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Effect of Thymectomy and Bursectomy on Migration Inhibition Test of Splenic Cells in Chickens
Effect of Thymectomy and Bursectomy on Migration Inhibition Test of Splenic Cells in Chickens CHIHARU MORITA AND MASAO SOEKAWA Research Center for Veterinary Science, The Kitasato Institute, Kashiwa, Chiba Pref., Japan (Received for publication October 12, 1971)
POULTRY SCIENCE 51: 1133-1136, 1972
INTRODUCTION
IGRATION inhibition test of splenic cells (SMIT) in mammals has been described as an in vitro indicator of delayed type hypersensitivity (Svejcar et al., 1971). However, there is no report concerning the effect of thymectomy on SMIT in chickens. Surgical thymectomy of chickens immediately after hatching, followed by sublethal X-irradiation reduces cell-mediated immunity such as delayed wattle reaction, homograft rejection and graft versus host reaction (Cooper et al., 1966). Glick et al. (1956) described the role of the bursa Fabricius on the development of humoral immunity in chickens. It is now established that hormonal bursectomy generally eliminates IgG immunoglobulins and reduces the antibody response to most antigens (Warner et al., 1969; Lerner et al., 1971). But this treatment does not significantly influence the cell-mediated immunity (Jankovic et al., 1963; Warner et al., 1971). Inhibition of splenic cell migration by the antigen in sensitized chickens has already been evidenced by the authors (Morita and Soekawa, 1971). The present study is intended to examine the influence of thymectomy and bursectomy on SMIT.
M
MATERIALS AND METHODS
Chickens: Goto strain 202 (Goto Hatch-
ery Inc.) of non-inbred White Leghorn chickens were used throughout the experiment. Thymectomy and bursectomy: Surgical thymectomy was carried out on the day of hatching. X-irradiation was given on the day after hatching. The dosage of X-irradiation was 650 R (200 kV, 20 mA, with 0.3 mm. copper and 1.0 mm. aluminum filter, half value layer 0.89 mm. Cu). Hormonal bursectomy was attained by the dipping of 2-day-old eggs into 2% testosterone-propionate ethanol solution for 5 seconds. One week after hatching, hormonally bursectomized chickens were examined for the lack of bursa Fabricius. Post mortem examinations were carried out on all experimental chickens. Sensitization: Forty-day-old chickens were injected with 8 Lf diphtheria toxoid (1818 Lf/mg. protein nitrogen) in Freund's complete adjuvant (Difco) in the foot pad. Delayed wattle reaction: On the 7th after sensitization, 2 Lf diphtheria toxoid in 0.1 ml. of phosphate buffered saline containing 1% normal chicken serum were injected intradermally into one wattle. The other wattle of the same chicken served as a control and was injected with phosphate buffered saline containing 1% normal chicken serum. When the thickness of the antigen injected wattle exceeded that of the
1133
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 11, 2015
ABSTRACT The migration of splenic cells from hormonally bursectomized chickens was inhibited by the antigen. On the other hand, the migration of splenic cells from thymectomized-irradiated chickens was not inhibited. The migration inhibition test of splenic cells is dependent on a function of thymusdependent cells.
1134
C. MOEITA AND M . SOEKAWA
Migration Index Migration in the presence of antigen Migration without antigen X 100 RESULTS
The results of the experiment are summarized in Table 1. In the non-sensitized group and in the sensitized, thymectomized-irradiated group, chickens were negative in delayed wattle reaction. In the X-irradiated and hormonally bursectomized group, chickens were positive in this reaction. But 2 out of 4 untreated, sensitized chickens were negative in this test. The results of the SMIT agreed well
with those of the delayed wattle reaction. The migration distances of the non-antigen control were not significantly different among each experimental group. The migration indexes of non-sensitized chickens and sensitized, thymectomized-irradiated chickens were more than 80. On the other hand, the migration indexes of other sensitized chickens were less than 75. The difference was significant at 1% level. No significant difference was observed among the groups of sensitized chickens except thymectomized-irradiated group. Migration indexes of 3 non-sensitized chickens were within a standard deviation of migration indexes of thymectomized sensitized chickens. DISCUSSION
In our previous report (Morita and Soekawa, 1971) it was revealed that the results of the SMIT were in satisfactory agreement with those of the delayed wattle reaction. Presently in the chicken, the wattle test is one of the methods available to determine cell-mediated immune responses against antigens. However, as seen in our studies this test can only be used when chickens have wattles of sufficient size. Recently, Warner et al. (1971) reported the use of capillary migration of peritoneal exudate cells for the studies of the delayed hypersensitivity in normal and bursectomized chickens. They observed the inhibition of migration of peritoneal cells by antigen in normal sensitized chickens. However, they could not obtain the sufficient numbers of peritoneal exudate cells from bursectomized chickens. In the present study, no significant difference was observed among the migration distances without antigen in each group of chickens. The migration distances obtained in the present study were less than those obtained in our previous study. This dis-
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 11, 2015
control wattle by 0.4 mm. in thickness after 24 hours the reaction was considered positive. Migration inhibition test of splenic cells: SMIT was carried out on the 8th day after sensitization. Spleens were removed from chickens and minced into about 1 mm. cubic fragments. Three fragments of each spleen were placed separately in 0.5 ml. of normal chicken plasma in a Petri dish and then clotted by the addition of 0.5 ml. of chicken embryo extract. Several minutes later, 3 ml. of Eagle's minimum essential medium supplemented by 20% bovine serum were added with or without 20 Lf diphtheria toxoid. At the 3rd day after cultivation, culture medium was changed with freshly prepared medium containing the same concentration of antigen as the previous medium. At the 4th day after cultivation, the distance of cellular migration from the edge of the fragment was measured by an ocular micrometer. In this test, the 35 ocular micrometer units correspond to 1 mm. length. Nine fragments were employed for each experiment. The migration index was calculated according to the following formula:
1135
SPLEEN CELLS MIGRATION INHIBITION TEST TABLE 1.—Delayed wattle reaction and migration inhibition test oj splenic cells • (SMIT) for diphtheria toxoid (DT) Migration Inhibition Test
Wattle Reaction Exp. Group
3
Chicken DT Control
Sensitized
DT
MD Without Antigen
Mean±SD6
MI 4
l . l 1 1.3 16.9 1.1 1.1 16.3 24.1 ND 2 1.1 1.1 28.1 24.1 ND 14.2 ND
20.8 18.6 22.1 30.0 26.2 14.1
81.3 87.6 109.0 93.7 92.0 100.7
y4.U3± y.Vo —
X-irradiated
1 2 3 4 5
1.4 1.8 1.7 1.4 1.6
1.0 11.8 1.3 19.2 1.1 11.1 0.9 15.4 1.1 19.9
22.7 30.0 16.8 26.9 31.3
52.0 64.0 66.1 57.2 63.6
60.58+ 5.84—
Hormonally bursectomized
1 2 3 4 5
1.6 1.9 1.7 1.6 1.7
1.2 22.0 1.2 25.9 1.1 8.9 1.0 29.4 1.3 18.2
46.7 34.7 15.2 42.5 26.2
47.1 74.6 58.6 69.2 69.5
Untreated
1 2 3 4 5
ND 19.8 1.6 1.2 28.7 1.7 1.3 24.8 1.0 0.7 18.3 1.0 0.7 10.8
27.7 38.4 38.7 27.8 IS.9
71.5 74.7 64.1 65.8 67.9
1 2
ND 0.8 0.7
14.0 15.6
14.2 17.2
98.6 90.7
1 2
1.2 1.6
1.1 27.8 1.7 41.0
25.2 47.3
110.3 86.7
S**
Non-Sensitized Thymectomized-irradiated Untreated
Q**
1
Thickness of wattle (mm.). The wattle was not suitable for the test because its size was not sufficient. Distance of cell migration in ocular micrometer units. * Migration index. 5 Standard deviation. ** Significant difference (P<0.01). 2 3
crepancy might come from the experimental condition such as the difference in the methods introduced in the SMIT and the age of chickens. The migration of splenic cells from the hormonally bursectomized chickens was inhibited by the antigen, whereas, the migration from the thymectomized-irradiated chickens was not inhibited. From the experimental results it is suggested that the mechanism of SMIT is dependent on the function of thymus depen-
dent cells and SMIT is an adequate indicator of cell-mediated immunity in the chicken. ACKNOWLEDGEMENT
The authors wish to express their gratitude to Dr. Y. Yamaguchi, Department of Microbiology, Faculty of Medicine, Nippon University, for his helpful guidance on the method of thymectomy and bursectomy.
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 11, 2015
Thymectomized-irradiated
1 2 3 4 5 6
1136
C. MORITA AND M . SOEKAWA
REFERENCES
NEWS AND NOTES (Continued from page 1132) animal health products and vaccines, professional veterinary products and agricultural chemicals throughout the world. BABCOCK NOTES A new company to handle the export of its products as well as those produced by other affiliated companies has been formed by Babcock Poultry Farm, Inc., Ithaca, New York. The new company, Babcock International Corporation, is a Domestic International Sales Corporation (DISC) as defined by the Internal Revenue Service. The officers are: President—B. M. Babcock, Vice President—W. R. Todd, Treasurer—H. B. Rumph, Jr., and Secretary—J. J. Clynes, Jr. Babcock International Corporation is a wholly owned subsidiary of Babcock Poultry Farm, Inc., with headquarters at Ithaca, New York. Its products for export include poultry breeding stock, eggs, vaccine, and swine breeding stock. SOUTHEASTERN POULTRY CLUB The Southeastern Poultry Science Club was formed by college students attending the Annual College Youth Program in January at the Southeastern-International Poultry Convention. The new organization will promote youth leadership in the poultry industry, attempt to stimulate industry, attempt to stimulate interest and give recognition to the outstanding future leaders of the industry.
Donald Wilburn, a senior at the University of Georgia, who is majoring in poultry, was elected President. Other officers appointed to serve until the election next year are: Jacque Atkins, North Carolina, and Scott Varner, Mississippi State University. The group will meet with Southeastern's Youth Committee to plan activities for next year's convention. A quarterly newsletter is planned to inform industry leaders about the graduates and to assist in finding them jobs. Tyron Spearman, Director of Public Relations, Abraham Baldwin Agricultural College, has been working on the idea of a new club for two years. He, Kirk Hale, University of Georgia, and Ralph Mobley, Chairman of Southeastern's Youth Committee, will serve as Advisors to the Club during the first year. TENNESSEE NOTES Dr. Sam L. Hansard has been named Acting Head of the Department of Animal Husbandry— Veterinary Science at the University of Tennessee. A departmental reorganization is taking place which will combine Animal Husbandry, Poultry Science and Dairy Science, with milk and meat processing going to Food Technology. CLEMSON NOTES The interdisciplinary nutrition program for graduate education, including the Departments of
(Continued on page 1174)
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 11, 2015
Cooper, M. D., R. D. A. Peterson, M. A. South and R. A. Good, 1966. The functions of the thymus system and the bursa system in the chicken. J. Exp. Med. 12: 75-102. Glick, B., T. S. Chang and R. G. Jaap, 1956. The bursa of Fabricius and antibody production. Poultry Sci. 35 : 224-225. Jankovic, B. D., M. Isvaneski, D. Milosevic and L. Popenskovic, 1963. Delayed hypersensitive reaction in bursectomized chickens. Nature, 198: 298-299. Lerner, K. G., B. Glick and F. C. McDuffie, 1971. Role of the bursa 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: 493-503. Morita, C , and M. Soekawa, 1971. Specific inhibi-
tion of splenic cells migration in chicken sensitized to delayed hypersensitivity. Poultry Sci. 50: 1503-1505. Svejcar, J., J. Johanovsky and J. Pekarek, 1971. In vitro technique of the cell migration from the spleen and/or artificial fragment. In: In Vitro Methods in Cell-mediated Immunity, pp. 263-272, edited by Bloom, B. R., and P. R. Glade, Academic Press, New York and London. Warner, N. L., J. W. Uhr, G. J. Thorbecke and Z. Ovary, 1969. Immunoglobulins, antibodies and the bursa of Fabricius: Induction of agammaglobulinemia and the loss of all antibody-forming capacity by hormonal bursectomy. J. Immunol. 103 : 1317-1330. Warner, S. L., Z. Ovary and F. S. Kantor, 1971. Delayed hypersensitivity reactions in normal and bursectomized chickens. Int. Arch. Allergy, 40: 719-728.
POULTRY SCIENCE 51: 1133-1136, 1972
INTRODUCTION
IGRATION inhibition test of splenic cells (SMIT) in mammals has been described as an in vitro indicator of delayed type hypersensitivity (Svejcar et al., 1971). However, there is no report concerning the effect of thymectomy on SMIT in chickens. Surgical thymectomy of chickens immediately after hatching, followed by sublethal X-irradiation reduces cell-mediated immunity such as delayed wattle reaction, homograft rejection and graft versus host reaction (Cooper et al., 1966). Glick et al. (1956) described the role of the bursa Fabricius on the development of humoral immunity in chickens. It is now established that hormonal bursectomy generally eliminates IgG immunoglobulins and reduces the antibody response to most antigens (Warner et al., 1969; Lerner et al., 1971). But this treatment does not significantly influence the cell-mediated immunity (Jankovic et al., 1963; Warner et al., 1971). Inhibition of splenic cell migration by the antigen in sensitized chickens has already been evidenced by the authors (Morita and Soekawa, 1971). The present study is intended to examine the influence of thymectomy and bursectomy on SMIT.
M
MATERIALS AND METHODS
Chickens: Goto strain 202 (Goto Hatch-
ery Inc.) of non-inbred White Leghorn chickens were used throughout the experiment. Thymectomy and bursectomy: Surgical thymectomy was carried out on the day of hatching. X-irradiation was given on the day after hatching. The dosage of X-irradiation was 650 R (200 kV, 20 mA, with 0.3 mm. copper and 1.0 mm. aluminum filter, half value layer 0.89 mm. Cu). Hormonal bursectomy was attained by the dipping of 2-day-old eggs into 2% testosterone-propionate ethanol solution for 5 seconds. One week after hatching, hormonally bursectomized chickens were examined for the lack of bursa Fabricius. Post mortem examinations were carried out on all experimental chickens. Sensitization: Forty-day-old chickens were injected with 8 Lf diphtheria toxoid (1818 Lf/mg. protein nitrogen) in Freund's complete adjuvant (Difco) in the foot pad. Delayed wattle reaction: On the 7th after sensitization, 2 Lf diphtheria toxoid in 0.1 ml. of phosphate buffered saline containing 1% normal chicken serum were injected intradermally into one wattle. The other wattle of the same chicken served as a control and was injected with phosphate buffered saline containing 1% normal chicken serum. When the thickness of the antigen injected wattle exceeded that of the
1133
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 11, 2015
ABSTRACT The migration of splenic cells from hormonally bursectomized chickens was inhibited by the antigen. On the other hand, the migration of splenic cells from thymectomized-irradiated chickens was not inhibited. The migration inhibition test of splenic cells is dependent on a function of thymusdependent cells.
1134
C. MOEITA AND M . SOEKAWA
Migration Index Migration in the presence of antigen Migration without antigen X 100 RESULTS
The results of the experiment are summarized in Table 1. In the non-sensitized group and in the sensitized, thymectomized-irradiated group, chickens were negative in delayed wattle reaction. In the X-irradiated and hormonally bursectomized group, chickens were positive in this reaction. But 2 out of 4 untreated, sensitized chickens were negative in this test. The results of the SMIT agreed well
with those of the delayed wattle reaction. The migration distances of the non-antigen control were not significantly different among each experimental group. The migration indexes of non-sensitized chickens and sensitized, thymectomized-irradiated chickens were more than 80. On the other hand, the migration indexes of other sensitized chickens were less than 75. The difference was significant at 1% level. No significant difference was observed among the groups of sensitized chickens except thymectomized-irradiated group. Migration indexes of 3 non-sensitized chickens were within a standard deviation of migration indexes of thymectomized sensitized chickens. DISCUSSION
In our previous report (Morita and Soekawa, 1971) it was revealed that the results of the SMIT were in satisfactory agreement with those of the delayed wattle reaction. Presently in the chicken, the wattle test is one of the methods available to determine cell-mediated immune responses against antigens. However, as seen in our studies this test can only be used when chickens have wattles of sufficient size. Recently, Warner et al. (1971) reported the use of capillary migration of peritoneal exudate cells for the studies of the delayed hypersensitivity in normal and bursectomized chickens. They observed the inhibition of migration of peritoneal cells by antigen in normal sensitized chickens. However, they could not obtain the sufficient numbers of peritoneal exudate cells from bursectomized chickens. In the present study, no significant difference was observed among the migration distances without antigen in each group of chickens. The migration distances obtained in the present study were less than those obtained in our previous study. This dis-
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 11, 2015
control wattle by 0.4 mm. in thickness after 24 hours the reaction was considered positive. Migration inhibition test of splenic cells: SMIT was carried out on the 8th day after sensitization. Spleens were removed from chickens and minced into about 1 mm. cubic fragments. Three fragments of each spleen were placed separately in 0.5 ml. of normal chicken plasma in a Petri dish and then clotted by the addition of 0.5 ml. of chicken embryo extract. Several minutes later, 3 ml. of Eagle's minimum essential medium supplemented by 20% bovine serum were added with or without 20 Lf diphtheria toxoid. At the 3rd day after cultivation, culture medium was changed with freshly prepared medium containing the same concentration of antigen as the previous medium. At the 4th day after cultivation, the distance of cellular migration from the edge of the fragment was measured by an ocular micrometer. In this test, the 35 ocular micrometer units correspond to 1 mm. length. Nine fragments were employed for each experiment. The migration index was calculated according to the following formula:
1135
SPLEEN CELLS MIGRATION INHIBITION TEST TABLE 1.—Delayed wattle reaction and migration inhibition test oj splenic cells • (SMIT) for diphtheria toxoid (DT) Migration Inhibition Test
Wattle Reaction Exp. Group
3
Chicken DT Control
Sensitized
DT
MD Without Antigen
Mean±SD6
MI 4
l . l 1 1.3 16.9 1.1 1.1 16.3 24.1 ND 2 1.1 1.1 28.1 24.1 ND 14.2 ND
20.8 18.6 22.1 30.0 26.2 14.1
81.3 87.6 109.0 93.7 92.0 100.7
y4.U3± y.Vo —
X-irradiated
1 2 3 4 5
1.4 1.8 1.7 1.4 1.6
1.0 11.8 1.3 19.2 1.1 11.1 0.9 15.4 1.1 19.9
22.7 30.0 16.8 26.9 31.3
52.0 64.0 66.1 57.2 63.6
60.58+ 5.84—
Hormonally bursectomized
1 2 3 4 5
1.6 1.9 1.7 1.6 1.7
1.2 22.0 1.2 25.9 1.1 8.9 1.0 29.4 1.3 18.2
46.7 34.7 15.2 42.5 26.2
47.1 74.6 58.6 69.2 69.5
Untreated
1 2 3 4 5
ND 19.8 1.6 1.2 28.7 1.7 1.3 24.8 1.0 0.7 18.3 1.0 0.7 10.8
27.7 38.4 38.7 27.8 IS.9
71.5 74.7 64.1 65.8 67.9
1 2
ND 0.8 0.7
14.0 15.6
14.2 17.2
98.6 90.7
1 2
1.2 1.6
1.1 27.8 1.7 41.0
25.2 47.3
110.3 86.7
S**
Non-Sensitized Thymectomized-irradiated Untreated
Q**
1
Thickness of wattle (mm.). The wattle was not suitable for the test because its size was not sufficient. Distance of cell migration in ocular micrometer units. * Migration index. 5 Standard deviation. ** Significant difference (P<0.01). 2 3
crepancy might come from the experimental condition such as the difference in the methods introduced in the SMIT and the age of chickens. The migration of splenic cells from the hormonally bursectomized chickens was inhibited by the antigen, whereas, the migration from the thymectomized-irradiated chickens was not inhibited. From the experimental results it is suggested that the mechanism of SMIT is dependent on the function of thymus depen-
dent cells and SMIT is an adequate indicator of cell-mediated immunity in the chicken. ACKNOWLEDGEMENT
The authors wish to express their gratitude to Dr. Y. Yamaguchi, Department of Microbiology, Faculty of Medicine, Nippon University, for his helpful guidance on the method of thymectomy and bursectomy.
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 11, 2015
Thymectomized-irradiated
1 2 3 4 5 6
1136
C. MORITA AND M . SOEKAWA
REFERENCES
NEWS AND NOTES (Continued from page 1132) animal health products and vaccines, professional veterinary products and agricultural chemicals throughout the world. BABCOCK NOTES A new company to handle the export of its products as well as those produced by other affiliated companies has been formed by Babcock Poultry Farm, Inc., Ithaca, New York. The new company, Babcock International Corporation, is a Domestic International Sales Corporation (DISC) as defined by the Internal Revenue Service. The officers are: President—B. M. Babcock, Vice President—W. R. Todd, Treasurer—H. B. Rumph, Jr., and Secretary—J. J. Clynes, Jr. Babcock International Corporation is a wholly owned subsidiary of Babcock Poultry Farm, Inc., with headquarters at Ithaca, New York. Its products for export include poultry breeding stock, eggs, vaccine, and swine breeding stock. SOUTHEASTERN POULTRY CLUB The Southeastern Poultry Science Club was formed by college students attending the Annual College Youth Program in January at the Southeastern-International Poultry Convention. The new organization will promote youth leadership in the poultry industry, attempt to stimulate industry, attempt to stimulate interest and give recognition to the outstanding future leaders of the industry.
Donald Wilburn, a senior at the University of Georgia, who is majoring in poultry, was elected President. Other officers appointed to serve until the election next year are: Jacque Atkins, North Carolina, and Scott Varner, Mississippi State University. The group will meet with Southeastern's Youth Committee to plan activities for next year's convention. A quarterly newsletter is planned to inform industry leaders about the graduates and to assist in finding them jobs. Tyron Spearman, Director of Public Relations, Abraham Baldwin Agricultural College, has been working on the idea of a new club for two years. He, Kirk Hale, University of Georgia, and Ralph Mobley, Chairman of Southeastern's Youth Committee, will serve as Advisors to the Club during the first year. TENNESSEE NOTES Dr. Sam L. Hansard has been named Acting Head of the Department of Animal Husbandry— Veterinary Science at the University of Tennessee. A departmental reorganization is taking place which will combine Animal Husbandry, Poultry Science and Dairy Science, with milk and meat processing going to Food Technology. CLEMSON NOTES The interdisciplinary nutrition program for graduate education, including the Departments of
(Continued on page 1174)
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 11, 2015
Cooper, M. D., R. D. A. Peterson, M. A. South and R. A. Good, 1966. The functions of the thymus system and the bursa system in the chicken. J. Exp. Med. 12: 75-102. Glick, B., T. S. Chang and R. G. Jaap, 1956. The bursa of Fabricius and antibody production. Poultry Sci. 35 : 224-225. Jankovic, B. D., M. Isvaneski, D. Milosevic and L. Popenskovic, 1963. Delayed hypersensitive reaction in bursectomized chickens. Nature, 198: 298-299. Lerner, K. G., B. Glick and F. C. McDuffie, 1971. Role of the bursa 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: 493-503. Morita, C , and M. Soekawa, 1971. Specific inhibi-
tion of splenic cells migration in chicken sensitized to delayed hypersensitivity. Poultry Sci. 50: 1503-1505. Svejcar, J., J. Johanovsky and J. Pekarek, 1971. In vitro technique of the cell migration from the spleen and/or artificial fragment. In: In Vitro Methods in Cell-mediated Immunity, pp. 263-272, edited by Bloom, B. R., and P. R. Glade, Academic Press, New York and London. Warner, N. L., J. W. Uhr, G. J. Thorbecke and Z. Ovary, 1969. Immunoglobulins, antibodies and the bursa of Fabricius: Induction of agammaglobulinemia and the loss of all antibody-forming capacity by hormonal bursectomy. J. Immunol. 103 : 1317-1330. Warner, S. L., Z. Ovary and F. S. Kantor, 1971. Delayed hypersensitivity reactions in normal and bursectomized chickens. Int. Arch. Allergy, 40: 719-728.