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Production of Mercaptoethanol Sensitive Antibody in Bursectomized Chicks
1358
H. R. BIRD AND D. W. OLSON
and Veterinary, University of Rio Grande do Sul; Dr. Luciano Machado, Secretary of Agriculture Rio Grande do Sul; Dr. Bento Pires Dias, Director of ASCAR; Dr. Dwayne Rohweder, Chief of Party, USAID —Wisconsin contract; and many members of the staffs of the four agencies mentioned.
REFERENCES Association of Official Agricultural Chemists, 1965. Official Methods of Analysis, 10th ed., pp. 1516. A.O.A.C., Washington, D.C. Bird, H. R., and D. W. Olson, 1970. Protein and amino acid content of yellow corn grown in southern Brazil: Effect of fertilizer. 14th World's Poultry Congress, in press. Henderson, L. M., and E. E. Snell, 1948. A uniform medium for determination of amino acids with various microorganisms. J. Biol. Chem. 172: 15-29. Keeney, D. R., 1969, Potassium boosts corn grain
quality. Better Crops with Plant Food, 53 ( 4 ) : 10-11. MacGregor, J. M., L. T. Tashovitch and W. P. Martin, 1961. Effect of nitrogen fertilizer and soil type on the amino acid content of corn grain. Agron. J. 53 : 211-214. National Research Council, 1969. United StatesCanadian tables of feed composition, 2nd rev., 92 pp. National Research Council, 1971. Nutrient requirements of poultry. 6th rev., 54 pp. Sauberlich, H. E., W. Y. Chang and W. D. Salmon, 1953. The amino acid and protein content of corn as related to variety and nitrogen fertilization. J. Nutrition 5 1 : 241-250. Spackman, D. H., W. H. Stein and S. Moore, 1958. Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30: 1190-1206. Sunde, M. L., 1970. Results of corn analysis—1969 crop. Feedstuffs, 42 (27): 36. Wilberg, E., B. Michael and W. G. Pollmer, 1968. Effect of nitrogen fertilization on opaque-2 corn rich in lysine. Z. Pflanzenernaehr. Bodenk. 121: 125-132.
Production of Mercaptoethanol Sensitive Antibody in Bursectomized Chicks K O J I SATO AND BRUCE GLICK Poultry Science Department, Mississippi State University, State College, Mississippi 39762 (Received for publication December 1, 1971) ABSTRACT The antibody titer and class of immunoglobulin was determined in surgically bursectomized birds following 1-4 immunizations with sheep-red-blood cells. While the primary immune response was significantly lower in bursectomized chicks, bursectomized chickens responsed normally to subsequent antigen injections; following the tertiary injection, the mercaptoethanol sensitive antibody was significantly increased in bursectomized chickens. POULTRY SCIENCE 5 1 : 1358-1360,
R
EMOVAL of the bursa of Fabricius in the embryo and the newly hatched chick disturbs subsequent development of immunological competence (Glick, 1970). The bursa appears to be necessary for the recovery of competence destroyed by x-irradiation (Sato and Suzuki, 1969). Bursectomy decreased serum IgG in the presence of normal levels of IgM (Cooper
1972
et al., 1969) or increased levels of IgM (Lerner et al., 1971). The antibody produced in bursectomized chicks after two immunizations was sensitive to mercaptoethanol (ME) (Jankovic and Isakovic, 1966; Cain et al, 1969; and Claflin et al, 1966). These results suggested that IgM synthesis in bursectomized chicks may not be impaired, even though the bursa is con-
1359
MERCAPTOETHANOL SENSITIVE ANTIBODY
sidered as a site for preparing precursors of the cells which eventually produce IgM and IgG (Cooper et al., 1969; and Cain et al., 1969). Experiments undertaken here confirm the presence of IgM synthesis in bursectomized birds following 1, 2, or 3 immunizations with sheep-red-blood cells and suggest an increase in specific IgM antibody. New Hampshire chicks of the Mississippi Agriculture Experiment Station were used throughout this study. A corn soybean ration fortified with vitamins and minerals was fed (Glick and Dreesen, 1967). Chicks were surgically bursectomized at 1 to 2 days of age (Glick, 1960). At 3, 5, 7, and 9 weeks of age, chicks were intravenously injected with 1 ml. of a 7% suspension of sheep red blood cells (S.R.B.C.). Seven days later, serum was secured and hemagglutinin titers determined by a microliter procedure (Sever, 1962). Before dilution one aliquot of each serum was treated with equal amounts of 0.2 M ME in phosphate buffered saline (pH 7.4) for 30 minutes at 37°C. (Delhanty and Solomon, 1966). Reduction of the titer by the
treatment was recorded as ME-sensitive antibody. The primary immune response was significantly lower in bursectomized chicks (Table 1, groups immunized only at 7 weeks). The second injection of S.R.B.C. raised the hemagglutinin titer approximately to the same level as that of intact control chicks (Table 1, S week and 7 week injections). After two to three injections of S.R.B.C, ME-sensitive antibody increased in bursectomized chicks, while it decreased in intact chicks. The IgG levels for BSX birds after 1 and 3 injections of S.R.B.C. were significantly lower than control values. Recently, Lerner et al. (1971) found that serum IgM of hyperimmunized hormonally bursectomized chicks exceeded the IgM level of intact chicks. The increased IgM levels were not accompanied by enhanced production of IgM antibody. In this experiment, the significant increase in IgM antibody in BSX birds following 3 injections of S.R.B.C. suggests that the high concentration of IgM observed in BSX birds may be related to specific antibody.
TABLE 1.—Mercaptoethanol {ME) sensitive and total antibody response of birds injected 1, 2, 3, or 4X with S.R.B.C. Age of Antigen Challenge 7w BSX Intact
Hemagglutinin Titers (logs) 1
2 3
4 5 6
2" 2 4 2
1 1
7 8 9 10 11 12 1 2
3.3 7.0*
2.5 6.0*
3
1 1 1 1
7.6 7.0
4.8 4.5
7.8 7.3
6.5* 3.3
7.0 7.7
4.0 2.9
1 1
3w, 5w, 7w BSX Intact
1 1 2 1 3 2
1 1
1 1 2 1 1 3
1 1
a
1
ME-sensitive Mean
3
5w, 7w BSX Intact
3w, 5w, 7w, 9w BSX Intact
Mean
1
No. of chicks. ** P < . 01 (all comparisons made between treatments within age groups titered at 7 days post injection).
1360
K. SATO AND B. GLICK
While the mechanisms underlying the immune disorder resulting from bursectomy are not entirely clear, it has been suggested that the bursa prepares immunological competent cells (Glick, 1960; and Mueller et al., 1960) and/or secretes some substance (Glick, 1960; St. Pierre and Ackerman, 1965; and Jankovic and Leskowitz, 1965) which affects immunological maturation. If the bursa controls IgM synthesis, as Cooper et al. (1969) have suggested, it is difficult to explain the high IgM antibody titers of this experiment and the high IgM levels reported by Lerner et al. (1971). High IgM production in bursectomized birds may reflect a failure of specific cells to pass through the bursa or be exposed to the humoral-bursal factor. These cells then retain a primitive cell function. On the other hand, potential IgM producing cells may migrate to other sites where they synthesise IgM. Therefore, bursaless birds would experience an unchecked synthesis of IgM as a result of the low levels of IgG. REFERENCES Cain, W. C , M. D. Cooper, P. J. Van Alten and R. A. Good, 1969. Development and function of the immunoglobulin producing system. II. Role of the bursa in the development of humoral immunological competence. J. Immunol. 102: 671-678. Claflin, A. J., O. Smithies and R. K. Meyers, 1966. Antibody responses in bursa-deficient chickens. J. Immunol. 97 : 693-700. Cooper, M. D., W. A. Cain, P. J. Van Alten and R. A. Good, 1969. Development and function of the immunoglobulin producing system. I. Effect of bursectomy at different stages of develop-
ment on germinal centers, plasma cells, immunoglobulins and antibody production. Int. Arch. Allergy, 35: 242-252. Delhanty, J. J., and J. B. Solomon, 1966. The nature of antibodies to goat erythrocytes in the developing chicken. Immunology, 11: 103-115. Glick, B., 1960. Extracts from the bursa of Fabricius—a lympho-epithelial gland of the chickenstimulate the production of antibodies in bursectomized chickens. Poultry Sci. 39: 10971101. Glick, B., 1970. The bursa of Fabricius: a central issue. BioScience, 20: 602-604. Glick, B., and L. J. Dreesen, 1967. The influence of selecting for large and small bursa size on adrenal, spleen, and thymus weights. Poultry Sci. 46: 396-402. lankovic, B. D., and S. Leskowitz, 1965. Restoration of antibody producing capacity in bursectomized chickens by bursal grafts in millipore chambers. Proc. Soc. Exptl. Biol. Med. 118: 1164-1166. Jankovic, B. D., and K. Isakovic, 1966. Antibody producing in bursectomized chickens given repeated injections of antigen. Nature, 211: 202-203. Lerner, K., B. Glick and F. C. McDuffie, 1971. Role of the bursa of Fabricius in IgG and IgM production in the chicken: evidence for the role of a non-bursal site in the development of humoral immunity. J. Immunol. 107: 493-511. Mueller, A. P., H. R. Wolfe and R. K. Meyer, 1960. Precipitin products in chickens. XXI. Antibody production in bursectomized chickens and in chickens injected with 19-nortesterone on the fifth day of incubation. J. Immunol. 85: 172-180. Sato, K., and K. Suzuki, 1969. Significance of the bursa after whole body x-irradiation. Poultry Sci. 48: 1195-1198. Sever, J. L., 1962. Application of a microtechnique to viral serological investigation. J. Immunol. 88: 320-329. St. Pierre, R. L., and G. A. Ackerman, 1965. Bursa of Fabricius in chickens: possible humoral factor. Science, 147: 1307.
MARCH 14-16, 1973. PACIFIC EGG AND POULTRY SUPERSHOW, TOWN AND COUNTRY HOTEL, SAN DIEGO, CALIFORNIA
H. R. BIRD AND D. W. OLSON
and Veterinary, University of Rio Grande do Sul; Dr. Luciano Machado, Secretary of Agriculture Rio Grande do Sul; Dr. Bento Pires Dias, Director of ASCAR; Dr. Dwayne Rohweder, Chief of Party, USAID —Wisconsin contract; and many members of the staffs of the four agencies mentioned.
REFERENCES Association of Official Agricultural Chemists, 1965. Official Methods of Analysis, 10th ed., pp. 1516. A.O.A.C., Washington, D.C. Bird, H. R., and D. W. Olson, 1970. Protein and amino acid content of yellow corn grown in southern Brazil: Effect of fertilizer. 14th World's Poultry Congress, in press. Henderson, L. M., and E. E. Snell, 1948. A uniform medium for determination of amino acids with various microorganisms. J. Biol. Chem. 172: 15-29. Keeney, D. R., 1969, Potassium boosts corn grain
quality. Better Crops with Plant Food, 53 ( 4 ) : 10-11. MacGregor, J. M., L. T. Tashovitch and W. P. Martin, 1961. Effect of nitrogen fertilizer and soil type on the amino acid content of corn grain. Agron. J. 53 : 211-214. National Research Council, 1969. United StatesCanadian tables of feed composition, 2nd rev., 92 pp. National Research Council, 1971. Nutrient requirements of poultry. 6th rev., 54 pp. Sauberlich, H. E., W. Y. Chang and W. D. Salmon, 1953. The amino acid and protein content of corn as related to variety and nitrogen fertilization. J. Nutrition 5 1 : 241-250. Spackman, D. H., W. H. Stein and S. Moore, 1958. Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30: 1190-1206. Sunde, M. L., 1970. Results of corn analysis—1969 crop. Feedstuffs, 42 (27): 36. Wilberg, E., B. Michael and W. G. Pollmer, 1968. Effect of nitrogen fertilization on opaque-2 corn rich in lysine. Z. Pflanzenernaehr. Bodenk. 121: 125-132.
Production of Mercaptoethanol Sensitive Antibody in Bursectomized Chicks K O J I SATO AND BRUCE GLICK Poultry Science Department, Mississippi State University, State College, Mississippi 39762 (Received for publication December 1, 1971) ABSTRACT The antibody titer and class of immunoglobulin was determined in surgically bursectomized birds following 1-4 immunizations with sheep-red-blood cells. While the primary immune response was significantly lower in bursectomized chicks, bursectomized chickens responsed normally to subsequent antigen injections; following the tertiary injection, the mercaptoethanol sensitive antibody was significantly increased in bursectomized chickens. POULTRY SCIENCE 5 1 : 1358-1360,
R
EMOVAL of the bursa of Fabricius in the embryo and the newly hatched chick disturbs subsequent development of immunological competence (Glick, 1970). The bursa appears to be necessary for the recovery of competence destroyed by x-irradiation (Sato and Suzuki, 1969). Bursectomy decreased serum IgG in the presence of normal levels of IgM (Cooper
1972
et al., 1969) or increased levels of IgM (Lerner et al., 1971). The antibody produced in bursectomized chicks after two immunizations was sensitive to mercaptoethanol (ME) (Jankovic and Isakovic, 1966; Cain et al, 1969; and Claflin et al, 1966). These results suggested that IgM synthesis in bursectomized chicks may not be impaired, even though the bursa is con-
1359
MERCAPTOETHANOL SENSITIVE ANTIBODY
sidered as a site for preparing precursors of the cells which eventually produce IgM and IgG (Cooper et al., 1969; and Cain et al., 1969). Experiments undertaken here confirm the presence of IgM synthesis in bursectomized birds following 1, 2, or 3 immunizations with sheep-red-blood cells and suggest an increase in specific IgM antibody. New Hampshire chicks of the Mississippi Agriculture Experiment Station were used throughout this study. A corn soybean ration fortified with vitamins and minerals was fed (Glick and Dreesen, 1967). Chicks were surgically bursectomized at 1 to 2 days of age (Glick, 1960). At 3, 5, 7, and 9 weeks of age, chicks were intravenously injected with 1 ml. of a 7% suspension of sheep red blood cells (S.R.B.C.). Seven days later, serum was secured and hemagglutinin titers determined by a microliter procedure (Sever, 1962). Before dilution one aliquot of each serum was treated with equal amounts of 0.2 M ME in phosphate buffered saline (pH 7.4) for 30 minutes at 37°C. (Delhanty and Solomon, 1966). Reduction of the titer by the
treatment was recorded as ME-sensitive antibody. The primary immune response was significantly lower in bursectomized chicks (Table 1, groups immunized only at 7 weeks). The second injection of S.R.B.C. raised the hemagglutinin titer approximately to the same level as that of intact control chicks (Table 1, S week and 7 week injections). After two to three injections of S.R.B.C, ME-sensitive antibody increased in bursectomized chicks, while it decreased in intact chicks. The IgG levels for BSX birds after 1 and 3 injections of S.R.B.C. were significantly lower than control values. Recently, Lerner et al. (1971) found that serum IgM of hyperimmunized hormonally bursectomized chicks exceeded the IgM level of intact chicks. The increased IgM levels were not accompanied by enhanced production of IgM antibody. In this experiment, the significant increase in IgM antibody in BSX birds following 3 injections of S.R.B.C. suggests that the high concentration of IgM observed in BSX birds may be related to specific antibody.
TABLE 1.—Mercaptoethanol {ME) sensitive and total antibody response of birds injected 1, 2, 3, or 4X with S.R.B.C. Age of Antigen Challenge 7w BSX Intact
Hemagglutinin Titers (logs) 1
2 3
4 5 6
2" 2 4 2
1 1
7 8 9 10 11 12 1 2
3.3 7.0*
2.5 6.0*
3
1 1 1 1
7.6 7.0
4.8 4.5
7.8 7.3
6.5* 3.3
7.0 7.7
4.0 2.9
1 1
3w, 5w, 7w BSX Intact
1 1 2 1 3 2
1 1
1 1 2 1 1 3
1 1
a
1
ME-sensitive Mean
3
5w, 7w BSX Intact
3w, 5w, 7w, 9w BSX Intact
Mean
1
No. of chicks. ** P < . 01 (all comparisons made between treatments within age groups titered at 7 days post injection).
1360
K. SATO AND B. GLICK
While the mechanisms underlying the immune disorder resulting from bursectomy are not entirely clear, it has been suggested that the bursa prepares immunological competent cells (Glick, 1960; and Mueller et al., 1960) and/or secretes some substance (Glick, 1960; St. Pierre and Ackerman, 1965; and Jankovic and Leskowitz, 1965) which affects immunological maturation. If the bursa controls IgM synthesis, as Cooper et al. (1969) have suggested, it is difficult to explain the high IgM antibody titers of this experiment and the high IgM levels reported by Lerner et al. (1971). High IgM production in bursectomized birds may reflect a failure of specific cells to pass through the bursa or be exposed to the humoral-bursal factor. These cells then retain a primitive cell function. On the other hand, potential IgM producing cells may migrate to other sites where they synthesise IgM. Therefore, bursaless birds would experience an unchecked synthesis of IgM as a result of the low levels of IgG. REFERENCES Cain, W. C , M. D. Cooper, P. J. Van Alten and R. A. Good, 1969. Development and function of the immunoglobulin producing system. II. Role of the bursa in the development of humoral immunological competence. J. Immunol. 102: 671-678. Claflin, A. J., O. Smithies and R. K. Meyers, 1966. Antibody responses in bursa-deficient chickens. J. Immunol. 97 : 693-700. Cooper, M. D., W. A. Cain, P. J. Van Alten and R. A. Good, 1969. Development and function of the immunoglobulin producing system. I. Effect of bursectomy at different stages of develop-
ment on germinal centers, plasma cells, immunoglobulins and antibody production. Int. Arch. Allergy, 35: 242-252. Delhanty, J. J., and J. B. Solomon, 1966. The nature of antibodies to goat erythrocytes in the developing chicken. Immunology, 11: 103-115. Glick, B., 1960. Extracts from the bursa of Fabricius—a lympho-epithelial gland of the chickenstimulate the production of antibodies in bursectomized chickens. Poultry Sci. 39: 10971101. Glick, B., 1970. The bursa of Fabricius: a central issue. BioScience, 20: 602-604. Glick, B., and L. J. Dreesen, 1967. The influence of selecting for large and small bursa size on adrenal, spleen, and thymus weights. Poultry Sci. 46: 396-402. lankovic, B. D., and S. Leskowitz, 1965. Restoration of antibody producing capacity in bursectomized chickens by bursal grafts in millipore chambers. Proc. Soc. Exptl. Biol. Med. 118: 1164-1166. Jankovic, B. D., and K. Isakovic, 1966. Antibody producing in bursectomized chickens given repeated injections of antigen. Nature, 211: 202-203. Lerner, K., B. Glick and F. C. McDuffie, 1971. Role of the bursa of Fabricius in IgG and IgM production in the chicken: evidence for the role of a non-bursal site in the development of humoral immunity. J. Immunol. 107: 493-511. Mueller, A. P., H. R. Wolfe and R. K. Meyer, 1960. Precipitin products in chickens. XXI. Antibody production in bursectomized chickens and in chickens injected with 19-nortesterone on the fifth day of incubation. J. Immunol. 85: 172-180. Sato, K., and K. Suzuki, 1969. Significance of the bursa after whole body x-irradiation. Poultry Sci. 48: 1195-1198. Sever, J. L., 1962. Application of a microtechnique to viral serological investigation. J. Immunol. 88: 320-329. St. Pierre, R. L., and G. A. Ackerman, 1965. Bursa of Fabricius in chickens: possible humoral factor. Science, 147: 1307.
MARCH 14-16, 1973. PACIFIC EGG AND POULTRY SUPERSHOW, TOWN AND COUNTRY HOTEL, SAN DIEGO, CALIFORNIA