Enhanced Immunosuppression in Bursectomized Chickens by Passive Transfer of Antibody 12

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1 WINDOWS IN EGG SHELLS 1203 ganic phases of the shell (Talbot and Taylor, 1974). REFERENCES Denison, J. W., The effect of mechanical disturbance of the egg cuticle on shell mottling. Poultry Sci. 46: Dorminey, R. W., J. E. Jones and H. R. Wilson, Influence of cage size and frightening on incidence of body checked eggs. Poultry Sci. 44: Jones, B., Exterior egg quality factors. Paper INTRODUCTION THE suppression of immune responses has received considerable attention in recent years. The increased interest in organ transplantations coupled with mounting knowledge of autoimmune diseases largely accounts for this interest. The chicken has played a pivotal role in this area of research because it is 1. Paper number 4524 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina. 2. A preliminary report of part of this paper was presented at the 63rd annual meeting of the Poultry Science Association, Morgantown, West Virginia. presented at S.P.E.A.-U.S.D.A. Egg Quality and Grading School, June 26-28, 1973, St. Petersburg Beach, FL. Roland, D. A., Sr., Do your eggs have windows? Proc. 32nd Annual Florida Poultry Institute, pp Talbot, C. J., and C. Tyler, A study of the fundamental cause of artificial translucent areas in egg shells. Br. Poultry Sci. 15: Tyler, C, and N. Standen, The artificial production of translucent streaks on egg shells and various factors influencing their development. Br. Poultry Sci. 10: Enhanced Immunosuppression in Bursectomized Chickens by Passive Transfer of Antibody 12 P. S. YOUNG, J. BRAKE, P. THAXTON, G. W. MORGAN, JR. AND F. W. EDENS Department of Poultry Science, North Carolina State University, Raleigh, North Carolina (Received for publication November 12, 1974) ABSTRACT Three trials were conducted using commercial broiler cockerels to determine the immunological consequences of passive transfers of immune sera to bursectomized chickens. The data illustrate that passive transfers of immune sera to juvenile chickens which had been surgically bursectomized immediately after hatching caused a suppression of the primary hemagglutination response which was greater than that caused by bursectomy or passive transfer of immune sera alone. However, when these same birds were given a secondary challenge of antigen the resulting secondary hemagglutination response was normal. These data indicate that the passive transfer of immune sera to bursectomized chickens greatly limits their ability to mount primary humoral responses, while not affecting the ability to develop anamnestic immunity. POULTRY SCIENCE 54: , 1975 an excellent model for immunological studies. The morphological and immunological clarity of the two known limbs of immunity, i.e. the humoral and cellular systems, are well known in the chicken. The B-cell system, associated with humoral immune responses, and the T-cell system, which is responsible for cellular immunity, are clearly related to the bursa of Fabricius and thymus, respectively (Cooper et al, 1966; Warner, 1967). Primary humoral immune responses in chickens are suppressed by surgical or chemical ablation of the bursa of Fabricius (Glick et al, 1956; Glick and Sadler, 1961) and by the passive transfer of specific immune sera prior to homologous antigen challenge (Thax-

2 1204 P. S. YOUNG, J. BRAKE, P. THAXTON, G. W. MORGAN, JR. AND F. W. EDENS ton and Young, 1974). However, Rose and Orlans (1968) reported a normal secondary immune response in surgically bursectomized chickens which were challenged with sheep erythrocytes or a soluble protein antigen. Although humoral immune responses in chickens are not eliminated entirely by bursectomy and not always by passive transfer of immune sera, the question remains whether these immunosuppressive methods, when applied in combination, will result in complete suppression of humoral immunity. Thus, the objective of the present investigation was to study primary and secondary immune responses in chickens which were bursectomized and then given passive transfers of immune sera. MATERIALS AND METHODS Three trials were conducted using broiler cockerels (Cobb x Arbor Acre). The chickens were housed in non-heated metal brooding batteries through four weeks of age. A brooding source of heat was not provided; however, the ambient room temperature was maintained at approximately 29.4 C. At four weeks of age the birds were transferred to growing cages, and the ambient temperature was lowered to 23.9 C. A starter-grower ration which met or exceeded the minimum nutritional requirements was formulated. Feed and water were available ad libitum throughout the experimental period. Non-immune (NIS) and immune (IS) sera for passive administration were collected from Cobb x Arbor Acre birds not included in the aforementioned trials. Birds were immunized with sheep red blood cells (SRBC) and seven days later the serum samples were collected and titered individually by a microhemagglutination procedure (Thaxton et al., 1971). The samples were pooled in the manner described previously (Thaxton and Young, 1974). NIS was collected in the same manner as the IS, except that the birds were not immunized. The mean log 2 titer of the pooled IS for all three trials was 7.5, while the NIS log 2 titer was 1.0. These sera were stored at -20 C. until needed. An antigen, which consisted of 1 ml. of a 15% saline suspension of SRBC, was employed exclusively in this study. All bleedings and injections were by venipuncture of the brachial veins. Additionally, the serum samples were prepared by allowing the blood to clot at room temperature, then incubating the samples for two hours at 37 C, and finally maximal serum yields were obtained by refrigerating the samples at 4 C. for at least two hours. Anti-SRBC antibody levels were determined by micro hemagglutinations. Trials 1 and 2. Each trial consisted of three groups of 50 chicks. One group of birds was surgically bursectomized (BSX) within 6 hours after hatching by the method of Glick (1960). A second group of chicks was sham bursectomized (SBSX) and a third group was maintained as non-operated controls (C). At four weeks of age 20 birds were selected from each of the three groups. The chicks were bled and the resulting serum samples were assayed individually to determine the levels of naturally occurring anti-srbc antibodies. Immediately following the pre-immunization bleeding, half of the birds in each of the three treatment groups were given 3 ml. of either IS or NIS intravenously. Twenty-four hours following the passive serum transfers each bird received a primary antigen challenge. Seven days following the challenge 2.5 ml. of blood was collected from each bird. Serum from each sample was collected and stored at 20 C. Prior to antibody titration the serum samples were heat inactivated in a water bath at 56 C. for 30 minutes to destroy the complement activity. The anti-srbc hemagglutinin levels were then determined serologically. Five weeks following the primary SRBC challenge the birds were bled again and given

3 BURSECTOMY AND IMMUNOSUPPRESSION 1205 a secondary SRBC challenge. A five week period has been indicated as necessary to allow dissipation of the primary anti-srbc hemagglutination response in chickens (Thaxton and Siegel, 1972). Following the secondary challenge, the birds were bled at weekly intervals for four consecutive weeks. Serum samples which were collected from the pre-treatment bleeding, as well as from the weekly bleedings, were assayed serologically to assess the secondary hemagglutination responses. Trial 3. At hatching 10 chicks were bursectomized and assigned to two groups of five birds each. An additional 10 chicks were assigned to two control groups of five birds FIG. 1. Anti-SRBC antibody levels produced by the chickens of Trials 1 and 2 at 7 days post-antigen challenge. BSX, SBSX and C represent surgical bursectomy, sham bursectomy and no surgery at hatching, while IS and NIS indicate that 3 ml. of immune or non-immune sera, respectively, were transferred passively 24 hours prior to challenge with SRBC. each. At four weeks of age all birds were bled to determine the levels of naturally occurring antibodies to SRBC. Following this bleeding the two groups of BSX birds were given 3 ml. of IS. In addition, one group of the BSX birds was given a primary challenge of SRBC 24 hours following the passive transfer of IS. The other group of BSX birds did not receive an antigen challenge. The two groups of non-operated controls were given 3 ml. of either IS or NIS. The control group which was treated with NIS was given a SRBC challenge and the other control group which was treated with IS was not challenged with SRBC. The birds were bled at two day intervals for eight days and then at 12 days following the antigen challenge to assess a time course evaluation of the primary hemagglutination response. Antibody titers were converted to log 2 values to facilitate statistical comparisons (Ambrose and Donner, 1973). Treatment differences were partitioned by analysis of variance and the means were compared by Kramer's modification of Duncan's new multiple range test (Kramer, 1956). Statements of significance are based on P s RESULTS Trials 1 and 2. The data of these two replicate trials did not exhibit significant replication effects, and therefore they were combined for collective presentation. The seven day mean anti-srbc primary antibody titers are presented in Figure 1. The SBSX and C birds which were given transfers of immune sera showed mean anti-srbc antibody levels which were reduced significantly when compared to the SBSX and C birds which received NIS. It is apparent that these birds experienced antibody mediated immunosuppression. The BSX birds which received NIS exhibited significantly reduced primary responses as compared to the SBSX and C birds which were given NIS. However, the

4 1206 P. S. YOUNG, J. BRAKE, P. THAXTON, G. W. MORGAN, JR. AND F. W. EDENS TABLE 1. Relationship of passively transferred antibody and bursectomy to secondary immunity' Treatment 0 Control + non-immune sera Control + immune sera Sham bursectomized + non-immune sera Sham bursectomized + immune sera Bursectomized + non-immune sera Bursectomized + immune sera 1.1 ± 1.1 ± ± ±.2 1 ± 1.1 ± Bleeding time (days post-srbc challenge) ± 6.9 ± 7.2 ± 6.8 ± 6 ± 7 ± ± 4.9 ±.2 5 ± ± ± 5.1 ± ± 4.1 ± ± 3 ± 3.9 ± ±.5 'Each mean (± S.E.M.) represents 20 birds. Statistical differences in the means of the treatment groups (within columns) did not occur ± 3.6 ± ±.2 3 ± ± 4.2 ±.5 TABLE 2. Mean anti-srbc antibody levels (± S.E.M.) of the primary responses of the chickens of Trial 3'- 2 Bleeding time (days post-srbc challenge) Treatment Bursectomized + immune sera + SRBC 0.0 ±.0 a 0 ±.2 a 0.6 ±.6 a 0.8 ±.6 a 1.2 ±,4 a 0.6 ±.2 a Control + non-immune sera + SRBC 0.0 ±.0 a 1.0 ± " 4.0 ±.5 b 5.8 ±.8 b 5.2 ±.9 b 2.8 ±.5 b Bursectomized + immune sera + no SRBC 0.0 ±.0 a 1.0 ±.0 a 0.0 ±.0 a 0.0 ±.0 a 0.0 ±.0 a 0.4 ±.2 a Control + immune sera + no SRBC 0.0 ±.0 a 1 ±.7 a 0.8 ±.5 a 0.8 ± a 0.0 ±.0 a 0.0 ±.0 a 1 Each mean represents 5 birds. 2 Means in a column possessing different superscripts differ significantly at P most notable finding in this segment of the pressed the primary hemagglutination restudy was that the BSX birds which were sponse to SRBC in the chickens. This result given IS exhibited primary anti-srbc levels was apparent when comparisons were made which were significantly lower than all the to the control birds which received NIS and other groups. SRBC. It is noteworthy that in the BSX and The data illustrating the effects of bursec- C birds which received IS in the absence of tomy and passive sera transfers on the sec- an antigen challenge only minimal levels of ondary hemagglutination responses are pre- anti-srbc antibody were found at any time sented in Table 1. Neither bursectomy nor during the 12 day period following the passive passive transfers of IS administered prior to transfers. These data suggest that the pasthe primary challenge were found to alter sively transferred antibody may not persist significantly the secondary hemagglutination in circulation in appreciable quantities. response. In fact, there were no significant differences among the treatment groups during the period in which the measurements were made. DISCUSSION The early classic work by Glick et al. (1956) suggested that neonatal bursectomy limited Trial 3. The data of this trial as presented the ability of juvenile chickens to express in Table 2 confirm and extend the finding humoral responses. Several reports have in Trials 1 and 2. Bursectomy in combination confirmed this original finding (Rose and with passive transfer of immune sera sup- Orlans, 1968; Bryant et al., 1973). However,

5 BURSECTOMY AND IMMUNOSUPPRESSION 1207 in all these reports bursectomized chickens which were immunologically deficient did exhibit a limited ability to express humoral antibody. Ivanyi (1970) and Thaxton and Young (1974) have demonstrated in chickens that the passive transfer of specific immune sera suppressed the ability of chickens to respond immunologically when a homologous antigen challenge was administered. The data of the present report indicate that antibody mediated immunosuppression of primary humoral immune responses in BSX chickens is more pronounced than in chickens which possessed intact bursae. Potential antibody producing cells, i.e. B- cells, give rise to two populations of cells when subjected to an antigen challenge. These two populations are the immunocompetent cells, which are responsible for primary antibody production and the memory cells, which function during anamnestic responses (Rowley et al., 1974). Several theories have been proposed to explain the mechanism of action of antibody mediated immunosuppression. However, the exact site of action of passively transferred antibody in antibody mediated suppression remains debatable. The theory of peripheral block whereby unprocessed antigen complexes with the passively transferred antibody to cause immunosuppression has been advanced. This theory has received criticism from many investigators because it fails to explain the condition in which non-proportional quantities of antigen and antibody exist (Ryder and Schwartz, 1969; Haughton and Adams, 1970; Rowley et al, 1973; Koros and Hamill, 1973). Ryder and Schwartz (1969) suggested that antibody inhibition occurs at the macrophage step in the immunization process. Alternatively, Rowley and Fitch (1964) suggested that passively transferred antibody acts at the level of the B-cell receptor site thus blocking antigen recognition and further antibody synthesis. Rowley and Fitch (1964) postulated that those receptor sites which are not blocked by the passively transferred antibody are able to actively synthesize antibody in the presence of an antigen. This minute quantity of actively synthesized antibody feeds back to the B-cell receptor site and functions concomitantly with the passively transferred antibody to enhance the suppressive effect. Rowley et al. (1969) suggested that antibody mediated suppression results from a reduction in activity of the initial numbers of antigentically respondent cells, rather than a reduced rate of cellular proliferation. Surgical bursectomy during the early production of B-cells also is involved in the reduction of antigentically respondent cells. Therefore, our finding of an additive degree of suppression when both bursectomy and passive transfer of antibody were employed simultaneously is explainable. The ablation of the bursa of Fabricius in day-old chicks removes the major population of B-cells responsible for antibody production. The remainder of the B-cell population which is not surgically removed and has specific receptor sites for the passive antibody may also be reduced in activity by treatment with IS. Therefore, the limiting factor in antibody production appears to be centered around B-cells, whether these B-cells are precursor cells, differentiating cells, or immunocompetent cells capable of antibody synthesis. Thus, it appears evident that in BSX birds treated with IS the primary humoral response is almost completely suppressed due to a deficiency of antibody synthesis by a limited population of B-cells. In Trial 3, where quantitative serological methods were utilized to determine the levels of passively administered anti-srbc antibodies in birds treated with IS and no antigen, only minimal levels of antibody were detected (Table 2). Leslie and Clem (1970) demonstrated that the biological half lives of the major chicken immunoglobulins range from one to four days. Therefore, the serologically demonstrable antibody observed in the birds

6 1208 P. S. YOUNG, J. BRAKE, P. THAXTON, G. W. MORGAN, JR. AND F. W. EDENS treated with IS and SRBC may have been a measure of both passively transferred and actively synthesized antibody. However, the majority of the immunoglobulins expressed in the primary response of the birds treated with IS and SRBC were actively synthesized antibody. This conclusion is supported by comparing the antibody levels of birds which were treated with IS and antigen to those which were treated with IS and no antigen. Minute quantities of a biologically active form of the passively transferred antibody must exist to be effective in causing antibody mediated suppression of the primary response (Ryder and Schwartz, 1969). Secondary responses are considerably more difficult to suppress than primary responses (Uhr and Baumann, 1961). Secondary responses are reported to be suppressed when large quantities of high avidity antibody are utilized in the passive transfers at the time of secondary immunization (Uhr and Moller, 1968). Rose and Orlans (1968) reported normal secondary hemagglutinin responses in BSX chickens. Therefore, the finding of a normal secondary response in BSX chickens treated with IS was not unexpected, since treatment with passively transferred antibody occurred prior to the primary challenge. It would be interesting to study the secondary responses of BSX chickens which were treated with IS prior to the secondary challenge. AC KNOWLEDGEMENTS The technical assistance of Ms. Jeannine Gilbert and Ms. Grace Brockman are greatly appreciated. REFERENCES Ambrose, C. T., and A. Donner, Application of the analysis of variance to hemagglutination titrations. J. Immunol. Meth. 3: Bryant, B. J., H. E. Adler, D. R. Cordy, M. Shifrine and A. J. Damassa, The avian bursa-independent humoral immune system: Serologic and morphologic studies. Europ. J. Immunol. 3: Cooper, M. D., R. D. A. Peterson, M. A. South and R. A. Good, The functions of the thymus system and bursa system in the chicken. J. Exp. Med. 123: Glick, B., Growth of the bursa of Fabricius and its relationship to the adrenal glands in the White Pekin duck, White Leghorn, outbred and inbred New Hampshire. Poultry Sci. 39: Glick, B., T. S. Chang and R. G. Japp, The bursa of Fabricius and antibody production. Poultry Sci. 35: Glick, B., and C. R. Sadler, The elimination of the bursa of Fabricius and antibody production in birds from eggs dipped in hormone solutions. Poultry Sci. 40: Houghton, G., and D. O. Adams, Specific immunosuppression by minute doses of passive antibody. 2. The site of action. J. Reticulo. Soc. 7: Ivanyi, J., Cytophilic antibodies in passive antibody induced immune suppression or enhancement. Nature, 226: Koros, A. N. C, and E. C. Hamill, Mechanisms of suppression of the immune response. 1. Differences in the effect of specific inhibitory antibody on distribution of "Cr-labelled sheep erythrocytes in different mouse strains. Immunol. 25: Kramer, C. Y., Extension of multiple range tests to groups means with unequal numbers of replications. Biometrics, 12: Leslie, G. A., and L. W. Clem, Chicken immunoglobulins: Biological half lives and normal adult serum concentrations of IgM and IgY. Proc. Soc. Exp. Biol. Med. 134: Rose, M. E., and E. Orlans, Normal immune responses of bursaless chickens to a secondary antigenic stimulus. Nature, 217: 1-5. Rowley, D. A., and F. W. Fitch, Homeostasis of antibody formation in the adult rat. J. Exp. Med. 120: Rowley, D. A., F. W. Fitch, M. A. Axelrod and C. W. Pierce, The immune response suppressed by specific antibody. Immunol. 16: Rowley, D. A., F. W. Fitch, F. P. Stuart, H. Kohler and H. Casenza, Specific suppression of immune responses. Science, 181: Ryder, R. J. W., and R. S. Schwartz, Immunosuppression by antibody: Localization of site of action. J. Immunol. 103: Thaxton, P., and H. S. Siegel, Depression of secondary immunity by high environmental temperature. Poultry Sci. 51: Thaxton, P., J. E. Williams and H. S. Siegel, 1971.

7 BURSECTOMY AND IMMUNOSUPPRESSION 1209 Microtitration of Salmonella pullorum agglutinins. Avian Dis. 14: Thaxton, P., and P. S. Young, Antibody mediated suppression of the primary hemagglutination response in young chickens. Poultry Sci. 53: Uhr, J. W., and J. B. Baumann, Antibody formation. 2. The specific anamnestic antibody response. J. Exp. Med. 113: Uhr, J. W., and G. Moller, Regulatory effect of antibody on the immune response. Adv. Immunol. 8: Warner, N. L., The immunological role of the avian thymus and bursa of Fabricius. Folia Biol. 13: The Effect of Time of Day of Insemination and Oviposition on the Fertility of Turkey Hens V. L. CHRISTENSEN AND N. P. JOHNSTON Department of Animal Science, Brigham Young University, Provo, Utah INTRODUCTION R ESEARCH has indicated that the fertility of the chicken hen is significantly higher if inseminated in the afternoon as opposed to the morning (Moore and Byerly, 1942; Malmstrom, 1943; Parker, 1945; Bornstein et al., 1960; Parker and Arscott, 1965; and Johnston and Parker, 1970). However, in the case of the turkey, Parker and Barton (1946) and Harper (1949) failed to observe significant differences between morning and afternoon inseminations. Interestingly, Wyne et al. (1959) and Smyth (1968) determined that a hard-shelled egg in the uterus at the time of insemination significantly reduced fertility as compared to the absence of a shell-egg in the oviduct. A hard-shelled egg is more likely to be present during the morning than during the afternoon (Smyth, 1968). The objectives of the study were to determine the effect of the hour of insemination (Received for publication November 12, 1974) ABSTRACT Turkey hens were inseminated at five different times of the day to determine the effect of time of day of insemination and the stage of egg formation on fertility. Results showed that fertility resulting from the 6 p.m. insemination was significantly greater than at 8 a.m., 10 a.m., 1 p.m. or 3 p.m. Conversely, fertility was significantly lower at 1 p.m. than at the other four times. Fertility was significantly lower if hens were inseminated during the last 10 hours that the egg was in the uterus and during the approximate time of ovulation than during the other times of egg development. POULTRY SCIENCE 54: , 1975 and the stage of egg formation at the time of insemination on fertility. PROCEDURE Fifty caged Orlopp Medium White females were randomly assigned into five groups according to time of day of insemination (8 a.m., 10 a.m., 1 p.m., 3 p.m., or 6 p.m.). Each female, at the appropriate time of insemination, was inseminated weekly with ml. of undiluted semen. Ten Orlopp Large White breeder males were also randomly assigned into five groups of two each under floor management. Semen was collected using the massage technique and syringe. The semen was inseminated immediately after collection. All hens were inseminated at a constant depth of approximately two centimeters. The five groups of toms were rotated weekly to insure that no one group of females received semen from