
5.3 ± .2 7.2 ± .2» 7.4 ± .2>
5.6 ± .2 6.2 ± .2b 6.5 ± .2b
2
3 5 7
4.8 ± .3 8.9 ± .2» 7.9 ± .2
4.7 ± .3 7.9 ± .2b 7.5 ± .2
4.3 ± .1 6.1 ± .3 6.0 ± .2
4.5 ± .1 5.5 ± .3 5.8 ± .2
3
3 5 7
3.6 ± .2 10.4 ± .3" 10.4 ± .2>
3.2 ± .2 7.8 ± .2b 7.8 ± .2b
3.0 ± .0 4.0 ± .1» 5.1 ± .1»
3.0 ± .0 2.9 ± .l b 4.0 ± .l b
a
-bMeans within a row and variable with no common superscript differ significantly (P £ .05).
Heumann, Y. Bouthillier, O. M. Ibanex, C. When carrageenan (van der Zijpp et al, StiffeL and M. Siqueira, 1979. Genetics of 1988) and colloidal carbon (van der Zijpp immunoresponsiveness to natural antigens in et al., 1989) were administered to duels the mouse. Curr. Top. Microbiol. Immunol. 85: from both lines, the interference caused by 31-98. these substances did not indicate that the Dunnington, E. A., C. T. Larsen, W. B. Gross, and P. B. Siegel, 1992. Antibody responses to combinacontribution of macrophages to the antitions of antigens in White Leghorn chickens of body responses was any greater in the different background genomes and major high line than the low line. With regard to histocompatibility complex genotypes. Poultry general phagocytic ability, Heller et al. Sci. 71:1801-1806. (1992) reported that their HC line of Heller, E. D., G. Leitner, A. Friedman, Z. Uni, M. Gutman, and A. Cahaner, 1992. Immunological chickens had faster carbon clearance than parameters in meat-type chicken lines divertheir LC line. Although neither the murine gently selected by antibody response to Eslines (Biozzi et al, 1979) nor the chicken cherichia coli vaccination. Vet. Immunol. Imlines of van der Zijpp and Nieuwland munopathol. 34:159-172. (1986) differed in their T cell involvement McCorkle, G., and B. Glick, 1980. The effect of aging on immune competence in the chicken. in immunity, in other experiments T cell Antibody-mediated immunity. Poultry Sci. 59: activity was greater in Line HA than LA 669-672. (Scott et al, 1991) and in HC than LC Mosier, D. E., and B. Subbarao, 1982. Thymuschickens (Heller et al, 1992). Further work independent antigens: complexity of Bwith the accessory cell component (i.e., lymphocyte activation revealed. Immunol. Today 38:217-223. macrophages) of these lines is needed to fully distinguish the contribution of this SAS Institute, 1985. SAS® User's Guide. Version 5 SAS Institute Inc., Cary, NC cell type to the immune response differ- Scott,Edition. T. R., E. A. Dunnington, and P. B. Siegel, 1991. ences among lines. T-cell activity of White Leghorn chickens
ACKNOWLEDGMENTS
Appreciation is expressed to Sandy Untch for her technical assistance. This is Technical Contribution Number 3439 of the South Carolina Agricultural Experiment Station, Clemson University. REFERENCES Biozzi, G., D. Mouton, O. A. SanfAnna, H. C. Passos, M. Gennari, M. H. Reis, V.C.A. Ferreira, A. M.
selected for high and low antibody responses to sheep erythrocytes. Poultry Sci. 70:1831-1834. Siegel, P. B., and W. B. Gross, 1980. Production and persistence of antibodies in chickens to sheep erythrocytes. 1. Directional selection. Poultry Sci. 59:1-5. van der Zijpp, A. J., M. B. Kmekniet, and M.G.B. Nieuwland, 1989. Responses to selection for high and low antibody production. Poultry Sci. 66(Suppl. l):188.(Abstr.) van der Zijpp, A. J., and M.G.B. Nieuwland, 1986. Immunological characterization of lines selected for high and low antibody production. Pages 211-215 in: Proceedings of the 7th European Poultry Conference, Paris, France. Vol. 1. World
RESEARCH NOTE Poultry Science Association, Branche Francaise, Tours, France. 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.
349
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 chickens. II. The effect of colloidal carbon. Vet. Immunol. Immunopathol. 23:187-194.
INTRODUCTION To achieve divergent separation of lines of chickens selected for antibody responsiveness, it has been common practice to utilize the polyvalent, T-dependent antigen SRBC to elicit a humoral immune response (Siegel and Gross, 1980; van der Zijpp et al, 1987). Biozzi et al. (1979), with their murine high and low antibody lines, showed that selection for erythrocyte stimulation resulted in a nonspecific effect for many types of antigens. The murine
Received for publication June 21, 1993. Accepted for publication October 14, 1993. 1 Clemson University. To whom correspondence should be addressed. 2 Virginia Polytechnic Institute and State University.
lines were high and low responders to various heterologous erythrocytes, proteins, haptens, bacterial polysaccharides, histocompatibility and tumor antigens, viruses, bacteria, and parasites. A recent paper by Heller et al. (1992) reports similar nonspecific effects for chicken lines selected for early (HC) or late (LC) antibody responses to Escherichia coli. The present experimentation was undertaken to examine antibody production in high (HA) and low (LA) antibody response lines of chickens (Siegel and Gross, 1980) following antigen stimulation with Brucella abortus, a Type-1 T-independent antigen (Mosier and Subbarao, 1982). MATERIALS AND METHODS Three experiments were conducted with chicks from parents selected for 17
346
RESEARCH NOTE
generations for high (HA) and low (LA) antibody response (Siegel and Gross, 1980). The chicks were housed in battery cages and provided ad libitum access to feed and water for the duration of each experiment. At 42 d of age in the first experiment, five males and five females from each line were immunized intravenously in the wing vein with .1 mL of B. abortus stock solution.3 Blood samples were drawn from the wing vein at 3, 5, and 7 d postimmunization. In Experiments 2 and 3, 15 males from each line were immunized and bled as described for Experiment 1. Serum samples were recovered from clotted blood following centrifugation, heat-inactivated at 56 C, and used in an assay to determined hemagglutination titers (McCorkle and Glick, 1980). Both total and 2-mercaptoethanol-resistant (IgG) titers were determined. Titers were expressed as the log2 of the reciprocal of the highest serum dilution showing visible agglutination. Data were analyzed by analysis of variance within days postimmunization using the General Linear Models (GLM) procedure of SAS® (SAS Institute, 1985). The model for the first experiment included the effects due to line, sex, and the line by sex interaction; but the model for the second and third experiments included only the line effect. Treatment means were further separated by least significant difference.
347
titers to B. abortus, but there was no line by sex interaction at any time postimmunization in Experiment 1. In Experiment 2, HA chicks had significantly higher (P £ .011) total titers against B. abortus than LA chicks at 5 d postimmunization (Table 1). The persistence observed in the first experiment was not evident in this experiment. The IgG titers were highest at 5 d postimmunization, with the difference between lines approaching significance (P <, .076). In Experiment 3, both total and IgG titers of HA chicks were higher than those of LA chicks at 5 and 7 d postimmunization (P < .001 for total and IgG titers at both sampling times). Total titers remained at the same level in both lines from 5 to 7 d postimmunization, whereas IgG titers of both lines continued to rise through 7 d postimmunization. Summarizing the three experiments, there were no differences between lines for titers at 3 d postimmunization and in no case were titers higher for LA than HA chicks at 5 and 7 d postimmunization. In 8 of 12 comparisons at 5 and 7 d postimmunization, titers were significantly greater for Line HA than LA chicks. These results are consistent with the previous observation in these lines that HA chicks had higher total titers to B. abortus at 27 d postimmunization than LA chicks (Dunnington et ah, 1992). Both that report and the present findings show that Line HA and LA chicks can exhibit respective high and low antibody responses against B. RESULTS AND DISCUSSION abortus even though they were selected for Antibody titers 3 d postimmunization antibody responsiveness to SRBC. Brucella abortus is classified as a Type-1 were similar between both lines within all experiments (Table 1). In Experiment 1 the T-independent antigen, which stimulates B mean total titer was lower (P < .008) for cells with little assistance from T helper LA than HA chicks at 7 but not 5 d cells, but does require macrophage-hke postimmunization. This pattern resulted adherent accessory cells to induce antifrom persistence of the antibody response body formation (Mosier and Subbarao, from 5 to 7 d postimmunization in HA but 1982). In lines of mice selected for antinot LA chicks. The IgG titers of HA chicks body responsiveness to erythrocyte antiwere higher at both 5 (P < .011) and 7 (P < gens, their divergent antibody responses .014) d postimmunization. A sex effect (P to the various T-dependent and T< .038) was present only at 7 d postimmu- independent antigens were due, in part, to nization with the males having the higher differences in activities of macrophages of total titers (9.9 ± .2) than females (9.3 ± .2). the respective lines (Biozzi et ah, 1979). The HA males had the highest antibody Van der Zijpp et ah (1988, 1989), on the other hand, did not believe that their lines of chickens selected for high and low antibody response differed in macrophage 3 ability to respond to antigenic challenge. Difco Laboratories, Detroit, MI 48232.
348
SCOTT ET AL.
TABLE 1. Mean ± SEM total and 2-mercaptoethanol (IgG)-resistant antibody titers (log2) of High (HA) and Low (LA) antibody response lines of chickens to Brucella abortus antigen at 5 and 7 d postimmunization
Experiment
Days . postimmunization HA
Total
IgG LA
HA
LA
1
3 5 7
5.7 ± .2 10.4 ± .2 10.0 ± .2"
5.9 ± .2 10.0 ± .2 9.2 ± .2b
5.3 ± .2 7.2 ± .2» 7.4 ± .2>
5.6 ± .2 6.2 ± .2b 6.5 ± .2b
2
3 5 7
4.8 ± .3 8.9 ± .2» 7.9 ± .2
4.7 ± .3 7.9 ± .2b 7.5 ± .2
4.3 ± .1 6.1 ± .3 6.0 ± .2
4.5 ± .1 5.5 ± .3 5.8 ± .2
3
3 5 7
3.6 ± .2 10.4 ± .3" 10.4 ± .2>
3.2 ± .2 7.8 ± .2b 7.8 ± .2b
3.0 ± .0 4.0 ± .1» 5.1 ± .1»
3.0 ± .0 2.9 ± .l b 4.0 ± .l b
a
-bMeans within a row and variable with no common superscript differ significantly (P £ .05).
Heumann, Y. Bouthillier, O. M. Ibanex, C. When carrageenan (van der Zijpp et al, StiffeL and M. Siqueira, 1979. Genetics of 1988) and colloidal carbon (van der Zijpp immunoresponsiveness to natural antigens in et al., 1989) were administered to duels the mouse. Curr. Top. Microbiol. Immunol. 85: from both lines, the interference caused by 31-98. these substances did not indicate that the Dunnington, E. A., C. T. Larsen, W. B. Gross, and P. B. Siegel, 1992. Antibody responses to combinacontribution of macrophages to the antitions of antigens in White Leghorn chickens of body responses was any greater in the different background genomes and major high line than the low line. With regard to histocompatibility complex genotypes. Poultry general phagocytic ability, Heller et al. Sci. 71:1801-1806. (1992) reported that their HC line of Heller, E. D., G. Leitner, A. Friedman, Z. Uni, M. Gutman, and A. Cahaner, 1992. Immunological chickens had faster carbon clearance than parameters in meat-type chicken lines divertheir LC line. Although neither the murine gently selected by antibody response to Eslines (Biozzi et al, 1979) nor the chicken cherichia coli vaccination. Vet. Immunol. Imlines of van der Zijpp and Nieuwland munopathol. 34:159-172. (1986) differed in their T cell involvement McCorkle, G., and B. Glick, 1980. The effect of aging on immune competence in the chicken. in immunity, in other experiments T cell Antibody-mediated immunity. Poultry Sci. 59: activity was greater in Line HA than LA 669-672. (Scott et al, 1991) and in HC than LC Mosier, D. E., and B. Subbarao, 1982. Thymuschickens (Heller et al, 1992). Further work independent antigens: complexity of Bwith the accessory cell component (i.e., lymphocyte activation revealed. Immunol. Today 38:217-223. macrophages) of these lines is needed to fully distinguish the contribution of this SAS Institute, 1985. SAS® User's Guide. Version 5 SAS Institute Inc., Cary, NC cell type to the immune response differ- Scott,Edition. T. R., E. A. Dunnington, and P. B. Siegel, 1991. ences among lines. T-cell activity of White Leghorn chickens
ACKNOWLEDGMENTS
Appreciation is expressed to Sandy Untch for her technical assistance. This is Technical Contribution Number 3439 of the South Carolina Agricultural Experiment Station, Clemson University. REFERENCES Biozzi, G., D. Mouton, O. A. SanfAnna, H. C. Passos, M. Gennari, M. H. Reis, V.C.A. Ferreira, A. M.
selected for high and low antibody responses to sheep erythrocytes. Poultry Sci. 70:1831-1834. Siegel, P. B., and W. B. Gross, 1980. Production and persistence of antibodies in chickens to sheep erythrocytes. 1. Directional selection. Poultry Sci. 59:1-5. van der Zijpp, A. J., M. B. Kmekniet, and M.G.B. Nieuwland, 1989. Responses to selection for high and low antibody production. Poultry Sci. 66(Suppl. l):188.(Abstr.) van der Zijpp, A. J., and M.G.B. Nieuwland, 1986. Immunological characterization of lines selected for high and low antibody production. Pages 211-215 in: Proceedings of the 7th European Poultry Conference, Paris, France. Vol. 1. World
RESEARCH NOTE Poultry Science Association, Branche Francaise, Tours, France. 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.
349
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 chickens. II. The effect of colloidal carbon. Vet. Immunol. Immunopathol. 23:187-194.